WO2024241208A1 - Cd3-targeting switchable car-t cells of which cd3 gene is edited - Google Patents

Abstract

The present invention relates to: CD3-targeting switchable CAR-T cells (chimeric antigen receptor T cells) of which a CD3 gene is edited; and use thereof for treating T cell-derived tumors. By using CAR-T cells with reduced or eliminated expression of CD3 and an adapter molecule that regulates the activity of the CAR-T cells in combination, the CAR-T cells exhibit anti-fratricide ability and the ability to avoid immunodeficiency toxicity, and thus can be effectively used in a therapeutic agent for T cell-derived tumors.

Description

【Specification】

【Title of invention】

CD3 gene-edited CD3 targeting switchable CAR-T cells {CD3 targeting switchable CAR-T cells whose CD3 genes are edited}

【Technical Field】 The present invention relates to a CD3 gene-edited CD3-targeted switchable CAR-T cell (chimeric antigen receptor T cell) and its use in treating T cell-derived tumors. By using a CAR-T cell with reduced or eliminated expression of CD3 and an adapter molecule that regulates the activity of the CAR-T cell in combination, the CAR-T cell can be effectively used in treating T cell-derived tumors.

【Invention Technology】 Chimeric antigen receptor (CAR) gene T-cell therapy (CAR-T therapy) is a gene-transferred T-cell therapy that converts T cells into cells that attack the desired target by loading an antibody gene (Chimeric Antigen Receptor) that recognizes cancer antigens and injects it into the patient. Compared to existing cancer-targeting antibodies or immune cell therapies, groundbreaking treatment results have been continuously reported in advanced countries such as the United States. However, successful cases of CAR-T therapy are still limited to CD19-positive B cell-derived hematological tumors, so the development of CAR gene T-cell therapy targeting various tumor antigen targets in addition to CD19 is expected to make a groundbreaking contribution to increasing the cure rate of hematological tumors. Peripheral T cell 1 lymphoma is a T cell-derived lymphoma and is known to be a cancer with a very poor prognosis with a 5-year survival rate of approximately 30%, but an appropriate treatment method has not been developed. Therefore, T-cell lymphoma may be an important target for the development of novel CAR-T cell therapies.

The ideal target antigen for CARs for T-cell lymphoma is CD3, which is widely expressed in T-cell lymphoma. However, it has the disadvantage of being expressed in normal T cells as well. Therefore, when producing T cells with CD3-targeting CAR genes, there is a technical barrier that CAR-T cells cannot be produced because they induce cellular suicide (fratricide) by attacking the CAR-T cells themselves. In addition, another technical barrier to CD3-targeting CAR-T cells is that when these cells are introduced into a patient's body, they may kill not only T-cell lymphoma but also normal T cells of the patient, causing immunodeficiency side effects in the patient. Therefore, in the present invention, a conjunctive CD3-targeting CAR-T cell is produced, which performs CD3-targeting function only during tumor killing and does not bind to CD3 after the killing function is finished. By overcoming the above two technical difficulties, the invention regarding CAR-T cell therapy for peripheral T-cell lymphoma was completed.

【Prior art literature】

【Patent Document】

(Patent Document 0001) Korean Patent Publication No. 2018-0031727 (Publication Date: 2018.03.28)

【Detailed description of the invention】

【Technical Problem】 The present invention provides an adapter molecule that regulates the activity of a chimeric antigen receptor T cell (CAR-T cell), comprising (a) a first moiety that binds to CD3 on a target cell; and (b) a second moiety that specifically binds to an extracellular domain of a chimeric antigen receptor. The present invention also provides a use of the adapter molecule for inducing activation of a chimeric antigen receptor T cell. The present invention also provides a chimeric antigen receptor T cell (CAR-T cell) in which CD3 expression is reduced or eliminated, wherein the CAR-T cell expresses a chimeric antigen receptor comprising (i) an extracellular domain comprising an antibody or an antigen-binding fragment thereof that specifically binds to the second moiety of the adapter molecule, (ii) a transmembrane domain, and (iii) an intracellular signaling domain. The present invention seeks to provide a complex comprising a chimeric antigen receptor T cell with reduced or eliminated expression of CD3 and an adapter molecule regulating the activity of the CAR-T cell. The present invention seeks to provide a kit for preventing or treating a T cell-derived tumor, comprising a chimeric antigen receptor T cell (CAR-T cell) with reduced or eliminated expression of CD3 and an adapter molecule regulating the activity of the CAR-T cell. The present invention seeks to provide a pharmaceutical composition for preventing or treating a T cell-derived tumor, comprising a chimeric antigen receptor T cell (CAR-T cell) with reduced or eliminated expression of CD3 and an adapter molecule regulating the activity of the CAR-T cell. The present invention seeks to provide a pharmaceutical composition for preventing or treating a T cell-derived tumor, comprising a chimeric antigen receptor T cell with reduced or eliminated expression of CD3 and co-administered with an adapter molecule regulating the activity of the CAR-T cell. The present invention provides a method for preventing or treating a T cell-derived tumor, comprising a step of administering the complex or pharmaceutical composition to a subject. The present invention provides a method for producing a chimeric antigen receptor T cell (CAR-T cell) in which the expression of CD3 is reduced or eliminated, comprising (a) a step of down-regulating the expression of CD3 or a T cell receptor (TCR) in a T cell, and (b) a step of introducing a chimeric antigen receptor (CAR) comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the steps (a) and (b) are performed regardless of the order.

【Technical Solution】 One aspect of the present invention provides an adapter molecule that regulates the activity of a chimeric antigen receptor T cell (CAR-T cell), comprising (a) a first moiety that binds to CD3 on a target cell; and (b) a second moiety that specifically binds to an extracellular domain of a chimeric antigen receptor. Another aspect of the present invention provides a use of the adapter molecule to induce activation of a chimeric antigen receptor T cell. As used herein, the term "adaptor molecule" means a molecule that binds to the extracellular domain of a CAR and, at the same time, binds to a target cell, for example, a target tumor cell, in a T cell therapy using the chimeric antigen receptor, i.e., a cell therapy agent called CAR-T. Since the adapter molecule has a specific binding ability to target T cell-mediated tumor cells or cancer, the tumor target attacked by the CAR-T cell can be changed by replacing the adapter molecule, and when side effects occur due to excessive activation of the CAR-T cell, the toxicity of the CAR cell can be alleviated by stopping the administration of the adapter molecule or reducing the administration dose (Cao et al., Angew Chem Int Ed Engl. 2016 June 20； 55(26) : 7520-7524.). As used herein, the term “antibody” refers to an antibody that specifically binds to a specific antigen, and includes not only a complete antibody form but also an antigen binding fragment of the antibody molecule. A complete antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to a heavy chain by a disulfide bond. The heavy chain constant region has gamma (X), mu (U), alpha (U), delta (6), and epsilon (心) types and has subclasses gamma 1 (X1), gamma 2 (x2), gamma 3 (x3), gamma 4 (x4), alpha l (al), and alpha 2 (a2). The light chain constant region has kappa (K) and lambda (入) types. (Cellular and Molecular Immunology, Wonsiewicz, M. J., Ed., Chapter 45, pp. 41—50, WB Saunders Co. Philadelphia, PA(1991) : Nisonof f, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4,pp. 45—65, Sinauer Associates, Inc., Sunder 1 and , MA (1984)). As used herein, the term “antigen binding fragment” means a fragment having antigen binding function, and includes Fab, F(ab'), and Fv, scFv, or sdAB (si ng 1 e-doma in antibody). Among antibody fragments, Fab (fragment antigen binding) has a structure having variable regions of the light and heavy chains, constant region of the light chain, and first constant region (CH1) of the heavy chain, and has one antigen binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. Fv is the minimum antibody fragment having only heavy chain variable region and light chain variable region, and recombinant technology for producing Fv fragments is disclosed in PCT International Patent Publication Nos. W0 88/10649, W0 88/106630, W0 88/07085, W0 88/07086, and W0 88/09344. As used herein, the term “heavy chain” refers to a full-length heavy chain and fragments thereof comprising an antibody variable domain VH and three constant domains CHI, CH2 and CH3, which comprise an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. In addition, the term “light chain” as used herein refers to a full-length light chain and fragments thereof comprising an antibody variable domain VL and constant domain CL, which comprise an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. As used herein, the term “complementarity determining region (CDR)” refers to an amino acid sequence of a hypervariable region of an immunoglobulin heavy chain or light chain (Rabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987)). The heavy chain (CDRH1, CDRH2 and CDRH3) and the light chain (CDRL1, CDRL2 and CDRL3) each contain three CDRs. The CDRs provide key contact residues for binding of the antibody to an antigen or epitope. In one embodiment, the first moiety may mean a protein having a specific binding ability to CD3. More specifically, the first moiety includes a protein that specifically binds to CD3 expressed on the surface of a target cell, for example, a T cell-derived tumor cell. In this case, the protein having a specific binding ability to CD3 may be any one selected from the group consisting of an antibody, an antigen-binding fragment, an affibody, a diabody and an aptamer. In addition, the protein that specifically binds to CD3 is a protein that binds to a CD3 molecule on the surface of a T cell-mediated tumor cell, and the protein may be an antibody, specifically, any one of a monoclonal antibody, a polyclonal antibody, or a recombinant antibody. In addition, the antibody may be a full-length antibody or an antibody fragment. At this time, the antibody fragment may include a part of an anti-CD3 antibody having the ability to bind to CD3. The antibody fragment may be Fab, Fab', F(ab')2, Fv, scFv, or a single-domain antibody (sdAB). The first moiety may be a known antibody or antigen fragment binding having the ability to specifically bind to CD3. More specifically, it may include a complementarity determining region (CDR), specifically, CDR1, CDR2, or CDR3, of a heavy chain variable region and/or a light chain variable region selected from the known anti-CD3 antibodies 0KT3, UCHT1, Tep 1 i zumab, Otel ixizumab, Vi si 1 i zumab, and Foralumab. The known anti-CD3 is only an example and is not limited thereto, and the antibody or binding fragment thereof may include a combination of CDR1, CDR2, or CDR3 of each of the known anti-CD3 antibodies. The first moiety may include a light chain variable region comprising CDR1, CDR2, and CDR3 represented by amino acid sequences of SEQ ID NOs: 23 to 25, respectively, or a heavy chain variable region comprising CDR1, CDR2, and CDR3 represented by amino acid sequences of SEQ ID NOs: 26 to 28, respectively. Additionally, it may include a light chain variable region comprising the amino acid sequence of SEQ ID NO: 29 and/or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 30. In one embodiment of the present invention, a linker may be included between the light chain variable region comprising the amino acid sequence of SEQ ID NO: 29 and the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 30. The linker may have an amino acid sequence of SEQ ID NO: 1 ((G4Sh linker), but the type of the linker is not limited thereto. The first moiety may include a light chain variable region comprising CDR1, CDR2, and CDR3 represented by amino acid sequences of SEQ ID NOs: 32 to 34, respectively, or CDR1, CDR2, and CDR3 represented by amino acid sequences of SEQ ID NOs: 35 to 37, respectively. In addition, it may include a light chain variable region comprising an amino acid sequence of SEQ ID NO: 38 and/or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 39. The first moiety may include a light chain variable region comprising CDR1, CDR2, and CDR3 represented by amino acid sequences of SEQ ID NOs: 67 to 69, respectively, or a heavy chain variable region comprising CDR1, CDR2, and CDR3 represented by amino acid sequences of SEQ ID NOs: 70 to 72, respectively. In addition, it ... A light chain variable comprising the amino acid sequence of 73 A heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 75 may be included. In one embodiment of the present invention, a linker may be included between the light chain variable region comprising the amino acid sequence of SEQ ID NO: 73 and the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 75. The linker may have the amino acid sequence of SEQ ID NO: 74, but the type of the linker is not limited thereto. In one embodiment of the present invention, the first moiety may be a scFv fragment having the amino acid sequences of SEQ ID NOs: 13, 22, 31, and 40, or a Fab fragment comprising the amino acid sequences of SEQ ID NOs: 41 and 42. Additionally, the anti-CD3 antibody or binding fragment thereof can comprise an amino acid sequence of SEQ ID NOS: 13, 22, 31, 40, 41, and 42, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto. In one embodiment, the second moiety can be a peptide tag or a hapten tag. The tag can be selected from the group consisting of a His tag, a Myc tag, a cotinine tag, a FITC tag, a Biotin tag, a Leucin zipper tag, a Flag tag, an Xpress tag, an Avi tag, a calmodulin binding peptide (CBP) tag, a polyglutamate tag, an HA tag, a Strep tag, a Softag 1, a Softag 3, and a V5 tag. In one embodiment of the present invention, the first moiety may be an anti-CD3 antibody or an antigen-binding fragment and/or the second moiety may be a His tag. In one embodiment of the present invention, the adapter molecule regulating the activity of a chimeric antigen receptor T cell comprising the first and second moieties may be a scFv fragment having amino acid sequences of SEQ ID NOs: 13, 22, 31, and 40, or a Fab fragment comprising amino acid sequences of SEQ ID NOs: 41 and 42, wherein 6XHis (HHHHHH) is further added. In addition, the adapter molecule may include a Fab in a form in which SEQ ID NOs: 42 and 44 are linked, a Fab in a form in which SEQ ID NOs: 46 and 48 are linked, a Fab in a form in which SEQ ID NOs: 50 and 52 are linked, or a Fab in a form in which SEQ ID NOs: 54 and 56 are linked. However, the present invention is not limited thereto. In one embodiment, the light chain variable region and the heavy chain variable region may be connected via a linker. For example, the linker may be a polypeptide consisting of any amino acid of 1 to 400, 1 to 200, or 2 to 200. The peptide linker may include Gly, Asn, and Ser residues, and may also include neutral amino acids such as Thr and Ala. Suitable amino acid sequences for the peptide linker are known in the art. Optimization of the linker may also be performed to achieve appropriate separation between functional moieties or to maintain essential inter-moiety interactions. The copy number " _n " can be controlled by considering. Other flexible linkers are known in the art, for example, G and S linkers which add amino acid residues such as T and A to maintain flexibility as well as adding polar amino acid residues to improve solubility. Thus, in one embodiment, the linker can be a flexible linker comprising G, S, and/or T residues. The linker can have a general formula selected from (GpSs) n and (SpGs) n , wherein independently, 日 is an integer from 1 to 10, s = 0 or an integer from 0 to 10, p + s is an integer less than or equal to 20, and n is an integer from 1 to 20. A more specific example of a linker is (GGGGS) n wherein n is an integer from 1 to 20, or from 1 to 10. In one embodiment of the present invention, when producing scFv as an antigen-binding fragment that specifically binds to CD3, the light chain variable region and the heavy chain variable region used a (G4S)3 linker (SEQ ID NO: 1). Another aspect of the present invention provides the use of the adapter molecule to induce activation of a chimeric antigen receptor T cell.

In CAR T cell therapy, after CAR T cells injected into a subject's body are used to selectively kill a tumor, and after the tumor is effectively reduced, the remaining residual CAR T cells show a side effect of killing normal cells. This is called CAR T immunodeficiency toxicity or normal T cell toxicity. The adapter molecule showed the effect of CAR T cell immunodeficiency toxicity or normal T cell toxicity evasion ability by regulating the activation of chimeric antigen receptor T cells. In one embodiment of the present invention, unlike existing CAR Ts that directly target existing tumors, the CAR T cell itself binds to the adapter molecule and binds to the tumor cell targeted by the adapter molecule, so that even when the CAR T cell was injected in the absence of the adapter molecule, it did not show toxicity to the target cell or tumor. That is, CAR T cells induce activation only in the presence of the adapter molecule, and thus show toxicity to the target cell or tumor. Therefore, the adapter molecule can induce activation or inactivation of CAR T cells. For this reason, the adapter molecule is also called a switchable molecule. Accordingly, the use of the adapter molecule to induce activation of chimeric antigen receptor T cells includes the use of preventing immunodeficiency by alleviating cytotoxicity of residual CAR T cells to normal cells and the resulting side effect of killing normal T cells after CAR T cell therapy. Another aspect of the present invention provides a chimeric antigen receptor T cell (CAR-T cell) in which CD3 expression is reduced or eliminated, wherein the CAR-T cell expresses a chimeric antigen receptor comprising (i) an extracellular domain comprising an antibody or an antigen-binding fragment thereof that specifically binds to a second moiety of an adaptor molecule, (ii) a transmembrane domain, and (iii) an intracellular signaling domain. The "first moiety", the "second moiety" or the "adaptor molecule" are as described above. The term "extracellular domain" as used herein means a domain that protrudes outside a cell and binds to a ligand or the like. The term "intracellular domain" as used herein means a domain that is located inside the cell membrane of a cell, i.e., in the cytoplasm, and transmits a signal transmitted by binding between the extracellular domain and the ligand into the cell. In one specific example, the chimeric antigen receptor (hereinafter also referred to as 'CAR') refers to a protein that fuses a cell membrane or intracellular signaling site of a T-cell activation protein (CD3-zeta chain, CD28, 41BBL, 0x40, I COS, high-affinity receptor for IgE (FCERI) and other T-cell activation proteins) with an antigen-binding site (single chain Fv fragment) of a cancer antigen-specific antibody. Specifically, the first-generation CAR includes an extracellular domain including an antigen recognition site specifically expressed in cancer cells, a transmembrane domain and an intracellular signaling domain, and may use only CD3 as the signaling domain. The second-generation CAR has a structure that combines a co-stimulatory domain (CD28 or CD137/4-1BB) and CD3《 to enhance responsiveness to immune cells, and may increase the number of immune cells containing CAR remaining in the body compared to the first-generation CAR. The third-generation CAR may have a structure that combines a co-stimulatory domain with 4-1BB, CD28 or 0X40, etc., using two or more co-stimulatory domains to achieve expansion and persistence of immune cells containing CAR in the body. The fourth-generation CAR may include an additional gene encoding a cytokine, such as IL-12 or IL-15, to enable additional expression of a CAR-based immune protein of the cytokine, and the fifth-generation CAR may additionally include an interleukin receptor chain, for example, IL-2RP, to strengthen immune cells. In one specific example, the chimeric antigen receptor may have the structure of a first-generation CAR to a fifth-generation CAR. In one specific example, the extracellular connecting portion of the chimeric antigen receptor may further include a hinge domain. In one specific example, the hinge domain may be characterized by being composed of an oligopeptide or a polypeptide and including 1 to 100 amino acid residues, specifically, 10 to 70 amino acid residues, but is not limited thereto. In one specific example, the intracellular signaling domain of the chimeric antigen receptor may be a portion located inside the cell membrane of an immune cell, i.e., in the cytoplasm, and may refer to a portion that transmits a signal within the cell when the antigen binding domain included in the extracellular domain binds to a target antigen and activates an immune response of the immune cell. In one specific embodiment, the intracellular signaling domain may be, but is not limited to, one or more intracellular signaling domains selected from the group consisting of CD3 zeta (《), CD3 gamma (Y), CD3 delta (6), CD3 epsilon (E), FcR gamma, FcR beta, CD5, CD22, CD79a, CD79b and CD66d, and in one embodiment, the intracellular signaling domain of the chimeric antigen receptor is CD3 zeta (《). In one specific embodiment, the intracellular signaling domain may be characterized by further comprising, but is not limited to, a costimulatory domain. The co-stimulatory domain according to the present invention may include one or more co-stimulatory domains selected from the group consisting of ligands that specifically bind to CD2, CD7, CD27, CD28, CD30, CD40, 4-lBB (CD137), 0X40 (CD134), I COS, LFA-1, GITR, MyD88, DAP1, PD-1, LIGHT, NKG2C, B7-H3 and CD83, but is not limited thereto. In one specific embodiment, the chimeric antigen receptor may be characterized by comprising one or more intracellular signaling domains and one or more co-stimulatory domains. In one specific embodiment, the co-stimulatory domain may be 4-lBB. The reduction or elimination of CD3 expression of the chimeric antigen receptor T cells may be achieved by removing CD3, specifically endogenous CD3, of the T cells or CAR-T cells introduced with the CAR gene before introducing the CAR gene, or by removing the TCR. In other words, the reduction or elimination of CD3 expression may be achieved by directly removing the CD3 gene or indirectly reducing, suppressing, or eliminating CD3 expression by removing the TCR. Reducing or eliminating the expression of the CD3 or TCR may be performed by a genome editing technique including a CRISPR system, specifically, CRISPR/Cas9, TALEN, Zinc fi nger nuclease, base-editing and prime-editing, or by a nucleic acid selected from the group consisting of antisense RNA, antagomir RNA, si RNA, shRNA and miRNA. The CAR T cell may be one that binds to the second moiety of the adaptor molecule. More specifically, the CAR T cell may exhibit cytotoxicity against a target cell or a tumor cell by binding to the second moiety of the adaptor molecule. That is, the extracellular domain of the CAR T cell, more specifically, the CAR T cell may include an antibody or an antigen-binding fragment thereof having binding ability to the second moiety of the adaptor molecule. The second moiety may be a His tag. Therefore, the CAR T cell, more specifically, the extracellular domain of the CAR T cell may include a protein having a specific binding ability to His tag, specifically, an antibody or an antigen-binding fragment thereof. In one embodiment of the present invention, the antibody or antigen-binding fragment thereof that specifically binds to the His tag may include an amino acid sequence of SEQ ID NO: 63, a part thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identity therewith. 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 having a part of the sequence deleted, modified, substituted or added may 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, in the present application, the gene sequence and the base sequence may be used interchangeably. For example, if it has the same or corresponding function as the CAR T cell, it is obvious that it is within the scope of the present application that a meaningless sequence is added to the sequence or the end of the sequence number, or a part of the sequence or the end of the sequence number is deleted. Homology and identity refer to the degree of relationship between two given base sequences and can be expressed as a percentage. The terms homology and identity are often used interchangeably. Whether any two sequences have homology or identity can be determined by, for example, using the default parameters such as Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 It can be determined using known computer algorithms such as the "FASTA" program. 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)), BLASTP, 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. Another aspect of the present invention provides a complex comprising a chimeric antigen receptor T cell in which the expression of CD3 is reduced or deleted and an adapter molecule that regulates the activity of the CAR-T cell. Another aspect of the present invention provides a kit for preventing or treating a T cell-derived tumor, comprising a chimeric antigen receptor T cell (CAR-T cell) in which the expression of CD3 is reduced or deleted and an adapter molecule that regulates the activity of the CAR-T cell. The "CAR T cell", the "first moiety", the "second moiety" or the "adapter molecule" are as described above. Another aspect of the present invention provides a composition comprising a chimeric antigen receptor T cell (CAR-T cell) in which the expression of CD3 is reduced or eliminated and an adapter molecule that regulates the activity of the CAR-T cell. The composition may be a pharmaceutical composition. The composition may be a pharmaceutical composition for preventing or treating a T cell-derived tumor. Another aspect of the present invention provides a pharmaceutical composition for preventing or treating a T cell-derived tumor, comprising a chimeric antigen receptor T cell in which the expression of CD3 is reduced or eliminated and is administered in combination with an adapter molecule that regulates the activity of the CAR-T cell. The "CAR T cell", the "first moiety", the "second moiety" or the "adapter molecule" As mentioned above. As used herein, the term "combination therapy" or "combined treatment" or "in combination" refers to any form of simultaneous or concurrent treatment using at least two separate therapeutic agents. The components of the combination therapy may be administered simultaneously, sequentially, in reverse order, or in any order. The components may be administered in different dosages, at different administration frequencies, or via different routes in an appropriate manner. As used herein, the term "administration" means introducing a given substance into a subject in an appropriate manner, and the "subject" means any living organism, including humans, such as rats, mice, and livestock, that may have a metabolic disease or adverse effects of a metabolic regulator. As a specific example, it may be a mammal, including humans. The term "administered simultaneously" as used herein is not particularly limited, and means that the components of the combination therapy are administered substantially simultaneously, for example, as a mixture or in an immediately subsequent order. The term "administered sequentially" as used herein is not particularly limited, and means that the components of the combination therapy are not administered simultaneously, but rather between administrations. Means administered one after another or in clusters with a specific time interval. The time interval may be the same or different between the administration of each of the components of the combination therapy and can be selected, for example, in the range of 2 minutes to 96 hours, 1 day to 7 days or 1 week, 2 weeks or 3 weeks. Typically, the time interval between administrations can be in the range of several minutes to several hours, for example, in the range of 2 minutes to 72 hours, 30 minutes to 24 hours or 1 to 12 hours. Further examples include time intervals in the range of 24 to 96 hours, 12 to 36 hours, 8 to 24 hours and 6 to 12 hours. The term "disease" as used herein can mean a pathological condition, particularly cancer, an infectious disease, an inflammatory disease, a degenerative disease, a disease involving apoptosis and graft rejection. The term "treatment" as used herein refers to or includes the alleviation, inhibition of progression, or prevention of a disease, disorder or condition, or one or more symptoms thereof, and "active ingredient" or "pharmaceutically effective amount" can mean any amount of a composition used in the course of practicing the invention provided herein sufficient to alleviate, inhibit progression, or prevent a disease, disorder or condition, or one or more symptoms thereof. The terms "administering,""introducing," and "implanting" as used herein are used interchangeably and refer to the placement of a composition according to one embodiment into a subject by a method or route that results in at least partial localization of the composition to a desired site according to one embodiment. can mean. The composition according to one embodiment can be administered by any suitable route that delivers the cells or at least a portion of the cellular components to a desired location within a viable organism. The survival period of the cells after administration to the organism can be as short as several hours, for example 24 hours, to several days, or as long as several years. The above T-cell derived tumors include T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, chronic lymphoprol i ferat ive disorder of NK cells, aggressive NK leukemia, systemic EBV-positive T-cell 1 lymphoprol i ferat ive disease of chi Idhood, hydroa vaccini forme- 1 ike lymphoma, adult T-cell leukemia/lymphoma, extranodal NK/T-cell 1 lympho (nasal type), Enteropathy-associated T-cel 1 lymphoma, hepatosplenic T-cel 1 lymphoma, subcutaneous panniculitis-like T-cel 1 lymphoma, mycosis fungoides, Se'zary syndrome, primary cutaneous CD30-positive T-cel 1 lymphoproliferative disorders, lymphomatoid papulosis, primary cutaneous anaplastic large cel 1 lymphoma, primary cutaneous gamma/delta T-cel 1 lymphoma, primary cutaneous CDS-positive aggressive epidermotropic cytotoxic It may be selected from the group consisting of primary cutaneous CD8-positive aggressive epidermotropic cytotoxic T-cel 1 lymphoma, primary cutaneous CD4-positive small 1/medium T-cel 1 lymphoma, peripheral T-cel 1 lymphoma NOS (not otherwise specified), angioimmunoblastic T-cel 1 lymphoma, ALK positive anaplastic large cel 1 lymphoma, and ALK negative anaplastic large cel 1 lymphoma. The administration may be in combination with an additional anticancer agent. Additional examples of anticancer agents may include alkylating agents, antimetabolites, spindle inhibitor plant alkaloids, cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Examples of such anticancer agents may include compounds used in targeted therapy and conventional chemotherapy. In addition, the antibodies Examples include alemtuzumab, apolizumab, acelizumab, atlizumab, bapineuzumab, bevacizumab, vivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, Pascolizumab, pekfucituzumab, pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resaivizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadoxizumab, talizumab, tefibazumab, tocilizumab, toralizumab, trastuzumab, tucotuzumab selmoleukin, tucusituzumab, umavizumab, urtoxazumab, and visilizumab may be included. The composition according to the present invention may additionally comprise a pharmaceutically acceptable carrier. In the case of injections, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used, and in the case of local administration, bases, excipients, lubricants, preservatives, etc. can be used. The formulation of the pharmaceutical composition of the present invention can be variously prepared by mixing it with the pharmaceutically acceptable carrier as described above. For example, in the case of injections, it can be prepared in the form of a unit dosage ampoule or a multiple dosage form. In addition, the anticancer composition may typically include a surfactant that facilitates movement through a membrane. Such surfactants include those derived from steroids, cationic lipids such as N-[l-(2,3-dioleoyl)propyl-N,N,N-trimethylammonium chloride (DOTMA), or various compounds such as cholesterol hemisuccinate and phosphatidyl glycerol. In one specific example, the composition can be administered in a pharmaceutically effective amount to treat cancer cells or their metastasis, or to inhibit the growth of cancer. It may vary depending on various factors such as cancer type, patient's age, weight, nature and degree of symptoms, type of current treatment, number of treatments, dosage form and route, and can be easily determined by a person skilled in the art. The composition according to the present invention can be administered together with the above-mentioned pharmacological or physiological components or administered sequentially, and can also be administered in combination with an additional conventional therapeutic agent, and can be administered sequentially or simultaneously with the conventional therapeutic agent. Such administration may be single or multiple administration. It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects considering all of the above factors, and this can be easily determined by a person skilled in the art. Another aspect of the present invention provides a method for preventing or treating a T cell-derived tumor, comprising administering to a subject the complex or pharmaceutical composition. Another aspect of the present invention provides a method for producing a chimeric antigen receptor T cell (CAR-T cell) having reduced or eliminated expression of CD3, comprising the steps of (a) down-regulating the expression of CD3 or a T cell receptor (TCR) in a T cell, and (b) introducing a chimeric antigen receptor (CAR) comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain comprises an antibody or an antigen-binding fragment thereof that specifically binds to a second moiety of an adaptor molecule, wherein steps (a) and (b) are performed in any order. The method for reducing or eliminating the expression of the CD3 or TCR may be performed by a genome editing technique including a CRISPR system, specifically, CRISPR/Cas9, TALEN, zinc finger nuclease, base editing and prime editing, or by a nucleic acid selected from the group consisting of antisense RNA, antagomir RNA, si RNA, shRNA and miRNA.

【Effect of the invention】 According to one aspect of the present invention, an adapter molecule comprising an anti-CD3 antigen-binding fragment and a T cell expressing a CD3-chimeric antigen receptor binding thereto exhibit anti-fratricide activity and immunodeficiency toxicity avoidance activity, and thus can be usefully used in a T cell-derived tumor treatment agent.

【Brief description of the drawings】 Fig. 1 is a graph showing the results of the first screening of guide genes for human CD3 gene editing. Fig. 2 is a graph showing the results of the second screening of guide genes for human CD3 gene editing. Fig. 3 is a graph showing the analysis of the phenotype of CD3 gene-edited human T cells. Fig. 4 is a graph showing the analysis of the phenotype of CAR T cells introduced with chemical and peptide tags for labeling anti-CD3 adapter antibodies (Cot: cotinine, His: 6XHis). Figure 5 is a graph analyzing the expression rate of 6xHis-CAR in human CAR T cells introduced with His tag. Figure 6 is a schematic diagram showing the structure of anti-CD3 adaptor antibody scFv. Figure 7 is an image showing the result of SDS-PAGE after purification of fusion proteins containing 0KT3-Ck-His scFv and 0KT3-Ck-linker-His. Figure 8 is a schematic diagram showing the structure of anti-CD3 adaptor antibody Fab. Figure 9 is an image showing the result of SDS-PAGE after purification of 1-4-2-Ck-His scFv/l-4-7-Ck-His scFv and 1-4-2-Fab-His/1-4-7-Fab-His fusion proteins. Figure 10 is a graph analyzing the binding ability of 0KT3-Ck-His, 0KT3- Ck- L- His, and UCHT1- Fab- His to the T cell tumor cell line Jurkat. Figure 11 is a graph analyzing the binding ability of 1- 4- 2- Ck- His/1- 4- 7- Ck- His and 1- 4- 2- Fab- Hi s/1- 4- 7- Fab- His to the T cell tumor cell line Jurkat. Figure 12 is a graph analyzing the antitumor activity and tumor killing ability of 0KT3- Ck- His scFv and human anti-6xHis CAR T cells. Figure 13 is a graph analyzing the tumor killing ability of 0KT3-His Fab and human anti-6xHis CAR T cells. Fig. 14 is a graph analyzing the antitumor activity and tumor killing capacity of UCHT1-His Fab and human anti-6xHis CAR T cells. Fig. 15 is a graph analyzing the antitumor activity and tumor killing capacity of 1-4-2-His Fab and 1-4-7-His Fab and human anti-6xHis CAR T cells. Fig. 16 is a graph showing the fratricidal activity of human anti-6xHis CAR T cells. Fig. 17 is a graph analyzing the phenotype of T cells after CD3 gene editing in human anti-6xHis CAR T cells. Fig. 18 is a graph confirming the fratricidal prevention capacity of human conjugated CAR T cells through anti-CD3 adapter antibody. Fig. 19 is a schematic diagram showing the proliferation conditions of CD3 gene-edited conjugated CAR T cells. Figure 20 is a graph analyzing the phenotype of T cells in the first and second CD3 gene-edited conjugated CAR T cell proliferation stages. Fig. 21 is a graph showing the number of CAR T cells measured at the CD3 gene-edited conjugated CAR T cell proliferation stage. Fig. 22 is a schematic diagram of an anti-tumor activity assay experiment of 0KT3- Ck- Hi s scFv and CD3- human Hi s CAR T cells in a mouse model. Fig. 23 is a graph analyzing the anti-tumor activity and the survival rate of a mouse model of 0KT3- Ck- Hi s scFv and CD3- human Hi s CAR T cells in a mouse model. Fig. 24 is a schematic diagram of an anti-tumor activity assay experiment of UCHT1- Fab- Hi s and CD3 -gene-edited human Hi s CAR T cells in a mouse model. Fig. 25 is a graph analyzing the anti-tumor activity and the survival rate of a mouse model of UCHT1- Fab- Hi s and CD3 -gene-edited human Hi s CAR T cells in a mouse model. Figure 26 is a graph analyzing the immunodeficiency prevention ability of human conjugated CAR T cells. Figure 27 shows the antigen affinity of humanized antibody anti-His. Figure 28 shows the results of confirming CAR expression on the surface of huHis CAR- T cells by flow cytometry. Figure 29 shows the results of verifying the in vitro activity level of conjugated CAR T cells using the produced huHis CAR- T cells and the UCHTl-Fab-His adapter.

【Form for carrying out the invention】 Hereinafter, preferred embodiments are presented to help understanding of the present invention. However, the following embodiments are provided only to more easily understand the present invention, and the content of the present invention is not limited by the following embodiments. The embodiments can be modified in various ways, and the embodiments are not limited to the embodiments disclosed below, but can be implemented in various forms. Example 1. Production of human conjugated CD3 edited CAR-T cells

In order for CAR-T cells to recognize the tumor surface antigen CD3 and selectively kill tumors, (1) conjugated CAR-T cells were created to avoid fratricide between CAR-T cells by making the D CAR-T cells themselves not express CD3, and (2) minimize the immunodeficiency side effect in patients caused by CAR-T cells from which CD3 has been removed killing normal T cells that express CD3. A schematic diagram of this concept is as follows.

[Conjugated CD3-targeted CAR-T cells] Experimental materials

(1) Mouse

C57BL/6CB6) mice were purchased from Orient Bio. NSG mice (NOD. Cg- Prkdcscid 112rgtmlWj 1/SzJ) were purchased from Jackson Laboratory. All mice were housed in a specific pathogen-free (SPF) animal facility at Seoul National University College of Medicine (Seoul, Republic of Korea) and maintained according to the guidelines of the Institutional Animal Care and Use Committee (IACUC).

(2) Cell line

Jurkat (human T-cell lymphoma) was purchased from ATCC (Manassas, VA USA). Phoenix GP was provided by Garry Nolan (Stanford University, USA). GFP-luciferase expressing cell line (Jurkat-Luc) was transduced into Jurkat cells with lentivirus for GFP-Luc expression (pLEF-Luc-GFP), and GFP-positive cells were isolated using a cell sorter (FACS Aria, Becton Dickinson), and then a single cell line was established through limiting dilution. CD3 The deficient Jurkat cell line was created by introducing an in-del mutation into the CD3 gene expressed by the cell line using the CRISPR-Cas9 system, then isolating CD3-negative cells using a cell sorter, and then establishing a single cell line through the limiting dilution method.

1-1. Production of CAR-expressing retrovirus and lentivirus In the case of scFv used in conjugated CAR- T cells, Cot CAR scFv was derived from rabbit-derived anti-cot inine antibody (Cl in Chim Acta 2010； 411：1238-1242), Hi s CAR scFv was derived from mouse-derived anti-Hi s antibody (mutated 3D5 clone； J . Mol . Biol . (2002) 318 , 135-147), and Myc CAR scFv was derived from mouse-derived anti-Myc antibody (9E10 clone； FEES Letters 414 (1997) 33-38). The mouse CAR protein coding region consists of the signal peptide of the mouse immunoglobulin kappa chain, the antibody scFv, and the previously reported CAR backbone region (mouse CD28 extracellular region-transmitter region-intracellular region, mouse CD3 zeta intracellular region; GenBank HM754222.1, Blood 2010: 116(20)：4099-4102). CAR cDNAs were produced by requesting DNA synthesis to Integrated DNA technologies (IDT) of Korea, USA. The retroviral vector for producing mouse CAR-T cells for CAR expression was produced by cloning the PuroR gene region downstream of the PGK promoter in the pMSCV-puro (Ckmtech, USA) retroviral vector and replacing it with the produced CAR cDNA. The human CAR protein coding region consists of the signal peptide of human GMCSF, the scFv of anti-His antibody, the extracellular region-transmitter region-intracellular region of human CD28, and the intracellular region of human CD3 zeta. The protein coding region cDNA was produced by requesting DNA synthesis to IDT, USA. The retroviral vector for human CAR- T cell expression was cloning using human CAR cDNA using pMSGV (Addgene Plasmid #64269). The lentiviral vector for human CAR- T cell expression was produced by cloning human CAR cDNA into a partially modified pCDH-EF1 vector (Addgene Plasmid #72266).

1-2. Production of retrovirus and lentivirus for CAR expression To obtain ecotrophic retrovirus for transduction of mouse T cells, the CAR construct was transfected into Phoenix GP cells using Lipofectamine 3000 (Invitrogen) together with the pMD2.G plasmid containing cDNA encoding vesicular stomatitis Indiana virus G protein (VSV-G) as a viral envelope protein. After 48 hours, VSV-G pseudotyped retrovirus was present. The supernatant was harvested and transduced into the Phenix Eco cell line. After 3–5 days, for Cot CAR, the cells were stained with anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA USA) and for His CAR & Myc CAR, anti-mouse I gG (Jackson ImmunoResearch, West Grove, PA USA). Then, CAR-high CAR-expressing Phoenix Eco cells were separated by FACS Ar ia (Becton Dickinson) to establish a retrovirus-producing cell line. The retrovirus culture supernatant produced from this cell line was concentrated 5–10 times using a centrifugal filter device (Amicon Ultra-100 kDa cutoff, Millipore, USA) and used. To obtain amphotropic retrovirus for transduction of human T cells, the PG13 cell line for virus production was used. Each retroviral plasmid was transformed into Phoenix Eco cells using Lipofectamine 3000 (Invitrogen). After 48 hours, the culture supernatant containing retrovirus was harvested and added to PG13 cells for transduction. Thereafter, the establishment of retrovirus production cell lines and virus production were performed in the same manner as for retrovirus for mouse cells. To obtain lentivirus for transduction of human T cells, each lentivirus plasmid was transfected into 293T cell 1 1 ine (ATCC) together with three types of packaging DNA (pMD.2G, pMDLg/pRRE, pRSV-rev) using Lipofectamine 3000 (Invitrogen). After 24 and 48 hours, the culture supernatant containing secreted lentivirus was harvested and filtered (0.45 × 1 filter) to remove cell residual particles. After 100-fold concentration using an ultracentrifuge, it was used as a lentivirus concentrate for producing CAR T cells.

1.3 Production of human CD3-edited T cells using the CRISPR/Cas9 system

The CRISPR/Cas9 system was used as a method for CD3 gene editing. In order to use this system efficiently, the cDNA of the human CD3 gene targeting guide RNA (sgRNAl~ 18) was cloned into the vector (PX458) containing the Cas9 cDNA. The CD3 gene editing was performed by transfecting Jurkat cells, which are CD3-positive cells, with the constructed plasmid through electroporation. For CD3 gene editing in human primary T cells, white blood cells obtained from normal individuals through leukapheresis were added together with anti-CD3 antibody (0KT3, 10//g/mt, BioXcell) to a 24-well plate coated with anti-CD28 antibody (CD28.2, 2//g/mt, BD Biosciences), and then cultured for 48 hours to activate T cells. Afterwards, a complex of Cas9 protein (Cas9 protein, Thermo) and target guide RNA Ribonucleoprotein (RNP) was produced and gene-edited into T cells using electroporation. The gene-edited T cells were cultured in a culture medium without antibiotics but with hIL-2 (200 U/ml) for 24 hours, and then transferred to a culture medium with antibiotics and hIL-2 to culture the T cells. The degree of CD3 protein removal through CD3 gene editing was confirmed 36 hours after gene editing by flow cytometry (FACS-Canto II, BD Biosciences) to determine the degree of CD3 protein expression or T cell receptor expression.

To remove human CD3 using the CRISPR-Cas9 system, 18 guide RNA sequences for the human CD3 ORF target region were designed. Then, a plasmid that simultaneously expresses the guide ORF and Cas9 nuclease in the human acute T cell leukemia cell line Jurkat was constructed, and the gene was introduced using electroporation. A primary screening was performed to verify whether CD3 was removed. The results are shown in Fig. 1.

In the first screening, four guide RNA sequences with good efficiency were selected, and a second screening was conducted using mRNA transfection with good T cell gene transfer efficiency. To this end, sgRNA (guide RNA + scaffold RNA) and Cas9 mRNA were newly produced through an in vitro transcription reaction, and then an experiment was conducted to verify whether CD3 was removed in Jurkat using mRNA electroporation, and the results are shown in Fig. 2. In addition, the sequences of sgRNA7 and sgRNA15 are shown in the following Table and Table 2, respectively.

【Table 11

도 1 및 도 2에 나타낸바와 같이 , 플라스미드 DNA transfection에 비해 높은 제거 (deletion) 효율을 얻을 수 있었으며 , 특히 2종의 sgRNA(sgRNA7 와 sgRNA15)의 경우 80% 이상의 높은 제거 효율이 관찰되었다. 이어서 제거 효율이 좋은 sgRNA7에 대하여 보다 간편하면서도 유전자편집효율이 좋은 것으로 알려진 RNP(r ibonucleoprotein) 전기천공법 방식을 통해 인간 T세포에서의 CD3제거를 시도하였으며 , 그 결과를 도 3에 나타내었다. 도 3에 나타낸 바와 같이, 재조합 Cas9단백질 및 sgRNA7의 복합체를 in

RNP형태로제작하여 활성화된 인간 T세포에 전기천공법을통해 CD3제거를 70% 이상의 효율로 획득하였다. 이상과 같이 , CRISPR/Cas9시스템을이용한인간 T세포에서의 CD3유전자편집 방법을 효과적으로 확립하였다. 【Table 2]

As shown in Figures 1 and 2, a high deletion efficiency was obtained compared to plasmid DNA transfection, and in particular, a high deletion efficiency of over 80% was observed for two types of sgRNA (sgRNA7 and sgRNA15). Next, CD3 deletion in human T cells was attempted using the RNP (ribonucleoprotein) electroporation method, which is known to be simpler and more efficient in gene editing, for sgRNA7 with good deletion efficiency, and the results are shown in Fig. 3. As shown in Fig. 3, the complex of recombinant Cas9 protein and sgRNA7 was in

CD3 deletion was achieved with an efficiency of over 70% in activated human T cells by electroporation produced in the form of RNP. As described above, a method for CD3 gene editing in human T cells using the CRISPR/Cas9 system was effectively established.

1.4 Exploration of the possibility of CD3 gene editing in various conjugated CAR-T cells For the production of conjugated CAR-T cells, cotinine, a nicotine metabolite, and peptide epitopes 6XHis tag and myc tag were selected as candidates for the epitope tags to be labeled on the anti-CD3 adapter antibody, and anti-cotinine CAR-T cells (Cot CAR-T cells), anti-6XHis tag CAR-T cells (His CAR-T cells), and anti-myc tag CAR-T cells (Myc CAR-T cells), respectively, were produced. Anti-cotinine CAR-T cells were produced by transducing a retrovirus containing a CAR gene including an anti-cotinine antibody scFv (Korean Patent Publication No. 2018-0031727) into activated mouse T cells. Using the same method, retroviruses carrying a CAR gene including the scFv of a previously reported anti-myc tag antibody (c 1 one 9E10, FEES Letters 414 (1997) 33-38) and a CAR gene including the scFv of an anti-6xHis tag antibody (mutated clone 3D5, J. Mol. Biol. (2002) 318, 135-147) were constructed, and these retroviruses were transduced into activated mouse T cells to construct Myc CAR-T cells and His CAR-T cells. Specifically, for mouse CAR T cells, spleen and lymph node cells obtained from normal mice were added together with a-CD28 antibody (37.51, 2//g/mt, BD Biosciences) to a 24-well plate coated with a-CD3 antibody (145-2C11, 10w/mt, BioXcell), and T cells were activated. After 24 h, T cells were transduced with concentrated retrovirus through spin infection in the presence of 6 ug/ml polybrene (polybrene, Sigma Aldrich) at 24 °C, 2500 rpm, and 90 min. This procedure was repeated once more on the same day. After spin infection, T cells were additionally cultured in the presence of mouse IL-2 (30 U/mt, Gibco) for 48 h. The retrovirus-transduced T cells were washed twice, and then proliferated for 2-3 days in fresh culture medium containing mouse IL-2 (20 U/mt) to be used as CAR T cells. The expression of CAR protein on the cell surface was performed using a-rabbit IgG (Cot CAR), Q-mouse IgG (His CAR, Myc CAR), His-biotin (biotin-labeled 6xHis peptide) and fluorescently labeled streptavidin were stained and measured using flow cytometry. Mouse CD3 gene-edited CAR- T cells were subjected to mouse CD3 gene editing using electroporation the day after retroviral transduction. The expression of CAR protein on the surface of each of these cells was confirmed, and this is shown in Fig. 4a. In addition, in order to confirm whether CD3-deleted CAR-T cells were possible for CAR-T cells for chemicals (cotinine) and peptides (6XHis tag or myc tag), transduction via CAR- retrovirus and CD3 deleting sgRNA/Cas9 mRNA transfection were performed sequentially, and CAR-T cells in which CAR expression and CD3 deletion were simultaneously induced were produced, and this is shown in Fig. 4b. As shown in Figures 4a and 4b, in the case of anti-cotinine CAR-T cells, it was observed at about 50-70% in CD4 and CD8 positive cells, and the highest expression rate was confirmed in His CAR T cells among the three CAR-T cells. Through the above results, it was confirmed that CD3 gene editing using the CRISPR-Cas9 system was possible in all three types of CAR-T cells (Cot CAR-T, His CAR-T, and Myc CAR- T cells).

1.5 Production of conjugated CAR-T cells To produce conjugated human CAR-T cells, the anti-His CAR gene used in the mouse CAR-T cell experiment was redesigned as a human CAR backbone, and then cloned into a retroviral vector for human CAR T cells to produce them. The produced retrovirus was genetically introduced into human T cells isolated from peripheral blood to produce His CAR-T cells. Specifically, leukocytes isolated from normal blood were added to a 24-well plate coated with anti-CD3 antibody together with anti-CD28 antibody, and then cultured for 48 hours to activate the T cells. The activated T cells were washed twice and then used for retroviral transduction. After coating with Retronectin (20w/mt, TaKaRa) overnight at 4°C, 2% BSA-DPBS was added to the washed 24-well plate, blocked at 37°C for 30 minutes, and washed. Retrovirus concentrate Imt was added, and centrifuged at 2000xg and 32°C for 2 hours to attach the retrovirus to the bottom of the well. After removing the virus concentrate and washing the well, activated T cells ( ^1X106 cells/mt) Imt were added to the well, and centrifuged for 10 minutes (lOOOxg, The cells were attached to the retrovirus via the 32°C medium. Subsequently, they were cultured for 48 hours in the presence of human IL-2 (200 U/mt, Pro leukin, Novartis). In the case of lentivirus transduction, lentivirus was added to the human T cell culture medium activated for 48 hours, and then cultured together for 48 hours. The T cells transduced with the virus in this way were washed twice to remove the virus remaining in the culture medium, and the culture medium containing human IL-2 (200 U/mt) was added twice every 3 days (day 3 + day 3) to produce CAR T cells. Figure 5 is a graph analyzing the expression rate of 6xHis-CAR in human CAR T cells introduced with the His tag. In addition, the amino acid sequence by domain of His CAR-T cells is shown in Table 3 below.

도 5에 나타낸 바와같이, 유세포분석을통하여 CAR발현율을분석한결과, 약 40%정도의 유전자 전달 효율으로 인간 항 6xHis CAR T세포가 성공적으로 제작됨을 확인하였다. 실시예 2. 항 CD3 어댑터 항체의 제조 【Table 3]

As shown in Fig. 5, the CAR expression rate was analyzed through flow cytometry, and it was confirmed that human anti-6xHis CAR T cells were successfully produced with a gene transfer efficiency of approximately 40%. Example 2. Production of anti-CD3 adapter antibody

2.1 Production of adapter antibodies via anti-CD3 antibodies - In the case of chemical tags such as 0KT3, UCHT1 cotinine, it is difficult to maintain a constant conjugation efficiency because they must be conjugated to the adapter through a chemical reaction, and there are disadvantages in that it is difficult to control the number of molecules of the chemical conjugated to one adapter (Drug Antibody Ratio, DAR). On the other hand, 6xHis In the case of peptide epitopes such as tag or myc tag, since the peptide can be linked to the recombinant antibody ORF gene through genetic recombination in the form of an epitope tag, there is an advantage of maintaining 100% conjugation efficiency and a 1:1 DAR in which one tag is bound to one adapter. In particular, the 6xHis peptide has been widely used as an epitope tag for protein purification, and therefore a system for mass purification of antibody drugs using it is well-established, so there is an advantage of being able to achieve two purposes at the same time, that is, the role of the adapter epitope tag for the conjugated His CAR T cell and the role of the epitope tag for adapter purification, using one epitope tag. In addition, there is an advantage of relatively low immunogenicity in vivo. Therefore, the 6xHis tag was used in the anti-CD3 adapter antibody. 6xHis-tagged adapter antibodies were produced in scFv form using the commercially available anti-human CD3 antibody 0KT3 (Muromonab) sequence or UCHT1 sequence. A construct was produced by connecting the VL region and the VH region of OKT antibody or UCHT1 antibody with a linker ((GGGGS)J, and then connecting the human immunoglobulin kappa constant region (CK) for separation and stability, and directly connecting a 6xHis tag to the C-terminus or connecting a short linker peptide and a 6xHis tag. A schematic diagram of the construct is shown in Fig. 6, and the amino acid sequences of the scFv of 0KT3 are shown in Tables 4 and 5, respectively. At this time, Table 4 shows the amino acid sequence of the scFv (0KT3-Ck-His) that does not include a linker after the Ck domain, and Table 5 shows the amino acid sequence of the scFv (0KT3-Ck-L-His) that includes a linker after the Ck domain. In addition, the amino acid sequence of the scFv (UCHTl-Ck-His) of UCHT1 is shown in Table 6 below. The above constructs After the expression plasmid was transformed into the HEK293K cell line, it was purified through affinity chromatography, and the results are shown in Figure 7.

【Table 4]

【Table 5]

scFv형태뿐 아니라, 어댑터 항체의 CAR결합력을 증가시키는 일환으로, 2개의 6xHis 태그를 보유하여 2가 (bi valent) 형태로 6xHis CAR에 결합할 수 있도록 Fab 형태로도 -His 어댑터 항체를 제작하였고, 이의 모식도를 도 8에 나타내었고, 각각 0KT3 및 UCHT1의 Fab (0KT3- Fab- His , UCHTl-Fab-His) 아미노산서열을 아래 표 7 및 8에 나타내었다. 【Table 6]

In addition to the scFv form, as part of an effort to increase the CAR binding affinity of the adapter antibody, a -His adapter antibody was also produced in a Fab form that possesses two 6xHis tags so that it can bind to 6xHis CAR in a bivalent form. A schematic diagram of this is shown in Fig. 8, and the amino acid sequences of the Fabs of 0KT3 and UCHT1 (0KT3-Fab-His, UCHT1-Fab-His), respectively, are shown in Tables 7 and 8 below.

【Table 7]

【Table 8]

2.2 Production of adapter antibodies using novel anti-CD3 antibodies - 1-4-2, 1-4-7 In addition to the above 0KT3 or UCHT1 antibodies, anti-human CD3 antibodies were additionally screened for CD3 binding antibodies using an animal immune antibody library. Specifically, human CD3 gamma/epsilon and delta/epsilon extracellular heterodimer proteins were mixed with an adjuvant and immunized in 10 chickens four times in total at two-week intervals. Then, the spleen, bone marrow, and synovial bursa of the chickens were collected, total RNA was isolated, cDNA was synthesized, variable light chain & heavy chain genes were PCR-processed, and cloned into _P Comb3XSS, a phage display vector, to produce a chicken immune antibody library. Thereafter, bio-panning was performed to screen for antibody clones that specifically bind to human CD3 protein. After purifying the positive antibody clones in the form of scFv, two antibodies (clones 1-4-2 and 1-4-7) that bind to CD3(+) Jurkat cells were finally selected. For the selected antibodies, they were cloned into an animal cell expression vector to produce them in the form of scFv-Ck-6xHis fusion proteins, and then transformed into HEK293F cells. After culturing with shaking for 5 days, the culture medium was harvested and affinity purified using KappaSelect beads, and the expression of the antibodies was confirmed through SDS-PAGE. That is, for the novel screening antibodies 1-4-2 and 1-4-7, scFv and Fab types of adapter antibodies were shown, and the amino acid sequences thereof are shown in Tables 9 to 12 below, and in particular, the results of SDS-PAGE analysis of scFv (1-4-2-Ck-His, 1-4-7-Ck-His) of 1-4-2 and 1-4-7 are shown in Figure 9.

【Table 2

【Table 10]

【Table 11]

구체적으로, 항 인간 CD3(UCHT1, 1-4-2, 1-4-7) Fab- His항체 및 항 인간 CD3(0KT3, UCHT1 , 1-4-2, 1-4-7) scFv- His항체는 항체 발현 벡터인 pCEP4에 유전자를 클로닝하고 293F세포에 형질도입하여 배양액을 통해 발현하였다. 발현된 항체는 친화도 정제를 통해 배양액 내에 존재하는 항체를 분리 정제하였다. 정제된 항체의 경우 SDS-폴리아크릴아마이드 겔 (SDS-polyacrylamide gel)을 이용한전기영동 방법과 인간 CD3를 발현하는 세포주에 유세포측정법을 통하여 세포표면 결합을 검증하였다. CD3 양성 세포와 어댑터 항체의 결합 검증은 lX10⁵cells/lug 어댑터를 4 °C에 1시간 배양하였다. 이후 자유 어댑터 항체를 제거하기 위해 세척 후, 어댑터 항체를 잡는 항 His항체 (J096G46, Biolegend)로 염색한 뒤 유세포측정법을 이용하여 분석하였다. 이상과 같이 , 발현 및 정제된 항 인간 CD3 항체가 T세포종양표면에 발현하는 CD3단백질과 결합하는지를 분석하기 위하여, 제조한 0KT3-His scFv (0KT3- Ck- His , 0KT3-Ck-L-His), UCHT1- Fab- His에 대해서는 Jurkat T세포에 결합여부를 유세포분석 (flow cytometry)한 결과를 도 10에 나타내었다. 동일한 방법으로 1-4 - 2의 scFv 및 Fab, 1-4- 7의 scFv 및 Fab 항체단백질에 대해서도 유세포분석 (flow cytometry)을수행하였고, 그 결과를 도 11에 나타내었다. 도 10에 나타낸 바와 같이 , 항 CD3 항체로 제조한 scFv, 및 Fab가 Jurkat 세포주에 특이적으로 결합하는 것을 확인하였다. 도 11에 나타낸 바와 같이, CD3음성 세포주 (CD3- deleted Jurkat)에는 결합하지 않고, Jurkat 세포주에만특이적으로 결합하는 것을 확인하였다. 실험예 1. 항 CD3 어댑터 항체와 인간 접합형 CAR-T 세포의 항종양 활성

상기 실시예 2에서 제작한 6xHis- tagged 항 CD3 어댑터 항체와 인간 항- 6xHis CAR-T 세포 (His CAR-T 세포) 복합체가 CD3양성 T세포 종양을 인지하여 활성화되는지를 in vitro에서 검증하기 위해, Jurkat leukemia세포와 0KT3 scFv-Ck- 6xHis(0KT3-Ck-His), 0KT3 scFv- Ck- 6xHis(0KT3- Ck- L- His)및 마우스 His CAR-T세포를 공배양한 후, CAR-T세포가분비하는 IFN- r의 양을 ELISA로측정하였다. 그결과, 두 가지 어댑터 존재하에서 T세포의 IFN- r생성이 증가함에 따라 CAR- T세포의 기능이 활성화됨을 확인하였다. 또한 어댑터 용량을 5배 증가함에 따라 그 효과가 더 증가하는 용량의존도를 보였다. 그 결과를 도 12a에 나타내었다. 이어서 , 두어댑터 중좀 더 활성이 좋은 0KT3 scFv- Ck- 6xHis(0KT3- Ck- His)를 Jurkat leukemia세포 및 인간 His CAR-T세포를공배양한후, CAR-T세포가분비하는 IFN- r의 양을 ELISA로 측정하였다. 또한, Jurkat 종양세포에 대한 세포살상능 (Cytotoxicity assay)을 확인하기 위한 실험을 진행하였고, 그 결과를 도 12b에 나타내었다. 동일한 방법으로, scFv어댑터 항체를마우스접합형 CAR T세포모델에서 검증된 Fab-His형태로 변환시켜 Jurkat 종양세포에 대한세포살상능실험을반복하였고, 그 결과를 도 13에 나타내었다. 또한, 항- CD3항체의 다른 클론인 UCHT1항체 및 스크리닝을 통해 선별한 두 종의 항체 (1-4- 2, 및 1-4- 7)를 이용하여 Fab-His 어댑터 항체를 제작하고, 이를 이용하여 접합형 CAR T세포의 in vitro 활성 및 세포살상능을 동일한 방법으로 분석하였다. 그 결과를 각각도 14(UCHT1) 및 도 15(1-4-2, 1-4- 7)에 나타내었다. 도 12내지 15에 나타낸바와같이 , 접합형 CAR T세포는항 CD3어댑터 항체의 존재하에서만 농도의존적으로 활성화되어 IFN- r를 다량 생산하고, 종양살상능을 보였다. 이상의 결과를통해 , 항 CD3어댑터 항체와 CAR T세포복합체의 암항원 인지력 , 특이적인 활성화 및 세포살상능력을 확인하였다. 접합형 CAR T세포의 종양살상능, 사이토카인 생성능 및 동족살상 방지기능 츺정 어댑터 항체를통해 종양세포를 인식하여 접합형 CAR- T세포가활성화되는지를 테스트하기 위해, 종양세포를 어댑터 항체와 1시간 동안 미리 배양 후, 붙지 않은 free 어댑터 항체를 세척을 통해 제거하였다. 이후 어댑터가 부착된 종양세포를 접합형 CAR- T세포와 함께 시험관내 실험에 사용하였다. Luciferase 기반종양살상능 측정의 경우, 레트로바이러스 또는 렌티바이러스 형질도입 후 증식된 CAR T세포 (1.2X10³〜 7.5 X10⁵ cells/100M/well)를, Jurkat— Luc세포 (3X104cel ls/50M/wel 1 )에 여러 비율로 (CAR- T：Jurkat=0.04〜 25：1)로 가하여 96웰 플레이트에서 밤새 공동배양 (co- culture)한 후, D-Luciferin (600/zg/mt, Pr omega) 50/z£를 가한 후 37°C에서 10분간 배양하여, 그 때까지 생존한 Jurkat- Luc세포에서의 luciferase 효소작용을 유발하였다. 이들 세포의 발광도를 Luminometer (Tecan)를 이용하여 측정하여, CAR T세포를 처리하지 않은 Jurkat- Luc세포의 발광도와 비교하여 종양세포의 생존율을 계산함으로써 CAR T세포의 종양살상능을 계측하였다 . 【Table 12]

Specifically, anti-human CD3 (UCHT1, 1-4-2, 1-4-7) Fab-His antibody and anti-human CD3 (0KT3, UCHT1, 1-4-2, 1-4-7) scFv-His antibody were cloned into the antibody expression vector pCEP4, transduced into 293F cells, and expressed in the culture medium. The expressed antibodies were separated and purified through affinity purification to separate the antibodies present in the culture medium. For the purified antibodies, cell surface binding was verified using an electrophoresis method using SDS-polyacrylamide gel and flow cytometry on a cell line expressing human CD3. The binding of CD3-positive cells to the adapter antibody was verified by incubating 1X10 ⁵ cells/lug adapter at 4 °C for 1 hour. After washing to remove free adapter antibodies, the cells were stained with anti-His antibody (J096G46, Biolegend) that captures the adapter antibodies, and then analyzed using flow cytometry. As described above, in order to analyze whether the expressed and purified anti-human CD3 antibody binds to the CD3 protein expressed on the surface of T cell tumors, the binding of the manufactured 0KT3-His scFv (0KT3-Ck-His, 0KT3-Ck-L-His) and UCHT1-Fab-His to Jurkat T cells was analyzed by flow cytometry, and the results are shown in Fig. 10. In the same way, flow cytometry was performed on the scFv and Fab of 1-4-2, and the scFv and Fab of 1-4-7, and the results are shown in Fig. 11. As shown in Fig. 10, it was confirmed that the scFv and Fab manufactured as anti-CD3 antibodies specifically bound to the Jurkat cell line. As shown in Fig. 11, it was confirmed that it specifically binds only to the Jurkat cell line, but not to the CD3-negative cell line (CD3-deleted Jurkat). Experimental Example 1. Antitumor activity of anti-CD3 adapter antibody and human conjugated CAR-T cells

To verify in vitro whether the complex of the 6xHis-tagged anti-CD3 adapter antibody and human anti-6xHis CAR-T cell (His CAR-T cell) produced in the above Example 2 recognizes and activates CD3-positive T cell tumors, Jurkat leukemia cells were co-cultured with 0KT3 scFv-Ck-6xHis (0KT3-Ck-His), 0KT3 scFv- Ck- 6xHis (0KT3- Ck- L- His), and mouse His CAR-T cells, and the amount of IFN- r secreted by the CAR-T cells was measured by ELISA. As a result, it was confirmed that the function of CAR-T cells was activated as the IFN- r production of T cells increased in the presence of the two adapters. In addition, it showed a dose-dependency in which the effect further increased as the adapter dose was increased fivefold. The results are shown in Fig. 12a. Next, 0KT3 scFv- Ck- 6xHis (0KT3- Ck- His), which has better activity among the two adapters, was co-cultured with Jurkat leukemia cells and human His CAR-T cells, and the amount of IFN- r secreted by CAR-T cells was measured by ELISA. In addition, an experiment was conducted to confirm the cytotoxicity assay for Jurkat tumor cells, and the results are shown in Fig. 12b. Using the same method, the scFv adapter antibody was converted to the Fab-His form verified in the mouse conjugated CAR T cell model, and the cytotoxicity experiment for Jurkat tumor cells was repeated, and the results are shown in Fig. 13. In addition, Fab-His adapter antibodies were produced using UCHT1 antibody, which is another clone of anti-CD3 antibody, and two antibodies (1-4-2 and 1-4-7) selected through screening, and the in vitro activity and cell killing ability of conjugated CAR T cells were analyzed using the same method. The results are shown in Fig. 14 (UCHT1) and Fig. 15 (1-4-2, 1-4-7), respectively. As shown in Figs. 12 to 15, the conjugated CAR T cells were activated in a concentration-dependent manner only in the presence of anti-CD3 adapter antibody, produced a large amount of IFN- r, and exhibited tumor killing ability. Through the above results, the cancer antigen recognition ability, specific activation, and cell killing ability of the anti-CD3 adapter antibody and CAR T cell complex were confirmed. To test whether conjugated CAR-T cells are activated by recognizing tumor cells via the adaptor antibody, tumor cells were pre-incubated with the adaptor antibody for 1 hour, and then unbound free adaptor antibody was removed by washing. After that, the tumor cells with the adaptor attached were used in in vitro experiments together with the conjugated CAR-T cells. For luciferase-based tumor killing activity measurement, CAR T cells (1.2 X 10 ³ to 7.5 X 10 ⁵ cells/100 M/well) expanded after retrovirus or lentivirus transduction were added to Jurkat-Luc cells (3 X 10 4 cells/50 M/well 1) at various ratios (CAR- T: Jurkat = 0.04 to 25:1) and co-cultured overnight in a 96-well plate. Then, D-Luciferin (600/zg/mt, Pro omega) 50/z£ was added and incubated at 37°C for 10 minutes to induce luciferase enzyme activity in Jurkat-Luc cells that survived until then. The tumor killing ability of CAR T cells was measured by measuring the luminescence of these cells using a Luminometer (Tecan) and comparing it with the luminescence of Jurkat-Luc cells that were not treated with CAR T cells to calculate the survival rate of tumor cells.

To measure the cytokine production ability of CAR T cells, CAR T cells and target cells (Jurkat cells) were mixed at a ratio of 1:5 (CAR- T: ^3X104 cells, Jurkat: ^1.5X105 cells) and co-cultured in a 96-well plate for 24 hours, and then the culture supernatant was harvested. The amount of IFN- x secreted into the supernatant was measured by ELISA (mouse IFNx, human IFNy ELISA kit, BD Biosciences). Experimental Example 2. Analysis of the fratricide phenomenon of human conjugated CAR-T cells When conjugated CAR T cells are combined with an anti-CD3 adapter antibody, the conjugated CAR T cells target CD3 on the surface of other CAR T cells and kill each other, which is a phenomenon called fratricide. We verified in = whether CD3 removal by gene editing can prevent this fratricide phenomenon. Specifically, anti-6xHis- CAR T cells were cultured alone (control group) or together with 6xHis-tagged anti-CD3 adapter antibodies and human anti-6xHis- CAR T cells (experimental group), and the viability of CAR T cells was analyzed by flow cytometry at 24 hours, 48 hours, and 72 hours after culture at 37°C, respectively. The viability analysis was performed using a method that analyzes the ratio of 7-AAD-negative cells (live cells) by utilizing 7-AAD, a fluorescent substance that binds to the surface of dead cells, and the results are shown in Fig. 16. As shown in Fig. 16, it was confirmed that fratricide was induced by confirming that the survival rate of anti-6xHis- CAR T cells was significantly reduced in the presence of anti-CD3 adapter antibodies. Therefore, it was experimentally confirmed that conjugated human CAR T cells require the removal of CD3 protein by CD3 gene editing through anti-CD3 adapter antibodies. Experimental Example 3. Production of human CD3-edited conjugated CAR-T cells and verification of anti-fratricide function As confirmed in the above Experimental Example 2, in order to produce CAR T cells that exclude fratricide, human T cells in which the CD3 gene was edited and which simultaneously express the conjugated CAR were produced. Specifically, CD3 deleting sgRNA/Cas9 protein RNP transfection and CAR retrovirus transduction were sequentially performed on human T cells isolated from peripheral blood, resulting in the production of human T cells in which CAR expression and CD3 deletion were simultaneously induced, and CAR expression and CD3 expression were confirmed, respectively, through flow cytometry. The results of flow cytometry analysis during the process of CD3 deleting and CAR introducing are shown in Fig. 17. In addition, to verify the anti-fratricide function of conjugated CAR T cells through CD3 deleting, a cell viability analysis experiment using 7AAD fluorescence staining was performed. Specifically, each was divided into ① (DHis CAR T cells, ② His CAR T cells + anti-CD3 adapter antibody (0KT3-His), ③ CD3 deleted His CAR T cells, @CD3 deleted His CAR T + anti-CD3 adapter antibody (0紅3-田8) cells and cultured at 37°C for 24 hours, and then cell viability was analyzed using 7AAD fluorescence staining. In addition, the CD3 expression level of (DHis CAR T cells, ② His CAR T cells + anti-CD3 adapter antibody (0KT3-His), ©CD3 deleted His CAR T + anti-CD3 adapter antibody (0紅3-田8) cells was analyzed using flow cytometry. The results are shown in Fig. 18. As shown in Fig. 17, it was confirmed that human CD3 gene-edited conjugated CAR T cells were successfully produced. As shown in Fig. 18, in the presence of anti-CD3 adapter antibodies, the survival rate of CD3 gene-edited CAR T cells (73.1%) was significantly higher than that of CAR T cells in which CD3 was not deleted (43.3%) (73.1% vs. 43.3% under 24-hour culture conditions). In addition, it was confirmed that CD3 expression was also significantly reduced in the @CD3 deleted His CAR T + anti-CD3 adapter antibody (0KT3-His) group. Through the above results, it was confirmed that anti-CD3 conjugated CAR T cells that excluded fratricide were successfully produced through CD3 gene editing. Experimental Example 4. Analysis of anti-tumor activity of anti-CD3 adapter antibodies and human conjugated CAR-T cells (in vivo)

4.1 Establishment of proliferation conditions for human CAR T cells

In order to confirm the tumor treatment effect using CD3 gene-edited conjugated human CAR T cells and anti-CD3 adapter antibodies in vivo (in r/w), it is essential to secure a sufficient number of T cells. Therefore, it is necessary to establish conditions for mass proliferation of human CAR T cells. First, CD3-positive cells were removed to increase the separation and purification efficiency of CD-negative cells. Then, in order to secure a sufficient number of CD3 gene-edited CAR T cells required for the experiment, secondary cell proliferation was induced by treating hlL-2 (300 U/ml) for 4 days after separation and purification. A schematic diagram of the established CD3 gene-edited conjugated CAR T cell proliferation conditions is shown in Fig. 19. The human CD3 gene-edited conjugated CAR T cells produced through CRISPR/Cas9 transfection and CAR transduction in the above Experimental Example 3 include some CD3-positive cells. To prevent fratricide and increase the targeting efficiency of CAR T, a process of purifying CD3-negative cells was required, and negative selection was performed using MACS (magnetic activation-associated cell sorting) using anti-human CD3 magnetic beads. Specifically, in the case of human CD3 gene-edited CAR T cells, 2 days after cell activation, CD3 protein gene-editing was performed using electroporation, and then retroviral or lentiviral transduction methods were used to produce CD3 gene-edited conjugated CAR-T cells. In the case of CD3-positive cells remaining after gene editing, only CD3-negative cells were sorted using magnetic-associated cell sorting (MACS). T cells Five days after CD3 was deleted using CRISPR/Cas9 gene editing technology, CD3 gene-edited T cells were cultured with anti-human CD3 antibodies attached to magnetic microspheres. Afterwards, cells cultured with magnetic microsphere-antibody (hCD3 microbead, Mil tenyi) were passed through a strong magnetic separator, and CD3-positive cells that were not gene-edited were attached to the magnetic microsphere-antibody and attached to the magnetic separator. On the other hand, CD3-negative cells passed through the separator without attaching to the separator and were separated. These separated CD3-negative cells were further cultured in a culture medium containing hIL-2 (200 U/ml). The expression of CAR protein on the cell surface was determined by flow cytometry (FACS-Canto II, BD Biosciences) after staining the expanded CAR T cells with Hi s-biotin and APC-labeled SA (SA-APC, BioLegend) 5 days after retroviral transduction. We confirmed that CD3-negative human CAR T cells could be effectively separated and purified by subjecting human CD3 gene-edited conjugated CAR T cells to the Ant i-CD3 MACS process, and we also confirmed whether the CD3-negative state was maintained in the T cell population after the secondary cell proliferation period. The results are shown in Figs. 20 and 21. As shown in Fig. 20, it was confirmed that most of the T cell population maintained the CD3-negative state and the level of CAR expression after the secondary cell proliferation period. As shown in Figure 21, CD3 gene-edited conjugated CAR T cells (day 1: approximately 2xlO ⁷ T cells) that had been expanded 20-fold over a total culture period of 11 days after the initial human T cell isolation and activation day (day 0: IxlO ⁶ T cells) were finally obtained.

4.2 Analysis of anti-tumor activity of human CAR T cells (0KT3- scFV- His) (in vivo) To verify the anti-cancer efficacy against T-cell lymphoma in vivo using anti-CD3 adapter antibody and CD3 gene-edited conjugated CAR T cells, a human-derived T-cell tumor •九»±31: cell0111厂コ31：-匕110) overexpressing luciferase was injected into immunodeficient NSG mice to establish a xenogeneic mouse tumor model. Specifically, CD3-positive Jurkat-Luc cell line (luciferase-transfected) 3xl0 ⁶ cells were intravenously injected into NSG mice. Three days after the Jurkat-Luc injection, CD3 gene-edited human CAR T cell 3xl0 ⁶ cells produced from human T cells were intravenously injected. The first anti-CD3 adapter antibody (0KT3(scFv)- Ck-His) was administered 4 hours before CAR T cell injection at a dose of 20 ug per mouse, and then administered at 2-day intervals for a total of 8 times (Fig. 22). Bioluminescence images were acquired once a week using IVIS 100 (PerkinElmer, Waltham, MA USA) to measure the size of tumor cells. Each mouse was injected intraperitoneally with 2 mg of D-Luci fer in 100 μl of saline. The size of the tumor cells was measured via IVIS 10 minutes after the intraperitoneal injection. The NSG mice were divided into three groups: ① a group injected with only the D Jurkat cell line, ② a group injected with only CD3 gene-edited conjugated CAR T cells without anti-CD3 adapter antibody, and ③ a group injected with anti-CD3 adapter antibody + CD3 gene-edited conjugated CAR T cells, and the experiment was conducted. The survival rate of the mouse model up to 60 days after tumor cell injection was analyzed. The results are shown in Fig. 23. As shown in Fig. 23, compared to when CD3 gene-edited conjugated CAR T cells were administered alone, when anti-CD3 adapter antibody was administered together, a significant decrease in the tumor was observed, and it was confirmed that the survival rate of the mice also increased significantly.

4.3 Analysis of anti-tumor activity of human CAR T cells (UCHT1- Fab- Hi s) (in vivo) In addition to the 0KT3(scFv)- Ck- Hi s adapter antibody performed in Experimental Example 4. 2, another type of anti-CD3 adapter antibody, UCHT1- Fab- Hi s, was used to demonstrate the anti-tumor activity in vivo. A xenogeneic mouse tumor model experimental model was created by injecting Jurkat-Luc cells, a T cell tumor derived from human origin, into DNLGO and immunodeficient NSG mice. Specifically, 3xl0 ⁶ cells, a CD3-positive Jurkat-Luc cell line, were intravenously injected into NSG mice. Three days after the Jurkat injection, 3xl0 ⁶ cells, a CD3 gene-edited human CAR T cell line produced using human T cells, were intravenously injected. The UCHTl-Fab-His adapter antibody was administered as the first anti-CD3 adapter antibody 4 hours before CAR T cell injection at 20 ug per mouse, and was then repeatedly administered 7 times at 2-day intervals. After a 2-week rest period, the anti-CD3 adapter antibody was administered 8 times at 2-day intervals again, for a total of 16 times (Fig. 24). The NSG mice were divided into ① a group injected with only the Jurkat cell line, ② a group injected with only CD3 gene-edited conjugated CAR T cells without anti-CD3 adapter antibody, ③ a group injected with only anti-CD3 adapter antibody without CD3 gene-edited conjugated CAR T cells, and © a group injected with anti-CD3 adapter antibody + CD3 gene-edited conjugated CAR T cells, and the experiment was conducted. The survival rate of the mouse models up to 80 days after tumor cell injection was analyzed. The results are shown in Fig. 25. As shown in Fig. 25, when the CD3 gene-edited conjugated CAR T cells were administered together with the anti-CD3 adapter antibody, a significant decrease in tumors was observed, similar to the results of the experiment using the 0KT3 scFv adapter, and the survival rate of the mice was also confirmed to be significantly increased. Through the above results, the excellent anti-tumor activity in vivo using the anti-CD3 antibody adapter antibody (0KT3-scFv and UCHT1-Fab) was confirmed, thereby demonstrating the efficacy of CD3-conjugated CAR T cells on T-cell tumors. It was confirmed that the anti-human CD3 adapter antibody and human conjugated CAR T cell complex can be provided as a therapeutic agent for the treatment of cancer. In addition, it was confirmed that various anti-CD3 antibodies can be used as adapter antibodies for CD3-conjugated CAR T cells. Experimental Example 5. Evasion of immunodeficiency toxicity (normal T cell toxicity) of human conjugated CAR T cells In order to confirm the evasion of toxicity against normal T cells of the anti-human CD3 adapter antibody and human conjugated CAR T cell complex, the killing ability against normal T cells was confirmed using normal T cells as target cells. At this time, since the immunodeficiency prevention ability could not be verified in vivo (九 r/ra), it was confirmed through an in vitro (九 vitro) experiment. Specifically, T cells of human peripheral blood mononuclear cells (PBMCs) were used as target cells, and after adding anti-human CD3 antibodies and/or CD3 gene-edited human conjugated CAR T cells, the killing of normal T cells was analyzed through flow cytometry, and the results are shown in Fig. 26. As shown in Fig. 26, only in the presence of anti-human CD3 adapter antibody, T cells expressing CD3 are rapidly reduced, thereby confirming the killing of normal T cells by CAR T cells, and in the absence of adapter antibody, the number of T cells positively expressing CD3 is maintained regardless of the presence or absence of CD3-conjugated CAR T cells, indicating that normal T cells are not killed. Through this, it was indirectly confirmed that the side effect of immunodeficiency toxicity of the CD3-conjugated CAR T cells of the present invention can be avoided by controlling the toxicity to normal T cells depending on the administration of adapter antibody in an actual living body. Experimental Example 6. Production of conjugated CAR-T cells using humanized antibodies

For commercialization of CD3 gene-edited conjugated CAR T cells, humanization and affinity maturation of the mouse-derived anti-6xHis scFv (mutated 3D5) used in the His CAR were performed. Antibody humanization and affinity maturation For humanization and affinity maturation of the mouse anti-His scFv antibody, first, six CDRs (complementarity determining regions) showing the most appropriate sequence similarity were grafted onto the IGHV1-18 and IGKV2-28 human germline genes. At the same time, several amino acid sites in the framework region (FR) were changed to remove post-translational modifications and perform reverse mutations. Subsequently, a sub-library of humanized antibodies containing site-directed mutations in the six CDRs was generated for affinity maturation. The antibody was produced using the Exp i293 expression system, affinity purified, and ELISA was performed to confirm the binding affinity to the antigen (WuXi Biologies). SPR binding affinity measurement of humanized antibodies

The binding kinetics and affinity constants were generated using a Biacore 8K SPR system (Cytiva) equipped with a CM5 sensor chip. To prepare the capture surface of anti-His antibody, streptavidin was immobilized and then biotin-conjugated 6xHis peptide was captured. The antibody was serially diluted 2-fold with 1x PBS and injected at a flow rate of 30 μL/min for 3 min followed by a 10-min dissociation step. The surface was regenerated using 10 mM glycine, pH 1.5. The kinetics were analyzed using Biacore 8K Evaluation Software Version 1.0 (Cytiva). Figure 27 shows the antigen affinity of the humanized antibody anti-His, and the final humanized antibody scFv showed an antigen affinity approximately 5.8-fold higher than that of the original scFv, demonstrating successful humanization. Next, using the humanized anti-6xHis scFv, a CAR gene similar to the existing His CAR was cloned into a lentiviral vector to produce a lentivirus, which was then transduced into human T cells to produce humanized His CAR- T cells (huHis CAR- T cells). The expression of the CAR on the surface of the huHis CAR- T cells was confirmed by flow cytometry, and the results are shown in Fig. 28. The in vitro activity of the conjugated CAR T cells using the produced huHis CAR- T cells and the UCHTl-Fab-His adapter was verified, and it was confirmed that the huHis CAR- T cells also showed T cell activation and tumor cell killing ability in the presence of the adapter antibody, similar to the existing His CAR- cells (Fig. 29). Therefore, by confirming that it is possible to produce a conjugated humanized CAR- T cell that can be applied clinically, the commercialization possibility of the present invention was proven.

이상의 결과를 통해 , 항 CD3 어댑터 항체와 CD3편집 접합형 CAR T세포의 복합투여가 T세포 종양에 대한 강력한 치료효과를 달성할 수 있을 뿐 아니라, 접합형이 CAR T를 구현하여 , 정상 T세포독성에 의한 면역결핍부작용의 회피 개연성을 입증하였다. 전술한 본 발명의 설명은 예시를 위한 것이며 , 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시 예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 【Table 13]

The results above demonstrate that combined administration of anti-CD3 adapter antibodies and CD3-edited conjugated CAR T cells can not only achieve a powerful therapeutic effect on T-cell tumors, but also demonstrate the possibility of avoiding immunodeficiency side effects caused by normal T-cell cytotoxicity by implementing conjugated CAR T. The above description of the present invention is for the purpose of illustration, and those skilled in the art to which the present invention pertains will understand that the present invention can be easily modified in other specific forms without changing the technical spirit or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all aspects and not limiting.

Claims

【Scope of Claims】 【Claim 11 A pharmaceutical composition for preventing or treating a T cell-derived tumor, comprising a chimeric antigen receptor T cell (CAR-T cell) in which CD3 expression is reduced or eliminated and an adapter molecule that regulates the activity of the CAR-T cell, wherein the adapter molecule comprises (a) a first moiety that binds to CD3 on a target cell and (b) a second moiety that specifically binds to an extracellular domain of a chimeric antigen receptor, and wherein the CAR-T cell expresses a chimeric antigen receptor comprising (i) an extracellular domain comprising an antibody or an antigen-binding fragment thereof that specifically binds to the second moiety of the adapter molecule, (ii) a transmembrane domain, and (iii) an intracellular signaling domain. 【Claim 2] A pharmaceutical composition for preventing or treating a T cell-derived tumor, comprising a chimeric antigen receptor T (CAR T) cell in which CD3 expression is reduced or eliminated, wherein the pharmaceutical composition is administered in combination with an adaptor molecule capable of modulating the activity of the CAR-T cell, wherein the adaptor molecule comprises (a) a first moiety that binds to CD3 on a target cell and (b) a second moiety that specifically binds to an extracellular domain of the chimeric antigen receptor, and wherein the chimeric antigen receptor has an extracellular domain, a transmembrane domain, and an intracellular signaling domain comprising an antibody or an antigen-binding fragment thereof that specifically binds to the second moiety of the adaptor molecule. 【Claim 3】 A pharmaceutical composition according to claim 1 or 2, wherein the first moiety is selected from the group consisting of an antibody, an antigen-binding fragment, an affibody, a diabody, and an aptamer. 【Claim 4】 A pharmaceutical composition according to any one of claims 1 to 3, wherein the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, scFv or a single-domain antibody (sdAB). 【Claim 5】 A pharmaceutical composition according to any one of claims 1 to 4, wherein the second moiety is a peptide tag or a hapten tag. 【Claim 6] A pharmaceutical composition according to any one of claims 1 to 5, wherein the tag is selected from the group consisting of a His tag, a Myc tag, a cotinine tag, a FITC tag, a biotin tag, a Leucin zipper tag, a Flag tag, an Xpress tag, an Avi tag, a calmodulin binding peptide (CBP) tag, a polyglutamate tag, an HA tag, a Strep tag, Softag 1, Softag 3, and a V5 tag. 【Claim 7】 A pharmaceutical composition according to any one of claims 1 to 6, wherein the first moiety is an anti-CD3 antibody or an antigen-binding fragment thereof, and/or the second moiety is a His tag. 【Claim 8】 An antibody according to any one of claims 1 to 7, wherein the first moiety comprises a light chain variable region comprising 0KT3, UCHT1, a CDR1 consisting of the amino acid sequence of SEQ ID NO: 23, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 24, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 25, and a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 27, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 28; A pharmaceutical composition selected from the group consisting of an antibody comprising a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 32, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 33, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 34 and a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 35, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 36, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 37; and a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 67, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 68, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 69 and a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 70, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 70, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 72, wherein the second moiety is a His tag. 【A pharmaceutical composition according to any one of claims 1 to 8, wherein the reduction or elimination of CD3 expression of CAR-T cells is achieved by removing CD3 or T cell receptor (TCR) of CAR-T cells. 【Claim 10】 The T-cell derived tumor of any one of claims 1 to 9 is T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, chronic lymphoprol i ferat ive disorder of NK cells, aggressive NK Leukemia (aggressive NK leukemia), systemic EBV-positive T-cell 1 lymphoprol i ferat ive disease of chi dhood, hydroa vacc inif orme- 1 i ke lymphoma, adult T-cell leukemia/lymphoma, extranodal NK/T-cell 1 lympho oma (nasal type), enteropathy-associated T-cell 1 lymphoma, hepatosplenic T-cell 1 lymphoma, subcutaneous panniculitis-like T-cell 1 lymphoma, mycosis fungoides fungoides, Se'zary syndrome, primary cutaneous CD30-positive T-cell lymphoproliferative disorders, lymphomatoid papulosis, primary cutaneous anaplastic large cel 1 lymphoma, primary cutaneous gamma/delta T-cell lymphoma, primary cutaneous CD8-positive aggressive epidermotropic cytotoxic T-cel 1 lymphoma, primary cutaneous CD4-positive small/medium T-cell 1 lymphoma, peripheral T-cell lymphoma NOS (not otherwise specified) A pharmaceutical composition selected from the group consisting of per ipheral T-cel 1 lymphoma N0S (not otherwise specified), angioimmunoblastic T-cel 1 lymphoma, ALK positive anaplastic large cel 1 lymphoma and ALK negative anaplastic large cel 1 lymphoma. 【Claim 11】 A complex comprising a chimeric antigen receptor (CAR-T cell) with reduced or deleted expression of CD3 and an adapter molecule that regulates the activity of the CAR-T cell, wherein the adapter molecule comprises (a) a first moiety that binds to CD3 on a target cell and (b) a second moiety that specifically binds to an extracellular domain of the chimeric antigen receptor, and wherein the CAR-T cell expresses a chimeric antigen receptor comprising (i) an extracellular domain comprising an antibody or an antigen-binding fragment thereof that specifically binds to the second moiety of the adapter molecule, (ii) a transmembrane domain, and (iii) an intracellular signaling domain. 【Claim 12】 A method for preventing or treating a T cell-derived tumor, comprising administering to a subject a pharmaceutical composition according to any one of claims 1 to 10 or a complex according to claim 11. 【Claim 13】 A chimeric antigen receptor T cell (CAR-T cell) in which CD3 expression is reduced or eliminated, wherein the CAR-T cell expresses a chimeric antigen receptor comprising (i) an extracellular domain comprising an antibody or an antigen-binding fragment thereof that specifically binds to a second moiety of an adaptor molecule, (ii) a transmembrane domain, and (iii) an intracellular signaling domain, wherein the adaptor molecule comprises (a) a first moiety that binds CD3 on a target cell and (b) a second moiety that specifically binds to the extracellular domain of the chimeric antigen receptor. 【Claim 14】 A CAR-T cell according to claim 13, wherein the first moiety is an anti-CD3 antibody or an antigen-binding fragment thereof and/or the second moiety is a His tag. 【Claim 15】 A CAR-T cell according to claim 13 or 14, wherein the second moiety is a His tag, and the antibody or antigen-binding fragment thereof that specifically binds to the His tag has an amino acid sequence of SEQ ID NO: 63. 【Claim 16】 A CAR-T cell having an amino acid sequence of SEQ ID NOs: 63 to 65 according to any one of claims 13 to 15. 【Claim 17】 (a) a step of down-regulating the expression of CD3 or T cell receptor (TCR) in T cells, and (b) a step of introducing a chimeric antigen receptor (CAR) comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the second moiety of an adapter molecule comprises an antibody or an antigen-binding fragment thereof that specifically binds to an adapter molecule, wherein steps (a) and (b) are performed in any order, a method for producing a chimeric antigen receptor T cell (CAR-T cell) in which CD3 expression is reduced or eliminated, wherein the adapter molecule comprises (i) a first moiety that binds to CD3 on a target cell and (ii) a second moiety that specifically binds to an extracellular domain of the chimeric antigen receptor. 【Claim 18】 In claim 17, the step of down-regulating the expression of CD3 or T cell receptor (TCR) in the (a) T cell is performed by a genome editing technique, shRNA, siRNA or miRNA. 【Claim 1】 A method according to claim 17 or 18, wherein the genome editing technique removes CD3 or T cell receptor (TCR) of CAR-T cells by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas 9, TALEN, zinc finger nuclease, base-editing, or prime— editing. 【Claim 20】 A method for removing CD3 or T cell receptor (TCR) of CAR-T cells by CRISPR/Cas9 technique using a guide gene comprising a polynucleotide sequence of SEQ ID NO: 57 or SEQ ID NO: 58, according to any one of claims 17 to 19. 【Claim 21] (a) a first moiety that binds to CD3 on a target cell; and (b) An adaptor molecule for regulating the activity of a chimeric antigen receptor T cell (CAR-T cell), comprising a second moiety that specifically binds to the extracellular domain of a chimeric antigen receptor. 【Claim 22】 An adapter molecule according to claim 21, wherein the first moiety is selected from the group consisting of an antibody, an antigen-binding fragment, an affibody, a diabody, and an aptamer. 【Claim 23】 An adapter molecule according to claim 21 or claim 22, wherein the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, scFv or a single-domain antibody (sdAB). 【Claim 24】 An adapter molecule according to any one of claims 21 to 23, wherein the second moiety is a tag, a polypeptide or a hapten. 【Claim 25】 An adapter molecule according to any one of claims 21 to 24, wherein the tag is selected from the group consisting of a His tag, a Myc tag, a cotinine tag, a FITC tag, a Biotin tag, a Leucin zipper tag, a Flag tag, an Xpress tag, an Avi tag, a calmodulin binding peptide (CBP) tag, a polyglutamate tag, an HA tag, a Strep tag, Softag 1, Softag 3, and a V5 tag. 【Claim 26] An adapter molecule according to any one of claims 21 to 25, wherein the first moiety is an anti-CD3 antibody or an antigen-binding fragment thereof, and/or the second moiety is a His tag. 【Claim 27】 An antibody according to any one of claims 21 to 26, wherein the first moiety comprises a light chain variable region comprising 0KT3, UCHT1, a CDR1 consisting of the amino acid sequence of SEQ ID NO: 23, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 24, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 25, and a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 26, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 27, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 28; An adapter molecule selected from the group consisting of an antibody comprising a light chain variable region comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 32, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 33, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 34, and a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 35, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 36, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 37; and an antibody comprising a light chain variable region comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 67, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 68, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 69, and a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 70, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 70, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 72, wherein the second moiety is a His tag.