Universal immunoregulatory t cells comprising chimeric antigen receptor and uses thereof

Abstract

The present invention relates to universal immunomodulatory T cells comprising a chimeric antigen receptor and uses thereof and, specifically, to universal immunomodulatory T cells in which immunogenicity of cells is removed so that general T cells obtainable in sufficient numbers from healthy people can be applied to all organ transplant patients and in which Foxp3 and a chimeric antigen receptor are both introduced, and a use thereof.

Description

Universal immunomodulatory T cells containing chimeric antigen receptors and uses thereof

[0001] The present invention relates to universal immunomodulatory T cells comprising a chimeric antigen receptor and their uses. Specifically, it relates to universal immunomodulatory T cells in which the immunogenicity of cells is removed and Foxp3 and a chimeric antigen receptor are simultaneously introduced so that general T cells, which can be obtained in sufficient numbers from healthy individuals, can be applied to all organ transplant patients, and their uses.

[0002]

[0003] T cells are cells that play a crucial role in mediating adaptive immunity. T cells are activated through the stimulation of antigen-recognizing receptors (T cell receptors, TCRs), co-stimulatory molecules, and cytokines. Furthermore, T cell TCRs trigger an immune response through major histocompatibility complex (MHC) molecules bound to antigens; however, cancer cells suppress the expression of MHC molecules as a mechanism to evade this immune response. Adoptive immune therapies (adoptive cellular therapies) are being developed by utilizing these characteristics of T cells.

[0004] The development of anticancer treatments using immune cells has progressed with a focus on T cells, and anticancer T cell therapy has shown tangible results as the in vitro culture and proliferation of tumor antigen-specific T cells became possible (Gattinoni L, et al., Nat Rev Immunol. 2006;6(5):383-93). In addition, existing immunosuppressants used to prevent rejection after organ transplantation have been problematic because they provide non-specific, generalized immunosuppression, which fails to induce immune tolerance and leads to increased infections and cancer. On the other hand, regulatory T cell (Treg) therapy has antigen-specific immunosuppressive efficacy and helps induce immune tolerance, resulting in fewer side effects such as increased infections and cancer, and allowing existing immunosuppressants to be discontinued or reduced. Conventional Treg cell therapy, which involves sorting Treg cells from a patient, proliferating them in vitro, and then re-injecting them into the transplant patient, has successfully completed or is currently conducting a safety study (ONE-STUDY, NCT02244801, NCT02091232) and has also started a efficacy study (TWO-STUDY, ISRCTN11038572).

[0005] Meanwhile, Tregs are scarce in the human body, and antigen-specific Tregs are even rarer. Consequently, the process of isolating and proliferating these cells is complex, and there are limitations such as reduced viability, proliferation, and immunosuppressive capacity after injection into the body. Recently, CAR T-cells—in which a chimeric antigen receptor (CAR) that recognizes specific antigens has been genetically introduced into T-cells—have achieved groundbreaking results in anticancer immunotherapy. Conversely, introducing a CAR into Tregs can confer antigen specificity and aid in maintaining survival, proliferation, and immunosuppressive capabilities, thus necessitating CAR Treg cell therapy. However, there is a practical problem in that the method of producing personalized CAR Treg cells using the patient's own autologous Tregs is difficult to commercialize. Therefore, for easy clinical application, universal CAR Treg (uCAR Treg) cell therapy technology is required, which involves producing cells from a select group of healthy individuals that can be applied uniformly to all patients. On the other hand, the issue of the low number of Tregs in the human body remains a challenge that must be addressed in CAR Treg cell therapy as well.

[0006] Therefore, while it is possible to commercialize CAR Tregs from healthy individuals by using them identically for multiple patients, there is a problem in that the recipient's immune rejection of different HLAs causes the immune-modulating ability of the CAR Treg to disappear. On the other hand, the method of individually producing CAR Tregs from the patient's own autologous CAR Treg to avoid immune rejection has limitations as it is impractical, so the development of universal CAR Treg cell therapy technology is required.

[0007] Under this technical background, the inventors of the present application have identified universal immunomodulatory T cells and their uses by obtaining general T cells in sufficient numbers from healthy individuals, removing the cellular immunity so that they can be applied to all organ transplant patients, and simultaneously introducing Foxp3 and a chimeric antigen receptor.

[0008] The information described above in the background section is intended solely to enhance understanding of the background of the present invention and may not include information that forms prior art already known to those skilled in the art to which the present invention belongs.

[0009]

[0010] Summary of the Invention

[0011] The object of the present invention is to provide recombinant immunoregulatory T cells (regulatory T: Treg cells).

[0012] Another objective of the present invention is to provide a method for producing the recombinant immunomodulatory T cells.

[0013] Another objective of the present invention is to provide a composition for the prevention or treatment of transplant rejection comprising the immunomodulatory T cells.

[0014] To achieve the above objective, the present invention introduces a nucleic acid encoding a chimeric antigen receptor, and

[0015] The β2M (β2-microglobin) coding gene is deleted, and

[0016] It provides recombinant immunoregulatory T cells (regulatory T: Treg cells) into which a non-classic HLA (Non-classic human leukocyte antigen) class I coding gene has been introduced.

[0017] The present invention also provides a method for producing recombinant immunoregulatory T cells (regulatory T: Treg cell) comprising the following steps:

[0018] (a) A step of introducing a nucleic acid encoding a chimeric antigen receptor;

[0019] (b) a step of deleting the β2M (β2-microglobin) coding gene; and

[0020] (c) Step of introducing a non-classical HLA (human leukocyte antigen) class I coding gene

[0021] The present invention also provides a composition for preventing or treating transplant rejection, comprising the immunomodulatory T cells.

[0022]

[0023] Figure 1 shows the results of constructing a vector for human anti-CD40 uCAR expression.

[0024] Figure 2 shows the results of confirming the activity of CR4 gene scissors in the T7E1 assay after applying CR4, which has the best gene scissors activity, to human primary lymphocytes.

[0025] Figure 3 shows the results of the construction and expression verification of the human Anti-CD40 uCAR TregFoxp3 vector.

[0026] Figure 4 shows the human anti-CD40 uCAR Treg Foxp3 This shows the results of production and expression verification.

[0027] Figure 5 shows the results of FACS analysis confirming the expression of anti-hCD40 uCAR TregFoxp3.

[0028] Figure 6 shows the results of confirming the expression levels of Myc, β2M, and HLA-E in cells using FACS.

[0029] Figure 7 shows the results of sequential transduction after virus production using hβ2M-CR4 gene scissors, followed by puromycin selection and a stabilization period under IL-2 supply, and then confirming expression by FACS.

[0030]

[0031] Detailed Description of the Invention and Preferred Embodiments

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0033] According to one embodiment of the present invention, a 'universal chimeric antigen receptor immunomodulatory T cell (uCAR Treg)' was produced that is applicable to all patients by using gene editing technology to remove the β2-microglobin (β2M) gene to eliminate the expression of MHC (HLA) I protein to evade T cell attacks, and by overexpressing nonclassic HLA Ib (mouse Qa-1b / human HLA-E) to block NK cell attacks.

[0034] Based on this, the present invention relates, in one aspect, to a recombinant regulatory T cell (regulatory T: Treg cell) having a nucleic acid encoding a chimeric antigen receptor introduced, a β2M (β2-microglobin) coding gene deleted, and a non-classic HLA (Non-classic human leukocyte antigen) class I coding gene introduced.

[0035] The present invention also relates to a method for producing recombinant immunoregulatory T cells (regulatory T: Treg cell) comprising the following steps:

[0036] (a) a step of introducing a nucleic acid encoding a chimeric antigen receptor; (b) a step of deleting a gene encoding β2M (β2-microglobin); and (c) a step of introducing a gene encoding non-classical HLA (human leukocyte antigen) class I.

[0037] The aforementioned immunomodulatory T cells or Treg cells can regulate the immune system and downregulate the induction and proliferation of effector T cells. Immunomodulatory T cells prevent autoimmune responses and assist the immune system in distinguishing between self and non-self. Immunomodulatory T cells produce repressive cytokines, including Transforming growth factor beta, Interleukin 35, and Interleukin 10, and can induce other cell types to express Interleukin 10. Immunomodulatory T cells can induce apoptosis of effector cells by producing Granzyme B. Furthermore, they function through reverse signaling via direct interaction with dendritic cells and the induction of immunosuppressive indoleamine 2,3-deoxygenase. Additionally, immunomodulatory T cells can downregulate immune responses through the exoenzymes CD39 and CD73 by producing immunosuppressive adenosine. Immunomodulatory T cells suppress immune responses through direct interaction with dendritic cells via LAG3 and TIGIT. Another control mechanism is through the IL-2 feedback loop. Another mechanism of immunosuppression by immunoregulatory T cells is the prevention of co-stimulation via CD28 in effector T cells by the action of the molecule CTLA-4.

[0038] A 'Chimeric antigen receptor (CAR)' is a synthetic construct designed to induce an immune response against a target antigen and cells expressing that antigen. A CAR includes an extracellular domain, a transmembrane domain, and an intracellular signaling domain.

[0039] First-generation CARs included an extracellular domain containing an antigen recognition site specifically expressed in cancer cells, a transmembrane domain, and an intracellular signaling domain, and used only CD3ζ as the signaling domain, but had problems such as minimal therapeutic effect on cancer and short duration.

[0040] To enhance responsiveness to immune cells, a second-generation CAR was manufactured by combining a co-stimulatory domain (CD28 or CD137 / 4-1BB) with CD3ζ, and compared to the first-generation CAR, the number of CAR-containing immune cells remaining in the body increased significantly. While the second-generation CAR utilized a single co-stimulatory domain, the third-generation CAR utilized two or more co-stimulatory domains. To achieve the expansion and persistence of CAR-containing immune cells in vivo, the co-stimulatory domain can be combined with 4-1BB, CD28, or OX40, etc.

[0041] 4th generation CARs include additional genes encoding cytokines such as IL-12 or IL-15 to allow for additional expression of CAR-based immune proteins of the cytokines, and 5th generation CARs additionally include an interleukin receptor chain, e.g., IL-2Rβ, to enhance immune cells.

[0042] The term “extracellular binding domain” above refers to a portion of the CAR comprising an antigen binding domain having the ability to specifically bind to a target antigen. The extracellular binding domain may comprise any protein, polypeptide, oligopeptide, or peptide having the ability to specifically recognize and bind to a biological molecule (e.g., cell surface receptor or tumor protein, lipid, polysaccharide, or other cell surface target molecule, or a component thereof). The binding domain comprises any naturally occurring, synthetic, semi-synthetic, or recombinated binding partner for the target biological molecule.

[0043] The term “specifically binding” in the present invention refers to the binding of a molecule to another molecule with a binding affinity greater than that of the background binding. For example, an extracellular binding domain specifically binds to a target molecule when it binds to or associates with the target molecule with an affinity of about 10⁻⁵ M or greater, or Ka (i.e., the equilibrium dissociation constant of a specific binding interaction having units of 1 / M). Alternatively, the affinity is the equilibrium dissociation constant (Kd) of a specific binding interaction having units of M (e.g., 10 -5 M to 10 -13 It can be defined as M or less.

[0044] The affinity of the extracellular binding domain and CAR according to the present invention can be easily measured by binding association or substitution analysis using conventional techniques, for example, competitive ELISA (enzyme-linked immunosorbent assay) or labeled ligands, or surface-plasmon resonance devices such as the Biacore T100 (available from Biacore Incorporated, Piscataway, NJ), or optical biosensor technologies such as EPIC systems or EnSpire available from Corning and Perkin Elmer, respectively.

[0045] In the present invention, the chimeric antigen receptor may include, for example, an antibody or an antigen-binding fragment thereof that specifically binds to an immunomodulatory molecule.

[0046] The above immunomodulatory molecules may be, for example, CD27, CD28, 4-1BB(CD137), OX40, CD30, CD40, IFNγ, PD-1, PD-L1, ICOS, TGF-β, CTLA-4Ig, IL-10, CD2, CD7, LIGHT, NKG2C, B7-H3, or CD83, but are not limited thereto.

[0047] The above chimeric antigen receptor may include, for example, an antibody that specifically binds to the immunomodulatory molecule CD40 or an antigen-binding fragment thereof.

[0048] The above "antibody" refers to a conjugate that specifically binds to an immunomodulatory molecule. The scope of the present invention includes not only the complete antibody form that specifically binds to an immunomodulatory molecule, but also the antigen-binding fragment of said antibody molecule.

[0049] As used herein, "antibody variable domain" refers to the light and heavy chain portions of an antibody molecule comprising the amino acid sequences of the complementarity determining region (CDR; i.e., CDR1, CDR2, and CDR3) and the backbone region (FR). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

[0050] "Complementary determining regions" (CDRs; i.e., CDR1, CDR2, and CDR3) refer to amino acid residues of the antibody variable domain that are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2, and CDR3.

[0051] A complete antibody has a structure consisting of two full-length light chains and two full-length heavy chains, with each light chain connected to the heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and has subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types.

[0052] In the present invention, the “fragment” of an antibody refers to a fragment possessing antigen-binding function and is used to include scFv, Fab, F(ab')2, and Fv fragments. Among the antibody fragments, Fab has a structure having a variable region of the light and heavy chains, a constant region of the light chain, and a first constant region (CH1) of the heavy chain, and possesses 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. The F(ab')2 antibody is generated when the cysteine ​​residues in the hinge region of Fab’ form disulfide bonds. Fv is the smallest antibody fragment having only a heavy chain variable region and a light chain variable region. Two-chain Fvs have a variable region of the heavy chain and a variable region of the light chain connected by non-covalent bonds, while single-chain Fvs (scFvs) generally have a variable region of the heavy chain and a variable region of the light chain connected by covalent bonds through a peptide linker or directly connected at the C-terminus, so they can form a dimer-like structure similar to two-chain Fvs. These antibody fragments can be obtained using proteolytic enzymes (for example, restriction cleavage of the whole antibody with papain yields Fab, and cleavage with pepsin yields the F(ab')2 fragment), and can also be produced through recombinant DNA technology.

[0053] A “single-strand Fv” or “scFv” antibody fragment contains the VH and VL domains of the antibody, which are present within a single polypeptide chain. The Fv polypeptide may further include a polypeptide linker between the VH domain and the VL domain to enable scFv to form a structure intended for antigen binding.

[0054] In the present invention, in a scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody, VH and VL can be connected through a linker.

[0055] The above linker may be a peptide linker and may have a length of about 10-25 aa. For example, it may include hydrophilic amino acids such as glycine and / or serine.

[0056] The above linker is, for example, (GS) n , (GGS) n , (GSGGS) n or (G n S) m (n and m may each be 1 to 10), but the linker may include, for example (G n S) m (n and m can be 1 to 10, respectively).

[0057] In a specific embodiment according to the present invention, the antibody that specifically binds to the immunomodulatory molecule CD40 may comprise a scFv in which VH and VL are linked by a (G4S)3 linker. The scFv may comprise the following sequence.

[0058]

[0059] The above scFv can be encoded by the following nucleic acid.

[0060]

[0061] The sequence of the antibody described herein may include not only the sequence of the antibody but also biological equivalents thereof. For example, additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, the deletion, insertion, and / or substitution of amino acid sequence residues of the antibody. Such amino acid modifications are made based on the relative similarity of amino acid side chain substituents, e.g., hydrophobicity, hydrophilicity, charge, size, etc. By analyzing the size, shape, and type of amino acid side chain substituents, it can be seen that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine may be considered biologically functional equivalents.

[0062] Considering the variant having the aforementioned biological equivalent activity, the antibody of the present invention or the nucleic acid molecule encoding it is interpreted to include a sequence that exhibits substantial identity with the sequence described in SEQ ID NO. The above substantial identity refers to a sequence that exhibits at least 90% homology, most preferably at least 95% homology, 96% or more, 97% or more, 98% or more, or 99% or more homology when any other sequence is aligned to correspond as much as possible with the sequence of the present invention described above and the aligned sequence is analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI, etc., and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the internet. BLAST is accessible at www.ncbi.nlm.nih.gov / BLAST / . The method for comparing sequence homology using this program can be found at www.ncbi.nlm.nih.gov / BLAST / blast_help.html.

[0063] Based on this, the antibody or its antigen-binding fragment of the present invention may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homology compared to the specified sequence or whole described in the specification. Such homology may be determined by sequence comparison and / or alignment by methods known in the art. For example, the percentage sequence homology of the nucleic acid or protein of the present invention may be determined using a sequence comparison algorithm (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection.

[0064] The above chimeric antigen receptor may be characterized by including a transmembrane domain.

[0065] The “transmembrane domain” mentioned above is a part of a CAR that fuses an extracellular binding portion and an intracellular signaling domain and anchors the CAR to the plasma membrane of an immune effector cell. The transmembrane domain may be derived from natural, synthetic, semi-synthetic, or recombinant sources. In the present invention, the transmembrane domain may be characterized as being selected from the group consisting of the alpha (α), beta (β), or zeta (ζ) chain of a T-cell receptor (TCR), CD28, CD3 epsilon (ε), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, but is not limited thereto.

[0066] The above transmembrane domain can be attached to the extracellular binding domain of the CAR through a linker. For example, the linker may be a short oligo- or polypeptide linker of 2 to 10 amino acids in length, preferably a glycine (G)-serine (S) doublet, but is not limited thereto.

[0067] The binding domain of a CAR is generally followed by one or more “hinge domains.” Accordingly, in the present invention, the extracellular domain may be characterized by additionally including a signal peptide (SP) and / or a hinge together with the binding domain. The extracellular domain is a site where the main signal is transmitted, located outside the cell membrane, and is a domain for specifically recognizing a target.

[0068] The term “hinge domain” in the present invention refers to a part of a CAR that plays a crucial role in localizing an extracellular binding domain, which contains an antigen binding site, away from the surface of the agent cell to enable appropriate cell / cell contact, antigen binding, and activation. A CAR generally comprises one or more hinge domains between an extracellular binding domain and a transmembrane domain. The hinge domain may be derived from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. The “altered hinge region” is (a) a naturally occurring hinge region having up to 30% amino acid change (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitution or deletion), (b) a portion of a naturally occurring hinge region of at least 10 amino acids (e.g., at least 12, 13, 14, or 15 amino acids) length having up to 30% amino acid change (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitution or deletion), or (c) a core hinge region (which is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids). It refers to a portion of a naturally occurring hinge region that includes (which may be of length). In certain embodiments, one or more cysteine ​​residues in the naturally occurring immunoglobulin hinge region may be substituted with one or more other amino acid residues (e.g., one or more serine residues). The altered immunoglobulin hinge region may alternatively or additionally have a proline residue of the wild-type immunoglobulin hinge region substituted with another amino acid residue (e.g., a serine residue).The hinge domain includes a hinge domain derived from the extracellular region of type 1 membrane proteins such as CD8, CD4, CD28, and CD7, which may be a wild-type hinge domain from these molecules or may be modified.

[0069] In the present invention, any transmembrane domain including the hinge domain can be used as long as it can connect the extracellular domain and the intracellular signaling domain across the cell membrane.

[0070] In some cases, the coupling domain and the transmembrane domain may be connected by a spacer domain. Preferably, the spacer domain may be a hinge domain.

[0071] In the present invention, the chimeric antigen receptor may be characterized by including an intracellular signaling domain.

[0072] In the present invention, the intracellular signal transduction domain refers to a portion located inside the cell membrane of an immune cell, i.e., in the cytoplasm, and is a site that activates the immune response of the immune cell by transducing a signal into the cell when a binding domain included in the extracellular domain binds to a target antigen.

[0073] In the present invention, the intracellular signaling domain may be characterized by including an intracellular signaling domain and / or a co-stimulatory signaling domain.

[0074] The above signaling domain can induce activation of normal effector functions in immune cells where the CAR is located. For example, it can induce cytolytic activation or helper activation through the secretion of cytokines. The above signaling domain may include a truncated fragment of an intracellular signaling domain sufficient to transduce effector function signals.

[0075] The intracellular signaling domain is a primary signaling domain selected from the group consisting of T-cell receptor (TCR) zeta (ζ), FcR gamma (γ), FcR beta (β), CD3 gamma (γ), CD3 delta (δ), CD3 epsilon (ε), CD3 zeta (ζ), CD5, CD22, CD79a, CD79b, and CD66d; It may be characterized by comprising a co-stimulatory signaling domain selected from the group consisting of ligands that specifically bind to CD2, CD7, CD27, CD28, CD30, CD40, 4-1BB(CD137), OX40(CD134), CDS, ICAM-1, ICOS(CD278), LFA-1(CD11a / CD18), GITR, MyD88, DAP10, DAP12, PD-1, LIGHT, NKG2C, B7-H3, and CD83.

[0076] The term “intracellular signaling domain” in the present invention refers to a portion of a CAR involved in transmitting a message of effective CAR binding to a target antigen into an immune effector cell to induce an effector cell function, for example, activation including the release of cytotoxic factors to CAR-bound target cells, cytokine production, proliferation, and cytotoxic activity, or other cellular responses induced by antigen binding to the extracellular binding domain of the CAR. An effector function refers to a specific function of a cell; for example, the effector function of an immune cell may be cytolytic activity, or may support or activate activities including the secretion of cytokines. Accordingly, an intracellular signaling domain refers to a portion of a protein that transmits effector function signals and directs the cell to perform a specific function.

[0077] Immune cell activation is mediated by two different classes of intracellular signaling domains. For example, immune cell activation is mediated by a primary signaling domain that initiates antigen-dependent primary activation and a co-stimulatory signaling domain that acts in an antigen-independent manner to provide a secondary signal. Accordingly, the intracellular signaling domain may be characterized by including a “primary signaling domain” and a “co-stimulatory signaling domain.”

[0078] The term “primary signaling domain” in the present invention refers to a signaling domain that regulates immune cell activation in a stimulating or inhibiting manner. A primary signaling domain acting in a stimulating manner may contain an immune receptor tyrosine-based activation motif or a signaling motif known as an ITAM. An ITAM containing a primary signaling domain may be selected from the group consisting of TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, and CD66d, but is not limited thereto.

[0079] The term “co-stimulatory signaling domain” in the present invention refers to an intracellular signaling domain of a co-stimulatory molecule. A co-stimulatory signaling domain refers to a part of a CAR that includes an intracellular signaling domain of a co-stimulatory molecule. It may include, but is not limited to, a co-stimulatory signaling domain selected from the group consisting of ligands that specifically bind to CD2, CD7, CD27, CD28, CD30, CD40, 4-1BB (CD137), OX40 (CD134), CDS, ICAM-1, ICOS (CD278), LFA-1 (CD11a / CD18), GITR, MyD88, DAP10, DAP12, PD-1, LIGHT, NKG2C, B7-H3, and CD83.

[0080] The above chimeric antigen receptor may be characterized by comprising two or more intracellular signaling domains, and in the case of comprising two or more intracellular signaling domains, the intracellular signaling domains may be connected in series with each other. They may be connected via an oligopeptide linker or polypeptide linker consisting of 2 to 10 amino acids, and an example of such a linker sequence may be a glycine-serine sequence. The above linker is, for example, (GS) n , (GGS) n , (GSGGS) n or (G n S) m It may include (n and m are each from 1 to 10), for example (G n S) m (n and m may be 1 to 10, respectively), but are not limited thereto.

[0081] In the present invention, the intracellular signaling domain may be characterized by comprising one or more selected from the group consisting of the co-stimulation signaling domain of CD28 or DAP10 and the intracellular signaling domain of CD3 zeta, but is not limited thereto.

[0082] Specifically, the chimeric antigen receptor according to the present invention may include human CD28 hinge-TM-human CD3 zeta. To confirm that the CAR is expressed on the cell surface, myc and hinge were expressed in the ectodomain.

[0083] The above myc may include the sequence of EQKLISEEDL (Sequence No. 10). The above myc may be encoded by the nucleic acid of GAGCAGAAGCTGATCTCCGAAGAGGACCTG (Sequence No. 11).

[0084] The above human CD28 hinge-TM-human CD3 zeta may include the following sequence.

[0085]

[0086] The above human CD28 hinge-TM-human CD3 zeta can be encoded by the following nucleic acid sequence.

[0087]

[0088] In the present invention, the chimeric antigen receptor may further include an immune function-promoting factor of an immune cell, and the immune function-promoting factor of the immune cell may be characterized as being an interleukin signal sequence. The interleukin signal sequence may be characterized as inducing the expression of IL-12, IL-8, or IL-2, but is not limited thereto. Additionally, IL-7 or CCL19 may be examples of immune function-promoting factors for T cells among the immune cells.

[0089] When designing a CAR, a signal peptide may be additionally included in front of the scFv. The nucleic acid encoding the chimeric antigen receptor may be characterized by further including a nucleic acid encoding a signal peptide. For example, the signal peptide may be GM-CSF, CD8α signal peptide, etc.

[0090] In a specific embodiment according to the present invention, the signal peptide may comprise the following sequence: HCLLQLCCCPWLCFCMLLD (SEQ No. 14). The signal peptide may be encoded by the following nucleic acid: CATTGCCTGTTACAGCTCTGCTGCTGCCCCTGGCTCTGCTTCTGCATGCTGCTAGACCT (SEQ No. 15).

[0091] In immunoregulatory T cells into which the nucleic acid encoding the chimeric antigen receptor has been introduced, the chimeric antigen receptor can be expressed on the surface of the immunoregulatory T cells.

[0092] The β2M (β2-microglobin) coding gene is deleted in the above immunomodulatory T cells. By removing the above β2M gene, the expression of MHC (HLA) I protein is eliminated, allowing for evasion of T cell attacks.

[0093] The above β2M is a serum protein, and the β2M gene is located on chromosome 15. The four exons each encode the major part of the protein (amino acids 3–95), the remaining four amino acids and part of the untranslated region, and the signal peptide, which is the remainder of the untranslated region. The β2M gene sequence has constant but limited similarity to the immunoglobulin constant region and the third extracellular region of major histocompatibility complex (MHC) class I molecules. Structurally, β2M consists of two β sheets connected by disulfide bonds and can form amyloid fibrils under certain pathological conditions.

[0094] β2M, an important subunit of MHC class I molecules, performs various functions under physiological and pathological conditions in both tumor and non-tumor cells, and plays a particularly important role in immune surveillance.

[0095] The above β2M may include the following amino acid sequence.

[0096] IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 2)

[0097] The above β2M can be encoded by the following nucleic acid.

[0098]

[0099] Although β2M knockout is a key factor in increasing the level of versatility, it hinders the stabilization of human HLA-E protein. Therefore, a trimer construction can be constructed and additionally included to express an HLA-E trimer, thereby functionally maintaining the β2M deletion while stabilizing and expressing the HLA-E protein.

[0100] The above trimer structure may include HLA class I histocompatibility antigen, HLA-G which is an alpha chain G isoform 1 precursor, and β2M signal peptide, and may include the following amino acid sequence.

[0101] HLA-G : VMA PRTLFL (Sequence No. 4)

[0102] β2M : MSRSVALAVLALLSLSGLEA (Sequence No. 5)

[0103] The above HLA-G and β2M signal peptides can each be encoded by the following nucleic acids.

[0104] HLA-G : caggaagagggttcggggcgccatgac (Sequence No. 6)

[0105] β2M : agcctccaggccagaaagagagagtagcgcgagcacagctaaggccacggagcgagacata (Sequence No. 7)

[0106] The above β2M coding gene can be deleted by gene editing. Specifically, the above β2M coding gene can be deleted by introducing (1) a nuclease or a nucleic acid encoding it and (2) a guide RNA (gRNA) or a nucleic acid encoding it that specifically recognizes the β2M coding gene.

[0107] The above nuclease is a means of cleaving the DNA double helix and may be ZNFN (zinc finger nuclease), TALEN (transcriptional activator-like effector nuclease), or Cas protein, but is not limited thereto. The above Cas proteins are Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cpf1 (Cas12a), Cas12b, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, Cas13a, Cas13b, Cas13c, Cas13d, Cas14, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, CsMT2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, It may be an endonuclease of Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, or Csf4, but is not limited thereto.

[0108] The above Cas protein is Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus (Streptococcus pyogenes), Lactobacillus, Mycoplasma, Bacteroides, Flaviflavibola, Flavobacterium, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus (Staphylococcus aureus), It may be derived from a microbial genus containing an ortholog of a Cas protein selected from the group consisting of Nitratifractor, Corynebacterium, and Campylobacter, and may be simply isolated or recombined from these.

[0109] The second class 2 (type V) effector protein, Cpf1, can be used for editing. Similar to Cas9, Cpf1 can be reprogrammed to target a DNA region of interest through complementarity with a guide RNA.

[0110] Unlike Cas9, which is guided by a single crRNA, recognizes T-rich PAM, and cleaves target and non-target DNA strands using its HNH and RuvC endonuclease domains, respectively, Cpf1 contains a RuvC domain but lacks a detectable second endonuclease domain.

[0111] The guide RNA may be one or more selected from the group consisting of, for example, CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and single guide RNA (sgRNA), and specifically, it may be a double-stranded crRNA:tracrRNA complex in which crRNA and tracrRNA are combined, or a single-stranded guide RNA (sgRNA) in which crRNA or a part thereof and tracrRNA or a part thereof are connected by an oligonucleotide linker.

[0112] Specifically, when Cas12a is included, the Cas12a guide RNA molecule comprises (a) a protospacer domain containing a targeting sequence and (b) a loop domain, respectively.

[0113] The above targeting sequence corresponds to a target domain within the genomic DNA sequence, and the target domain is adjacent to a protospacer-adjacent motif (PAM) recognized by the Cas12a protein.

[0114] The loop domain is a component of the Cas12a gRNA of the present disclosure comprising a stem-loop structure recognized by the Cas12a protein. The loop domain may comprise a nucleotide sequence of a naturally occurring stem-loop sequence recognized by the Cas12a protein, or may comprise a engineered nucleotide sequence forming a stem-loop structure recognized by the Cas12a protein.

[0115] The above PAM refers to a region of a Cas12a gRNA molecule containing a targeting sequence. The protospacer domain is sometimes referred to as crRNA. The above PAM refers to a genomic DNA sequence that is 5' for the target domain and is generally 4 nucleotides long, which is required for the cleavage of genomic DNA by a Cas12a protein that recognizes the PAM. An exemplary PAM sequence may be TTTV (where V is A, C, or G).

[0116] hB2M-CR4 : ATCCATCCGACATTGAAGTT (Sequence No. 1)

[0117] We confirmed that the best gene editing activity was observed in CR4, and as a result of applying it to human primary lymphocytes, we confirmed that the CR4 gene editing tool showed activity in the T7E1 assay.

[0118] A non-classical HLA (human leukocyte antigen) class I coding gene has been introduced into the above immunomodulatory T cells.

[0119] The aforementioned non-classical HLA class I may be, for example, human HLA-E. HLA-E belongs to the HLA class I heavy chain analogs. The HLA class I molecule is a heterodimer composed of a heavy chain and a light chain (beta-2 microglobulin). The heavy chain is anchored to a membrane. The heavy chain is approximately 45 kDa, and the gene contains eight exons. Exon 1 codes for the leader peptide, exons 2 and 3 code for the alpha1 and alpha2 domains that bind to the peptide, exon 4 codes for the alpha3 domain, exon 5 codes for the transmembrane region, and exons 6 and 7 code for the cytoplasmic tail.

[0120] The above human HLA-E can interact with the inhibitory CD94 / NKG2 receptor found in most NK cells and some T cells. The above human HLA-E includes, for example, the following amino acid sequence.

[0121]

[0122] The above human HLA-E can be encoded, for example, by the following nucleic acid sequence.

[0123]

[0124] The Foxp3 (forkhead box P3) coding gene can be additionally introduced into the immunomodulatory T cells according to the present invention.

[0125] 'Universal Foxp3-introduced chimeric antigen receptor immunomodulatory T cells (uCAR Treg)' which combines Foxp3-induction technology—which converts the much more numerous non-Treg into Tregs by introducing the Foxp3 gene—with uCAR Tregs. Foxp3 Foxp3-introduced Treg technology, which converts non-Tregs—which are easier to obtain than Tregs—into Tregs by introducing Foxp3, which determines the Treg lineage, can increase the number of scarce Tregs.

[0126] The above Foxp3 may include the following amino acid sequence.

[0127]

[0128] The above Foxp3 can be encoded by the following nucleic acid.

[0129]

[0130] "Nucleic acid" has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acids, include not only natural nucleotides but also analogues in which sugar or base sites are modified. The sequence of nucleic acid encoding the heavy chain and light chain variable regions of the present invention may be modified. Such modification includes the addition, deletion, non-conservative, or conservative substitution of nucleotides.

[0131] The above nucleic acid (polynucleotide) can be modified by codon optimization, which is due to the degeneracy of codons, and it will be well understood by a person skilled in the art that there exist many nucleotide sequences encoding polypeptides or fragments of variants thereof. Some of these polynucleotides (nucleic acids) possess minimal homology with the nucleotide sequences of any naturally occurring gene.

[0132] In particular, due to differences in codon utilization, variable polynucleotides (nucleic acids), for example, polynucleotides (nucleic acids) optimized for codon selection in humans, primates, and / or mammals, are desirable.

[0133] It may include an expression vector containing the above nucleic acid and a virus containing the above expression vector.

[0134] The term “vector” in the present invention refers to a nucleic acid molecule capable of transferring or transporting other nucleic acid molecules. The transferred nucleic acid is generally linked to the vector nucleic acid molecule, for example, inserted into the vector nucleic acid molecule. The vector may include a sequence directing autonomous replication in a cell or may include a sequence sufficient to enable integration into the host cell DNA. The vector may be characterized as being selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, and retroviral vectors, but is not limited thereto.

[0135] Vector components generally include, but are not limited to, one or more of the following: a signal sequence, a replication origin, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0136] In the above vector, the nucleic acid encoding the antibody is operatively linked to the promoter.

[0137] “Operational linkage” refers to a functional linkage between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, thereby allowing the regulatory sequence to regulate the transcription and / or translation of the other nucleic acid sequence.

[0138] In the case of a prokaryotic cell as the host, it is common to include a powerful promoter capable of proceeding transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. In addition, for example, when a eukaryotic cell is used as a host, a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rhoese's sarcoma virus (RSV) promoter) may be used, and generally has a polyadenylation sequence as a transcription termination sequence.

[0139] In some cases, the vector may be fused with other sequences to facilitate the purification of the antibody expressed therefrom. The fused sequences include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA).

[0140] The above vector contains antibiotic resistance genes commonly used in the industry as selection markers, such as resistance genes for ampicillin, gentamicin, cabbageillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0141] In the present invention, “virus” means genetically modified to express the composition of the present invention. Genetically modified means adding external genetic material in the form of DNA or RNA to the entire genetic material in a cell.

[0142] The above nucleic acid or the above vector is transfected or transfected into a virus. Various techniques commonly used to introduce exogenous nucleic acid (DNA or RNA) into a prokaryotic or eukaryotic host cell to “transfect” or “transfect” may be used, such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection.

[0143] The present invention also relates to a composition for the prevention or treatment of transplant rejection comprising the immunomodulatory T cells. The present invention relates to the use of the immunomodulatory T cells in the preparation of a composition for the prevention or treatment of transplant rejection. The present invention relates to a method for prevention or treatment comprising the step of administering the immunomodulatory T cells to an individual.

[0144] Graft rejection is a risk factor for multiple graft loss, with the observation of graft rejection in recipients within the first year after transplantation. Acute rejection is also a known risk factor for progression to chronic rejection; therefore, the detection and treatment of acute rejection episodes as quickly as possible are necessary to minimize graft damage and prevent downstream rejection. An adaptive immune response to the transplanted tissue is a major obstacle to successful transplantation. Rejection is triggered by an immune response to alloantigens on the graft, which are proteins that vary within the species and are therefore recognized as foreign by the recipient.

[0145] "Acute rejection" or "acute cellular rejection" refers to an immune response caused by an allogeneic transplanted organ. Generally, such acute rejection begins on days 2 to 60 after transplantation and possibly other cell-specific antigens are expressed by the tubular epithelium and tubular endothelium. This is triggered by mismatched HLA antigens and possibly other cell-specific antigens expressed by the tubular epithelium and tubular endothelium. Both delayed hypersensitivity and cytotoxic mechanisms are believed to be involved. Acute rejection is characterized by the infiltration of the transplanted tissue by recipient immune cells that perform effector functions and destroy the transplanted tissue. This includes interstitial vascular endothelial cell swelling, interstitial accumulation of lymphocytes, protoplasmic cells, immunoblasts, macrophages, and neutrophils; tubular separation accompanied by edema / necrosis of the tubular epithelium; It may be characterized by swelling and vacuolation of endothelial cells, angioedema, hemorrhage and inflammation, renal tubular necrosis, sclerotic glomeruli, and tubular 'thyroidization'.

[0146] "Chronic rejection" generally occurs within months to years after engraftment, even in the presence of successful immunosuppression of acute rejection. Fibrosis is a common factor in chronic rejection of all types of organ transplants. Chronic rejection can typically be initiated by a series of specific diseases characteristic of a particular organ. For example, in lung transplants, such diseases include fibroproliferative destruction of the airways (obstructive bronchiolitis); in heart transplants or transplants of heart tissue, e.g., valve replacements, such diseases include fibrotic atherosclerosis; in kidney transplants, such diseases include obstructive nephropathy, nephrosclerosis, and tubulointerstitial nephropathy; and in liver transplants, such diseases include disappearing cholangiocarcinoma. Chronic rejection may also be characterized by ischemic attacks associated with immunosuppressive drugs, denervation of transplanted tissue, hyperlipidemia, and hypertension.

[0147] "Transplant rejection reaction" includes both the acute and chronic transplant rejection mentioned above.

[0148] "Transplantation" refers to the process of taking a cell, tissue, or organ, referred to as a "graft" or "plant," from one individual and placing it (usually) on another individual. The individual providing the graft is referred to as the "donor," and the individual receiving the graft is referred to as the "host" (or "recipient"). An organ or graft transplanted between two genetically different individuals of the same species is referred to as an "allograft." A graft transplanted between individuals of different species is referred to as a "xenograft."

[0149] The therapeutic composition of the present invention is a composition for the prevention or treatment of transplant rejection, wherein the term “prevention” of the present invention refers to any act of suppressing or delaying the progression of transplant rejection by administering the composition of the present invention, and “treatment” refers to the suppression of the development of transplant rejection, or the alleviation or elimination of symptoms.

[0150] "Transplant rejection" refers to the functional and structural deterioration of an organ resulting from an active immune response exhibited by the recipient, and is unrelated to non-immunological causes of organ failure. Acute transplant rejection can arise from the activation of the recipient's T cells and / or B cells; rejection primarily caused by T cells is classified as T-cell-mediated acute rejection or acute cellular rejection (ACR), while rejection primarily caused by B cells is classified as antibody-mediated acute rejection (AMR).

[0151] It is preferable that the above composition contains 1 to 10 times the number of tumor cells in the treatment target, but is not limited thereto.

[0152] The composition according to the present invention may be a pharmaceutical composition. The pharmaceutical composition may additionally include pharmaceutically acceptable excipients. Examples of such excipients may include, but are not limited to, surfactants, preferably nonionic surfactants of the polysorbate series; buffers such as neutral buffered saline, human salt buffered saline, etc.; sugars or sugar alcohols such as glucose, mannose, sucrose or dextran, mannitol, etc.; amino acids or proteins or polypeptides such as glycine, histidine, etc.; antioxidants; chelating agents such as EDTA or glutathione, e.g.; penetrating agents; adjuvants; and preservatives.

[0153] According to one embodiment of the present invention, the pharmaceutical composition of the present invention may contain, in a single dose, the number of recombinant immunomodulatory T cells in an amount of 1 to 10 times the number of cells by transplantation in the treatment target.

[0154] The composition of the present invention may be formulated using methods known in the art. The formulation may be in the form of powder, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, or sterile powders.

[0155] The above pharmaceutical composition may be in various dosage forms for oral or parenteral administration. When formulated, it is prepared using diluents or excipients such as commonly used fillers, volume expanders, binders, wetting agents, disintegrants, and surfactants. Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules, and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, or gelatin, with one or more compounds. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid dosage forms for oral administration include suspensions, liquids, emulsions, and syrups, and may contain various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspension solvents. Witepsol, macrogol, Tween 61, cocoa paste, laurin paste, glycerogelatin, etc. may be used as bases for suppositories.

[0156] The recombinant immunomodulatory T cells according to the present invention or a composition containing the same may be administered orally, infusion, intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intrarectal administration, topical administration, intranasal injection, etc., but are not limited thereto.

[0157] The dosage of the active ingredient can be appropriately selected according to various factors such as the route of administration, the patient's age, gender, weight, and severity of the patient, and the therapeutic composition according to the present invention can be administered in combination with a known compound having the effect of preventing, improving, or treating symptoms of transplant rejection.

[0158]

[0159] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0160]

[0161] Example 1. Fabrication of anti-CD40 CAR vector

[0162]

[0163] A vector for human anti-CD40 uCAR expression was constructed (Fig. 1). An EF1 promoter was used, and a Myc-tag was attached to the signal sequence to enable FACS confirmation of CAR expression. CAR activation was induced by linking human anti-CD40 ScFv and linking human CD28 and human CD3ζ.

[0164]

[0165] [Sequence No. 21] CAR signal peptide

[0166] CATTGCTGTTACAGCTCTGCTGCTGCCCCTGGCTCTGCTTCTGCATGCTGCTAGACCT

[0167] [Sequence No. 11] Myc-tag

[0168] GAGCAGAAGCTGATCTCCGAAGAGGACCTG

[0169]

[0170]

[0171]

[0172] Example 2. Selection of gene editing tools effectively removing β2M and verification of activity

[0173]

[0174] After constructing a gene editing tool that effectively removes human β2M, two gene editing tools utilizing AsCpf1 were designed for screening and validation, and activity tests were conducted on Jurkat cells, a human T lymphoblast cell line. CR1, which targets intron and exon junctions and had worked well in human iPSCs and other cell lines, and CR4, which showed the best gene editing activity, were applied to human primary lymphocytes. As a result, it was confirmed that the CR4 gene editing tool exhibited activity in the T7E1 assay (Fig. 2).

[0175]

[0176] Example 3. Construction and expression verification of human Anti-CD40 uCAR TregFoxp3 vector

[0177]

[0178] The expression of anti-CD40 CAR and Foxp3 was constructed by replacing the ORF of the pLenti-P2V2-PuroR vector, which was constructed as a bidirectional vector using the EF1 promoter for expression and the PGK promoter for trimer construction, with that of human CD40 CAR, human Foxp3, HLA-E, and beta-2-microglobulin / peptide (Qdm) fusion trimer (SEQ No. 22), and it was confirmed that the three proteins were well expressed (Fig. 3).

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185] Example 4. Human anti-CD40 uCAR Treg Foxp3 produce

[0186]

[0187] A human modified, bidirectional, 2-promoter, single vector (hP2V2) was introduced into primary T cells via lentiviral transduction. When the expression of Bidirectional_hP2V2 in primary T cells was confirmed via Single (MOI 4.18) / Sequential (MOI 2) Transduction, FACS analysis did not detect Myc expression, but higher HLA-E expression and lower Foxp3 expression compared to the NT group were confirmed (Fig. 4).

[0188] The expression of anti-hCD40 uCAR TregFoxp3 was confirmed by sequential transduction of non-freezing viruses (MOI6, MOI60). In FACS analysis, Myc expression (4–5%) was low, but HLA-E and Foxp3 expression were confirmed (Fig. 5).

[0189] To achieve higher expression efficiency, 10–20 μg of Bidirectional_hP2V2 was transfected into human T cells (1 x 10⁶) via electroporation, a gene transfer method using electrical stimulation, and cell expression was confirmed by FACS 48 hours later. As a result, the expression levels of Myc, β2M, and HLA-E were confirmed (Fig. 6).

[0190]

[0191] Example 5. Verification of the activity of Human β2M knockout (KO) gene editing in primary T cells

[0192]

[0193] After producing the virus using hβ2M-CR4 gene editing tools, sequential transduction was performed. When expression was confirmed by FACS following puromycin selection and a stabilization period under IL-2 supply, it was confirmed that the expression of β2M and HLA was reduced. Unlike the PCR results which showed complete KO, FACS showed a KO efficiency of approximately 30%, which is interpreted as being due to the fact that there were a significant number of cells that did not undergo transduction, and expression may remain visible in FACS confirmation due to β2M already expressed on the cell membrane (Fig. 7).

[0194]

[0195] The recombinant immunomodulatory T cells according to the present invention are the first uCAR Treg for transplant rejection that seek to resolve the limitations of Treg therapy, such as numerical scarcity and difficulties in maintaining antigen specificity, survival, and activity, through the co-introduction of Foxp3 / CAR. Foxp3 This is a study on uCAR Tregs for rejection, the strongest immune response, through universal CAR Tregs applicable to all patients. Foxp3 The technology can be applied to the regulation of other immune diseases.

[0196]

[0197] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0198]

[0199] I have attached the electronic file.

Claims

1. A nucleic acid encoding a chimeric antigen receptor has been introduced, and The β2M (β2-microglobin) coding gene is deleted, and Recombinant immunoregulatory T cells (regulatory T: Treg cells) into which a non-classic HLA (non-classic human leukocyte antigen) class I coding gene has been introduced.

2. An immunomodulatory T cell according to claim 1, characterized by the additional introduction of a Foxp3 (forkhead box P3) coding gene.

3. An immunomodulatory T cell according to claim 1, wherein the chimeric antigen receptor comprises an antibody or an antigen-binding fragment thereof that specifically binds to an immunomodulatory molecule.

4. An immunomodulatory T cell according to claim 3, wherein the chimeric antigen receptor comprises an antibody that specifically binds to CD40 or an antigen-binding fragment thereof.

5. An immunomodulatory T cell according to claim 1, characterized in that the β2M coding gene is deleted by gene editing.

6. An immunomodulatory T cell according to claim 5, characterized in that the β2M coding gene is deleted by introducing (1) a nuclease or a nucleic acid encoding the same and (2) a guide RNA (gRNA) or a nucleic acid encoding the same that specifically recognizes the β2M coding gene.

7. An immunomodulatory T cell according to claim 6, characterized in that the gRNA comprises the sequence of SEQ ID NO.

1.

8. An immunomodulatory T cell according to claim 1, characterized in that HLA class I is human HLA-E.

9. A method for preparing recombinant immunoregulatory T cells (regulatory T: Treg cell) comprising the following steps: (a) A step of introducing a nucleic acid encoding a chimeric antigen receptor; (b) a step of deleting the β2M (β2-microglobin) coding gene; and (c) Step of introducing a non-classical HLA (human leukocyte antigen) class I coding gene.

10. A manufacturing method according to claim 9, characterized by including the step of additionally introducing a Foxp3 (forkhead box P3) coding gene.

11. A composition for the prevention or treatment of transplant rejection comprising immunomodulatory T cells according to any one of claims 1 to 8.

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