Dual gene-transduced immune cells using CTLA-4 mutant and chimeric antigen receptor and their uses

Abstract

The present invention relates to doubly transfected T cells using a CTLA-4 mutant and a chimeric antigen receptor, and an anti-cancer immunotherapy composition comprising the same. According to one aspect of the present invention, the doubly transfected T cells using a CTLA-4 mutant and a chimeric antigen receptor, and the anti-cancer immunotherapy composition comprising the same, the introduction of a CTLA-4 mutant induces enhanced T cell function without causing side effects such as autoimmune diseases, and the injection of a small amount of T cells shows improved tumor treatment efficiency and tumor recurrence suppression effects, making them useful for immune cell therapy for cancer treatment.

Description

[Technical Field]

[0001] The present invention relates to dual gene-transduced immune cells using a CTLA-4 mutant and a chimeric antigen receptor (CAR) and uses thereof. When a CTLA4-CD28 fusion protein is co-expressed with a chimeric antigen receptor, tumor specificity and antitumor effect are improved, and the cells can be effectively used as an immunoanticancer agent. [Background technology]

[0002] Conventional anti-cancer T cell therapy has shown promise as a useful immune cell therapy in early clinical trials, but difficulties in securing sufficient numbers of tumor-specific T cells and the need for enhanced efficacy have emerged. Blocking antibodies against the T cell inhibitory receptor CTLA-4 are commercially available under FDA approval due to their ability to enhance the efficacy of tumor-specific T cells in patients, but autoimmune side effects caused by the activation of autoantigen-specific T cells when administered systemically have been raised as a concern.

[0003] In previous research, the inventors devised a CTLA4-CD28 chimera (CTC28) molecule, a CTLA-4 function-suppressing mutant, to enhance the efficacy of tumor-specific T cell therapy, and proposed an enhanced T cell therapy in which CTLA-4 function was inhibited without autoimmune side effects by generating CTC28 transfected T cells (Patent Document 0001). However, securing a sufficient number of tumor-specific T cells remained a challenge.

[0004] Chimeric antigen receptors are recombinant receptors that link an antibody moiety that binds to a tumor surface antigen with a T cell internal activation signal moiety, and have the advantage that when a CAR gene is introduced into a patient's peripheral blood T cells, a large number of tumor-specific T cells (CAR-T cells) can be generated in a short period of time. Taking advantage of the fact that multiple genes can be carried in a single retroviral or lentiviral vector, the present invention aims to develop CAR-T cells that simultaneously achieve functional enhancement and generate a large number of tumor-specific CAR-T cells through a single gene transfer by generating double-transfected T cells in which both the CAR gene and the CTC28 gene are introduced. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Korean Patent Publication No. 2013-0045284 (Publication Date: 2013.05.06) Summary of the Invention [Problem to be solved by the invention]

[0006] The inventors have confirmed that, unlike second-generation CAR molecules containing the CD28 intracellular region, first-generation CAR molecules or second-generation CAR molecules containing the 4-1BB intracellular region exhibit synergistic tumor specificity and anti-tumor effects when expressed together with CTC28, and have completed an invention related to CTC28-loaded CAR-T cells. [Means for solving the problem]

[0007] One embodiment of the present invention relates to a method for producing a CTLA4-CD28 fusion protein comprising: (i) a first gene encoding a fusion protein (CTLA4-CD28 fusion protein) comprising a CTLA4 (Cytotoxic T Lymphocyte Antigen-4) protein or a domain thereof and CD28 or a domain thereof; (ii) a second gene encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain, an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain; The present invention provides a construct comprising:

[0008] Another aspect provides a vector comprising the construct. Another aspect provides an immune cell into which the construct or vector has been introduced. Another aspect provides an immune cell comprising a protein expressed from the construct.

[0009] Another aspect provides an immune cell comprising: (i) a first gene encoding a fusion protein comprising a CTLA-4 protein or a domain thereof and CD28 or a domain thereof (CTLA4-CD28 fusion protein); and (ii) a second gene encoding a chimeric antigen receptor comprising an antigen-binding domain, an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain.

[0010] Another aspect provides an immune cell that expresses both (i) a fusion protein comprising a CTLA-4 protein or a domain thereof and CD28 or a domain thereof (CTLA4-CD28 fusion protein), and (ii) a chimeric antigen receptor comprising an antigen-binding domain, an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain.

[0011] Another aspect provides a composition comprising said construct, said vector, or said immune cell.

[0012] Another aspect provides a pharmaceutical composition for preventing or treating a disease (e.g., cancer), comprising the construct, the vector, or the immune cell, and a pharmaceutically acceptable carrier.

[0013] Another aspect provides the use of the construct, the vector, the immune cell, or the composition in the prevention or treatment of a disease (e.g., cancer).

[0014] Another aspect provides the use of the construct, the vector, the immune cell, or the composition for the manufacture of a medicament for the prevention or treatment of a disease (e.g., cancer).

[0015] Another aspect provides a method for preventing or treating a disease (e.g., cancer), comprising administering the construct, the vector, the immune cell, or the composition to an individual in need thereof.

[0016] Another aspect provides a method for producing immune cells that express a CTLA4-CD28 fusion protein and a chimeric antigen receptor, the method comprising the step of introducing the first gene and the second gene into immune cells.

[0017] Another aspect provides a method for producing immune cells that express a CTLA4-CD28 fusion protein and a chimeric antigen receptor, comprising the step of introducing the construct or the vector into immune cells. [Effects of the Invention]

[0018] In one embodiment, dual gene-transduced T cells using a CTLA-4 mutant and a chimeric antigen receptor and an anti-cancer immunotherapy composition containing the same induce enhanced T cell function by introducing a CTLA-4 mutant, and the injection of a small amount of T cells shows improved tumor treatment efficiency and tumor recurrence suppression effects, making them useful for immune cell therapy for cancer treatment. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 is a schematic diagram showing double transgenic T cells using CTLA-4 mutants and chimeric antigen receptors. [Figure 2]Figure 2 is an image showing the structure of the gene constructs of first-generation CAR (mCD19z) and first-generation CAR and CTLA4-CD28 (mCD19z-CTC28). [Figure 3] Figure 3 is a graph analyzing the expression levels of CAR and CTLA4 proteins on the surface of T cells transfected with first-generation CAR (mCD19z) and first-generation CAR CTC28 (mCD19z-CTC28). [Figure 4] Figure 4 is a graph analyzing the tumor-killing ability of T cells transfected with the first-generation CAR (mCD19z) and the first-generation CAR CTC28 (mCD19z-CTC28) against A20 cells. [Figure 5] Figure 5 is a graph showing the amount of IFN-γ secreted by T cells introduced with the first generation CAR (mCD19z) and the first generation CAR CTC28 (mCD19z-CTC28) when co-cultured with A20 cells. [Figure 6] Figure 6 is a graph showing the change in tumor size in mice injected with T cells transduced with the first-generation CAR (mCD19z) and the first-generation CAR CTC28 (mCD19z-CTC28) (tumor size = major axis 2 × minor axis / 2). [Figure 7] Figure 7 is an image showing the structure of the gene constructs of CD28-based second-generation CAR (mCD1928z) and second-generation CAR and CTLA4-CD28 (mCD1928z-CTC28). [Figure 8] Figure 8 is a graph analyzing the expression levels of CAR and CTLA4 proteins on the surface of T cells transfected with CD28-based second-generation CAR (mCD1928z) and second-generation CAR CTC28 (mCD1928z-CTC28). [Figure 9] Figure 9 is a graph showing the amount of IFN-γ secreted when T cells transfected with CD28-based second-generation CAR (mCD1928z) and second-generation CAR CTC28 (mCD1928z-CTC28) were co-cultured with A20 cells. [Figure 10]Figure 10 is a graph showing the change in tumor size in mice injected with T cells transfected with CD28-based second-generation CAR (mCD1928z) and second-generation CAR CTC28 (mCD1928z-CTC28) (tumor size = major axis 2 × minor axis / 2). [Figure 11] Figure 11 is an image showing the structure of the gene constructs of 4-1BB-based second-generation CAR (mCD19BBz) and second-generation CAR and CTLA4-CD28 (mCD19BBz-CTC28). [Figure 12] Figure 12 is a graph analyzing the expression levels of CAR and CTLA4 proteins on the surface of T cells transfected with 4-1BB-based second-generation CAR (mCD19BBz) and second-generation CAR CTC28 (mCD19BBz-CTC28). [Figure 13] Figure 13 is a graph showing the amount of IFN-γ secreted when T cells transfected with 4-1BB-based second-generation CAR (mCD19BBz) and second-generation CAR CTC28 (mCD19BBz-CTC28) were co-cultured with A20 cells. [Figure 14] Figure 14 is a graph showing the change in tumor size in mice injected with T cells transfected with 4-1BB-based second-generation CAR (mCD19BBz) and second-generation CAR CTC28 (mCD19BBz-CTC28) (tumor size = major axis 2 × minor axis / 2). [Figure 15] Figure 15 is an image showing the structure of the gene constructs for second-generation CAR (mCD19BBz) containing a T cell tracking marker (Thy1.1) and second-generation CAR and CTLA4-CD28 (mCD19BBz-CTC28). [Figure 16] Figure 16 is a graph analyzing the expression levels of Thy1.1 and CTC28 proteins on the surface of CAR-T cells into which mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28) were introduced. [Figure 17] Figure 17 is a graph analyzing the tumor-killing ability of CAR-T cells introduced with mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28) against A20 cells. [Figure 18] Figure 18 is a graph showing the amounts of IFN-γ, IL-2, and TNF-α secreted by CAR-T cells transfected with mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28) when co-cultured with A20 cells. [Figure 19] Figure 19 is a graph analyzing changes in the division ability of CD8 CAR-T cells into which mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28) were introduced. [Figure 20] Figure 20 is a graph showing the change in tumor size in mice injected with CAR-T cells transfected with mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28) (tumor size = major axis 2 × minor axis / 2). [Figure 21] Figure 21 is a graph showing the survival rate of mice injected with CAR-T cells transfected with mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28). [Figure 22] Figure 22 is a graph measuring the number of CD4 and CD8 CAR-T cells transduced with mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28) in an A20 cell tumor mouse model. [Figure 23] Figure 23 is a graph showing the amounts of IFN-γ, IL-2, and TNF-α secreted by CD4 and CD8 CAR-T cells transduced with mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28) in an A20 cell tumor mouse model. [Figure 24] Figure 24 is a graph quantifying the amounts of IFN-γ, IL-2, and TNF-α secreted by CD4 and CD8 CAR-T cells transduced with mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28) in an A20 cell tumor mouse model. [Figure 25]Figure 25 is a graph showing the change in tumor size in mice when CTC28-introduced CD4 or CD8 mCD19BBz CAR-T cells (indicated by "(+)") and CD4 or CD8 mCD19BBz CAR-T cells not infused with CTC28 (indicated by "(-)") were mixed at a 1:1 ratio and co-administered in an A20 cell tumor mouse model (tumor size = major axis 2 × minor axis / 2). [Figure 26] Figure 26 is a graph showing the number of CAR-T cells in mice when CTC28-introduced CD4 or CD8 mCD19BBz CAR-T cells (indicated by "(+)") and CD4 or CD8 mCD19BBz CAR-T cells not infused with CTC28 (indicated by "(-)") were mixed at a 1:1 ratio and co-administered in an A20 cell tumor mouse model. [Figure 27] Figure 27 is a graph showing RNA-seq analysis in mice injected with CD4 and CD8 CAR-T cells transduced with mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28) in an A20 cell tumor mouse model. [Figure 28] Figure 28 is a graph showing the change in tumor size in mice in an A20 cell tumor mouse model when mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28)-introduced CAR-T cells were co-treated with an IL-2 neutralizing antibody (α-IL2) (tumor size = major axis 2 × minor axis / 2). [Figure 29] Figure 29 is a graph showing the number of CAR-T cells in mice treated with mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28)-introduced CAR-T cells and an IL-2 neutralizing antibody (α-IL2) in an A20 cell tumor mouse model. [Figure 30] Figure 30 is a graph measuring the number of CD4 and CD8 CAR-T cells in peripheral blood after administration of CAR T cells transduced with mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28) in a mouse model. [Figure 31]Figure 31 is a graph measuring changes in mouse body weight after administration of CAR T introduced with mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28) in a mouse model. [Figure 32] Figure 32 is a graph showing the number of CAR-T cells and B cells in the blood in a mouse model treated with α-CTLA4 antibody after administration of CAR T cells incorporating mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28). [Figure 33] Figure 33 is an image showing the structures of the human 4-1BB-based CAR (hCD19BBz) gene construct and the human CTC28 and CAR co-expression gene construct (hCD19BBz-CTC28). [Figure 34] Figure 34 is a graph analyzing the expression levels of CAR and CTC28 proteins on the surface of T cells (CD19-CTC28 CAR) into which human CAR (hCD19BBz) and human CTC28 were co-introduced. [Figure 35] FIG. 35 is a graph showing the analysis of the tumor-killing ability of T cells transfected with human hCD19BBz (CD19) and hCD19BBz-CTC28 (CD19-CTC28) against Raji cells. [Figure 36] Figure 36 is a graph showing the amounts of IFN-γ, IL-2, and TNF-α secreted by T cells transfected with human hCD19BBz (CD19) and hCD19BBz-CTC28 (CD19-CTC28) when cocultured with Raji cells. [Figure 37] Figure 37 is a graph showing changes in tumor size in mice injected with T cells transfected with human hCD19BBz (CD19) and hCD19BBz-CTC28 (CD19-CTC28) (tumor size = major axis 2 × minor axis / 2). [Figure 38] Figure 38 is a graph measuring the number of CD4 and CD8 CAR-T cells in mice transfected with human hCD19BBz (CD19) and hCD19BBz-CTC28 (CD19-CTC28) in the NSG immunodeficient mouse model. DETAILED DESCRIPTION OF THE INVENTION

[0020] One embodiment of the present invention relates to a method for producing a CTLA4-CD28 fusion protein comprising: (i) a first gene encoding a fusion protein (CTLA4-CD28 fusion protein) comprising a CTLA4 (Cytotoxic T Lymphocyte Antigen-4) protein or a domain thereof and CD28 or a domain thereof; (ii) a second gene encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain, an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain; The present invention provides a construct comprising:

[0021] As used herein, the term "construct" refers to a macromolecule or molecular complex containing a polynucleotide that is delivered to a host cell either in vitro, in vivo, or ex vivo. As used herein, the term "vector" refers to any nucleic acid construct capable of delivering or directing the movement of exogenous genetic material to a target cell where the polynucleotide can be replicated and / or expressed. As used herein, the term "vector" includes the delivered construct. A vector may be a linear or circular molecule. A vector may be integrating or non-integrating. Major types of vectors include, but are not limited to, plasmids, episomal vectors, viral vectors, non-viral vectors, cosmids, and artificial chromosomes. Viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, Sendai viral vectors, etc.

[0022] As used herein, the term "encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to act as a template for the synthesis of other polymers and macromolecules in biological processes that have either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties that result therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene in a cell or other biological system produces the protein. Both the coding strand, which is the nucleotide sequence identical to the mRNA sequence and usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0023] As used herein, the term "extracellular domain" refers to a domain that protrudes to the outside of a cell and binds to a ligand or the like. As used herein, the term "transmembrane domain" refers to a domain that connects the extracellular domain with the intracellular domain and is located in the cell membrane. As used herein, the term "intracellular domain" refers to a domain located inside the cell membrane, i.e., in the cytoplasm, that transmits signals transmitted by the binding of an extracellular domain to a ligand or the like into the cell.

[0024] One embodiment provides a fusion protein in which the intracellular signaling domain of a T cell inhibitory receptor has been removed and fused with the intracellular signaling domain of the T cell activation surface protein CD28.

[0025] In one embodiment, the fusion protein can be an immunomodulatory related protein.

[0026] In one embodiment, the fusion protein may comprise an extracellular domain, a transmembrane domain, and an intracellular domain.

[0027] In one embodiment, the CTLA4-CD28 fusion protein can comprise, but is not limited to, the extracellular domain of CTLA4 and the intracellular domain of CD28.

[0028] In one embodiment, the CTLA4-CD28 fusion protein may be, but is not limited to, one fused via the transmembrane domain of CTLA4 or CD28.

[0029] In one embodiment, the CTLA4-CD28 fusion protein may comprise, but is not limited to, the extracellular domain of CTLA4-the transmembrane domain of CTLA4-the intracellular domain of CD28, or the extracellular domain of CTLA4-the transmembrane domain of CD28-the intracellular domain of CD28.

[0030] Specifically, a CTLA-4 molecule is a molecule comprising the cytotoxic T-lymphocyte-associated antigen 4 extracellular domain. The CTLA-4 extracellular domain comprises a portion of the CTLA-4 protein that recognizes and binds to at least one B7 (CD80 / 86) antigen, as well as the B7 antigen expressed on B cells and antigen-presenting cells (APCs). The extracellular domain may also comprise a fragment or derivative of CTLA-4 that binds to the B7 antigen. The CTLA-4 extracellular domain may recognize and bind to CD80 (B7-1) and / or CD86 (B7-2). The extracellular domain may comprise a fragment or derivative of CTLA-4 that binds to CD80 and / or CD86.

[0031] In one embodiment, the fusion protein may comprise, but is not limited to, the extracellular domain of CTLA4-the transmembrane domain of CTLA4-the intracellular domain of CD28, or the extracellular domain of CTLA4-the transmembrane domain of CD28-the intracellular domain of CD28.

[0032] In one embodiment, the amino acid sequence encoding the CTLA4-CD28 chimeric protein may include an amino acid sequence of SEQ ID NO: 1 to 7, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0033] [Table 1]

[0034] In one embodiment, the CTLA-4 may comprise the amino acid sequence of SEQ ID NO: 1 derived from human, the amino acid sequence of SEQ ID NO: 2 derived from mouse, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, the amino acid sequence from positions 1 to 161 in the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2 may represent the extracellular domain, i.e., the site that binds to a ligand such as B7, the amino acid sequence from positions 162 to 189 may represent the transmembrane domain, and the amino acid sequence from positions 190 to 223 may represent the intracellular domain.

[0035] In one embodiment, the CD28 may comprise the amino acid sequence of SEQ ID NO: 3 from a human, the amino acid sequence of SEQ ID NO: 4 from a mouse, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0036] In one embodiment, the amino acid sequence from positions 1 to 152 in the amino acid sequence of SEQ ID NO: 3 represents the extracellular domain, i.e., the site that binds to a ligand such as B7, the amino acid sequence from positions 153 to 178 represents the transmembrane domain, and the amino acid sequence from positions 179 to 220 represents the intracellular domain; the amino acid sequence from positions 1 to 150 in the amino acid sequence of SEQ ID NO: 4 represents the extracellular domain, i.e., the site that binds to a ligand such as B7, the amino acid sequence from positions 151 to 176 represents the transmembrane domain, and the amino acid sequence from positions 177 to 218 represents the intracellular domain.

[0037] In one embodiment, the CTLA4-CD28 fusion protein (hereinafter also referred to as "CTLA4-CD28") may be fused to the transmembrane domain of CTLA-4 or CD28. Specifically, when the transmembrane domain of CTLA-4 is used, it will be obvious to those skilled in the art that the scope of the present invention also includes a partial sequence of the CTLA-4 intracellular domain in addition to the extracellular and transmembrane domains of CTLA-4, or a partial sequence of the CD28 transmembrane domain in the intracellular domain of CD28, as long as the intracellular inhibitory signal of CTLA-4 is not transmitted.

[0038] In one embodiment, the CTLA4-CD28 fusion protein (hereinafter also referred to as "CTLA4") can be fused to the transmembrane domain of CTLA4 or CD28. Specifically, when the transmembrane domain of CTLA4 is used, it will be obvious to those skilled in the art that the scope of the present invention also includes a case in which the extracellular domain and transmembrane domain of CTLA4 additionally contain a partial sequence of the CTLA4 intracellular domain, or a case in which the intracellular domain of CD28 additionally contains a partial sequence of the CD28 transmembrane domain, as long as the intracellular inhibitory signal of CTLA4 is not transmitted.

[0039] In one embodiment, when the transmembrane domain of CD28 is used in the CTLA4-CD28 chimeric protein, it will be obvious to those skilled in the art that the scope of the present invention also includes a case in which a portion of the extracellular domain of CD28 is further included in the intracellular domain and transmembrane domain of CD28, or a case in which a partial sequence of the CTLA-4 transmembrane domain is further included in the extracellular domain of CTLA-4, within the range that does not affect the binding of CTLA-4 to a ligand.

[0040] In one embodiment, an example of the CTLA4-CD28 chimeric protein may include any one of the amino acid sequences of SEQ ID NO: 5 to SEQ ID NO: 7, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0041] In one embodiment, the CTLA-4 extracellular domain-CTLA-4 transmembrane domain-CD28 intracellular domain may be, but is not limited to, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0042] In one embodiment, the CTLA-4 extracellular domain-CD28 transmembrane domain-CD28 intracellular domain may be, but is not limited to, the amino acid sequence of SEQ ID NO: 7, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0043] In one embodiment, the CTLA4-CD28 chimeric protein may comprise the fusion protein disclosed in Korean Patent Publication No. 10-2013-0045824. Specifically, the CTLA4-CD28 chimeric protein may comprise the amino acid sequences of SEQ ID NOs: 1 to 7 of the document, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0044] In one embodiment, the CTLA4-CD28 chimeric protein may be a fusion protein in which the intracellular inhibitory signaling domain of CTLA-4 has been removed and the intracellular activating signaling domain of the CD28 protein has been fused in its place.

[0045] When a ligand binds to CTLA-4 in the fusion protein, the inhibitory signal for T cell activity generated by the binding of CTLA-4 to the ligand may be converted into an activating signal by the action of the intracellular activation signaling domain of CD28 contained in the CTLA4-CD28 chimeric protein.

[0046] Therefore, the CTLA4-CD28 chimeric protein can not only overcome T cell tolerance caused by cancer cells, but also maximize the effect of improving anti-cancer ability through T cell activation.

[0047] In one embodiment, the chimeric antigen receptor (hereinafter also referred to as "CAR") is a general term for proteins in which the cell membrane or intracellular signaling domain of a T cell activation protein (CD3-zeta chain, CD28, 4-1BBL, OX40, ICOS, high-affinity receptor for IgE (FcεRI), and other T cell activation proteins) is fused with an antigen binding domain.

[0048] In one embodiment, the chimeric antigen receptor may be (a) a chimeric antigen receptor comprising an antigen-binding domain, an extracellular binding domain, a transmembrane domain, and a CD3zeta intracellular signaling domain (first-generation CAR), or (b) a chimeric antigen receptor comprising an antigen-binding domain, an extracellular binding domain, a transmembrane domain, a 4-1BB intracellular domain, and a CD3zeta intracellular signaling domain (4-1BB-based second-generation CAR).

[0049] Specifically, first-generation CARs may contain an extracellular domain containing an antigen recognition site specifically expressed in cancer cells, a transmembrane domain, and an intracellular signaling domain, and may use only CD3ζ as the signaling domain. Second-generation CARs may combine a costimulatory domain (CD28 or CD137 / 4-1BB) with CD3ζ to enhance immune cell reactivity, potentially resulting in a higher number of CAR-bearing immune cells remaining in the body compared to first-generation CARs. Third-generation CARs may use two or more costimulatory domains, combining a costimulatory domain with 4-1BB, CD28, or OX40, for example, to achieve the expansion and persistence of CAR-bearing immune cells in the body. Fourth-generation CARs may contain additional genes encoding cytokines such as IL-12 or IL-15, allowing for the further expression of cytokine-based CAR-based immune proteins. Fifth-generation CARs may further include an interleukin receptor chain, such as IL-2Rβ, to enhance immune cell activation.

[0050] In one embodiment, the chimeric antigen receptor may have the structure of a first-generation CAR to a fifth-generation CAR.

[0051] In one embodiment, the chimeric antigen receptor may not comprise the CD28 intracellular site.

[0052] In one embodiment, the extracellular linkage portion of the chimeric antigen receptor may further comprise a hinge domain.

[0053] In one embodiment, the hinge domain may be characterized as consisting of an oligopeptide or polypeptide and containing 1 to 100 amino acid residues, specifically 10 to 70 amino acid residues, but is not limited thereto.

[0054] In one embodiment, the chimeric antigen receptor may comprise the gene sequence or amino acid sequence of SEQ ID NO: 8 to 15, a portion thereof, or a gene sequence or amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0055] [Table 2] JPEG2025540660000004.jpg248170JPEG2025540660000005.jpg215170

[0056] In one embodiment, the intracellular signaling domain of the chimeric antigen receptor (CAR) refers to a portion located inside the cell membrane, i.e., in the cytoplasm, of an immune cell, which transmits a signal intracellularly to activate an immune response of the immune cell when the antigen-binding domain included in the extracellular domain binds to a target antigen. In one embodiment, the CAR may be one or more CARs selected from the group consisting of, but not limited to, CD3 zeta (ζ), CD3 gamma (γ), CD3 delta (δ), CD3 epsilon (ε), FcR gamma, FcR beta, CD5, CD22, CD79a, CD79b, and CD66d. In one example, the CAR is CD3 zeta (ζ).

[0057] In one embodiment, the intracellular signaling domain may further comprise, but is not limited to, a costimulatory domain, which may include one or more costimulatory domains selected from the group consisting of CD2, CD7, CD27, CD28, CD30, CD40, 4-1BB (CD137), OX40 (CD134), ICOS, LFA-1, GITR, MyD88, DAP1, PD-1, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83, but is not limited thereto.

[0058] In one embodiment, the chimeric antigen receptor may be characterized by comprising one or more intracellular signaling domains and one or more costimulatory domains. In one embodiment, the costimulatory domain can be 4-1BB.

[0059] In one embodiment, the chimeric antigen receptor may comprise the amino acid sequence of SEQ ID NO:9, SEQ ID NO:13, or SEQ ID NO:15, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0060] In one embodiment, the antigen-binding domain may be, but is not limited to, an antibody, an antigen-binding fragment thereof, a ligand protein or domain thereof that binds to an antigen.

[0061] In one embodiment, the antigen binding domain may comprise an antibody or antigen binding fragment thereof that specifically binds to one or more antigens selected from the group consisting of, but not limited to: 4-1BB, BCMA, BAFF, B7-H3, B7-H6, CA9, CTAGIB, CEA, cyclin, cyclin A2, cyclin B1, CCL-1, CCR4, CD3, CD4, CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD52, CD58, CD62, CD79A, CD79B, CD80, CD123, CD133, CD138, CD171, CSPG4, CLDN18, CLDN18.2, CLDN6, CTLA-4, c-Met, DLL3, EGFR, tEGFR, EGFRvIII, EPG-2, EPG-40, ephrin B2, EPH A2, estrogen receptor, Fc receptor, FCRL5, FGF23, FBP, FOLR1, FOLR2, GD2, ganglioside GD3, gp100, GPC3, GPCR5D, GM-CSF, Her2 / neu, Her3, Her4, erbB dimers, HMW-MAA, HBsAg, HLA-A1, HLA-A2, IL-22Ra, IL-13Ra2, ICOS, IGF-1 receptor, integrin αvβ6, interferon receptor, IFNγ, IL-2R, IL-4R, IL-5R, IL-6R, IL-17RA, IL-31R, IL-36R, kdr, L1-CAM, CE7 epitope of L1-CAM, LRRC8A, Lewis Y, LAG3, MAGEA1, MAGEA3, MAGEA6, MAGEA10, MSLN, CMV, MUC1, NKG2D ligand, MART-1, NGF, NCAM, NRP-1, NRP-2, carcinoembryonic antigen, PD-L1, PRAME, progesterone receptor, prostate-specific antigen, PSCA, PSMA, RANKL, ROR1, SLAMF7, survivin, TPBG, TAG72, TRP1, TRP2, and Wilms' tumor 1 (WT1).

[0062] In one embodiment, the antigen-binding fragment refers to a fragment that retains antigen-binding function, and may be, but is not limited to, a single-chain variable fragment (scFv) of an antibody, (scFv)2, Fv, Fab, Fab', F(ab')2, nanobody, or a combination thereof.

[0063] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0064] An "Fv" fragment is an antibody fragment that contains a complete antigen recognition and binding site. Such region consists of a dimer of one heavy- and one light-chain variable domain in tight, substantially covalent association, e.g., as an scFv.

[0065] A "Fab" fragment contains the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. A "F(ab')2" antibody fragment generally comprises a pair of Fab fragments covalently linked near their carboxy termini by hinge cysteines between them.

[0066] A "nanobody" is a fragment containing a monomeric variable antibody domain. It consists of low-molecular-weight fragments derived from antibody domains such as camelids, which primarily exhibit target specificity through the heavy monomeric antibody chain alone.

[0067] In one embodiment, the chimeric antigen receptor may further comprise, but is not limited to, a signal peptide at the N-terminus of the antigen-binding domain. In the present invention, the signal peptide may be derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, Ig-kappa, and IgG1 heavy chain, but is not limited thereto, and is preferably a CD8α signal peptide. The CD8α signal peptide may comprise the amino acid sequence set forth in SEQ ID NO: 25, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0068] In one embodiment, the construct may further comprise a promoter sequence, and in this case, the first gene and the second gene may be operably linked to the promoter sequence, but is not limited thereto.

[0069] In one embodiment, the first gene encoding the CTLA4-CD28 fusion protein and the second gene encoding the chimeric antigen receptor are linked to each other via a 2A peptide sequence or an IRES sequence, or the first gene and the second gene can each be operably linked to an independent promoter, but are not limited to this.

[0070] As used herein, the term "operably linked" means that a first gene and a second gene are functionally linked to their respective promoters or to a single promoter so that a nucleic acid sequence having promoter activity initiates and mediates transcription of the first gene or the second gene. Operable linkage can be achieved using recombinant DNA techniques known in the art.

[0071] Another aspect provides a vector comprising the construct.

[0072] In one embodiment, the vector may be, but is not limited to, a viral vector or a non-viral vector.

[0073] In one embodiment, the viral vector may be any one selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus, and vaccinia virus, but is not limited thereto.

[0074] The viral or non-viral vector can be used without limitation as long as it can transduce or transfect animal cells, particularly T cells, etc., through infection. The virus may be capable of carrying two or more genes, and may be a retrovirus, a lentivirus, an adenovirus, or an adeno-associated virus.

[0075] In this example, retrovirus and lentivirus were used, but the present invention is not limited thereto. Specifically, two gene groups, including a gene encoding a CTLA4-CD28 chimeric protein and a gene encoding a chimeric antigen receptor, were carried by retrovirus and lentivirus.

[0076] The non-viral vector is preferably one that utilizes a transposon system (Hackett et al., US 6,489,458 B), but is not limited thereto, and it will be obvious to those skilled in the art that any commonly used non-viral vector that is suitable for the purposes of the present invention can be used.

[0077] In one embodiment, the viral or non-viral vector may comprise genes for a CTLA4-CD28 chimeric protein and a chimeric antigen receptor. Specifically, the vector may comprise, but is not limited to, the gene sequence of SEQ ID NO: 16, SEQ ID NO: 20, or SEQ ID NO: 22, a portion thereof, or a gene sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0078] In one embodiment, the construct of the present invention may comprise, but is not limited to, the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 21, or SEQ ID NO: 23, a portion thereof, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0079] [Table 3] JPEG2025540660000007.jpg248170JPEG2025540660000008.jpg248170JPEG2025540660000009.jpg133170

[0080] Although the present application states that a gene sequence / amino acid sequence of a particular SEQ ID NO is included or that a gene sequence / amino acid sequence of a particular SEQ ID NO is included, it is obvious that a gene sequence / amino acid sequence in which a part of the sequence is deleted, modified, substituted or added may also be used in the present application, as long as it has the same or corresponding function as the gene sequence / amino acid sequence of the relevant SEQ ID NO. Furthermore, the terms gene sequence and nucleotide sequence may be used interchangeably in the present application.

[0081] For example, it is obvious that, as long as the product has the same or equivalent function as the CTLA4-CD28 chimeric protein, the chimeric antigen receptor, or the construct, a nonsensical sequence may be added to the interior or end of the sequence of the corresponding SEQ ID NO, or a partial sequence may be deleted from the interior or end of the sequence of the corresponding SEQ ID NO, and this also falls within the scope of the present application.

[0082] Homology and identity refer to the degree to which two given base sequences are related and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0083] Whether any two sequences have homology or identity can be determined using known computer algorithms such as the "FASTA" program using default parameters, for example, as in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, it can be determined using 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) (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 et al. (1988) SIAM J Applied Math 48:1073. For example, BLAST from the National Database Center for Biotechnology Information, or ClustalW, can be used to determine sequence homology or identity.

[0084] One aspect provides an immune cell into which the construct or a vector containing the construct has been introduced.

[0085] In one embodiment, the viral or non-viral vectors can be used to provide transduced T cells. Another aspect provides an immune cell comprising a protein expressed from the construct or a vector comprising the construct.

[0086] In one embodiment, the immune cells may be, but are not limited to, T cells, NK cells, NKT cells, macrophages, or a combination thereof.

[0087] In one embodiment, the immune cells may be, but are not limited to, CD4 T cells, CD8 T cells, or a combination thereof. T cells containing the CTLA4-CD28 chimeric gene and first-generation CAR or 4-1BB-based second-generation CAR not only have excellent cancer therapeutic efficacy, but also have been proven safe because they can be easily eliminated by α-CTLA4, and can be usefully used in T cell immunotherapy.

[0088] Another aspect provides an immune cell comprising: (i) a first gene encoding a fusion protein comprising a CTLA-4 protein or a domain thereof and CD28 or a domain thereof (CTLA4-CD28 fusion protein); and (ii) a second gene encoding a chimeric antigen receptor comprising an antigen-binding domain, an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain.

[0089] In one embodiment, the first gene and the second gene may be introduced into immune cells independently and simultaneously, sequentially, or in reverse order.

[0090] Another aspect provides an immune cell that expresses both (i) a fusion protein comprising a CTLA-4 protein or a domain thereof and CD28 or a domain thereof (CTLA4-CD28 fusion protein), and (ii) a chimeric antigen receptor comprising an antigen-binding domain, an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain. Other aspects provide compositions comprising the construct, a vector comprising the construct, or the immune cell.

[0091] Another aspect provides a composition comprising the construct, a vector comprising the construct, or the immune cell, and a pharmaceutically acceptable carrier.

[0092] In one embodiment, the construct, the vector, the immune cell, or the composition may be for use in the prevention or treatment of a disease (e.g., cancer).

[0093] Another aspect provides the use of said construct, said vector, said immune cell, or said composition for the manufacture of a medicament for the prevention or treatment of a disease (e.g., cancer).

[0094] As used herein, the term "disease" may refer to a pathological condition, in particular cancer, infectious diseases, inflammatory diseases, degenerative diseases, cell death-related diseases and transplant rejection.

[0095] As used herein, the term "treatment" refers to or includes the alleviation, slowing down 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 utilized in the course of practicing the inventions provided herein sufficient to alleviate, slow down or prevent a disease, disorder, or condition, or one or more symptoms thereof.

[0096] As used herein, the terms "administer," "introduce," and "implant" are used interchangeably and may refer to the placement of a composition according to an embodiment into an individual by a method or route that results in at least partial localization of the composition according to an embodiment at a desired site. Administration may be by any suitable route that delivers at least a portion of the cells or cellular components of a composition according to an embodiment to a desired location within a living individual. The survival period of the cells after administration to an individual may be as short as a few hours, e.g., 24 hours to a few days, or as long as several years.

[0097] The administration may be in combination with an additional anti-cancer agent. Examples of the additional anti-cancer agent may include alkylating agents, antimetabolites, spindle inhibitor plant alkaloids, cytotoxic / anti-tumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Examples of the anti-cancer agent may include compounds used in targeted therapy and conventional chemotherapy. Further, examples of the antibody include alemtuzumab, apolizumab, aselizumab, atlizumab, bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidutuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motavizumab, natalizumab, and nimotuzumab. , norobizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resivizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, trastuzumab, tucotuzumab celmoleukin, tuxituzumab, umavizumab, urtoxazumab, and visilizumab.

[0098] The cancer or carcinoma is not particularly limited and includes solid cancers and blood cancers, specifically gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, pancreatic cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, parathyroid cancer, kidney cancer, esophageal cancer, biliary tract cancer, testicular cancer, rectal cancer, head and neck cancer, cervical cancer, ureteral cancer, osteosarcoma, neurocytoma, melanoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, glioma, etc., more preferably, gastric cancer, ovarian cancer, pancreatic cancer, breast cancer, etc. The composition of the present invention may further comprise a pharmaceutically acceptable carrier. For oral administration, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes, flavorings, etc. may be used. For injections, buffers, preservatives, soothing agents, solubilizers, isotonicity agents, stabilizers, etc. may be mixed. For topical administration, bases, excipients, lubricants, preservatives, etc. may be used. The pharmaceutical composition of the present invention may be prepared in various forms by mixing with the above-mentioned pharmaceutically acceptable carriers. For example, for oral administration, the composition may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, the composition may be prepared in the form of unit-dose ampoules or multi-dose forms. The anticancer composition may also typically comprise a surfactant to facilitate transport across membranes. Such surfactants are derived from steroids, cationic lipids such as N-[1-(2,3-dioleoyl)propyl-N,N,N-trimethylammonium chloride (DOTMA), or various compounds such as cholesterol hemisuccinate and phosphatidylglycerol.

[0099] Another aspect provides a method for preventing or treating a disease (e.g., cancer), comprising administering the construct, vector, immune cell, or composition to an individual in need thereof. For example, the method may be, but is not limited to, a method for suppressing cancer growth.

[0100] In one embodiment, the composition may be administered in a pharmaceutically effective amount to treat cancer cells or their metastasis or to inhibit cancer growth. This may vary depending on various factors, such as the type of cancer, the patient's age and weight, the characteristics and severity of symptoms, the type of current treatment, the number of treatments, and the form and route of administration, and can be easily determined by one skilled in the art. The composition according to the present invention may be administered together with the pharmacological or physiological components or sequentially. It may also be administered in combination with additional conventional therapeutic agents, and may be administered sequentially or simultaneously with the conventional therapeutic agents. Such administration may be single or multiple. It is important to administer an amount that can achieve maximum efficacy with the minimum amount without side effects, taking all of the above factors into consideration, and this can be easily determined by one skilled in the art.

[0101] Another embodiment is a method for producing a CTLA-4 antibody comprising: (i) a first gene encoding a fusion protein comprising a CTLA-4 protein or a domain thereof and a CD28 protein or a domain thereof (a CTLA4-CD28 fusion protein); (ii) a second gene encoding a chimeric antigen receptor comprising an antigen-binding domain, an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain; and The present invention provides a method for producing immune cells that express a CTLA4-CD28 fusion protein and a chimeric antigen receptor, the method comprising the step of introducing the above into immune cells.

[0102] In one embodiment, the step may include, but is not limited to, introducing the construct or a vector containing the construct into immune cells. In one embodiment, the process may include, but is not limited to, introducing the first gene and the second gene into immune cells independently and simultaneously, sequentially, or in reverse order. In this case, the independent introduction of the first gene and the second gene may be performed by a method known in the art. For example, but not limited to, a vector containing the first gene and a vector containing the second gene may be used independently.

[0103] Preferred examples are described below to aid in understanding the present invention. However, the following examples are provided to facilitate understanding of the present invention, and are not intended to limit the scope of the present invention. Various modifications can be made to the examples, and the examples are not limited to the examples disclosed below, and can be implemented in various forms.

[0104] Example 1. Anti-cancer efficacy of first-generation CAR-T cells loaded with CTC28 1.1. Generation of CTC28 first-generation CAR retrovirus and T cell transduction Currently, the primary preclinical animal model for evaluating the anti-cancer efficacy of CAR-T cells involves administering human CAR-T cells to immunodeficient mice inoculated with human tumors. However, the absence of endogenous immune cells makes it difficult to verify the immunological efficacy of CAR-T cells. Therefore, the inventors first attempted to verify the concept of enhancing the anti-cancer efficacy of CAR-T cells by introducing CTC28 using normal mice with preserved immune function. To this end, they generated mouse CAR-T cells targeting CD19 and established a mouse syngeneic tumor model to test the efficacy of these CAR-T cells in CD19-positive mouse lymphoma cells.

[0105] To produce CAR-T cells co-transduced with CTC28 (see Figure 1), a retrovirus for expressing CAR was produced.

[0106] Specifically, we created a first-generation CAR gene (mCD19z) by linking the antigen-binding site of the 1D3 antibody, an antibody against mouse CD19, to the intracellular signaling site of the mouse T cell receptor signaling subunit (CD3zeta), and cloned it into a retroviral vector.

[0107] The first-generation mouse CAR (mCD19z) protein coding region consists of a mouse immunoglobulin kappa chain signal peptide, an anti-mouse CD19 antibody (1D3) scFv, the extracellular and transmembrane domains of mouse CD8, and the intracellular domain of mouse CD3 zeta. These CAR cDNAs were synthesized by Bionear Korea and Integrated DNA Technologies (IDT, USA). The retroviral vector for CAR expression was constructed by substituting the constructed CAR cDNA for the PuroR gene site under the PGK promoter in the pMSCV-puro retroviral vector (Clontech, USA). The mouse CTLA4-CD28 chimera (CTC28) protein coding region, which combines the extracellular and transmembrane domains of mouse CTLA-4 and the intracellular domain of mouse CD28, was constructed as previously reported (Blood 2012;119(24):5678-87). A schematic diagram is shown in Figure 2.

[0108] To produce ecotropic retrovirus for mouse CAR expression, the mouse CAR retroviral vector and a VSV-G envelope expression plasmid (pMD2.G, Addgene plasmid #12259) were co-transfected into the Phoenix GP cell line using Lipofectamin 3000 (Invitrogen). After 48 hours, the VSV-G pseudotyped retrovirus secreted into the culture supernatant was transduced into the Phoenix Eco cell line. After 3–5 days, cells stably expressing CAR protein on the cell surface were isolated using a cell sorter (FACS Aria, Becton Dickinson), and these cells were used as ecotropic retrovirus-producing cell lines. The retroviral culture supernatant produced from this cell line was concentrated 5-10 times using a centrifugal filter device (Amicon Ultra-100 kDa cut-off, Millipore, USA) and used to produce mouse CAR-T cells.

[0109] Flow cytometry analysis (FACS, fluorescence activated cell sorter) was performed to confirm the expression of CAR (L-protein) and CTC28 in the transduced T cells, and the results are shown in Figure 3.

[0110] FIG. 1 is a schematic diagram showing double transgenic T cells using CTLA-4 mutants and chimeric antigen receptors.

[0111] Figure 2 is an image showing the structure of the gene constructs of first-generation CAR (mCD19z) and first-generation CAR and CTLA4-CD28 (mCD19z-CTC28).

[0112] Figure 3 is a graph analyzing the expression levels of CAR and CTLA4 proteins on the surface of T cells transfected with first-generation CAR (mCD19z) and first-generation CAR CTC28 (mCD19z-CTC28).

[0113] As shown in Figure 3, the CAR-positive cells were measured as 66.0% in T cells transfected with the first-generation CAR (mCD19z), and 40.4% and 78.9% in T cells transfected with the first-generation CAR and CTLA4-CD28 (mCD19z-CTC28), confirming good CAR and CTC28 expression in all cases.

[0114] 1.2. Anti-cancer efficacy of first-generation CAR-T cells loaded with CTC28 - in vitro study To evaluate the in vitro anticancer efficacy of T cells co-expressing first-generation CAR and CTC28, the tumor-killing ability of the manufactured T cells co-expressing first-generation CAR and CTC28 against A20 cells, a mouse B-cell lymphoma cell line, was analyzed.

[0115] Specifically, A20 (BALB / c-derived lymphoma cell line) was purchased from ATCC (USA), and a cell line (A20-Luc) transfected with the GFP-luciferase gene was prepared by transducing A20 cells with a retrovirus (pMP-LucGFP) for GFP-Luc expression, followed by isolating GFP-positive cells using a cell sorter (FACS Aria, Becton Dickinson). To measure the tumor-killing ability of CAR-T cells, CAR-T cells (effector cells, 1.2 × 10 ) expanded after retroviral transduction were used. 3 ~7.5×10 5 cells / 100 μl / well) and A20-Luc cells (target cells, 3 × 10 4A20-Luc cells were added to CAR-T cells at various ratios (0.04 to 25:1) in a 96-well plate and co-cultured overnight. 50 μL of D-luciferin (600 μg / ml, Promega) was then added and incubated at 37°C for 10 minutes to induce luciferase activity in the viable A20-Luc cells. The luminescence of these cells was measured using a luminometer (Tecan) and compared with that of untreated A20-Luc cells to calculate the tumor cell viability. The tumor-killing ability of CAR-T cells was measured, and the results are shown in Figure 4.

[0116] In addition, to evaluate the cytokine production ability of T cells co-expressing first-generation CAR and CTC28, the amount of IFN-γ secreted from the manufactured T cells co-expressing first-generation CAR and CTC28 was measured when co-cultured with A20 cells.

[0117] Specifically, CAR-T cells and target cells A20 were administered in equal numbers (3 × 10 4 The cells were mixed with the IFN-γ receptor activator and co-cultured in a 96-well plate for 24 hours, and the culture supernatant was harvested. The amount of IFN-γ secreted into the supernatant was measured by ELISA (mouse IFNγ ELISA kit, BD Biosciences). The results are shown in Figure 5.

[0118] Figure 4 is a graph analyzing the tumor-killing ability of T cells transfected with the first-generation CAR (mCD19z) and the first-generation CAR CTC28 (mCD19z-CTC28) against A20 cells.

[0119] Figure 5 is a graph showing the amount of IFN-γ secreted when T cells introduced with first-generation CAR (mCD19z) and first-generation CAR CTC28 (mCD19z-CTC28) were co-cultured with A20 cells.

[0120] As shown in Figure 4, when co-cultured with A20 cells, first-generation CTC28 CAR-T cells exhibited similar or slightly enhanced tumor-killing ability compared to first-generation CAR-T cells.

[0121] In contrast, as shown in Figure 5, the amount of IFN-γ secreted by first-generation CTC28 CAR-T cells was significantly increased compared to first-generation CAR-T cells, confirming that expression of CTC28 significantly increases the cytokine activity of CAR-T cells.

[0122] 1.3. Anti-cancer efficacy of first-generation CAR-T cells loaded with CTC28 - in vivo study To evaluate the in vivo anti-cancer efficacy of T cells co-expressing a first-generation CAR and CTC28, CAR-T cells were intravenously injected into Balb / C mice subcutaneously inoculated with A20 cells, and tumor growth over time was measured.

[0123] Specifically, BALB / c mice were purchased from Orient Bio (Seongnam, Republic of Korea), housed in the specific pathogen-free (SPF) animal facility at Seoul National University College of Medicine (Seoul, Republic of Korea), and maintained in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC). To simultaneously evaluate the in vivo efficacy and toxicity of CAR-T cells in a syngeneic mouse model, BALB / c mice were inoculated with A20 cells (5 × 10 per mouse). 6 CAR-T cells (1 × 10 per mouse), which had expanded for 4–5 days after retroviral transduction, were injected subcutaneously. Seven days later, low-dose total-body irradiation (2.5 Gy) was administered for lymphodepletion. Two days after irradiation, CAR-T cells (1 × 10 per mouse), which had expanded for 4–5 days after retroviral transduction, were injected subcutaneously. 6 cells or 5 × 10 6 The tumor size was then measured twice a week using a caliper to evaluate the in vivo efficacy of CAR-T. The results are shown in Figure 6.

[0124] FIG. 6 is a graph showing the change in tumor size in mice injected with T cells transfected with the first-generation CAR (mCD19z) and the first-generation CAR CTC28 (mCD19z-CTC28) (tumor size = long diameter 2 × Short diameter / 2).

[0125] As shown in Figure 6, for first-generation CAR-T cells, 5 × 10 6 When CAR-T cells were administered, a significant tumor growth suppression effect was observed, but when 5 × 10 5 When 5 × 10 CAR-T cells were administered, almost no antitumor effect was observed. However, when 5 × 10 1st-generation CTC28 CAR-T cells were administered, 5 Even when 5 × 10 cells were administered, 6 The administration of 100 first-generation CAR-T cells demonstrated a significant tumor-suppressing effect, demonstrating that the antitumor effect of CTC28 CAR-T cells is approximately 10 times stronger than that of first-generation CAR-T cells.

[0126] Based on these results, we were able to confirm that co-expression of CTC28 significantly enhances the antitumor activity of first-generation CARs.

[0127] Example 2. Anti-cancer efficacy of second-generation CAR-T cells loaded with CTC28 2.1. Generation of CTC28-loaded second-generation CAR retrovirus and transduction of T cells CAR-T cells currently used as therapeutic agents in clinical trials are second-generation CAR-T cells that contain the CD28 or 4-1BB intracellular signaling domain in the intracellular signaling domain of the CAR molecule (e.g., axicabtagene ciloleucel is a CD28-based second-generation CAR-T cell therapy, and tisagenlecleucel is a CTC28-1BB-based second-generation CAR-T cell therapy). Therefore, to examine whether co-expression of CTC28 can enhance the efficacy of second-generation CAR-T cells, we generated CD28-based and 4-1BB-based second-generation mouse CAR-T cells loaded with CTC28.

[0128] Specifically, the protein coding region of the CD28-based second-generation mouse CAR (mCD1928z) is composed of the rat immunoglobulin kappa chain signal peptide, the scFv of the anti-mouse CD19 antibody (1D3), the mouse CD28 extracellular domain-transmembrane domain-intracellular domain, and the mouse CD3 zeta intracellular domain, as previously reported (GenBank HM754222.1, Blood 2010;116(20): 4099-4102).

[0129] The 4-1BB-based second-generation mouse CAR (mCD19BBz) protein coding region consists of a mouse CD8 signal peptide, an anti-mouse CD19 antibody (1D3) scFv, mouse CD8 extracellular and transmembrane domains, a human 4-1BB intracellular domain, and a mouse CD3 zeta intracellular domain. The human 4-1BB intracellular domain was used based on previous reports that the human 4-1BB sequence is more functional than the mouse 4-1BB sequence in mouse CAR-T cells (JCI Insight. 2018;3(18):e121322). These CAR cDNAs were synthesized by Bionear Korea and Integrated DNA Technologies (IDT, USA). The retroviral vector for CAR expression was constructed by substituting the constructed CAR cDNA for the PuroR gene site under the PGK promoter in the pMSCV-puro retroviral vector (Clontech, USA) and cloning it using the same method as in Example 1.1. The mouse CTLA4-CD28 chimera (CTC28) protein coding region and mouse CAR-T cells using the same were prepared in the same manner as in Example 1.1.

[0130] 2.2. Anti-cancer efficacy of CD28-based second-generation CAR-T cells loaded with CTC28 First, to verify the effect of co-carrying CTC28 in CD28-based second-generation CAR-T cells, mouse T cells were transduced with a retrovirus expressing the gene construct shown in the schematic diagram in Figure 7.

[0131] Flow cytometry analysis (FACS, fluorescence activated cell sorter) was performed in the transduced T cells to confirm the expression of CAR (L-protein) and CTC28 using the same method as in Example 1.2. The results are shown in Figure 8.

[0132] To evaluate the in vitro anti-cancer efficacy of T cells co-expressing second-generation CAR and CTC28, the amount of IFN-γ secreted from the prepared T cells co-expressing second-generation CAR and CTC28 when co-cultured with A20 tumor cells was measured using the same method as in Example 1.2. The results are shown in Figure 9.

[0133] To evaluate the in vivo anti-cancer efficacy of T cells co-expressing second-generation CARs and CTC28, CAR-T cells were intravenously injected into Balb / C mice subcutaneously inoculated with A20 cells, and tumor growth over time was measured.

[0134] Specifically, Balb / C mice were treated with low-dose whole-body irradiation (3 Gy) for lymphodepletion and A20 cells (5 × 10 per mouse). 6 Three days later, CAR-T cells were intravenously injected, and tumor growth over time was measured using the same method as in Example 1.3. The results are shown in Figure 10.

[0135] Figure 7 is an image showing the structure of the gene constructs of CD28-based second-generation CAR (mCD1928z) and second-generation CAR and CTLA4-CD28 (mCD1928z-CTC28).

[0136] Figure 8 is a graph analyzing the expression levels of CAR and CTLA4 proteins on the surface of T cells transfected with CD28-based second-generation CAR (mCD1928z) and second-generation CAR CTC28 (mCD1928z-CTC28).

[0137] Figure 9 is a graph showing the amount of IFN-γ secreted when T cells transfected with CD28-based second-generation CAR (mCD1928z) and second-generation CAR CTC28 (mCD1928z-CTC28) were co-cultured with A20 cells.

[0138] FIG. 10 is a graph showing the change in tumor size in mice injected with T cells transfected with CD28-based second-generation CAR (mCD1928z) and second-generation CAR CTC28 (mCD1928z-CTC28) (tumor size = long diameter 2 × Short diameter / 2).

[0139] As shown in Figure 8, the CAR-positive cells were measured at 78.1% in T cells transfected with the second-generation CAR (mCD1928z), and 85.3% and 88.3% in T cells transfected with the second-generation CAR and CTLA4-CD28 (mCD1928z-CTC28), confirming good CAR and CTC28 expression in all cases.

[0140] As shown in Figure 9, contrary to the pattern observed with first-generation CAR-T cells, the amount of IFN-γ secreted by CD28-based second-generation CTC28 CAR-T cells was significantly lower than that of second-generation CAR-T cells.

[0141] As shown in Figure 10, in a mouse tumor model, CD28-based second-generation CAR-T cells showed a significant antitumor effect at a cell count (1 × 10 6 When the same number of second-generation CTC28 CAR-T cells was administered as in the control group, the antitumor activity was significantly reduced. Therefore, in the case of CD28-based second-generation CAR-T cells, unlike first-generation CAR-T cells, it was observed that co-expression of CTC28 actually inhibited the anti-cancer efficacy of CAR-T cells.

[0142] Therefore, this suggests that the increased anti-cancer efficacy of CAR-T cells co-loaded with CTC28 may not be observed for all CAR forms, but may only be effective for certain CAR forms.

[0143] 2.3. Anti-cancer efficacy of 4-1BB-based second-generation CAR-T cells loaded with CTC28 Next, to verify the effect of co-loading CTC28 with 4-1BB-based second-generation CAR-T cells, mouse T cells were transduced with a retrovirus expressing the gene construct shown in the schematic diagram in Figure 11.

[0144] Flow cytometry analysis (FACS, fluorescence activated cell sorter) was performed to confirm the expression of CAR (α-Rat IgG) and CTC28 in the transduced T cells, and the results are shown in Figure 12.

[0145] To evaluate the in vitro anti-cancer efficacy of T cells co-expressing 4-1BB-based second-generation CAR and CTC28, the amount of IFN-γ secreted from the prepared T cells co-expressing second-generation CAR and CTC28 when co-cultured with A20 tumor cells was measured using the same method as in Example 1.2. The results are shown in Figure 13.

[0146] To evaluate the in vivo anti-cancer efficacy of T cells co-expressing 4-1BB-based second-generation CAR and CTC28, CAR-T cells were intravenously injected into Balb / C mice subcutaneously inoculated with A20 cells, and tumor growth was measured over time.

[0147] Specifically, Balb / C mice were inoculated with A20 cells (5 × 10 per mouse). 6 Twelve days later, the mice were treated with low-dose total-body irradiation (2.5 Gy) for lymphodepletion. Two days later, CAR-T cells (1 × 10 per mouse) were injected subcutaneously. 6 cells or 5 × 106 After intravenous injection of the 1000 mg / kg / day mice, tumor growth was measured over time in the same manner as in Example 1.3. The results are shown in FIG.

[0148] Figure 11 is an image showing the structure of the gene constructs of 4-1BB-based second-generation CAR (mCD19BBz) and second-generation CAR and CTLA4-CD28 (mCD19BBz-CTC28).

[0149] Figure 12 is a graph analyzing the expression levels of CAR and CTLA4 proteins on the surface of T cells transfected with 4-1BB-based second-generation CAR (mCD19BBz) and second-generation CAR CTC28 (mCD19BBz-CTC28).

[0150] Figure 13 is a graph showing the amount of IFN-γ secreted when T cells transfected with 4-1BB-based second-generation CAR (mCD19BBz) and second-generation CAR CTC28 (mCD19BBz-CTC28) were co-cultured with A20 cells.

[0151] FIG. 14 is a graph showing the change in tumor size in mice injected with T cells transfected with 4-1BB-based second-generation CAR (mCD19BBz) and second-generation CAR CTC28 (mCD19BBz-CTC28) (tumor size = long diameter 2 × Short diameter / 2).

[0152] As shown in Figure 12, CAR-positive cells were measured at 40.2% in T cells transfected with the 4-1BB-based second-generation CAR (mCD19BBz), and CAR-positive cells were measured at 61.5% and CTC28-positive cells at 88.3% in T cells transfected with the second-generation CAR and CTLA4-CD28 (mCD19BBz-CTC28), confirming good CAR and CTC28 expression in all cases.

[0153] As shown in Figure 13, it was confirmed that the amount of IFN-γ produced by 4-1BB-based second-generation CTC28 CAR-T cells was increased compared to second-generation CAR (4-1BB) T cells.

[0154] As shown in Figure 14, in the mouse tumor model, the cell dose at which 4-1BB-based second-generation CAR-T cells were unable to completely eliminate tumors and tumors recurred (5 × 10 5 ) also demonstrated that 4-1BB-based second-generation CTC28 CAR-T cells were able to completely eliminate tumors, indicating that co-transfection of CTC28 with 4-1BB-based CAR-T cells significantly enhanced the efficacy of CAR-T cells.

[0155] This confirmed that the efficacy of CTC28 co-loading differs depending on the type of intracellular signaling site in the CAR construct. Specifically, in the case of first-generation CAR-T cells and 4-1BB-based second-generation CAR-T cells, the efficacy of CTC28 CAR-T cells was enhanced.

[0156] These results demonstrate that when CTC28 is added to clinically used 4-1BB-based second-generation CAR-T cells, it is possible to produce CAR-T cells with significantly improved functionality.

[0157] Example 3. Analysis of the functional improvement and mechanism of action of combined treatment of CD4 and CD8 T cells in 41BB-based CAR-T cell therapy loaded with CTC28 3.1. Activation Effect of CTC28 CD4 and CD8 CAR-T Cells - In Vitro Study In previous studies of classical T cell therapy using CTC28, it was found that the functional enhancing effect of CTC28 was stronger on CD4 T cells than on CD8 T cells among T cell subsets, and that when equal numbers of CTC28 CD4 T cells and CTC28 CD8 T cells were infused together, the therapeutic effect was maximized (Blood 2012;119(24):5678-87). In this CAR-T cell study, we sought to confirm whether the CD4 bias of the CTC28 effect and the enhancing effect of CTC28 would be observed in CD4 and CD8 T cell combination therapy.

[0158] Specifically, to analyze the mechanism of functional improvement of 4-1BB-based CTC28 CAR-T cells separately for CD4 and CD8 T cells, we constructed retroviral vectors for CAR expression, incorporating the Thy1.1 marker for tracking mouse CAR-T cells in the blood. The Thy1.1 protein coding region and the T2A-CAR or T2A-CAR-P2A-CTC28 protein coding region were amplified by PCR, and the amplified fragments were cloned into the pMSCV-puro vector after blunt-end ligation. The mouse CTLA4-CD28 chimera (CTC28) protein coding region and mouse CAR-T cells using it were constructed using the same method as in Example 1.1. A schematic diagram is shown in Figure 15.

[0159] Specifically, for mouse CAR-T cells, spleen and lymph node cells from normal Balb / C mice were stained with anti-CD4 and anti-CD8 microbeads (Miltenyi Biotech), respectively, and CD4 and CD8 T cells were isolated and purified using a MACS LS column (Miltenyi Biotech). T cells were then activated by adding anti-CD3 antibody (145-2C11, 10 μg / ml, BioXcell) and anti-CD28 antibody (37.51, 2 μg / ml, BD Biosciences) to a 24-well plate coated with anti-CD3 antibody. After 24 hours of activation, T cells were transduced with the concentrated retrovirus by centrifugation at 2500 rpm for 90 minutes in the presence of 6 μg / ml polybrene (Sigma-Aldrich) at 24°C. This retroviral transduction process by centrifugation was repeated once each day. The T cells were then cultured for 48 hours in the presence of mouse IL-2 (30 U / ml, Gibco). Retrovirally transduced T cells were washed twice and expanded for 2–3 days in fresh culture medium containing mouse IL-2 (20 U / ml). These cells were then used as CAR-T cells. After CAR-T cell generation, cell surface CAR protein expression was measured using flow cytometry (FACS-Canto II, BD Biosciences) after staining with biotinylated protein L (Thermo) and PE-labeled streptavidin or FITC-labeled anti-rat IgG Fab fragment (Jackson ImmunoResearch). Expression of CTC28 and Thy1.1 was analyzed by flow cytometry after staining with fluorescently labeled anti-CTLA4 antibody (UC10-4B9, Biolegend) and anti-Thy1.1 antibody (OX-7, Biolegend), respectively. The results are shown in Figure 16.

[0160] Specifically, to confirm the cell killing ability through co-culture of CAR-transduced CD4 and CD8 T cells with A20 cells, the tumor killing ability of CAR-T cells was measured by calculating the tumor cell viability in comparison with the luminescence intensity of A20-Luc cells that were not treated with CAR-T cells in the same manner as in Example 1.2. The results are shown in Figure 17.

[0161] Specifically, to evaluate the cytokine production ability of CAR-transduced CD4 and CD8 T cells, the amounts of IFN-γ, IL-2, and TNF-α secreted from CAR-transduced CD4 and CD8 T cells when co-cultured with A20 cells were measured using the same method as in Example 1.2.

[0162] To analyze the division potential of CAR-transduced T cells, we performed a dye dilution assay to measure the degree to which the fluorescence intensity of the dye stained on the cells decreases as the CAR-T cells stained with CTV dye divide. Specifically, CAR-T cells were labeled with CTV (2.5 μM, eBioscience) and then transfected with the same number of target cells (A20 cells) (3 × 10 4 The cells were mixed with Thy1.1 and added to a 96-well plate. After 48 or 72 hours, the cells were harvested and stained with an anti-Thy1.1 antibody (OX-7). The percentage of Thy1.1-positive CAR-T cells, whose CTV fluorescence intensity had decreased due to cell division, was then measured using flow cytometry (FACS Fortessa X-20, BD Bioscience). The results are shown in Figure 19.

[0163] Figure 15 is an image showing the structure of the gene constructs for second-generation CAR (mCD19BBz) containing a T cell tracking marker (Thy1.1) and second-generation CAR and CTLA4-CD28 (mCD19BBz-CTC28).

[0164] Figure 16 is a graph analyzing the expression levels of Thy1.1 and CTC28 proteins on the surface of CAR-T cells into which mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28) were introduced.

[0165] Figure 17 is a graph analyzing the tumor-killing ability of CAR-T cells introduced with mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28) against A20 cells.

[0166] Figure 18 is a graph showing the amounts of IFN-γ, IL-2, and TNF-α secreted by CAR-T cells transfected with mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28) when co-cultured with A20 cells.

[0167] Figure 19 is a graph analyzing changes in the division ability of CD8 CAR-T cells into which mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28) were introduced.

[0168] As shown in Figure 16, in CD4 T cells transfected with the second-generation CAR (mCD19BBz), Thy1.1-positive cells were 93.0% and CTC28-positive cells were 82.2%, and in CD8 T cells, Thy1.1-positive cells were 81.9% and CTC28-positive cells were 71.8%, confirming good CAR and CTC28 expression in all cases.

[0169] As shown in Figure 17, we confirmed that CD8 CAR-T cells, which are known to mediate tumor killing, exhibited a higher tumor-killing ability than CD4 CAR-T cells. Co-loading with CTC28 did not significantly increase the tumor-killing ability of either CD8 or CD4 CAR-T cells.

[0170] However, as shown in FIG. 18, it was confirmed that CTC28 significantly increased cytokine production in both CD4 and CD8 T cells.

[0171] As shown in Figure 19, CD8 CAR-T cells co-loaded with CTC28 showed a more pronounced decrease in CTV staining compared to conventional CD8 CAR-T cells, confirming their enhanced cell division ability.

[0172] These results confirmed that Thy1.1-labeled CTC28 CAR-T cells also showed improved in vitro reactivity, and that the improved reactivity of CAR-T cells produced from isolated CD4 and CD8 T cells was also maintained.

[0173] 3.2. Efficacy evaluation of the combination therapy of CTC28 CD4 CAR-T cells and CD8 CAR-T cells - in vivo study To evaluate the anti-cancer efficacy of combined administration of CD4 and CD8 CAR-T cells in an in vivo tumor model using CTC28-loaded second-generation 4-1BB-based CAR-T cells, CAR-T cells were intravenously injected into Balb / C mice subcutaneously inoculated with A20 cells, and tumor growth over time was measured.

[0174] Specifically, BALB / c mice were inoculated with A20 cells (5 × 10 per mouse). 6 CD4 CAR-T cells and CD8 CAR-T cells (1 × 10 per mouse) were injected subcutaneously. 12 days later, low-dose total-body irradiation (2.5 Gy) was administered as a conditioning treatment for lymphodepletion before CAR-T cell infusion. 2 days after irradiation, CD4 CAR-T cells and CD8 CAR-T cells expanded for 4–5 days after retroviral transduction were mixed at a 1:1 ratio and injected intravenously (1 × 10 per mouse). 6 cells or 5 × 10 6Tumor size was then measured twice weekly using a caliper, and the number of CAR-T cells in the peripheral blood was measured once weekly. Viable CD4 or CD8 CAR-T cells in the blood were stained with anti-CD4 antibody (GK1.5, BioLegend), anti-CD8 antibody (53.6.7, BioLegend), anti-Thy1.1 antibody (OX-7, BioLegend), and 7-AAD (BioLegend), and the number of CD4(+)Thy1.1(+)7AAD(-) or CD8(+)Thy1.1(+)7AAD(-) cells was calculated by flow cytometry using counting beads (123count eBeads, ThermoFisher). The results are shown in Figures 20 and 21, respectively.

[0175] Figure 20 is a graph showing the change in tumor size in mice injected with CAR-T cells transfected with mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28) (tumor size = long diameter 2 × Short diameter / 2).

[0176] Figure 21 is a graph showing the survival rate of mice injected with CAR-T cells transfected with mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28).

[0177] As shown in Figure 20, the high dose (5 × 10 6 ) and low dose (1 × 10 6 We confirmed the enhanced tumor suppressive ability of second-generation CTC28 CAR-T cells over 4-1BB-based second-generation CAR-T cells at both low and high cell doses. 6 ) and confirmed that even when the efficacy of 4-1BB-based second-generation CAR-T cells was barely evident, the second-generation CTC28 CAR-T cells largely eliminated tumors.

[0178] As shown in FIG. 21, the survival rate of mice was significantly improved, demonstrating that CTC28 exhibited a therapeutic efficacy that was enhanced by more than 5 times.

[0179] In vivo characterization of CTC28-loaded CD4 and CD8 CAR-T cells For in vivo monitoring of the CD4 and CD8 CAR-T cells administered in the CTC28 CD4 and CD8 CAR-T cell combination therapy, a mouse model was performed using the same method as in Example 3.2. After CAR-T cell administration, 35 μl of blood was collected from the mouse tail once a week to measure the number of CAR-T cells in the blood. The results are shown in Figure 22.

[0180] CAR-T cells isolated from mouse spleens on day 7, the time point of maximum CD8 CAR-T cell proliferation, were co-cultured with A20 cells for 6 hours, and the levels of IFN-γ, IL-2, and TNF-α secreted by CD4 and CD8 CAR-T cells were analyzed ex vivo using flow cytometry analysis plots. The results are shown in Figure 23, and a quantified graph is shown in Figure 24.

[0181] Figure 22 is a graph measuring the number of CD4 and CD8 CAR-T cells transduced with mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28) in an A20 cell tumor mouse model.

[0182] Figure 23 is a graph showing the amounts of IFN-γ, IL-2, and TNF-α secreted by CD4 and CD8 CAR-T cells transduced with mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28) in an A20 cell tumor mouse model.

[0183] Figure 24 is a graph quantifying the amounts of IFN-γ, IL-2, and TNF-α secreted by CD4 and CD8 CAR-T cells transduced with mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28) in an A20 cell tumor mouse model.

[0184] As shown in Figure 22, it was observed that the proliferation of CTC28-loaded CAR-T cells was significantly increased compared to pre-existing CAR-T cells for both CD4 and CD8 CAR-T cells. While CTC28-loaded CD8 CAR-T cells showed dramatic proliferation in the blood initially, CTC28-loaded CD4 CAR-T cells showed characteristic late-phase proliferation and ultimately showed much greater cell proliferation than CD8 CAR-T cells, indicating CD4 bias.

[0185] As shown in Figures 23 and 24, it was observed that the cytokine secretion ability of all CD8 CAR-T cells and CD4 CAR-T cells into which CTC28 had been introduced was enhanced on the initial 7th day, confirming that they were functionally highly activated cells.

[0186] 3.4. Analysis of the antitumor mechanisms of CTC28-loaded CD4 and CD8 CAR-T cells In previous studies of classical T cell therapy using CTC28, it was observed that when CTC28 was introduced into CD4 T cells, the activation of CD8 T cells by CD4 T cells (so-called CD4 help) was significantly enhanced (Blood 2012;119(24):5678-87). Therefore, to confirm whether the enhancement of CD4 help by CTC28 also contributes to the enhanced antitumor effect in this CAR T cell setting, CTC28-loaded CD4 CAR T cells were administered in combination with CD8 CAR T cells that were not loaded with CTC28.

[0187] Specifically, A20 cells (5 × 10 per mouse) were inoculated into Balb / C mice in the same manner as in Experimental Example 3.2. 6 cells) were injected subcutaneously, and after irradiation, CD4 and CD8 CAR T cells were injected intravenously (1 × 10 per mouse). 6 The tumor growth and the number of CD4 and CD8 CAR T cells were measured, and the results are shown in Figures 25 and 26, respectively.

[0188] Figure 25 is a graph showing the change in tumor size in mice when CTC28-introduced CD4 or CD8 mCD19BBz CAR T cells (indicated by "(+)") and CD4 or CD8 mCD19BBz CAR T cells not infused with CTC28 (indicated by "(-)") were mixed at a 1:1 ratio and co-administered in an A20 cell tumor mouse model (Tumor size = long diameter 2 × Short diameter / 2).

[0189] Figure 26 is a graph measuring the number of CAR T cells in mice when CTC28-introduced CD4 or CD8 mCD19BBz CAR T cells (indicated by "(+)") and CD4 or CD8 mCD19BBz CAR T cells (indicated by "(-)") not introduced with CTC28 were mixed at a 1:1 ratio and co-administered in an A20 cell tumor mouse model.

[0190] As shown in Figure 25, the presence of CTC28 alone on CD4 CAR T cells significantly enhanced the antitumor activity of CD8 CAR T cells lacking CTC28 (CD4(-)+CD8(-) vs CD4(+)+CD8(-)), confirming that CTC28 significantly increased CD4 help. However, a certain degree of enhanced antitumor activity was also observed when CTC28 was expressed only on CD8 CAR T cells (CD4(-)+CD8(-) vs CD4(-)+CD8(+)), confirming that CTC28 also enhanced the intrinsic activity of CD8 CAR T cells.

[0191] As shown in Figure 26, in terms of cell proliferation, when CTC28 was introduced only into CD4 CAR T cells, the initial proliferation of CD8+ CAR T cells was significantly increased compared to CD4 CAR T cells without CTC28 (CD4(-)+CD8(-) vs CD4(+)+CD8(-)), confirming that CTC28 on CD4 CAR T cells enhances the CD4 help function of CD8 CAR T cells. However, a direct increase in proliferation of CD8 CAR T cells by CTC28 was also observed (CD4(-)+CD8(-) vs CD4(-)+CD8(+)). In particular, when CTC28 was introduced into both CD4 and CD8 CAR T cells, the greatest CD8 CAR T cell proliferation was confirmed (CD4(+)+CD8(+)), confirming that introduction of CTC28 into both CD4 and CD8 CAR T cells is most effective.

[0192] These results confirmed that the functional improvement power of CTC28 extends to both CD4 and CD8 CAR T cell populations. To investigate the factors mediating the functional enhancement of CAR T cells in CTC28, CD4 and CD8 CAR T cells were isolated from the spleen of mice 4 days after CAR T cell infusion, and RNA sequencing was performed using Gene Set Enrichment Analysis (GSEA).

[0193] Specifically, A20 cells were subcutaneously injected in the same manner as in Experimental Example 3.2. On the 14th day, when the tumor size reached approximately 200 mm, low-dose whole-body irradiation was administered. Then, 5 × 10 CAR T cells (mCD19BBz(CD19)) (CD4:CD8=1:1) without CTC28 transfection or CAR T cells (mCD19BBz-CTC28(CD19-CTC28)) (CD4:CD8=1:1) with CTC28 transfection were administered. 6Each CAR T cell was administered intravenously. Four days after CAR T cell infusion, spleens were harvested from mice and stained as single cell suspensions with anti-CD4 (GK1.5), anti-CD8 (53.6.7), anti-Thy1.1 (OX-7), and DAPI (Thermofisher). CD4(+)Thy1.1(+)DAPI(-) or CD8(+)Thy1.1(+)DAPI(-) cells were isolated and purified using a cell sorter. CAR T cell RNA was extracted using the RNeasy Mini kit (Qiagen), and RNA sequencing analysis was performed by Macrogen. Gene set enrichment analysis (GSEA) was performed using GSEA software (gsea-v4.3.0, http: / / software.broadinstitute.org / gsea / downloads.jsp). The duplicated RNA sequencing data were subjected to GSEA after batch effect correction between the two samples using Combat-seq (Bioconductor sva v3.36.0, https: / / bioconductor.org / packages / release / bioc / html / sva.html). p-values ​​were calculated using the Kolmogorov-Smirnov test (threshold = 0.01), and the FDR (False Discovery Rate)-q value is the estimated probability that a given NES indicates a false positive result for a gene set. The q-value threshold for GSEA was 0.25, and the Hallmark gene set was used for the GSEA analysis. The results are shown in Figure 27.

[0194] As shown in Figure 27, the analysis confirmed that RNAs involved in IL-2-STAT5 signaling were enriched in CTC28 CD4 and CD8 CAR T cells, suggesting that IL-2 may have contributed to the enhanced function of CTC28 CAR T cells.

[0195] To confirm whether IL-2 mediates the enhancement of CTC28 CAR T cell function, A20 cells (5 × 10 per mouse) were injected into Balb / C mice in the same manner as in Experimental Example 3.2. 6 cells) were injected subcutaneously, followed by irradiation and intravenous injection of CD4 and CD8 CAR T cells (1 × 10 per mouse). 6 Then, as the experimental group, 200 μg of an IL-2 neutralizing antibody (α-IL-2: JES6-1A12, BioXcell) or an isotype control antibody (iso: Rat IgG2a, BioXcell) was intraperitoneally injected at 2-3 day intervals from the time of CAR T cell injection. Tumor size and the number of CD4 and CD8 CAR T cells were measured, and the results are shown in Figure 28 and Figure 29, respectively.

[0196] Figure 27 is a graph showing RNA-seq analysis in mice injected with mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28) transduced CD4 and CD8 CAR T cells in an A20 cell tumor mouse model.

[0197] Figure 28 is a graph showing the change in tumor size in mice in an A20 cell tumor mouse model when mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28)-introduced CAR T cells were co-treated with an IL-2 neutralizing antibody (Tumor size = diameter × minor diameter / 2).

[0198] Figure 29 is a graph showing the number of CAR T cells in mice treated with mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28)-introduced CAR T cells and an IL-2 neutralizing antibody in an A20 cell tumor mouse model.

[0199] As shown in Figures 28 and 29, the tumor suppression ability and blood proliferation ability of CTC28-mediated CAR T cells were both reduced in the IL-2 neutralizing antibody-treated group, confirming that IL-2 contributes in part to the enhancement of CAR T cell function by CTC28.

[0200] These results confirmed that the mechanism by which CTC28 improves CAR T cell function is due to enhanced function of CD4 help and CD8 T cells, and that IL-2 contributes in part to this functional enhancement.

[0201] Example 4. Safety analysis of CTC28 CAR-T cells

[0202] In the above example, a characteristic of the CTC28-loaded CAR-T cells in the blood was a significant increase in cell count. To verify the safety of this increase in cell count, we tracked the increase or decrease in CAR-T cell count in the peripheral blood over a long period of approximately 5 months after CAR-T cell administration. We also tracked weight loss in mice as an indicator of toxicity.

[0203] Specifically, A20 cells (5×10 per mouse) were inoculated into Balb / C mice in the same manner as in Example 3.2. 6 After subcutaneous injection of CAR-T cells (1 × 10 per mouse), irradiation was performed and CAR-T cells were intravenously injected (1 × 10 per mouse). 6 or 5×10 6 The mice were then injected with CAR-T cells (Figure 30 and Figure 31), and 35 μl of blood was collected from the tail of each mouse once a week to measure the number of CAR-T cells in the blood. To evaluate CAR-T toxicity, each mouse was continuously monitored for weight loss and survival twice a week after CAR-T injection, and the mice were weighed 28 days and 142 days after CAR-T cell injection. The results are shown in Figure 30 and Figure 31.

[0204] Figure 30 is a graph measuring the number of CD4 and CD8 CAR-T cells in peripheral blood after administration of CAR T cells transduced with mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28) in a mouse model.

[0205] Figure 31 is a graph measuring changes in mouse body weight after administration of CAR T introduced with mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28) in a mouse model.

[0206] As shown in Figure 30, CD8 CTC28 CAR-T cells showed a decrease in cell count after the initial mass proliferation, but maintained a stable high level of cell count compared to conventional CAR-T cells. CD4 CTC28 CAR-T cells showed a more accelerated proliferation in the latter half of the treatment, showing a sustained increase in cell count after about 3 weeks, but maintained a stable cell count after about 2 months.

[0207] As shown in Figure 31, the CTC28 CAR-T cell-administered group did not show any weight loss during the first 1-2 weeks, which could be a manifestation of acute toxicity. Furthermore, the weight measured after 5 months showed no significant difference compared to the classical CAR-T cell-administered group. This suggests that CTC28-loaded CAR-T cells are unlikely to exhibit acute or chronic toxicity.

[0208] Nevertheless, if chronic toxicity due to the long-term presence of a large number of CTC28-loaded CAR-T cells is a concern, elimination of these CTC28-loaded CAR-T cells would ensure safety. As part of this effort, it has been reported that the currently available α-CTLA4 antibody (ipilimumab) has the function of eliminating CTLA4-expressing cells. Therefore, if an α-CTLA4 antibody binds to the extracellular CTLA4 site of CTC28, it could play a role in eliminating CTC28-loaded CAR-T cells. In this case, CTC28 can act as both a hyperactivation receptor and a suicide receptor.

[0209] Therefore, we investigated whether the CTC28 CAR-T cells that had increased in the blood could be eliminated using the α-CTLA4 antibody. Specifically, A20 cells (5 × 10 per mouse) were injected into Balb / C mice in the same manner as in Example 3.2. 6cells) were injected subcutaneously, followed by irradiation and intravenous injection of CAR-T cells (5 × 10 per mouse). 6 The mice were then treated with CAR-T cell therapy (Figure 32). 42 days after CAR-T cell administration, 200 μg of α-CTLA4 antibody (UC10-4B9, BioXcell) was intraperitoneally injected. Starting three days later, 100 μg of α-CTLA4 antibody was injected twice a day for a total of three booster injections. 35 μl of blood was collected from the mouse tail once a week, and the number of Thy1.1-positive CAR-T cells and CD19-positive B cells in the peripheral blood was monitored by flow cytometry. The results are shown in Figure 32.

[0210] Figure 32 is a graph showing the number of CAR-T cells and B cells in the blood in a mouse model treated with α-CTLA4 antibody after administration of CAR T cells incorporating mCD19BBz (CD19) and mCD19BBz-CTC28 (CD19-CTC28).

[0211] As shown in Figure 32, α-CTLA4 antibody treatment confirmed complete elimination of CTC28-loaded CAR-T cells in the blood. CD19-targeted CAR-T cells are known to have the side effect of eliminating not only CD19-positive tumor cells but also CD19-positive normal B cells in the blood. Therefore, while CTC28-loaded CAR-T cells were present, B cells were not detected in the blood (untreated group). However, after CAR-T cell elimination by α-CTLA4 antibody treatment, B cells were gradually detected in the blood, confirming complete elimination of CAR-T cells. This suggests that when CTC28-loaded CAR-T cells are actually administered clinically, it may be possible to eliminate CTC28-loaded CAR-T cells by administering a commercially available α-CTLA4 antibody (ipilimumab) if necessary. These results confirmed that CTC28 CAR-T cells that increase in the blood over the long term are not toxic and can be removed with anti-CTLA4 antibodies if necessary, confirming the safety of CTC28-loaded CAR-T cell therapy.

[0212] Example 5. Anti-cancer efficacy of CAR-T cells loaded with human CTC28 5.1. Construction of human CTC28 CAR lentivirus and transduction of cells To confirm the effect of CTC28 on enhancing CAR-T cell function in human CAR-T cells, we produced human CTC28-loaded CAR-T cells.

[0213] Specifically, the cDNA encoding the protein coding region of 4-1BB-based CAR (hCD19BBz) using the human anti-CD19 antibody (FMC63) was prepared by requesting DNA synthesis from IDT, Inc., USA, according to the previously published sequence (US Patent US_2013_0287748_A1). The lentiviral vector for human CAR expression was constructed by cloning human CAR cDNA into a partially modified pCDH-EF1 vector (Addgene Plasmid #72266). The human CTC28 protein coding region was designed by linking the corresponding regions of human CTLA4 and CD28, and the DNA sequence was codon-optimized and then DNA synthesis was requested from IDT. The lentiviral vector for dual expression of CAR and CTC28 was constructed by cloning human CTC28 cDNA downstream of the human CAR protein coding region via the P2A sequence. A schematic diagram is shown in Figure 33.

[0214] To produce lentivirus for human CAR expression, each lentiviral vector was transfected into 293T cells (ATCC, USA) using Lipofectamine 3000 (Invitrogen) along with three types of packaging DNA (pMD.2G; pMDLg / pRRE, addgene plasmid #12251; pRSV-rev, addgene plasmid #12253). After 24-48 hours, the culture supernatant containing the secreted lentivirus was harvested and filtered (0.45 μm filter) to remove residual cellular particles. The supernatant was then concentrated 100-fold using an ultra-high-speed centrifuge and used as the lentivirus concentrate for human CAR-T cell production.

[0215] For human CAR-T cells, leukocytes obtained from normal subjects by leukapheresis were stained with anti-CD4 or anti-CD8 microbeads (Miltenyi Biotech) and then CD4 and CD8 T cells were isolated using a MACS LS column (Miltenyi Biotech). The isolated T cells were added to a 24-well plate coated with anti-CD3 antibody (OKT3, 10 μg / ml, BioXcell) together with anti-CD28 antibody (CD28.2, 2 μg / ml, BD Biosciences) and cultured for 48 hours for T cell activation. Activated T cells were washed twice and then used for lentiviral transduction. After coating with Retronectin (20 μg / ml, TaKaRa) overnight at 4°C, the washed 24-well plate was blocked with 2% BSA-DPBS at 37°C for 30 minutes and washed. Lentivirus concentrate was then added and centrifuged at 2000 × g and 32°C for 2 hours to allow the lentivirus to adhere to the bottom of the wells. After removing the virus concentrate and washing the wells, activated T cells (1 × 10 6One ml of 1000 cells / ml of T cells was added to each well and centrifuged for 10 minutes at 1,000 × g and 32°C to allow the cells to attach to the lentivirus. The cells were then cultured for 48 hours in the presence of human IL-2 (300 IU / ml, Proleukin, Novartis). The lentiviral-transduced T cells were washed twice and expanded for 3–6 days in fresh culture medium containing human IL-2 (200 IU / ml). These cells were then used as CAR-T cells. Cell surface CAR protein expression was measured by staining CAR-T cells expanded 3–6 days after lentiviral transduction with CD19-Ck protein (a fusion protein of a self-produced CD19 extracellular domain and the human immunoglobulin kappa chain constant domain (Ck)) and an APC-labeled anti-Ck antibody (anti-Ck-APC, BioLegend) using flow cytometry (FACS-Canto II, BD Biosciences). The expression of CTC28 on the cell surface was confirmed by staining with a fluorescently labeled anti-CTLA4 antibody (L3D10, BioLegend). The results are shown in Figure 34.

[0216] Figure 33 is an image showing the structures of the human 4-1BB-based CAR (hCD19BBz) gene construct and the human CTC28 and CAR co-expression gene construct (hCD19BBz-CTC28).

[0217] Figure 34 is a graph analyzing the expression levels of CAR and CTC28 proteins on the surface of T cells (CD19-CTC28 CAR) into which human CAR (hCD19BBz) and human CTC28 were co-introduced.

[0218] As shown in Figure 34, among human CD19-CTC28 CD4 T cells, hCD19-Ck positive cells were 88.2% and CTC28 positive cells were 88.4%, and among human CD19-CTC28 CD8 T cells, hCD19-Ck positive cells were 53.5% and CTC28 positive cells were 57.6%, confirming good CAR and CTC28 expression.

[0219] 5.2. Anti-cancer efficacy of CAR-T cells loaded with human CTC28 - in vitro study To evaluate the in vitro anti-cancer efficacy of human CTC28-loaded CAR-T cells, the tumor-killing ability of the produced human CAR-T cells against human tumor cells, Raji cells, was analyzed.

[0220] Specifically, Raji cells (human B-cell lymphoma cell line) were purchased from ATCC (USA). The Raji cell line (Raji-Luc) transduced with the GFP-luciferase gene was prepared by transducing the Raji cells with a lentivirus (pLEF-LucGFP) for GFP-Luc expression, followed by isolating GFP-positive cells using a cell sorter (FACS Aria, Becton Dickinson). To measure the tumor-killing ability of CAR-T cells, CAR-T cells (effector cells, 1.2 × 103 to 7.5 × 106) grown for 3 days after CAR lentivirus transduction were used. 5 cells / 100 μl / well) were added to Raji-Luc cells (target cells, 3 × 10 4 The Raji-Luc cells were added to CAR-T cells (50 μl / well) at various ratios (0.04 to 25:1) and co-cultured overnight in a 96-well plate. 50 μl of D-luciferin (600 μg / ml, Promega) was then added and incubated at 37°C for 10 minutes to induce luciferase activity in the surviving Raji-Luc cells. The luminescence of these cells was measured using a luminometer (Tecan), and the tumor cell viability was calculated by comparing it with the luminescence of Raji-Luc cells not treated with CAR-T cells. The tumor-killing ability of CAR-T cells was measured, and the results are shown in Figure 35.

[0221] To evaluate the cytokine production ability of CAR-transduced CD4 and CD8 T cells, the amounts of IFN-γ, IL-2, and TNF-α secreted when CAR-transduced CD4 and CD8 T cells were cocultured with Raji cells were measured using the same method as in Example 3.1 above, and the results are shown in Figure 36.

[0222] FIG. 35 is a graph showing the analysis of the tumor-killing ability of T cells transfected with human hCD19BBz (CD19) and hCD19BBz-CTC28 (CD19-CTC28) against Raji cells.

[0223] Figure 36 is a graph showing the amounts of IFN-γ, IL-2, and TNF-α secreted by T cells transfected with human hCD19BBz (CD19) and hCD19BBz-CTC28 (CD19-CTC28) when cocultured with Raji cells.

[0224] As shown in Figure 35, the increase in the tumor-killing ability of CAR-T cells by human CTC28 was not significantly observed in both CD4 and CD8 CAR-T cells.

[0225] However, as shown in Figure 36, it was confirmed that the cytokine production ability of human CD4 and CD8 CAR-T cells was significantly increased by CTC28 transfection.

[0226] 5.3. Anti-cancer efficacy of human CTC28-loaded CAR-T cells – in vivo study The in vivo efficacy of human CAR-T cells is typically assessed by inoculating immunodeficient mice (e.g., NSG mice) with human tumor cells, followed by administration of human CAR-T cells to confirm that tumor growth is inhibited.

[0227] Therefore, to evaluate the in vivo anticancer efficacy of combined administration of human CTC28-loaded CD4 and CD8 CAR-T cells, CAR-T cells were intravenously injected into NSG mice subcutaneously inoculated with Raji cells, and the time course of tumor growth and changes in the number of CD4 and CD8 CAR-T cells in the blood were measured.

[0228] Specifically, NSG mice were inoculated with Raji-Luc cells (5 × 10 per mouse). 5 Seven days later, CD4 CAR-T cells and CD8 CAR-T cells expanded for 7–10 days after CAR lentiviral transduction were mixed at a 1:1 ratio and injected intravenously (1 × 10 per mouse). 6 cells or 5 × 10 6 Tumor size was then measured twice weekly using a caliper, and the number of CAR-T cells in the peripheral blood was measured weekly. Viable CD4 or CD8 CAR-T cells in the blood were stained with anti-CD3ε antibody (UCHT1, BioLegend), anti-CD4 antibody (SK3, BioLegend), anti-CD8 antibody (SK1, BioLegend), anti-FMC63 scFv antibody (FM3-HPY53, Acrobio), and 7-AAD (BioLegend), and the number of CD3(+)CD4(+)CAR(+)7AAD(-) or CD3(+)CD8(+)CAR(+)7AAD(-) cells was calculated by flow cytometry using counting beads (123count eBeads, ThermoFisher). The results are shown in Figures 37 and 38.

[0229] Figure 37 is a graph showing the change in tumor size in mice injected with T cells transfected with human hCD19BBz (CD19) and hCD19BBz-CTC28 (CD19-CTC28) (tumor size = long diameter 2 × Short diameter / 2).

[0230] Figure 38 is a graph measuring the number of CD4 and CD8 CAR-T cells in mice transfected with human hCD19BBz (CD19) and hCD19BBz-CTC28 (CD19-CTC28) in the NSG immunodeficient mouse model.

[0231] As shown in Figure 37, it was confirmed that CTC28-loaded CAR-T cells exhibited a significant tumor-suppressing effect compared to general CAR-T cells.

[0232] As shown in Figure 38, a significant numerical increase in CTC28 CAR-T cells in the blood was also observed.

[0233] This demonstrated the ability of CTC28 to enhance CAR-T cell function in a human CAR-T cell system, confirming its potential for future application in CAR-T cell therapy with enhanced function.

[0234] Based on these results, CTC28 CAR-T cell therapy demonstrates significantly enhanced CAR-T cell functionality compared to existing CAR-T cell therapies, which may contribute significantly to improving the efficacy of CAR-T cell therapy.

[0235] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential characteristics of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and not limiting.

Claims

1. (i) a first gene encoding a fusion protein (CTLA4-CD28 fusion protein) containing a CTLA4 (Cytotoxic T Lymphocyte Antigen-4) protein or a domain thereof and CD28 or a domain thereof; (ii) a second gene encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain, an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain; and A construct comprising:

2. The construct of claim 1, wherein the CTLA4-CD28 fusion protein comprises the extracellular domain of CTLA4 and the intracellular domain of CD28.

3. The construct of claim 1 or 2, wherein the CTLA4-CD28 fusion protein comprises the extracellular domain of CTLA4, the transmembrane domain of CTLA4, and the intracellular domain of CD28, or the extracellular domain of CTLA4, the transmembrane domain of CD28, and the intracellular domain of CD28.

4. The construct of any one of claims 1 to 3, wherein the CTLA4 comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; or an amino acid sequence having at least 90% identity thereto.

5. The construct of any one of claims 1 to 4, wherein the CD28 comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4; or an amino acid sequence having at least 90% identity thereto.

6. The construct of any one of claims 3 to 5, wherein the CTLA4 extracellular domain-CTLA4 transmembrane domain-CD28 intracellular domain comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6; or an amino acid sequence having at least 90% identity thereto.

7. The construct of any one of claims 3 to 5, wherein the CTLA4 extracellular domain-CD28 transmembrane domain-CD28 intracellular domain comprises the amino acid sequence of SEQ ID NO: 7; or an amino acid sequence having at least 90% identity thereto.

8. The chimeric antigen receptor is (a) a chimeric antigen receptor comprising an antigen-binding domain, an extracellular binding domain, a transmembrane domain, and a CD3 zeta intracellular signaling domain; or (b) the construct according to any one of claims 1 to 7, which is a chimeric antigen receptor comprising an antigen-binding domain, an extracellular binding domain, a transmembrane domain, a 4-1BB intracellular domain, and a CD3zeta intracellular signaling domain.

9. 9. The construct of claim 8, wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO:9, SEQ ID NO:13, or SEQ ID NO:15; or an amino acid sequence having at least 90% identity thereto.

10. The construct of any one of claims 1 to 9, wherein the antigen-binding domain comprises an antibody or antigen-binding fragment thereof that specifically binds to one or more antigens selected from the group consisting of: 4-1BB, BCMA, BAFF, B7-H3, B7-H6, CA9, CTAG1B, CEA, cyclin, cyclin A2, cyclin B1, CCL-1, CCR4, CD3, CD4, CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44 v7 / 8, CD52, CD58, CD62, CD79A, CD79B, CD80, CD123, CD133, CD138, CD171, CSPG4, CLDN18 , CLDN18.2, CLDN6, CTLA-4, c-Met, DLL3, EGFR, tEGFR, EGFRvIII, EPG-2, EPG-40, EphrinB2 , EPHA2, estrogen receptor, Fc receptor, FCRL5, FGF23, FBP, FOLR1, FOLR2, GD2, ganglioside GD3, gp100, GPC3, GPCR5D, GM-CSF, Her2 / neu, Her3, Her4, erbB dimer, HMW-MAA, HBsAg, HLA-A1, HLA-A 2, IL-22Ra, IL-13Ra2, ICOS, IGF-1 receptor, integrin αvβ6, interferon receptor, IFNγ, IL-2R, IL-4R, IL-5R, IL-6R, IL-17RA, IL-31R, IL-36R, kdr, L1-CAM, CE7 epitope of L1-CAM, LRRC8A, Lewis Y, LAG3, MAGEAl, MAGEA3, MAGEA6, MAGEAlO, MSLN, CMV, MUC1, NKG2D ligand, MART-1, NGF, NCAM, NRP-1, NRP-2, carcinoembryonic antigen, PD-L1, PRAME, progesterone receptor, prostate-specific antigen, PSCA, PSMA, RANKL, ROR1, SLAMF7, survivin, TPBG, TAG72, TRP1, TRP2, and Wilms' tumor 1 (WT1).

11. The antigen-binding domain is an antibody, an antigen-binding fragment thereof, a ligand protein that binds to an antigen, or a domain thereof, and the antigen-binding fragment is a single chain variable fragment (scFv) of an antibody, (scFv) ２ , Fv, Fab, Fab', F(ab') ２ 11. The construct of any one of claims 1 to 10, which is a polypeptide, a polypeptide chain, a polypeptide fragment ...

12. The construct of any one of claims 1 to 11, wherein the chimeric antigen receptor further comprises a signal peptide at the N-terminus of the antigen-binding domain.

13. The construct according to any one of claims 1 to 12, wherein the first gene encoding the CTLA4-CD28 fusion protein and the second gene encoding the chimeric antigen receptor are linked to each other via a 2A peptide sequence or an IRES sequence, or the first gene and the second gene are each operably linked to an independent promoter.

14. 14. The construct of any one of claims 1 to 13, wherein the construct comprises the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 21, or SEQ ID NO: 23; or an amino acid sequence having at least 90% identity thereto.

15. A vector comprising the construct of any one of claims 1 to 14.

16. The vector of claim 15 , wherein the vector is a viral vector or a non-viral vector.

17. 17. The vector of claim 16, wherein the viral vector is any one selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus, and vaccinia virus.

18. An immune cell introduced with the construct according to any one of claims 1 to 14 or a vector containing said construct.

19. An immune cell comprising a protein expressed from the construct of any one of claims 1 to 14 or a vector comprising said construct.

20. (i) a first gene encoding a fusion protein (CTLA4-CD28 fusion protein) comprising a CTLA4 protein or a domain thereof and CD28 or a domain thereof; (ii) a second gene encoding a chimeric antigen receptor comprising an antigen-binding domain, an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain; and immune cells, including

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

22. The immune cell according to any one of claims 18 to 21, wherein the immune cell is a T cell, a NK cell, a NKT cell, a macrophage, or a combination thereof.

23. The immune cell according to any one of claims 18 to 22, wherein the immune cell is a CD4 T cell, a CD8 T cell, or a combination thereof.

24. A composition comprising the construct according to any one of claims 1 to 14, a vector comprising the construct, an immune cell into which the construct or the vector has been introduced, or the immune cell according to any one of claims 18 to 23.

25. A pharmaceutical composition for cancer treatment, comprising the construct according to any one of claims 1 to 14, a vector comprising said construct, an immune cell into which said construct or said vector has been introduced, or the immune cell according to any one of claims 18 to 23, and a pharmaceutically acceptable carrier.

26. (i) a first gene encoding a fusion protein (CTLA4-CD28 fusion protein) comprising a CTLA4 protein or a domain thereof and CD28 or a domain thereof; (ii) a second gene encoding a chimeric antigen receptor comprising an antigen-binding domain, an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain; and A method for producing immune cells that express a CTLA4-CD28 fusion protein and a chimeric antigen receptor, comprising the step of introducing the above into immune cells.

27. The method according to claim 26, wherein the step comprises introducing a construct according to any one of claims 1 to 14 or a vector containing the construct into immune cells.

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