Immune cells overexpressing externally introduced cell signaling regulators and uses thereof

Abstract

The present invention relates to immune cells overexpressing externally introduced cell signaling pathway modulators and uses thereof. As a specific example, immune cells expressing a fusion protein comprising a chimeric antigen receptor and a cell signaling pathway modulator perform an immune response by selecting target cancer cells through the chimeric antigen receptor expressed on the cell membrane. In this case, the cell signaling pathway modulator is overexpressed in the cytoplasm, thereby being able to modulate the activity of the immune cells. Accordingly, the fusion protein comprising a chimeric antigen receptor and a cell signaling pathway modulator of the present invention, and the immune cells overexpressing the cell signaling pathway modulator can be used for the treatment of cancer.

Description

Technical Field

[0001] This invention relates to engineered immune cells that overexpress cell signaling pathway regulators and their uses. Background Technology

[0002] Cancer is the second leading cause of illness and death worldwide. Commonly occurring cancers include breast cancer, lung and bronchial cancer, prostate cancer, colorectal cancer, bladder cancer, melanoma, non-Hodgkin's lymphoma, thyroid cancer, kidney and renal pelvis cancer, endometrial cancer, leukemia, and pancreatic cancer. Various methods have been tried to treat cancer, such as surgery, radiation therapy, and chemotherapy. However, due to reported side effects, immunotherapy utilizing the patient's immune function has been developed in recent years. Research is actively underway on treatments involving the expression of chimeric antigen receptors (CARs) or T cell receptors that can recognize specific cancer cells in immune cells via viral vectors, followed by transplantation into patients with cancer (CAR-T-based / TCR T-based adoptive cell therapy).

[0003] Specifically, chimeric antigen receptors (CARs) consist of antibody fragments, hinge regions, transmembrane domains, and intracellular signaling domains. For example, CAR-T cells (T cells expressing chimeric antigen receptors) or CAR-NK cells (natural killer cells expressing chimeric antigen receptors) are immune cell-specific recognition cells (such as cancer cells). The antibody fragments recognize cells expressing target molecules to activate the cytotoxicity of the immune cells, thereby inducing cell death in the target cells. Therefore, immune cells expressing chimeric antigen receptors are used in genetically engineered cell therapy. Specifically, chimeric antigen receptor T cells have been reported to exhibit very high therapeutic efficacy against CD19-expressing hematologic malignancies.

[0004] However, unlike hematologic malignancies, in the case of solid tumors, it is not easy for gene-transmitted immune cells, such as CAR-T cells, to migrate to the tumor tissue where cancer cells are growing. Furthermore, even if immune cells infiltrate the tumor tissue, the immune response of CAR-T cells is reduced due to several types of immune checkpoint proteins and various cytokines expressed in cancer cells or surrounding cells, thus it has not shown therapeutic efficacy in solid tumors to date.

[0005] TGF-β is known to be a representative cytokine that suppresses the CAR-T cell immune response. The TGF-β signaling pathway is mediated by two types of receptors: TGF-βR1 and TGF-βR2, both located on the cell membrane. First, when TGF-β binds to TGF-βR2, TGF-βR1 is phosphorylated and activated; one activated TGF-βR1 phosphorylates a SMAD protein. The phosphorylated SMAD protein forms a trimer and translocates to the nucleus to induce the expression of specific genes (Andres Rojas et al., Biochimica et Biophysica Acta 1793 (2009) 1165-1173). Therefore, much research is underway to overcome the problem of suppressing the CAR-T cell immune response, thereby generating immune cells with better activity. Summary of the Invention

[0006] Technical issues

[0007] Therefore, the inventors have researched and developed a method that not only prevents the inhibition of the activity of gene-introduced immune cells but also increases the cellular activity of gene-introduced immune cells. This experiment was conducted on CAR-T cells, a representative example of gene-introduced immune cells. Thus, by designing a mechanism that not only prevents the inhibition of CAR-T cells but also increases their cellular activity, the inventors have completed this invention.

[0008] Solution to the problem

[0009] To address the aforementioned problems, one aspect of the present invention provides a polynucleotide encoding a fusion protein, said fusion protein comprising (i) an antigen-binding domain; (ii) a transmembrane domain; (iii) an intracellular signal transduction domain comprising at least one co-stimulatory domain; (iv) a self-cleaving peptide; and (v) a signaling pathway regulator.

[0010] Another aspect of the invention provides polynucleotides comprising (i) polynucleotides encoding an antigen-binding domain; (ii) polynucleotides encoding a transmembrane domain; (iii) polynucleotides encoding an intracellular signal transduction domain comprising at least one co-stimulatory domain; (iv) polynucleotides encoding an IRES (Internal Ribosome Entry Site); and (v) polynucleotides encoding a signaling pathway regulator.

[0011] Another aspect of the invention provides a vector comprising a polynucleotide encoding a fusion protein, said fusion protein comprising (i) an antigen-binding domain; (ii) a transmembrane domain; (iii) an intracellular signal transduction domain comprising at least one co-stimulatory domain; and (iv) a signaling pathway modulator.

[0012] Another aspect of the present invention provides immune cells expressing chimeric antigen receptors, characterized in that the immune cells express a fusion protein, the fusion protein comprising (i) an antigen-binding domain; (ii) a transmembrane domain; (iii) an intracellular signal transduction domain comprising at least one co-stimulatory domain, and overexpressing an externally introduced signaling pathway regulator.

[0013] Another aspect of the present invention provides immune cells expressing externally introduced signaling pathway modulators.

[0014] Effects of the present invention

[0015] This invention engineered immune cells that overexpress chimeric antigen receptors and externally introduced signaling pathway modulators to specifically trigger an immune response against specific cancer cells via chimeric antigen receptors expressed on the cell membrane. Furthermore, since the externally introduced signaling pathway modulators are overexpressed in the cytoplasm of immune cells, it is possible to attenuate arbitrary specific immunosuppressive signaling pathways. In addition, it is possible to enhance the overall ability to kill tumor cells by modulating the activity of immune cells. Therefore, the immune cells engineered to overexpress chimeric antigen receptors and the externally introduced signaling pathway modulators of this invention can be effectively used to treat cancer. Attached Figure Description

[0016] Figure 1 This is a view showing the domain structure of the 19bbz CAR experimental group constructed in one implementation scheme.

[0017] Figure 2 This is a view showing the domain structure of the 19bbz CAR experimental group constructed in one implementation scheme.

[0018] Figure 3 This is a view showing the domain structure of the Hbbz CAR experimental group constructed in one implementation scheme.

[0019] Figure 4 This is a view showing the domain structure of the 43bbz CAR experimental group constructed in one implementation scheme.

[0020] Figure 5 This is a view showing the domain structure of the 47bbz CAR experimental group constructed in one implementation scheme.

[0021] Figure 6This is a view showing the domain structure of the Pbbz-CAR experimental group constructed in one implementation scheme.

[0022] Figure 7 Images to compare the antitumor efficacy of CAR-T cell experimental groups constructed in one implementation scheme to evaluate antitumor therapeutic efficacy in animal models of hematologic malignancies (II).

[0023] Figure 8 A view showing the spectrum of the lentiviral vector expressing 19bbz used in one embodiment.

[0024] Figure 9 A view showing the spectrum of the lentiviral vector expressing 19bbz#F used in one implementation.

[0025] Figure 10 A view showing the spectrum of the lentiviral vector expressing 19bbz#M used in one embodiment.

[0026] Figure 11 A view showing the spectrum of the lentiviral vector expressing 19bbz#N used in one embodiment.

[0027] Figure 12 A view showing the spectrum of the lentiviral vector expressing 19bbz#C used in one implementation.

[0028] Figure 13 A view showing the spectrum of the lentiviral vector expressing 19bbz#S1 used in one embodiment.

[0029] Figure 14 A view showing the spectrum of the lentiviral vector expressing 19bbz#S2 used in one implementation.

[0030] Figure 15 A view showing the spectrum of the lentiviral vector expressing 19bbz#TC used in one implementation.

[0031] Figure 16 A view showing the spectrum of the lentiviral vector expressing 19bbz#RG used in one implementation.

[0032] Figure 17 A view showing the spectrum of the lentiviral vector expressing 19bbzT used in one embodiment.

[0033] Figure 18 A view showing the spectrum of the lentiviral vector expressing 19bbz#FCS2 used in one implementation.

[0034] Figure 19A view showing the spectrum of the lentiviral vector expressing Hbbz used in one implementation.

[0035] Figure 20 A view showing the spectrum of the lentiviral vector expressing Hbbz#F used in one implementation.

[0036] Figure 21 A view showing the spectrum of the lentiviral vector expressing Hbbz#C used in one implementation.

[0037] Figure 22 A view showing the spectrum of the lentiviral vector expressing Hbbz#S2 used in one implementation.

[0038] Figure 23 A view showing the spectrum of the lentiviral vector expressing Hbbz#FCS2 used in one implementation.

[0039] Figure 24 A view showing the spectrum of a lentiviral vector expressing 43bbz used in one embodiment.

[0040] Figure 25 A view showing the spectrum of the lentiviral vector expressing 43bbz#F used in one implementation.

[0041] Figure 26 A view showing the spectrum of the lentiviral vector expressing 43bbz#C used in one implementation.

[0042] Figure 27 A view showing the spectrum of the lentiviral vector expressing 43bbz#S2 used in one embodiment.

[0043] Figure 28 A view showing the spectrum of the lentiviral vector expressing 43bbz#FCS2 used in one implementation.

[0044] Figure 29 A view showing the spectrum of a lentiviral vector expressing 47bbz used in one embodiment.

[0045] Figure 30 A view showing the spectrum of the lentiviral vector expressing 47bbz#F used in one implementation.

[0046] Figure 31 A view showing the spectrum of the lentiviral vector expressing 47bbz#C used in one embodiment.

[0047] Figure 32A view showing the spectrum of the lentiviral vector expressing 47bbz#S2 used in one embodiment.

[0048] Figure 33 A view showing the spectrum of the lentiviral vector expressing 47bbz#FCS2 used in one implementation.

[0049] Figure 34 A view showing the spectrum of the lentiviral vector expressing Pbbz used in one implementation.

[0050] Figure 35 A view showing the spectrum of the lentiviral vector expressing Pbbz#F used in one implementation.

[0051] Figure 36 A view showing the spectrum of the lentiviral vector expressing Pbbz#C used in one implementation.

[0052] Figure 37 A view showing the spectrum of the lentiviral vector expressing Pbbz#S2 used in one implementation.

[0053] Figure 38 A view showing the spectrum of the lentiviral vector expressing Pbbz#FCS2 used in one implementation.

[0054] Figure 39 A view showing the spectrum of lentiviral vectors expressing HERV-E-specific TCRs used in one embodiment.

[0055] Figure 40 A view showing the spectrum of a lentiviral vector expressing HERV-E-specific TCR#F used in one embodiment.

[0056] Figure 41 A view showing the spectrum of a lentiviral vector expressing HERV-E-specific TCR#C used in one embodiment.

[0057] Figure 42 A view showing the spectrum of a lentiviral vector expressing HERV-E-specific TCR#S2 used in one embodiment.

[0058] Figure 43 A view showing the spectrum of a lentiviral vector expressing HERV-E-specific TCR#FCS2 used in one embodiment.

[0059] Figure 44 A view showing the spectrum of a lentiviral vector expressing an NY-ESO-1 specific TCR used in one embodiment.

[0060] Figure 45 A view showing the spectrum of a lentiviral vector expressing NY-ESO-1 specific TCR#F used in one embodiment.

[0061] Figure 46 A view showing the spectrum of a lentiviral vector expressing NY-ESO-1 specific TCR#C used in one embodiment.

[0062] Figure 47 A view showing the spectrum of a lentiviral vector expressing NY-ESO-1 specific TCR#S2 used in one embodiment.

[0063] Figure 48 A view showing the spectrum of a lentiviral vector expressing NY-ESO-1 specific TCR#FCS2 used in one embodiment.

[0064] Figure 49 A table summarizing the CAR-T experimental groups used in one implementation scheme to evaluate the efficacy (I) of antitumor therapy in animal models of hematologic malignancies.

[0065] Figure 50 Images for comparing the antitumor efficacy of constructed CAR-T cell experimental groups in one implementation scheme to evaluate the antitumor therapeutic efficacy in animal models of hematologic malignancies (I).

[0066] Figure 51 A view showing the co-crystal structure of the type 1 receptor kinase (Kinase) domain of TGF-β that binds to FKBP12.

[0067] Figure 52 To show the view of the tetrad aromatic amino acids (aromatic residues) of FKBP12, which participate in the binding of FKBP12 to the type 1 receptor of TGF-β in a pivotal manner.

[0068] Figure 53 A view showing how aromatic amino acids of FKBP12 participate in the binding of FKBP12 to the TGF-1 receptor via a pivotal mechanism.

[0069] Figure 54 This was to confirm a flow cytometry view of the expression of CD19-specific CAR in CAR-T cells constructed in one implementation scheme.

[0070] Figure 55 These are immunofluorescence microscopy images revealing the characteristics of surface CAR expression in CD19-specific CAR-T cells constructed in one embodiment.

[0071] Figure 56 Images obtained using immunofluorescence microscopy show the pairing of CAR-T cells with target tumor cells in one implementation scheme.

[0072] Figure 57 A view of flow cytometry analysis in CAR(+) cell phylogenetics to evaluate a subset of T cells from CD19-specific CAR-T cells expanded in vitro in one implementation.

[0073] Figure 58 A flow cytometry analysis of CD19 expression in K562, K562-CD19, and Daudi cells.

[0074] Figure 59 A Western blot image showing protein expression of the N-terminal SH2 domain of FKBP12, cyclophilin A, and SHP2 proteins in CAR-T cells constructed in one embodiment.

[0075] Figure 60 A table illustrating RNA expression of the N-terminal SH2 domains of FKBP12, cyclophilin A, and SHP2 proteins in CAR-T cells constructed in one embodiment. RNA expression was quantified by real-time polymerase chain reaction using cDNA derived from the same amount of total RNA.

[0076] Figure 61 A diagram showing the amount of IFNγ released by CD19-specific CAR-T cells upon antigen stimulation.

[0077] Figure 62 A graph showing the amount of TNFα released by CD19-specific CAR-T cells upon antigen stimulation.

[0078] Figure 63 A graph showing the amount of IL-2 released by CD19-specific CAR-T cells upon antigen stimulation.

[0079] Figure 64 A graph showing the amount of IFNγ released by CD19-specific CAR-T cells upon antigen stimulation in the presence of TGF-β1 in one embodiment.

[0080] Figure 65 A graph showing the amount of TNFα released by CD19-specific CAR-T cells upon antigen stimulation in the presence of TGF-β1 in one embodiment.

[0081] Figure 66 This figure shows the results of an in vitro cell migration assay performed after activating CD19-specific CAR-T cells with anti-CD3 / anti-CD28 beads.

[0082] Figure 67 Images showing the intrinsic motility of CAR-T cells cultured in the presence of antigen stimulation in one embodiment.

[0083] Figure 68 The figure shows the results of CAR-T-mediated tumor cell lysis assays, measured against K562-CD19 cells (bottom) and Daui-Fluc-eGFP cells (top), which are target cells for CD19-specific CAR-T cells.

[0084] Figure 69 The flow cytometry analysis confirmed CAR expression in Her2-specific CAR-T cells constructed in one implementation scheme. Specifically, the expression of Her2-specific CARs in T cells transduced with Hbbz, Hbbz#F, Hbbz#C, Hbbz#S2, or Hbbz#FCS2 lentiviruses was analyzed by flow cytometry.

[0085] Figure 70 These are immunofluorescence microscopy images revealing the characteristics of surface CAR expression in Her2-specific CAR-T cells constructed in one embodiment.

[0086] Figure 71 A diagram showing the amount of IFNγ released by Her2-specific CAR-T cells upon antigen stimulation.

[0087] Figure 72 A graph showing the amount of TNFα released by Her2-specific CAR-T cells upon antigen stimulation.

[0088] Figure 73 A graph showing the amount of IL-2 released by Her2-specific CAR-T cells upon antigen stimulation.

[0089] Figure 74 This figure shows the results of an in vitro cell migration assay performed after Her2-specific CAR-T cells were activated with anti-CD3 / anti-CD28 beads.

[0090] Figure 75 A graph showing the results of CART-mediated tumor cell lysis assays performed on SKBR3-Luc cells, which are target cells for Her2-specific CAR-T cells.

[0091] Figure 76 Images comparing the antitumor efficacy of Her2-specific CAR-T cells used in one implementation scheme.

[0092] Figure 77The flow cytometry analysis confirmed the expression of PSMA-specific CARs in CAR-T cells constructed in one embodiment.

[0093] Figure 78 These are immunofluorescence microscopy images revealing the characteristics of surface CAR expression in PSMA-specific CAR-T cells constructed in one embodiment.

[0094] Figure 79 This is a flow cytometry analysis of CAR(+) cell phylogenetics, used to assess the T cell subsets of PSMA-specific CAR-T cells expanded in vitro.

[0095] Figure 80 This is a flow cytometry view of CAR(+) cells used to assess T cell exhaustion in in vitro expanded PSMA-specific CAR-T cells.

[0096] Figure 81 This figure shows the results of an in vitro cell migration assay performed after PSMA-specific CAR-T cells were activated with anti-CD3 / anti-CD28 beads.

[0097] Figure 82 and Figure 83 A graph showing the results of CAR T-mediated tumor cell lysis assays on target tumor cells against PSMA-specific CAR-T cells. Detailed Implementation

[0098] The present invention will now be described in detail.

[0099] Polynucleotides containing self-cleaving peptides

[0100] One aspect of the invention provides a polynucleotide encoding a fusion protein comprising (i) an antigen-binding domain; (ii) a transmembrane domain; (iii) an intracellular signaling domain comprising at least one co-stimulatory domain; (iv) a self-cleaving peptide; and (v) a signaling pathway regulator.

[0101] In this case, a spacer region can also be provided between (i) the antigen-binding domain and (ii) the transmembrane domain.

[0102] In this case, polynucleotides can specifically take the following forms:

[0103] (a) a polynucleotide, including a polynucleotide encoding a fusion protein comprising (i) an antigen-binding domain, (ii) a spacer region and a transmembrane domain, and (iii) an intracellular signaling domain comprising at least one co-stimulatory domain; (iv) a polynucleotide encoding a self-cleaving peptide; and (v) a signaling pathway regulator; or

[0104] (b) Polynucleotides, including (i) polynucleotides encoding antigen-binding domains; (ii) polynucleotides encoding spacer regions and transmembrane domains; (iii) polynucleotides encoding intracellular signaling domains containing at least one co-stimulatory domain; (iv) polynucleotides encoding self-cleaving peptides; and (v) polynucleotides encoding signaling pathway regulators.

[0105] In this configuration, (i) the polynucleotide encoding the antigen-binding domain; (ii) the polynucleotide encoding the transmembrane domain; (iii) the polynucleotide encoding the intracellular signaling domain containing at least one co-stimulatory domain; (iv) the polynucleotide encoding the self-cleaving peptide; and (v) the polynucleotide encoding the signaling pathway regulator can be sequentially linked in a 5′ to 3′ order. However, the order can be appropriately adjusted as long as the signaling pathway regulator can be expressed in the cytoplasm. For example, the signaling pathway regulator can be located upstream of the antigen-binding domain. In this case, the self-cleaving sequence can be located downstream of the signaling pathway regulator.

[0106] Externally introduced signaling pathway modulators

[0107] In addition, signaling pathway regulators may be selected from any of the following groups: a) proteins located in immunosuppressive signaling pathways, b) immunoprophylactic proteins or fragments thereof, c) proteins involved in antigen loss-mediated relapse, d) proteins located in T cell stimulation signaling pathways, e) proteins involved in suppressing negative feedback, and f) combinations thereof.

[0108] In this context, signaling pathway regulators are characterized by their function in the cytoplasm.

[0109] Proteins located in the immunosuppressive signaling pathway: TGF-β / SMAD signaling pathway

[0110] Proteins located in immunosuppressive signaling pathways can be proteins or fragments thereof located in the TGF-β / SMAD (FKBP12-FK506 / rapamycin, FKBP12-FK506 / rapamycin) signaling pathway. Specifically, proteins located in the TGF-β / SMAD (FKBP12-FK506 / rapamycin) signaling pathway can be selected from FKBP12 (FK506-binding protein 12, hereinafter, FKBP12 after the self-cleaved peptide is referred to as #F, SEQ ID NO:13), the C-terminal MH2 domain of SMAD4 protein (hereinafter, the C-terminal MH2 domain of SMAD4 protein after the self-cleaved peptide is referred to as #M, SEQ ID NO:20), and the N-terminal domain of SKI protein (N-SKI, hereinafter, N-SKI after the self-cleaved peptide is referred to as #N, SEQ ID NO:22).

[0111] As used in this article, the term "transforming growth factor β (TGF-β)" is a member of the transforming growth factor β superfamily, referring to cytokines that perform various cellular functions, including cell growth, differentiation, cell death, and development.

[0112] As used herein, the term "TGF-β / SMAD signaling pathway" refers to a signaling pathway activated by TGF-β. During TGF-β signaling, ligand binding promotes the formation of heterotetramers composed of TGF-β type 1 and type 2 receptors, and the TGF-β type 1 receptor is phosphorylated by the TGF-β type 2 receptor. Once the TGF-β type 1 receptor is phosphorylated, the cytoplasmic signaling protein SMAD is phosphorylated, enabling subsequent signal transduction. Phosphorylated SMAD forms SMAD trimers via its C-terminal domain, which then translocate to the nucleus, where they bind transcription factors / cofactors and induce up / down regulation of specific target genes. TGF-β / SMAD signaling in T cells leads to a significant reduction in cytokine production, cell proliferation, and various immune-related functions. Specifically, proteins located in the TGF-β / SMAD signaling pathway may include FKBP12, the C-terminal MH2 domain of SMAD4, and N-SKI, among others.

[0113] As used herein, the term "FKBP12" refers to a member of the FK506-binding protein family with a molecular weight of 12 kDa, involved in the regulation of various cellular activities, such as protein folding and transport, as well as immune regulation. Specifically, FKBP12 binds to the type 1 receptor of TGF-β and spatially blocks the phosphorylation of the type 1 receptor of TGF-β mediated by the type 2 receptor of TGF-β, thereby preventing TGF-β-mediated signaling. Interestingly, it is known that even in the absence of TGF-β, the type 1 receptor of TGF-β can form a complex with the type 2 receptor of TGF-β to some extent through its innate affinity. Therefore, this leaky signaling can occur at any time, regardless of the presence or absence of TGF-β. FKBP12, which can inhibit the phosphorylation of the type 1 receptor of TGF-β, is therefore considered a "molecular guardian that blocks leaky signaling of the type 1 receptor of TGF-β." On the other hand, two well-known immunosuppressive drugs, rapamycin and FK506, have been found to bind to FKBP12, thereby competitively interfering with the binding of FKBP12 to the type 1 receptor of TGF-β. Therefore, rapamycin and FK506 promote immunosuppressive signaling that relies on phosphorylation of the type 1 receptor of TGF-β.

[0114] Furthermore, the FKBP12 and FK506 complex, acting as an immunosuppressive drug, inhibits calcineurin activity by binding to it. Calcineurin is a form of calcium (Ca) 2+ T-cell-dependent protein phosphatases activate inflammatory immune responses by mediating the dephosphorylation of NFAT (nuclear factor of activated T cells). NFAT is a transcription factor expressed in most immune cells. In particular, NFAT enhances T cell-mediated immune responses by upregulating the transcription of interleukin-2 (IL-2) in T cells.

[0115] FKBP12 may be derived from human and may include the amino acid sequence disclosed in the NCBI reference sequence: NP_000792.1 or NP_001186715.1. Alternatively, FKBP12 may include the amino acid sequence disclosed in NPU004107 (FKBP12.6).

[0116] Furthermore, FKBP12 may comprise the amino acid sequence shown in SEQ ID NO:13 or 19. Additionally, FKBP12 may have approximately 80%, 90%, 95%, or 99% or higher homology with the amino acid sequence shown in SEQ ID NO:13 or 19. Furthermore, the nucleotide sequence encoding FKBP12 may be the nucleotide sequence shown in SEQ ID NO:14. Furthermore, the nucleotide sequence encoding FKBP12 may have approximately 95%, 97%, or 99% or higher homology with the nucleotide sequence shown in SEQ ID NO:14. Furthermore, the fragment of FKBP12 may comprise amino acids from amino acid 27 to amino acid 100 of SEQ ID NO:13. Figure 52 As shown, amino acids 27, 47, 60, and 100 of SEQ ID NO:13 are amino acids involved in TGF-β receptor binding.

[0117] As used herein, the term "SMAD" refers to a key protein transduction signal derived from the TGF-β superfamily receptors and is involved in cell growth, differentiation, cell death, and development. There are three types of SMADs: receptor-regulated SMADs (R-SMADs), common partner SMADs (Co-SMADs), and inhibitory SMADs (I-SMADs). A trimer composed of two receptor-regulated SMADs and one common partner SMAD acts as a transcription factor regulating the expression of specific genes. Receptor-regulated SMADs include SMAD1, SMAD2, SMAD3, SMAD5, and SMAD8 / 9; common partner SMADs include SMAD4; and inhibitory SMADs include SMAD6 and SMAD7. The receptor-regulated / common partner SMAD complex is primarily located in the cytoplasm, but upon receiving TGF-β signals, it accumulates and acts in the nucleus, while I-SMADs are primarily located in the nucleus and act as transcription regulators.

[0118] As used herein, the term "SMAD4" refers to the common pathway receptor as described above, which forms a complex with receptor-regulated SMAD to support the function of receptor-regulated SMAD. On the other hand, SMAD4 mediates the reduction of TGF-β-dependent c-myc expression in T cells, T cell proliferation, and T cell immune responses. "c-myc" is a proto-oncogene, a transcription factor that regulates cell proliferation and growth. Specifically, SMAD4 can be a protein having the amino acid sequence disclosed in NP_005350. The SMAD4 fragment used in one embodiment of the invention (the C-terminal MH2 domain of the SMAD4 protein) is a domain involved in "SMAD trimerization." Therefore, when this fragment is overexpressed in immune cells, the immunosuppressive TGF-β signaling is blocked due to the lack of DNA-binding activity, preventing the formation of a non-functional SMAD complex as a transcription factor, thereby increasing overall immune responsiveness. This SMAD4 fragment may consist of the amino acid sequence shown in SEQ ID NO:20. Furthermore, the SMAD4 fragment may have approximately 80%, 90%, 95%, or 99% or higher homology with the amino acid sequence shown in SEQ ID NO:20.

[0119] As used herein, the term "SKI" refers to a proto-oncogene product that acts as a transcriptional regulatory protein, enhancing the transcription of specific genes in the cell nucleus. One of the known activities of SKI proteins is to block TGF-β signaling by inhibiting the formation of functional SMAD trimers. In one embodiment, an N-SKI is used to participate in the function of a SKI protein that blocks TGF-β signaling. Specifically, the N-terminal region of the SKI protein binds directly to SMAD and inhibits the action of TGF-β by disrupting SMAD-mediated transcriptional regulation of target genes. In this case, the N-terminal region of the SKI protein that binds to SMAD can be referred to as an N-SKI. The N-SKI binds to the trimerization domain of the SMAD protein and interferes with the formation of functional SMAD trimers. Therefore, when this fragment is overexpressed in immune cells, immunosuppressive TGF-β signaling is blocked, thereby increasing overall immune responsiveness. Specifically, the SKI can be a protein comprising the amino acid sequence disclosed in NP_003027. In one embodiment, the fragment of the N-SKI used can be the amino acid sequence shown in SEQ ID NO:22. In addition, the N-SKI fragment may have approximately 80%, 90%, 95%, or 99% or higher homology with the amino acid sequence shown in SEQ ID NO:22.

[0120] Proteins located in the immunosuppressive signaling pathway: Proteins located in the immune checkpoint pathway

[0121] Proteins located in immunosuppressive signaling pathways can be proteins or fragments thereof located in the inhibitory immune checkpoint pathway. Specifically, proteins located in the inhibitory immune checkpoint pathway can be the N-terminal SH2 (N-SH2) domain of SHP-1 protein (Src homology 2 domain-containing phosphatase 1, hereinafter referred to as #S1, SEQ ID NO:26) or the N-SH2 domain of SHP-2 protein (hereinafter referred to as #S2, SEQ ID NO:28).

[0122] As used in this article, the term "inhibitory immune checkpoint pathway" refers to an intracellular signaling pathway that induces immune tolerance or inhibits immune stimulation signaling pathways. Proteins involved in the inhibitory immune checkpoint pathway in T cells include programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4).

[0123] Specifically, the binding of PD-1 to its ligands PD-L1 (PD-1 ligand) or PD-L2 leads to phosphorylation of ITIM (immunoreceptor tyrosine-based inhibitory motif) and ITSM (immunoreceptor tyrosine-based switch motif) in the PD-1 cytoplasmic domain, and subsequently to activation of SHP-2 / SHP-1 and inactivation of immune stimulation signaling in T cells. CTLA-4 binds to CD80 / 86 to inhibit CD28 signaling and activates SHP-2 via the YVKM motif in its cytoplasmic domain to inhibit RAS, thereby suppressing immune stimulation signaling in T cells.

[0124] As used herein, the term "SHP-1" refers to one of the PTP (protein tyrosine phosphatase) families, specifically PTPN6 (tyrosine-protein phosphatase non-receptor type 6). PTPs are signal transduction molecules that regulate various cellular processes, including cell growth, differentiation, the mitotic cycle, and oncogenic transformation. SHP-1 comprises two tandem Src homologous regions (SH2, N-SH2, and C-SH2) involved in phosphotyrosine binding and a PTP domain (catalytic domain) near the C-terminus that possesses protein tyrosine phosphatase activity. The N-terminal SH2 (N-SH2) domain regulates SHP-1 activity by binding the PTP domain in an inactive state and inhibiting enzyme activity. However, when the Tyr residue of the N-SH2 domain is phosphorylated, the N-SH2 domain is released from the PTP domain, and the free PTP domain interacts with the substrate, thereby converting SHP-1 to a dephosphorylating state. Therefore, when the N-SH2 domain of the SHP-1 protein is overexpressed in immune cells, inhibitory immune checkpoint signals are blocked by binding to the PTP domain of the SHP-1 protein and remaining in an inactive state. Consequently, the overall immune activity of the corresponding immune cells increases. SHP-1 can be a protein comprising the amino acid sequence disclosed in NP_002822. In one embodiment, the SHP-1 fragment used can be the amino acid sequence shown in SEQ ID NO:26. Furthermore, the SHP-1 fragment may have approximately 80%, 90%, 95%, or 99% or higher homology to the amino acid sequence shown in SEQ ID NO:26.

[0125] As used herein, the term "SHP-2" refers to one of the PTP family members and is also known as PTPN11 (tyrosine-protein phosphatase non-receptor type 11), PTP-1D (protein-tyrosine phosphatase 1D), or PTP-2C (protein-tyrosine phosphatase 2C). Similar to SHP-1, SHP-2 also consists of two tandem Src homologous regions (SH2, N-SH2, and C-SH2) that act as the protein phosphotyrosine binding domain at the N-terminus and a PTP domain (catalytic domain) with phosphatase activity at the C-terminus. When SHP-2 is in an inactive state, the N-SH2 domain blocks the active site of the PTP domain of SHP-2. When the N-SH2 domain binds to a phosphotyrosine-containing peptide region, the PTP domain is released from the N-SH2 domain and becomes catalytically active. Therefore, when the N-SH2 domain of the SHP-2 protein is overexpressed in immune cells, inhibitory immune checkpoint signals are blocked by binding to the PTP domain of the SHP-2 protein and remaining inactive. Consequently, the overall immune activity of the corresponding immune cells is increased through extracellular expression of the N-SH2 domain. SHP-2 can be a protein having the amino acid sequence disclosed in NP_002825. In one embodiment, the SHP-2 fragment used can be the amino acid sequence shown in SEQ ID NO:28. Furthermore, the SHP-2 fragment can have about 80%, 90%, 95%, or 99% or higher homology with the amino acid sequence shown in SEQ ID NO:28.

[0126] Immunoplasmin: FKBP12 and Cyclophilin A

[0127] As used herein, the term "immunophile protein" refers to the target proteins of well-known immunosuppressants such as FK506, rapamycin, and cyclosporine. One known class of immunophiles binds to FK506; there are 16 such proteins, one of which is FKBP12. Another known class of immunophiles binds to cyclosporine; there are 16 such proteins, one of which is cyclophilin A. They typically possess peptidyl prolylisomerase (PPI) activity. This activity promotes peptide isomerization and mediates protein folding. This regulates various intracellular signaling pathways. When they bind to immunosuppressants and their original function is disrupted, they induce immunosuppression. Therefore, these immunophiles are considered to have important immunomodulatory functions. As examples of such immune-related functions, one of the known functions performed by FKBP12 and cyclophilin A (CYPA) is the regulation of T cell adhesion and migration.

[0128] It is known that immunoglobulins in T cells bind to CrkII (the CT10 regulator of kinase II) to promote CrkII activation. "Crk" is an adaptor protein that mediates signal transduction by transmitting signals formed by the T cell antigen receptor (TCR) in response to external stimuli to downstream proteins of the receptor, including CrkI and CrkII.

[0129] In T cells, CrkII binds to ZAP70, a protein located downstream of the TCR signaling pathway, and transmits the signal to C3G (Crk SH3 domain-binding guanine-nucleotide releasing factor), thereby inducing the activity of RAP1 (Ras-related protein 1). The TCR is a receptor located on the surface of T cells that recognizes antigens presented by the major histocompatibility complex (MHC) of antigen-presenting cells, thereby activating the T cell immune response.

[0130] ZAP70 is a component protein of the TCR and transmits the TCR activation signal to downstream proteins to induce T cell activity. RAP1 is a small GTPase belonging to the Ras superfamily. GTPases are activated upon binding to GTP and inactivated upon binding to GDP. The activity of this GTPase is regulated by the active GTPase protein (GAP) and GEF (guanine nucleotide exchange factor). GAP promotes the formation of GDP-bound GTPases, while GEF promotes the formation of GTPases bound to GTP.

[0131] "C3G" is a type of GEF that increases RAP1 binding to GTP in T cells, thereby inducing the activity of LFA-1 (lymphocyte function-associated antigen 1) to increase T cell adhesion. LFA-1 is one of the integrins expressed in T cells that binds to ICAM-1 (intercellular adhesion molecule 1), a ligand expressed on target cells during migration from the bloodstream to body tissues, mediating T cell adhesion to target cells. It is known that immunoglobulins such as FKBP12 and CYPA bind to CrkII and increase the binding of CrkII and C3G by binding to CrkII, thereby increasing the activity of downstream C3G signaling pathways and thus increasing LFA-1-regulated T cell adhesion.

[0132] In this context, one embodiment of the FKBP family could be FKBP12. Furthermore, the sequences of the FKBP12 protein and its fragments are as described above in "Proteins in Immunosuppressive Signaling Pathways." Additionally, one embodiment of cyclophilin could be cyclophilin A. The protein in the cyclophilin A-mediated signaling pathway could be CYPA (cyclophilin A, hereinafter, the CYPA bound after self-cleavage peptide is referred to as #C, SEQ ID NO:24).

[0133] As used herein, the term "cyclosporine A (CsA)" is an immunosuppressant derived from a natural product. Administered orally or intravenously, it is used to prevent rheumatoid arthritis, psoriasis, Crohn's disease, nephrotic syndrome, and organ transplant rejection. Cyclosporine A is a cytoplasmic binding protein that binds to CsA. The CsA / CYPA complex inhibits the phosphatase activity of calcineurin, thereby suppressing the lymphocyte immune response. Furthermore, CYPA participates in protein folding through peptidylprolyl isomerase (PPI) activity and regulates biological processes such as intracellular signaling, transcription, inflammation, and cell death. In addition to its intracellular effects, CYPA is known to be secreted in response to inflammatory stimuli, hypoxia, infection, and oxidative stress, and acts as a chemoattractant in viral infections, periodontitis, and atherosclerosis, thereby promoting inflammatory responses. Specifically, in one embodiment, cyclophilin A is a protein comprising the amino acid sequence disclosed in NP_066953. Specifically, cyclophilin A may comprise the amino acid sequence shown in SEQ ID NO:24. In this case, cyclophilin A may have approximately 80%, 90%, 95%, or 99% or higher homology to the amino acid sequence shown in SEQ ID NO:24.

[0134] Proteins involved in antigen loss-mediated relapse

[0135] Proteins involved in antigen loss-mediated relapse can be proteins or fragments thereof involved in trogocytosis. Specifically, proteins involved in trogocytosis can be TC21 (teratocarcinomaoncogene 21, hereinafter referred to as #TC, SEQ ID NO:32) or RhoG (Ras homology growth-related, hereinafter referred to as #RG, SEQ ID NO:34).

[0136] As used herein, the term "TC21" is also referred to as R-Ras2 and is one of the Ras GTPases superfamily. It binds to the cell membrane and mediates signal transduction associated with cell proliferation. Furthermore, it is known to bind to the TCR and activate the downstream protein PI3K (phosphoinositide 3-kinase) in the signaling pathway, thereby inducing the internalization of the immune synapse and mediating membrane molecule transport. TC21 can be a protein having the amino acid sequence disclosed in NP_036382. Additionally, TC21 can be a variant, and in one embodiment, TC21 used may include the amino acid sequence shown in SEQ ID NO:32. TC21 may have approximately 80%, 90%, 95%, or 99% or higher homology to the amino acid sequence shown in SEQ ID NO:32.

[0137] As used herein, the term "RhoG" is a monomeric GTP-binding protein (G protein) involved in the regulation of cell motility, transcription, endocytosis, dendritic growth, etc. Furthermore, it is known to mediate TCR-induced membrane molecule transport through TC21 and PI3K activation. RhoG can be a protein having the amino acid sequence disclosed in NP_001656. Additionally, RhoG can be a variant, and the RhoG fragment used in one embodiment may include the amino acid sequence shown in SEQ ID NO:34. The RhoG fragment may have about 80%, 90%, 95%, or 99% or higher homology to the amino acid sequence shown in SEQ ID NO:34.

[0138] As used herein, the term "membrane molecular transport" refers to the phenomenon of T cells and antigen-presenting cells binding through immune synapses, in which surface molecules of one cell are detached and transported to other cells. This is well known to occur to regulate the suppression or amplification of the immune response. An "immune synapse" is a molecular structure formed during the adhesion and recognition process between T cells and antigen-presenting cells.

[0139] When TC21 and RhoG proteins are overexpressed in immune cells, cytokinesis is disrupted in the response to cancer cells, thus inhibiting the immune evasion process mediated by antigen loss.

[0140] Proteins located in the T cell stimulation signaling pathway: adaptor proteins of the TCR / ZAP70 pathway

[0141] Proteins located in the T cell stimulation signaling pathway can be adaptor proteins or fragments of the TCR / ZAP70 pathway. Specifically, adaptor proteins in the TCR / ZAP70 pathway can be any one of the following groups: NCK1 (hereinafter, NCK1 that binds after peptide cleavage is referred to as #K), LAT (Linker for activation of T cells, hereinafter, LAT that binds after peptide cleavage is referred to as #L), and NEMO (NF-κB essential modulator, hereinafter, NEMO that binds after peptide cleavage is referred to as #I).

[0142] As used herein, the term "TCR / ZAP70 pathway" refers to the signaling pathway that occurs when the T cell antigen receptor of a naive T cell binds to the MHC / antigen complex of an antigen-presenting cell and activates the T cell's immune response. Specifically, when the TCR of a naive T cell binds to the MHC / antigen complex, the γ, δ, ε, and ζ chains of the TCR's accessory protein CD3 are phosphorylated, and ZAP70 binds to the phosphorylated CD3 ζ chain. Then, LCKs that bind to the T cell's accessory receptors CD4 or CD8 are phosphorylated and activate ZAP70.

[0143] Activated ZAP70 phosphorylates LAT and SLP-76 (SH2 domain-containing leukocyte protein 76kDa) to induce signaling responses activating transcription factors such as NF-κB (nuclear factor kappa-light-chain enhancer of activated B cells), AP-1 (activator protein 1), and NFAT. NF-κB is a transcription factor that regulates inflammatory responses, immune modulation, cell death, cell proliferation, and differentiation, and is composed of p50, p52, RelA (p65), RelB, c-Rel, and v-Rel. AP-1 is a transcription factor that regulates inflammatory responses, cell death, cell proliferation, and differentiation, and forms dimers with other transcription factors such as c-Fos, c-Jun, ATF (activating transcription factor), and JDP, thereby playing a role in regulating transcription. Transcription factors such as NF-κB, AP-1, and NFAT promote the expression of interleukin-2, thereby activating T cell division, differentiation, and immune responses.

[0144] As used herein, the term "NCK1" is a signaling mediator including SH2 and SH3 domains and mediating signaling of tyrosine kinase receptors. Furthermore, NCK1 can regulate cytoskeletal rearrangement by binding to the WASP / Arp2 / 3 complex. The WASP / Arp2 / 3 complex is a protein that induces the formation of actin filaments, which are part of the cytoskeleton. In T cells, NCK1 promotes the formation of immune synapses induced by TCR activity. NCK1 can be a protein including the amino acid sequence disclosed in NP_006144.

[0145] As used herein, the term "LAT" refers to a 34 kDa transmembrane protein that is phosphorylated by ZAP70 / Syk (spleen-associated tyrosine kinase) during TCR signaling pathway activation. Adaptor proteins, including those with an SH2 domain, involved in the TCR signaling pathway directly or indirectly bind to phosphorylate LAT to mediate signal transduction. Adaptor proteins may include PLCγ1 (phospholipase Cγ1), Grb2 (growth factor receptor-bound protein 2), Gads (Grb2-related adapter protein downstream of Shc), GRP (Grb2-related adaptor protein), SH3BP2 (SH3 domain-binding protein 2), Shb (SH2 domain-containing adapter protein B), SOS1 (son of sevenless homolog 1), c-Cbl (casitas B lymphoma), VAV, SLP-76 (SH2 domain-containing leukocyte protein of 76kDa), and Itk (IL-2-inducible T cell kinase). (e.g., kinase). LAT can be a protein that includes the amino acid sequence disclosed in NP_055202.

[0146] As used herein, the term "NEMO" refers to IKKγ (IκB kinase γ) and forms a complex with IKKα / IKKβ to induce phosphorylation and degradation of IκB (inhibitor of nuclear factor κ-B kinase), thereby promoting NF-κB activity. NEMO can be a protein including the amino acid sequence disclosed in NP_001093326.

[0147] Proteins located in the T cell stimulation pathway; proteins located in the TNFR / TLR receptor pathway

[0148] Proteins located in the T cell stimulation pathway can be proteins or fragments thereof located in the TNFR / TLR receptor (TRAF / NF-κB) pathway. Specifically, the TNFR / TLR receptor (TRAF / NF-κB) pathway protein can be TLR4, and preferably, it can be the intracellular signal transduction domain of the TLR4 protein (hereinafter, the intracellular signal transduction domain of the TLR4 protein that binds after self-cleavage is referred to as #T, SEQ ID NO:30).

[0149] As used in this article, the term "TNFR (tumor necrosis factor receptor)" refers to the receptor for tumor necrosis factor α (TNFα). TNFα is an inflammatory cytokine mainly produced in activated macrophages, helper T cells, natural killer cells, etc., and regulates various biological activities such as cell growth, differentiation, cell death, and inflammatory responses.

[0150] As used herein, the term "TNFR signaling pathway" refers to an intracellular signaling pathway induced by TNFR binding to its ligands. When TNFα binds to TNFR1, a complex forms involving TRADD (TNF receptor-associated death domain), RIP1 (receptor-interacting protein kinase 1), TRAF (tumor necrosis factor receptor (TNFR)-associated factors)2 or TRAF5 (TRAF2 / 5), cIAP1 (apoptosis inhibitor 1), and cIAP2 to promote TAK1 activation. Activated TAK1 (transforming growth factor-β-activated kinase 1) can phosphorylate IKKβ to induce IκB phosphorylation and proteolysis, thereby inducing NF-κB activity.

[0151] As used in this article, the term "TLR (Toll-like receptor)" refers to proteins that play an important role in innate immunity. TLRs are non-catalytic, single-protein receptors embedded in the cell membrane, primarily expressed on the surface of macrophages, dendritic cells, mucosal epithelial cells, neutrophils, etc., during innate immunity. There are 13 types of TLRs: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13. Of these, TLR11, TLR12, and TLR13 are not expressed in humans.

[0152] As used herein, the term "TLR signaling pathway" refers to an intracellular signaling pathway induced by TLR binding to its ligand, and TLR signaling is typically activated via MyD88 (myeloid differentialfactor 88) and TRIF (Toll / IL-1R domain-containing adaptor inducing IFN-γ). MyD88 binds to the TIR (Toll / IL-1R) domain of TLR to induce the activity of IRAK-4 (IL-1 receptor-associate kinase 4), and activated IRAK-4 phosphorylates IRAK-1 to induce the phosphorylation of TRAF6. Phosphorylated TRAF6 activates IKK to induce the phosphorylation and proteolysis of IκB, thereby inducing NF-κB activity.

[0153] “TLR4” is a receptor belonging to the TLR family and is activated by recognizing LPS (lipopolysaccharide). “LPS,” also known as endotoxin, is a molecule composed of lipids and polysaccharides and is a component of the outer membrane of Gram-negative bacteria. TLR4 can be a protein having the amino acid sequence disclosed in NP_003257. In one embodiment, the TLR4 fragment used can be the amino acid sequence shown in SEQ ID NO:30. The TLR4 fragment can have approximately 80%, 90%, 95%, or 99% or higher homology with the amino acid sequence shown in SEQ ID NO:30.

[0154] Proteins located in the T cell stimulation pathway; proteins located in the cytokine receptor (JAK-STAT) pathway.

[0155] Proteins located in the T cell stimulation pathway can be proteins or fragments of proteins located in the cytokine receptor (JAK-STAT) pathway.

[0156] As used herein, the term "cytokine" refers to polypeptides or glycoproteins secreted by several types of cells in living organisms that participate in cell proliferation, differentiation, activation, and play important roles in immune and inflammatory responses. As used herein, the term "cytokine receptor signaling pathway" refers to intracellular signaling pathways induced by cytokines. Intracellular signaling processes undergo similar pathways compared to the various types of cytokines present. When a cytokine binds to each specific receptor, it induces oligomerization (e.g., dimerization) of the receptor, and the JAK (janus kinase) downstream of the receptor is phosphorylated and activated. Tyrosine residues in the receptor's cytoplasmic domain are phosphorylated by the activated JAK to bind STAT (signal transducers and activators of transcription). Furthermore, receptor-bound STAT is activated by JAK phosphorylation and dissociates from the receptor. Activated STAT forms homodimers or heterodimers in the cytoplasm and then translocates to the nucleus to induce the expression of target genes.

[0157] Proteins located in the T cell stimulation pathway; proteins located in the MAP kinase pathway

[0158] Proteins located in the T cell stimulation pathway can be proteins or fragments thereof located in the MAP kinase (mitogen-activated protein kinase) pathway. Specifically, proteins located in the MAP kinase pathway can be any one of the following groups: GADD45α (growth arrest and DNA-damage-inducible gene 45α, hereinafter, GADD45β bound after self-cleavage peptide is referred to as #A), CDC42 (cell division cycle 42, hereinafter, CDC42 bound after self-cleavage peptide is referred to as #B), and HRAS (hereinafter, HRAS bound after self-cleavage peptide is referred to as #H).

[0159] As used herein, the term "MAPK signaling pathway" refers to a signaling pathway activated by various mitogens (such as growth factors, cytokines, or hormones) and involved in a wide range of cellular functions, including cell proliferation, differentiation, and migration. This pathway includes many proteins, including MAPK, called ERK (extracellular signal-regulated kinase), which transmits signals via phosphorylation of neighboring proteins. The MAPK signaling pathway regulates cellular responses such as inflammation, cellular stress response, cell differentiation, cell division, cell proliferation, metabolism, movement, and cell death. In mammals, MAPK is divided into four groups: ERK-1 / 2, JNK1 / 2 / 3 (Jun amino-terminal kinases 1,2,3), p38 proteins (p38α,β,γ,δ), and ERK5.

[0160] As used herein, the term "GADD45" refers to a protein found in the cell nucleus, of three types: GADD45α, β, and γ. It acts as a sensor for environmental and physiological stresses and binds to or regulates the activity of proteins associated with the cell cycle, cell survival, cell death, maintenance of genome stability, and DNA repair. GADD45α inhibits the G2 / M transition of the cell cycle, induces cell death, and stabilizes the genome through DNA demethylation. Furthermore, it is known to inhibit T cell activity by binding to p38MAPK in type 1 helper T cells (Th1) and dendritic cells, thereby inhibiting p38MAPK phosphorylation (Tyr323). GADD45 can be a protein having the amino acid sequence disclosed in NP_001915.

[0161] As used herein, the term "CDC42" refers to a Rho family GTPase that regulates cell shape, migration, endocytosis, and the cell cycle. CDC42 activity promotes cytoskeleton rearrangement to induce cell adhesion and migration. Furthermore, it is activated by downstream signaling proteins of the TCR to promote the activation of JNK and p38MAPK, thereby inducing the production of inflammatory cytokines and T cell proliferation. CDC42 can be a protein including the amino acid sequence disclosed in NP_001782.

[0162] As used herein, the term "HRAS" stands for GTPase, also known as transforming protein 21, and regulates cell division through growth factors. Activated HRAS activates signaling proteins such as c-Raf and PI3K, which are downstream proteins in the signaling pathway. HRAS binds to GTP in its active state and is inactivated by cleaving the phosphate terminus of the nucleotide to convert GTP to GDP. HRAS can be a protein including the amino acid sequence disclosed in NP_005334.

[0163] Proteins involved in suppressing negative feedback

[0164] In addition, proteins involved in inhibiting negative feedback can be proteins or fragments thereof involved in inhibiting negative feedback in cytokine signaling pathways. Specifically, signal regulators involved in inhibiting negative feedback of cytokine signaling pathways can be the C-terminal domain of SOCS1 protein (suppressor of cytokine signaling 1; hereinafter, the C-terminal domain of SOCS1 protein after self-cleavage peptide binding is referred to as #Q, NP_003736).

[0165] As used herein, the term "cytokine signaling pathway" is as described in the cytokine receptor signaling pathway. The JAK-STAT signaling pathway comprises a negative feedback regulatory mechanism composed of three types of regulatory proteins: PIAS (protein inhibitors of activated STAT), PTP, and SOCS. PIAS inhibit STAT activity by binding to STAT with SUMO (small ubiquitin-like modifier) ​​or by inhibiting STAT binding to DNA. PIAS can be PIAS1, PIAS3, PIASx, and PIASγ. SUMO is a protein covalently linked to or dissociated from a protein and binds to a target protein to regulate its function. It primarily performs functions such as nucleocytoplasmic transport, transcriptional regulation, and protein stability regulation. PTP binds to cytokine receptors JAK and STAT and removes their phosphate groups, thereby inhibiting the cytokine signaling pathway. PTP can be SHP-1, SHP-2, and CD45.

[0166] As used herein, the term "SOCS" refers to a family of proteins comprising an SOCS box motif with an SH2 domain at the C-terminus and a 40-amino acid region. This family includes eight proteins: CISH (cytokine-inducible SH2 domain-containing protein), SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, and SOCS7. SOCS belongs to the same protein family as JAB (JAK-binding protein), CIS (cytokine-inducible STAT inhibitor), and SSI (STAT-induced STAT inhibitor). Cytokine activity in the JAK-STAT signaling pathway induces the expression of CISH, SOCS1, and SOCS3, and these proteins interfere with JAK activation or inhibit STAT activation by blocking STAT binding through binding to the phosphorylated domain of the receptor. In particular, SOCS-1 induces the proteolysis of phosphorylated JAK by binding to phosphorylated JAK.

[0167] The C-terminal domain of the SOCS1 protein can bind to ElonginB / C, a mediator of ubiquitin ligase, competing with intact SOCS1. Therefore, when the C-terminal domain of SOCS1 is overexpressed, even if intact SOCS1 binds to phosphorylated JAK, the unubiquitinated JAK is rescued and not degraded. This mechanism can thus enhance the immune activity of immune cells. The signaling regulator involved in suppressing negative feedback is the C-terminal domain of the SOCS1 protein, and can be a protein with the amino acid sequence disclosed in NP_003736.

[0168] Combination of signaling pathway modulators

[0169] Signal pathway regulators may include one or more signal pathway regulators. In this case, a self-cleaving peptide may be included between each signal pathway regulator. Specifically, a signal pathway regulator may include two signal pathway regulators. Alternatively, a signal pathway regulator may include three signal pathway regulators.

[0170] Combination of two signaling pathway modulators

[0171] Signal pathway modulators can have the structure shown in the following formula (I):

[0172] N'-X-L1-Y-C'(I)

[0173] In structural formula (I),

[0174] N' is the N-terminus of the fusion protein.

[0175] C' is the C-terminus of the fusion protein, and

[0176] X and Y can each be any of the following groups: a) proteins or fragments thereof located in immunosuppressive signaling pathways; b) pro-immunogenic proteins or fragments thereof; c) proteins or fragments thereof involved in antigen loss-mediated relapse; d) proteins or fragments thereof located in T cell stimulation pathways; e) proteins or fragments thereof involved in suppressing negative feedback.

[0177] L1 can be a self-cleaving peptide or an IRES (Internal Ribosome EntrySite).

[0178] Specifically, X can be selected from proteins located in immunosuppressive signaling pathways. In one embodiment, X can be selected from any one of the following groups: FKBP12 protein or a fragment thereof; the C-terminal MH2 domain of SMAD4 or a fragment thereof; N-SKI or a fragment thereof; the N-SH2 domain of SHP-1 protein or a fragment thereof; and the N-SH2 domain of SHP-2 protein or a fragment thereof. Alternatively, X can be an immunophile protein. In one embodiment, X can be FKBP12 protein or a fragment thereof; or cyclin A (CYPA) or a fragment thereof. Alternatively, X can be selected from proteins involved in antigen loss-mediated relapse. In one embodiment, X can be selected from any one of the following groups: TC21 or a fragment thereof; and RHOG or a fragment thereof. Alternatively, X can be selected from proteins located in T cell stimulation pathways. In one embodiment, X can be selected from any one of the following groups: NCK1 or a fragment thereof; LAT or a fragment thereof; NEMO or a fragment thereof; the intracellular domain of TLR4 or a fragment thereof; GADD45α or a fragment thereof; CDC42 or a fragment thereof; and HRAS or a fragment thereof. Alternatively, X can be selected from proteins involved in suppressing negative feedback. In one implementation, X may be the C-terminal domain of the SOCS1 protein or a fragment thereof.

[0179] Specifically, Y can be selected from proteins located in immunosuppressive signaling pathways. In one embodiment, Y can be selected from any one of the following groups: FKBP12 protein or a fragment thereof; the C-terminal MH2 domain of SMAD4 protein or a fragment thereof; N-SKI or a fragment thereof; the N-SH2 domain of SHP-1 protein or a fragment thereof; and the N-SH2 domain of SHP-2 protein or a fragment thereof. Alternatively, Y can be an immunophile protein. In one embodiment, Y can be FKBP12 protein or a fragment thereof; or cyclin A (CYPA) or a fragment thereof. Alternatively, Y can be selected from proteins involved in antigen loss-mediated relapse. In one embodiment, Y can be selected from any one of the following groups: TC21 or a fragment thereof; and RhoG or a fragment thereof. Alternatively, Y can be selected from proteins located in T cell stimulation pathways. In one embodiment, Y can be selected from any one of the following groups: NCK1 or a fragment thereof; LAT or a fragment thereof; NEMO or a fragment thereof; the intracellular domain of TLR4 or a fragment thereof; GADD45α or a fragment thereof; CDC42 or a fragment thereof; and HRAS or a fragment thereof. Additionally, Y can be selected from proteins involved in suppressing negative feedback. In one embodiment, Y can be the C-terminal domain of the SOCS1 protein or a fragment thereof.

[0180] Both signaling pathway regulators may include immunophilic proteins. Specifically, FKBP12 protein or fragments thereof; or cyclophilin A may be included.

[0181] Specific examples of a combination of two signaling pathway regulators could be FKBP12 protein or a fragment thereof and cyclin A. Alternatively, in one embodiment, it could be FKBP12 protein or a fragment thereof, and the N-SH2 domain of SHP-1 protein or a fragment thereof. Another embodiment could be FKBP12 protein or a fragment thereof, and the N-SH2 domain of SHP-2 protein or a fragment thereof. Another embodiment could be FKBP12 protein or a fragment thereof, and the C-terminal domain of SOCS1 protein or a fragment thereof. Another embodiment could be cyclin A and the N-SH2 domain of SHP-1 protein or a fragment thereof. Another embodiment could be cyclin A and the N-SH2 domain of SHP-2 protein or a fragment thereof.

[0182] Combination of three signaling pathway modulators

[0183] Signal pathway modulators can have the structure shown in the following formula (II):

[0184] N'-X-L1-Y-L2-Z-C'(II)

[0185] In structural formula (II),

[0186] N' is the N-terminus of the fusion protein.

[0187] C' is the C-terminus of the fusion protein, and

[0188] X, Y, and Z can each be freely selected from the following groups: a) proteins or fragments located in immunosuppressive signaling pathways; b) pro-immunogenic proteins or fragments; c) proteins or fragments involved in antigen loss-mediated relapse; d) proteins or fragments located in T cell stimulation pathways; and e) any of the following groups consisting of proteins or fragments involved in suppressing negative feedback.

[0189] Each of L1 and L2 can be a self-cleaving peptide or an IRES.

[0190] Specifically, X can be selected from proteins located in immunosuppressive signaling pathways. In one embodiment, X can be selected from any one of the following groups: FKBP12 protein or a fragment thereof; the C-terminal MH2 domain of SMAD4 or a fragment thereof; N-SKI or a fragment thereof; the N-SH2 domain of SHP-1 protein or a fragment thereof; and the N-SH2 domain of SHP-2 protein or a fragment thereof. Alternatively, X can be an immunophile protein. In one embodiment, X can be FKBP12 protein or a fragment thereof; or cyclin A (CYPA) or a fragment thereof. Alternatively, X can be selected from proteins involved in antigen loss-mediated relapse. In one embodiment, X can be selected from any one of the following groups: TC21 or a fragment thereof; and RHOG or a fragment thereof. Alternatively, X can be selected from proteins located in T cell stimulation pathways. In one embodiment, X can be selected from any one of the following groups: NCK1 or a fragment thereof; LAT or a fragment thereof; NEMO or a fragment thereof; the intracellular domain of TLR4 or a fragment thereof; GADD45α or a fragment thereof; CDC42 or a fragment thereof; and HRAS or a fragment thereof. Alternatively, X can be selected from proteins involved in suppressing negative feedback. In one implementation, X may be the C-terminal domain of the SOCS1 protein or a fragment thereof.

[0191] Specifically, Y can be selected from proteins located in immunosuppressive signaling pathways. In one embodiment, Y can be selected from any one of the following groups: FKBP12 protein or a fragment thereof; the C-terminal MH2 domain of SMAD4 protein or a fragment thereof; N-SKI or a fragment thereof; the N-SH2 domain of SHP-1 protein or a fragment thereof; and the N-SH2 domain of SHP-2 protein or a fragment thereof. Alternatively, Y can be an immunophile protein. In one embodiment, Y can be FKBP12 protein or a fragment thereof; or cyclin A (CYPA) or a fragment thereof. Alternatively, Y can be selected from proteins involved in antigen loss-mediated relapse. In one embodiment, Y can be selected from any one of the following groups: TC21 or a fragment thereof; and RhoG or a fragment thereof. Alternatively, Y can be selected from proteins located in T cell stimulation pathways. In one embodiment, Y can be selected from any one of the following groups: NCK1 or a fragment thereof; LAT or a fragment thereof; NEMO or a fragment thereof; the intracellular domain of TLR4 or a fragment thereof; GADD45α or a fragment thereof; CDC42 or a fragment thereof; and HRAS or a fragment thereof. Additionally, Y can be selected from proteins involved in suppressing negative feedback. In one embodiment, Y can be the C-terminal domain of the SOCS1 protein or a fragment thereof.

[0192] Specifically, Z can be selected from proteins located in immunosuppressive signaling pathways. In one embodiment, Z can be selected from any one of the following groups: FKBP12 protein or a fragment thereof; the C-terminal MH2 domain of SMAD4 protein or a fragment thereof; N-SKI or a fragment thereof; the N-SH2 domain of SHP-1 protein or a fragment thereof; and the N-SH2 domain of SHP-2 protein or a fragment thereof. Alternatively, Z can be an immunophile protein. In one embodiment, Z can be FKBP12 protein or a fragment thereof; or cyclin A (CYPA) or a fragment thereof. Alternatively, Z can be selected from proteins involved in antigen loss-mediated relapse. In one embodiment, Z can be selected from any one of the following groups: TC21 or a fragment thereof; and RhoG or a fragment thereof. Alternatively, Z can be selected from proteins located in T cell stimulation pathways. In one embodiment, Z can be selected from any one of the following groups: NCK1 or a fragment thereof; LAT or a fragment thereof; NEMO or a fragment thereof; the intracellular domain of TLR4 or a fragment thereof; GADD45α or a fragment thereof; CDC42 or a fragment thereof; and HRAS or a fragment thereof. Additionally, Z can be selected from proteins involved in suppressing negative feedback. In one embodiment, Z can be the C-terminal domain of the SOCS1 protein or a fragment thereof.

[0193] The three signaling pathway regulators can be in various combinations. Preferably, at least one of the three signaling pathway regulators can be an immunoglobulin.

[0194] Specific examples of a combination of three signaling pathway regulators could be the FKBP12 protein or a fragment thereof, and the N-SH2 domains or fragments thereof of cyclophilin A and SHP-2 proteins. Alternatively, in one embodiment, it could be the FKBP12 protein or a fragment thereof, and the N-SH2 domains or fragments thereof of cyclophilin A and SHP-1 proteins.

[0195] Chimeric antigen receptor

[0196] The term "antigen-binding domain" as used in this invention refers to an antibody site that binds to an antigen. The antigen-binding domain can be an antibody or an antigen-binding fragment thereof. Preferably, the antigen-binding domain can be an antigen-binding fragment. Alternatively, the antigen-binding fragment can be a fragment having an antigen-binding site formed by linking a heavy chain and a light chain of an antibody via disulfide bonds. The antigen-binding fragment can be selected from the group consisting of scFv, Fab, and Fab'. Preferably, the antigen-binding fragment can be scFv. In one embodiment, scFv is used as the antigen-binding domain.

[0197] The antigen-binding domain can specifically bind to α-folate receptors (examples of target tumors: ovarian cancer, gastric cancer, etc.), 5T4 (renal cancer, prostate cancer, etc.), αvβ6 integrin (ovarian cancer, pancreatic cancer, etc.), BCMA (multiple myeloma), B7-H3 (brain cancer, osteosarcoma, etc.), B7-H6 (lymphoma, melanoma, etc.), CAIX (renal cancer, glioblastoma, etc.), and CD16 (IgG opsonized tumor). CD19 (leukemia, lymphoma), CD20 (leukemia, lymphoma), CD22 (leukemia, lymphoma), CD30 (leukemia, lymphoma), CD33 (leukemia), CD43 (leukemia), CD44 (liver cancer), CD44v6 (leukemia, multiple myeloma, ovarian cancer, etc.), CD44v7 / 8 (uterine cancer), CD47 (leukemia, lymphoma, ovarian cancer, pancreatic cancer, etc.), CD70 (leukemia, lymphoma, neuroma), CD79a (leukemia, lymphoma), CD79b (leukemia, lymphoma), CD123 (leukemia... CD138 (multiple myeloma), CD171 (neuroblastoma), CEA (colorectal cancer, gastric cancer, lung cancer), CSPG4 (leukemia, glioblastoma), EGFR (breast cancer, lung cancer), the EGFR family includes ErbB2 (HER2) (glioblastoma, sarcoma), EGFRvIII (glioblastoma), EGP2 (colorectal cancer), EGP40 (colorectal cancer), EPCAM (colorectal cancer), EphA2 (esophageal cancer), FAP (mesothelioma, lung cancer, colorectal cancer), fetal AchR (rhabdomyosarcoma), FRα (ovarian cancer). Ovarian cancer, breast cancer), GD2 (neuroblastoma), GD3 (melanoma, neuroma, etc.), phosphatidylinositol proteoglycan-3 (GPC3) (liver cancer), HLA-A1+MAGE1 (melanoma), HLA-A2+MAGE1 (melanoma), HLA-A3+MAGE1 (melanoma), HLA-A1+NY-ESO-1 (synovial sarcoma, melanoma), HLA-A2+NY-ESO-1 (synovial sarcoma, melanoma), HLA-A3+NY-ESO-1 (synovial sarcoma, melanoma), IL-11Rα (osteosarcoma), I L-13Rα2 (neuroma), λ, Lewis-Y, κ (lymphoma), mesothelin (mesothelioma, pancreatic cancer, etc.), Muc1 (breast cancer), Muc16 (ovarian cancer), NCAM (neuroblastoma, lung cancer), NKG2D ligand (leukemia, multiple myeloma), NY-ESO-1 (multiple myeloma), PRAME (myeloid cell tumor), PSCA (prostate cancer), PSMA (prostate cancer), ROR1 (lung cancer, breast cancer), SSX (synovial sarcoma), survivin (lung cancer), TAG72 (ovarian cancer), TEM.The antigen can bind to any one of the antigens in the group consisting of VEGFR2 (melanoma, renal cell carcinoma) and WT-1 (leukemia). Specifically, the antigen-binding domain can specifically bind to CD19, HER2, PSMA, CD43, and CD47.

[0198] Chimeric antigen receptor: CD19

[0199] The CD19-specific antigen-binding domain may include an antibody fragment that specifically binds to CD19. In one embodiment of the invention, the CD19-specific antigen-binding domain may include a light chain variable region comprising L-CDR1 (SEQ ID NO:73), L-CDR2 (SEQ ID NO:74), and L-CDR3 (SEQ ID NO:75). Furthermore, the CD19-specific antigen-binding domain may include a heavy chain variable region comprising H-CDR1 (SEQ ID NO:76), H-CDR2 (SEQ ID NO:77), and H-CDR3 (SEQ ID NO:78). Additionally, the CD19-specific antigen-binding domain may include the amino acid sequence shown in SEQ ID NO:3. Furthermore, the nucleotide sequence encoding the CD19-specific antigen-binding domain may be the nucleotide sequence shown in SEQ ID NO:4.

[0200] Chimeric antigen receptor: HER2

[0201] The Her2-specific antigen-binding domain may include an antibody fragment that specifically binds to Her2. In one embodiment of the invention, the Her2-specific antigen-binding domain may include a light chain variable region comprising L-CDR1 (SEQ ID NO: 79), L-CDR2 (SEQ ID NO: 80), and L-CDR3 (SEQ ID NO: 81). Furthermore, the Her2-specific antigen-binding domain may include a heavy chain variable region comprising H-CDR1 (SEQ ID NO: 82), H-CDR2 (SEQ ID NO: 83), and H-CDR3 (SEQ ID NO: 84). Additionally, the Her2-specific antigen-binding domain may include the amino acid sequence shown in SEQ ID NO: 45. Furthermore, the nucleotide sequence encoding the Her2-specific antigen-binding domain may be the nucleotide sequence shown in SEQ ID NO: 46.

[0202] Chimeric antigen receptor: PSMA

[0203] The PSMA-specific antigen-binding domain may include an antibody fragment that specifically binds to PSMA. In one embodiment of the invention, the PSMA-specific antigen-binding domain may include a light chain variable region comprising L-CDR1 (SEQ ID NO: 85), L-CDR2 (SEQ ID NO: 86), and L-CDR3 (SEQ ID NO: 87). Furthermore, the PSMA-specific antigen-binding domain may include a heavy chain variable region comprising H-CDR1 (SEQ ID NO: 88), H-CDR2 (SEQ ID NO: 89), and H-CDR3 (SEQ ID NO: 90). Additionally, the PSMA-specific antigen-binding domain may include the amino acid sequence shown in SEQ ID NO: 54. Furthermore, the nucleotide sequence encoding the PSMA-specific antigen-binding domain may be the nucleotide sequence shown in SEQ ID NO: 55.

[0204] Chimeric antigen receptor: CD43

[0205] The CD43-specific antigen-binding domain may include an antibody fragment that specifically binds to CD43. In one embodiment of the invention, the CD43-specific antigen-binding domain may include a light chain variable region comprising L-CDR1 (SEQ ID NO: 91), L-CDR2 (SEQ ID NO: 92), and L-CDR3 (SEQ ID NO: 93). Furthermore, the CD43-specific antigen-binding domain may include a heavy chain variable region comprising H-CDR1 (SEQ ID NO: 94), H-CDR2 (SEQ ID NO: 95), and H-CDR3 (SEQ ID NO: 96). Additionally, the CD43-specific antigen-binding domain may include the amino acid sequence shown in SEQ ID NO: 47. Furthermore, the nucleotide sequence encoding the CD43-specific antigen-binding domain may be the nucleotide sequence shown in SEQ ID NO: 48.

[0206] Chimeric antigen receptor: CD47

[0207] The CD47-specific antigen-binding domain may include an antibody fragment that specifically binds to CD47. In one embodiment of the invention, the CD47-specific antigen-binding domain may include a light chain variable region, which includes L-CDR1 (SEQ ID NO: 100), L-CDR2 (SEQ ID NO: 101), and L-CDR3 (SEQ ID NO: 102). Furthermore, the CD47-specific antigen-binding domain may include a heavy chain variable region, which includes H-CDR1 (SEQ ID NO: 97), H-CDR2 (SEQ ID NO: 98), and H-CDR3 (SEQ ID NO: 99). Additionally, the CD47-specific antigen-binding domain may include the amino acid sequence shown in SEQ ID NO: 50. Furthermore, the nucleotide sequence encoding the CD47-specific antigen-binding domain may be the nucleotide sequence shown in SEQ ID NO: 51.

[0208] Transmembrane domain

[0209] The term "transmembrane domain" as used in this invention refers to a site in a protein structure located in the cell membrane that connects an antigen-binding domain and a domain that transmits intracellular signals and crosses the cell membrane, thereby anchoring the protein to the cell membrane. A transmembrane domain can be derived from any one of the following groups: T cell receptor, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN, and PD-1. Specifically, a transmembrane domain can be derived from CD8α.

[0210] In one embodiment of the invention, the spacer region and transmembrane domain are composed of the amino acid sequence shown in SEQ ID NO:5, which is derived from CD8α. Alternatively, the nucleotide sequences encoding the spacer region and transmembrane domain may be the nucleotide sequences shown in SEQ ID NO:6.

[0211] Interval

[0212] As described above, the antigen-binding domain and the transmembrane domain can be connected by a spacer region. The spacer region refers to a linker, which can be a protein or peptide. Furthermore, it can consist of 1 to 1000 amino acids, or 10 to 300 amino acids. It can also consist of 15 to 100 amino acids, or 15 to 60 amino acids. Additionally, the spacer region can consist of 15 to 45 amino acids. Furthermore, the linker can be a fragment of a protein in the human body, such as the Fc region. Furthermore, the linker can be derived from any of the following: CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN, and PD-1. Furthermore, the spacer region can include an amino acid sequence derived from the CD8α hinge.

[0213] Intracellular signal transduction domains

[0214] The term "intracellular signal transduction domain" as used in this invention refers to the region within the cell that transmits signals to induce responses such as cell activation, release of cytotoxic factors, cytokine production, and proliferation when an antigen receptor (antigen-binding domain) on the cell surface recognizes an extracellular antigen. Furthermore, since the signal transmitted through a single antigen receptor (antigen-binding domain) is typically insufficient to activate the cell, secondary or co-stimulatory signals are required. Therefore, intracellular signal transduction domains may include primary signal transduction domains, secondary signal transduction domains, and / or co-stimulatory domains. Specifically, intracellular signal transduction domains may include co-stimulatory domains and primary signal transduction domains.

[0215] The co-stimulatory domain can be derived from at least one molecule selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70. Specifically, the co-stimulatory domain can be derived from CD137 (4-1BB).

[0216] In one embodiment of the invention, a co-stimulatory domain is used, consisting of the amino acid sequence shown in SEQ ID NO:7, which is derived from CD137(4-1BB). Alternatively, the nucleotide sequence encoding the co-stimulatory domain may be the nucleotide sequence shown in SEQ ID NO:8.

[0217] Primary signaling domains may be derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, or CD66d. Specifically, T cells transmit signals intracellularly via the γ, δ, ε, and ζ chains of CD3. When constructing CAR-T cells (chimeric antigen receptor T cells), the γ, δ, ε, and ζ chains of CD3 can be used as primary signaling domains. Specifically, the primary signaling domain can be derived from CD3ζ.

[0218] In one embodiment of the invention, a primary signal transduction domain is used, consisting of the amino acid sequence shown in SEQ ID NO:9, derived from CD3ζ. Alternatively, the nucleotide sequence encoding the primary signal transduction domain may be the nucleotide sequence shown in SEQ ID NO:10.

[0219] Intracellular signaling domains can be appropriately combined. In one embodiment, the intracellular signaling domain may include CD137(4-1BB) and CD3ζ.

[0220] Self-cleaving peptides

[0221] As used in this invention, the term "self-cleaving peptide" refers to a peptide composed of 10 to 50, 12 to 42, 14 to 34, 16 to 26, or 18 to 22 amino acids that can induce the cleavage of proteins synthesized in the cell. Self-cleaving peptides can be derived from the 2A region of viral genes. Self-cleaving peptides can be derived from P2A, E2A, F2A, or T2A. Specifically, self-cleaving peptides can be derived from P2A. In addition to self-cleaving peptides, protease-cleaving peptides present in the cytoplasm can be used.

[0222] In one embodiment of the invention, a self-cleaving peptide is used, consisting of an amino acid sequence derived from P2A, represented by SEQ ID NO:11 or 56. Alternatively, the nucleotide sequence encoding the self-cleaving peptide may be the nucleotide sequence shown in SEQ ID NO:12.

[0223] Specific examples of fusion proteins

[0224] Specific examples of fusion proteins are as follows:

[0225] (1) N-terminal signal peptide-antigen binding domain-hinge-transmembrane domain-co-stimulatory domain-primary signal domain-autocleaving peptide-signal pathway regulator (FKBP12 or its fragment)-C-terminus;

[0226] (2) N-terminal signal peptide-antigen binding domain-hinge-transmembrane domain-co-stimulatory domain-primary signal domain-co-stimulatory domain-self-cleaving peptide-signal pathway regulator (cyclophilin A)-C-terminus;

[0227] (3) N-terminal signal peptide-antigen binding domain-hinge-transmembrane domain-co-stimulatory domain-primary signal domain-autocleaving peptide-signal pathway regulator (N-SH2 domain of SHP-1 protein)-C-terminus;

[0228] (4) N-terminal signal peptide-antigen binding domain-hinge-transmembrane domain-co-stimulatory domain-primary signal domain-autocleaving peptide-signal pathway regulator (N-SH2 domain of SHP-2 protein)-C-terminus;

[0229] (5) N-terminal signal peptide-antigen binding domain-hinge-transmembrane domain-co-stimulatory domain-primary signal domain-autocleaving peptide-signal pathway regulator (C-terminal MH2 domain of SMAD4 protein or a fragment thereof)-C-terminus;

[0230] (6) N-terminal signal peptide-antigen binding domain-hinge-transmembrane domain-co-stimulatory domain-primary signal domain-autocleaving peptide-signal pathway regulator (N-SKI or its fragment)-C-terminus;

[0231] (7) N-terminal signal peptide-antigen binding domain-hinge-transmembrane domain-co-stimulatory domain-primary signal transduction domain-autocleaving peptide-signal pathway regulator (FKBP12 or its fragment)-autocleaving peptide-signal pathway regulator (cyclin A)-C-terminus;

[0232] (8) N-terminal signal peptide-antigen binding domain-hinge-transmembrane domain-co-stimulatory domain-primary signal domain-autocleaving peptide-signal pathway regulator (FKBP12 fragment)-autocleaving peptide-signal pathway regulator (N-SH2 domain of SHP-2 protein)-C-terminus;

[0233] (9) N-terminal signal peptide-antigen binding domain-hinge-transmembrane domain-co-stimulatory domain-primary signal domain-autocleaving peptide-signal pathway regulator (cyclophilin A)-autocleaving peptide-signal pathway regulator (N-SH2 domain of SHP-2 protein)-C-terminus;

[0234] (10) N-terminal - signal peptide - antigen-binding domain - hinge - transmembrane domain - co-stimulatory domain - primary signal domain - autocleaving peptide - signal pathway regulator (FKBP12 or its fragment) - autocleaving peptide - signal pathway regulator (cyclophilin A) - autocleaving peptide - signal pathway regulator (N-SH2 domain of SHP-2 protein) - C-terminus;

[0235] (11) N-terminus - signaling pathway regulator (FKBP12 or its fragment) - self-cleaving peptide - signal peptide - antigen-binding domain - hinge - transmembrane domain - co-stimulatory domain - primary signaling domain - C-terminus;

[0236] (12) N-terminus - signaling pathway regulator (cyclophilin A) - self-cleaving peptide - signal peptide - antigen-binding domain - hinge - transmembrane domain - co-stimulatory domain - primary signaling domain - co-stimulatory domain - C-terminus;

[0237] (13) N-terminus - signaling pathway regulator (N-SH2 domain of SHP-1 protein) - self-cleaving peptide - signal peptide - antigen binding domain - hinge - transmembrane domain - co-stimulatory domain - primary signaling domain - C-terminus.

[0238] (14) N-terminus - signaling pathway regulator (N-SH2 domain of SHP-2 protein) - self-cleaving peptide - signal peptide - antigen binding domain - hinge - transmembrane domain - co-stimulatory domain - primary signaling domain - C-terminus.

[0239] (15) N-terminus - signaling pathway regulator (C-terminal MH2 domain of SMAD4 protein or a fragment thereof) - self-cleaving peptide - signal peptide - antigen-binding domain - hinge - transmembrane domain - co-stimulatory domain - primary signaling domain - C-terminus;

[0240] (16) N-terminus - signaling pathway regulator (N-SKI or its fragment) - self-cleaving peptide - signal peptide - antigen-binding domain - hinge - transmembrane domain - co-stimulatory domain - primary signaling domain - C-terminus;

[0241] (17) N-terminus - signaling pathway regulator (N-SKI or its fragment) - self-cleaving peptide - signaling pathway regulator (cyclophilin A) - self-cleaving peptide - signal peptide - antigen-binding domain - hinge - transmembrane domain - co-stimulatory domain - primary signaling domain - C-terminus;

[0242] (18) N-terminus - signaling pathway regulator (FKBP12 or its fragment) - self-cleaving peptide - signaling pathway regulator (N-SH2 domain of SHP-2 protein) - self-cleaving peptide - signal peptide - antigen-binding domain - hinge - transmembrane domain - co-stimulatory domain - primary signaling domain - C-terminus;

[0243] (19) N-terminus - signaling pathway regulator (cyclophilin A) - self-cleaving peptide - signaling pathway regulator (N-SH2 domain of SHP-2 protein) - self-cleaving peptide - signal peptide - antigen-binding domain - hinge - transmembrane domain - co-stimulatory domain - primary signaling domain - C-terminus.

[0244] (20) N-terminus - signaling pathway regulator (FKBP12 or its fragment) - self-cleaving peptide - signaling pathway regulator (cyclophilin A) - self-cleaving peptide - signaling pathway regulator (N-SH2 domain of SHP-2 protein) - self-cleaving peptide - signal peptide - antigen-binding domain - hinge - transmembrane domain - co-stimulatory domain - primary signaling domain - C-terminus.

[0245] In this case, in the above formulas (1) to (20), the antigen-binding domain, hinge, transmembrane domain, co-stimulatory domain, primary signaling domain, self-cleaving peptide, and signaling pathway regulator are as described above. Specifically, in the above formulas (1) to (20), the antigen-binding domain can be a CD19-specific scFv, a Her2-specific scFv, a PSMA-specific scFv, a CD43-specific scFv, or a CD47-specific scFv; the transmembrane domain can be derived from CD8α; the co-stimulatory domain can be derived from CD137(4-1BB); the primary signaling domain can be derived from CD3ζ; and the self-cleaving peptide can be derived from P2A.

[0246] In one embodiment of the invention, the design includes an antigen-binding domain (CD19 scFv), a transmembrane domain (CD8α), intracellular signal transduction domains (4-1BB and CD3ζ), a self-cleaving peptide (P2A), and FKBP12, referred to as "19bbz#F". Additionally, the signaling pathway regulators and symbols are as described above.

[0247] Polynucleotides encoding fusion proteins

[0248] As used herein, the term "polynucleotide" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA / RNA hybrids. Polynucleotides are single-stranded or double-stranded and can be recombined, synthesized, or isolated. Polynucleotides include, but are not limited to, pre-mRNA, messenger RNA (mRNA), RNA, genomic RNA (gRNA), positive-strand RNA (RNA(+)), negative-strand RNA (RNA(-)), synthetic RNA, synthetic mRNA, genomic DNA (gDNA), PCR-amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.

[0249] Polynucleotides can be codon-optimized. As used herein, the term “codon optimization” refers to replacing codons in a polynucleotide encoding a polypeptide to increase the polypeptide’s expression, stability, and / or activity. Factors influencing codon optimization include, but are not limited to, (i) modifications to the degree of codon preference in an organism, gene, or set of genes, (ii) systematic modifications including codons of neighboring sequences, (iii) modifications to codons based on their decoding tRNA, (iv) modifications to codons based on the overall or one of the three GC% values, (v) modifications to the similarity to a reference sequence, such as a naturally derived sequence, (vi) modifications to the codon frequency cutoff, (vii) structural characteristics of mRNA transcribed from a DNA sequence, (viii) prior knowledge of the function of a DNA sequence designed based on codon replacement sets, (ix) systematic modifications to the codon set for each amino acid, and / or (x) the removal of non-logical translation initiation sites.

[0250] Vectors containing polynucleotides encoding fusion proteins

[0251] Another aspect of the present invention provides a carrier comprising polynucleotides.

[0252] Polynucleotides are as described above. Polynucleotides can be prepared, engineered, expressed, and delivered using any of the various established techniques known and available in the art. To express the desired fusion protein, the polynucleotide encoding the fusion protein can be inserted into a suitable vector.

[0253] This vector can be used with various vectors known in the art, expressing regulatory sequences such as promoters, terminators, and enhancers, as well as membrane-targeting or secretion sequences. It can be appropriately selected based on the host cell type producing the antigen receptor and can be combined in various ways according to the purpose. The vectors of this invention include, but are not limited to, plasmid vectors, granulosome vectors, phage vectors, and viral vectors. Suitable vectors include expression regulatory elements such as promoters, operons, start codons, stop codons, polyadenylation signals, and enhancers, as well as signal peptides or leader sequences for membrane targeting or secretion, and can be prepared according to different purposes.

[0254] Preferably, the vector can be a viral vector, and the viral vector can be derived from retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpesviruses, poxviruses, baculoviruses, papillomaviruses, and parvoviruses. In one embodiment of the invention, a lentiviral vector is used.

[0255] The vector may also include a sequence encoding a signal peptide to expose the antigen-binding domain to the cell membrane. In this case, the sequence encoding the signal peptide may be inserted before the sequence encoding the antigen-binding domain. The signal peptide may consist of the amino acid sequence shown in SEQ ID NO:1, and the nucleotide sequence encoding this amino acid sequence may be the sequence shown in SEQ ID NO:2.

[0256] Viruses containing polynucleotides encoding fusion proteins

[0257] Another aspect of the invention provides a virus comprising polynucleotides. In this case, the polynucleotides are as described above. The virus may be a retrovirus, lentivirus, adenovirus, adeno-associated virus, herpesvirus, poxvirus, baculovirus, papillomavirus, or parvovirus.

[0258] Cells expressing polynucleotides encoding fusion proteins

[0259] Another aspect of the present invention provides an immune cell delivery vector. In this case, the vector is as described above. The immune cell can be a T cell or a natural killer cell.

[0260] The vector can be introduced into immune cells using methods known in the art, and the vector can be introduced into cells by, but is not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE-Dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, or other known methods of introducing nucleic acids into cells (Wu et al., J. Bio. Chem., 267:963-967, 1992; Wu and Wu, J. Bio. Chem., 263:14621-14624, 1988).

[0261] After the vector is introduced, the transduced or transfected immune cells proliferate in vitro. In one embodiment, the transfected immune cells can be cultured for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days to proliferate, and preferably for 12 to 14 days.

[0262] Methods for confirming whether a vector has been successfully introduced into immune cells include, for example, molecular biological analyses well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; and biochemical analyses, for example, detecting the presence or absence of a specific peptide by immunological methods such as ELISA and Western blot.

[0263] When the fusion protein is expressed in immune cells, the self-cleaving peptide of (iv) allows for the dissociation of signaling pathway regulators (e.g., FKBP12 or fragments thereof) corresponding to the chimeric antigen receptor (CAR) in the cytoplasm from (i) to (iii). For the chimeric antigen receptor, it can be immobilized on the cell membrane to recognize extracellular antigens and transduce signals into the cell. Furthermore, signaling pathway regulators, such as FKBP12, can block the TGF-β signaling pathway by binding to TGFβR1.

[0264] Polynucleotides including IRES

[0265] Another aspect of the present invention provides a polynucleotide comprising (i) a polynucleotide encoding an antigen-binding domain; (ii) a polynucleotide encoding a transmembrane domain; (iii) a polynucleotide encoding an intracellular signaling domain comprising at least one co-stimulatory domain; (iv) a polynucleotide encoding an IRES (Internal Ribosome EntrySite); and (v) a polynucleotide encoding a signaling pathway regulator.

[0266] In this case, the polynucleotide encoding the spacer region can be further included between the polynucleotide encoding the antigen-binding domain and the polynucleotide encoding the transmembrane domain.

[0267] Antigen-binding domains, transmembrane domains, intracellular signaling domains, and signaling pathway regulators are as described above.

[0268] As used in this article, the term "IRES" is an abbreviation for internal ribosome entry site. IRES is a nucleic acid used to simultaneously express two genes.

[0269] Another aspect of the invention provides an expression vector comprising polynucleotides. In this case, the vector and the components included herein are as described above.

[0270] Another aspect of the invention provides a virus comprising polynucleotides. In this case, the virus and its constituent components are as described above.

[0271] Another aspect of the present invention provides the introduction of immune cells containing polynucleotides. In this case, the method for introducing immune cells and polynucleotides is as described above.

[0272] Polynucleotides excluding self-cleaving sequences or IRES

[0273] Another aspect of the invention provides a polynucleotide encoding a fusion protein comprising (i) an antigen-binding domain; (ii) a transmembrane domain; (iii) an intracellular signaling domain including at least one co-stimulatory domain; and (iv) a signaling pathway regulator.

[0274] In this context, the fusion protein may further include (i) an antigen-binding domain and (ii) a spacer region between the transmembrane domain.

[0275] In this context, the signaling pathway regulator can be a protein located in the T cell stimulation pathway. Specifically, the protein located in the T cell stimulation pathway can be a protein or fragment thereof located in the TNFR / TLR receptor pathway. More specifically, the protein located in the TNFR / TLR receptor pathway can be the TLR4 intracellular domain (hereinafter referred to as T).

[0276] In this context, the protein located in the T cell stimulation pathway can be a protein or fragment thereof located in the cytokine receptor (JAK-STAT) pathway. Specifically, the protein located in the cytokine receptor (JAK-STAT) pathway can be γc (hereinafter referred to as γ).

[0277] In this context, when the self-cleaving peptide is not included, intracellular signaling can be modulated when the signaling pathway regulator binds to CAR.

[0278] Another aspect of the invention provides an expression vector comprising a polynucleotide. The expression vector is as described above. The vector may further include a sequence encoding a signal peptide to expose an antigen-binding domain to the cell membrane. In this case, the sequence encoding the signal peptide may be inserted prior to the sequence encoding the antigen-binding domain. The signal peptide is as described above.

[0279] Another aspect of the present invention provides a virus comprising polynucleotides. The virus is as described above.

[0280] Another aspect of the invention provides immune cells infused with polynucleotides. In this case, the immune cells are as described above.

[0281] Immune cells engineered to overexpress signaling pathway regulators

[0282] Another aspect of the invention provides transformed immune cells, characterized in that the transformed immune cells are engineered to overexpress signaling pathway regulators.

[0283] In this case, the immune cells can be T cells or NK cells. Furthermore, the immune cells can be transformed T cells or NK cells. In this case, the transformed T cells can be CAR-T cells or TCR-T cells.

[0284] Signaling pathway regulators are as described above. Specifically, signaling pathway regulators may be selected from any of the following groups: FKBP12 protein or a fragment thereof; the C-MH2 domain of SMAD4 protein or a fragment thereof; N-SKI or a fragment thereof; cyclophilin A (CYPA) or a fragment thereof; the N-SH2 domain of SHP-1 protein or a fragment thereof; and the N-SH2 domain of SHP-2 protein or a fragment thereof; TC21 or a fragment thereof; and RhoG or a fragment thereof; NCK1 or a fragment thereof; LAT or a fragment thereof; NEMO or a fragment thereof; the intracellular domain of TLR4 or a fragment thereof; GADD45α or a fragment thereof; CDC42 or a fragment thereof; HRAS or a fragment thereof; and the C-terminal domain of SOCS1 protein or a fragment thereof.

[0285] In this context, signaling pathway regulators are characterized by their function in the cytoplasm. Furthermore, the polynucleotides encoding these regulators are expressed in the cytoplasm, and the regulators themselves are not secreted extracellularly. Therefore, signaling pathway regulators may reside in immune cells and potentially regulate intracellular signaling pathways.

[0286] In addition, it can express two or three signaling pathway regulators.

[0287] CAR-T immune cells

[0288] Chimeric antigen receptors (CARs) are prepared based on the scFv (single-chain variable fragment) nucleotide sequence of monoclonal antibodies that recognize characteristic antigens expressed on the surface of cancer cells. Therefore, T cells expressing these antigens specifically bind to the tumor antigen, thus allowing only tumor cells to be eliminated. The CAR structure consists of a receptor region that binds to the tumor antigen, a co-stimulatory domain that activates T cells after receptor-antigen binding, and a spacer region and transmembrane domain connecting these two regions.

[0289] In this context, CAR-T cells are characterized by expressing a fusion protein comprising (i) an antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain containing at least one co-stimulatory domain.

[0290] In one embodiment, the transformed CAR-T immune cells can be prepared by transducing a polynucleotide comprising (i) an antigen-binding domain; (ii) a spacer region and a transmembrane domain; (iii) an intracellular signaling domain comprising at least one co-stimulatory domain; (iv) a self-cleaving peptide; and (v) a signaling pathway regulator as described above.

[0291] Furthermore, transformed immune cells can be prepared by transducing a first polynucleotide and a second polynucleotide separately. The first polynucleotide includes (i) an antigen-binding domain; (ii) a spacer region and a transmembrane domain; and (iii) an intracellular signaling domain containing at least one co-stimulatory domain. The second polynucleotide includes a signaling pathway regulator. In this case, the first and second polynucleotides can be introduced via a single virus, but they can also be introduced into immune cells via different viruses.

[0292] In this context, signaling pathway regulators can be selected from any of the following groups: a) proteins located in immunosuppressive signaling pathways, b) pro-immunogenic proteins, c) proteins involved in antigen loss-mediated relapse, d) proteins located in T cell stimulation pathways, e) proteins involved in suppressing negative feedback, and f) combinations of the above proteins. Details of signaling pathway regulators and their combinations are as described above.

[0293] In one embodiment, the immune cells may be cells that overexpress immunophile proteins. Preferably, they may be CAR-T cells that overexpress FKBP12 or fragments thereof and / or cyclin A.

[0294] In this case, the polynucleotide is as described above. Furthermore, the various methods described above can be used to introduce vectors containing polynucleotides.

[0295] TCR-T immune cells

[0296] TCR-T cells refer to T cell receptor-engineered T cells (TCR-T). TCR-T is an immunotherapy that involves introducing T cell receptor genes that recognize tumor-specific antigen peptides presented on the surface of tumor cells via MHC molecules. This allows the expressed TCRs to recognize specific tumor antigens and selectively attack targets. TCR-T cells are then expanded in vitro and administered to the body.

[0297] TCRs typically comprise two polypeptide chains, such as the α chain, β chain, γ chain, δ chain, or a combination thereof. The polypeptide chains of TCRs described above are known in the art. Specifically, a TCR-T may be a polypeptide chain expressing both an antigen-recognizable α and β chain.

[0298] For example, the α-chain or β-chain can comprise any amino acid sequence, as long as it can specifically bind to disease-associated antigens or their epitopes for immune recognition. Furthermore, viral vectors can serve as a method for introducing the TCR gene into T cells. In this case, the viral vector is as described above.

[0299] In one embodiment, the transformed TCR-T immune cells can be prepared by transducing a polynucleotide comprising (i) a polynucleotide encoding the TCRα chain; (ii) a polynucleotide encoding a self-cleaving peptide; (iii) a polynucleotide encoding the TCRβ chain; (iv) a polynucleotide encoding a self-cleaving peptide; and (v) a polynucleotide encoding a signaling pathway regulator as described above.

[0300] Furthermore, the transformed immune cells can be prepared by transducing a first polynucleotide and a second polynucleotide, respectively. The first polynucleotide includes (i) a polynucleotide encoding the α chain of the TCR; (ii) a polynucleotide encoding a self-cleaving peptide; and (iii) a polynucleotide encoding the β chain of the TCR. The second polynucleotide includes a signaling pathway regulator. In this case, the first and second polynucleotides can be introduced into the immune cells using different viruses.

[0301] In this context, signaling pathway regulators can be selected from any of the following groups: a) proteins located in immunosuppressive signaling pathways, b) pro-immunogenic proteins, c) proteins involved in antigen loss-mediated relapse, d) proteins located in T cell stimulation pathways, e) proteins involved in suppressing negative feedback, and f) combinations thereof. Details regarding signaling pathway regulators and their combinations are as described above.

[0302] In one embodiment, the immune cells may be cells that overexpress immunophile proteins. Preferably, they may be TCR-T cells that overexpress FKBP12 or fragments thereof and / or cyclophilin A.

[0303] In this case, the transduction of polynucleotides can be achieved by introducing a vector containing polynucleotides as described above using the various methods described above.

[0304] Pharmaceutical compositions including immune cells engineered to overexpress signaling pathway regulators

[0305] Another aspect of the present invention provides a pharmaceutical composition for treating cancer, comprising immune cells engineered to overexpress signaling pathway modulators as an active ingredient.

[0306] In this context, the immune cells can be T cells, NK cells, preferably CD8+ T cells or CD4+ T cells, or a mixture of these cells in a certain proportion. Alternatively, immune cells can be prepared by transducing a vector containing externally introduced regulators of the aforementioned signaling pathways.

[0307] In this context, the cancer can be selected from any of the following groups: stomach cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, leukemia, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, lymphoma, kidney cancer, melanoma, multiple myeloma, brain cancer, osteosarcoma, glioblastoma, IgG-opsonized tumor, lymphoma, neuroma, mesothelioma, and esophageal cancer.

[0308] The type of cancer can be determined based on the antigen-binding domain. In one embodiment, CD19-specific immune cells can be used to treat hematologic malignancies. Preferably, the hematologic malignancy can be leukemia.

[0309] Cancer can be targeted when the antigen-binding domain specifically binds to a protein that is specifically overexpressed in cancer. Specifically, when a protein specifically overexpressed in a solid tumor is specifically recognized, it can be used to treat various solid tumors. In one implementation, Her2-specific CAR-T cells can be used to treat Her2-overexpressing cancers, particularly effective for Her2(+) breast cancer. Furthermore, PSMA-specific CAR-T cells are effective in treating prostate cancer.

[0310] The "pharmaceutical composition" described herein for use in immunotherapy of human patients includes immune cells. In addition to cells, other pharmaceutically acceptable salts, carriers, excipients, vehicles, and other additives that can further improve the immune response may be added to the pharmaceutical composition.

[0311] The terms “effective amount” or “therapeutic effective amount” are used interchangeably herein and refer to the amount of a compound, formulation, material, or composition, such as immune cells, that effectively achieves a specific biological outcome as described herein. In one embodiment, CAR-T cells may include, but are not limited to, 1 × 10⁻⁶ cells when administered as a single dose. 2 Up to 1×10 10 1×10 3 Up to 1×10 8 and 1×10 4 Up to 1×10 6 Each cell.

[0312] The cell therapy composition of the present invention can be administered in a conventional manner via rectal, intravenous, intra-arterial, intraperitoneal, intramuscular, intrasternal, transdermal, local, intracranial, intraocular, or intradermal routes.

[0313] Methods of cancer therapy or prevention using immune cells expressing externally introduced signaling pathway modulators.

[0314] Another aspect of the present invention provides a method for treating or preventing cancer, comprising the step of administering immune cells engineered to overexpress signaling pathway regulators to a subject.

[0315] In this context, signaling pathway regulators are as described above. Furthermore, immune cells are as described above. Specifically, immune cells may include CAR-T cells and TCR-T cells.

[0316] Uses of immune cells expressing externally introduced signaling pathway modulators

[0317] Another aspect of the present invention provides the use of immune cells overexpressing externally introduced signaling pathway modulators in the treatment or prevention of cancer.

[0318] In this context, signaling pathway regulators are as described above. Furthermore, immune cells are as described above. Specifically, immune cells may include CAR-T cells and TCR-T cells.

[0319] The characteristics of CAR-T cells prepared in one embodiment and designed to overexpress signaling pathway regulators will be described in detail below.

[0320] Preparation of CD19-specific CAR-T cells expressing signal pathway modulators Figure 2 and Figure 54 )

[0321] Generally, CD19-specific CAR-T cells (19bbz) representing the most commonly used and proven traditional second-generation CAR-T cells in clinical practice were prepared and used in experiments. Furthermore, immuno-regulatory cytoplasmic small-sized proteins (hereinafter referred to as signaling pathway regulators), which are expected to enhance the anti-cancer function of CAR-T cells when overexpressed in CAR-T cells, were expressed co-with the CAR gene via lentivirus. As a specific example, two types of CAR-T cells (19bbz#F and 19bbz#C) expressing two immunophilin proteins, FKBP12 (12kDa) and cyclophilin A (18kDa), were prepared, respectively.

[0322] The inventors have designed a signaling pathway modulator that functions as a useful small-sized immune modulator, capable of inhibiting the signaling pathways of various inhibitory immune checkpoint molecules. In one embodiment, a small protein was designed to effectively inhibit pTyr dephosphorylation, which occurs very early and frequently in the signaling pathways of various inhibitory immune checkpoint molecules. Specifically, CAR-T cells (19bbz#S2) overexpressing the N-terminal SH2 domain (12kDa) of SHP-2 in the cytoplasm of CAT-T cells were prepared as a small-sized modulator.

[0323] SHP2 protein (Src homology-2 domain-containing protein tyrosine phosphatase-2), together with SHP1 protein, plays a crucial role in mediating approximately 100 different inhibitory immune checkpoint signals. Based on crystal structure data of SHP2 protein published in 1998, it was noted that the N-terminal SH2 domain of SHP2 protein is a tyrosine dephosphorylase, effectively acting as a "lid" to block the accessibility of the catalytic active site. The inventors prepared CAR-T cells (19bbz#S2) that overexpressed a small N-terminal SH2 domain in the cytoplasm.

[0324] Furthermore, it was confirmed that when CAR-T cells (19bbz#FCS2) that simultaneously overexpress small signaling pathway regulators with these other immune activation mechanisms are prepared, super-armed CAR-T cells that can exert anti-cancer activity even in an immunosuppressive environment can be prepared through the complementary synergistic effect of these other signaling pathway regulators.

[0325] As shown by FACS analysis, when CAR expression on the cell surface was confirmed by binding of FITC-labeled recombinant CD19 protein to CAR-T cells on day 4 post-lentiviral transduction, CAR production was confirmed as expected in all CAR-T cells. Generally, it is known that the efficiency of genome delivery via lentiviral vectors decreases with increasing transgene size. Therefore, in cases where three types of signaling pathway regulator genes are delivered together with the CAR gene via lentiviral transduction, a relatively low transduction rate was observed compared to transduction with only one type of regulator delivered with the CAR gene. However, in cell culture, specifically 19bbz#FCS2, a CAR(+)-T cell ratio of 40-50% was observed.

[0326] As observed in this embodiment, in order to correct for differences in transduction rates in each CAR-T cell preparation, an appropriate amount of untransduced T cells (non-transduction mock T cells) (i.e., CAR(-)-T cells obtained from the same donor peripheral blood cells through the same culture process, but without lentivirus treatment) were added immediately before in vitro functional analysis, and the "T cell count" and "total T cell count" were equalized in all CAR-T cell samples before reaction with cancer cells.

[0327] In vivo assessment of anti-tumor efficacy of CD 19-specific CAR-T cells (II) Figure 7 )

[0328] The antitumor efficacy of CAR-T cells_19bbz#F, CAR-T cells_19bbz#C, CAR-T cells_19bbz#M, CAR-T cells_19bbz#N, CAR-T cells_19bbz#S1, CAR-T cells_19bbz#S2, CAR-T cells_19bbz#TC, CAR-T cells_19bbz#RG and CAR-T cells_19bbzT was evaluated.

[0329] The results are as follows Figure 7 As shown, the control group treated with conventional second-generation CAR-T cells (19bbz) did not effectively inhibit tumor growth, and therefore rapid tumor growth was observed in all three subjects in the control CAR-T group.

[0330] On the other hand, groups treated with CAR-T cells overexpressing FKBP12 (an immunoglobulin) (19bbz#F) and groups treated with CAR-T cells overexpressing another immunoglobulin and cyclin A (19bbz#C) showed excellent effects in inhibiting tumor progression. These results indicate that overexpression of immunoglobulins is a very useful technique for enhancing the anticancer activity of immune cells.

[0331] Furthermore, in the groups treated with CAR-T cells overexpressing FKBP12 (19bbz#F), CAR-T cells overexpressing the C-terminal MH2 domain of SMAD4 protein (19bbz#M), and CAR-T cells overexpressing N-SKI (19bbz#N), FKBP12, the C-terminal MH2 domain of SMAD4 protein, and N-SKI, as cytoplasmic immune modulators capable of inhibiting TGF-β signaling, also exhibited excellent anti-tumor effects. Therefore, these results demonstrate that overexpression of signaling pathway modulators that block immunosuppressive cytokines is a very useful technique for enhancing the anti-cancer activity of immune cells.

[0332] In the groups treated with CAR-T cells overexpressing the N-SH2 domain of the SHP1 protein (19bbz#S1) and those treated with CAR-T cells overexpressing the N-SH2 domain of the SHP2 protein (19bbz#S2), the N-SH2 domains of both proteins, acting as cytoplasmic immunomodulators capable of blocking various immunosuppressive checkpoint signaling pathways, were also observed to exhibit excellent inhibitory effects on tumor progression. Therefore, these results demonstrate that overexpression of these specific regulators that can inhibit the activity of pTyr phosphatase in various immunosuppressive checkpoint signaling pathways is a very useful technique for enhancing the anticancer activity of immune cells.

[0333] Excellent tumor-progression-inhibiting effects were also observed in the groups treated with CAR-T cells overexpressing TC21 (19bbz#TC) and those treated with CAR-T cells overexpressing RG (19bbz#RG). TC21 and RG are cytoplasmic immune modulators that inhibit cytokinesis, which leads to the loss of target antigens in cancer cells. Therefore, these results indicate that overexpression of these specific modulators that inhibit cytokinesis is a very useful technique for enhancing the anticancer activity of immune cells.

[0334] In the group treated with CAR-T cells (19bbzT) overexpressing the intracellular domain of the TLR4 protein, which possesses a TIR domain (Toll / IL-1 receptor homology domain), excellent efficacy in inhibiting tumor progression was also observed, as an example of a specific regulator belonging to the immune enhancement signaling system. Therefore, these results demonstrate that the technique of overexpressing regulators of immune enhancement signaling is a very useful technique for enhancing the anticancer activity of immune cells.

[0335] In vivo assessment of anti-tumor efficacy of CD 19 specific CAR-T cells (I) Figure 49 and Figure 50 )

[0336] In the specified multiple doses (1×10) 6 3×10 6 and 1×10 7 The antitumor efficacy of CAR-T cells 19bbz and CAR-T cells 19bbz#F was evaluated.

[0337] The results are as follows Figure 50 As shown, the control group treated with conventional second-generation CAR-T cells (19bbz) did not effectively inhibit tumor growth, thus rapid tumor growth was observed. On the other hand, a dose-dependent anti-cancer effect of CAR-T cells was observed in the group treated with CAR-T cells overexpressing FKBP12 (an immunoglobulin) as a cytoplasmic immunomodulator (19bbz#F). Specifically, a high dose (1×10⁻⁶) was used... 7 The group treated with CAR-T cells (19bbz#F) showed a remarkable anti-cancer effect in rapidly controlling the growth of invasive tumors. Therefore, these results indicate that overexpression of the immunoglobulin FKBP12 is a very useful technique for enhancing the anti-cancer activity of immune cells.

[0338] FKBP12 binding to TGF-receptors Figures 51 to 53 )

[0339] Figure 51 The figure shows the co-crystal structures (protein co-crystals) of the TGF-β type 1 receptor kinase domains binding to FKBP12, as revealed by three TGF-β type 1 receptors. This figure is based on data from the following protein databases: TGFβR1 (PDB ID: 1b6c), ACVR1A (PDB ID: 3h9r), and BMPR1B (PDB ID: 3mdy). The gray surface structures represent the kinase domains of the TGF-β type 1 receptor, and the red banded backbone structures correspond to the FKBP12 protein. Figure 51As shown, the binding sites of FKBP12 in these three TGF-β type 1 receptor proteins are structurally highly conserved. In fact, this is very consistent with the mechanism by which FKBP12 binding negatively affects TGF-β signaling in all seven TGF-β type 1 receptors identified to date. These structures consistently reveal the fact that FKBP12 binding spatially blocks the phosphorylation site of the TGF-β type 2 receptor.

[0340] Figure 52 The four aromatic amino acids (aromatic residues) of FKBP12 are shown, which participate in the binding of FKBP12 to the type 1 receptor of TGF-β via a pivoting mechanism, as illustrated in the co-crystal structures of the three type 1 receptors of TGF-β. The aromatic amino acids of FKBP12 considered crucial for binding are tyrosine at position 27, phenylalanine at position 47, tryptophan at position 60, and phenylalanine at position 100, and their positions are highly conserved in the three-dimensional structure.

[0341] Figure 53 The positions of aromatic amino acids (tyrosine at position 27, phenylalanine at position 47, tryptophan at position 60, and phenylalanine at position 100) that are involved in the binding of TGF-β to the type 1 receptor in a pivotal manner are shown on the amino acid sequence of the FKBP12 protein.

[0342] Characterization of CD 19-specific CAR-T cell surface CAR expression by immunofluorescence microscopy Figure 55 )

[0343] For CAR-T, the ability of CAR-T to screen specific target tumor cells depends on the "surface expression level" of CAR molecules. However, when CARs are overexpressed above the appropriate level, "abnormal CAR clustering without antigen (Ag)" occurs, which is usually caused by inherent aggregation. In this case, CAR molecules may not be uniformly distributed on the cell surface, but exhibit discontinuity, repetitive aggregation, and absence (punctate phenotype). This phenomenon has been reported to be associated with antigen-independent T cell signaling, leading to an exhausted T cell phenotype that loses the effector functions required for antitumor efficacy.

[0344] In this embodiment, the aim was to investigate whether the anticipated and forced expression of CARs with cytoplasmic immunomodulators via lentiviruses would lead to "CAR clustering in the absence of antigen" and aberrant CAR distribution (discontinuous distribution) (punctate phenotype). For this purpose, CAR-T cells were immunofluorescently analyzed using a Cy3-labeled goat anti-mouse IgG antibody to label CD19-specific CARs. CAR-T cells obtained from the culture were stained on day 7 following initial stimulation. As the results showed, CAR molecules were confirmed to be uniformly distributed on the cell surface of these CAR T cells, which were considered to be in a stable state of CAR expression sufficient to represent a representative distribution of CARs.

[0345] Immunofluorescence analysis confirms CAR-mediated interaction between CAR-T cells and target tumor cells Figure 56 )

[0346] In this embodiment, to confirm whether the prepared CAR-T cells directly interact with CD19-positive cancer cells through CAR molecules on the surface of CAR-T cells, the CAR-T cells were co-incubated with Daudi cells expressing eGFP for 1 hour to show green fluorescence. Subsequently, the CAR molecules were immunostained on a glass slide using Cy3-labeled goat anti-mouse IgG antibody and observed using a fluorescence microscope.

[0347] As shown in the figure, it has been confirmed that the CAR molecule labeled with cyanin 3 (red) actually mediates the close contact between CAR-T cells and the target cancer cells (green) indicated by eGFP fluorescence. Furthermore, the green fluorescence intensity of Daudi cells that closely interact with CAR-T cells is slightly reduced compared to that of free Daudi cells.

[0348] T cell subset analysis of CAR-T cells Figure 57 )

[0349] Recent clinical applications of CAR-T cells in patients and numerous non-clinical animal model studies have demonstrated that the design of adoptive CAR-T tumor therapies should ensure the persistence of anti-cancer immunity to prevent tumor recurrence. Therefore, to elucidate the potential impact of CAR-T cell subsets on clinical outcomes, many studies have been conducted, ultimately highlighting the advantages of T cells rich in relatively high T cell stemness, such as stem cell-like memory T cells (Tscm, CD45RA). + CCR7 + ) and central memory T cells (CD45RA) - CCR7 +This allows cells to proliferate rapidly in vivo and remain viable for a longer period. Therefore, in the field of preparing CAR-T cells for cancer therapy, there is increasing interest in techniques such as using IL-7, IL-15, IL-21, or similar substances instead of IL-2 to induce highly stem cells.

[0350] According to recent literature, in the characterization of engineered GD2-specific CAR-T cells (HA-28z) with higher affinity for the target antigen, phenotypic T cell analysis showed that T cell exhaustion was induced, accompanied by a significant reduction in T cells with high T cell stemness.

[0351] Therefore, the aim was to investigate whether CAR-T cells prepared in one implementation scheme could effectively maintain T cell stemness, which is important for the long-term sustainability of anti-cancer immunity, while simultaneously achieving high-effector T cell activity through overexpression of cytoplasmic immunomodulators. To this end, functional analyses were performed on the prepared CAR-T cells, including tumor cell killing activity, cytokine release activity, and T cell motility. Phenotypic analysis was also conducted to confirm T cell stemness.

[0352] For these analyses, non-transduced control T cells (NTDs) were used as negative control cells. These cells were derived from the same donor and cultured in parallel with CAR-T culture using the same immunobead-based stimulation and T cell expansion procedures, but without lentiviral transduction. When T cell subset analysis was performed on the negative control T cells, the results showed that the effector T cell population, corresponding to differentiated cells with minimal stemness (renewal potential), was approximately half (49%), and the Tscm subset was approximately 47%.

[0353] On the other hand, in the case of control CAR-T cells (19bbz) expressing only conventional second-generation CAR, Tscm was approximately 70%, which was found to remain above NTD by 20% or more. In the case of CAR-T cells (19bbz#S2) overexpressing the N-terminal SH2 domain of SHP2, a T cell population most similar to that of control CAR-T cells (19bbz) expressing only second-generation CAR was observed.

[0354] On the other hand, for CAR-T cells overexpressing immunoglobulins such as FKBP12 or cyclin A (19bbz#F, 19bbz#C), the proportion of the Tscm subset was approximately 76%, which was about 6% higher than that of the second-generation control CAR-T cells (19bbz). Furthermore, for CAR-T cells simultaneously expressing two immunoglobulins and the N-terminal SH2 domain of SHP2 (19bbz#FCS2), the proportion of the Tscm subset was approximately 80%, which was 10% or more higher than that of the second-generation control CAR-T. Therefore, CAR-T cells (19bbz#FCS2) appear to have the highest stemness among the tested cells.

[0355] Therefore, it is shown that overexpression of the cell signaling pathway regulator introduced in this invention is unlikely to lead to a reduction in T cell stemness, which is an ideal characteristic for the anti-cancer efficacy of CAR-T cells.

[0356] Analysis of CD19 expression in target tumor cells Figure 58 )

[0357] To evaluate the in vitro tumor cell killing ability and cytokine release activity of the prepared CD19-specific CAR-T cells, tumor cells expressing CD19 were cultured. Furthermore, the expression level of CD19 was analyzed using APC-labeled mouse anti-CD19 antibody and flow cytometry.

[0358] Based on the average fluorescence units obtained from flow cytometry, the expression level of CD19 in Daudi cells was approximately twice that in Nalm6. Furthermore, it was found that CD19 expression in Nalm6 was approximately three times that of K562-CD19 (CD19 expression levels: Daudi > Nalm6 > K562-DD19).

[0359] Specifically, Daudi cells stably expressing the emerald green fluorescent protein (eGFP) gene were used as target cells for fluorescence microscopy analysis. Furthermore, since Daudi cells also express the firefly luciferase (Fluc) gene, these cells were used for in vitro tumor cell killing activity analysis using bioluminescence assays. Additionally, Daudi-Fluc-Puro cells were used as target cells in previous animal models of hematologic malignancies to evaluate the efficacy of CAR-T therapy in vivo.

[0360] As a negative control lacking CD19 expression, K562 cells were used for other in vitro functional analyses of CD19-specific CAR-T cells. Furthermore, K562-CD19 cells, due to their larger cell size, are easily identifiable from T cells and have also been used as target cells, particularly in experiments using microscopy and flow cytometry.

[0361] Expression levels of FKBP12, cyclophilin A and SH2 domain of N-terminal of SHP2 protein in CAR-T Figure 59 and Figure 60 )

[0362] The overexpression level of exogenous genes of specific immunomodulators transduced into cells via lentiviral vectors was confirmed by comparing with intrinsic levels of related intracellular proteins. Specifically, the assessment was performed using qRT-PCR and antibody immunoblotting analysis.

[0363] like Figure 60 As shown, according to published T lymphocyte proteomic data (Hukelmann et al., Nature Immunology, 2015), the intrinsic intracellular protein levels of FKBP12 were 1,623,586 copies / cell, cyclophilin A was 28,065,984 copies / cell, and SHP2 was 23,530 copies / cell. These data indicate that, based on protein copy number, the intrinsic protein levels are in the order of cyclophilin A >> FKBP12 >> SHP2. Clearly, the actual amount of SHP2 protein, which transduces various inhibitory immune checkpoint signals, is very small.

[0364] Therefore, although all these signaling pathway regulators are expressed via the same promoter as the CAR gene, the fold increase in overexpression varies significantly among them. Specifically, in CAR-T cells (19bbz#F) with overexpression of FKBP12, the forced overexpression of FKBP12 was approximately six-fold higher than the endogenous expression. Furthermore, in CAR-T cells (19bbz#C) with overexpression of cyclin A, the forced overexpression of cyclin was approximately twice the endogenous expression. On the other hand, in CAR-T cells (19bbz#S2) with overexpression of the N-terminal SH2 domain of SHP2, the forced overexpression of the N-terminal SH2 domain of SHP2 was confirmed to be approximately 38-fold higher than the endogenous level of the SHP2 protein.

[0365] As mentioned above, endogenous protein levels vary considerably. In all CAR-T samples analyzed, the proportion of CAR-positive cells was approximately 40%. As shown in the immunoblot images, the degree of overexpression obtained varied significantly compared to endogenous levels. Specifically, in CAR-T cells overexpressing the N-terminal SH2 domain of SHP2 (19bbz#S2), the observed overexpression was very pronounced (at least 30-fold compared to endogenous SHP2 levels). Furthermore, in CAR-T cells overexpressing FKBP12 (19bbz#F), the observed overexpression level was lower than the aforementioned level and ranged from 2 to 7-fold compared to endogenous FKBP12 levels. In CAR-T cells overexpressing cyclin A (19bbz#C), due to the excessively high levels of endogenous protein, the differences caused by overexpression generally fell within the error range of typical immunoblot analysis, making quantification less reliable.

[0366] In CAR-T cells (19bbz#FCS2) simultaneously expressing three types of signaling pathway regulators, the overexpression level of FKBP12 was observed to be similar to that in CAR-T cells (19bbz#F). However, the overexpression level of the N-terminal SH2 domain of SHP2, possibly limited by the requirement for three consecutive autocleavage events to occur successfully on three P2A sites, was about one-third of the expression level observed in CAR-T cells (19bbz#S2), where the overexpression of the N-terminal SH2 domain of SHP2 depends on one autocleavage activity event on one P2A site.

[0367] Determination of IFNy, TNFa and IL-2 secreted by CAR-T cells after antigen stimulation Figures 61 to 63 )

[0368] Generally, it is well known that the anticancer effect of CAR-T cells may derive from i) tumor cell killing activity dependent on perforin / granzyme granules secreted after CAR-T cells form an immune synapse with antigen-positive target tumor cells; ii) tumor cell killing and bystander killing activity dependent on increased Fas ligand expression on the surface of activated CAR-T cells; and iii) tumor cell killing activity based on cytokine-mediated indirect mechanisms (secondary mechanisms).

[0369] IFNγ is one of the key cytokines secreted by activated CAR-T cells, and it has the following functions: i) By increasing the expression of IFNγ receptors on the surface of stromal cells, it induces increased secretion of chemokines (such as IP10 (CXCL10), MIC (CXCL9), etc.), thereby leading to increased immune cell infiltration in tumor tissues, such as T cells, NK cells, dendritic cells, monocytes / macrophages, etc. Furthermore, ii) it may also directly cause tumor stromal cell destruction. Furthermore, iii) it is known to exhibit a growth-inhibiting effect (cytostatic effect) by directly acting on tumor cells. Furthermore, iv) it induces polarization of tumor-associated macrophages and tumor-resident T cells, relieving them from an immunosuppressive state. Therefore, it is well known that it can indirectly confer anticancer activity capable of killing tumor cells, and exert secondary anticancer effects in this way.

[0370] TNFα (tumor necrosis factor alpha) is another important cytokine secreted by activated CAR-T cells. i) It directly induces tumor cell death through signal transduction from TNFα receptors expressed on the surface of tumor cells. ii) TNFα activates effector T cells and NK cells by blocking regulatory T cells. iii) TNFα acts on endothelial cells, inducing tumor microvascular collapse and the destruction of angiogenesis. iv) TNFα induces polarization in M2 macrophages, which lack anticancer activity, while M1 macrophages possess anticancer activity. v) TNFα can attract and activate neutrophils and monocytes to the tumor site, thereby exhibiting anticancer activity. Finally, it is known that TNFα in cancerous tissues interferes with the differentiation of monocytes into immunosuppressive cells by downregulating the expression of IL-13 in eosinophilic-like cells.

[0371] IL-2 (interleukin-2) is another important cytokine secreted by activated CAR-T cells and has been successfully used in anti-cancer immunotherapy. Therefore, in addition to its well-known activity of stimulating T cell proliferation, it can also promote anti-cancer immunity by greatly enhancing the cytolytic activity of NK cells and lymphokine-activated killer cells.

[0372] Therefore, in this embodiment, the cytokine release activities of CAR-T (19bbz#F), CAR-T (19bbz#C), CAR-T (19bbz#S2) and CAR-T (19bbz#FCS2) prepared by the present invention are compared in parallel to test whether the invented CAR-T is superior to the traditional second-generation CAR-T.

[0373] As shown in the figure, when assessing three types of cytokines (IFNγ) Figure 61 ), TNFα Figure 62 ), IL-2 Figure 63 When the cytokine was secreted, no significant level of cytokine secretion was detected in any co-culture of CAR-T cells with K562 cells lacking target CD19 expression 24 hours later.

[0374] On the other hand, after 24 hours, a large number of cytokines were detected in all co-cultures of CAR-T cells and CD19-expressing K562-CD19 cells.

[0375] As the design concept anticipated, overexpression of pro-immunogenic proteins known to be involved in the basic immune activity of T cells, or overexpression of the N-terminal SH2 domain of tyrosine dephosphorylation (SHP2) by three types of CAR-T cells (19bbz#F), CAR-T cells (19lbz#C), and CAR-T cells (19bbz#S2), which can block tyrosine dephosphorylation (SHP2) through immune checkpoint signaling, appeared to slightly increase cytokine secretion compared to conventional control CAR-T cells (19bbz).

[0376] Interestingly, when stimulated by target tumor cells, CAR-T cells (19bbz#FCS2) that simultaneously overexpressed these three signaling pathway regulators showed significantly enhanced cytokine secretion levels compared to conventional control CAR-T cells (19bbz).

[0377] Based on clinical experience with human CAR-T to date, it is well known that solid tumors are more difficult to access tumor sites than hematologic malignancies. Even when CAR-T reaches the tumor site, its efficacy is hindered by several pre-established mechanisms within the immunosuppressive tumor microenvironment (TME). Therefore, it is necessary to design a super-armed CAR-T that can be tolerated even in the presence of immunosuppressive signals within this TME.

[0378] Based on the results of this embodiment, it is expected that CAR-T (19bbz#FCS2) may secrete a large number of cytokines even in an immunosuppressive environment. Therefore, by secreting a large number of cytokines to transform the tumor microenvironment from an immunosuppressive environment to a more immunofriendly one, it may exert a considerable anti-cancer effect.

[0379] In the presence of TGF-β1, IFNγ and TNFα secreted by CAR-T cells were measured based on antigenicity. Figure 64 and Figure 65 )

[0380] Generally speaking, it is well known that high concentrations of TGF-β in tumor tissue can trigger immunosuppressive signals through TGFβ receptors (TGFβRI, TGFβRII) expressed on the surface of T cells, thereby greatly inhibiting the normal functions of T cells, such as T cell activation, proliferation and differentiation.

[0381] However, studies using mouse models have shown that disruption of TGF-β signaling in naïve T cells can induce autoimmune diseases. Therefore, while there is a need to develop a method to inhibit TGF-β signaling to enhance the anti-cancer effect of CAR-T cells, it is best not to design a new device to completely shut down TGF-β signaling in the T cells used, given the potential risks of autoimmunity.

[0382] Within the TGF-β1 concentration range used in this embodiment, the half maximal effective concentration (EC50) for a significant concentration-dependent in vivo response is considered to be 0.04–0.2 ng / mL. Furthermore, the experimental concentration range was set to exceed the upper limit of high concentrations accumulated in pathological environments.

[0383] Furthermore, it is believed that tumor cell microheterogeneity frequently occurs during tumor development and progression. Therefore, to illustrate this, CAR-T activity was tested on three types of cancer cells that differed in CD19 expression levels, target antigens, and cell size. The order of cell size was: K562-CD19 >> Nalm6, Daudi; the order of Ag expression levels was: Daudi > Nalm6 > K562-DD19.

[0384] As an evaluation method, we attempted to assess the secretory activity of key cytokines in the CAR-T anti-cancer immune mechanism. The results showed that the effect of TGF-β1 concentration on CAR-T cells appeared to vary depending on the target cell and the secreted cytokines.

[0385] However, across a wide concentration range of TGFβ1 (0 ng / mL–5 ng / mL), CAR-T cells overexpressing FKBP12 (19bbz#F) showed higher or equal activity compared to conventional second-generation CAR-T cells (19bbz) in all three types of target cell responses.

[0386] In reactions with Nalm6 or K562-CD19, antigen stimulation of CAR-T cells is expected to be less likely and mild due to relatively low antigen expression levels, while the difference in CAR-T activity caused by FKBP12 overexpression is clearly more pronounced. In contrast to the Daudi reaction, antigen stimulation of CAR-T cells can be easily and forcefully ensured due to high levels of antigen expression on the target surface.

[0387] Typically, reduced antigen levels are one of the immune evasive mechanisms employed by cancer cells during tumor progression. Taking this information and results into account, overexpression of small-sized regulatory proteins such as FKBP12 could help modify CAR-T cells to maintain their ability to fight against Ag-low tumor cells, which may potentially drive tumor growth.

[0388] T cells are thought to maintain low basal activity through tetanic signaling (TGFβ-independent dimerization-mediated signaling), in which TGFβRII remains active even in the absence of TGFβ and binds to TGFβRI to induce TGFβ signaling. However, when the receptor isomerization competitive inhibitor FKBP12 is overexpressed, the basal activity of these T cells is suppressed to some extent. Therefore, T cells are relatively easily activated even when responding to cancer cells with low antigen expression levels.

[0389] Confirmation of CAR-T cell migration ability Figure 66 and Figure 67 )

[0390] For adoptive CAR-T cells, the ability to effectively migrate to the tumor site in vivo is an important factor to consider when designing the overall efficacy of CAR-T therapy.

[0391] Generally, the transport of T cells into the TME (tumor microenvironment) is accomplished through a series of tightly controlled steps. It is well known that these steps in T cell transport, particularly integrin-mediated T cell adhesion and downstream events leading to T cell migration, are sensitive to immunophilic proteins such as FKBP12 and cyclophilin A.

[0392] In this embodiment, to compare the in vitro migration activity of CAR-T cells, CAR-T cells were activated using anti-CD3 / CD28 Dynabeads during culture. Debeaded cells were then collected and washed, and suspended in serum-free medium supplemented with 0.25% human serum albumin for transwell migration. Generally, animal cells migrate through the pores of the biological matrix via amoeboid motion.

[0393] As shown in the figure below, the migration ability of CAR(+)T cells was compared by assessing the percentage of CAR(+) cells that migrated to the lower chamber 1 hour prior to migration. Although the total T cell and CAR(+)T cell concentrations of all tested CAR-T cells were adjusted to be the same before migration, the migration ability of a specific CAR-T (19bbz#FCS2) was significantly higher than that of other CAR-T cells.

[0394] Furthermore, T cell migration was assessed in the presence of an EGF, a cytokine known to interfere with T cell infiltration when cytokines are added to the upper chamber. Even under these conditions, as shown in the middle figure, when comparing the number of CAR(+) cells migrating to the lower chamber, a specific CAR-T cell (19bbz#FCS2) was found to have significantly superior migration ability compared to other CAR-T cells.

[0395] On the other hand, as shown in the figure above, we found that CAR(-) cells in the CAR-T (19bbz#FCS2) sample did not exhibit superior migration ability. Therefore, it was confirmed that the high migration ability in these CAR-T (19bbz#FCS2) samples was limited to the activity of CAR(+) cells overexpressing the three cytoplasmic regulators.

[0396] Similarly, it can be clearly observed that in photographs taken after co-culturing with target cells in 96-well plates for 48 hours, CAR-T cells (19bbz#FCS2) simultaneously overexpressing three types of cytoplasmic modulators exhibit significantly superior intrinsic motility. Figure 67 ).

[0397] As shown in the photo, the corresponding CAR-T cells are most active when co-cultured with K562-CD19 cells. K562-CD19 cells can stimulate T cells by expressing the antigen CD19 on their surface.

[0398] In vitro tumor cell killing activity of CD19-specific CAR-T cells ( Figure 68 )

[0399] In this embodiment, the following two different methods were used to evaluate the tumor cell killing activity of CAR-T cells.

[0400] When evaluating K562-CD19 cells, as a method for observing tumor cell lysis, a method was used to quantify TDA (2,2':6',2"-terpyridine-6,6"-dicarboxylic acid) secreted into the culture medium by cell lysis using TRF (time-resolved fluorescence).

[0401] Therefore, as shown in the bottom chart, the results of CAR(+)-T cell assessment of tumor cell killing activity were found to be slightly consistent with the results of the above assessment of cytokine secretion activity.

[0402] Three types of CAR-T cells—CAR-T cells (19bbz#F), CAR-T-cells (19bbz#C), and CAR-T cells (19bbz#S2)—overexpressed known immunogenic proteins involved in the basic immune activity of T cells, or overexpressed the N-terminal SH2 domain of SHP2, which can inhibit pTyr dephosphorylation via immune checkpoint signaling. Compared with conventional control CAR-T cells (19bbz), CAR-T cells (19bbz#F), CAR-T-cells (19bbz#C), and CAR-T cells (19bbz#S2) appeared to have relatively high tumor cell killing activity.

[0403] On the other hand, it has been confirmed that CAR-T cells (19bbz#FCS2) simultaneously overexpress these three types of small-sized cytoplasmic immune modulators, and their activity is significantly higher compared with conventional control CAR-T cells (19bbz).

[0404] In another group, the cytotoxic activity of CAR-T cells against tumor cells expressing luciferase Daudi cells was examined. The degree of bioluminescence reduction was used to compare the tumor cell lysis of CAR(+)-T cells. As shown in the figure above, the results were similar to those for the assessment of Eu-TDA release.

[0405] Compared to conventional control CAR-T cells (19bbz), three types of CAR-T cells overexpressing either immunoglobulin or the N-terminal SH2 domain of SHP2—CAR-T cells (19bbz#F), CAR-T cells (19bbz#C), and CAR-T cells (19bbz#S2)—exhibited slightly higher tumor cell killing activity. In particular, CAR-T cells (19bbz#FCS2) were confirmed to exhibit the highest activity across the entire E:T ratio assay.

[0406] Preparation of Her2-specific CAR-T cells Figure 3 and Figure 69 )

[0407] As another example of the present invention, for CAR-T therapy of Her2-positive cancer cells, “Her2-specific CAR-T cells (Hbbz)” representing conventional second-generation CAR-T were prepared. Furthermore, two types of CAR-T cells (Hbbz#F and Hbbz#C) were prepared, expressing two known immunophile proteins, FKBP12 (12 kDa) and cyclophilin A (18 kDa), respectively, which are expected to enhance the anti-cancer function of the prepared CAR-T cells.

[0408] In addition, CAR-T cells overexpressing the N-terminal SH2 domain of SHP2 (Hbbz#S2) were prepared, which can inhibit pTyr dephosphorylation (tyrosine dephosphorylation) through immune checkpoint signaling. Furthermore, CAR-T cells simultaneously overexpressing these three signaling pathway regulators (Hbbz#FCS2) were also prepared. CAR expression levels measured on day 4 after lentiviral transduction are shown below. Figure 69 As shown. Subsequently, through the following expanded culture, the proportion of CAR(+) cells in CAR-T (Hbbz#FCS2) was observed to reach 40-50%.

[0409] Immunofluorescence microscopy reveals the characteristics of CAR expression on the surface of Her2-specific CAR-T cells. Figure 70 )

[0410] The results show that when expressed in the prepared CAR-T cells, CAR molecules are evenly distributed on the cell surface.

[0411] Measurement of IFNγ, TNFα and IL-2 secreted by Her2-specific CAR-T cells after antigen stimulation ( Figures 71 to 73 )

[0412] In this embodiment, the cytokine release activities of CAR-T (Hbbz#F), CAR-T (Hbbz#C), CAR-T (Hbbz#S2), and CAR-T (Hbbz#FCS2) prepared by the present invention were compared in parallel to test whether the invented CAR-T is superior to the traditional second-generation CAR-T.

[0413] As shown in the figure, when assessing the secretion of three types of cytokines (IFNγ, TNFα, and IL-2), after 24 hours, almost no significant levels of TNFα and IL-2 cytokine secretion were detected in all co-cultures of CAR-T cells with Daudi cells lacking target Her2 expression. However, for IFNγ, unlike TNFα and IL-2, the amount of IFNγ secreted corresponded to less than one-third of the amount secreted by SKBR3 cells expressing the target antigen Her2. This is thought to be because Daudi cells do not express Her2, but instead express some costimulatory molecules as B lymphocytes. Therefore, Daudi cells may serve as potential antigen-presenting cells from different populations, capable of inducing T cell activation in certain TCRs that respond to specific histocompatibility antigens.

[0414] On the other hand, after 24 hours, a large number of cytokines were detected in all co-cultures of CAR-T cells and SKBR3 cells expressing Her2.

[0415] Therefore, compared with the traditional second-generation control CAR-T cells (Hbbz), the three types of CAR-T cells: CAR-T cells (Hbbz#F), CAR-T cells (Hbbz#C) and CAR-T cells (Hbbz#S2) showed almost the same or slightly increased cytokine secretion.

[0416] Specifically, after stimulation by target tumor cells, the cytokine secretion of CAR-T cells (Hbbz#FCS2) was significantly enhanced compared with that of conventional second-generation control CAR-T cells (Hbbz).

[0417] In vitro migration of Her2-specific CAR-T cells ( Figure 74 )

[0418] In this embodiment, to compare the in vitro migration activity of Her2-specific CAR-T cells, CAR-T cells were activated using anti-CD3 / CD28 Dynabeads during culture, and then debeaded cells were collected, washed, and suspended in serum-free medium supplemented with 0.25% human serum albumin for transwell migration.

[0419] Therefore, although the total T cell and CAR(+) T cell concentrations of all subjects were adjusted to be the same before migration, a particular CAR-T (Hbbz#FCS2) showed significantly higher migration ability than other CAR-Ts.

[0420] In vitro tumor-killing activity of Her2-specific CAR-T cells ( Figure 75 )

[0421] It has been confirmed that, compared with the traditional second-generation control CAR-T cells (Hbbz), all three types of CAR-T cells—CAR-T cells (Hbbz#F), CAR-T cells (Hbbz#C), and CAR-T cells (Hbbz#S2)—exhibited slightly higher tumor cell killing activity.

[0422] On the other hand, it was confirmed that CAR-T cells (Hbbz#FCS2) that simultaneously overexpressed these three regulators exhibited significantly high activity across the entire E:T ratio test range.

[0423] In vivo assessment of the antitumor efficacy of Her2-specific CAR-T cells ( Figure 76 )

[0424] The usefulness of the CAR-T cell technology introduced in this invention was tested in a solid tumor model. SKBR3 Luc cells (2 × 10⁶ cells per subject) were used. 6 1 × 10⁻⁶ cells were injected into immunocompromised NSGA mice to induce intraperitoneal xenograft tumors for 14 days. Ten subjects in each group received the same number of xenograft cells intravenously.6 Each designated CAR-T cell was selected. Subsequently, tumor burden was monitored weekly using an IVIS device via bioluminescence imaging.

[0425] Therefore, as shown in the figure, the group treated with conventional second-generation control CAR-T cells (Hbbz) did not effectively inhibit the growth of peritoneal tumors. On the other hand, in the group treated with CAR-T cells overexpressing the immunoglobulin FKBP12 (Hbbz#F), a signaling pathway regulator, and in the group treated with CAR-T cells simultaneously overexpressing three signaling pathway regulators (Hbbz#FCS2), the effects of inhibiting and eliminating intra-abdominal solid tumor growth could be rapidly and clearly observed.

[0426] Furthermore, the results were consistent with those observed when evaluating several in vitro CAR-T activities (cytokine secretion, in vitro migration, and in vitro tumor cell killing ability), as illustrated in other examples. Additionally, CAR-T cells (Hbbz#FCS2) simultaneously overexpressing three signaling pathway regulators were confirmed to exhibit significantly superior antitumor activity against solid cancers established in animal model studies in vivo.

[0427] Furthermore, the results were consistent with those observed in the antitumor efficacy assessment of hematologic malignancy models in other examples, and it was also confirmed that even in the antitumor efficacy assessed in solid tumor animal models, the group treated with CAR-T cells (Hbbz#F) that overexpressed only one immunoglobulin FKBP12 showed superior anticancer activity compared to conventional second-generation control CAR-T cells (Hbbz).

[0428] Furthermore, in the solid tumor model results of this embodiment, CAR-T cells (Hbbz#FCS2) that simultaneously overexpress three signaling pathway regulators showed significantly superior anti-cancer effects in vivo, which is due to the enhanced expected synergistic effect mediated by multiple signaling pathway regulators based on different mechanisms.

[0429] Preparation of PSMA-specific CAR-T cells Figure 6 and Figure 77 )

[0430] As another embodiment of the present invention, for CAR-T targeting Her2-positive cancer cells, “PSMA-specific CAR-T cells (Pbbz)” representing conventional second-generation CAR-T cells were prepared. Furthermore, in this case, two types of CAR-T cells (Pbbz#F and Pbbz#C) were prepared, expressing two immunophile proteins, FKBP12 (12kDa) and cyclophilin A (18kDa), respectively, which are expected to enhance anti-cancer effects.

[0431] In addition, CAR-T cells overexpressing the N-terminal SH2 domain of SHP2 (Pbbz#S2) were prepared, which can inhibit pTyr dephosphorylation (tyrosine dephosphorylation) through immune checkpoint signaling. Furthermore, CAR-T cells simultaneously overexpressing these three signaling pathway regulators (Pbbz#FCS2) were also prepared. CAR expression rates measured on day 4 after lentiviral transduction are shown below. Figure 77 As shown. Subsequently, through the following expanded culture, it was observed that the proportion of CAR(+) cells in CAR-T cells (Pbbz#FCS2) reached 40-50%.

[0432] Immunofluorescence microscopy reveals the characteristics of CAR expression on the surface of PSMA-specific CAR-T cells. Figure 70 )

[0433] As shown in the figure, when expressed in the prepared CAR-T cells, CAR molecules are evenly distributed on the cell surface.

[0434] T cell subset analysis of PSMA-specific CAR(+)-T cells Figure 79 )

[0435] For NTD (non-transduced control T cells), the effector T cell population corresponding to differentiated cells with minimal stemness (renewal potential) was almost half (47%), and the Tscm subset was approximately 45%. Conversely, in control CAR-T cells (Pbbz) expressing only conventional second-generation CAR, the Tscm ratio was approximately 53%, about 8% higher than in NTD.

[0436] The study found that CAR-T cells overexpressing the N-terminal SH2 domain of SHP2 (Pbbz#S2) had the most similar T cell population to control CAR-T cells expressing only conventional second-generation CARs (Pbbz).

[0437] On the other hand, in CAR-T cells overexpressing immunoglobulins such as FKBP12 or cyclin A (Pbbz#F, Pbbz##C), the proportion of the Tscm subset was 63% and 64%, respectively, and was found to be about 10% higher than that of the second-generation control CAR-T cells (Pbbz). In CAR-T cells simultaneously expressing two immunoglobulins and the N-terminal SH2 domain of SHP2 (Pbbz#FCS2), the proportion of the Tscm subset was about 66%, and was found to be at least 10% higher than that of the second-generation control CAR-T cells, confirming that it had the highest stemness.

[0438] The results of the T cell subset analysis in this embodiment show that even when the functional activity of CAR-T cells is enhanced by overexpression of cell signaling pathway regulators, T cell stemness (the preferred characteristic of therapeutic CAR-T cells) is not impaired and remains good.

[0439] Phenotypic analysis of T cell exhaustion Figure 80 )

[0440] Flow cytometry analysis using specific antibodies showed that within the CAR(+) cell phylum, the proportion of PD-1 positive cells was within the range of 10+ / -3%, the proportion of LAG-3 positive cells was less than 3%, the proportion of TIGIT positive cells was within the range of 12+ / -3%, and the proportion of CTLA-4 positive cells was around 2-3%. Typically, the expression levels of these T cell exhaustion markers are not high. Therefore, this indicates that the overexpression of the signaling pathway regulators introduced in this invention is unlikely to be accompanied by CAR-T cell exhaustion.

[0441] In vitro migration of PSMA-specific CAR-T cells ( Figure 81 )

[0442] As shown in the figure below, to compare the motility of CAR(+)-T cells, the total number of cells that migrated to the lower chamber 1 hour ago and the CAR(+)% of the migrated cells were measured. Although the total T cell and CAR(+)T cell concentrations of all tested CAR-T cells were adjusted to be the same before migration, the migration ability of the specific CAR-T (Pbbz#FCS2) was significantly higher than that of other CAR-T cells.

[0443] On the other hand, no significant difference was found between CAR-T samples in terms of the number of CAR(-)-T cells that migrated to the lower chamber at the same time (top). This suggests that the high migration capacity in these CAR-T (Pbbz#FCS2) samples is limited to CAR(+) cells that overexpress all three signaling pathway regulators.

[0444] In vitro tumor cell killing activity of PSMA-specific CAR-T cells ( Figure 82 and Figure 83 )

[0445] For example, the 2-hour time point of the reaction ( Figure 82 ) and the evaluation results at the 3-hour time point ( Figure 83 As shown in the figure, the tumor cell killing activity of all three types of CAR-T cells (Pbbz#F, Pbbz#C, and Pbbz#S2) was significantly higher than that of conventional second-generation control CAR-T lymphocytes (Pbbz).

[0446] On the other hand, it has been confirmed that CAR-T cells (Pbbz#FCS2) that simultaneously overexpress these three small-sized cytoplasmic immune modulators exhibit significantly enhanced activity across the entire E:T ratio test range.

[0447] Example

[0448] Preferred embodiments are given below to aid in understanding the invention. However, the following embodiments are provided merely for easier understanding of the invention, and the scope of the invention is not limited to these embodiments.

[0449] I. Preparation of CAR-T cells with signaling pathway modulators

[0450] Example 1. Design of fusion protein constructs

[0451] Example 1.1. FKBP12 Structural Design

[0452] The construct containing the chimeric antigen receptor and FKBP12 was designed to include a signal peptide, an antigen-binding domain, a hinge and transmembrane domain, an intracellular signaling domain, a self-cleaving peptide, and FKBP12.

[0453] For example, such as Figure 1 As shown, its design includes a signal peptide (ss), an antigen-binding domain (CD19 scFv), a hinge and transmembrane domain (H+TM), intracellular signaling domains (4-1BB and CD3Z), a self-cleaving peptide (P2A), and FKBP12; its overall structure is called "19bbz#F". Conversely, the chimeric antigen receptor containing the signal peptide (ss), antigen-binding domain (CD19 scFv), hinge and transmembrane domain (H+TM), and intracellular signaling domains (4-1BB and CD3ζ) is called "19bbz".

[0454] Specifically, the CD19-specific CAR was constructed by linking i) a nucleotide sequence from the CD8 signal peptide (Uniprot: P01732-1, 1-21aa, SEQ ID NO: 1) and ii) a nucleotide sequence from the scFv fragment of the CD19-specific FMC63 antibody (Nichoison et al., 1997) (SEQ ID NO: 3), and then linking nucleotide sequences derived from: iii) the CD8 hinge transmembrane domain (Uniprot: P01732-1, 138-206aa, SEQ ID NO: 5), iv) the cytoplasmic region of the human 4-1BB costimulatory factor domain (Uniprot: Q07011, 214-255aa, SEQ ID NO: 7), and v) the human CD3ζ intracellular domain (GenBank: NP000725.1, 52-163aa, SEQ ID NO: 9).

[0455] Example 1.2 Design of carrier constructs including other signaling pathway modulators

[0456] In the same manner as in Example 1.1, it is designed to include cyclophilin A (#C), the C-terminal MH2 domain of SMAD4 protein (#M), N-SKI (#N), the N-terminal SH2 domain of SHP-1 protein (#S1), the N-terminal NH2 domain of SHP-2 protein (#S2), TC21 (#TC), RhoG (#RG), and the N-terminal SH2 domain of FKBP12 / cyclophilin A / SHP-2 protein (#F#C#S2, abbreviated as #FCS2), instead of FKBP12. These are respectively referred to as "19bbz#C", "19bbz#M", "19bbz#N", "19bbz#S1", "19bbz#S2", "19bbz#TC", "19bbz#RG", and "19bbz#FCS2". Furthermore, the construct was prepared in the same manner as in Example 1.1, except that FKBP12 was replaced by a TLR4 intracellular domain (T) without a self-cleaving sequence, and was termed "19bbzT". Figures 1 to 2 ).

[0457] Example 2. Construction of a vector encoding a fusion protein

[0458] Example 2.1. Construction of a vector containing FKBP12

[0459] The pPVLV5 vector was used as the vector, which is a third-generation self-inactivated lentiviral vector containing the human elongation factor α (EF1α: 531bp or 212bp) promoter. The gene encoding the chimeric antigen receptor (including the signal peptide (ss), the CD19-specific single-stranded variable fragment (CD19 scFv), the hinge and transmembrane domain of human CD8 (H+TM), intracellular signaling domains (4-1BB and CD3ζ), and the self-cleaving peptide (P2A) and FKBP12) was inserted into the pPVLV5 vector, named "p_19bbz#F".

[0460] In addition, a gene encoding a chimeric antigen receptor was inserted into the pPVLV5 vector. This chimeric antigen receptor includes a signal peptide (ss), a single-stranded variable fragment that specifically binds to CD19 (CD19 scFv), the hinge and transmembrane domain of human CD8 (H+TM), and intracellular signaling domains (4-1BB and CD3ζ), and is referred to as "p_19bbz".

[0461] The amino acid and nucleotide sequences of the chimeric antigen receptors used in the experiment are shown in Table 1 below, and the amino acid and nucleotide sequences of FKBP12(#F) are shown in Table 2.

[0462] Table 1

[0463]

[0464] Table 2

[0465]

[0466]

[0467] As a vector, a third-generation lentiviral vector modified with pLenti-EF1a-Backbone(NG)(Addgene, #27963) was used, and the gene encoding CAR was loaded into the vector using DNA assembly Master Mix(NEB, #E2621).

[0468] Furthermore, to express the immunomodulatory protein in the cytoplasm, a nucleotide sequence encoding the immunomodulatory protein was inserted downstream of the CAR sequence. The immunomodulatory protein is FKBP12 (#F, SEQ ID NO:14).

[0469] Example 2.2. Construction of vectors encoding CAR and other signaling pathway regulator genes

[0470] Vectors were constructed using lentiviral vectors in the same manner as in Example 2.1, including cyclophilin A (#C, SEQ ID NO:25), the C-terminal MH2 domain of SMAD4 protein (#M, SEQ ID NO:21), N-SKI (#N, SEQ ID NO:23), the N-SH2 domain of SHP-1 protein (#S1, SEQ ID NO:27), the N-SH2 domain of SHP-2 protein (#S2, SEQ ID NO:29), TC21 (#TC, SEQ ID NO:33), RhoG (#RG, SEQ ID NO:35), or the N-SH2 domain of FKBP12 / cyclophilin A / SHP2 protein (#FCS2, SEQ ID NO:14, SEQ ID NO:25, SEQ ID NO:29). These are respectively named "p_19bbz#F", "p_19bbz#C", "p_19bbz#M", "p_19bbz#N", "p_19bbz#S1", "p_19bbz#S2", "p_19bbz#TC", "p_19bbz#RG", and "p_19-bbz#F#CS#2". Furthermore, in the same manner as in Example 2.1, a vector comprising the TLR4 cytoplasmic domain (T, SEQ ID NO:31) instead of FKBP12 without the self-cutting sequence was constructed and named "p_19bbzT".

[0471] The amino acid and nucleotide sequences corresponding to the proteins or fragments used to prepare the p_19bbz#C, p_19bbz#M, p_19bbz#N, p_19bbz#S1, p_19bbz#S2, "p_19bbz#TC", p_19bbz#RG, p_19bbz#F#C#S2, and p_19bbzT vectors are shown in Tables 3 to 10 below. Furthermore, the structural diagram of each plasmid is shown below. Figures 8 to 18 As shown.

[0472] Table 3

[0473]

[0474]

[0475] Table 4

[0476]

[0477] Table 5

[0478]

[0479]

[0480] Table 6

[0481]

[0482] Table 7

[0483]

[0484] Table 8

[0485]

[0486]

[0487] Table 9

[0488]

[0489] Table 10

[0490]

[0491] Example 3. Generation of Lentivirals

[0492] Example 3.1 Generation of lentiviruses containing CAR and FKBP12 genes

[0493] To produce each batch of lentivirus, an optimized lentivirus production kit, the LV-MAX Lentiviral Production System (Gibco, #A35684), was used. Lentiviral viruses were prepared by co-transfecting pMD2.G (Addgene, #12259), pMDLg / pRRE (Addgene, #12251), and pRSV-Rev (Addgene, #12253) with the constructed third-generation lentivirus transfer plasmid, modified lentivirus envelope plasmid, and packaging plasmid. After transforming the vector into HEK293F cells adapted to serum-free medium and culturing for 50 to 54 hours, the virus-containing medium was collected and concentrated using Lenti-X concentrator (Lenti-X concentrator, Takara, #631232). The concentrated virus was stored at -80°C for later use. The resulting lentivirus was designated "Lenti_19bbz#F".

[0494] Example 3.2 Generation of lentiviruses containing CAR and other signaling pathway regulator genes

[0495] Lentivirals were generated using the p_19bbz#C, p_19bbz#M, p_19-bbz#N, p_19bbz#S1, p_19bbz#S2, p_19bbz#TC, p_19bbz#RG, "p_19bbz#F#CS#2", and p_19bbzT lentivirus transfer plasmids prepared in Example 2.2, in the same manner as in Example 3.1. These were respectively named "Lenti_19bbz#C", "Lenti_19bbz#M", "Lenti_19bbz#N", "Lenti_19bbz#S1", "Lenti_19bbz#S2", "Lenti_19bbz#TC", "Lenti_19bbz#RG", "Lenti_19bbz#F#C#S2", and "Lenti_19bbzT".

[0496] Example 4. Construction of CAR-T cells

[0497] Example 4.1. Construction of CAR-T cells infused with FKBP12

[0498] CAR-T cells were constructed using peripheral blood mononuclear cells (PBMCs) from healthy donors. Adherent cells were removed from the PBMCs and then cultured in X-VIVO 15 medium (Lonza) containing 200 IU / mL rhIL-2 (BMI, Korea) and anti-CD3 / CD28 Dynabeads (Life Technologies) to induce T cell activation (beads:CD3+ T cells = 1:1).

[0499] Two days later, the two lentiviruses (Lenti_19bbz#F and Lenti_19bbz) generated in Example 3.1 were treated with activated T cells. The next day, the supernatant, including the lentiviruses, was separated by centrifugation. The T cells were then centrifuged at 3 × 10⁻⁶. 5 Cells / mL were resuspended in X-VIVO 15 medium containing 200 IU / mL rhIL-2 and cultured for 3 days. Subsequently, anti-CD3 / CD28 Dynabeads were isolated and cultured, and T cells were proliferated by changing the medium to fresh rhIL-2 every 2 or 3 days. In this case, CAR-T cells prepared using Lenti_19bbz#F are referred to as "CAR-T cells_19bbz#F", while PAR-T cells prepared using Lenti_19bbz are referred to as "CAR-T cells_19bbz".

[0500] Example 4.2. Construction of CAR-T cells infused with other signaling pathway regulators

[0501] In the same manner as in Example 4.1, the lentiviruses “Lenti_19bbz#C”, “Lenti_19bbz#M”, “Lenti_19bbz#N”, “Lenti_19bbz#S1”, “Lenti_19bbz#S2”, “Lenti_19bbz#TC”, “Lenti_19bbz#RG”, “Lenti_19bbz#F#C#S2” and “Lenti_19bbzT” prepared in Example 3.2 were used for transformation and... CAR-T cells were constructed and named "CAR-T cell_19bbz#C", "CAR-T cell_19bbz#M", "CAR-T cell_19bbz#N", "CAR-T cell_19bbz#S1", "CAR-T cell_19bbz#S2", "CAR-T cell_19bbz#TC", "CAR-T cell_19bbz#RG", "CAR-T cell_19bbz#FCS2" and "CAR-T cell_19bbzT".

[0502] Example 5. Preparation of Her2-specific CAR-T

[0503] The preparation method of Her2-specific CAR-T is the same as that in Examples 1 to 4, except that the antigen-binding CD19 scFv domain is replaced by Her2 scFv.

[0504] Specifically, the Her2-specific CAR was constructed by linking i) a nucleotide sequence derived from the CD8 signal peptide (Uniprot: P01732-1, 1-21aa, SEQ ID NO: 1), and ii) a nucleotide sequence derived from the scFv fragment (SEQ ID NO: 45) (SEQ ID NO: 46) of the Her2-specific antibody trastuzumab (IMGT database), and then linking iii) the CD8 hinge transmembrane domain (SEQ ID NO: 5), iv) the cytoplasmic region of the human 4-1BB costimulatory factor domain (SEQ ID NO: 7), and v) a nucleotide sequence derived from the human CD3ζ intracellular domain (SEQ ID NO: 9) (Table 11).

[0505] In addition, various signaling pathway regulators expressed in conjunction with Her2-specific CARs were prepared. Specifically, Her2-specific CAR-Ts expressing FKBP12 protein, cyclin A, and SHP2-nSH2 were prepared, respectively. Furthermore, CAR-Ts expressing all FKBP12 proteins, cyclin A, and SHP2-nSH2 were also prepared. The vectors used in this case include... Figures 19 to 23 As shown.

[0506] Table 11

[0507]

[0508]

[0509] Example 6. Preparation of PSMA-specific CAR-T

[0510] PSMA-specific CAR-Ts were prepared in the same manner as in Examples 1 to 4, except that the antigen-binding CD19 scFv domain was replaced by PSMAscFv.

[0511] Specifically, the PSMA-specific CAR was constructed by linking: i) the nucleotide sequence of a signal peptide derived from the Ig heavy chain (GenBank AAA51634.1, 1-19aa, SEQ ID NO:52), and ii) the nucleotide sequence of an scFv fragment (SEQ ID NO:55) derived from a PSMA-specific J591 antibody (WO2002 / 098897A2), and then linking iii) the CD8 hinge transmembrane domain (SEQ ID NO:5), iv) the cytoplasmic region of the human 4-1BB costimulatory factor domain (SEQ ID NO:7) and v) the nucleotide sequence derived from the human CD3ζ intracellular domain (SEQ ID NO:9) (Table 12).

[0512] In addition, various signaling pathway regulators expressed in conjunction with PSMA-specific CARs were prepared. Specifically, PSMA-specific CAR-T cells expressing FKBP12 protein, cyclin A, and SHP2-nSH2 were prepared, respectively. Furthermore, CAR-T cells expressing all FKBP12 proteins, cyclin A, and SHP2-nSH2 were also prepared. The vectors used in this case include... Figures 34 to 38 As shown.

[0513] Table 12

[0514]

[0515]

[0516] Example 7. Preparation of CD43-specific CAR-T

[0517] The preparation method of CD43-specific CAR-T cells was the same as in Examples 1 to 4, except that the antigen-binding CD19 scFv domain was replaced by CD43 scFv. The sequences used in this case are shown in Table 13 below. In addition, various signaling pathway regulators expressed in conjunction with the CD43-specific CAR were also prepared. Specifically, CD43-specific CAR-T cells expressing FKBP12 protein, cyclin A, and SHP2-nSH2 were prepared, respectively. Furthermore, CAR-T cells expressing all FKBP12 proteins, cyclin A, and SHP2-nSH2 were also prepared. The vectors used in this case are as follows... Figures 24 to 28 As shown.

[0518] Table 13

[0519]

[0520]

[0521] Example 8. Preparation of CD47-specific CAR-T

[0522] The preparation method of CD47-specific CAR-T cells was the same as in Examples 1 to 4, except that the antigen-binding CD19 scFv domain was replaced by CD47 scFv. The sequences used in this example are shown in Table 14 below. In addition, various signaling pathway regulators expressed in conjunction with the CD47-specific CAR were also prepared. Specifically, CD47-specific CAR-T cells expressing FKBP12 protein, cyclin A, and SHP2-nSH2 were prepared, respectively. Furthermore, CAR-T cells expressing all FKBP12 proteins, cyclin A, and SHP2-nSH2 were also prepared. The vectors used in this case are as follows... Figures 29 to 33 As shown.

[0523] Table 14

[0524]

[0525]

[0526] Example 9. Preparation of HERV-E TCR-T

[0527] The preparation of HERV-E-specific TCR-T cells targeting HLA-A11-specific HERV-E epitopes was performed in the same manner as in Examples 1 to 4, except that the CAR region was replaced by a HERV-E-specific TCR. Various signaling pathway regulators expressed in conjunction with the HERV-E-specific TCRs were also prepared. Specifically, HERV-E-specific TCR-T cells expressing FKBP12 protein, cyclin A, and SHP2-nSH2 were prepared, respectively. Furthermore, TCR-T cells expressing all FKBP12 proteins, cyclin A, and SHP2-nSH2 were prepared. The sequences used in this example are shown in Tables 15 to 24 below. The vector used in this case is as follows... Figures 39 to 43 As shown.

[0528] Table 15

[0529]

[0530]

[0531] Table 16

[0532]

[0533]

[0534] Table 17

[0535]

[0536] Table 18

[0537]

[0538]

[0539] Table 19

[0540]

[0541] Table 20

[0542]

[0543]

[0544] Table 21

[0545]

[0546] Table 22

[0547]

[0548]

[0549] Table 23

[0550]

[0551]

[0552] Table 24

[0553]

[0554]

[0555] Example 10. Preparation of NY-ESO-1 specific TCR-T

[0556] NY-ESO-1-specific TCR-Ts targeting HLA-A2-specific NY-ESO-1 epitopes were prepared in the same manner as in Examples 1 to 4, except that the CAR region was replaced by an NY-ESO-1-specific TCR. Various signaling pathway regulators expressed in conjunction with the NY-ESO-1-specific TCRs were also prepared. Specifically, NY-ESO-1-specific TCR-Ts expressing FKBP12 protein, cyclin A, and SHP2-nSH2 were prepared, respectively. Furthermore, TCR-Ts expressing all FKBP12 proteins, cyclin A, and SHP2-nSH2 were prepared. The sequences used in this example are shown in Tables 25 to 34 below. The vectors used in this case are as follows... Figures 44 to 48 As shown.

[0557] Table 25

[0558]

[0559] Table 26

[0560]

[0561] Table 27

[0562]

[0563]

[0564] Table 28

[0565]

[0566]

[0567] Table 29

[0568]

[0569] Table 30

[0570]

[0571]

[0572] Table 31

[0573]

[0574]

[0575] Table 32

[0576]

[0577]

[0578] Table 33

[0579]

[0580] Table 34

[0581]

[0582]

[0583]

[0584] II. Confirmation of CAR-T activity after delivery of signaling pathway modulators: in vitro

[0585] Experimental Example 1. Confirmation of Antigen Receptor Expression in CAR-T Cells

[0586] Experimental Example 1.1. Evaluation of CAR Expression by Flow Cytometry

[0587] To confirm the expression of CAR on the cell surface of CAR-T cells and the antigen expression of target tumor cells, CAR-T lymphocytes were collected 4 days after viral infection and stained with FITC-labeled recombinant human CD19 (Acro, #CD9-HF2H2) to analyze the expression of CD19-specific CAR. Figure 54 Furthermore, staining with Alexa Fluor 488-labeled AffiniPure F(ab')2 fragment goat anti-human IgG (H+L) antibody (Jackson ImmunoResearch, #109-546-003) also confirmed the expression of Her2-specific CAR. Figure 69 ).

[0588] Furthermore, the expression of PSMA-specific CARs was confirmed by staining with biotin-labeled AffiniPure F(ab')2 fragment goat anti-mouse IgG antibody (F(ab')2 fragment specific antibody) (Jackson ImmunoResearch, #115-066-006) and PE-labeled streptavidin (BDPharmingen, #554061). Figure 77 ).

[0589] For target tumor cells, CD19 expression was confirmed by staining with APC-labeled anti-human CD19 antibody (BD Pharmingen, #555415). Figure 58 The expression levels of these proteins on the surface of stained cells were determined using CytoFLEX S (Beckman Coulter) flow cytometry fluorescence sorting (FACS) technology, and analyzed using CytExpert software.

[0590] Experimental Example 1.2. Assessment of CAR Expression Using Immunofluorescence Staining

[0591] Eight days after the start of stimulation, the CD19-specific CAR-T cells constructed in Example 4 were stained with a Cyanine 3-labeled goat anti-mouse IgG antibody (Invitrogen, #A10521). Figure 55 The Her-specific CAR-T cells constructed in Example 5 were stained with an Alexa Fluor 488-labeled AffiniPure F(ab')2 fragment goat anti-human IgG (H+L) antibody (Jackson ImmunoResearch, #109-546-003). Figure 70 In addition, the PSMA-specific CAR-T cells constructed in Example 6 were stained with a goat anti-mouse IgG antibody labeled with cyanin 3 (Invitrogen, #A10521). Figure 78 CAR-stained cells were fixed and permeated in eBioscience solution (eBioschience, #00-5123) for 20 minutes. Subsequently, the samples were blocked and the nuclei were stained with DAPI containing ProLong Gold Antifade Mountant solution (Invitrogen, #P36931).

[0592] In another embodiment, 9 days after the start of stimulation, CD19-specific CAR-T cells were co-cultured with Daudi-Fluc-eGFP cells at a 1:1 ratio for 1 hour, followed by staining with cyanin 3-labeled goat anti-mouse IgG antibody and DAPI. Cell images were captured at 400x magnification using an Optivity KI-2000 microscope equipped with an Optivity HDMI 4K C-Mount Camera KCX-80. Figure 56 Green fluorescence of EGFP was observed in the cytoplasm of Daudi Fluc EGFP cells.

[0593] Experimental Example 2. T Cell Subpopulation Analysis

[0594] Eight days after the start of stimulation, the T cell subsets of the CAR-T cells constructed in Example 8 were analyzed by flow cytometry: based on the expression or absence of CD45RA and CCR7, these cells were classified as stem cell-like memory T cells (CD45RA). + CCR7 + ), central memory T cells (CD45RA) - CCR7 + Effector memory T cells (CD45RA) - CCR7 - ) and effector T cells (CD45RA) + CCR7 - In analyzing CD19-specific CAR(+) cells, CARs were stained with FITC-labeled recombinant human CD19 protein (Acro, #CD9-HF2H2). Similarly, in analyzing PSMA-specific CAR(+) cells, CARs were stained with FITC-labeled recombinant human PSMA protein (Acro, #PSA-HF244). In both cases, common T cell subsets were analyzed after staining with the specific antibodies shown in Table 35 below. Figure 57 ).

[0595] Table 35

[0596] target protein Manufacturer, # Item Number Fluorescent dyes Species CD45RA BD Pharmingen, #554061 Alexa Fluor 700 mice CCR7 BD Pharmingen, #566743 BV421 mice

[0597] Experimental Example 3. Confirmation of Signal Pathway Modulator Expression

[0598] Experimental Example 3.1. Assessment of protein expression by Western blotting

[0599] To confirm protein expression in the CAR-T cells constructed in the above examples, CAR-T cells were lysed using PROPREP protein extraction buffer (iNtRON Biotechnology, #17081) containing a protease inhibitor. Protein concentration was quantified using the BCA Protein Assay kit (Pierce).

[0600] After electrophoresis of the same amount of protein using SDS-PAGE, the protein was transferred to a PVDF membrane using the XCell SureLock Mini-Cell Electrophoresis System and the XCell II Blot Module (Invitrogen). The membrane was then blocked with a blocking solution (5% skim milk solution), and protein expression was confirmed by sequential incubation with primary and secondary antibodies. Goat anti-mouse IgG HRP (Invitrogen, #62-6520) or goat anti-rabbit IgG HRP (Abcam, #ab6721) were used as secondary antibodies.

[0601] Protein expression images were captured using the Azure C400 Gel Imaging System and analyzed using AzureSpot analysis software (Azure Biosystems). Figure 59 The primary antibodies used in the experiment are shown in Table 36 below.

[0602] Table 36

[0603] Primary antibody (target protein) Manufacturer, # Item Number Species FKBP12 Santa Cruz#sc-133067 mice Cyclophilin A Sino Biological #106317-T36 rabbit N-terminal SH2 of the human SHP2 domain MyBioSource#MBS1751917 mice Human GAPDH Santa Cruz#sc-32233 mice

[0604] Experimental Example 3.2. Assessment of mRNA expression using real-time polymerase chain reaction

[0605] To confirm the expression of mRNA in the CAR-T cells constructed in the above examples, RNA was isolated from CAR-T cells 8 days after stimulation using the ISOLATEII RNA Mini Kit (Bioline, #52072) and quantified using a Nanodrop 1000 spectrophotometer (Thermo Scientific). After synthesizing cDNA using 0.5 μg or 1 μg of RNA, real-time quantitative PCR (Q-PCR) was performed using the SensiFAST Probe Hi-ROX One-Step Kit (Bioline, #77001).

[0606] The GAPDH gene was used as an internal reference. All experiments were repeated twice. Gene expression was analyzed using the ΔΔCt method. Figure 60 The primers and TaqMan probes used in Q-PCR are shown in Table 37 below.

[0607] Table 37

[0608]

[0609] Experimental Example 4. Confirmation of CAR-T cell activity

[0610] Experimental Example 4.1. Target Tumor Cell Lines and Their Culture

[0611] To perform in vitro functional analysis of CD19-specific CAR-T cells, a target tumor cell line (K562-CD19) was established by stably expressing human CD19 protein in human myeloid leukemia K562 cells (ATCC, #CCL-243) using a lentiviral vector. The K562 parental cell line served as a negative control.

[0612] In addition, in vitro functional analysis of CD19-specific CAR-T cells was performed using human B lymphoblast cells (Daudi cells) stably expressing firefly luciferase and emerald green fluorescent protein (GFP, Imanis, #CL158). Furthermore, human B lymphoblast cells (Daudi cells) stably expressing firefly luciferase were used to analyze the in vivo anticancer effect of CD19-specific CAR-T cells. Additionally, in vitro functional analysis of CD19-specific CAR-T cells was performed using the human B-cell precursor leukemia cell line Nalm6 (ATCC, #CCL-3273).

[0613] The human breast cancer cell line SKBR3, stably expressing firefly luciferase (JCRB, #1627.1), was used for in vitro functional analysis and in vivo anticancer efficacy analysis of Her2-specific CAR-T cells. The human prostate cancer cell line LNCaP (ATCC, #CRL-1740-LUC2), stably expressing firefly luciferase, was used for in vitro functional analysis of PSMA-specific CAT-T cells.

[0614] Each of the K562, K562-CD19, Nalm6, and Daudi Fluc eGFP target tumor cell lines was cultured in RPMI-1640 medium (Gibco #11875-085) containing 10% FBS (fetal bovine serum) and an antibiotic antimicrobial agent (Gibco, #15240096). SKBR3-Luc cells were cultured in McCoy's 5a medium (Gibco, #16600-082) containing 10% FBS and an antibiotic antimicrobial agent. LnCap-Luc cells were cultured in RPMI-1640 medium containing 10% FBS, an antibiotic antimicrobial agent, and 2 μg / mL Blastidin (Gibco, #A1113903).

[0615] Experimental Example 4.2. Assessment of the ability of CAR-T cells to secrete cytokines

[0616] The CAR-T cells (4×10) constructed in the above embodiments were used. 4 The cells were co-cultured with target tumor cells at a 1:1 ratio for 24 hours. Afterward, the culture medium was collected, and the concentrations of various cytokines, such as IFNγ, TNFα, and IL-2, in the medium were measured using ELISA (R&D Systems). The results are shown as the average of two replicates. Figures 61 to 63 / Figures 71 to 73 ).

[0617] In addition, when assessing the ability of cytokines to release in the presence of TGF-β1, a medium containing a specific concentration (0 ng / mL to 5 ng / mL) of TGF-β1 was used, and CAR-T cells were co-cultured at a 1:1 ratio (1 × 10⁻⁶ cells / mL). 5 ) and target tumor cells for 24 hours. The results are shown as the average of two repeated experiments ( Figure 64 and Figure 65 ).

[0618] To correct for differences in CAR-T cell transduction efficiency, untransduced T cells were used to adjust the number of transduced CAR-T cells and the total number of T cells.

[0619] Experimental Example 5. Confirmation of CAR-T cell killing activity

[0620] Experimental Example 5.1. Assay of CAR-T cell killing activity based on EuTDA

[0621] The cytotoxic activity of the CD19-specific CAR-T cells constructed in the above examples against target tumor cells was evaluated by measuring EuTDA release after 4 hours using the DELFIA cytotoxicity measurement kit (PerkinElmer, #AD0116). Specifically, target tumor cells were reacted with DELFIA BATDA reagent at 37°C for 15 minutes, followed by washing three times with RPMI 1640 medium. Subsequently, the target tumor cells were resuspended in the medium to a concentration of 5 × 10⁶ cells / mL. 4 Cells / mL. CAR-T cells versus target tumor cells (5 × 10⁻⁶ cells / mL). 3 The culture medium (20 μL) was co-cultured in a predetermined ratio in a 96-well V-bottom plate. Subsequently, the culture medium was transferred to a white bottom plate, mixed with europium solution (200 μL), and reacted for 15 minutes with gentle stirring.

[0622] TDA released into the culture medium was assessed by measuring fluorescence using a SpectraMax iD5 MultiMode microplate reader (Molecular Device). Each experiment was performed in triplicate, and solubility was calculated using the fluorescence measurements according to the following equation:

[0623] <Mathematical Equation 1>

[0624] % solubility = {(experimental group release value - background release value) / (maximum release value - background release value)} × 100;

[0625] Among them, the background release value is the value measured during the spontaneous release of target tumor cells, and the maximum release value is the value measured after the target tumor cells are completely dissolved with DELFIA lysis buffer.

[0626] Experimental Example 5.2. Determination of CAR-T cell cytotoxic activity based on luciferase

[0627] The CAR-T cells constructed in the above embodiments and the target tumor cells expressing firefly luciferase (3×10⁻⁶) 4CAR-T cells were co-cultured in 96-well U-bottom plates at a predetermined ratio. Each experiment was repeated twice. Specifically, 75 μg / mL of D-luciferin potassium salt (PerkinElmer, #122799) was added, and each target tumor cell was cultured at 37°C for an optimized time. Subsequently, the luciferase activity of live cells was measured using a SpectraMax iD5 multimodal microplate reader (Molecular Device). The ability of CAR(+)-T cells to lyse target tumor cells was assessed by comparing the differences in luciferase activity induced by CAR-T cells and untransduced T cells at each E:T (effector (CAR-T):target (target tumor cell) ratio). Results are shown as the average of two replicates. Figure 68 (top), Figure 75 In each experiment, to correct for differences in transduction efficiency, the number of transduced CAR-T cells and the total number of T cells were adjusted using untransduced T cells.

[0628] Experimental Example 6. Measurement of Cell Migration

[0629] The migration ability of the CAR-T cells constructed in the above examples was determined using a Transwell membrane insert with 8 μm pores (24 wells, Corning, #3422). Beads were removed from the CAR-T cells previously reacted with anti-CD3 / anti-CD28 Dynabeads, and the bead-free CAR-T cells were resuspended in serum-free RPMI 1640 medium (containing 0.25% human serum albumin (GreenCross)) containing 0.2 μg / mL recombinant human EGF (Sino Biological, #10605-HNAE). The CAR-T cells were loaded into the upper chamber of the Transwell, while the lower chamber was filled with RPMI 1640 medium (GreenCross) containing 0.25% human serum albumin. After 1 hour of incubation, the number of cells that migrated to the lower chamber was determined by flow cytometry and trypan blue staining. Figure 66 , Figure 74 ).

[0630] In addition, to compare and evaluate the intrinsic motility of CAR-T cells after antigen stimulation, CAR-T and target tumor cells were cultured at a 1:1 ratio at 37°C for 48 hours (using a 96-well U-bottom plate), and then cell images were taken. In this case, the experimental group culturing only CAR-T cells served as a negative control. Figure 67 ).

[0631] To correct for differences in transduction efficiency, experiments were conducted after adjusting the number of transduced CAR-T cells (CAR(+)-T cells) and the total number of T cells using untransduced T cells. Culture media 1 and 2 were not treated with cytokines, and the serum compositions of culture media 1 and 2 differed from each other.

[0632] II. Confirmation of CAR-T activity after delivery of signaling pathway modulators: in vivo

[0633] Experimental Example 7. Evaluation of the anti-cancer effect of CAR-T cells incorporating signal pathway modulators using a hematologic cancer cell model (I)

[0634] Using the following method, according to the dosage (1×10) 6 3×10 6 and 1×10 7 The anticancer effects of CAR-T cells_19bbz and CAR-T cells_19bbz#F were compared and evaluated. The Daudi cancer cell line (Daudi Fluc) with the introduction of the firefly luciferase gene was used as a hematologic cancer cell model.

[0635] To induce blood cancer, 100 μL of 1×10 6 One Daudi-Fluc cell was injected into a healthy NPG (NOD-Prkdc) cell that had undergone a certain period of domestication. scid IL2rg null (Vital Star, China) D-luciferin (D-luciferin, PerkinElmer, USA) solution was injected intraperitoneally into the left and right tail veins of female mice (7 weeks old). Following transplantation of the Daudi Fluc cancer cell line, D-luciferin was injected intraperitoneally on day 10, and images were taken 10 minutes later using an IVIS imaging device (PerkinElmer, USA). Subsequently, luciferase expression levels were measured for each subject using IVIS LuminaSeries Software, and groups were formed by calculating the average value. To assess the anti-cancer effect of each CAR-T cell, 100 μL of a specific dose (1 × 10⁻⁶) was injected into the left and right tail veins of mice using a 1 mL syringe (BD REF328820, 31G). 6 3×10 6 and 1×10 7 CAR-T cells were injected with cancer cells. Following injection of cancer cells, D-luciferin solution was intraperitoneally injected on days 14, 21, 28, 35, and 42 in the same manner as described above, and tumor growth was monitored by IVIS imaging. Figure 50 CAR-Tcell_19bbz was set as the control.

[0636] The results are as follows Figure 50 As shown, when CAR-T cells were administered at the highest dose, a significant increase in tumor suppression rate was confirmed only in the CAR-T_19bbz#F treatment group.

[0637] Experimental Example 8. Evaluation of the anti-cancer effect of CAR-T cells incorporating signal pathway modulators using a hematologic cancer cell model (II)

[0638] The anticancer effects of CAR-T cells_19bbz#F, CAR-T cells_19bbz#C, CAR-T cells_19-bbz#M, CAR-T cells_19bbz#N, CAR-T cells_19bbz#S1, CAR-T cells_19bbz#S2, CAR-T cells_19bbz#TC, CAR-T cells_19bbz#RG, and CAR-T cells_19bbzT were evaluated using the following methods. The Daudi cancer cell line (Daudi-Fluc), infused with the firefly luciferase gene, was used as a hematologic cancer cell model.

[0639] To induce blood cancer, 100 μL of 1×10 6 One Daudi-Fluc cell was injected into a healthy NPG (NOD-Prkdc) cell that had undergone a certain period of domestication. scid IL2rg null (Vital Star, China) D-luciferin (D-luciferin, PerkinElmer, USA) solution was injected intraperitoneally into the left and right tail veins of female mice (7 weeks old). Ten minutes after transplantation of the Daudi Fluc cancer cell line, images were taken using an IVIS imaging device (PerkinElmer, USA). Subsequently, the luciferase expression level of each subject was measured using IVIS Lumina Series Software, and groups were formed by calculating the average value.

[0640] To evaluate the anti-cancer effect of each CAR-T cell after grouping, 100 μL of a 1×10⁻⁶ CAR-T cell was injected into the left and right tail veins of mice using a 1 mL syringe (BD REF328820, 31G). 7 CAR-T cells were injected. Following injection of cancer cells, D-luciferin solution was intraperitoneally injected on days 14, 17, 21, 28, and 35 in the same manner as described above, and tumor growth was monitored by IVIS imaging. Figure 7CAR-T cells (19bbz) were used as a control. The results confirmed that CAR-T cells overexpressing any cellular signaling cytokine had a superior anti-cancer effect compared to the control.

[0641] Experimental Example 9. Evaluation of the anti-cancer effect of CAR-T cells infused with signaling pathway modulators using a solid tumor model.

[0642] The anticancer effects of CAR-T cells_Hbbz, CAR-T cells_Hbbz#F, and CAR-T cells_Hbz#FCS2 were evaluated using the following methods. The SKBR3 cancer cell line (SKBR3-Luc) infused with the firefly luciferase gene was used as a solid tumor cell model expressing Her2.

[0643] 200 μL of 2×10 6 SKBR3-Luc cells were injected into healthy NSGA (NOD-Prkdc) cells that had undergone a certain period of acclimatization. scid IL2rg null (JABio, Korea) Intraperitoneal administration of D-luciferin (D-luciferin, PerkinElmer, USA) to female mice (7 weeks old) after transplantation of the SKBR3-Luc cancer cell line. On day 14, D-luciferin (PerkinElmer, USA) solution was injected intraperitoneally, and images were taken 10 minutes later using an IVIS imaging device (PerkinElmer, USA). Subsequently, luciferase expression levels were measured for each subject using IVIS LuminaSeries Software, and groups were formed by calculating the average values.

[0644] To evaluate the anticancer effect of each CAR-T cell after grouping, 100 μL of prepared CAR T cells (dose: 1 × 10⁻⁶) were injected into the left and right tail veins of mice using a 1 mL syringe (BD REF328820, 31G). 6 (1 CAR(+)-T cells). Following injection of cancer cells, D-luciferin solution was intraperitoneally injected on days 21 (1 week after CAR-T injection), 28 (2 weeks after CAR-T injection), and 35 (3 weeks after CAR-T injection) in the same manner as described above, and tumor growth was monitored by IVIS imaging. Quantitative image data were obtained using LuminaSeries Software. The PBS-treated group served as a control.

[0645] The results are as follows Figure 76 As shown in the figure, CAR-T cells prepared in one embodiment have demonstrated excellent efficacy even in the treatment of solid tumors. SEQUENCE LISTING <110> Korea Immunotechnology Biopharmaceutical Co., Ltd. <120> Immune cells overexpressing externally introduced cell signaling regulators and their applications <130> P22115442WP <150> KR 2020-0018169 <151> 2020-02-14 <160> 114 <170> PatentIn version 3.5 <210> 1 <211> twenty one <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of signal peptide <400> 1 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 2 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of signal peptide <400> 2 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccg 63 <210> 3 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of scFv(CD19) <400> 3 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu 115 120 125 Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys 130 135 140 Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg 145 150 155 160 Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser 165 170 175 Glu Thr Tyr Asn Serves With Lys Serves Arg With Thr Ile Ile 180 185 190 Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Leu Met Asn Ser Leu Gln 195 200 205 Thr Asp Asp Thr Ala Ile Tyr Cys Ala Lys His Tyr Tyr Gly 210 215 220 Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val 225 230 235 240 Ser Ser <210> 4 <211> 726 <212> DNA <213> Artificial Sequence <220> <223> scFv(CD19) <400> 4 gataccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60 atcagttgca gggcaagtca ggacattagt aaatatta attggtatca gcagaaacca 120 gatgaactg ttaaactcct gatctaccat acatcagat tacactcagg agtcccatca 180 aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240 gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300 gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360 ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420 tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480 cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540 tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600 ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660 tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 720 tcctca 726 <210> 5 <211> 69 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of hinge and transmembrane domains <400> 5 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile 35 40 45 Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val 50 55 60 Ile Thr Leu Tyr Cys 65 <210> 6 <211> 207 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequences of hinges and transmembrane domains <400> 6 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120 gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 180 ctgtcactgg ttatcaccct ttactgc 207 <210> 7 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of the co-stimulatory domain <400> 7 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 8 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of the co-stimulatory domain <400> 8 aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60 actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120 gaactg 126 <210> 9 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of the first signal transduction domain <400> 9 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 10 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> I want to be a part of the company. <400> 10 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgc 336 <210> 11 <211> twenty two <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of the self-cleaving peptide (P2A) <400> 11 Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5 10 15 Glu Glu Asn Pro Gly Pro 20 <210> 12 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of the self-cleaving peptide (P2A) <400> 12 ggatccggcg caacaaactt ctctctgctg aaacaagccg gagatgtcga agagaatcct 60 ggaccg 66 <210> 13 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of FKBP12 <400> 13 Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe 1 5 10 15 Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu 20 25 30 Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys 35 40 45 Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val 50 55 60 Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp 65 70 75 80 Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala 85 90 95 Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu 100 105 <210> 14 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> FKBP12 is the most popular <400> 14 atgggagtgc aggtggaaac catctcccca ggagacgggc gcaccttccc caagcgcggc 60 cagacctgcg tggtgcacta caccgggatg cttgaagatg gaaagaaatt tgattcctcc 120 cgggacagaa acaagccctt taagttatg ctaggcaagc aggaggtgat ccgaggctgg 180 gaagaagggg ttgcccagat gagtgtgggt cagagagcca aactgactat atctccagat 240 tatgcctatg gtgccactgg gcacccaggc atcatcccac cacatgccac tctcgtcttc 300 gatgtggagc ttctaaaact ggaa 324 <210> 15 <211> 486 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of chimeric antigen receptor (19bbz) <400> 15 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu 20 25 30 Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln 35 40 45 Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr 50 55 60 Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile 85 90 95 Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly 100 105 110 Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 130 135 140 Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser 145 150 155 160 Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly 165 170 175 Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly 180 185 190 Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser 195 200 205 Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys 210 215 220 Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys 225 230 235 240 His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly 245 250 255 Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 260 265 270 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 275 280 285 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 290 295 300 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 305 310 315 320 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 325 330 335 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 340 345 350 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 355 360 365 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 370 375 380 Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 385 390 395 400 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 405 410 415 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 420 425 430 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 435 440 445 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 450 455 460 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 465 470 475 480 Gln Ala Leu Pro Pro Arg 485 <210> 16 <211> 1458 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of chimeric antigen receptor (19bbz) <400> 16 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc 120 accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180 ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240 tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300 caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 360 ggggggacca agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420 ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc 480 ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt 540 cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca 600 tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa 660 gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa 720 cattattact acggtggtag ctatgctatg gactactggg gccaaggaac ctcagtcacc 780 gtctcctcaa ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 840 cagcccctgt ccctgcgcc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 900 agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 960 gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 1020 tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1080 agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1140 agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200 ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260 ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320 atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380 gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440 caggccctgc cccctcgc 1458 <210> 17 <211> 616 <212> PRT <213> Artificial Sequence <220> <223> Relatively unique (19bbz:F) <400> 17 Put Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Wing Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu 20 25 30 Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln 35 40 45 Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr 50 55 60 Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile 85 90 95 Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly 100 105 110 Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 130 135 140 Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser 145 150 155 160 Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly 165 170 175 Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly 180 185 190 Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser 195 200 205 Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys 210 215 220 Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys 225 230 235 240 His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly 245 250 255 Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 260 265 270 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 275 280 285 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 290 295 300 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 305 310 315 320 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 325 330 335 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 340 345 350 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 355 360 365 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 370 375 380 Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 385 390 395 400 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 405 410 415 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 420 425 430 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 435 440 445 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 450 455 460 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 465 470 475 480 Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu 485 490 495 Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Gly Val Gln 500 505 510 Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly 515 520 525 Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys 530 535 540 Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly 545 550 555 560 Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser 565 570 575 Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly 580 585 590 Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe 595 600 605 Asp Val Glu Leu Leu Lys Leu Glu 610 615 <210> 18 <211> 1848 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of fusion protein (19bbz#F) <400> 18 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc 120 accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180 ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240 tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300 caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 360 ggggggacca agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420 ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc 480 ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt 540 cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca 600 tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa 660 gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa 720 cattattact acggtggtag ctagctatg gactactggg gccaaggaac ctcagtcacc 780 gtctcctcaa ccacgacgcc agcgccgcga ccacacac cggcgcccac catcgcgtcg 840 cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 900 agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 960 gtccttctcc tgtcactggt tatcacctt tactgcaac ggggcagaaa gaaactcctg 1020 tatatattca aaaaccatt tatgagacca gtashaacta ctcagagga agatggctgt 1080 agctgccgat ttccagaga agagaagga ggatgtgaac tgagagtgaa gttcagcagg 1140 agcgcagacg cccccgcgta ccagcaggc cagaaccagc tctataacga gctcaatcta 1200 ggacgaagg aggagtacga tgttttggac agagacgtg gccgggaccc tgagatgggg 1260 ggaaagccga gaggagaa cccctcaggaa ggcctgtaca atgaacctgca gaagataag 1320 atggcggagg cctacagtga gattgggatg aaggcgagc gccggagggg caggggcac 1380 gatggccttt accagggctct cagtacagcc accaaggaca cctacgacgc ccttcacacatg 1440 caggccctgc cccctcgcgg atccggcgca acaaacttct ctctgctgaa acaagccgga gatgtcgaag agaatcctgg accgatgggga gtgcaggtgg aaaccatctc cccaggagac 1560 gggcgcacct tccccaagcg cggccagacc tgcgtggtgc actacaccgg gatgcttgaa 1620 gatggaaaga aatttgattc ctcccgggac agaaacaagc cctttaagtt tatgctaggc aagcaggagg tgatccgagg ctgggaagaa ggggttgccc agatgagtgt gggtcagaga gccaaactga ctatatctcc agattatgcc tatggtgcca ctgggcaccc aggcatcatc ccaccacatg ccactctcgt cttcgatgtg gagcttctaa aactggaa <210> 19 <211> 97 <212> PRT <213> Artificial Sequence <220> <223> FKBP12(FKBP12)FKBP12 (FKBP12)FKBP12 <400> 19 Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe 1 5 10 15 Pro Lys Arg Gly Glyn Thr Cys Val Val His Tyr Thr Asp Glu Cys Gly 20 25 30 Ser Glu Ser Gln Thr Asp Tyr Ile Ser Arg Leu Cys Leu Trp Cys His 35 40 45 Trp Ala Pro Arg His His Pro Thr Thr Cys His Ser Arg Leu Arg Cys 50 55 60 Gly Ala Ser Lys Thr Gly Met Thr Gly Met Ala Ser Ser Leu Ser Ser 65 70 75 80 Leu Phe Leu Asp Leu Pro Trp Arg Asp Leu Val Pro Pro Asp Met Cys 85 90 95 Thr <210> 20 <211> 234 <212> PRT <213> Artificial Sequence <220> <223> MH2 domain of SMAD4 <400> 20 Ala Pro Glu Tyr Trp Cys Ser Ile Ala Tyr Phe Glu Met Asp Val Gln 1 5 10 15 Val Gly Glu Thr Phe Lys Val Pro Ser Ser Cys Pro Ile Val Thr Val 20 25 30 Asp Gly Tyr Val Asp Pro Ser Gly Gly Asp Arg Phe Cys Leu Gly Gln 35 40 45 Leu Ser Asn Val His Arg Thr Glu Ala Ile Glu Arg Ala Arg Leu His 50 55 60 Ile Gly Lys Gly Val Gln Leu Glu Cys Lys Gly Glu Gly Asp Val Trp 65 70 75 80 Val Arg Cys Leu Ser Asp His Ala Val Phe Val Gln Ser Tyr Tyr Leu 85 90 95 Asp Arg Glu Ala Gly Arg Ala Pro Gly Asp Ala Val His Lys Ile Tyr 100 105 110 Pro Ser Ala Tyr Ile Lys Val Phe Asp Leu Arg Gln Cys His Arg Gln 115 120 125 Met Gln Gln Gln Ala Ala Thr Ala Gln Ala Ala Ala Ala Ala Gln Ala 130 135 140 Ala Ala Val Ala Gly Asn Ile Pro Gly Pro Gly Ser Val Gly Gly Ile 145 150 155 160 Ala Pro Ala Ile Ser Leu Ser Ala Ala Ala Gly Ile Gly Val Asp Asp 165 170 175 Leu Arg Arg Leu Cys Ile Leu Arg Met Ser Phe Val Lys Gly Trp Gly 180 185 190 Pro Asp Tyr Pro Arg Gln Ser Ile Lys Glu Thr Pro Cys Trp Ile Glu 195 200 205 Ile His Leu His Arg Ala Leu Gln Leu Leu Asp Glu Val Leu His Thr 210 215 220 Met Pro Ile Ala Asp Pro Gln Pro Leu Asp 225 230 <210> 21 <211> 702 <212> DNA <213> Artificial Sequence <220> <223> SMAD4 or MH2-mediated smooth muscle activation <400> 21 gctcctgagt attggtgttc cattgcttac tttgaaatgg atgttcaggt aggagagaca tttaaggttc cttcaagctg ccctattgtt actgttgatg gatacgtgga cccttctgga ggagatcgct tttgtttggg tcaactctcc aatgtccaca ggacagaagc cattgagaga gcaaggttgc acataggcaa aggtgtgcag ttggaatgta aaggtgaagg tgatgtttgg gtcaggtgcc ttagtgacca cgcggtcttt gtcagagtt actacttaga cagagaagct gggcgtgcac ctggagatgc tgttcataag atctacccaa gtgcatatat aaaggtcttt 360 gatttgcgtc agtgtcatcg acagatgcag cagcaggcgg ctactgcaca agctgcagca 420 gctgcccagg cagcagccgt ggcaggaaac atccctggcc caggatcagt aggtggaata gctccagcta tcagtctgtc agctgctgct ggaattggtg ttgatgacct tcgtcgctta 540 tgcatactca ggatgagttt tgtgaaaggc tggggaccgg attacccaag acagagcatc 600 aaagaaacac cttgctggat tgaaattcac ttacaccggg ccctccagct cctagacgaa 660 gtacttcata ccatgccgat tgcagaccca caacctttag ac 702 <210> 22 <211> 315 <212> PRT <213> Artificial Sequence <220> <223> SKI boarding <400> 22 Met Glu Ala Ala Ala Gly Gly Arg Gly Cys Phe Gln Pro His Pro Gly 1 5 10 15 Leu Gln Lys Thr Leu Glu Gln Phe His Leu Ser Ser Met Ser Ser Leu 20 25 30 Gly Gly Pro Ala Ala Phe Ser Ala Arg Trp Ala Gln Glu Ala Tyr Lys 35 40 45 Lys Glu Ser Ala Lys Glu Ala Gly Ala Ala Ala Val Pro Ala Pro Val 50 55 60 Pro Ala Ala Thr Glu Pro Pro Pro Val Leu His Leu Pro Ala Ile Gln 65 70 75 80 Pro Pro Pro Pro Val Leu Pro Gly Pro Phe Phe Met Pro Ser Asp Arg 85 90 95 Ser Thr Glu Arg Cys Glu Thr Val Leu Glu Gly Glu Thr Ile Ser Cys 100 105 110 Phe Val Val Gly Gly Glu Lys Arg Leu Cys Leu Pro Gln Ile Leu Asn 115 120 125 Ser Val Leu Arg Asp Phe Ser Leu Gln Gln Ile Asn Ala Val Cys Asp 130 135 140 Glu Leu His Ile Tyr Cys Ser Arg Cys Thr Ala Asp Gln Leu Glu Ile 145 150 155 160 Leu Lys Val Met Gly Ile Leu Pro Phe Ser Ala Pro Ser Cys Gly Leu 165 170 175 Ile Thr Lys Thr Asp Ala Glu Arg Leu Cys Asn Ala Leu Leu Tyr Gly 180 185 190 Gly Ala Tyr Pro Pro Pro Cys Lys Lys Glu Leu Ala Ala Ser Leu Ala 195 200 205 Leu Gly Leu Glu Leu Ser Glu Arg Ser Val Arg Val Tyr His Glu Cys 210 215 220 Phe Gly Lys Cys Lys Gly Leu Leu Val Pro Glu Leu Tyr Ser Ser Pro 225 230 235 240 Ser Ala Ala Cys Ile Gln Cys Leu Asp Cys Arg Leu Met Tyr Pro Pro 245 250 255 His Lys Phe Val Val His Ser His Lys Ala Leu Glu Asn Arg Thr Cys 260 265 270 His Trp Gly Phe Asp Ser Ala Asn Trp Arg Ala Tyr Ile Leu Ser 275 280 285 Gln Asp Tyr Thr Gly Lys Glu Glu Gln Ala Arg Leu Gly Arg Cys Leu 290 295 300 Asp Asp Val Lys Glu Lys Phe Asp Tyr Gly Asn 305 310 315 <210> 23 <211> 945 <212> DNA <213> Artificial Sequence <220> <223> Skiing is a snowmobile trail <400> 23 atggaggcgg cggcaggcgg ccgcggctgt ttccagccgc acccggggct gcagaagacg ctggagcagt tccacctgag ctccatgagc tcgctgggcg gcccggccgc tttctcggcg 120 cgctgggcgc aggaggccta caagaaggag agcgccaagg aggcggcgc ggccgcggtg 180 ccggcgccgg tgcccgcagc caccgagccg ccgcccgtgc tgcacctgcc cgccatccag 240 ccgccgccgc ccgtgctgcc ccggcccttc ttcatgccgt ccgaccgctc caccgagcgc 300 tgcgagaccg tactggaagg cgagaccatc tcgtgcttcg tggtgggagg cgagaagcgc 360 ctgtgtctgc cgcagattct caactcggtg ctgcgcgact tctcgctgca gcagatcaac gcggtgtgcg acgagctcca catctactgc tcgcgctgca cggccgacca gctggagatc 480 ctcaaagtca tgggcatcct gcccttctcg gcgccctcgt gcgggctcat caccaagacg 540 gacgccgagc gcctgtgcaa cgcgctgctc tacggcggcg cctacccgcc gccctgcaag 600 aaggagctgg ccgccagcct ggcgctgggc ctggagctca gcgagcgcag cgtccgcgtg 660 720. taccacgagt gcttcggcaa gtgtaagggg ctgctggtgc ccgagctcta cagcagcccg agcgccgcct gcatccagtg cctggactgc cgcctcatgt acccgccgca caagttcgtg 780 gtgcactcgc acaaggccct ggagaaccgg acctgccact ggggcttcga ctcggccaac tggcgggcct acatcctgct gagccaggat tacacgggca aggaggagca ggcgcgcctc 900 ggccgctgcc tggacgacgt gaggagaa ttcgactatg gcaac <210> 24 <211> 165 <212> PRT <213> Artificial Sequence <220> <223> CypA (PPIA) <400> 24 Met Val Asn Pro Thr Val Phe Phe Asp Ile Ala Val Asp Gly Glu Pro 1 5 10 15 Leu Gly Arg Val Ser Phe Glu Leu Phe Ala Asp Lys Val Pro Lys Thr 20 25 30 Ala Glu Asn Phe Arg Ala Leu Ser Thr Gly Glu Lys Gly Phe Gly Tyr 35 40 45 Lys Gly Ser Cys Phe His Arg Ile Ile Pro Gly Phe Met Cys Gln Gly 50 55 60 Gly Asp Phe Thr Arg His Asn Gly Thr Gly Gly Lys Ser Ile Tyr Gly 65 70 75 80 Glu Lys Phe Glu Asp Glu Asn Phe Ile Leu Lys His Thr Gly Pro Gly 85 90 95 Ile Leu Ser Met Ala Asn Ala Gly Pro Asn Thr Asn Gly Ser Gln Phe 100 105 110 Phe Ile Cys Thr Ala Lys Thr Glu Trp Leu Asp Gly Lys His Val Val 115 120 125 Phe Gly Lys Val Lys Glu Gly Met Asn Ile Val Glu Ala Met Glu Arg 130 135 140 Phe Gly Ser Arg Asn Gly Lys Thr Ser Lys Lys Ile Thr Ile Ala Asp 145 150 155 160 Cys Gly Gln Leu Glu 165 <210> 25 <211> 495 <212> DNA <213> Artificial Sequence <220> <223> CypA (PPIA) <400> 25 atggtcaacc ccaccgtgtt cttcgacatt gccgtcgacg gcgagccctt gggccgcgtc 60 tcctttgagc tgtttgcaga caaggtccca aagacagcag aaaatttcg tgctctgagc 120 actggagaga aaggatttgg ttataagggt tcctgctttc acagaattat tccagggttt 180 atgtgtcagg gtggtgactt cacacgccat aatggcactg gtggcaagtc catctatggg 240 gagaaatttg aagatgagaa cttcatccta aagcatacgg gtcctggcat cttgtccatg 300 gcaaatgctg gacccaacac aaatggttcc cagtttttca tctgcactgc caagactgag 360 tggttggatg gcaagcatgt ggtgtttggc aaagtgaaag aaggcatgaa tattgtggag 420 gccatggagc gctttgggtc caggaatggc aagaccagca agaagatcac cattgctgac 480 tgtggacaac tcgaa 495 <210> 26 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> N-terminal SH2 domain of SHP-1 <400> 26 Met Val Arg Trp Phe His Arg Asp Leu Ser Gly Leu Asp Ala Glu Thr 1 5 10 15 Leu Leu Lys Gly Arg Gly Val His Gly Ser Phe Leu Ala Arg Pro Ser 20 25 30 Arg Lys Asn Gln Gly Asp Phe Ser Leu Ser Val Arg Val Gly Asp Gln 35 40 45 Val Thr His Ile Arg Ile Gln Asn Ser Gly Asp Phe Tyr Asp Leu Tyr 50 55 60 Gly Gly Glu Lys Phe Ala Thr Leu Thr Glu Leu Val Glu Tyr Tyr Thr 65 70 75 80 Gln Gln Gln Gly Val Leu Gln Asp Arg Asp Gly Thr Ile Ile His Leu 85 90 95 Lys Tyr Pro Leu Asn Cys Ser Asp Pro Thr Ser 100 105 <210> 27 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> SHP‐1 antibodies and SH2 antibodies are actively active <400> 27 atggtgaggt ggtttcaccg agacctcagt gggctggatg cagagaccct gctcaagggc cgaggtgtcc acggtagctt cctggctcgg cccagtcgca agaaccaggg tgacttctcg ctctccgtca gggtggggga tcaggtgacc catattcgga tccagaactc aggggatttc 180 tatgacctgt atggagggga gaagtttgcg actctgacag agctggtgga gtactacact 300. cagcagcagg gtgtcctgca ggaccgcgac ggcaccatca tccacctcaa gtacccgctg aactgctccg atcccactag t 321 <210> 28 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> SHP‐2 N                2 or <400> 28 Met Thr Ser Arg Arg Trp Phe His Pro Asn Ile Thr Gly Val Glu Ala 1 5 10 15 Glu Asn Leu Leu Leu Thr Arg Gly Val Asp Gly Ser Phe Leu Ala Arg 20 25 30 Pro Ser Lys Ser Asn Pro Gly Asp Phe Thr Leu Ser Val Arg Arg Asn 35 40 45 Gly Ala Val Thr His Ile Lys Ile Gln Asn Thr Gly Asp Tyr Tyr Asp 50 55 60 Leu Tyr Gly Gly Glu Lys Phe Ala Thr Leu Ala Glu Leu Val Gln Tyr 65 70 75 80 Tyr Met Glu His His Gly Gln Leu Lys Glu Lys Asn Gly Asp Val Ile 85 90 95 Glu Leu Lys Tyr Pro Leu Asn Cys Ala Asp Pro 100 105 <210> 29 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> SHP‑2 is not a SH2 system. <400> 29 atgacatcgc ggagatggtt tcacccaaat atcactggtg tggaggcaga aaacctactg 60 ttgacaagag gagttgatgg cagttttttg gcaaggccta gtaaaagtaa ccctggagac 120 ttcacacttt ccgttagaag aaatggagct gtcacccaca tcaagattca gaacactggt 180 gattactatg acctgtatgg aggggagaaa tttgccactt tggctgagtt ggtccagtat 240 tacatggaac atcacgggca attaaaagag aagaatggag atgtcattga gcttaaatat 300 cctctgaact gtgcagatcc t 321 <210> 30 <211> 187 <212> PRT <213> Artificial Sequence <220> <223> The internal cytoplasmic domain of TLR4 <400> 30 Lys Phe Tyr Phe His Leu Met Leu Leu Ala Gly Cys Ile Lys Tyr Gly 1 5 10 15 Arg Gly Glu Asn Ile Tyr Asp Ala Phe Val Ile Tyr Ser Ser Gln Asp 20 25 30 Glu Asp Trp Val Arg Asn Glu Leu Val Lys Asn Leu Glu Glu Gly Val 35 40 45 Pro Pro Phe Gln Leu Cys Leu His Tyr Arg Asp Phe Ile Pro Gly Val 50 55 60 Ala Ile Ala Ala Asn Ile Ile His Glu Gly Phe His Lys Ser Arg Lys 65 70 75 80 Val Ile Val Val Val Ser Gln His Phe Ile Gln Ser Arg Trp Cys Ile 85 90 95 Phe Glu Tyr Glu Ile Ala Gln Thr Trp Gln Phe Leu Ser Ser Arg Ala 100 105 110 Gly Ile Ile Phe Ile Val Leu Gln Lys Val Glu Lys Thr Leu Leu Arg 115 120 125 Gln Gln Val Glu Leu Tyr Arg Leu Leu Ser Arg Asn Thr Tyr Leu Glu 130 135 140 Trp Glu Asp Ser Val Leu Gly Arg His Ile Phe Trp Arg Arg Leu Arg 145 150 155 160 Lys Ala Leu Leu Asp Gly Lys Ser Trp Asn Pro Glu Gly Thr Val Gly 165 170 175 Thr Gly Cys Asn Trp Gln Glu Ala Thr Ser Ile 180 185 <210> 31 <211> 561 <212> DNA <213> Artificial Sequence <220> <223> TLR4 is a high-frequency transcription factor <400> 31 aagttctatt ttcacctgat gcttcttgct ggctgcataa agtatggtag aggtgaaaac atctatgatg cctttgttat ctactcaagc caggatgagg actgggtaag gaatgagcta gtaagaatt tagaagaagg ggtgcctcca tttcagctct gccttcacta cagagacttt attcccggtg tggccattgc tgccaacatc atccatgaag gtttccataa aagccgaaag 240 gtgattgttg tggtgtccca gcacttcatc cagagccgct ggtgtatctt tgaatatgag attgctcaga cctggcagtt tctgagcagt cgtgctggta tcatcttcat tgtcctgcag 360 aaggtggaga agaccctgct caggcagcag gtggagctgt accgccttct cagcaggac 420 acttacctgg agtggagga cagtgtcctg gggcggcaca tcttctggag acgactcaga 480 aaagccctgc tggatggtaa atcatggaat ccagaagga cagtgggtac aggatgcaat tggcaggaag caacatctat c <210> 32 <211> 204 <212> PRT <213> Artificial Sequence <220> <223> TC21(RRAS2)G23V Configuration <400> 32 Met Ala Ala Ala Gly Trp Arg Asp Gly Ser Gly Gln Glu Lys Tyr Arg 1 5 10 15 Click Download to save Leu Val Val Gly Lys Ser Ala Leu Thr Ile mp3 youtube com 20 25 30 Gln Phe Ile Gln Ser Tyr Phe Val Thr Asp Tyr Asp Pro Thr Ile Glu 35 40 45 Asp Ser Tyr Thr Lys Gln Cys Val Ile Free Download Asp Asp Arg Ala Ala Arg Leu 50 55 60 Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Phe Gly Ala Met Arg Glu 65 70 75 80 Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Leu Val Phe Ser Val Thr 85 90 95 Asp Arg Gly Ser Phe Glu Glu Ile Tyr Lys Phe Gln Arg Gln Ile Leu 100 105 110 Arg Val Lys Asp Arg Asp Glu Phe Pro Met Ile Leu Ile Gly Asn Lys 115 120 125 Ala Asp Leu Asp His Gln Arg Gln Val Thr Gln Glu Glu Gly Gln Gln 130 135 140 Leu Ala Arg Gln Leu Lys Val Thr Tyr Met Glu Ala Ser Ala Lys Ile 145 150 155 160 Arg Met Asn Val Asp Gln Ala Phe His Glu Leu Val Arg Val Ile Arg 165 170 175 Lys Phe Gln Glu Gln Glu Cys Pro Pro Ser Pro Glu Pro Thr Arg Lys 180 185 190 Glu Lys Asp Lys Lys Gly Cys His Cys Val Ile Phe 195 200 <210> 33 <211> 612 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of TC21(RRAS2) G23V mutant <400> 33 atggccgcgg ccggctggcg ggacggctcc ggccaggaga agtaccggct cgtggtggtc 60 ggcggggtcg gcgtgggcaa gtcggcgctc accatccagt tcatccagtc ctattttgta 120 acggattatg atccaaccat tgaagattct tacacaaagc agtgtgtgat agatgacaga 180 gcagcccggc tagatatttt ggatacagca ggacaagaag agtttggagc catgagagaa 240 cagtatatga ggactggcga aggcttcctg ttggtctttt cagtcacaga tagaggcagt 300 tttgaagaaa tctataagtt tcaaagacag attctcagag taaaggatcg tgatgagttc 360 ccaatgattt taattggtaa taaagcagat ctggatcatc aaagacaggt aacacaggaa 420 gaaggacaac agttagcacg gcagcttaag gtaacataca tggaggcatc agcaaagatt 480 aggatgaatg tagatcaagc tttccatgaa cttgtccggg tttcaggaa atttcaagag 540 caggaatgtc ctccttcacc agaaccaaca cggaaagaaa aagacaagaa aggctgccat 600 tgtgtcattt tc 612 <210> 34 <211> 191 <212> PRT <213> Artificial Sequence <220> <223> RhoGQ61V <400> 34 Met Gln Ser Ile Lys Cys Val Val Val Gly Asp Gly Ala Val Gly Lys 1 5 10 15 Thr Cys Leu Leu Ile Cys Tyr Thr Thr Asn Ala Phe Pro Lys Glu Tyr 20 25 30 Ile Pro Thr Val Phe Asp Asn Tyr Ser Ala Gln Ser Ala Val Asp Gly 35 40 45 Arg Thr Val Asn Leu Asn Leu Trp Asp Thr Ala Gly Val Glu Glu Tyr 50 55 60 Asp Arg Leu Arg Thr Leu Ser Tyr Pro Gln Thr Asn Val Phe Val Ile 65 70 75 80 Cys Phe Ser Ile Ala Ser Pro Pro Ser Tyr Glu Asn Val Arg His Lys 85 90 95 Trp His Pro Glu Val Cys His His Cys Pro Asp Val Pro Ile Leu Leu 100 105 110 Val Gly Thr Lys Lys Asp Leu Arg Ala Gln Pro Asp Thr Leu Arg Arg 115 120 125 Leu Lys Glu Gln Gly Gln Ala Pro Ile Thr Pro Gln Gln Gly Gln Ala 130 135 140 Leu Ala Lys Gln Ile His Ala Val Arg Tyr Leu Glu Cys Ser Ala Leu 145 150 155 160 Gln Gln Asp Gly Val Lys Glu Val Phe Ala Glu Ala Val Arg Ala Val 165 170 175 Leu Asn Pro Thr Pro Ile Lys Arg Gly Arg Ser Cys Ile Leu Leu 180 185 190 <210> 35 <211> 576 <212> DNA <213> Artificial Sequence <220> <223> RhoG Q61V in the right-hand side <400> 35 atgcagagca tcaagtgcgt ggtggtgggt gatggggctg tgggcaagac gtgcctgctc 60 atctgctaca caactaacgc tttccccaaa gagtacatcc ccaccgtgtt cgacaattac 120 agcgcgcaga gcgcagttga cgggcgcaca gtgaacctga acctgtggga cactgcgggc 180 gtggaggagt atgaccgcct ccgtacactc tcctaccctc agaccaacgt ttcgtcatc 240 tgttctcca ttgccagtcc gccgtcctat gagaacgtgc ggcacaagtg gcatccagag 300 gtgtgccacc actgccctga tgtgcccatc ctgctggtgg gcaccaagaa ggacctgaga 360 gcccagcctg acaccctacg gcgcctcaag gagcagggcc aggcgcccat cacaccgcag 420 cagggccagg cactggccaa gcagatccac gctgtgcgct acctcgaatg ctcagccctg 480 caacaggatg gtgtcaagga agtgttcgcc gaggctgtcc gggctgtgct caaccccacg 540 ccgatcaagc gtgggcggtc ctgcatcctc ttgtga 576 <210> 36 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> FKBP12 positive <400> 36 gacctgcgtg gtgcacta 18 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FKBP12 Reverse <400> 37 cccgggagga atcaaatttc 20 <210> 38 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> FKBP12 TaqMan probe <400> 38 cgggatgctt gaagat 16 <210> 39 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Cyclophilic A positive <400> 39 gcaaagtgaa agaaggcatg aa 22 <210> 40 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Cyclophilic A reverse <400> 40 ccattcctgg acccaaagc 19 <210> 41 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> TaqMan probe for cyclin A <400> 41 tccatggcct ccacaat 17 <210> 42 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> The N-terminal SH2 domain of SHP2 is positively oriented. <400> 42 cgggatgctt gaagat 16 <210> 43 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> The N-terminal SH2 domain of SHP2 is reversed. <400> 43 ttctcccctc catacaggtc at 22 <210> 44 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> N-terminal SH2 domain probe of SHP2 <400> 44 tcagaacact ggtgattac 19 <210> 45 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of scFv (Her2) <400> 45 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu 115 120 125 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 130 135 140 Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Arg 145 150 155 160 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr 165 170 175 Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 180 185 190 Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp 210 215 220 Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 225 230 235 240 Ser Ser <210> 46 <211> 726 <212> DNA <213> Artificial Sequence <220>[[ID=A3]] <223> Nucleotide sequence of scFv (Her2) <400> 46 gacatccaga tgacccagtc tccgtcttct ctgtctgctt ctgttggtga ccgtgttacc 60 atcacctgcc gtgcttctca ggacgttaac accgctgttg cttggtacca gcagaaaccg 120 ggtaaagctc cgaaactgct gatctactct gcttctttcc tgtactctgg tgttccgtct 180 cgtttctctg gttctcgttc tggtaccgac ttcaccctga ccatctcttc tctgcagccg 240 gaagacttcg ctacctacta ctgccagcag cactacacca ccccgccgac cttcggtcag 300 ggtaccaaag ttgaaatcaa aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360 ggatctgaag ttcagctggt tgaatctggt ggtggtctgg ttcagccggg tggttctctg 420 cgtctgtctt gcgctgcttc tggtttcaac atcaaagaca cctacatcca ctgggttcgt 480 caggctccgg gtaaaggtct ggaatgggtt gctcgtatct acccgaccaa cggttacacc 540 cgttacgctg actctgttaa aggtcgtttc accatctctg ctgacacctc taaaaacacc 600 gcttacctgc agatgaactc tctgcgtgct gaagacaccg ctgtttacta ctgctctcgt 660 tggggtggtg acggtttcta cgctatggac tactggggtc agggtaccct ggttaccgtt 720 tcttct 726 <210> 47 <211> 244 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of scFv (CD43) <400> 47 Asp Thr Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Met Phe Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln 115 120 125 Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys 130 135 140 Lys Ala Ser Gly Tyr Thr Phe Asn Gly Tyr Phe Met Asn Trp Val Arg 145 150 155 160 Gln Ala Pro Gly Gln Gly Leu Glu Arg Met Gly Arg Ile Asn Pro Asn 165 170 175 Asn Gly Asp Ser Phe Tyr Asn Gln Lys Phe Gln Gly Arg Val Thr Met 180 185 190 Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Pro Ser Leu 195 200 205 Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Tyr Tyr 210 215 220 Gly Gly Arg Gly Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 225 230 235 240 Thr Val Ser Ser <210> 48 <211> 732 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of scFv (CD43) <400> 48 gacacccaga tgacacagag ccctagctcc gtgtctgcca gcgtgggaga cagagtgacc 60 atcacctgta gagccagcca ggacatcagc aactacctga actggtatca gcagaagccc 120 ggcaaggccc ctaagctgct gatctacgcc acaagcagac tgcacagcgg cgtgccaagc 180 agattttctg gcagcggctc tggcaccgac ttcaccctga ccatatctag cctgcagcct 240 gaggacttcg ccacctacta ctgccagcag agcaacatgt tcccctacac ctttggccag 300 ggcaccaagc tggaaatcaa aggcggcgga ggatctggcg gaggtggaag tggcggaggc 360 ggatctcaag ttcagctggt tcagtctggc gccgaagtga agaaacctgg cgcctctgtg 420 aaggtgtcct gcaaggccag cggctacacc ttcaacggct acttcatgaa ctgggtccga 480 caggcccctg gacagggact cgaaagaatg ggcagaatca accccaacaa cggcgacagc 540 ttctacaacc agaaattcca gggccgcgtg accatgacca gagacaccag cacaagcacc 600 gtgtacatgg aactgcccag cctgagaagc gaggacaccg ccgtgtacta ctgtgccaga 660 gagggctatt acggcggcag aggctacgcc ctggattatt ggggacaggg cacactggtc 720 accgtgtcta gc 732 <210> 49 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of the signal peptide (CD47) <400> 49 atggccctgc ccgtgacagc cctgctgctg cccctggccc tgctgctgca cgccgcccgg 60 ccc 63 <210> 50 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of scFv (CD47) <400> 50 Ala Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 Gly Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Thr Ile Thr Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Ser Leu Ala Gly Asn Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala 130 135 140 Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala 145 150 155 160 Ser Gln Ser Ile Ser Asp Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly 165 170 175 Gln Ala Pro Arg Leu Leu Ile Tyr Phe Ala Ser Gln Arg Ala Thr Gly 180 185 190 Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 195 200 205 Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln 210 215 220 Gln Gly His Gly Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu 225 230 235 240 Ile Lys <210> 51 <211> 726 <212> DNA <213> Artificial Sequence <220> <223> scFv promoter gene(CD47) . <400> 51 gcctccgagg tgcagctggt ggagagcggg ggggggctcg tgcagcctgg gggctccctg aggctcagct gcgccgcctc cggggttcacc ttctccggggt acgggatgag ctgggtgagg 120 caggccccg gcaagggcct ggagtgggtc gccaccatca cctccggcgg cacatacacc 180 tactaccccg acagcgtgaa gggccggttc acaattagcc gggataacgc caagaacagc ctgtacctgc agatgaacag cctgagggcc gaggacactg ccgtgtacta ctgcgccagg agcctggccg gcaacgcaat ggattactgg gggcagggca cactggtgac agtgagcagc 360 ggcggggggcg ggagcggggc cggggggc ggcgggggg gcagcgagat tgtgctcacc 420 cagtcccccg ccaccctgag cctgagcccc ggcgagaggg ccacactgtc gtgccggggcc 480 agccagtcca tctccgacta cctgcactgg tatcagcaga agcccggcca ggcccctagg 540 ctgctgatct acttcgccag ccagagggcc accgggattc ctgccaggtt cagcggcagc 600 gggagcggga ctgattttac actgacaatt tctagtttgg aacctgaaga tttcgctgtg 660 tattactgcc agcagggaca cggctttcct agaacatttg gaggaggaac aaaggtggag 720 attaag 726 <210> 52 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence (PSMA) of the signal peptide <400> 52 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser <210> 53 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Signal peptide nucleotide sequence (PSMA) <400> 53 atgggatggt catgtatcat cctttttcta gtagcaactg caactggagt acattca 57 <210> 54 <211> 238 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence (PSMA) of scFv <400> 54 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Ile Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Met Leu Asp Leu Lys Arg Gly Gly Gly Gly 100 105 110 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln 115 120 125 Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Thr Ser Val Arg Ile Ser 130 135 140 Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr Thr Ile His Trp Val 145 150 155 160 Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly Asn Ile Asn Pro 165 170 175 Asn Asn Gly Gly Thr Tyr Asn Gln Lys Phe Glu Asp Lys Ala Thr 180 185 190 Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser 195 200 205 Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Ala Gly Trp Asn 210 215 220 Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 225 230 235 <210> 55 <211> 714 <212> DNA <213> Artificial Sequence <220> <223> scFv protein receptor (PSMA) <400> 55 gatacgtga tgacccagag ccacaagttc atgagcacca gcgtggggcga cagagtgtcc 60 atcatctgca aggccagcca ggacgtggga accgccgtgg actggtacca gcagaaacct 120 ggccagagtc ctaagctgct gatctactgg gcctctacaa vakacaccgg cgtgcctgat 180 agatcaccg gctctggcag cggcaccgac ttcaccctga caatcaccaa cgtgcagagc 240 gaggacctgg ccgactactt ctgccagcaa tacacagct accccctgac cttcggcgcc 300 ggcacaatgc tggacctgaa gcggggtggc ggtggctcgg gcggtggtgg gtcgggtggc 360 ggcggatctg aggtgcagct gcagcagtct ggtcctgagc tgaagaagcc cggcacaagc 420 gtgcggatca gctgtaaaac cagcggctac accttcaccg agtacaccat ccactgggtc 480 aagcagagcc acggcaagtc cctggaatgg atcggcaaca tcaaccccaa caacggcgga 540 acaacctaca accagaagtt cgaggacaag gccacactga ccgtggacaa gagcagcagc 600 accgcctaca tggaactgag aagcctgaca agtgaagata gcgccgtgta ctactgcgcc 660 gctggctgga acttcgacta ctggggccag ggcaccaccc tgaccgtgtc cagc 714 <210> 56 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> P2A (P2A) <400> 56 Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn 1 5 10 15 Pro Gly Pro <210> 57 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of the TCRα signal peptide (HERV-E) <400> 57 Met Lys Arg Ile Leu Gly Ala Leu Leu Gly Leu Leu Ser Ala Gln Val 1 5 10 15 Cys Cys Val Arg Gly 20 <210> 58 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of the TCRα signal peptide (HERV-E) <400> 58 atgaagagga tattggggagc tctgctgggg ctcttgagtg cccaggtttg ctgtgtgaga 60 gga 63 <210> 59 <211> 250 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of TCRα (HERV-E) <400> 59 Ile Gln Val Glu Gln Ser Pro Pro Asp Leu Ile Leu Gln Glu Gly Ala 1 5 10 15 Asn Ser Thr Leu Arg Cys Asn Phe Ser Asp Ser Val Asn Asn Leu Gln 20 25 30 Trp Phe His Gln Asn Pro Trp Gly Gln Leu Ile Asn Leu Phe Tyr Ile 35 40 45 Pro Ser Gly Thr Lys Gln Asn Gly Arg Leu Ser Ala Thr Thr Val Ala 50 55 60 Thr Glu Arg Tyr Ser Leu Leu Tyr Ile Ser Ser Ser Gln Thr Thr Asp 65 70 75 80 Ser Gly Val Tyr Phe Cys Ala Val Arg Gly Gly Ala Asp Gly Leu Thr 85 90 95 Phe Gly Lys Gly Thr His Leu Ile Ile Gln Pro Tyr Ile Gln Asn Pro 100 105 110 Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser 115 120 125 Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser 130 135 140 Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg 145 150 155 160 Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser 165 170 175 Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp 180 185 190 Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu 195 200 205 Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val 210 215 220 Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu 225 230 235 240 Met Thr Leu Arg Leu Trp Ser Ser Thr Gly 245 250 <210> 60 <211> 750 <212> DNA <213> Artificial Sequence <220> <223> TCRα receptor (HERV‐E) <400> 60 atacaagtgg agcagagtcc tccagacctg attctccagg agggagccaa ttccacgctg 60 cggtgcaatt tttctgactc tgtgaacaat ttgcagtggt ttcatcaaaa cccttgggga 120 cagctcatca acctgtttta cattccctca gggacaaaac agaatggaag attaagcgcc 180 acgactgtcg ctacggaacg ctacagctta ttgtacattt ccagcagcca gaccacagac 240 tcaggcgttt atttctgtgc tgtcgagga ggtgctgacg gactcacctt tggcaaaggg 300 actcatctaa tcatccagcc ctatatccag aaccctgacc ctgccgtgta ccagctgaga 360 gactctaaat ccagtgacaa gtctgtctgc ctattcaccg atttgattc tcaaacaaat 420 gtgtcacaat caaaggattc tgatgtgtat atcacagaca aaactgtgct agacatgagg 480 tctatggact tcaagagcaa cagtgctgtg gcctggagca acaaatctga ctttgcatgt 540 gcaaacgcct tcaacaacag cattattcca gaagacacct tcttccccag cccagaaagt 600 tcctgtgatg tcaagctggt cgagaaaagc tttgaaacag atacgaacct aaactttcaa 660 aacctgtcag tgattgggtt ccgaatcctc ctcctgaaag tggccgggtt taatctgctc 720 atgacgctgc ggctgtggtc cagcaccggt 750 <210> 61 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> TCRβ-related human receptor (HERV‐E) <400> 61 Gly Pro Gly Cys Arg Leu Leu Cys Cys Ala Val Leu Cys Leu Leu Gly 1 5 10 15 Ala Val Pro Ile Asp Thr 20 <210> 62 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> TCRβ-responsive region (HERV‐E) <400> 62 gggcccggct gcaggctgct ctgctgtgcg gttctctgtc tcctgggagc agttcccata 60 gacact 66 <210> 63 <211> 288 <212> PRT <213> Artificial Sequence <220> <223> TCRβ-receptor (HERV‐E) <400> 63 Glu Val Thr Gln Thr Pro Lys His Leu Val Met Gly Met Thr Asn Lys 1 5 10 15 Lys Ser Leu Lys Cys Glu Gln His Met Gly His Arg Ala Met Tyr Trp 20 25 30 Tyr Lys Gln Lys Ala Lys Lys Pro Pro Glu Leu Met Phe Val Tyr Ser 35 40 45 Tyr Glu Lys Leu Ser Ile Asn Glu Ser Val Pro Ser Arg Phe Ser Pro 50 55 60 Glu Cys Pro Asn Ser Ser Leu Leu Asn Leu His Leu His Ala Leu Gln 65 70 75 80 Pro Glu Asp Ser Ala Leu Tyr Leu Cys Ala Ser Ser Pro Pro Asn Glu 85 90 95 Lys Leu Phe Phe Gly Ser Gly Thr Gln Leu Ser Val Leu Glu Asp Leu 100 105 110 Asn Lys Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala 115 120 125 Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly 130 135 140 Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu 145 150 155 160 Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro 165 170 175 Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser 180 185 190 Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln 195 200 205 Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys 210 215 220 Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys 225 230 235 240 Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile 245 250 255 Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val 260 265 270 Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp Phe Glu Phe 275 280 285 <210> 64 <211> 864 <212> DNA <213> Artificial Sequence <220> <223> HERV‐E TCRβ <400> 64 gaagttaccc agacaccaaa acacctggtc atgggaatga caaataagaa gtctttgaaa 60 tgtgaacaac atatggggca cagggctatg tattggtaca agcagaaagc taagaagcca 120 ccggagctca tgtttgcta cagctatgag aaactctcta taaatgaaag tgtgccaagt 180 cgcttctcac ctgaatgccc caacagctct ctcttaaacc ttcacctaca cgccctgcag 240 ccagaagact cagccctgta tctctgcgcc agcagccctc ccaatgaaaa actgtttttt 300 ggcagtggaa cccagctctc tgtcttggag gacctgaaca aggtgttccc acccgaggtc 360 gctgtgtttg agccatcaga agcagagatc tcccacaccc aaaaggccac actggtgtgc 420 ctggccacag gcttcttccc tgaccacgtg gagctgagct ggtgggtgaa tgggaaggag 480 gtgcacagtg gggtcagcac ggacccgcag cccctcaagg agcagcccgc cctcaatgac 540 tccagatact gcctgagcag ccgcctgagg gtctcggcca ccttctggca gaacccccgc 600 aaccacttcc gctgtcaagt ccagttctac gggctctcgg agaatgacga gtggacccag 660 gatagggcca aacccgtcac ccagatcgtc agcgccgagg cctggggtag agcagactgt 720 ggcttacct cggtgtccta ccagcaaggg gtcctgtctg ccaccatcct ctatgagatc 780 ctgctaggga aggccaccct gtatgctgtg ctggtcagcg cccttgtgtt gatggcaatg 840 gtcaagagaa aggatttcga attc 864 <210> 65 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> TCR α-receptor (NY‑ESO‑1) <400> 65 With Glu Thr Slow Slow Gly Slow Slow Ile Slow Trp Slow Gln Slow Gln Trp 1 5 10 15 Val Dear Dear <210> 66 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of the TCRα signal peptide (NY-ESO-1) <400> 66 atggagaccc tcttgggcct gcttatcctt tggctgcagc tgcaatgggt gagcagc 57 <210> 67 <211> 255 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of TCRα (NY-ESO-1) <400> 67 Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val Pro Glu Gly 1 5 10 15 Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala Ile Tyr Asn 20 25 30 Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr Ser Leu Leu 35 40 45 Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg Leu Asn Ala 50 55 60 Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile Ala Ala Ser 65 70 75 80 Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Arg Pro Thr Ser 85 90 95 Gly Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser Leu Ile Val 100 105 110 His Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp 115 120 125 Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser 130 135 140 Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp 145 150 155 160 Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala 165 170 175 Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn 180 185 190 Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser 195 200 205 Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu 210 215 220 Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys 225 230 235 240 Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 245 250 255 <210> 68 <211> 765 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of TCRα (NY-ESO-1) <400> 68 aaacaggagg tgacgcagat tcctgcagct ctgagtgtcc cagaaggaga aaacttggtt 60 ctcaactgca gtttcactga tagcgctatt tacaacctcc agtggtttag gcaggaccct 120 gggaaaggtc tcacatctct gttgcttatt cagtcaagtc agagagagca aacaagtgga 180 aggcttaatg cctcgctgga taaatcatca ggacgtagta ctttatacat tgcagcttct 240 cagcctggtg actcagccac ctacctctgt gctgtgaggc ccacatcagg aggaagctac 300 atacctacat ttggaagagg aaccagcctt attgttcatc cgtatatcca gaaccctgat 360 cctgccgtgt accagctgcg ggacagcaag agcagcgaca agagcgtgtg cctgttcacc 420 gacttcgaca gccagaccaa cgtgtcccag agcaaggaca gcgacgtgta catcaccgac 480 aagaccgtgc tggacatgcg gagcatggac ttcaagagca acagcgccgt ggcctggtcc 540 aacaagagcg atttcgcctg cgccaacgcc ttcaacaaca gcattatccc cgaggacaca 600 ttcttcccaa gccccgagag cagctgcgac gtgaagctgg tggaaaagag cttcgagaca 660 gacaccaacc tgaacttcca gaacctgagc gtgatcggct tccggatcct gctgctgaag 720 gtggccggct tcaacctgct gatgaccctg agactgtggt ccagc 765 <210> 69 <211> twenty one <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of TCRβ signal peptide (NY-ESO-1) <400> 69 Met Ser Ile Gly Leu Leu Cys Cys Ala Ala Leu Ser Leu Leu Trp Ala 1 5 10 15 Gly Pro Val Asn Ala 20 <210> 70 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of TCRβ signal peptide (NY-ESO-1) <400> 70 atgagcatcg gcctcctgtg ctgtgcagcc ttgtctctcc tgtgggcagg tccagtgaat 60 gct 63 <210> 71 <211> 288 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of TCRβ (NY-ESO-1) <400> 71 Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr Gly Gln Ser 1 5 10 15 Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met Ser Trp 20 25 30 Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His Tyr Ser Val 35 40 45 Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Gly Tyr Asn Val 50 55 60 Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser Ala Ala 65 70 75 80 Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Ser Tyr Val Gly Asn 85 90 95 Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu Glu 100 105 110 Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser 115 120 125 Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala 130 135 140 Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly 145 150 155 160 Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys Glu 165 170 175 Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg 180 185 190 Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln 195 200 205 Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg 210 215 220 Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala 225 230 235 240 Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly Val Leu Ser Ala 245 250 255 Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val 260 2,65 270 Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp Phe 275 280 285 <210> 72 <211> 864 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of TCRβ (NY-ESO-1) <400> 72 ggtgtcactc agaccccaaa attccaggtc ctgaaaacag gacagagcat gacactgcag 60 tgtgcccagg atatgaacca tgaatacatg tcctggtatc gacaagaccc aggcatgggg 120 ctgaggctga ttcattactc agttggtgct ggtatcactg accaaggaga agtccccaat 180 ggctacaatg tctccagatc aaccacagag gatttcccgc tcaggctgct gtcggctgct 240 ccctcccaga catctgtgta cttctgtgcc agcagttacg tcgggaacac cggggagctg 300 ttttttggag aaggctctag gctgaccgta ctggaggatc tgaacaaggt gttcccccca 360 gaggtggccg tgttcgagcc ttctgaggcc gagatcagcc acacccagaa agccaccctc 420 gtgtgcctgg ccaccggctt tttccccgac cacgtggaac tgtcttggtg ggtcaacggc 480 aaagaggtgc acagcggcgt gtccaccgat ccccagcctc tgaaagaaca gcccgccctg 540 aacgacagcc ggtactgcct gagcagcaga ctgagagtgt ccgccacctt ctggcagaac 600 ccccggaacc acttcagatg ccaggtgcag ttctacggcc tgagcgagaa cgacgagtgg 660 acccaggaca gagccaagcc cgtgacccag atcgtgtctg ccgaagcctg gggcagagcc 720 gattgcggct ttaccagcgt gtcctatcag cagggcgtgc tgagcgccac catcctgtac 780 gagatcctgc tgggcaaggc caccctgtac gccgtgctgg tgtctgccct ggtgctgatg 840 gccatggtca agcggaaggacttc 864 <210> 73 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of LCDR1(CD19) <400> 73 Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn 1 5 10 <210> 74 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of LCDR2(CD19) <400> 74 Tyr His Thr Ser Arg Leu His Ser 1 5 <210> 75 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of LCDR3(CD19) <400> 75 Gln Gln Gly Asn Thr Leu Pro Tyr Thr 1 5 <210> 76 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR1 (CD19) <400> 76 Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser 1 5 10 <210> 77 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR2 (CD19) <400> 77 Val Ile Trp Gly Ser Glu Thr Thr Tyr 1 5 <210> 78 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR3(CD19) <400> 78 His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr 1 5 10 <210> 79 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of LCDR1(Her2) <400> 79 Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala 1 5 10 <210> 80 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of LCDR2 (Her2) <400> 80 Tyr Ser Ala Ser Phe Leu Tyr Ser 1 5 <210> 81 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of LCDR3(Her2) <400> 81 Gln Gln His Tyr Thr Thr Pro Pro Thr 1 5 <210> 82 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR1 (Her2) <400> 82 Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His 1 5 10 <210> 83 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR2 (Her2) <400> 83 Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg 1 5 10 <210> 84 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR3 (Her2) <400> 84 Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr 1 5 10 <210> 85 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of LCDR1 (PSMA) <400> 85 Lys Ala Ser Gln Asp Val Gly Thr Ala Val Asp 1 5 10 <210> 86 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of LCDR2 (PSMA) <400> 86 Tyr Trp Ala Ser Thr Arg His Thr 1 5 <210> 87 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of LCDR3 (PSMA) <400> 87 Gln Gln Tyr Asn Ser Tyr Pro Leu Thr 1 5 <210> 88 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR1 (PSMA) <400> 88 Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr Thr Ile His 1 5 10 <210> 89 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR2 (PSMA) <400> 89 Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr 1 5 10 <210> 90 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR3 (PSMA) <400> 90 Gly Trp Asn Phe Asp Tyr 1 5 <210> 91 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of LCDR1(CD43) <400> 91 Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn 1 5 10 <210> 92 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of LCDR2(CD43) <400> 92 Tyr Ala Thr Ser Arg Leu His Ser 1 5 <210> 93 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of LCDR3(CD43) <400> 93 Gln Gln Ser Asn Met Phe Pro Tyr Thr 1 5 <210> 94 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR1(CD43) <400> 94 Lys Ala Ser Gly Tyr Thr Phe Asn Gly Tyr Phe Met Asn 1 5 10 <210> 95 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR2(CD43) <400> 95 Arg Ile Asn Pro Asn Asn Gly Asp Ser Phe 1 5 10 <210> 96 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR3 (CD43) <400> 96 Glu Gly Tyr Tyr Gly Gly Arg Gly Tyr Ala Leu Asp Tyr 1 5 10 <210> 97 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR1 (CD47) <400> 97 Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr Gly Met Ser 1 5 10 <210> 98 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR2 (CD47) <400> 98 Thr Ile Thr Ser Gly Gly Thr Tyr Thr Tyr 1 5 10 <210> 99 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of HCDR3 (CD47) <400> 99 Ser Leu Ala Gly Asn Ala Met Asp Tyr 1 5 <210> 100 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of LCDR1(CD47) <400> 100 Arg Ala Ser Gln Ser Ile Ser Asp Tyr Leu His 1 5 10 <210> 101 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of LCDR2(CD47) <400> 101 Tyr Phe Ala Ser Gln Arg Ala Thr 1 5 <210> 102 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of LCDR3(CD47) <400> 102 Gln Gln Gly His Gly Phe Pro Arg Thr 1 5 <210> 103 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of ACDR1 (NY-ESO-1) <400> 103 Ser Phe Thr Asp Ser Ala Ile Tyr Asn Leu Gln 1 5 10 <210> 104 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of ACDR2 (NY-ESO-1) <400> 104 Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser 1 5 10 <210> 105 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of ACDR3 (NY-ESO-1) <400> 105 Ala Val Arg Pro Thr Ser Gly Gly Ser Tyr Ile Pro Thr 1 5 10 <210> 106 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of BCDR1 (NY-ESO-1) <400> 106 Ala Gln Asp Met Asn His Glu Tyr Met Ser 1 5 10 <210> 107 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of BCDR2 (NY-ESO-1) <400> 107 Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly 1 5 10 <210> 108 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of BCDR3 (NY-ESO-1) <400> 108 Ala Ser Ser Tyr Val Gly Asn Thr Gly Glu Leu Phe 1 5 10 <210> 109 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of ACDR1 (HERV-E) <400> 109 Asn Phe Ser Asp Ser Val Asn Asn Leu Gln 1 5 10 <210> 110 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of ACDR2 (HERV-E) <400> 110 Tyr Ile Pro Ser Gly Thr Lys Gln Asn 1 5 <210> 111 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> The amino acid sequence of ACDR3 (HERV-E) <400> 111 Ala Val Arg Gly Gly Ala Asp Gly Leu Thr 1 5 10 <210> 112 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of BCDR1 (HERV-E) <400> 112 Glu Gln His Met Gly His Arg Ala Met Tyr 1 5 10 <210> 113 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of BCDR2 (HERV-E) <400> 113 Val Tyr Ser Tyr Glu Lys Leu Ser Ile Asn Glu 1 5 10 <210> 114 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of BCDR3 (HERV-E) <400> 114 Ala Ser Ser Pro Pro Asn Glu Lys Leu Phe 1 5 10

Claims

1. A polynucleotide encoding a fusion protein, said fusion protein comprising the following (i) to (v): (i) Antigen-binding domain; (ii) Transmembrane domains; (iii) Intracellular signaling domains containing at least one co-stimulatory domain; (iv) self-cleaving peptides; and, (v) Signaling pathway modulators; The signal pathway modulator described herein has the following structural formula (II): N'-X-L1-Y-L2-Z-C'(II); in, In structural formula (II), N' is the N-terminus of the fusion protein. C' is the C-terminus of the fusion protein. L1 and L2 are both self-cleaving peptides. X represents FKBP12 protein, Y represents cyclophilin A (CYPA), and Z represents the N-terminal SH2 domain of SHP-2 protein. The antigen-binding domain binds to any one of the antigens selected from CD19, ErbB2 (HER2), and PSMA; The transmembrane domain is derived from CD8α; The intracellular signaling domains include co-stimulatory domains and primary signaling domains; The costimulatory domain is derived from CD137 (4-1BB); and, The primary signal structure domain is derived from CD3ζ.

2. The polynucleotide according to claim 1, wherein, further, the spacer region is disposed between the (i) antigen-binding domain and the (ii) transmembrane domain.

3. The polynucleotide of claim 1, wherein the signaling pathway regulator functions in the cytoplasm.

4. The polynucleotide of claim 1, wherein the antigen-binding domain specifically binds to CD19.

5. The polynucleotide according to claim 1, wherein the antigen-binding domain is composed of the amino acid sequence shown in SEQ ID NO:

3.

6. The polynucleotide according to claim 1, wherein the transmembrane domain consists of the amino acid sequence shown in SEQ ID NO:

5.

7. The polynucleotide according to claim 1, wherein the co-stimulatory domain consists of the amino acid sequence shown in SEQ ID NO:

7.

8. The polynucleotide according to claim 1, wherein the primary signaling domain consists of the amino acid sequence shown in SEQ ID NO:

9.

9. The polynucleotide of claim 1, wherein the self-cleaving peptide is derived from P2A, E2A, F2A or T2A.

10. The polynucleotide of claim 1, wherein the self-cleaving peptide is P2A.

11. An expression vector comprising a polynucleotide according to any one of claims 1 to 10.

12. The expression vector of claim 11, wherein the vector further comprises a sequence encoding a signal peptide.

13. The expression vector of claim 12, wherein the sequence encoding the signal peptide is inserted before the sequence encoding the antigen-binding domain.

14. A virus comprising a polynucleotide according to any one of claims 1 to 10.

15. An immune cell wherein a polynucleotide according to any one of claims 1 to 10 is introduced.

16. The immune cell according to claim 15, wherein the immune cell is a T cell or a natural killer cell.

17. A pharmaceutical composition for treating or preventing cancer, comprising immune cells as an active ingredient according to claim 15.

18. The pharmaceutical composition according to claim 17, wherein the cancer is selected from the group consisting of: gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, kidney cancer, melanoma, multiple myeloma, osteosarcoma, lymphoma, neuroma, mesothelioma, and esophageal cancer.

19. The pharmaceutical composition according to claim 17, wherein the cancer is an IgG-opsonized tumor.

20. The pharmaceutical composition of claim 18, wherein the cancer is selected from the group consisting of acute myeloid leukemia, laryngeal cancer, salivary gland cancer, and glioblastoma.

21. The use of an immune cell according to claim 15 or a pharmaceutical composition according to claim 17 in the preparation of a medicament for treating cancer; The cancer is selected from any one of blood cancer, breast cancer, or prostate cancer.

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