Hybrid costimulatory signal structure and use thereof

Abstract

Provided is a fusion protein, comprising a first costimulatory structure and a second costimulatory structure, wherein the first costimulatory structure comprises the full length of a first costimulatory molecule or an intracellular domain thereof, and the second costimulatory structure comprises the full length of a second costimulatory molecule or an intracellular domain thereof. Further provided are a modified immune cell comprising the fusion protein, a nucleic acid molecule, a vector, a cell, and a composition, and a use.

Description

Hybrid co-stimulatory signal structures and their applications Technical Field

[0001] This application relates to the field of biomedicine, specifically to a fusion protein comprising a first costimulatory structure and a second costimulatory structure and its applications. Background Technology

[0002] Currently, two types of engineered T cells have been successfully used to treat cancer patients: CAR-T and TCR-T. CAR uses a high-affinity antigen-binding domain and a co-stimulatory domain to induce T cell activation, but it typically responds to target cells with relatively high antigen concentrations. TCR, on the other hand, recognizes antigens by activating T cells through contact with a single pHLA / antigen on the target cell. TCR has a much lower affinity for its antigen but can respond to target cells displaying small amounts of antigen molecules. TCR consists of TCRα and TCRβ chains, which are non-covalently bound to CD3ε, CD3γ, CD3δ, and CD3ζ chains containing the tyrosine activation motif domain of the intracellular immune receptor responsible for TCR signaling. However, further improvements are needed to enhance its function for more effective sensing and more persistent killing of target cells, especially those with low-density antigens.

[0003] Therefore, the effectiveness of existing engineered immune cell technologies still needs to be improved. There is an urgent need to develop new co-stimulatory structures that can better enhance the killing ability of immune cells against tumor cells and improve the duration of killing, and can also overcome the limitations of engineered immune cells on antigen density, so as to solve related problems. Summary of the Invention

[0004] This application provides a fusion protein comprising a first co-stimulatory structure and a second co-stimulatory structure. The first co-stimulatory structure comprises a full-length or intracellular domain of a first co-stimulatory molecule, and the second co-stimulatory structure comprises a full-length or intracellular domain of a second co-stimulatory molecule. In this application, the fusion protein can be expressed on the cell surface and exhibits good expression performance on immune cells. It can activate immune cells at low antigen concentrations and demonstrate stronger killing power against tumors expressing rare antigens, reducing tumor escape. Furthermore, immune cells expressing the fusion protein can specifically bind to and kill target cells, and after re-inoculation experiments, they can continuously reduce tumor volume, exhibiting a sustained in vivo tumor-suppressing effect and effectively inhibiting tumor growth in the long term.

[0005] On one hand, this application provides a fusion protein comprising a first costimulatory structure and a second costimulatory structure, wherein the first costimulatory structure comprises a full-length or intracellular domain of a first costimulatory molecule, and the second costimulatory structure comprises a full-length or intracellular domain of a second costimulatory molecule, and the fusion protein is expressed on immune cells.

[0006] In some implementations, the first costimulatory structure comprises the full length of the first costimulatory molecule.

[0007] In some embodiments, the first co-stimulatory molecule is OX40 or 4-1BB.

[0008] In some embodiments, the second co-stimulatory structure includes an intracellular domain of the second co-stimulatory molecule.

[0009] In some embodiments, the second co-stimulatory molecule is OX40 or 4-1BB.

[0010] In some embodiments, the first costimulatory structure comprises the full length of a first costimulatory molecule, which is OX40, and the second costimulatory structure comprises an intracellular domain of a second costimulatory molecule, which is 4-1BB.

[0011] In some embodiments, the amino acid sequence of the first co-stimulatory structure is shown in SEQ ID NO:35, and the amino acid sequence of the second co-stimulatory structure is shown in SEQ ID NO:38.

[0012] In some embodiments, the first costimulatory structure comprises the full length of a first costimulatory molecule, which is 4-1BB, and the second costimulatory structure comprises an intracellular domain of a second costimulatory molecule, which is OX40.

[0013] In some embodiments, the amino acid sequence of the first co-stimulatory structure is shown in SEQ ID NO:37, and the amino acid sequence of the second co-stimulatory structure is shown in SEQ ID NO:36.

[0014] In some embodiments, the second costimulatory structure is connected to the C-terminus of the first costimulatory structure.

[0015] In some embodiments, the fusion protein is capable of providing a co-stimulatory signal.

[0016] In some embodiments, the immune cells are T cells.

[0017] In some embodiments, the immune cells are iNKT cells.

[0018] In some embodiments, the immune cells are TCR-T cells and / or CAR-T cells.

[0019] In some embodiments, the fusion protein can be used for adoptive cell therapy (ACT).

[0020] In some embodiments, the adoptive cell therapy is TCR-T therapy and / or CAR-T therapy.

[0021] In some embodiments, the fusion protein is co-expressed with an antigen recognition receptor.

[0022] In some embodiments, the antigen recognition receptor is a T-cell receptor (TCR) and / or a chimeric antigen receptor (CAR).

[0023] In some embodiments, the T cell receptor and / or chimeric antigen receptor specifically bind to the target antigen.

[0024] In some embodiments, the target antigen is a tumor antigen.

[0025] In some embodiments, the target antigens are: PD-L1, AFP, ASCL2, B melanoma antigen (BAGE) family members, BORIS, cancer-testis antigen, cancer-testis antigen 83 (CT-83), CAIX, carcinoembryonic antigen (CEA), EBV, EPHB2, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, glycoprotein 100 (GP100), hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) NS3, human papillomavirus (HPV) E6, HPV-E7, IGF2BP3, IGF2BP1, K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, LMP2, LY6G6D, melanoma antigen (MAGE) family members, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), NYESO-1, P53, PAP, PLAC1, melanoma preferred expression antigen (PRAME), PSA, SSX1, SSX2, SSX3, SSX4, SSX5, SSX8, thyroglobulin, tyrosinase, TRP-1, TRP-2, WT-1, Wnt10A, CMV, MCPyV, ROPN1, TP53 R175H, TP53 R248Q, XAGE-1 and / or XAGE-2.

[0026] In some embodiments, the target antigen is NY-ESO-1.

[0027] In some implementations, the target antigen is PRAME.

[0028] In some implementations, the TCR comprises an α chain and a β chain, the α chain comprising an α chain variable region (TRAV) and an α chain constant region (TRAC), and the β chain comprising a β chain variable region (TRBV) and a β chain constant region (TRBC).

[0029] In some implementations, the TRAV includes complementary decision regions CDR1, CDR2, and CDR3.

[0030] In some embodiments, the TRAV includes complementarity-determining regions CDR1, CDR2, and CDR3, the amino acid sequences of which are selected from any combination of the following:

[0031] a) The amino acid sequence of CDR1 is shown in SEQ ID NO:12, the amino acid sequence of CDR2 is shown in SEQ ID NO:13, and the amino acid sequence of CDR3 is shown in SEQ ID NO:14; and

[0032] b) The amino acid sequence of CDR1 is shown in SEQ ID NO:28, the amino acid sequence of CDR2 is shown in SEQ ID NO:29, and the amino acid sequence of CDR3 is shown in SEQ ID NO:30.

[0033] In some embodiments, the amino acid sequence of the TRAV is as shown in SEQ ID NO:1 or SEQ ID NO:18.

[0034] In some implementations, the TRBV includes complementary determination regions CDR1, CDR2, and CDR3.

[0035] In some embodiments, the TRBV includes complementarity-determining regions CDR1, CDR2, and CDR3, the amino acid sequences of which are selected from any combination of the following:

[0036] a) The amino acid sequence of CDR1 is shown in SEQ ID NO:15, the amino acid sequence of CDR2 is shown in SEQ ID NO:16, and the amino acid sequence of CDR3 is shown in SEQ ID NO:17; and

[0037] b) The amino acid sequence of CDR1 is shown in SEQ ID NO:31, the amino acid sequence of CDR2 is shown in SEQ ID NO:32, and the amino acid sequence of CDR3 is shown in SEQ ID NO:33.

[0038] In some embodiments, the amino acid sequence of the TRBV is as shown in SEQ ID NO:4 or SEQ ID NO:21.

[0039] In some implementations, the TRAC and / or TRBC are mouse and / or human constant regions.

[0040] In some embodiments, the CAR includes an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain.

[0041] In some embodiments, the antigen-binding domain is an antibody or its antigen-binding fragment, or the antigen-binding domain is the PD-1 extracellular domain.

[0042] In some embodiments, the amino acid sequence of the PD-1 extracellular domain is shown in SEQ ID NO:44.

[0043] In some embodiments, the transmembrane domain is a transmembrane domain derived from any of the following proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

[0044] In some embodiments, the intracellular signaling domain is an intracellular signaling domain derived from any of the following proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (KSHV), DAP10, DAP-12, and a domain containing at least one ITAM.

[0045] In some embodiments, the chimeric antigen receptor includes a hinge region derived from any of the following proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

[0046] In some embodiments, the chimeric antigen receptor comprises a signal peptide.

[0047] On the other hand, this application provides a modified immune cell containing the fusion protein.

[0048] In some embodiments, the modified immune cells also contain antigen recognition receptors.

[0049] In some embodiments, the antigen recognition receptor is a T-cell receptor (TCR) and / or a chimeric antigen receptor (CAR).

[0050] In some embodiments, the T cell receptor and / or chimeric antigen receptor specifically bind to the target antigen.

[0051] In some embodiments, the target antigen is a tumor antigen.

[0052] In some embodiments, the target antigen is PD-L1, AFP, ASCL2, B melanoma antigen (BAGE) family members, BORIS, cancer-testis antigen, cancer-testis antigen 83 (CT-83), CAIX, carcinoembryonic antigen (CEA), EBV, EPHB2, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, glycoprotein 100 (GP100), hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) NS3, human papillomavirus (HPV) E6, HPV-E7, IGF2BP3, IGF2BP1, K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, LMP2, LY6G6D, melanoma antigen (MAGE) family members, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), NYESO-1, P53, PAP, PLAC1, melanoma preferred expression antigen (PRAME), PSA, SSX1, SSX2, SSX3, SSX4, SSX5, SSX8, thyroglobulin, tyrosinase, TRP-1, TRP-2, WT-1, Wnt10A, CMV, MCPyV, ROPN1, TP53 R175H, TP53 R248Q, XAGE-1 and / or XAGE-2.

[0053] In some embodiments, the target antigen is NY-ESO-1.

[0054] In some implementations, the target antigen is PRAME.

[0055] In some implementations, the TCR comprises an α chain and a β chain, the α chain comprising an α chain variable region (TRAV) and an α chain constant region (TRAC), and the β chain comprising a β chain variable region (TRBV) and a β chain constant region (TRBC).

[0056] In some implementations, the TRAV includes complementary decision regions CDR1, CDR2, and CDR3.

[0057] In some embodiments, the TRAV includes complementarity-determining regions CDR1, CDR2, and CDR3, the amino acid sequences of which are selected from any combination of the following:

[0058] a) The amino acid sequence of CDR1 is shown in SEQ ID NO:12, the amino acid sequence of CDR2 is shown in SEQ ID NO:13, and the amino acid sequence of CDR3 is shown in SEQ ID NO:14; and

[0059] b) The amino acid sequence of CDR1 is shown in SEQ ID NO:28, the amino acid sequence of CDR2 is shown in SEQ ID NO:29, and the amino acid sequence of CDR3 is shown in SEQ ID NO:30.

[0060] In some embodiments, the amino acid sequence of the TRAV is as shown in SEQ ID NO:1 or SEQ ID NO:18.

[0061] In some implementations, the TRBV includes complementary determination regions CDR1, CDR2, and CDR3.

[0062] In some embodiments, the TRBV includes complementarity-determining regions CDR1, CDR2, and CDR3, the amino acid sequences of which are selected from any combination of the following:

[0063] a) The amino acid sequence of CDR1 is shown in SEQ ID NO:15, the amino acid sequence of CDR2 is shown in SEQ ID NO:16, and the amino acid sequence of CDR3 is shown in SEQ ID NO:17; and

[0064] b) The amino acid sequence of CDR1 is shown in SEQ ID NO:31, the amino acid sequence of CDR2 is shown in SEQ ID NO:32, and the amino acid sequence of CDR3 is shown in SEQ ID NO:33.

[0065] In some embodiments, the amino acid sequence of the TRBV is as shown in SEQ ID NO:4 or SEQ ID NO:21.

[0066] In some implementations, the TRAC and / or TRBC are mouse and / or human constant regions.

[0067] In some embodiments, the CAR includes an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain.

[0068] In some embodiments, the antigen-binding domain is an antibody or its antigen-binding fragment, or the antigen-binding domain is the PD-1 extracellular domain.

[0069] In some embodiments, the amino acid sequence of the PD-1 extracellular domain is shown in SEQ ID NO:44.

[0070] In some embodiments, the transmembrane domain is a transmembrane domain derived from any of the following proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

[0071] In some embodiments, the intracellular signaling domain is an intracellular signaling domain derived from any of the following proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (KSHV), DAP10, DAP-12, and a domain containing at least one ITAM.

[0072] In some embodiments, the chimeric antigen receptor includes a hinge region derived from any of the following proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

[0073] In some embodiments, the chimeric antigen receptor comprises a signal peptide.

[0074] In some embodiments, the immune cells are T cells.

[0075] In some embodiments, the immune cells are iNKT cells.

[0076] In some embodiments, the immune cells are TCR-T cells and / or CAR-T cells.

[0077] On the other hand, this application provides isolated nucleic acid molecules that encode the fusion protein.

[0078] In some embodiments, the nucleic acid molecule also encodes an antigen recognition receptor.

[0079] In some embodiments, the antigen recognition receptor is a T-cell receptor (TCR) and / or a chimeric antigen receptor (CAR).

[0080] In some embodiments, the nucleic acid molecule also encodes a self-cleaving peptide.

[0081] In some embodiments, the sequence encoding the self-cleaving peptide is located between the sequences encoding the fusion protein and the antigen recognition receptor.

[0082] In some embodiments, the self-cleaving peptide comprises a 2A peptide.

[0083] In some embodiments, the self-cleaving peptide is P2A, T2A, E2A, or F2A.

[0084] In some embodiments, the self-cleaving peptide is P2A.

[0085] On the other hand, this application provides a vector containing the nucleic acid molecule.

[0086] On the other hand, this application provides cells that contain the nucleic acid molecules and / or the vector.

[0087] On the other hand, this application provides methods for preparing the fusion protein, the modified immune cells, the nucleic acid molecules, the vector, and / or the cells.

[0088] On the other hand, this application provides a pharmaceutical composition comprising the fusion protein, the modified immune cell, the nucleic acid molecule, the carrier and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0089] On the other hand, this application provides the use of the fusion protein, the modified immune cell, the nucleic acid molecule, the carrier, the cell and / or the pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of diseases and / or conditions.

[0090] On the other hand, this application provides a method for preventing and / or treating diseases and / or conditions, comprising administering the fusion protein, the modified immune cells, the nucleic acid molecules, the carrier, the cells, and / or the pharmaceutical composition to a subject in need.

[0091] On the other hand, this application provides the fusion protein, the modified immune cell, the nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition for the prevention and / or treatment of diseases and / or conditions.

[0092] In some implementations, the disease and / or symptom is a tumor.

[0093] In some embodiments, the tumor is a solid tumor and / or a hematoma.

[0094] In some embodiments, the tumor is lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, stomach cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer, and / or skin cancer.

[0095] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0096] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0097] Figure 1 shows a schematic diagram of the reinforcement structure and NY-ESO-1 TCR described in this application.

[0098] Figure 2 shows the results of the mixed co-stimulatory signal structure described in this application and the expression of NY-ESO-1 TCR after transfection into cells.

[0099] Figure 3A shows the killing effect of the hybrid co-stimulation signaling structure described in this application on positive target cells A-375; Figure 3B shows the killing effect of the hybrid co-stimulation signaling structure described in this application on negative target cells MB-231.

[0100] Figure 4 shows the IFN-γ secretion level results of the hybrid co-stimulatory signaling structure described in this application.

[0101] Figure 5A shows the in vivo antitumor experimental structure of the hybrid co-stimulation signaling structure described in this application; Figure 5B shows the in vivo sustained antitumor experimental results of the hybrid co-stimulation signaling structure described in this application.

[0102] Figure 6 shows a schematic diagram of the reinforcement structure and PRAME TCR described in this application.

[0103] Figure 7 shows the results of the mixed co-stimulatory signal structure described in this application and the expression of PRAME TCR after transfection into cells.

[0104] Figure 8A shows the killing effect of the hybrid co-stimulation signaling structure described in this application on positive target cells A-375; Figure 8B shows the killing effect of the hybrid co-stimulation signaling structure described in this application on negative target cells MB-231.

[0105] Figure 9 shows the results of the mixed co-stimulatory signal structure described in this application and the expression of NY-ESO-1 TCR after transfection into iNKT cells.

[0106] Figure 10 shows the killing effect of the hybrid co-stimulatory signaling structure described in this application on positive target cells A-375.

[0107] Figure 11A shows a schematic diagram of the enhancement structure and ECD-CAR described in this application; Figure 11B shows the results of D4 expression of the ECD-CAR described in this application; Figure 11C shows the results of D8 expression of the ECD-CAR described in this application; Figure 11D shows the killing effect of the mixed co-stimulatory signaling structure described in this application on positive target cells A-375; Figure 11E shows the killing effect of the mixed co-stimulatory signaling structure described in this application on positive target cells MB-231; Figure 11F shows the killing effect of the mixed co-stimulatory signaling structure described in this application on positive target cells HCC827; Figure 11G shows the killing effect of the mixed co-stimulatory signaling structure described in this application on positive target cells LS174T; Figure 11H shows the killing effect of the mixed co-stimulatory signaling structure described in this application on target cells LO2; Figure 11I shows the killing effect of the mixed co-stimulatory signaling structure described in this application on target cells 293T. Detailed Implementation

[0108] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0109] Terminology Definition

[0110] In this application, the term "co-stimulatory molecule" generally refers to a molecule involved in co-stimulatory signal transduction. In this application, the co-stimulatory molecule can be a cell surface molecule and its ligand that provides a co-stimulatory signal for the complete activation of T (or B) cells. In this application, the co-stimulatory molecule can be a full-length co-stimulatory molecule or a truncated co-stimulatory molecule. In this application, the terms "full-length" and "FL (full length)" are generally used interchangeably and usually refer to the full size of the original molecule. In this application, the full-length co-stimulatory molecule can be a co-stimulatory molecule comprising an extracellular domain, a transmembrane domain, and an intracellular domain. For example, the co-stimulatory molecule can be the complete structure of a co-stimulatory molecule. For example, the co-stimulatory molecule can be a truncated co-stimulatory molecule that retains the function of providing a co-stimulatory signal. In this application, the co-stimulatory molecule can be OX40 or 4-1BB. In this application, the term "co-stimulatory structure" generally refers to a structure containing the full length or a portion thereof of a co-stimulatory molecule. In this application, the co-stimulatory structure can provide a co-stimulatory signal. For example, the co-stimulatory structure may include the full length of the co-stimulatory molecule. For example, the co-stimulatory structure may include an intracellular domain of the co-stimulatory molecule. For example, the co-stimulatory structure may include the full length and / or intracellular domain of OX40 and / or 4-1BB.

[0111] In this application, the term "fusion protein" generally refers to a protein composed of two or more polypeptides that are typically not bound in their native state, forming a continuous polypeptide linked together by peptide bonds at the amino and carboxyl ends of the protein. In this application, the binding can refer to the direct or indirect binding of the two or more polypeptide components. In this application, the indirect binding can be through linker binding.

[0112] The protein and / or amino acid sequences involved in this application should also be understood to include at least the following range: variants or homologs having the same or similar functions as the protein. In this application, the variant can be a protein or polypeptide that has undergone substitution, deletion, or addition of one or more amino acids in the amino acid sequence of the protein. For example, the functional variant may comprise a protein or polypeptide that has undergone amino acid alterations through substitution, deletion, and / or insertion of at least one, such as 1-30, 1-20, or 1-10, or even 1, 2, 3, 4, or 5 amino acids. The functional variant may substantially retain the biological properties of the protein or polypeptide before the alteration (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least about 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the biological activity (e.g., the ability to provide co-stimulatory signals) of the protein or polypeptide before the alteration. For example, the substitution can be a conservative substitution.

[0113] In this application, the homolog can be a protein or polypeptide that has at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology with the amino acid sequence of the protein and / or the polypeptide.

[0114] In this application, the term "immune cell" generally refers to an immune cell that participates in the immune response and performs effector functions. For example, the immune cell can clear foreign antigens or promote immune effector responses. In this application, the immune cell can be a modified immune cell. In this application, the immune cell can be a T cell. In this application, the immune cell can be an iNKT cell. In this application, the modified immune cell is also called an engineered immune cell, which generally refers to an immune cell that has been genetically modified by transcribing additional genetic material in the form of DNA or RNA. For example, the modified immune cell can express a T cell receptor (TCR). In this application, the modified immune cell can express a TCR. For example, the modified immune cell can be a TCR-T cell.

[0115] In this application, the term "antigen recognition receptor" generally refers to a protein present on the cell surface that triggers a response in the immune system by binding to a target antigen. In this application, the antigen recognition receptor may be a B cell receptor or a T cell receptor (TCR). In this application, the antigen recognition receptor may be wild-type or artificially modified. For example, the antigen recognition receptor may be a TCR.

[0116] In this application, the terms "T cell receptor" and "TCR" are generally used interchangeably and typically refer to a specific receptor on the surface of T cells. In this application, the TCR may be responsible for recognizing antigens presented by the major histocompatibility complex (MHC). In this application, the TCR may be a heterodimer composed of two distinct subunits. For example, the TCR may be composed of an α subunit and / or a β subunit. For example, the TCR may contain an α chain and / or a β chain. In this application, the α chain may contain an α chain variable region (TRAV / Vα) and / or an α chain constant region (TRAC / Cα), and the β chain may contain a β chain variable region (TRBV / Vβ) and / or a β chain constant region (TRBC / Cβ). For example, the TCR may be composed of a γ subunit and / or a δ subunit. In this application, the TCR may be wild-type or artificially modified. For example, the TCR may be a synthetically produced TCR. In this application, the TCR may specifically bind to a target antigen. For example, the TCR can specifically bind to NY-ESO-1. For example, the TCR can specifically bind to PRAME. In this application, the TCR may include a complementarity-determining region (CDR). In this application, the CDR can be determined in various ways. In this application, the CDR covers CDR sequences partitioned according to any CDR partitioning method. In this application, the CDR may cover its variants. For example, the amino acid sequence of the CDR may be substituted, deleted, and / or added with one or more amino acids, such as 1-30, 1-20, or 1-10, or for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids substituted, deleted, and / or inserted. For example, the CDR covers homologs. For example, the homolog can be an amino acid sequence that has at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology with the amino acid sequence of the CDR.

[0117] In this application, the terms "chimeric antigen receptor" and "CAR" are generally used interchangeably, typically referring to a recombinant polypeptide comprising at least an extracellular domain, a transmembrane domain, and an intracellular domain that specifically bind to an antigen or target. In this application, the extracellular domain may include an antigen-binding domain. In this application, the extracellular domain may also include a hinge region and / or a signal peptide. In this application, the intracellular domain may include an intracellular signal transduction domain. In this application, the intracellular domain may also include one or more co-stimulatory domains. In this application, the CAR may be artificially synthesized.

[0118] In this application, the term "target antigen" generally refers to any molecule that can elicit an immune response or can be bound by antibodies or antigen-binding molecules. In this application, the target antigen can be a macromolecule, peptide, polypeptide, or amino acid. In this application, the target antigen can be endogenously expressed or recombinantly expressed. For example, the target antigen can be expressed from genomic DNA. For example, the target antigen can be generated by mutations in normal proteins. For example, the target antigen can be generated by protein cleavage or hydrolysis. In this application, the target antigen can be a tumor antigen. For example, the target antigen can be a tumor-associated antigen. For example, the target antigen can be a tumor-specific antigen. In this application, the target antigen may be PD-L1, AFP, ASCL2, B melanoma antigen (BAGE) family members, BORIS, cancer-testis antigen, cancer-testis antigen 83 (CT-83), CAIX, carcinoembryonic antigen (CEA), EBV, EPHB2, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, glycoprotein 100 (GP100), hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) NS3, human papillomavirus (HPV) E6, HPV-E7, IGF2BP3, IGF2BP1, K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, LMP2, LY6G6D, members of the melanoma antigen (MAGE) family, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), NYESO-1, P53, PAP, PLAC1, melanoma preferred expression antigen (PRAME), PSA, SSX1, SSX2, SSX3, SSX4, SSX5, SSX8, thyroglobulin, tyrosinase, TRP-1, TRP-2, WT-1, Wnt10A, CMV, MCPyV, ROPN1, TP53 R175H, TP53 R248Q, XAGE-1 and / or XAGE-2. For example, the target antigen may be NY-ESO-1. For example, the target antigen may be PRAME.

[0119] In this application, the term "self-cleaving peptide" generally refers to a class of polypeptides capable of cleaving proteins. For example, the self-cleaving peptide can achieve protein cleavage via ribosome jumping. In this application, the self-cleaving peptide can be a 2A peptide. For example, the self-cleaving peptide can be T2A, F2A, P2A, and / or E2A. For example, the self-cleaving peptide can be P2A.

[0120] In this application, the term "pharmaceuticalally acceptable carrier" generally refers to a non-toxic material that does not interfere with the effectiveness of the bioactivity of the active ingredient. For example, a pharmaceutically acceptable carrier includes pharmaceutically acceptable excipients or stabilizers that are non-toxic to cells or mammals exposed to them at the doses and concentrations used. For example, a physiologically acceptable carrier may be water, salt, protein, polysaccharide, lipid, or inactive viral particles.

[0121] In this application, the term "prevention and / or treatment" generally refers to the prevention and / or treatment of a disease. For example, prevention and / or treatment may include preventing the onset of the disease, slowing or reversing the disease progression, preventing or slowing the onset of one or more symptoms associated with the disease, reducing or alleviating one or more symptoms associated with the disease, reducing the severity and duration of the disease and any symptoms associated with it, or preventing further increase in the severity of the disease and any symptoms associated with it. In this application, the disease may be a tumor and / or a virus. For example, the disease may be a tumor.

[0122] Invention Details

[0123] Fusion protein

[0124] On one hand, this application provides a fusion protein comprising a first co-stimulatory structure and a second co-stimulatory structure, wherein the fusion protein is expressed on immune cells. In this application, the fusion protein is capable of providing a co-stimulatory signal.

[0125] In this application, the first co-stimulatory structure may comprise the full length of the first co-stimulatory molecule or an intracellular domain. For example, the first co-stimulatory structure may comprise the full length of the first co-stimulatory molecule.

[0126] In this application, the co-stimulatory molecule can be OX40 and / or 4-1BB. For example, the amino acid sequence of OX40 can be as shown in SEQ ID NO:35. For example, the amino acid sequence of 4-1BB can be as shown in SEQ ID NO:37.

[0127] In this application, the first co-stimulatory molecule may be OX40 and / or 4-1BB. For example, the first co-stimulatory molecule may be OX40. For example, the first co-stimulatory molecule may contain the amino acid sequence shown in SEQ ID NO:35. For example, the first co-stimulatory molecule may be 4-1BB. For example, the first co-stimulatory molecule may contain the amino acid sequence shown in SEQ ID NO:37.

[0128] In this application, the first co-stimulatory structure may comprise a full length of 0.40. In this application, the first co-stimulatory structure may comprise a full length of 4-1BB.

[0129] In this application, the second co-stimulatory structure may comprise the full-length or intracellular domain of the second co-stimulatory molecule. For example, the second co-stimulatory structure may comprise the intracellular domain of the second co-stimulatory molecule.

[0130] In this application, the second co-stimulatory molecule can be OX40 and / or 4-1BB. For example, the second co-stimulatory molecule can be OX40. For example, the second co-stimulatory molecule can contain the amino acid sequence shown in SEQ ID NO:35. For example, the second co-stimulatory molecule can be 4-1BB. For example, the second co-stimulatory molecule can contain the amino acid sequence shown in SEQ ID NO:37.

[0131] In this application, the second co-stimulatory structure may include an intracellular domain of OX40. For example, the second co-stimulatory structure may include the amino acid sequence shown in SEQ ID NO:36. In this application, the second co-stimulatory structure may include an intracellular domain of 4-1BB. For example, the second co-stimulatory structure may include the amino acid sequence shown in SEQ ID NO:38.

[0132] In this application, the fusion protein comprises a first costimulatory structure and a second costimulatory structure, wherein the first costimulatory structure comprises the full length of the first costimulatory molecule, and the second costimulatory structure comprises the intracellular domain of the second costimulatory molecule.

[0133] For example, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure, wherein the first co-stimulatory structure comprises the full length of OX40, and the second co-stimulatory structure comprises an intracellular domain of 4-1BB.

[0134] For example, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure, wherein the first co-stimulatory structure comprises the full length of OX40, and the second co-stimulatory structure comprises an intracellular domain of 4-1BB, the amino acid sequence of OX40 being as shown in SEQ ID NO:35, and the amino acid sequence of 4-1BB being as shown in SEQ ID NO:37. For example, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure, wherein the first co-stimulatory structure comprises the full length of 4-1BB, and the second co-stimulatory structure comprises an intracellular domain of OX40, the amino acid sequence of OX40 being as shown in SEQ ID NO:35, and the amino acid sequence of 4-1BB being as shown in SEQ ID NO:37.

[0135] For example, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure, wherein the first co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:35 and the second co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:38. Alternatively, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure, wherein the first co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:37 and the second co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:36.

[0136] In this application, the second co-stimulatory structure can be connected to either the N-terminus or the C-terminus of the first co-stimulatory structure. For example, the second co-stimulatory structure can be connected to the C-terminus of the first co-stimulatory structure.

[0137] In this application, the first co-stimulatory structure and the second co-stimulatory structure can be directly connected or indirectly connected. For example, the first co-stimulatory structure and the second co-stimulatory structure can be directly connected. For example, the second co-stimulatory structure can be directly connected to the C-terminus of the first co-stimulatory structure. For example, the first co-stimulatory structure and the second co-stimulatory structure can be indirectly connected. For example, the second co-stimulatory structure and the first co-stimulatory structure can be connected via a connector. For example, the second co-stimulatory structure can be indirectly connected to the C-terminus of the first co-stimulatory structure.

[0138] For example, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:39. For example, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:40.

[0139] In this application, the fusion protein can be a protein or polypeptide whose amino acid sequence has been substituted, deleted, or added to one or more amino acids. For example, the fusion protein may contain a protein or polypeptide with amino acid alterations caused by substitution, deletion, and / or insertion of at least one, such as 1-30, 1-20, or 1-10, or even 1, 2, 3, 4, or 5 amino acids. In this application, the fusion protein can substantially retain the biological properties of the protein or polypeptide before the alteration (e.g., substitution, deletion, or addition). For example, the fusion protein can retain at least about 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the biological activity (e.g., the ability to provide co-stimulatory signals) of the protein or polypeptide before the alteration. For example, the substitution can be a conserved substitution.

[0140] In this application, the fusion protein may be a homolog thereof. In this application, the homolog may be a protein or polypeptide having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology with the amino acid sequence of the protein and / or the polypeptide.

[0141] In this application, the fusion protein may contain a sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher homology with the amino acid sequence shown in SEQ ID NO:39. In this application, the fusion protein may contain a sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher homology with the amino acid sequence shown in SEQ ID NO:40.

[0142] In this application, the fusion protein can be used in adoptive cell therapy (ACT). For example, the adoptive cell therapy can be TCR-T therapy. For example, the adoptive cell therapy can be CAR-T therapy.

[0143] In this application, the fusion protein can be co-expressed with other proteins, peptides, or polypeptides. For example, the fusion protein can be co-expressed with an antigen recognition receptor. For example, the fusion protein can be co-expressed with a TCR. For example, the fusion protein can be co-expressed with a CAR. In this application, the antigen recognition receptor can specifically bind to the target antigen. In this application, the antigen recognition receptor can specifically bind to a target antigen, which may be PD-L1, AFP, ASCL2, B melanoma antigen (BAGE) family members, BORIS, cancer-testis antigen, cancer-testis antigen 83 (CT-83), CAIX, carcinoembryonic antigen (CEA), EBV, EPHB2, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, glycoprotein 100 (GP100), hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) NS3, human papillomavirus (HPV) E6, HPV-E7, IGF2BP3, IGF2BP1, K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, LMP2, LY6G6D, melanoma antigen (MAGE) family members, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), NYESO-1, P53, PAP, PLAC1, melanoma preferred expression antigen (PRAME), PSA, SSX1, SSX2, SSX3, SSX4, SSX5, SSX8, thyroglobulin, tyrosinase, TRP-1, TRP-2, WT-1, Wnt10A, CMV, MCPyV, ROPN1, TP53 R175H, TP53 R248Q, XAGE-1 and / or XAGE-2. For example, the TCR can specifically bind to the target antigen. For example, the target antigen may be a tumor and / or viral antigen. For example, the tumor antigen may be a tumor-associated antigen. For example, the tumor antigen may be a tumor-specific antigen. In this application, the TCR may specifically bind to a tumor antigen. For example, the TCR may specifically bind to NY-ESO-1. For example, the TCR may specifically bind to PRAME. In this application, the TCR may specifically bind to a viral antigen.

[0144] In this application, the TCR may comprise an α chain and / or a β chain. In this application, the α chain comprises an α chain variable region (TRAV / Vα) and / or an α chain constant region (TRAC / Cα), and the β chain comprises a β chain variable region (TRBV / Vβ) and / or a β chain constant region (TRBC / Cβ).

[0145] In this application, the TCR may include a complementarity-determining region (CDR). In this application, the α chain and / or β chain may include a CDR. In this application, the TRAV and / or TRBV may include a CDR. In this application, the CDR can be determined in various ways. In this application, the CDR encompasses the CDR sequence partitioned according to any CDR partitioning method. In this application, the CDR may encompass its variants. For example, the amino acid sequence of the CDR may be substituted, deleted, and / or added with one or more amino acids, such as 1-30, 1-20, or 1-10, or for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids substituted, deleted, and / or inserted. For example, the CDR encompasses homologs. For example, the homolog can be an amino acid sequence that has at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology with the amino acid sequence of the CDR.

[0146] In this application, the TCR may include a constant region. In this application, the constant region may be a wild-type sequence or a mutated or optimized sequence. In this application, the constant region may be a constant region of any species origin; for example, the constant region may be a constant region derived from rats, mice, rabbits, goats, alpacas, or humans. For example, the constant region may be a mouse constant region. For example, the constant region may be a human constant region. In this application, the constant region may be a TRAC and / or TRBC.

[0147] In this application, the TCR may include a TRAV. In this application, the TRAV may include CDR1, CDR2, and / or CDR3. For example, the TRAV may include CDR1, CDR2, and CDR3.

[0148] For example, the TRAV may contain CDR1, CDR2, and CDR3, where CDR1 may contain the amino acid sequence shown in SEQ ID NO:12, CDR2 may contain the amino acid sequence shown in SEQ ID NO:13, and CDR3 may contain the amino acid sequence shown in SEQ ID NO:14. For example, the amino acid sequence of the TRAV may be as shown in SEQ ID NO:1.

[0149] For example, the TRAV may contain CDR1, CDR2, and CDR3, where CDR1 may contain the amino acid sequence shown in SEQ ID NO:28, CDR2 may contain the amino acid sequence shown in SEQ ID NO:29, and CDR3 may contain the amino acid sequence shown in SEQ ID NO:30. For example, the amino acid sequence of the TRAV may be as shown in SEQ ID NO:18.

[0150] In this application, the TCR may further comprise a TRAC. The TRAC may contain the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:19. In this application, the TRAC may be a mouse TRAC. For example, the TRAC may be an mTRAC. In this application, the TRAC may be a human TRAC. For example, the TRAC may be an hTRAC.

[0151] In this application, the TCR may comprise an α chain, which may comprise the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:20.

[0152] In this application, the TCR may include a TRBV. In this application, the TRBV may include CDR1, CDR2, and / or CDR3. For example, the TRBV may include CDR1, CDR2, and CDR3.

[0153] For example, the TRBV may contain CDR1, CDR2, and CDR3, where CDR1 may contain the amino acid sequence shown in SEQ ID NO:15, CDR2 may contain the amino acid sequence shown in SEQ ID NO:16, and CDR3 may contain the amino acid sequence shown in SEQ ID NO:17. For example, the amino acid sequence of the TRBV may be as shown in SEQ ID NO:4.

[0154] For example, the TRBV may contain CDR1, CDR2, and CDR3, where CDR1 may contain the amino acid sequence shown in SEQ ID NO:31, CDR2 may contain the amino acid sequence shown in SEQ ID NO:32, and CDR3 may contain the amino acid sequence shown in SEQ ID NO:33. For example, the amino acid sequence of the TRBV may be as shown in SEQ ID NO:21.

[0155] In this application, the TCR comprises an α chain and a β chain, the α chain comprises a TRAV, the TRAV comprises CDR1-3, the amino acid sequence of CDR1 is shown in SEQ ID NO:12, the amino acid sequence of CDR2 is shown in SEQ ID NO:13, and the amino acid sequence of CDR3 is shown in SEQ ID NO:14; the β chain comprises a TRBV, the TRBV comprises CDR1-3, the amino acid sequence of CDR1 is shown in SEQ ID NO:15, the amino acid sequence of CDR2 is shown in SEQ ID NO:16, and the amino acid sequence of CDR3 is shown in SEQ ID NO:17. In this application, the TCR comprises an α chain and a β chain, the α chain comprises a TRAV, the TRAV comprises CDR1-3, the amino acid sequence of CDR1 is shown in SEQ ID NO:28, the amino acid sequence of CDR2 is shown in SEQ ID NO:29, and the amino acid sequence of CDR3 is shown in SEQ ID NO:30; the β chain comprises a TRBV, the TRBV comprises CDR1-3, CDR1 may comprise the amino acid sequence shown in SEQ ID NO:31, CDR2 may comprise the amino acid sequence shown in SEQ ID NO:32, and CDR3 may comprise the amino acid sequence shown in SEQ ID NO:33.

[0156] In this application, the TCR comprises an α chain and a β chain, the α chain comprises TRAV, and the β chain comprises TRBV. The amino acid sequence of the TRAV may be as shown in SEQ ID NO:1, and the amino acid sequence of the TRBV may be as shown in SEQ ID NO:4. In this application, the TCR comprises an α chain and a β chain, the α chain comprises TRAV, and the β chain comprises TRBV. The amino acid sequence of the TRAV may be as shown in SEQ ID NO:18, and the amino acid sequence of the TRBV may be as shown in SEQ ID NO:21.

[0157] In this application, the TCR may further comprise a TRBC. The TRBC may contain the amino acid sequence shown in SEQ ID NO:5. In this application, the TRBC may be a mouse TRBC. For example, the TRBC may be an mTRBC. In this application, the TRBC may be a human TRBC. For example, the TRBC may be an hTRBC.

[0158] In this application, the TCR may comprise a β chain, which may comprise the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:22.

[0159] In this application, the TCR comprises an α chain and a β chain, the amino acid sequence of the α chain being shown in SEQ ID NO:3, and the amino acid sequence of the β chain being shown in SEQ ID NO:6. In this application, the TCR comprises an α chain and a β chain, the amino acid sequence of the α chain being shown in SEQ ID NO:20, and the amino acid sequence of the β chain being shown in SEQ ID NO:22.

[0160] In this application, the CAR may include an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In this application, the antigen-binding domain can specifically bind to a target antigen.

[0161] In this application, the antigen-binding domain may be an antibody or an antigen-binding fragment thereof. For example, the antigen-binding fragment may be a Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH, or dAb fragment.

[0162] In this application, the antigen-binding domain can specifically bind to PD-L1. In this application, the antigen-binding domain can be the PD-1 extracellular domain. In this application, the PD-1 can be of any species origin. In this application, the amino acid sequence of the PD-1 extracellular domain is shown in SEQ ID NO:44.

[0163] In this application, the transmembrane domain may be a transmembrane domain derived from any of the following proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM. For example, the transmembrane domain may be a transmembrane domain of CD28. For example, the amino acid sequence of the transmembrane domain may be as shown in SEQ ID NO:46.

[0164] In this application, the intracellular signal transduction domain is an intracellular signal transduction domain derived from any of the following proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (KSHV), DAP10, DAP-12, and a domain containing at least one ITAM. For example, the intracellular signal transduction domain may be the intracellular signal transduction domain of CD3ζ. For example, the amino acid sequence of the intracellular signal transduction domain may be as shown in SEQ ID NO:47.

[0165] In this application, the chimeric antigen receptor includes a hinge region, which is derived from any of the following proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT. For example, the hinge region may be the hinge region of CD8. For example, the amino acid sequence of the hinge region may be as shown in SEQ ID NO:45.

[0166] In this application, the chimeric antigen receptor may further comprise a signal peptide. In this application, the signal peptide of the chimeric antigen receptor may be a CD8α signal peptide. For example, the amino acid sequence of the signal peptide may be as shown in SEQ ID NO:42.

[0167] In this application, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure. The first co-stimulatory structure comprises the full-length OX40, and the second co-stimulatory structure comprises a 4-1BB intracellular domain. The fusion protein is co-expressed with a TCR, and the TCR targets NY-ESO-1.

[0168] In this application, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure. The first co-stimulatory structure comprises the full length of OX40, and the second co-stimulatory structure comprises an intracellular domain of 4-1BB. The fusion protein is co-expressed with a TCR, which targets NY-ESO-1. The TCR comprises TRAV and TRBV. The TRAV may comprise CDR1, CDR2, and CDR3. CDR1 may comprise the amino acid sequence shown in SEQ ID NO:12, CDR2 may comprise the amino acid sequence shown in SEQ ID NO:13, and CDR3 may comprise the amino acid sequence shown in SEQ ID NO:14. The TRBV may comprise CDR1, CDR2, and CDR3. CDR1 may comprise the amino acid sequence shown in SEQ ID NO:15, CDR2 may comprise the amino acid sequence shown in SEQ ID NO:16, and CDR3 may comprise the amino acid sequence shown in SEQ ID NO:17. In this application, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure. The first co-stimulatory structure comprises the full length of 4-1BB, and the second co-stimulatory structure comprises an intracellular domain of OX40. The fusion protein is co-expressed with a TCR. The TCR targets the amino acid sequence shown in SEQ ID NO:12 for CDR1, SEQ ID NO:13 for CDR2, and SEQ ID NO:14 for CDR3. The TRBV comprises CDR1, CDR2, and CDR3. CDR1 comprises the amino acid sequence shown in SEQ ID NO:15, CDR2 comprises the amino acid sequence shown in SEQ ID NO:16, and CDR3 comprises the amino acid sequence shown in SEQ ID NO:17.

[0169] In this application, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure. The first co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:35, and the second co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:38. The fusion protein is co-expressed with a TCR, and the TCR targets NY-ESO-1. In this application, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure. The first co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:37, and the second co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:36. The fusion protein is co-expressed with a TCR, and the TCR targets NY-ESO-1.

[0170] In this application, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure. The first co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:35, and the second co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:38. The fusion protein is co-expressed with a TCR, which targets NY-ESO-1. The TCR comprises a TRAV and a TRBV. The TRAV may comprise CDR1, CDR2, and CDR3. CDR1 may comprise the amino acid sequence shown in SEQ ID NO:12, CDR2 may comprise the amino acid sequence shown in SEQ ID NO:13, and CDR3 may comprise the amino acid sequence shown in SEQ ID NO:14. The TRBV may comprise CDR1, CDR2, and CDR3. CDR1 may comprise the amino acid sequence shown in SEQ ID NO:15, CDR2 may comprise the amino acid sequence shown in SEQ ID NO:16, and CDR3 may comprise the amino acid sequence shown in SEQ ID NO:17. In this application, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure. The first co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:37, and the second co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:36. The fusion protein is co-expressed with a TCR, which targets NY-ESO-1. The TCR comprises a TRAV and a TRBV. The TRAV may comprise CDR1, CDR2, and CDR3. CDR1 may comprise the amino acid sequence shown in SEQ ID NO:12, CDR2 may comprise the amino acid sequence shown in SEQ ID NO:13, and CDR3 may comprise the amino acid sequence shown in SEQ ID NO:14. The TRBV may comprise CDR1, CDR2, and CDR3. CDR1 may comprise the amino acid sequence shown in SEQ ID NO:15, CDR2 may comprise the amino acid sequence shown in SEQ ID NO:16, and CDR3 may comprise the amino acid sequence shown in SEQ ID NO:17.

[0171] In this application, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:39, the fusion protein is co-expressed with a TCR, the TCR targets NY-ESO-1, the TCR comprises an α chain and a β chain, the α chain comprises the amino acid sequence shown in SEQ ID NO:3, and the β chain comprises the amino acid sequence shown in SEQ ID NO:6. In this application, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:40, the fusion protein is co-expressed with a TCR, the TCR targets NY-ESO-1, the TCR comprises an α chain and a β chain, the α chain comprises the amino acid sequence shown in SEQ ID NO:3, and the β chain comprises the amino acid sequence shown in SEQ ID NO:6.

[0172] In this application, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure. The first co-stimulatory structure comprises the full-length OX40, and the second co-stimulatory structure comprises a 4-1BB intracellular domain. The fusion protein is co-expressed with a TCR, and the TCR targets PRAME.

[0173] In this application, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure. The first co-stimulatory structure comprises the full length of OX40, and the second co-stimulatory structure comprises a 4-1BB intracellular domain. The fusion protein is co-expressed with a TCR, which targets PRAME. The TCR comprises TRAV and TRBV. The TRAV may comprise CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO:28, the amino acid sequence of CDR2 is shown in SEQ ID NO:29, and the amino acid sequence of CDR3 is shown in SEQ ID NO:30. The TRBV may comprise CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO:31, the amino acid sequence of CDR2 is shown in SEQ ID NO:32, and the amino acid sequence of CDR3 is shown in SEQ ID NO:33. In this application, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure. The first co-stimulatory structure comprises a full-length 4-1BB structure, and the second co-stimulatory structure comprises an intracellular domain of OX40. The fusion protein is co-expressed with a TCR, which targets PRAME. The TCR comprises a TRAV and a TRBV. The TRAV may comprise CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO:28, the amino acid sequence of CDR2 is shown in SEQ ID NO:29, and the amino acid sequence of CDR3 is shown in SEQ ID NO:30. The TRBV may comprise CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO:31, the amino acid sequence of CDR2 is shown in SEQ ID NO:32, and the amino acid sequence of CDR3 is shown in SEQ ID NO:33.

[0174] In this application, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure. The first co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:35, and the second co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:38. The fusion protein is co-expressed with a TCR, and the TCR targets PRAME. In this application, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure. The first co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:37, and the second co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:36. The fusion protein is co-expressed with a TCR, and the TCR targets PRAME.

[0175] In this application, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure. The first co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:35, and the second co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:38. The fusion protein is co-expressed with a TCR, the TCR targets PRAME, and the TCR comprises a TRAV and a TRBV. The TRAV may comprise CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO:28, the amino acid sequence of CDR2 is shown in SEQ ID NO:29, and the amino acid sequence of CDR3 is shown in SEQ ID NO:30. The TRBV may comprise CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO:31, the amino acid sequence of CDR2 is shown in SEQ ID NO:32, and the amino acid sequence of CDR3 is shown in SEQ ID NO:33. In this application, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure. The first co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:37, and the second co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:36. The fusion protein is co-expressed with a TCR, the TCR targets PRAME, and the TCR comprises a TRAV and a TRBV. The TRAV may comprise CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO:28, the amino acid sequence of CDR2 is shown in SEQ ID NO:29, and the amino acid sequence of CDR3 is shown in SEQ ID NO:30. The TRBV may comprise CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO:31, the amino acid sequence of CDR2 is shown in SEQ ID NO:32, and the amino acid sequence of CDR3 is shown in SEQ ID NO:33.

[0176] In this application, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:39, the fusion protein is co-expressed with a TCR, the TCR targets PRAME, the TCR comprises an α chain and a β chain, the α chain comprises the amino acid sequence shown in SEQ ID NO:20, and the β chain comprises the amino acid sequence shown in SEQ ID NO:22. In this application, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:40, the fusion protein is co-expressed with a TCR, the TCR targets PRAME, the TCR comprises an α chain and a β chain, the α chain comprises the amino acid sequence shown in SEQ ID NO:20, and the β chain comprises the amino acid sequence shown in SEQ ID NO:22.

[0177] In this application, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure. The first co-stimulatory structure comprises the full-length OX40, and the second co-stimulatory structure comprises a 4-1BB intracellular domain. The fusion protein is co-expressed with a CAR, and the CAR targets PD-L1.

[0178] In this application, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure. The first co-stimulatory structure comprises the full length of OX40, and the second co-stimulatory structure comprises an intracellular domain of OX40. The fusion protein is co-expressed with a CAR, and the CAR comprises an extracellular domain of PD-1.

[0179] In this application, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure. The first co-stimulatory structure comprises the full length of OX40, and the second co-stimulatory structure comprises a 4-1BB intracellular domain. The fusion protein is co-expressed with a CAR, and the CAR comprises a CD8 signal peptide, a PD-1 extracellular domain, a CD8 hinge region, a CD28 transmembrane domain, and a CD3ζ intracellular signal transduction domain. In this application, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure. The first co-stimulatory structure comprises the full length of 4-1BB, and the second co-stimulatory structure comprises an OX40 intracellular domain. The fusion protein is co-expressed with a CAR, and the CAR comprises a CD8 signal peptide, a PD-1 extracellular domain, a CD8 hinge region, a CD28 transmembrane domain, and a CD3ζ intracellular signal transduction domain.

[0180] In this application, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure. The first co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:35, and the second co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:38. The fusion protein is co-expressed with a CAR, and the CAR comprises an extracellular domain of PD-1, the amino acid sequence of which is shown in SEQ ID NO:44. In this application, the fusion protein comprises a first co-stimulatory structure and a second co-stimulatory structure. The first co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:37, and the second co-stimulatory structure comprises the amino acid sequence shown in SEQ ID NO:36. The fusion protein is co-expressed with a CAR, and the CAR comprises an extracellular domain of PD-1, the amino acid sequence of which is shown in SEQ ID NO:44.

[0181] immune cells

[0182] In this application, the immune cell can be an immune cell capable of participating in an immune response and performing effector functions. For example, the immune cell can be a T cell. For example, the immune cell can be an iNKT cell.

[0183] In this application, the immune cells can be modified immune cells. For example, the immune cells can be engineered immune cells. For example, the immune cells can be modified T cells. For example, the immune cells can be modified iNKT cells. For example, the immune cells can be immune cells expressing engineered TCRs. For example, the immune cells can be immune cells expressing CARs. For example, the immune cells can be TCR-T cells. For example, the immune cells can be CAR-T cells.

[0184] For example, the immune cells may be effector cells. For example, the immune cells may be T cells and / or iNKT cells. For example, the immune cells may be a mixture, and the mixture may contain different types of immune cells; for example, the mixture may contain one or more types of immune cells.

[0185] On the other hand, this application provides a modified immune cell, wherein the immune cell may contain the fusion protein. In this application, the immune cell may also contain an antigen recognition receptor.

[0186] In this application, the immune cells may contain and / or express one or more antigen recognition receptors and / or fusion proteins.

[0187] In this application, the immune cells may contain and / or express one or more TCRs and / or fusion proteins.

[0188] In this application, the immune cells may contain and / or express one or more CARs and / or fusion proteins.

[0189] In this application, the antigen recognition receptor can be as described above. For example, the TCR can be as described above. For example, the CAR can be as described above.

[0190] Nucleic acid molecules, vectors, and host cells

[0191] On the other hand, this application provides isolated nucleic acid molecules that encode the fusion protein. For example, the isolated nucleic acid molecule may encode the complete fusion protein or a portion thereof.

[0192] On the other hand, this application provides isolated nucleic acid molecules that encode the fusion protein and the antigen recognition receptor.

[0193] For example, the isolated nucleic acid molecule may encode the complete fusion protein and / or antigen recognition receptor, or it may encode a portion of them. For example, the isolated nucleic acid molecule may encode the fusion protein and the antigen recognition receptor separately. For example, the isolated nucleic acid molecule may encode both the fusion protein and the antigen recognition receptor simultaneously.

[0194] For example, the nucleic acid molecule encoding the fusion protein may be located before the nucleic acid molecule encoding the antigen recognition receptor. Alternatively, the nucleic acid molecule encoding the fusion protein may be located after the nucleic acid molecule encoding the antigen recognition receptor.

[0195] In this application, the nucleic acid molecule may contain a sequence encoding a self-cleaving peptide.

[0196] In this application, the sequence encoding the self-cleaving peptide may be located between the sequences encoding the fusion protein and the antigen recognition receptor. For example, the sequence encoding the self-cleaving peptide may be located between the sequences encoding the fusion protein and the TCR. For example, the sequence encoding the self-cleaving peptide may be located between the sequences encoding the fusion protein and the CAR. For example, the sequence encoding the self-cleaving peptide may be located between the sequences encoding the fusion protein and the NY-ESO-1 TCR. For example, the sequence encoding the self-cleaving peptide may be located between the sequences encoding the fusion protein and the PRAME TCR. In this application, the sequence encoding the self-cleaving peptide may be located between the sequences encoding the TCR α chain and the β chain. For example, the sequence encoding the self-cleaving peptide may be located between the sequences encoding the fusion protein and the PD-1ECD CAR.

[0197] In this application, the self-cleaving peptide may comprise a 2A peptide. For example, the self-cleaving peptide may be P2A, T2A, E2A, or F2A. For example, the self-cleaving peptide may be T2A. For example, the self-cleaving peptide may be P2A.

[0198] In this application, the nucleic acid molecule may contain a sequence encoding a signal peptide.

[0199] In this application, the nucleic acid molecule may sequentially contain the following nucleotide sequences from the 5' end to the 3' end: a gene encoding NYESO1TRBV, a gene encoding mTRBC, a gene encoding P2A, a gene encoding NYESO1 TRAV, a gene encoding mTRAC, a gene encoding P2A, a gene encoding OX40 FL, and a gene encoding 4-1BB ICD.

[0200] In this application, the nucleic acid molecule may sequentially contain the following nucleotide sequences from the 5' end to the 3' end: a gene encoding NYESO1TRBV, a gene encoding mTRBC, a gene encoding P2A, a gene encoding NYESO1 TRAV, a gene encoding mTRAC, a gene encoding P2A, a gene encoding 4-1BB FL, and a gene encoding OX40 ICD.

[0201] In this application, the nucleic acid molecule may sequentially contain the following nucleotide sequences from the 5' end to the 3' end: a gene encoding PRAME TRBV, a gene encoding hTRBC, a gene encoding P2A, a gene encoding PRAME TRAV, a gene encoding hTRAC, a gene encoding P2A, a gene encoding OX40 FL, and a gene encoding 4-1BB ICD.

[0202] In this application, the nucleic acid molecule may sequentially contain the following nucleotide sequences from the 5' end to the 3' end: a gene encoding PRAME TRBV, a gene encoding hTRBC, a gene encoding P2A, a gene encoding PRAME TRAV, a gene encoding hTRAC, a gene encoding P2A, a gene encoding 4-1BB FL, and a gene encoding OX40 ICD.

[0203] In this application, the nucleic acid molecule may sequentially include the following nucleotide sequences from the 5' end to the 3' end: a gene encoding the CD8 signal peptide, a gene encoding PD-1ECD, a gene encoding the CD8 hinge region, a gene encoding the CD28 transmembrane domain, a gene encoding CD3ζ, a gene encoding P2A, a gene encoding PRAME TRAV, a gene encoding hTRAC, a gene encoding P2A, a gene encoding OX40 FL, and a gene encoding 4-1BB ICD.

[0204] In this application, the nucleic acid molecule may sequentially include the following nucleotide sequences from the 5' end to the 3' end: a gene encoding the CD8 signal peptide, a gene encoding PD-1ECD, a gene encoding the CD8 hinge region, a gene encoding the CD28 transmembrane domain, a gene encoding CD3ζ, a gene encoding P2A, a gene encoding PRAME TRAV, a gene encoding hTRAC, a gene encoding P2A, a gene encoding 4-1BB FL, and a gene encoding OX40 ICD.

[0205] In this application, the nucleic acid molecule may be generated or synthesized by: (i) in vitro amplification, such as by polymerase chain reaction (PCR); (ii) clonal recombination; (iii) purification, such as by enzyme digestion and gel electrophoresis fractionation; or (iv) synthesis, such as by chemical synthesis. In this application, the nucleic acid molecule may be DNA and / or RNA. In this application, the nucleic acid molecule may be an artificially synthesized nucleic acid analog. In this application, the nucleic acid molecule may be a modified nucleic acid molecule.

[0206] On the other hand, this application provides a vector containing the nucleic acid molecule.

[0207] In this application, the vector may contain one or more of the aforementioned nucleic acid molecules. In this application, the vector may contain one or more of the aforementioned nucleic acid molecules. In this application, the vector may be an expression vector or a cloning vector. In this application, the vector may be a viral vector or a non-viral vector. In this application, the vector may be a viral vector, a plasmid vector, a bacteriophage vector, or other vectors commonly used in, for example, genetic engineering. For example, the viral vector may be adenovirus, adeno-associated virus, or retrovirus (including lentivirus). In this application, the vector may be a fusion vector or a non-fusion vector.

[0208] In this application, the vector may also contain other genes. For example, the other genes may be marker genes.

[0209] On the other hand, this application provides a cell that contains the nucleic acid molecule or the vector.

[0210] In this application, the cell may be a prokaryotic cell (e.g., a bacterial cell), a CHO cell, an NS / O cell, a HEK293T cell or a HEK293A cell, or other eukaryotic cells, such as fungal or yeast cells.

[0211] In this application, the cell may contain one or more of the aforementioned nucleic acid molecules and / or one or more vectors. In this application, the vector may contain one or more of the aforementioned nucleic acid molecules and / or one or more vectors.

[0212] In this application, the vector can be introduced into the cells using methods known in the art. For example, the method may be electroporation, lipofectine transfection, or lipofectamin transfection.

[0213] On the other hand, this application provides a method for preparing the fusion protein, the method comprising transfecting or transducing the nucleic acid molecule into cells. For example, the DNA sequence of the fusion protein is cloned into the vector and then transfected into immune cells for expression.

[0214] On the other hand, this application provides a method for preparing the fusion protein and the antigen recognition receptor, the method comprising transfecting or transducing the nucleic acid molecule into cells.

[0215] In this application, the DNA sequences of the fusion protein and the antigen recognition receptor are cloned into the vector and then transfected into immune cells for expression.

[0216] Pharmaceutical Composition

[0217] On the other hand, this application provides a pharmaceutical composition comprising the fusion protein, the modified immune cell, the nucleic acid molecule, the carrier and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0218] In this application, the pharmaceutical composition may comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical compositions of the present invention may include liquid, freeze-dried, and lyophilized compositions.

[0219] In this application, the pharmaceutically acceptable carrier may comprise any and all solvents, dispersion media, coatings, isotonic agents and absorption delay agents compatible with drug administration, generally safe and non-toxic, and neither biologically nor otherwise undesirable.

[0220] In this application, the pharmaceutical composition may be administered parenterally, percutaneously, intracavitarily, intra-arterially, intrathecally, and / or intranasally, or directly injected into tissues. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, the pharmaceutical composition may be administered in various ways, such as intravenously, intraperitoneally, subcutaneously, intramuscularly, locally, or intradermally.

[0221] On the other hand, this application provides a method for preparing the fusion protein.

[0222] On the other hand, this application provides a method for preparing the modified immune cells.

[0223] On the other hand, this application provides a method for preparing the pharmaceutical composition.

[0224] use

[0225] On the other hand, this application provides the use of the fusion protein, the modified immune cell, the nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of diseases and / or conditions.

[0226] On the other hand, this application provides a method for preventing and / or treating diseases and / or conditions, comprising administering the fusion protein, the modified immune cells, the nucleic acid molecules, the carrier, the cells, and / or the pharmaceutical composition to a subject in need.

[0227] On the other hand, this application provides the fusion protein, the modified immune cell, the nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition for the prevention and / or treatment of diseases and / or conditions.

[0228] In this application, the prevention and / or treatment may be to prevent the onset of the disease and / or condition, slow down or reverse the course of the disease and / or condition, prevent or slow down the onset of one or more symptoms associated with the disease and / or condition, reduce or alleviate one or more symptoms associated with the disease and / or condition, reduce the severity and duration of the disease and / or condition and any symptoms associated therewith, or prevent further increase in the severity of the disease and / or condition and any symptoms associated therewith.

[0229] In this application, the disease and / or condition can be a tumor and / or a virus. In this application, the tumor can be a hematologic malignancy and / or a blood tumor. For example, the tumor can be an NY-ESO-1 positive tumor. For example, the tumor can be a PRAME positive tumor. For example, the disease and / or condition can be lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, stomach cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer, and / or skin cancer. For example, the tumor can be lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, stomach cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer, and / or skin cancer.

[0230] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the fusion protein, preparation method and use of this application, and are not intended to limit the scope of the invention.

[0231] Example

[0232] Experimental materials

[0233] Example 1: Preparation and expression of NY-ESO-1 TCR structure

[0234] 1.1 Cell Culture

[0235] Human lymphoblastoid cells (T2), human embryonic kidney cells (HEK-293T), breast cancer cells (MDA-MB-231), and malignant melanoma cells (A-375) were all purchased from ATCC (American Society for the Study of Cell Killing). To facilitate cell killing assays, all target cells stably expressed GFP. The T2 cell line was maintained in complete medium (RPMI-1640 containing 20% ​​heat-inactivated FBS, 100 U / mL penicillin / streptomycin, and 2 mM L-glutamine). The other cell lines were maintained in complete medium (IMDM containing 10% heat-inactivated FBS, 100 U / mL penicillin / streptomycin, and 2 mM L-glutamine). PBMCs were isolated from whole blood of healthy donors using Ficoll-Paque and cultured at 2 × 10⁻⁶ cells / mL. 7 The sample was aliquoted into 1 mL aliquots at a concentration of 10 cells / mL and frozen in a liquid nitrogen tank. The culture medium was heat-inactivated FBS with 10% DMSO (vol / vol).

[0236] 1.2 Clone Construction

[0237] The NY-ESO-1 structure and its associated OX40 and / or 4-1BB enhancement structures were inserted into the pALD expression plasmid via XhoI and NheI cloning sites. The related enhancement structure combinations are shown in Table 1 and Figure 1. The mixed co-stimulatory signal structures described in this application are #9035 and #9036. After double enzyme digestion and sequencing verification, the expression plasmid was amplified extensively for lentiviral packaging.

[0238] Table 1. Schematic diagram of reinforced structural combination design

[0239] The amino acid sequence of NYESO1 TCR TRBV is shown in SEQ ID NO:4, the amino acid sequence of NYESO1 TCR mTRBC is shown in SEQ ID NO:5, the amino acid sequence of NYESO1 TCR TRB is shown in SEQ ID NO:6, the amino acid sequence of NYESO1 TCR TRAV is shown in SEQ ID NO:1, the amino acid sequence of NYESO1 TCR mTRAC is shown in SEQ ID NO:2, and the amino acid sequence of NYESO1 TCR TRA is shown in SEQ ID NO:3. The amino acid sequences of TRA and TRB also contain a Furin protease cleavage site sequence, which is shown in SEQ ID NO:41 and is located between TRB and P2A. The amino acid sequence of 4-1BB FL is shown in SEQ ID NO:37, the amino acid sequence of 4-1BB ICD is shown in SEQ ID NO:38, the amino acid sequence of OX40 FL is shown in SEQ ID NO:35, the amino acid sequence of OX40 ICD is shown in SEQ ID NO:36, the amino acid sequence of P2A is shown in SEQ ID NO:34, the amino acid sequence of 9030 is shown in SEQ ID NO:7, the amino acid sequence of 9033 is shown in SEQ ID NO:8, the amino acid sequence of 9034 is shown in SEQ ID NO:9, the amino acid sequence of 9035 is shown in SEQ ID NO:10, and the amino acid sequence of 9036 is shown in SEQ ID NO:11.

[0240] 1.3 Preparation of Lentiviral Viruses

[0241] All lentiviruses were prepared from HEK-293T cells. Freshly resuspended HEK-293T cells were washed in PBS buffer and seeded in 10cm culture dishes at a cell density of 70%-80% of the dish surface area. A mixture of lentiviral packaging plasmids and transfection plasmids was transiently transfected into the seeded HEK-293T cells using Lipofectamine-3000 transfection agent. After 48 hours of culture, the supernatant was collected, filtered through a 0.45μm filter, aliquoted into 1ml tubes, and stored at -80°C.

[0242] 1.4 Activation and transduction of T cells

[0243] To transfect lentiviruses encoding the corresponding structures into PBMCs, frozen PBMCs were thawed and added to 24-well empty plates. CD3 and CD28-conjugated magnetic beads and IL-2 (500 IU / ml) were added for activation for 24-72 hours. The activated PBMCs were then aliquoted into multiple-well 24-well plates, and the corresponding lentivirus and polybrene (8 g / ml) were added. The plates were centrifuged at 32°C and 2000g for 2 hours, then transferred to an incubator at 37°C and 5% CO2 for culture. In the control group, the lentivirus was replaced with complete culture medium, while the remaining steps were the same. Transfected cells were cultured in X-VIVO-15 cell culture medium containing IL-2 (500 IU / ml) for 48-72 hours before subsequent experiments and analyses.

[0244] 1.5 Detection of NY-ESO-1 TCR-T cell structural expression levels

[0245] After 72 hours and 168 hours of infection with lentiviruses of different structures, small samples were collected and NY-EOS-1 fused with the PE fluorophore was used to detect the expression levels of related structures by flow cytometry.

[0246] 1.6 Flow Cytometry Analysis

[0247] Flow cytometry was performed using a Beckman Coulter instrument in semi-automatic or plate mode, and data were analyzed using FlowJo software. Cells were washed once with FACS buffer (PBS containing 2% FBS) and resuspended to 1–5 × 10⁶ cells / mL. 6 Cells / mL, placed on ice before staining. Add antibody or recombinant protein containing a fluorophore for staining, incubate at 4°C for 40 minutes in the dark, then add 7-AAD and continue staining for 5 minutes. After staining, wash away unbound antibody with a large amount of FACS buffer, centrifuge at 300xg for 3 minutes, and remove the supernatant. After washing and resuspending with FACS buffer, run the sample on a flow cytometer. Flow cytometry data were analyzed and processed using FlowJo software.

[0248] Experimental results:

[0249] The results of NY-ESO-1 TCR structure expression levels are shown in Figure 2. The various structural combinations prepared in this application all exhibited good positive expression rates and remained stable during long-term culture. Meanwhile, conventionally, the size of the transgenic fragment directly affects gene integration and expression, and its expression rate is inversely proportional to the size of the exogenous gene fragment. However, the TCR expression rates of the co-stimulatory signals OX40, 4-1BB, and their mixed combinations were not affected; in fact, they were expressed at higher levels (#9033, #9034, #9035, #9036).

[0250] Experimental results show that the hybrid co-stimulatory signaling structure of this application has a good expression effect on T cells and is suitable for preparing TCR-T cells.

[0251] Example 2: Comparison of in vitro killing effects of NY-ESO-1 related TCR structural T cells

[0252] 2.1 Tumor cell lines

[0253] Breast cancer cells MDA-MB-231 were used as negative control cells, while malignant melanoma cells A-375 were used as positive cells. GFP was stably expressed in these tumor cell lines for subsequent experiments.

[0254] 2.2 Co-culture of NY-ESO-1 TCR-T cells with tumor cells (Incucyte method)

[0255] Different structures of NY-ESO-1 TCR (#9030), NY-ESO-1 TCR-4-1BB FL (#9033), NY-ESO-1 TCR-OX40 FL (#9034), NY-ESO-1 TCR-OX40 FL-4-1BB ICD (#9035), and NY-ESO-1 TCR-4-1BB FL-OX40 ICD (#9036) were transduced into T cells and cultured in an incubator (37°C, 5% CO2) for 72–120 hours. Before co-culture, a small amount of transduced cells was taken and stained with NY-ESO-1-PE peptide to determine the expression levels of different structures. After adjusting the expression levels to a common level, the transfected T cells and tumor cells were resuspended in 200 μl of cell culture medium at an effector-to-target ratio of 1:1 and seeded in flat-bottomed 96-well plates for continuous observation using an Incucyte instrument.

[0256] Experimental results:

[0257] The cell killing results are shown in Figures 3A-3B. Against the positive target cells A-375, compared to the Mock-T group, both the mixed co-stimulatory signaling molecules NY-ESO-1 TCR-OX40 FL-4-1BB ICD (#9035) and NY-ESO-1 TCR-4-1BB FL-OX40 ICD (#9036) significantly enhanced the ability to kill target cells. Among them, NY-ESO-1 TCR-OX40 FL-4-1BB ICD (#9035) was more effective in inhibiting tumor growth and exhibited the strongest target cell killing ability. However, for the negative control cell group (MDA-MB-231), neither NY-ESO-1 TCR-OX40 FL-4-1BB ICD (#9035) nor NY-ESO-1 TCR-4-1BB FL-OX40 ICD (#9036) produced significant non-specific killing.

[0258] Experimental results show that the mixed co-stimulatory signaling structures NY-ESO-1 TCR-OX40 FL-4-1BB ICD(#9035) and NY-ESO-1 TCR-4-1BB FL-OX40 ICD(#9036) of this application can specifically bind to and kill target cells, and have a strong ability to kill target cells.

[0259] Example 3: Comparison of antigen recognition effects of NY-ESO-1 related TCR structure T cells

[0260] 3.1 Related cell lines

[0261] HLA A02-restricted human lymphoblastic T2 cells were used as target cells and activated with different concentrations of NY-ESO-1 peptide for subsequent related experiments.

[0262] 3.2 Comparison of minimum activating antigen concentrations in NY-ESO-1 TCR-T cells (CD25 activation signal)

[0263] T2 cells were washed twice with PBS and once with serum-free RPMI-1640, then resuspended in serum-free RPMI-1640. The resuspended cells were accurately counted, and 3000 cells per well were added to each well of a 96-well plate. Different concentrations of NY-ESO-1 peptide were added to the wells, ranging from 0 ng / ml to 100 ng / ml, and the plates were incubated at 37°C for at least 4 hours. The peptide-stimulated T2 cells were washed twice with serum-containing RPMI-1640 and resuspended. The resuspended T2 cells and transduced TCR-T cells were resuspended at a ratio of 1:3 in 200 μl of cell culture medium and seeded in flat-bottomed 96-well plates. After 24 hours of co-culture, the cells were centrifuged, washed with PBS, stained with CD25 antibody containing the PE fluorescein group, washed and resuspended with FACS buffer, and then run on a flow cytometer. Flow cytometry data were analyzed and processed using FlowJo software.

[0264] 3.3 Comparison of the lowest activating antigen concentrations in NY-ESO-1 TCR-T cells (IFN-γ detection)

[0265] T2 cells were washed twice with PBS and once with serum-free RPMI-1640, then resuspended in serum-free RPMI-1640. The resuspended cells were accurately counted, and 3000 cells per well were added to each well of a 96-well plate. Different concentrations of NY-ESO-1 peptide were added to the wells, ranging from 0 ng / ml to 100 ng / ml, and the plates were incubated at 37°C for at least 4 hours. The peptide-stimulated T2 cells were washed twice with serum-containing RPMI-1640 and resuspended. The resuspended T2 cells and transduced TCR-T cells were resuspended at a ratio of 1:3 in 200 μl of cell culture medium and seeded in flat-bottomed 96-well plates. After 24 hours of culture, the cells were centrifuged, and the supernatant was used for enzyme-linked immunosorbent assay (ELISA) to determine interferon-gamma secretion. #9020 was the PRAME TCR-T cell group, serving as a negative T cell control group.

[0266] 3.4 Detection of Cytokine Secretion Levels

[0267] After simple centrifugation, the supernatant was carefully aspirated, diluted appropriately, and the secretion level of cytokines in the culture medium was measured using the ELISA MAX™ Deluxe Set Human IFN-γ Kit and the ELISA MAX™ Standard Set Human IL-2 Kit.

[0268] Experimental results:

[0269] The results for minimum activation antigen concentrations showed that the mean fluorescence intensity (MFI) of CD25 activation signaling indicated that the minimum T cell activation concentration was between 0.1-1 ng / ml. NY-ESO-1 TCR-4-1BB FL (#9033) and NY-ESO-1 TCR-OX40 FL (#9034), containing a single co-stimulatory structure, and NY-ESO-1 TCR-OX40 FL-4-1BB ICD (#9035) and NY-ESO-1 TCR-4-1BB FL-OX40 ICD (#9036), containing a mixed co-stimulatory structure, all lowered the effective T cell stimulation threshold, especially the mixed co-stimulatory signal structure NY-ESO-1 TCR-4-1BB FL-OX40 ICD (#9036).

[0270] The IFN-γ secretion results are shown in Figure 4. At different concentrations, both NY-ESO-1 TCR-OX40 FL-4-1BB ICD(#9035) and NY-ESO-1 TCR-4-1BB FL-OX40 ICD(#9036) effectively stimulated IFN-γ secretion. At concentrations between 0.1-10 nM, compared to the original TCR structure or TCRs with a single co-stimulatory signal, both NY-ESO-1 TCR-OX40 FL-4-1BB ICD(#9035) and NY-ESO-1 TCR-4-1BB FL-OX40 ICD(#9036) with mixed co-stimulatory signals enhanced the stimulation activity and efficiency of T cells to low-density surface antigens, showing a 2-3 fold increase in activating efficiency (at concentrations of 1-10 nM). Experimental data show that this application...

[0271] Experimental results show that TCR-T cells containing the hybrid co-stimulatory signaling structure of this application can be activated at low antigen concentrations, exhibit stronger killing power against tumors expressing rare antigens, and reduce tumor escape. Furthermore, the hybrid co-stimulatory signaling structure of this application can effectively stimulate cytokine secretion, effectively improving the activity and efficiency of T cells.

[0272] Example 4: Comparison of in vivo killing effects of NY-ESO-1 related TCR structural T cells

[0273] 4.1 Culture and Inoculation of Tumor Cells

[0274] Human melanoma A375 cells were cultured in IMDM medium supplemented with 10% FBS at 37°C in an incubator containing 5% CO2. Before ten consecutive passages of cell culture, approximately 1×10⁻⁶ cells were... 6A-375 cells were resuspended in 100 μL of PBS and mixed with an equal volume of Matrigel matrix (v / v = 1:1). The mixture was then subcutaneously injected into the right side of NCG mice in a volume of 200 μL. Mice were anesthetized with 3-4% isoflurane prior to inoculation.

[0275] 4.2 Drug delivery regimen for NY-ESO-1 TCR-T cells

[0276] The day of tumor cell inoculation was defined as day 0. Three days after tumor cell inoculation, mice were divided into groups of six based on body weight and administered the appropriate drug treatment. The dosage for each animal was 2E6 cells per animal (NY-ESO-1 TCR-T positive cell percentage ≈ 80%), adjusted to 200 μL per animal. One day after TCR-T cell injection, i.e., four days post-inoculation, each animal received an intraperitoneal injection of 6 × 10⁶ cells three times a week. 4 IU IL-2 was administered to maintain TCR-T cell growth in vivo for 4 weeks. Animal condition was observed daily, and tumor diameter and animal weight were measured twice weekly. At the end of the experiment, tumors from surviving animals were removed and weighed. Survival curves were plotted, and survival rates were statistically analyzed.

[0277] 4.3 Tumor cell re-inoculation experiment

[0278] After the first round of in vivo killing experiments was completed in mice, on day 35, no tumors were observed or the tumor volume was less than 200 mm. 3 The mice will have approximately 1×10 6 A-375 cells were resuspended in 100 μL of PBS and mixed with an equal volume of Matrigel matrix (v / v = 1:1). The mixture was then injected subcutaneously into the left side of NCG mice in a volume of 200 μL.

[0279] Experimental results:

[0280] The results of the in vivo antitumor assay are shown in Figure 5A. Compared with the Mock-T group, the groups containing the mixed co-stimulatory signaling molecules NY-ESO-1TCR-OX40 FL-4-1BB ICD(#9035) and NY-ESO-1 TCR-4-1BB FL-OX40 ICD(#9036) were able to effectively kill tumor cells, reduce tumor volume, and inhibit tumor growth in vivo. Among them, NY-ESO-1 TCR-OX40 FL-4-1BB ICD(#9035) had stronger tumor-killing and tumor-suppressing effects.

[0281] The results of the in vivo sustained antitumor experiment are shown in Figure 5B. On day 35, we performed a tumor cell re-inoculation experiment on the other side of these mice. Then, at day 35+21, we found that the mixed co-stimulatory signals NY-ESO-1 TCR-OX40 FL-4-1BB ICD (#9035) and NY-ESO-1 TCR-4-1BB FL-OX40 ICD (#9036) had a stronger sustained tumor-suppressing effect than the group with a single co-stimulatory signal structure and the group without a co-stimulatory signal structure.

[0282] Experimental results show that only the mixed co-stimulatory signaling structures NY-ESO-1 TCR-OX40 FL-4-1BB ICD(#9035) and NY-ESO-1 TCR-4-1BB FL-OX40 ICD(#9036) of this application, when prepared as TCR-T cells, can continuously reduce tumor volume after tumor cell re-inoculation, and have a sustained tumor-suppressing effect in vivo, and can effectively inhibit tumor growth in the long term.

[0283] Example 5: Comparison of the in vitro killing effects of PRAME TCR structure and its reinforced structure

[0284] 5.1 PRAME TCR-T cell structural expression

[0285] Figure 6 shows the PRAME TCR and its associated enhancement structures. After 72 hours of infection with lentiviruses of different structures, a small number of samples were extracted, stained with PRAME tetramers fused to the PE fluorophore, and the expression levels of related structures were detected by flow cytometry.

[0286] The amino acid sequences of PRAME TCR TRBV are shown in SEQ ID NO:21, PRAME TCR mTRBC are shown in SEQ ID NO:5, PRAME TCR TRB are shown in SEQ ID NO:22, PRAME TCR TRAV are shown in SEQ ID NO:18, PRAME TCR mTRAC are shown in SEQ ID NO:19, and PRAME TCR TRA are shown in SEQ ID NO:20. The amino acid sequences of TRA and TRB also contain a Furin protease cleavage site located between TRB and P2A. The amino acid sequences of 9020 are shown in SEQ ID NO:23, 9023 in SEQ ID NO:24, 9024 in SEQ ID NO:25, 9025 in SEQ ID NO:26, and 9026 in SEQ ID NO:27.

[0287] 5.2 Detection of PRAME TCR-T cell expression rate (flow cytometry analysis)

[0288] Flow cytometry was performed using a Beckman Coulter instrument in semi-automatic or plate mode, and data were analyzed using FlowJo software. Cells were washed once with FACS buffer (PBS containing 2% FBS) and resuspended to 1–5 × 10⁶ cells / mL. 6 Cells / mL, placed on ice before staining. Add antibody or recombinant protein containing a fluorophore for staining, incubate at 4°C for 40 minutes in the dark, then add 7-AAD and continue staining for 5 minutes. After staining, wash away unbound antibody with a large amount of FACS buffer, centrifuge at 300xg for 3 minutes, and remove the supernatant. After washing and resuspending with FACS buffer, run the sample on a flow cytometer. Flow cytometry data were analyzed and processed using FlowJo software.

[0289] 5.3 Tumor Cell Lines

[0290] Breast cancer cells MDA-MB-231 were used as negative control cells, while malignant melanoma cells A-375 were used as positive cells. GFP was stably expressed in these tumor cell lines for subsequent experiments.

[0291] 5.4 Co-culture of PRAME TCR-T cells with tumor cells (Incucyte method)

[0292] Different structures of PRAME TCR (#9020), PRAME TCR-4-1BB (#9023), PRAME TCR-OX40 (#9024), PRAME TCR-4-1BB FL-OX40 ICD (#9025), and PRAME TCR-OX40 FL-4-1BB ICD (#9026) were transduced into T cells and cultured in an incubator (37°C, 5% CO2) for 72 hours. Before co-culture, a small amount of transduced cells was taken and stained with PRAME-PE peptide to determine the expression levels of different structures. After adjusting the expression levels to a common level, the transfected T cells and tumor cells were resuspended in 200 μl of cell culture medium at an effector-to-target ratio of 3:1 and seeded in flat-bottomed 96-well plates for continuous observation using an Incucyte instrument.

[0293] Experimental results:

[0294] The results of PRAME TCR structure expression level are shown in Figure 7. PRAME TCR-4-1BB FL-OX40 ICD(#9025) and PRAME TCR-OX40 FL-4-1BB ICD(#9026) both showed good positive expression rates on T cells and are suitable for preparing TCR-T cells.

[0295] The cell-killing effects are shown in Figures 8A-B. Compared with the groups with a single co-stimulatory signal structure and the groups without a co-stimulatory signal structure, the groups with PRAME TCR-4-1BB FL-OX40 ICD (#9025) and PRAME TCR-OX40 FL-4-1BB ICD (#9026) showed outstanding killing ability against positive target cells. Among them, PRAME TCR-OX40 FL-4-1BB ICD (#9026) had a stronger ability to inhibit tumor cell growth. However, the two groups with mixed co-stimulatory signal structures did not produce significant specific killing of the negative control cell group MB-231.

[0296] Experimental results show that the mixed co-stimulatory signaling structures PRAME TCR-4-1BB FL-OX40 ICD (#9025) and PRAME TCR-OX40 FL-4-1BB ICD (#9026) of this application can specifically bind to and kill target cells, and have a strong ability to kill target cells.

[0297] Example 6: Hybrid co-stimulatory structure for iNKT cells

[0298] 6.1 Expression of NY-ESO-1 TCR structure in iNKT cells

[0299] The NY-ESO-1 TCR structure and related OX40 and / or 4-1BB enhancing structures shown in Example 1 were prepared and expressed on iNKT cells. iNKT cells were obtained by stimulating, culturing, and purifying peripheral blood mononuclear cells using aGalcer. The iNKT cells were then transduced using a lentiviral vector. The expression rate of NY-ESO-1 TCR on iNKT cells was detected by flow cytometry on days 5 and 8 after viral transduction.

[0300] 6.2 Co-culture of NY-ESO-1 TCR-iNKT cells with tumor cells (Incucyte method)

[0301] After determining the expression efficiency of NY-ESO-1 TCR by flow cytometry, NY-ESO-1 TCR-iNKT cells were co-cultured with A375 tumor cells carrying GFP fluorescence at a 1:1 effector-target ratio and then continuously observed the killing effect of NY-ESO-1 TCR-iNKT cells on A375 tumor cells in an Incucyte instrument.

[0302] Experimental results:

[0303] The expression levels of the NY-ESO-1 TCR structure on iNKT cells are shown in Figure 9. The NY-ESO-1 TCR (#9030) structure prepared in this application achieved an expression efficiency of approximately 49%, while the NY-ESO-1 TCR-OX40 FL-4-1BB ICD (#9035) structure achieved an expression efficiency of approximately 53%, indicating higher expression efficiency. Furthermore, the TCR structure maintained relatively stable expression efficiency on iNKT cells on days 5 (post-TD) and 8 (post-TD) after lentiviral transduction.

[0304] Figure 10 shows the killing effect of NY-ESO-1 TCR-iNKT cells on A375 target cells. iNKT cells expressing NY-ESO-1 TCR(#9030) structure and NY-ESO-1 TCR-OX40 FL-4-1BB ICD(#9035) structure can effectively inhibit the growth of A375 tumor cells and have a good killing effect on target cells.

[0305] Experimental results show that the hybrid co-stimulatory signaling structure NY-ESO-1 TCR-OX40 FL-4-1BB ICD(#9035) of this application can achieve high and relatively durable expression efficiency on iNKT cells, and can continuously reduce tumor volume after being prepared into TCR-iNKT cells, demonstrating a strong ability to kill target cells.

[0306] Example 7: Hybrid co-stimulation structure for ECD-CAR-T cells

[0307] 7.1 Expression of ECD-CAR structure on T cells

[0308] The ECD-CAR structure and its associated OX40 and / or 4-1BB enhancement structures are shown in Figure 11A. The encoded gene structure was inserted into the pALD expression plasmid through the XhoI and NheI cloning sites. After double enzyme digestion and sequencing verification, the expression plasmid was amplified extensively for lentiviral packaging. The plasmid was prepared and expressed on T cells. Flow cytometry was used to detect ECD-CAR expression on days 4 and 8 after viral transduction.

[0309] The amino acid sequences of the CD8 signal peptide are shown in SEQ ID NO:42, the Flag Tag amino acid sequence is shown in SEQ ID NO:43, the PD-1 extracellular domain amino acid sequence is shown in SEQ ID NO:43, the CD8 hinge region amino acid sequence is shown in SEQ ID NO:45, the CD28 transmembrane domain amino acid sequence is shown in SEQ ID NO:46, the CD8ζ amino acid sequence is shown in SEQ ID NO:47, the 8460 amino acid sequence is shown in SEQ ID NO:48, the 8610 amino acid sequence is shown in SEQ ID NO:49, the 8611 amino acid sequence is shown in SEQ ID NO:50, the 8612 amino acid sequence is shown in SEQ ID NO:51, and the 8613 amino acid sequence is shown in SEQ ID NO:52.

[0310] 7.2 Co-culture of ECD-CAR-T cells with tumor cells

[0311] A375, MB231, HCC827, LS174T, LO2, and 293T were all purchased from ATCC (American Academy of Sciences). All target cells stably expressed GFP. PBMCs were isolated from whole blood of healthy donors using Ficoll-Paque and cultured at 2 × 10⁻⁶ cells / year. 7 The sample was aliquoted into 1 mL aliquots at a concentration of 10 cells / mL and frozen in a liquid nitrogen tank. The culture medium was heat-inactivated FBS with 10% DMSO (vol / vol).

[0312] After co-culturing ECD-CAR-T cells with tumor cells at a 1:1 effector-target ratio, the cells were placed in an Incucyte instrument to detect the killing effect of ECD-CAR-T cells on tumor cells.

[0313] Experimental results:

[0314] The expression levels of the ECD-CAR-T structure on ECD-CAR-T cells are shown in Figures 11B-C. The expression efficiencies of the ECD-CAR-4-1BBFL+OX40 (#8612) and ECD-CAR-OX40FL+4-1BB (#8613) prepared in this application are comparable to those of ECD-CAR (#8460). Furthermore, the ECD-CAR structure maintained relatively stable expression efficiency on T cells on days 4 and 8 after lentiviral transduction.

[0315] The killing effect of ECD-CAR-T cells on A375 and MB231 target cells is shown in Figures 11D-E. T cells expressing ECD-CAR-4-1BBFL+OX40(#8612) and ECD-CAR-OX40FL+4-1BB(#8613) structures can effectively inhibit the growth of tumor cells with high PD-L1 expression and have a good effect on killing target cells.

[0316] The killing effect of ECD-CAR-T cells on HCC827 and LS174 T target cells is shown in Figure 11F-G. T cells expressing ECD-CAR-4-1BBFL+OX40(#8612) and ECD-CAR-OX40FL+4-1BB(#8613) structures can effectively inhibit the growth of tumor cells with low PD-L1 expression and have a good killing effect on target cells.

[0317] The killing effect of ECD-CAR-T cells on LO2 and 293T target cells is shown in Figure 11H-I. T cells expressing ECD-CAR-4-1BBFL+OX40(#8612) and ECD-CAR-OX40FL+4-1BB(#8613) structures did not show significant killing effect on non-tumor cell lines without PD-L1 expression, indicating that they have a specific killing effect on target cells.

[0318] Experimental results show that the hybrid co-stimulatory signaling structures ECD-CAR-4-1BBFL+OX40(#8612) and ECD-CAR-OX40FL+4-1BB(#8613) of this application can achieve high and relatively durable expression efficiency on T cells and exhibit strong ability to kill target cells. Combined with Examples 1-6, it is evident that the hybrid co-stimulatory signaling structures of this application can effectively enhance the ability of both TCR-T and CAR-T cells to kill target cells, demonstrating broad application potential.

Claims

1. A fusion protein comprising a first costimulatory structure and a second costimulatory structure, the first costimulatory structure comprising a full-length or intracellular domain of a first costimulatory molecule, the second costimulatory structure comprising a full-length or intracellular domain of a second costimulatory molecule, the fusion protein being expressed on immune cells.

2. The fusion protein of claim 1, wherein the first co-stimulatory structure comprises the full length of the first co-stimulatory molecule.

3. The fusion protein according to any one of claims 1-2, wherein the first co-stimulatory molecule is OX40 or 4-1BB.

4. The fusion protein according to any one of claims 1-3, wherein the second co-stimulatory structure comprises an intracellular domain of the second co-stimulatory molecule.

5. The fusion protein according to any one of claims 1-4, wherein the second co-stimulatory molecule is OX40 or 4-1BB.

6. The fusion protein according to any one of claims 1-5, wherein the first co-stimulatory structure comprises the full length of the first co-stimulatory molecule, the first co-stimulatory molecule being OX40, and the second co-stimulatory structure comprises an intracellular domain of the second co-stimulatory molecule, the second co-stimulatory molecule being 4-1BB.

7. The fusion protein according to any one of claims 1-6, wherein the amino acid sequence of the first co-stimulatory structure is as shown in SEQ ID NO:35, and the amino acid sequence of the second co-stimulatory structure is as shown in SEQ ID NO:

38.

8. The fusion protein according to any one of claims 1-7, wherein the first co-stimulatory structure comprises the full length of the first co-stimulatory molecule, the first co-stimulatory molecule being 4-1BB, and the second co-stimulatory structure comprises an intracellular domain of the second co-stimulatory molecule, the second co-stimulatory molecule being OX40.

9. The fusion protein according to any one of claims 1-8, wherein the amino acid sequence of the first co-stimulatory structure is as shown in SEQ ID NO:37, and the amino acid sequence of the second co-stimulatory structure is as shown in SEQ ID NO:

36.

10. The fusion protein according to any one of claims 1-9, wherein the second co-stimulatory structure is attached to the C-terminus of the first co-stimulatory structure.

11. The fusion protein according to any one of claims 1-10, wherein the fusion protein is capable of providing a co-stimulatory signal.

12. The fusion protein according to any one of claims 1-11, wherein the immune cell is a T cell.

13. The fusion protein according to any one of claims 1-12, wherein the immune cell is an iNKT cell.

14. The fusion protein according to any one of claims 1-13, wherein the immune cell is a TCR-T cell and / or a CAR-T cell.

15. The fusion protein according to any one of claims 1-14, wherein the fusion protein is capable of being used in adoptive cell therapy (ACT).

16. The fusion protein of claim 15, wherein the adoptive cell therapy is a TCR-T therapy and / or a CAR-T therapy.

17. The fusion protein according to any one of claims 1-16, wherein the fusion protein is co-expressed with an antigen recognition receptor.

18. The fusion protein of claim 17, wherein the antigen recognition receptor is a T-cell receptor (TCR) and / or a chimeric antigen receptor (CAR).

19. The fusion protein of claim 18, wherein the T cell receptor and / or chimeric antigen receptor specifically binds to the target antigen.

20. The fusion protein of claim 19, wherein the target antigen is a tumor antigen.

21. The fusion protein according to any one of claims 19-20, wherein the target antigen is: PD-L1, AFP, ASCL2, B melanoma antigen (BAGE) family member, BORIS, cancer-testis antigen, cancer-testis antigen 83 (CT-83), CAIX, carcinoembryonic antigen (CEA), EBV, EPHB2, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, glycoprotein 100 (GP100), hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) NS3, human papillomavirus (HPV) E6, HPV-E7, IGF2BP3, IGF2BP1, K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, LMP2, LY6G6D, melanoma antigen (MAGE) family members, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), NYESO-1, P53, PAP, PLAC1, melanoma preferred expression antigen (PRAME), PSA, SSX1, SSX2, SSX3, SSX4, SSX5, SSX8, thyroglobulin, tyrosinase, TRP-1, TRP-2, WT-1, Wnt10A, CMV, MCPyV, ROPN1, TP53R175H, TP53 R248Q, XAGE-1 and / or XAGE-2.

22. The fusion protein according to any one of claims 19-21, wherein the target antigen is NY-ESO-1.

23. The fusion protein according to any one of claims 19-22, wherein the target antigen is PRAME.

24. The fusion protein according to any one of claims 18-23, wherein the TCR comprises an α chain and a β chain, the α chain comprising an α chain variable region (TRAV) and an α chain constant region (TRAC), and the β chain comprising a β chain variable region (TRBV) and a β chain constant region (TRBC).

25. The fusion protein of claim 24, wherein the TRAV comprises complementarity-determining regions CDR1, CDR2, and CDR3.

26. The fusion protein according to any one of claims 24-25, wherein the TRAV comprises complementarity-determining regions CDR1, CDR2, and CDR3, the amino acid sequences of said CDR1, CDR2, and CDR3 being selected from any group of the following combinations: a) The amino acid sequence of CDR1 is shown in SEQ ID NO:12, the amino acid sequence of CDR2 is shown in SEQ ID NO:13, and the amino acid sequence of CDR3 is shown in SEQ ID NO:14; and b) The amino acid sequence of CDR1 is shown in SEQ ID NO:28, the amino acid sequence of CDR2 is shown in SEQ ID NO:29, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

30.

27. The fusion protein according to any one of claims 24-26, wherein the amino acid sequence of the TRAV is as shown in SEQ ID NO:1 or SEQ ID NO:

18.

28. The fusion protein according to any one of claims 24-27, wherein the TRBV comprises complementarity-determining regions CDR1, CDR2, and CDR3.

29. The fusion protein according to any one of claims 24-28, wherein the TRBV comprises complementarity-determining regions CDR1, CDR2, and CDR3, and the amino acid sequences of CDR1, CDR2, and CDR3 are selected from any group of the following combinations: a) The amino acid sequence of CDR1 is shown in SEQ ID NO:15, the amino acid sequence of CDR2 is shown in SEQ ID NO:16, and the amino acid sequence of CDR3 is shown in SEQ ID NO:17; and b) The amino acid sequence of CDR1 is shown in SEQ ID NO:31, the amino acid sequence of CDR2 is shown in SEQ ID NO:32, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

33.

30. The fusion protein according to any one of claims 24-29, wherein the amino acid sequence of the TRBV is as shown in SEQ ID NO:4 or SEQ ID NO:

21.

31. The fusion protein according to any one of claims 24-30, wherein the TRAC and / or TRBC are mouse and / or human constant regions.

32. The fusion protein according to any one of claims 18-31, wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain.

33. The fusion protein according to claim 32, wherein the antigen-binding domain is an antibody or its antigen-binding fragment, or the antigen-binding domain is the PD-1 extracellular domain.

34. The fusion protein according to claim 33, wherein the amino acid sequence of the PD-1 extracellular domain is shown in SEQ ID NO:

44.

35. The fusion protein according to any one of claims 32-34, wherein the transmembrane domain is a transmembrane domain derived from any of the following proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

36. The fusion protein according to any one of claims 32-35, wherein the intracellular signal transduction domain is an intracellular signal transduction domain derived from any of the following proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (KSHV), DAP10, DAP-12, and a domain comprising at least one ITAM.

37. The fusion protein according to any one of claims 32-36, wherein the chimeric antigen receptor comprises a hinge region, the hinge region of the chimeric antigen receptor being derived from the hinge region of any one of the following proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

38. The fusion protein according to any one of claims 32-37, wherein the chimeric antigen receptor comprises a signal peptide.

39. A modified immune cell comprising the fusion protein of any one of claims 1-38.

40. The modified immune cell of claim 39, further comprising an antigen recognition receptor.

41. The modified immune cell of claim 40, wherein the antigen recognition receptor is a T cell receptor (TCR) and / or a chimeric antigen receptor (CAR).

42. The modified immune cell of claim 41, wherein the T cell receptor and / or chimeric antigen receptor specifically bind to the target antigen.

43. The modified immune cell according to claim 42, wherein the target antigen is a tumor antigen.

44. The modified immune cells according to any one of claims 42-43, wherein the target antigen is PD-L1, AFP, ASCL2, B melanoma antigen (BAGE) family member, BORIS, cancer-testis antigen, cancer-testis antigen 83 (CT-83), CAIX, carcinoembryonic antigen (CEA), EBV, EPHB2, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, glycoprotein 100 (GP100), hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) NS3, human papillomavirus (HPV) E6, HPV-E7, IGF2BP3, IGF2BP1, K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, LMP2, LY6G6D, melanoma antigen (MAGE) family members, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), NYESO-1, P53, PAP, PLAC1, melanoma preferred expression antigen (PRAME), PSA, SSX1, SSX2, SSX3, SSX4, SSX5, SSX8, thyroglobulin, tyrosinase, TRP-1, TRP-2, WT-1, Wnt10A, CMV, MCPyV, ROPN1, TP53 R175H, TP53 R248Q, XAGE-1 and / or XAGE-2.

45. The modified immune cell according to any one of claims 42-44, wherein the target antigen is NY-ESO-1.

46. ​​The modified immune cell according to any one of claims 42-45, wherein the target antigen is PRAME.

47. The modified immune cell according to any one of claims 41-46, wherein the TCR comprises an α chain and a β chain, the α chain comprising an α chain variable region (TRAV) and an α chain constant region (TRAC), and the β chain comprising a β chain variable region (TRBV) and a β chain constant region (TRBC).

48. The modified immune cell of claim 47, wherein the TRAV comprises complementarity-determining regions CDR1, CDR2 and CDR3.

49. The modified immune cell according to any one of claims 47-48, wherein the TRAV comprises complementarity-determining regions CDR1, CDR2, and CDR3, the amino acid sequences of said CDR1, CDR2, and CDR3 being selected from any group of the following combinations: a) The amino acid sequence of CDR1 is shown in SEQ ID NO:12, the amino acid sequence of CDR2 is shown in SEQ ID NO:13, and the amino acid sequence of CDR3 is shown in SEQ ID NO:14; and b) The amino acid sequence of CDR1 is shown in SEQ ID NO:28, the amino acid sequence of CDR2 is shown in SEQ ID NO:29, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

30.

50. The modified immune cells according to any one of claims 47-49, wherein the amino acid sequence of the TRAV is as shown in SEQ ID NO:1 or SEQ ID NO:

18.

51. The modified immune cell according to any one of claims 47-50, wherein the TRBV comprises complementarity-determining regions CDR1, CDR2 and CDR3.

52. The modified immune cell according to any one of claims 47-51, wherein the TRBV comprises complementarity-determining regions CDR1, CDR2, and CDR3, and the amino acid sequences of CDR1, CDR2, and CDR3 are selected from any group of the following combinations: a) The amino acid sequence of CDR1 is shown in SEQ ID NO:15, the amino acid sequence of CDR2 is shown in SEQ ID NO:16, and the amino acid sequence of CDR3 is shown in SEQ ID NO:17; and b) The amino acid sequence of CDR1 is shown in SEQ ID NO:31, the amino acid sequence of CDR2 is shown in SEQ ID NO:32, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

33.

53. The modified immune cell according to any one of claims 47-52, wherein the amino acid sequence of the TRBV is as shown in SEQ ID NO:4 or SEQ ID NO:

21.

54. The modified immune cells according to any one of claims 47-53, wherein the TRAC and / or TRBC are mouse and / or human constant regions.

55. The modified immune cell according to any one of claims 41-54, wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain.

56. The modified immune cell according to claim 55, wherein the antigen-binding domain is an antibody or an antigen-binding fragment thereof, or the antigen-binding domain is a PD-1 extracellular domain.

57. The modified immune cell according to claim 56, wherein the amino acid sequence of the extracellular domain of PD-1 is shown in SEQ ID NO:

44.

58. The modified immune cell according to any one of claims 55-57, wherein the transmembrane domain is a transmembrane domain derived from any of the following proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

59. The modified immune cell according to any one of claims 55-58, wherein the intracellular signal transduction domain is an intracellular signal transduction domain derived from any of the following proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (KSHV), DAP10, DAP-12, and a domain comprising at least one ITAM.

60. The modified immune cell according to any one of claims 55-59, wherein the chimeric antigen receptor comprises a hinge region, the hinge region of the chimeric antigen receptor being a hinge region derived from any of the following proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

61. The modified immune cell according to any one of claims 55-60, wherein the chimeric antigen receptor comprises a signal peptide.

62. The modified immune cell according to any one of claims 39-61, wherein the immune cell is a T cell.

63. The modified immune cell according to any one of claims 39-62, wherein the immune cell is an iNKT cell.

64. The modified immune cells according to any one of claims 39-63, wherein the immune cells are TCR-T cells and / or CAR-T cells.

65. An isolated nucleic acid molecule encoding a fusion protein as described in any one of claims 1-38.

66. The nucleic acid molecule according to claim 65, further encoding an antigen recognition receptor.

67. The nucleic acid molecule of claim 66, wherein the antigen recognition receptor is a T cell receptor (TCR) and / or a chimeric antigen receptor (CAR).

68. The nucleic acid molecule of claim 67, wherein the T cell receptor and / or chimeric antigen receptor specifically binds to the target antigen.

69. The nucleic acid molecule according to claim 68, wherein the target antigen is a tumor antigen.

70. The nucleic acid molecule according to any one of claims 68-69, wherein the target antigen is PD-L1, AFP, ASCL2, a member of the B-melanoma antigen (BAGE) family, BORIS, cancer-testis antigen, cancer-testis antigen 83 (CT-83), CAIX, carcinoembryonic antigen (CEA), EBV, EPHB2, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, glycoprotein 100 (GP100), hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) NS3, human papillomavirus (HPV) E6, HPV-E7, IGF2BP3, IGF2BP1, K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, LMP2, LY6G6D, melanoma antigen (MAGE) family members, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), NYESO-1, P53, PAP, PLAC1, melanoma preferred expression antigen (PRAME), PSA, SSX1, SSX2, SSX3, SSX4, SSX5, SSX8, thyroglobulin, tyrosinase, TRP-1, TRP-2, WT-1, Wnt10A, CMV, MCPyV, ROPN1, TP53 R175H, TP53 R248Q, XAGE-1 and / or XAGE-2.

71. The nucleic acid molecule according to any one of claims 68-70, wherein the target antigen is NY-ESO-1.

72. The nucleic acid molecule according to any one of claims 68-71, wherein the target antigen is PRAME.

73. The nucleic acid molecule according to any one of claims 68-72, wherein the TCR comprises an α chain and a β chain, the α chain comprising an α chain variable region (TRAV) and an α chain constant region (TRAC), and the β chain comprising a β chain variable region (TRBV) and a β chain constant region (TRBC).

74. The nucleic acid molecule of claim 73, wherein the TRAV comprises complementarity-determining regions CDR1, CDR2, and CDR3.

75. The nucleic acid molecule according to any one of claims 73-74, wherein the TRAV comprises complementarity-determining regions CDR1, CDR2, and CDR3, and the amino acid sequences of CDR1, CDR2, and CDR3 are selected from any group of the following combinations: a) The amino acid sequence of CDR1 is shown in SEQ ID NO:12, the amino acid sequence of CDR2 is shown in SEQ ID NO:13, and the amino acid sequence of CDR3 is shown in SEQ ID NO:14; and b) The amino acid sequence of CDR1 is shown in SEQ ID NO:28, the amino acid sequence of CDR2 is shown in SEQ ID NO:29, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

30.

76. The nucleic acid molecule according to any one of claims 73-75, wherein the amino acid sequence of the TRAV is as shown in SEQ ID NO:1 or SEQ ID NO:

18.

77. The nucleic acid molecule according to any one of claims 73-76, wherein the TRBV comprises complementarity-determining regions CDR1, CDR2 and CDR3.

78. The nucleic acid molecule according to any one of claims 73-77, wherein the TRBV comprises complementarity-determining regions CDR1, CDR2, and CDR3, and the amino acid sequences of CDR1, CDR2, and CDR3 are selected from any group of the following combinations: a) The amino acid sequence of CDR1 is shown in SEQ ID NO:15, the amino acid sequence of CDR2 is shown in SEQ ID NO:16, and the amino acid sequence of CDR3 is shown in SEQ ID NO:17; and b) The amino acid sequence of CDR1 is shown in SEQ ID NO:31, the amino acid sequence of CDR2 is shown in SEQ ID NO:32, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

33.

79. The nucleic acid molecule according to any one of claims 73-78, wherein the amino acid sequence of the TRBV is as shown in SEQ ID NO:4 or SEQ ID NO:

21.

80. The nucleic acid molecule according to any one of claims 73-79, wherein the TRAC and / or TRBC are mouse and / or human constant regions.

81. The nucleic acid molecule according to any one of claims 68-70, wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain.

82. The nucleic acid molecule according to claim 81, wherein the antigen-binding domain is an antibody or an antigen-binding fragment thereof, or the antigen-binding domain is a PD-1 extracellular domain.

83. The nucleic acid molecule according to claim 82, wherein the amino acid sequence of the PD-1 extracellular domain is shown in SEQ ID NO:

44.

84. The nucleic acid molecule according to any one of claims 81-83, wherein the transmembrane domain is a transmembrane domain derived from any of the following proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

85. The nucleic acid molecule according to any one of claims 81-84, wherein the intracellular signal transduction domain is an intracellular signal transduction domain derived from any of the following proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (KSHV), DAP10, DAP-12, and a domain comprising at least one ITAM.

86. The nucleic acid molecule according to any one of claims 81-85, wherein the chimeric antigen receptor comprises a hinge region, the hinge region of the chimeric antigen receptor being a hinge region derived from any one of the following proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

87. The nucleic acid molecule according to any one of claims 81-86, wherein the chimeric antigen receptor comprises a signal peptide.

88. The nucleic acid molecule according to any one of claims 65-87, further encoding a self-cleaving peptide.

89. The nucleic acid molecule of claim 88, wherein the sequence encoding the self-cleaving peptide is located between the sequences encoding the fusion protein and the antigen recognition receptor.

90. The nucleic acid molecule according to any one of claims 88-89, wherein the self-cleaving peptide comprises a 2A peptide.

91. The nucleic acid molecule according to any one of claims 88-90, wherein the self-cleaving peptide is P2A, T2A, E2A or F2A.

92. The nucleic acid molecule according to any one of claims 88-91, wherein the self-cleaving peptide is P2A.

93. A vector comprising any one of claims 65-92.

94. A cell comprising the nucleic acid molecule of any one of claims 65-92 and / or the vector of claim 93.

95. A method for preparing the fusion protein of any one of claims 1-38, the modified immune cell of any one of claims 39-64, the nucleic acid molecule of any one of claims 65-92, the vector of claim 93, and / or the cell of claim 94.

96. A pharmaceutical composition comprising a fusion protein according to any one of claims 1-38, a modified immune cell according to any one of claims 39-64, a nucleic acid molecule according to any one of claims 65-92, a carrier according to claim 93 and / or a cell according to claim 94, and optionally a pharmaceutically acceptable carrier.

97. Use of the fusion protein of any one of claims 1-38, the modified immune cell of any one of claims 39-64, the nucleic acid molecule of any one of claims 65-92, the carrier of claim 93, the cell of claim 94, and / or the pharmaceutical composition of claim 96 in the preparation of a medicament for the prevention and / or treatment of diseases and / or conditions.

98. The use according to claim 97, wherein the disease and / or condition is a tumor.

99. The use according to claim 98, wherein the tumor is a solid tumor and / or a hematoma.

100. The use according to any one of claims 98-99, wherein the tumor is lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, gastric cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer and / or skin cancer.

101. A method for preventing and / or treating diseases and / or conditions, comprising administering to a subject in need a fusion protein of any one of claims 1-38, a modified immune cell of any one of claims 39-64, a nucleic acid molecule of any one of claims 65-92, a carrier of claim 93, a cell of claim 94, and / or a pharmaceutical composition of claim 96.

102. The method of claim 101, wherein the disease and / or symptom is a tumor.

103. The method of claim 102, wherein the tumor is a solid tumor and / or a hematoma.

104. The method according to any one of claims 102-103, wherein the tumor is lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, gastric cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer and / or skin cancer.

105. The fusion protein of any one of claims 1-38, the modified immune cell of any one of claims 39-64, the nucleic acid molecule of any one of claims 65-92, the vector of claim 93, the cell of claim 94, and / or the pharmaceutical composition of claim 96, for the prevention and / or treatment of diseases and / or conditions.

106. The fusion protein, modified immune cell, nucleic acid molecule, carrier, cell and / or pharmaceutical composition according to claim 105, wherein the disease and / or condition is a tumor.

107. The fusion protein, modified immune cell, nucleic acid molecule, carrier, cell and / or pharmaceutical composition according to claim 106, wherein the tumor is a solid tumor and / or hematologic malignancy.

108. The fusion protein, modified immune cell, nucleic acid molecule, carrier, cell and / or pharmaceutical composition according to any one of claims 106-107, wherein the tumor is lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, gastric cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer and / or skin cancer.

Images