Methods and reagents for generating chimeric antigen receptors using split-inteins

Abstract

This invention relates to methods and reagents for generating chimeric antigen receptors using splitting integuments, and the use of said chimeric antigen receptors, particularly in diseases requiring cell-mediated immune responses against target antigens. The invention also relates to cage-locked integuments and methods for generating chimeric antigen receptors using these cage-locked integuments.

Description

Technical Field

[0001] This invention relates to methods and reagents for generating chimeric antigen receptors using split inteins, and the uses of said chimeric antigen receptors, particularly in diseases requiring cell-mediated immune responses against target antigens. The invention also relates to caged inteins and methods for generating recombinant proteins, particularly chimeric antigen receptors, using these caged inteins. Background Technology

[0002] Chimeric antigen receptors (CARs) are used in many clinical applications, including cancer therapy. A CAR is a recombinant receptor consisting of an extracellular antigen-binding domain and an intracellular T-cell signaling domain. Using CAR-modified T cells is an innovative immunotherapy approach. CAR cell therapy relies on reprogramming T cells to express a receptor that allows the cell to recognize target cells. Typically, CAR therapy involves collecting T cells from a patient, introducing a chimeric antigen into the collected cells, expanding the transfected cells, and subsequently infusing them into the patient.

[0003] Immunotherapy has generated unprecedented hope in cancer treatment, relying on the immune system as a powerful weapon against cancer. In recent years, adoptive cell immunotherapy based on chimeric antigen receptors (CARs) has shown great potential. T cells, or T lymphocytes, constantly seek out foreign antigens and distinguish abnormal cells (cancer cells or infected cells) from normal cells. Genetically modifying T cells with CARs is the most common method for designing tumor-specific T cells. CAR-T cells targeting tumor-associated antigens (TAAs) can be infused into patients (a process known as adoptive cell transfer or ACT), representing an effective immunotherapy approach.

[0004] Recent advancements in the use of chimeric antigen receptor (CAR) modified T-cell (CAR) therapy, which relies on redirecting T cells to appropriate cell surface molecules on cancer cells, have shown promising results in harnessing the power of the immune system to treat cancer.

[0005] Given the ongoing need for improved strategies for targeting diseases such as cancer, there is a high expectation for new compositions and methods to improve CAR-T therapy. Summary of the Invention

[0006] In a first aspect, the present invention relates to a polynucleotide, hereinafter referred to as a first polynucleotide of the present invention, encoding a fusion protein comprising, from the N-terminus to the C-terminus:

[0007] i) A cleaved C-inteptide comprising a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 or a functionally equivalent variant thereof.

[0008] ii) Transmembrane structural domains, and

[0009] iii) Intracellular signal transduction domains and / or co-stimulatory domains from the receptor.

[0010] In a second aspect, the present invention relates to a carrier, hereinafter referred to as the first carrier of the present invention, which comprises the first polynucleotide of the present invention.

[0011] In another aspect, the present invention relates to a host cell, hereinafter referred to as the first host cell of the present invention, which comprises the first polynucleotide of the present invention, the first vector of the present invention, or a fusion protein encoded by the first polynucleotide of the present invention.

[0012] In another aspect, the present invention relates to fusion proteins, hereinafter referred to as the first fusion protein of the present invention, which is encoded by the first polynucleotide of the present invention.

[0013] In another aspect, the present invention relates to a fusion protein, hereinafter referred to as the second fusion protein of the present invention, comprising, from the N-terminus to the C-terminus:

[0014] i) Heterologous peptides and

[0015] ii) Split N-intein comprising a sequence selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 or a functionally equivalent variant thereof.

[0016] In another aspect, the present invention relates to a polynucleotide, hereinafter referred to as the second polynucleotide of the present invention, which encodes the second fusion protein of the present invention.

[0017] In another aspect, the present invention relates to a carrier, hereinafter referred to as a second carrier of the present invention, which comprises a second polynucleotide of the present invention.

[0018] In another aspect, the present invention relates to a host cell, hereinafter referred to as the second host cell of the present invention, which contains the second polynucleotide of the present invention or the second vector of the present invention.

[0019] In another aspect, the present invention relates to a chimeric receptor, hereinafter referred to as the chimeric receptor of the present invention, comprising from the N-terminus to the C-terminus:

[0020] i) An extracellular domain comprising a polypeptide that specifically binds to a target molecule, or a polypeptide capable of forming an oligomer in the presence of one or more additional subunits, wherein the oligomer specifically binds to the target molecule.

[0021] ii) The linker region, whose sequence is the result of protein splicing.

[0022] iii) Transmembrane domains, and

[0023] iv) Intracellular signal transduction domains from the receptor,

[0024] The protein splicing reaction occurs between two complementary parts of the splitting inteptide, and the splitting inteptide is selected from Gp41.1 splitting inteptide, IMPDH splitting inteptide, NrdJ1 splitting inteptide and Gp41.8 splitting inteptide.

[0025] In another aspect, the present invention relates to a host cell, hereinafter referred to as the third host cell of the present invention, which contains the chimeric receptor of the present invention.

[0026] In another aspect, the present invention relates to compositions or kits, hereinafter referred to as a first composition or first kit, comprising a first component and a second component, wherein

[0027] i) The first component is the first polynucleotide of the present invention, the first vector of the present invention, the first host cell of the present invention, or the first fusion protein of the present invention, and

[0028] ii) The second component is the second fusion protein of the present invention or the second polynucleotide of the present invention, wherein

[0029] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0030] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0031] - The C-integer contains a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the N-integer contains a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0032] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0033] In another aspect, the present invention relates to an in vitro method for generating cells comprising chimeric receptors, hereinafter referred to as the first method of the invention, wherein the method comprises:

[0034] i) Providing cells comprising: a first polynucleotide of the present invention, a polypeptide encoded by said polynucleotide, or a first vector of the present invention, wherein the provision is carried out under conditions suitable for expression of a fusion protein encoded by said polynucleotide in said cells, and

[0035] ii) Contact the cells from step i) with the second fusion protein of the present invention.

[0036] The contact step ii) is performed under conditions that allow a splicing reaction to occur between a cleaved C-intein that forms a portion of the fusion protein produced in step (i) and a cleaved N-intein that forms a portion of the second fusion protein of the present invention, and wherein...

[0037] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0038] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0039] - The cleaved C-inteptide comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the cleaved N- comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0040] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0041] In another aspect, the present invention relates to an in vitro method for generating cells comprising chimeric receptors, hereinafter referred to as a second method of the invention, wherein the method comprises:

[0042] (i) Provide cells containing the following:

[0043] a. The first polynucleotide of the present invention, the first vector of the present invention, or a polypeptide encoded by said polynucleotide, wherein said polynucleotide further comprises a nucleotide sequence encoding a signal peptide, wherein said signal peptide is in-frame and located at the N-terminus of a cleaved C-inteptide, and said signal peptide allows the fusion protein to insert into the membrane, and

[0044] b. The second polynucleotide of the present invention, the second vector of the present invention, or the second fusion protein of the present invention, wherein the fusion protein encoded by said polynucleotide or said fusion protein further comprises a nucleotide sequence encoding a signal peptide, wherein said signal peptide is within a frame and located at the N-terminus of a polypeptide that specifically binds to a target molecule, and said signal peptide allows said fusion protein to be secreted from cells, and

[0045] (ii) The cells are maintained under conditions suitable for the expression of the two fusion proteins in the cells, suitable for the insertion of the fusion protein encoded by the first polynucleotide of the present invention into the membrane, suitable for the secretion of the fusion protein encoded by the second polynucleotide of the present invention, and suitable for splicing reactions between the cleavage of C-inte and the cleavage of N-inte, wherein...

[0046] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0047] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0048] - The cleaved C-inteptide comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the cleaved N- comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0049] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0050] In another aspect, the present invention relates to cells, hereinafter referred to as the first cell of the invention, which comprises a chimeric receptor obtained by the first or second method of the invention.

[0051] In another aspect, the present invention relates to a third host cell or a first cell of the present invention for treating diseases requiring a cell-mediated immune response against a target molecule, preferably against a target antigen.

[0052] In another aspect, the present invention relates to a second fusion protein, a second polynucleotide, a second vector, or a second host cell of the present invention for treating diseases requiring a cell-mediated immune response against a target molecule, preferably against a target antigen, wherein the patient to be treated is characterized by containing cells comprising: a first polynucleotide, a first vector, or a fusion protein encoded by the first polynucleotide of the present invention, wherein...

[0053] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0054] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0055] - The C-integer contains a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the N-integer contains a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0056] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0057] In another aspect, the present invention relates to a first polynucleotide, a first vector, a first host cell, or a first fusion protein of the present invention for treating diseases requiring a cell-mediated immune response against a target molecule, preferably against a target antigen, wherein the patient to be treated is characterized by containing cells comprising: a second fusion protein, a second polynucleotide, a second vector, or a second host cell of the present invention, wherein...

[0058] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0059] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0060] - The C-integer contains a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the N-integer contains a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0061] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0062] In another aspect, the present invention relates to a polynucleotide, hereinafter referred to as the third polynucleotide of the present invention, encoding a fusion protein comprising, from the N-terminus to the C-terminus:

[0063] (i) A modified N-inpeptide selected from modified GP41.1 N-inpeptide, modified IMPDH N-inpeptide, modified NrdJ1 N-inpeptide, or modified GP41.8 N-inpeptide.

[0064] The modified N-inpeptide comprises a fragment of an N-inpeptide protein that prevents trans-splicing in the presence of the corresponding C-inpeptide, and

[0065] The modified N-inpeptide contains at least one mutation that reduces the affinity between the modified N-inpeptide and the corresponding C-inpeptide.

[0066] (ii) A cleaved C-inteptide comprising a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 or a functionally equivalent variant thereof, wherein

[0067] - If the modified N-inpeptide is a modified GP41.1 N-inpeptide, then the split C-inpeptide contains the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof.

[0068] - If the modified N-inpeptide is a modified IMPDH N-inpeptide, then the split C-inpeptide contains the sequence of SEQ ID NO: 2 or a functionally equivalent variant thereof, wherein the modified N-inpeptide is a modified IMPDH N-inpeptide.

[0069] - If the modified N-inpeptide is a modified NrdJ1 N-inpeptide, then the split C-inpeptide contains the sequence of SEQ ID NO: 3 or a functionally equivalent variant thereof, or

[0070] - If the modified N-inpeptide is a modified GP41.8 N-inpeptide, then the split C-inpeptide comprises the sequence of SEQ ID NO: 4 or a functionally equivalent variant thereof, and

[0071] (iii) Heterologous polypeptides.

[0072] In another aspect, the present invention relates to a carrier, hereinafter referred to as the third carrier of the present invention, which comprises the third polynucleotide of the present invention.

[0073] In another aspect, the present invention relates to a host cell, hereinafter referred to as the fourth host cell of the present invention, which comprises the third polynucleotide of the present invention, the third vector of the present invention, or a fusion protein encoded by the third polynucleotide of the present invention.

[0074] In another aspect, the present invention relates to fusion proteins, hereinafter referred to as the third fusion protein of the present invention, which is encoded by the third polynucleotide of the present invention.

[0075] In another aspect, the present invention relates to a polynucleotide, hereinafter referred to as the fourth polynucleotide of the present invention, encoding a fusion protein comprising, from the N-terminus to the C-terminus:

[0076] (i) A modified N-inpeptide selected from modified GP41.1 N-inpeptide, modified IMPDH N-inpeptide, modified NrdJ1 N-inpeptide, or modified GP41.8 N-inpeptide.

[0077] The modified N-inpeptide comprises a fragment of an N-inpeptide protein that prevents trans-splicing in the presence of the corresponding C-inpeptide, and

[0078] The modified N-inpeptide contains at least one mutation that reduces the affinity between the modified N-inpeptide and the corresponding C-inpeptide.

[0079] (ii) A cleaved C-inteptide comprising a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 or a functionally equivalent variant thereof, wherein

[0080] - If the modified N-inpeptide is a modified GP41.1 N-inpeptide, then the split C-inpeptide contains the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof.

[0081] - If the modified N-inpeptide is a modified IMPDH N-inpeptide, then the split C-inpeptide contains the sequence of SEQ ID NO: 2 or a functionally equivalent variant thereof, wherein the modified N-inpeptide is a modified IMPDH N-inpeptide.

[0082] - If the modified N-inpeptide is a modified NrdJ1 N-inpeptide, then the split C-inpeptide contains the sequence of SEQ ID NO: 3 or a functionally equivalent variant thereof, or

[0083] - If the modified N-inpeptide is a modified GP41.8 N-inpeptide, then the split C-inpeptide comprises the sequence of SEQ ID NO: 4 or a functionally equivalent variant thereof.

[0084] (iii) Transmembrane domains, and

[0085] (iv) Intracellular signal transduction domains and / or co-stimulatory domains from the receptor.

[0086] In another aspect, the present invention relates to a carrier, hereinafter referred to as the fourth carrier of the present invention, which contains the fourth polynucleotide of the present invention.

[0087] In another aspect, the present invention relates to a host cell, hereinafter referred to as the fifth host cell of the present invention, which comprises the fourth polynucleotide of the present invention, the fourth vector of the present invention, or a fusion protein encoded by the fourth polynucleotide of the present invention.

[0088] In another aspect, the present invention relates to fusion proteins, hereinafter referred to as the fourth fusion protein of the present invention, which is encoded by the fourth polynucleotide of the present invention.

[0089] In another aspect, the present invention relates to a composition or kit, hereinafter referred to as a second composition or second kit, comprising a first component and a second component, wherein

[0090] i) The first component is the third or fourth polynucleotide of the present invention, the third or fourth vector of the present invention, the fourth or fifth host cell of the present invention, or the third or fourth fusion protein of the present invention, and

[0091] ii) The second component is the second fusion protein of the present invention or the second polynucleotide of the present invention, wherein

[0092] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0093] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0094] - The C-integer contains a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the N-integer contains a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0095] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0096] In another aspect, the present invention relates to a method for cleaving a heterologous polypeptide from a third fusion protein of the invention, the method comprising incubating the fusion protein together with a cleaving N-intepid under conditions allowing intepid splicing, wherein

[0097] - If the cleaved C-inpeptide forming part of the fusion protein comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, then the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0098] - If the cleaved C-inpeptide forming part of the fusion protein comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, then the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0099] - If the cleaved C-inpeptide forming part of the fusion protein comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, then the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0100] - If the split C-inpeptide forming part of the fusion protein contains a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, then the split N-inpeptide contains a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0101] In another aspect, the present invention relates to a method for covalently linking the N-terminus of a first polypeptide to the C-terminus of a second polypeptide, the method comprising incubating a second composition of the invention or associating components of a second kit of the invention under conditions allowing inteptide splicing, wherein the first polypeptide is a heterologous polypeptide forming part of a third fusion protein of the invention, and the second polypeptide is a heterologous polypeptide forming part of a second fusion protein of the invention.

[0102] In another aspect, the present invention relates to an in vitro method for generating cells comprising chimeric receptors, hereinafter referred to as the third method of the invention, wherein the method comprises:

[0103] (i) Providing cells comprising: the fourth polynucleotide of the present invention, a polypeptide encoded by said polynucleotide, or the fourth carrier of the present invention, wherein the provision is carried out under conditions suitable for expression of a fusion protein encoded by said polynucleotide in said cells, and

[0104] (ii) Contacting the cells of step i) with the second fusion protein of the present invention, wherein this contacting step ii) is carried out under conditions that allow a splicing reaction to occur between the cleaving C-intein that forms part of the fusion protein produced in step (i) and the cleaving N-intein that forms part of the second fusion protein of the present invention, and wherein

[0105] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0106] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0107] - The cleaved C-inteptide comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the cleaved N- comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0108] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0109] In another aspect, the present invention relates to an in vitro method for generating cells comprising chimeric receptors, hereinafter referred to as the fourth method of the invention, wherein the method comprises:

[0110] (i) Provide cells containing the following:

[0111] a. The fourth polynucleotide of the present invention, the fourth vector of the present invention, or a polypeptide encoded by said polynucleotide, wherein said polynucleotide further comprises a nucleotide sequence encoding a signal peptide, wherein said signal peptide is within a frame and located at the N-terminus of said modified N-integral peptide, and said signal peptide allows said fusion protein to insert into a membrane, and

[0112] b. The second polynucleotide of the present invention, the second vector of the present invention, or the second fusion protein of the present invention, wherein the fusion protein encoded by said polynucleotide or said fusion protein further comprises a nucleotide sequence encoding a signal peptide, wherein said signal peptide is within a frame and located at the N-terminus of a polypeptide that specifically binds to a target molecule, and said signal peptide allows said fusion protein to be secreted from cells, and

[0113] (ii) The cells are maintained under conditions suitable for the expression of the two fusion proteins in the cells, suitable for the insertion of the fusion protein encoded by the fourth polynucleotide of the present invention into the membrane, suitable for the secretion of the fusion protein encoded by the second polynucleotide of the present invention, and suitable for splicing reactions between the cleavage of C-inte and the cleavage of N-inte, wherein...

[0114] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0115] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0116] - The cleaved C-inteptide comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the cleaved N- comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0117] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0118] In another aspect, the present invention relates to cells, hereinafter referred to as the second cell of the invention, which contains a chimeric receptor obtained by the third or fourth method of the invention.

[0119] In another aspect, the present invention relates to a third host cell or a second cell of the present invention for treating diseases requiring a cell-mediated immune response against a target molecule, preferably against a target antigen.

[0120] In another aspect, the present invention relates to the second fusion protein of the present invention, the second polynucleotide of the present invention, the second vector of the present invention, or the second host cell of the present invention for treating diseases requiring a cell-mediated immune response against a target molecule, preferably against a target antigen, wherein the patient to be treated is characterized by containing cells comprising: the fourth polynucleotide of the present invention, the fourth vector of the present invention, or the fusion protein encoded by the fourth polynucleotide of the present invention, wherein...

[0121] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0122] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0123] - The cleaved C-inteptide comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the cleaved N- comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0124] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0125] In another aspect, the present invention relates to the fourth polynucleotide of the present invention, the fourth vector of the present invention, the fifth host cell of the present invention, or a fusion protein encoded by the fourth polynucleotide of the present invention, for treating diseases requiring a cell-mediated immune response against a target molecule, preferably against a target antigen, wherein the patient to be treated is characterized by containing cells comprising: the second fusion protein of the present invention, the second polynucleotide of the present invention, the second vector of the present invention, or the second host cell of the present invention, wherein...

[0126] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0127] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0128] - The C-integer contains a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the N-integer contains a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0129] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof. Attached Figure Description

[0130] Figure 1 . Expression, purification, and trans-splicing activity analysis of the αCD19 scFv-gp41.1-N-ST inteptide fusion. A) Western blot analysis of α-Strep tag expression of the αCD19 scFv-gp41.1-N-ST inteptide fusion in HEK293F cells at hour post-transfection (hpt). B) Coomassie affinity purification analysis of culture supernatant containing the αCD19 scFv-gp41.1-N-ST inteptide fusion. This analysis shows the protein content of the chromatographic input (in), flow-through (FT), wash (w), and recovered elution fractions (11 to 27). C) Coomassie staining analysis of the trans-splicing reaction between the αCD19 scFv-gp41.1-N-ST inteptide fusion and the gp41.1-C-Trx fusion. Lane descriptions: M, protein molecular weight marker; IN, pure αCD19 scFv-gp41.1-N-ST inteptide fusion at the initial concentration added to the reaction; IC, pure gp41.1-C-Trx inteptide fusion at the initial concentration added to the reaction; t0, aliquot corresponding to the start of the reaction; 1h to 4h, aliquots extracted from the reaction after 1, 2, and 4 hours of incubation, with or without DTT. The formation of the splicing product (SP) αCD19 scFv-Trx is shown in the reaction with DTT.

[0131] Figure 2 .Expression, purification, and trans-splicing activity analysis of the αBCMA scFv-IMPDH.1-N-His inteptide fusion. A) Western blot analysis of α-His tag expression in HEK293F cells at 144 and 168 hours post-transfection (hpt). B) Coomassie staining affinity purification analysis of culture supernatant containing the αBCMA scFv-IMPDH.1-N-His inteptide fusion. This analysis shows the protein content of the chromatographic loading (in), flow-through (FT), washing (w), and recovered elution fractions (T13 to T21). C) Coomassie staining analysis of the trans-splicing reaction between the αBCMA scFv-IMPDH.1-N-His inteptide fusion and the model ST-SUMO-His-IMPDH.1-C-VHH IntC fusion. Lane descriptions: M, protein molecular weight label; IN, pure αBCMA scFv-IMPDH.1-N-His inteptide fusion, concentration equivalent to the initial concentration added to the reaction; IC, pure ST-SUMO-His-IMPDH.1-C-VHH inteptide fusion, concentration equivalent to the initial concentration added to the reaction; t0, aliquots corresponding to the start of the reaction; 2h and 4h, aliquots extracted from the reaction after 2 hours and 4 hours of incubation, respectively; FT and res, aliquots extracted from the flow-through and elution buffers of the negative IMAC procedure, with or without DTT. The formation of the splice product (SP) αBCMA scFv-VHH in the flow-through buffer of the negative IMAC reaction with DTT was observed.

[0132] Figure 3 .Expression, purification, and trans-splicing activity analysis of the αCD22 scFv-gp41.8-N-His intima-peptide fusion. A) Western blot analysis of α-His tag expression in HEK293F cells at 120, 144, and 168 h (hpt) post-transfection. B) Coomassie staining affinity purification analysis of culture supernatant containing the αCD22 scFv-gp41.8-N-His intima-peptide fusion. This analysis shows the protein content of the chromatographic loading (in), flow-through (FT), washing (w), and recovered elution fractions (T6 to T10). C) Coomassie staining analysis of the trans-splicing reaction between the αCD22 scFv-gp41.8-N-His intima-peptide fusion and the gp41.8-C-LipTla fusion. Lane descriptions: M, protein molecular weight label; IN, pure αCD22 scFv-gp41.8-N-His inteptide fusion, concentration equivalent to the initial concentration added to the reaction; IC, pure gp41.8-C-LipTla inteptide fusion, concentration equivalent to the initial concentration added to the reaction; t0, aliquots corresponding to the start of the reaction; 1h to 4h, aliquots extracted from the reaction after 1, 2, and 4 hours of incubation, with or without DTT. The formation of the splice product (SP) αCD22 scFv-LipTla occurs in the presence of DTT.

[0133] Figure 4 .Expression, purification, and trans-splicing activity analysis of the αCD1a scFv-NrJ.1-N-His inteptide fusion. A) Western blot analysis of α-His tag expression in HEK293F cells at 120, 144, and 168 h (hpt) post-transfection. B) Coomassie staining affinity purification analysis of culture supernatant containing the αCD1a scFv-NrdJ.1-N-His inteptide fusion. This analysis shows the protein content of the chromatographic loading (in), flow-through (FT), washing (w), and recovered elution fractions (T14 to T22). C) Coomassie staining analysis of the trans-splicing reaction between the αCD1a scFv-NrdJ.1-N-His inteptide fusion and the NrdJ.1-C-LipTla fusion. Lane descriptions: M, protein molecular weight label; IN, pure αCD1a scFv-NrdJ.1-N-His inteptide fusion, concentration equivalent to the initial concentration added to the reaction; IC, pure NrdJ.1-C-LipTla inteptide fusion, concentration equivalent to the initial concentration added to the reaction; t0, aliquots corresponding to the start of the reaction; 1h to 4h, aliquots extracted from the reaction after 1, 2, and 4 hours of incubation, with or without DTT. The formation of the splice product (SP) αCD1a scFv-LipTla occurs in the presence of DTT.

[0134] Figure 5 . Expression of IntC-CAR fusions in HEK293F cells. A) Schematic diagram of IntC-CAR constructs. SP: signal peptide; His: 6×His tag; INTC: IntC domain corresponding to gp41.1-C, IMPDH.1-C, gp41.8-C, or NrdJ.1-C; CD8-HR: CD8 hinge region; CD8 TM: CD8 transmembrane domain; 4-1BB and CD3z: intracellular signal transduction domains. B) Flow cytometry analysis of expression of different IntC-CAR constructs using APC-labeled anti-His antibody. C) Anti-CD3z Western blot analysis of the integrity of IntC-CAR fusions containing peptides of gp41.1 (G1), IMPDH.1 (I1), NrdJ.1 (N1), and gp41.8 (G8). m: monomer; m + PTM: monomer + post-translational modification; glyco m: glycosylated monomer.

[0135] Figure 6 .Expression of cage-locked IntC-CAR fusions in HEK293F cells. A) Schematic diagram of the cage-locked IntC-CAR construct. SP: signal peptide; ST: Strep tag; INTNcage: C-terminal fragment of the corresponding IntN; adapter: peptide adapter; His: 6×His tag; INTC: IntC domain corresponding to gp41.1-C, IMPDH.1-C, gp41.8-C, or NrdJ.1-C; CD8-HR: CD8 hinge region; CD8 TM: CD8 transmembrane domain; 4-1BB and CD3z: intracellular signal transduction domains. B) Expression levels of non-cage-locked and cage-locked IntC-CAR constructs obtained by flow cytometry analysis using APC-labeled α-His antibody. G1: gp41.1-CAR; I1: IMPDH.1-C-CAR; N1: NrdJ.1-C-CAR; G8: gp41.8-C-CAR. C) Comparison of flow cytometry analysis of expression of caged and non-caged IntC-CAR constructs of gp41.1, IMPDH.1, NrdJ.1 and gp41.8 inteptides obtained using APC-labeled α-His antibody.

[0136] Figure 7 . Flow cytometry analysis of the trans-splicing reaction between HEK293F cells expressing the gp41.1-C-CAR construct and the α-CD19 scFv-gp41.1-N inteptide fusion. A) Schematic diagram of the trans-splicing reaction between cells expressing IntC-CAR on their surface and the scFv-IntN fusion to produce a full-length scFv-CAR. B) Flow cytometry analysis of the trans-splicing reaction between gp41.1-C-CAR and α-CD19 scFv-gp41.1-N-ST exposed on the surface of transfected HEK293F cells. This analysis used an APC-labeled α-His antibody to determine the expression level of gp41.1-C-CAR and a PE-labeled α-FLAG antibody to detect cells containing the reconstructed α-CD19 scFv-CAR. C) Flow cytometry analysis of the trans-splicing reaction between gp41.1-C-CAR and α-CD19 scFv-gp41.1-N-ST exposed to the surface of transfected HEK293F cells, using PE-labeled CD19 antigen. D) αCD3z Western blot analysis of gp41.1-C-CAR-expressing cells before (-) and after (+) trans-splicing with the α-CD19 scFv-gp41.1-N inteptide fusion.

[0137] Figure 8 .Flow cytometry analysis of the trans-splicing reaction between HEK293F cells expressing the IMPDH.1-C-CAR construct and the α-BCMA scFv-IMPDH.1-N inteptide fusion. A) Flow cytometry analysis of the trans-splicing reaction between IMPDH.1-C-CAR and α-BCMA scFv-IMPDH.1-N-His exposed on the surface of transfected HEK293F cells. This analysis used an APC-labeled α-His antibody to determine the expression level of IMPDH.1-C-CAR and a PE-labeled α-FLAG antibody to detect cells containing reconstructed α-BCMA scFv-CAR. B) αCD3z Western blot analysis of IMPDH.1-C-CAR-expressing cells that have undergone trans-splicing with the α-BCMA scFv-IMPDH.1-N inteptide fusion. C) Schematic diagram of the IMPDH.1-C-connector-CAR construct. SP: Signal peptide; His: 6×His tag; INTC: IntC domain corresponding to the IMPDH.1-C inteptide; Linker: Amino acid peptide; CD8-HR: CD8 hinge region; CD8 TM: CD8 transmembrane domain; 4-1BB and CD3z: Intracellular signal transduction domains. D) Flow cytometry analysis of the trans-splicing reaction between IMPDH.1-C-linker-CAR and α-BCMA scFv-IMPDH.1-N-His exposed on the surface of transfected HEK293F cells. This analysis used APC-labeled α-His antibody to determine the expression level of IMPDH.1-C-linker-CAR and PE-labeled α-FLAG antibody to detect cells containing reconstructed α-BCMA scFv-linker-CAR.

[0138] Figure 9 .Flow cytometry analysis of the trans-splicing reaction between HEK293F cells expressing the caged gp41.1-C-CAR construct and the α-CD19 scFv-gp41.1-N inteptide fusion. A) Flow cytometry analysis of the trans-splicing reaction between caged gp41.1-C-CAR and α-CD19 scFv-gp41.1-N-ST exposed on the surface of transfected HEK293F cells. This analysis used APC-labeled α-His antibody to determine the expression level of gp41.1-C-CAR and PE-labeled α-FLAG antibody to detect cells containing the reconstructed α-CD19 scFv-CAR. B) Flow cytometry analysis of the trans-splicing reaction between caged gp41.1-C-CAR and α-CD19 scFv-gp41.1-N-ST exposed to the surface of transfected HEK293F cells, using PE-labeled CD19 antigen. C) αCD3z Western blot analysis of cells expressing caged gp41.1-C-CAR before (-) and after (+) trans-splicing with the α-CD19 scFv-gp41.1-N inteptide fusion.

[0139] Figure 10 . Flow cytometry analysis of the trans-splicing reaction between HEK293F cells expressing the caged IMPDH.1-C-CAR construct and the α-BCMA scFv-IMPDH.1-N inteptide fusion. A) Flow cytometry analysis of expression of the caged IMPDH.1-C-CAR in HEK293F cells transfected with an APC-labeled α-His antibody to detect the His tag in the construct; and flow cytometry analysis of the trans-splicing reaction between the caged IMPDH.1-C-CAR and α-BCMA scFv-IMPDH.1-N-His using a PE-labeled α-FLAG antibody to detect cells containing the reconstructed α-BCMA scFv-CAR. B) αCD3z Western blot analysis of cells expressing the caged IMPDH.1-C-CAR that have undergone trans-splicing with the α-BCMA scFv-IMPDH.1-N inteptide fusion.

[0140] Figure 11 .Comparison of the percentage of cells with high levels of reconstructed scFv-CAR on their surface between cells expressing caged IntC-CAR and cells expressing non-caged IntC-CAR. A) Comparison of the percentage of cells with high levels of reconstructed αCD19 scFv-CAR between cells expressing caged gp41.1-C-CAR and cells expressing non-caged gp41.1-C-CAR. Flow cytometry analysis of the trans-splicing reaction was performed using PE-labeled CD19 antigen. B) Comparison of the percentage of cells with high levels of reconstructed αBCMA scFv-CAR between cells expressing caged IMPDH.1-C-CAR and cells expressing non-caged IMPDH.1-C-CAR. Flow cytometry analysis of the trans-splicing reaction was performed using PE-labeled α-FLAG antibody.

[0141] Figure 12 . Western blot analysis of HEK293F cells co-transfected with plasmids encoding gp41.1-C-CAR fusions (cage-locked or non-cage-locked) and scFv-gp41.1-N fusions. A) αCD3z Western blot analysis of cells co-transfected with gp41.1-C-CAR. B) αCD3z Western blot analysis of cells co-transfected with cage-locked gp41.1-C-CAR. In both gels, lanes 1 and 2 correspond to co-transfection with the scFv-IMPDH.1-N construct, which cannot undergo trans-splicing; and lanes 3 and 4 correspond to co-transfection with the scFv-gp41.1-N construct. The gels show the formation of reconstructed scFv-CAR at 48 and 72 hours post-transfection. The figure below shows the bands corresponding to the precursor IntC-CAR fusion (gp41.1-C-CAR in gel A and cage-type gp41.1-C-CAR in gel B), and the figure above shows the trans-splicing products (scFv-CAR in both gels).

[0142] Figure 13 .Western blot analysis of HEK293F cells co-transfected with plasmids encoding different IntC-CAR fusions (cage-locked or non-cage-locked) and scFv-IntN fusions. A) αCD3z Western blot analysis of cells co-transfected with plasmid combinations described in the table below the Western blot membrane. Trans-splicing products obtained from co-transfection are labeled as shown in the table. B) αFLAG Western blot analysis of cells co-transfected with plasmid combinations described in the table below the Western blot membrane. For each sample, cell lysates and growth medium containing secreted proteins were loaded onto the gel as shown in the table. C) Flow cytometry analysis of cells co-transfected with plasmid combinations shown below each figure. Cells were incubated with a combination of two antibodies: an APC-labeled α-His antibody for detecting the expression of the gp41.1-C-CAR construct; and a PE-labeled α-FLAG antibody for detecting the expression of reconstructed α-CD19 scFv-CAR.

[0143] Figure 14 . Western blot analysis of HEK293F cells co-transfected with plasmids encoding IMPDH.1-C-CAR fusions (cage-locked or non-cage-locked) and scFv-gp41.1-N fusions. A) αCD3z Western blot analysis of cells co-transfected with IMPDH.1-C-CAR. B) αCD3z Western blot analysis of cells co-transfected with cage-locked IMPDH.1-C-CAR. In both gels, lanes 1, 2, and 3 correspond to co-transfection with the CD19scFv-gp41.1-N construct, which cannot undergo trans-splicing; lanes 3, 4, and 5 correspond to co-transfection with the CD19scFv-IMPDH.1-N construct; and lanes 6, 7, and 8 correspond to co-transfection with the BCMAscFv-IMPDH.1-N construct. The gels show the formation of reconstructed scFv-CAR at 24, 48, and 72 hours post-transfection. The figure below shows the bands corresponding to the precursor IntC-CAR fusion (IMPDH.1-C-CAR in gel A and cage-type IMPDH.1-C-CAR in gel B), and the figure above shows the trans-splicing products (scFv-CAR in both gels).

[0144] Figure 15 .αCD3z Western blot analysis was performed on HEK293F cells co-transfected with plasmids encoding gp41.1-C-CAR and αPSMA IgG fused to the C-terminus of the heavy chain. Lane 1 corresponds to co-transfection with the CD19scFv-IMPDH.1-N construct, which does not undergo trans-splicing. Lane 2 corresponds to co-transfection with the PSMAIgG-HC-gp41.1-N construct, which produces a reconstructed CAR with an αPSMA-targeting molecule (PSMA-HC-CAR).

[0145] Figure 16 . Integration of IntC-CAR did not affect T cell activation and proliferation. A) Schematic diagram of the IntC-CAR construct used for T cell transduction. SP: signal peptide; His: 6×His tag; INTC: IntC domain corresponding to IMPDH.1-C or NrdJ.1-C; CD8-HR: CD8 hinge region; CD8 TM: CD8 transmembrane domain; 4-1BB and CD3z: intracellular signal transduction domains; T2A sequence; and GFP: green fluorescent protein. B) Proliferation assay of T cells expressing the IntC-CAR construct. Two different IntC-CAR constructs were analyzed: NrdJ.1-C-CAR and IMPDH.1-CAR. No significant difference in cell proliferation was observed compared with untransduced activated T cells (sample number = 3).

[0146] Figure 17 . IntC-CAR gene delivery to activated T cells does not affect T cell memory phenotype over time. A) Time-resolved memory T cell phenotypes of NrdJ.1-C-CAR and IMPDH.1-C-CAR T cells. Data from a representative donor are shown. B) Pooled data of uninfected T cells, C) NrdJ.1-C-CAR T cells, and D) IMPDH.1-C-CAR T cells (n=5). Initial / SCM: CCR7+CD45RA+; Central memory: CCR7+CD45RA+; Effector memory: CCR7-CD45RA-; and Terminal differentiation effector memory or TEMRA: CCR7-CD45RA-.

[0147] Figure 18 .Trans-splicing reaction between IMPDH.1-C-CAR T cells and purified αCD1a scFv-IMPDH.1-N fusion. A) The trans-splicing reaction between IMPDH.1-C-CAR T cells and αCD1a scFv-IMPDH.1-N fusion is specific for IMPDH.1 inteptide pairs because when αCD1a scFv-IMPDH.1-N fusion is combined with NrdJ.1-C-CAR T cells, these cells do not incorporate αCD1a scFv onto their surface. B) 72.4% of activated T cells transduced with the IMPDH.1-C-CAR construct expressed the construct on their surface. After trans-splicing reaction with reduced αCD1a scFv-IMPDH.1-N, 63.2% of the cells contained αCD1a scFv on their surface, resulting in a trans-splicing efficiency of 87%. C) Trans-splicing efficiency depends on the T cell donor but is always above 60%. A summary of three independent T cell donors shows the initial percentage of His+ cells and the percentage of αCD1a scFv+ cells after trans-splicing. Trans-splicing efficiency is calculated as follows: .

[0148] Figure 19 .αCD1a scFv-CAR T cells reconstituted via IMPDH.1 inteptide trans-splicing exhibited cytotoxicity against CD1a+ T-ALL cells. A) Gating strategy for MOLT4 cell death analysis. Equal numbers of MOLT4 cell lines expressing wild-type CD1a (T-ALL cell lines) pre-stained with Cell Violet tracer were mixed with CD1ako MOLT4 cell lines expressing GFP, also pre-stained with Cell Violet tracer. GFP expression was used as a surrogate indicator for CD1a negativity. To analyze the effect of αCD1a scFv-CAR T cells on the MOLT4 mixture, GFP signals (CD1a negative) obtained by flow cytometry after gating the CellViolet+ cell population were used. B) A representative example of results obtained after co-incubation of αCD1ascFv-CAR T cells with a MOLT4 mixture for 24 hours following a trans-splicing reaction between IMPDH.1-C-CAR cells and a reduced αCD1a scFv-IMPDH.1-N fusion. An asterisk indicates a reduction in GFP- (i.e., CD1a+) MOLT4 cells after incubation. C) Specific cytotoxicity of αCD1a scFv-CAR T cells against CD1a+ MOLT4 cells after 24 hours (left) and 48 hours (right) under different trans-splicing conditions. D) IFN-γ secretion obtained at 24 hours (left) and 48 hours (right) under each condition shown in Figure C. The figures in Figures C and D summarize results from three independent donors. Detailed Implementation

[0149] The first polynucleotide of the present invention

[0150] In a first aspect, the present invention relates to a polynucleotide, hereinafter referred to as a first polynucleotide of the present invention, encoding a fusion protein comprising, from the N-terminus to the C-terminus:

[0151] i) A cleaved C-inteptide comprising a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 or a functionally equivalent variant thereof.

[0152] ii) Transmembrane structural domains, and

[0153] iii) Intracellular signal transduction domains and / or co-stimulatory domains from the receptor.

[0154] As used herein, the terms “nucleic acid,” “polynucleotide,” or “nucleic acid molecule” refer to polymeric compounds composed of covalently linked subunits (called nucleotides). Nucleic acids include polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which can be single-stranded or double-stranded. DNA includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA. Polynucleotides may contain deoxyribonucleotides, ribonucleotides, and / or their analogues. Nucleotides may have any three-dimensional structure and may perform any known or unknown function. The term “polynucleotide” includes, for example, single-stranded, double-stranded, and triple-stranded molecules, genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. In addition to natural nucleic acid molecules, the nucleic acid molecules of the present invention may also include modified nucleic acid molecules.

[0155] The first polynucleotide of the present invention encodes a fusion protein. The term "fusion protein" or "fusion polypeptide" refers to a protein or polypeptide consisting of at least two polypeptides and optionally a linking sequence for efficiently linking the two polypeptides into a single continuous polypeptide. The two polypeptides linked in a fusion polypeptide typically originate from two independent sources, and therefore the fusion polypeptide comprises two linking polypeptides that are not normally found in nature in a linked form. The two polypeptides can be directly and operably linked by peptide bonds, or indirectly linked by a linker described herein or other linkers known in the art.

[0156] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to an amino acid polymer of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. These terms also cover amino acid polymers modified in a natural manner or by intervention; for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other operation or modification, such as conjugation with a labeled component. This definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids), and other modifications known in the art. In a preferred embodiment, the polypeptide is formed solely of amino acids.

[0157] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid simulants that function in a manner similar to naturally occurring amino acids. Furthermore, the term "amino acid" includes both D- and L-amino acids (stereoisomers). Preferably, the amino acid is an L-amino acid.

[0158] The term “natural amino acids” or “naturally occurring amino acids” includes 20 naturally occurring amino acids; those that are usually modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine, and phosphothreonine; and other less common amino acids, including but not limited to 2-aminoadipic acid, hydroxylysine, isodesin, valine, leucine, and ornithine.

[0159] The first polynucleotide of the present invention encoding a fusion protein comprises i) a split C-inteptide, said split C-inteptide comprising a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 or a functionally equivalent variant thereof.

[0160] The term "intrapeptide" refers to a protein element that can be self-cleaved from a host protein and catalyze flanking sequences linked by peptide bonds.

[0161] The term "split intein" refers to any intein in which the N-terminal domain and C-terminal domain are not directly linked by peptide bonds. Naturally occurring split inteins have been identified in cyanobacteria and archaea, but they can also be artificially generated by splitting the intein sequence into two parts. The split inteins used in this invention may contain six conserved protein splicing motifs from the HINT (Hog / Intein) family.

[0162] The term "split C-inpeptide" refers to the C-terminal domain of an inpeptide. The "C-terminal domain of an inpeptide" refers to an inpeptide sequence containing a C-terminal amino acid sequence that is functional for trans-splicing and / or C-terminal self-cleavage. The C-terminal domain of an inpeptide may contain C1 and / or C2 motifs of the HINT (Hog / Intein) family.

[0163] The first polynucleotide of the present invention encoding the fusion protein comprises a split C-inteptide, said split C-inteptide comprising a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 or a functionally equivalent variant thereof.

[0164] This refers to the C-terminal domain of the GP41.1 cleavage inteptide.

[0165] This refers to the C-terminal domain of the IMPDH1 cleavage inner peptide.

[0166] This refers to the C-terminal domain of the NrdJ1 cleavage peptide.

[0167] This refers to the C-terminal domain of the GP41.8 cleavage inteptide.

[0168] The term “functionally equivalent variant of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4” refers to all sequences resulting from modifications, insertions, and / or deletions of one or more amino acids in the above sequences (including post-translational modifications such as glycosylation, phosphorylation, hydroxylation, acylation, alkylation, γ-carboxylation, nucleotide addition, iodination, propionylation, S-glutathioneylation, S-nitrosylation, succinylation, and sulfation), provided that the function of the integument is substantially maintained. Suitable assays for determining the integument activity of functionally equivalent variants are described in patent application disclosed in WO2013045632A1.

[0169] Post-translational modification (PTM) refers to the alteration of a protein's covalent bonds after biosynthesis. PTM can involve enzymes or occur spontaneously. Proteins are produced by ribosomes translating mRNA into polypeptide chains, which can then undergo changes to form the mature protein product. PTM can occur on amino acid side chains or at the C- or N-terminus of a protein. Sites frequently undergoing PTM are those with functional groups that can act as nucleophiles in the reaction: hydroxyl groups of serine, threonine, and tyrosine; amine forms of lysine, arginine, and histidine; thiolate anions of cysteine; carboxyl groups of aspartic acid and glutamate; and N- and C-terminal sites. Additionally, although the amide group of asparagine is a weak nucleophile, it can act as a linker for glycans. Less common modifications occur at oxidized methionine and some methylene groups in the side chain. PTM can be experimentally detected using various techniques, including mass spectrometry, Eastern blotting, and Western blotting.

[0170] Preferably, variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 are (i) polypeptides in which one or more amino acid residues are replaced by conserved or non-conserved amino acid residues (preferably conserved amino acid residues), and such replaced amino acids may or may not be encoded by the genetic code; (ii) polypeptides containing one or more modified amino acid residues, such as residues modified by substituent bonding; (iii) polypeptides generated by alternative processing of similar mRNAs; (iv) polypeptide fragments; and / or (v) polypeptides generated by fusing the polypeptides defined in (i) to (iii) with other polypeptides (e.g., secretory leader sequences or sequences for purification (e.g., His tags) or sequences for detection (e.g., Sv5 epitope tags)). Fragments include polypeptides generated by proteolytic cleavage (including multisite proteolysis) of the original sequence. Variants may be post-translationally modified or chemically modified. Such variants will be apparent to those skilled in the art.

[0171] Those skilled in the art will recognize that the value of nucleotide sequence identity can be appropriately adjusted by taking into account codon degeneracy, conserved amino acid substitutions, and reading frame positioning to determine the corresponding sequence identity of the two nucleotide sequences encoding the polypeptide of the present invention.

[0172] In the context of this invention, "conservative amino acid alteration" and "conservative amino acid substitution" are used synonymously. "Conservative amino acid substitution" refers to the interchangeability of residues with similar side chains, and means replacing one or more amino acids in a natural amino acid sequence with other amino acids having similar side chains, resulting in a silent change that does not alter protein function. Conservative substitutes for amino acids in a natural amino acid sequence can be selected from other members of the group to which naturally occurring amino acids belong. For example, the group of amino acids with aliphatic side chains includes glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic-hydroxy side chains includes serine and threonine; the group of amino acids with amide-containing side chains includes asparagine and glutamine; the group of amino acids with aromatic side chains includes phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains includes lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains includes cysteine ​​and methionine. In some embodiments of the invention, preferred conserved amino acid substitutions are: valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. Therefore, the invention relates to functionally equivalent variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4; and whose amino acid sequences differ from a given sequence by one or more amino acids, the difference being the result of one or more conserved amino acid substitutions. It is known in the art that one or more amino acids in a polypeptide sequence can be replaced by at least one other amino acid having similar charge and polarity, such that the substitution results in a silencing change in the modified polypeptide, which does not alter the function of the modified polypeptide relative to the unmodified sequence. This invention relates to any polypeptide sequence that differs from the sequence given in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 by one or more amino acids (whether as a result of conserved or non-conserved substitutions, and / or as a result of sequence insertion or deletion), provided that the further provided polypeptide sequence has the same or similar intein C-terminal domain as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0173] In some embodiments, functionally equivalent variants of the split C-integral peptide are characterized by containing a C2 box (F box) sequence. The C2 box is a non-strictly conserved sequence. The C2 box may contain, for example, the sequence... (SEQ ID NO: 9), where X is any amino acid, h is a hydrophobic amino acid, and p is a polar amino acid. In some embodiments, the C-terminal domain of the peptide contains a sequence (SEQ ID NO: 10), where X1 is N, E, L, K, Q, D, P, or R; X2 is V, L, or T; X3 is Y, I, V, H, or F; X4 is D; X5 is I or L; X6 is G, E, T, Q, or K; X7 is V or T; X8 is E, S, T, D, N, or K; X9 is R, G, D, N, Q, S, or K; X 10 It is D, E, N, T, or K; X 11 It is H, R, S, I, or N; X 12 It is N, L, S, I, or N; X 13 It is F, Y, L, or I; and X 14 It is A, Y, F, N, C, or S. In some embodiments, the C-terminal domain of the peptide contains a sequence... (SEQ ID NO: 11), where X1 is E, L, K, Q, D, P, or R; X2 is V, L, or T; X3 is Y, I, V, H, or F; X4 is D; X5 is I or L; X6 is G, E, T, Q, or K; X7 is V or T; X8 is E, S, T, D, N, or K; X9 is G, D, N, Q, S, or K; X 10 It is D, E, N, T, or K; X11 is H, R, S, I, or N; X 12 It is N, L, S, I, or N; X 13 It is F, Y, L, or I; and X 14 It is A, Y, F, N, C, or S.

[0174] Based on their chemical properties, amino acids can be grouped as follows: (i) charged (D, E, K, R, H), (ii) acidic (D, E), (iii) basic (K, R, H), (iv) small (V, C, S, T, P, G, D, A), (v) polar (N, Q, S, T), (vi) large (E, Q, R, K, H, Y, W, F, M, L, I), (vii) hydrophobic (V, I, L, M, F, Y, W, A) and (viii) nucleophilic (S, T, C).

[0175] In some embodiments, functionally equivalent variants of the split C-integral peptide are characterized by containing a portion of a C1 box (G box) sequence. The C1 box is a non-strictly conserved sequence. The C1 box may contain, for example, the sequence... (SEQ ID NO:12), where X is any amino acid, h is a hydrophobic amino acid, and n is a nucleophilic amino acid. In some embodiments, the C-terminal domain of the peptide contains the sequence (SEQ ID NO: 13), wherein X1 is L, A, V, I, or C; X2 is N or R; X3 is G, D, A, or N; X4 is I, F, or T; X5 is L, I, or V; X6 is V, I, T, or A; X7 is H or S; X8 is N; and X9 is S, T, or C. In some embodiments, the C-terminal domain of the peptide contains a sequence (SEQ ID NO: 14), wherein X1 is A, V, I, or C; X2 is N or R; X3 is G, D, A, or N; X4 is I, F, or T; X5 is L or V; X6 is V, I, or T; X7 is H; X8 is N; and X9 is S, T, or C. In the C1 box sequence, amino acids X1 to X8 correspond to the inteptide sequence, and X9 corresponds to the first amino acid of the expeptide.

[0176] The last two amino acids in the C-terminal domain of an inpeptide are highly conserved and important for protein splicing. Therefore, in some embodiments, the last amino acid in the C-terminal domain is asparagine. In some embodiments, the last amino acid in the C-terminal domain is glutamine. In some embodiments, the penultimate amino acid in the C-terminal domain is histidine. In some embodiments, the last amino acid in the C-terminal domain is an amino acid other than asparagine or glutamine. In some embodiments, the penultimate amino acid in the C-terminal domain is an amino acid other than histidine. In some embodiments, the penultimate amino acid in the C-terminal domain is an amino acid other than serine. For example, the last amino acid and / or the penultimate amino acid in the C-terminal domain can be alanine.

[0177] In some embodiments, the C-terminal domain of the peptide is about 10 to about 80 amino acids. In some embodiments, the C-terminal domain of the peptide is about 20 to about 70 amino acids. In some embodiments, the C-terminal domain of the peptide is about 30 to about 60 amino acids. In some embodiments, the C-terminal domain of the peptide is about 25 to about 35, about 30 to about 40, about 35 to about 45, about 40 to about 50, about 45 to about 55, or about 55 to about 65 amino acids.

[0178] In the context of two or more amino acid or nucleotide sequences, the terms "identity," "identical," or "percentage identity" refer to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid or nucleotide residues when compared and aligned (with gaps introduced if necessary) to obtain maximum correspondence, regardless of any conserved amino acid substitutions as part of sequence identity. Percentage identity can be measured using sequence comparison software or algorithms or through visual inspection. Various algorithms and software are known in the art for obtaining amino acid or nucleotide sequence alignments.

[0179] The sequence identity percentage can be determined by comparing two optimally aligned sequences within a comparison window. The alignment sequences can be polynucleotide or polypeptide sequences. For optimal alignment of two sequences, the portion of the polynucleotide or amino acid sequence within the comparison window may contain insertions or deletions (i.e., vacancies) compared to a reference sequence (which does not contain insertions or deletions). The sequence identity percentage is calculated by determining the number of positions where the same nucleotide or amino acid residues appear in both comparison sequences to generate the number of matching positions, then dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Sequence identity between two polypeptide sequences or two polynucleotide sequences can be determined, for example, by using the Gap program in WISCONSINPACKAGE version 10.0-UNIX of Genetics Computer Group, Inc., based on the Needleman and Wunsch method (J. Mol. Biol. 48:443-453, 1970), using the default parameter set for pairwise comparisons (for amino acid sequence comparisons: Gap Creation Penalty = 8, Gap Extension Penalty = 2; for nucleotide sequence comparisons: Gap Creation Penalty = 50; Gap Extension Penalty = 3); or by using the TBLASTN program in the BLAST 2.2.1 software suite (Altschul et al., Nucleic Acids Res. 25:3389-3402), using the BLOSUM62 matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA). 89:10915-10919, 1992) and the default parameter set used for pairwise comparisons (gap creation cost = 11, gap extension cost = 1).

[0180] Identity may exist in sequence regions of at least about 10, about 20, about 40 to 60 residues or any integer value therebetween, and may exist in regions longer than 60 to 80 residues (e.g., at least about 90 to 100 residues), and in some embodiments, the sequences are substantially identical along the full length of the sequences to be compared (e.g., coding regions of nucleotide sequences).

[0181] Functionally equivalent variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 also include sequences having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0182] In some implementations, a functionally equivalent variant of SEQ ID NO: 1 is a sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1.

[0183] In some implementations, a functionally equivalent variant of SEQ ID NO: 2 is a sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 2.

[0184] In some implementations, a functionally equivalent variant of SEQ ID NO: 3 is a sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 3.

[0185] In some implementations, a functionally equivalent variant of SEQ ID NO: 4 is a sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 4.

[0186] In some embodiments, the C-terminal domain of the inteptide contains at least about 10, at least about 20, at least about 30, at least about 40, or at least about 50 amino acids selected from the sequences of SEQ ID NO: 1, 2, 3, and 4. In some embodiments, the C-terminal domain of the inteptide contains at least about 10, at least about 20, at least about 30, at least about 40, or at least about 50 consecutive amino acids selected from the sequences of SEQ ID NO: 1, 2, 3, and 4. In some embodiments, the C-terminal domain of the inteptide contains a deletion of no more than about 5, about 10, about 15, about 20, or about 25 amino acids selected from the sequences of SEQ ID NO: 1, 2, 3, and 4. In some embodiments, the C-terminal domain of the inteptide contains a deletion of no more than about 5, about 10, about 15, about 20, or about 25 consecutive amino acids selected from the sequences of SEQ ID NO: 1, 2, 3, and 4.

[0187] In one embodiment, the functionally equivalent variants of SEQ ID NO: 1, 2, 3, or 4 have at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the corresponding sequence SEQ ID NO: 1, 2, 3, or 4, and said sequence identity is determined over the entire length of the sequence SEQ ID NO: 1, 2, 3, or 4.

[0188] The first polynucleotide of the present invention encoding the fusion protein includes ii) a transmembrane domain.

[0189] The term "transmembrane domain" (TMD) as used in this article refers to a transmembrane protein domain. A transmembrane domain is a transmembrane domain of a transmembrane protein (e.g., a type I transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. A TMD may consist of one or more α-helices or transmembrane β-barrels. Since the interior of the lipid bilayer is hydrophobic, the amino acid residues in a TMD are generally hydrophobic; however, proteins such as membrane pumps and ion channels may contain polar residues. A transmembrane domain may contain one or more additional amino acids adjacent to the transmembrane region, such as one or more amino acids associated with the extracellular region of the protein from which the transmembrane domain originates (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to a maximum of 15 amino acids in the extracellular region) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein originates (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to a maximum of 15 amino acids in the intracellular region).

[0190] Transmembrane domains can be derived from natural or recombinant sources. When the source is natural, the domain can originate from any membrane-bound or transmembrane protein. Some non-limiting examples of transmembrane domains particularly used in this invention may include at least the following transmembrane regions: for example, the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD3ζ, KIRDS2, OX40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, CTLA4, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160, CD19, IL2Rβ, IL2Rγ, IL7Ra, ITGA1, VLA1, C D49a, ITGA4, IA4CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDGA, CDGA, CD103, ITGAL, CDLa, LFA-1, ITGAM, CDllb, ITGAX, CDlc, ITGB1, CD29, ITGB2, CD18, LFA-1, LGA ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A), Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, Kp30, NKp46, including NKG2D; and / or transmembrane domains selected from the NKG2C transmembrane domain.

[0191] In one specific implementation, the transmembrane domain is selected from the transmembrane domains of CD28, CD8α, 4-1BB, CD4, CTLA4, CD27, or CD3ζ.

[0192] The first polynucleotide of the present invention encoding the fusion protein comprises iii) an intracellular signaling domain and / or a co-stimulatory domain derived from the receptor. Therefore, in one embodiment, the first polynucleotide of the present invention encoding the fusion protein comprises iii) an intracellular signaling domain derived from the receptor. In another embodiment, the first polynucleotide of the present invention encoding the fusion protein comprises iii) a co-stimulatory domain.

[0193] As used in this article, “intracellular signal transduction domain” or “signal transduction domain” refers to the intracellular portion of a molecule, and more specifically to any oligopeptide or polypeptide known to function as a domain that transduces signals to induce activation or inhibition of biological processes in the cell.

[0194] In one specific implementation, the intracellular signal transduction domain from the receptor is the intracellular signal transduction domain from the antigen receptor, i.e., the antigen receptor signal transduction domain. In a more specific implementation, the antigen receptor signal transduction domain is selected from: CD3ζ, CD28, 4-1BB, NKG2D, DAP10, OX40, CD70, CD27, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP 12 antigen receptor signal transduction domain, or combinations thereof.

[0195] Antigen receptor signaling domains generate signals that stimulate the immune effector functions of cells containing chimeric antigen receptors (CARs), such as CAR-T cells. For example, the effector functions of T cells can be cytolytic functions or helper activities, including the secretion of cytokines. Therefore, intracellular signaling domains can be part of proteins that transduce effector function signals and direct cells (such as T cells) to perform specialized functions.

[0196] Typically, the entire intracellular signal transduction domain can be used; however, it should be understood that the entire domain need not be used, provided that any portion of the signal transduction domain used is still capable of transducing effector signals. It should also be understood that variants of such intracellular signal transduction domains having substantially the same or greater functional capabilities can also be used. Therefore, the invention includes the following meaning: the variant should have substantially the same or greater effector signal transduction. Typically, substantially the same or greater signal transduction includes at least 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, or 120% or more of the signal transduction of the unmodified intracellular signal transduction domain, wherein the signal transduction of the unmodified intracellular signal transduction domain corresponds to 100%. Methods for assessing effector signal transduction are well known to those skilled in the art and include, for example, assessing the amount and / or activity of molecules (e.g., proteins such as cytokines) indicating the transduced signal. Thus, when the signal is a cytolytic function of T cells, the method may involve measuring one or more cytokines secreted by T cells that are known to have cytolytic activity (e.g., IFNγ). Other methods for assessing cytolytic function include CFSE staining and counting of positive cells by flow cytometry, or chromium release assays known in the art.

[0197] Some examples of intracellular signal transduction domains include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors (which work together after antigen-receptor binding to initiate signal transduction), as well as any derivatives or variants of these sequences and any recombinant sequences with the same functional capabilities.

[0198] Sometimes, the signal generated by the TCR alone is insufficient to fully activate T cells, and secondary and / or co-stimulatory signals may also be required. Therefore, T cell activation can be considered to be mediated by two distinct classes of intracellular signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary intracellular signaling domains) and those that function in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary intracellular signaling domains, such as co-stimulatory domains). Co-stimulatory domains promote the activation of effector functions and may also contribute to the persistence of effector functions and / or cell survival.

[0199] Primary intracellular signaling domains regulate primary activation of the TCR complex in a stimulatory or inhibitory manner. Primary intracellular signaling domains acting in a stimulatory manner may contain signaling motifs referred to as immune receptor tyrosine-based activation motifs or ITAMs (e.g., 2, 3, 4, 5, or more ITAMs). Therefore, an intracellular signaling domain may contain one or more ITAMs. Some examples of ITAM-containing primary intracellular signaling domains particularly used in this invention include those of CD3ζ, Fc receptor γ, Fc receptor β, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0200] "Co-stimulatory signal transduction domain" refers to a portion of a chimeric receptor that contains the intracellular domain of a co-stimulatory molecule.

[0201] The term "co-stimulatory molecule" refers to a identifiable T cell binding partner that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response generated by the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules are cell surface molecules other than antigen-specific receptors or their ligands that are essential for an effective immune response. Co-stimulatory molecules can be cell surface molecules other than antigen receptors or their ligands that are essential for an effective response of immune cells (e.g., lymphocytes) to antigens. Co-stimulatory molecules can be classified into the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules (SLAM proteins), and NK cell activation receptors. Examples of such molecules include, but are not limited to, 4-1BB (CD137), OX40, ICOS, DAP10, CD27, CD28, CDS, CD30, CD137 (4-1BB), CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, GITR, NKG2C, SLAMF7, NKp80, BAFFR, HVEM, BTLA, ICAM-1, LFA-1 (CD11a / CD18), B7-H3, and ligands that specifically bind to CD83.

[0202] In one specific embodiment, the first polynucleotide of the present invention comprises a nucleotide sequence encoding a signal peptide, wherein the signal peptide is within a frame and located at the N-terminus of a split C-integer, and the signal peptide allows the fusion protein to insert into the membrane.

[0203] The term "signal peptide" refers to a relatively short peptide, typically 5 to 40 amino acid residues, that guides proteins synthesized in the cell to specific locations, particularly allowing fusion proteins encoded by the first polynucleotide of the present invention to insert into the membrane.

[0204] Any signal peptide that allows the fusion protein encoded by the first polynucleotide of the present invention to insert into the membrane can be used in the present invention. In one specific embodiment, the signal peptide is selected from: CD8α, IL-2, GM-CSF receptor (GM-CSF) α chain, mouse Ig-κ (IgK), mouse IgK V-III region (IgKVIII), human IgKVIII, CD33, human tissue plasminogen activator (TPA), and naturally occurring secretory alkaline phosphatase (SEAP).

[0205] In another specific embodiment, the first polynucleotide of the present invention further includes a linker between the split C-integer and the transmembrane domain.

[0206] The term "linker" refers to a suitable peptide that allows two or more functional domains in a fusion protein to be linked together. Linkers can be flexible or rigid. In a preferred embodiment, the linker is a flexible linker. As used herein, "flexible linker" means that the linked domains require a degree of mobility or interaction. They are typically composed of small, nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The small size of these amino acids provides flexibility and allows the linked functional domains to be mobile. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thereby reducing unfavorable interactions between the linker and the protein moieties.

[0207] In some embodiments, the linker is a peptide containing 1 to 150 amino acid residues, 1 to 90 amino acid residues, 2 to 85 amino acid residues, 3 to 80 amino acid residues, 5 to 75 amino acid residues, 5 to 70 amino acid residues, 5 to 65 amino acid residues, 10 to 60 amino acid residues, 10 to 50 amino acid residues, 30 to 50 amino acids, 10 to 150 amino acids, 10 to 125 amino acids, 50 to 125 amino acids, or 75 to 125 amino acids. In a preferred embodiment, the linker between the cleaving C-integer and the transmembrane domain comprises 40 to 45 amino acids.

[0208] In one specific implementation, the linker between the split C-integument and the transmembrane domain is a hinge region or a flexible linker.

[0209] The terms "hinge domain," "hinge region," or "spacer" used in this article refer to a flexible amino acid region that allows the splitting C-integer to separate from the transmembrane domain.

[0210] In one specific implementation, the hinge region is selected from: IgG1 hinge region, IgG2 hinge region, IgG4 hinge region, FcγR hinge region, CD3ε hinge region, CD3ζ hinge region, CD8α hinge region, CD4 hinge region, CD28 hinge region, CD7 hinge region and CD19 hinge region.

[0211] Exemplary flexible linkers include glycine- and serine-rich linkers, such as (GGP)n, (GGGGS)n (SEQ ID NO: 30), or (GGGS)n (SEQ ID NO: 19), where n is 1 to 5. The most commonly used flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues (“GS” linkers). The length of the GS linker can be optimized by adjusting the copy number “n” to achieve proper separation of functional domains or to maintain necessary interdomain interactions. In a preferred embodiment, the linker that splits the C-integer from the transmembrane domain is a glycine- and serine-rich linker.

[0212] In some embodiments, the first amino acid of the linker is serine, cysteine, or threonine. In some embodiments, the first, second, third, fourth, and / or fifth amino acids of the linker are serine, cysteine, or threonine.

[0213] In another specific implementation, the transmembrane domain and the intracellular signal transduction domain are directly linked by peptide bonds or by amino acid linkers.

[0214] The term "peptide bond" refers to an amide-type covalent chemical bond that connects two consecutive α-amino acids along a peptide chain or protein chain, specifically connecting the C1 (carbon number one) of one α-amino acid to the N2 (nitrogen number two) of another α-amino acid.

[0215] In one specific embodiment, the fusion protein encoded by the first polynucleotide of the present invention further comprises a modified N-intein, wherein the modified N-intein is located at the N-terminus relative to the cleaved C-intein, wherein:

[0216] - If the cleaved C-inpeptide contains the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof, then the modified N-inpeptide is the modified GP41.1 N-inpeptide.

[0217] - If the cleaved C-inpeptide contains the sequence of SEQ ID NO: 2 or a functionally equivalent variant thereof, then the modified N-inpeptide is a modified IMPDH N-inpeptide.

[0218] - If the cleaved C-inpeptide contains the sequence of SEQ ID NO: 3 or a functionally equivalent variant thereof, then the modified N-inpeptide is a modified NrdJ1 N-inpeptide, or

[0219] - If the cleaved C-inpeptide contains the sequence of SEQ ID NO: 4 or a functionally equivalent variant thereof, then the modified N-inpeptide is the modified GP41.8 N-inpeptide.

[0220] The modified N-inpeptide comprises a fragment of an N-inpeptide protein, which prevents trans-splicing in the presence of a corresponding C-inpeptide, and

[0221] The modified N-intein contains at least one mutation that reduces the affinity between the modified N-intein and the corresponding C-intein.

[0222] The modified N-intein comprises a fragment of an N-intein protein that prevents trans-splicing in the presence of a corresponding C-intein.

[0223] In one specific embodiment, the modified N-intein does not contain the complete sequence of the N-intein protein.

[0224] According to the present invention, the complete sequence of the N-inteptide protein is selected from the following inteptides: GP41.1 N-inteptide, IMPDH N-inteptide, NrdJ1 N-inteptide, and GP41.8 N-inteptide. The sequences of the N-terminal domains of GP41.1 inteptide, IMPDH inteptide, NrdJ1 inteptide, and GP41.8 inteptide are respectively described in SEQ ID NO: 5, 6, 7, and 8:

[0225] This refers to the N-terminal domain of the GP41.1 cleavage inteptide.

[0226] This refers to the N-terminal domain of the IMPDH1 cleavage peptide.

[0227] This refers to the N-terminal domain of the NrdJ1 cleavage peptide.

[0228] This refers to the N-terminal domain of the GP41.8 cleavage inteptide.

[0229] In one embodiment, if the cleaved C-integrin contains the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof, the modified GP41.1 N-integrin contains a fragment of SEQ ID NO: 5 that prevents trans-splicing in the presence of the GP41.1 C-integrinin. In one embodiment, the modified GP41.1 N-integrinin does not contain the complete sequence of SEQ ID NO: 5.

[0230] In another specific embodiment, if the cleaved C-integrin contains the sequence of SEQ ID NO: 2 or a functionally equivalent variant thereof, the modified IMPDH1 N-integrin contains the fragment of SEQ ID NO: 6, which prevents trans-splicing in the presence of the IMPDH1 C-integrin. In one specific embodiment, the modified IMPDH1 N-integrin does not contain the complete sequence of SEQ ID NO: 6.

[0231] In another specific embodiment, if the cleaved C-inteptide contains the sequence of SEQ ID NO: 3 or a functionally equivalent variant thereof, the modified NrdJ1 N-inteptide contains a fragment of SEQ ID NO: 7 that prevents trans-splicing in the presence of the NrdJ1 C-inteptide. In one specific embodiment, the modified NrdJ1 N-inteptide does not contain the complete sequence of SEQ ID NO: 7.

[0232] In another specific embodiment, if the cleaved C-integrin contains the sequence of SEQ ID NO: 4 or a functionally equivalent variant thereof, the modified GP41.8 N-integrin contains a fragment of SEQ ID NO: 8 that prevents trans-splicing in the presence of the GP41.8 C-integrinin. In one specific embodiment, the modified GP41.8 N-integrinin does not contain the complete sequence of SEQ ID NO: 8.

[0233] The term "fragment" refers to a portion or subset of a longer amino acid sequence that retains certain defined features or functions. In one specific embodiment, the fragment comprises or consists of 10 to 100 amino acids, preferably 20 to 65 amino acids, more preferably 45 to 60 amino acids. The N-intein protein is truncated to obtain a modified N-intein, which prevents trans-splicing; that is, the fragment of the N-intein protein prevents trans-splicing in the presence of the corresponding C-intein.

[0234] In one specific embodiment, the modified GP41.1 N-inpeptide is a fragment of the sequence SEQ ID NO: 5, the fragment comprising or consisting of 18 to 83 amino acids, preferably 30 to 60 amino acids, more preferably 40 to 50 amino acids. The fragment of sequence SEQ ID NO: 5 contains at least one mutation that reduces the affinity between the modified GP41.1 N-inpeptide and the GP41.1 C-inpeptide.

[0235] In one specific embodiment, the modified GP41.1 N-intein does not contain the first 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65 amino acids of the polypeptide as defined in SEQ ID NO: 5.

[0236] In another specific embodiment, the modified GP41.1 N-inteptide does not contain the last 5, last 10, last 15, or last 20 amino acids of the polypeptide as defined in SEQ ID NO:5.

[0237] The removal of amino acids at the N-terminus of the GP41.1 N-inpeptide (i.e., the first amino acid of the polypeptide as defined in SEQ ID NO: 5) and the C-terminus of the GP41.1 N-inpeptide (i.e., the last amino acid of the polypeptide as defined in SEQ ID NO: 5) can be combined. In one specific embodiment, the modified GP41.1 N-inpeptide does not contain the first 5, first 10, first 15, first 20, first 25, first 30, first 35, first 40, first 45, or first 50 amino acids of the polypeptide as defined in SEQ ID NO: 5, and does not contain the last 5, last 10, last 15, or last 20 amino acids of the polypeptide as defined in SEQ ID NO: 5.

[0238] In a more specific embodiment, the modified GP41.1 N-intein does not contain the first 5, first 10, first 15, first 20, first 25, first 30, first 35, or first 40 amino acids of the polypeptide as defined in SEQ ID NO: 5, and does not contain the last 5, last 10, last 15, or last 20 amino acids of the polypeptide as defined in SEQ ID NO: 5.

[0239] In a preferred embodiment, the modified GP41.1 N-intein does not contain the first 41 amino acids of the polypeptide as defined in SEQ ID NO: 5.

[0240] In another specific embodiment, the modified IMPDH N-intein is a fragment of SEQ ID NO: 6, the fragment comprising or consisting of 21 to 96 amino acids, preferably 40 to 70 amino acids, more preferably 50 to 60 amino acids. The fragment of SEQ ID NO: 6 contains at least one mutation that reduces the affinity between the modified IMPDH N-intein and the IMPDH C-intein.

[0241] In one specific embodiment, the modified IMPDH N-intein does not contain the first 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65 amino acids of the polypeptide as defined in SEQ ID NO: 6.

[0242] In another specific embodiment, the modified IMPDH N-inteptide does not contain the last 5, last 10, last 15, or last 20 amino acids of the polypeptide as defined in SEQ ID NO: 6.

[0243] The removal of amino acids at the N-terminus of the IMPDH N-inpeptide (i.e., the first amino acid of the polypeptide as defined in SEQ ID NO: 6) and at the C-terminus of the IMPDH N-inpeptide (i.e., the last amino acid of the polypeptide as defined in SEQ ID NO: 6) can be combined. In one specific embodiment, the modified IMPDH N-inpeptide does not contain the first 5, first 10, first 15, first 20, first 25, first 30, first 35, first 40, first 45, first 50, first 55, or first 60 amino acids of the polypeptide as defined in SEQ ID NO: 6, and does not contain the last 5, last 10, last 15, or last 20 amino acids of the polypeptide as defined in SEQ ID NO: 6.

[0244] In a more specific embodiment, the modified IMPDH N-intein does not contain the first 5, first 10, first 15, first 20, first 25, first 30, first 35, first or first 40 amino acids of the polypeptide as defined in SEQ ID NO: 6, and does not contain the last 5, last 10, last 15 or last 20 amino acids of the polypeptide as defined in SEQ ID NO: 6.

[0245] In a preferred embodiment, the modified IMPDH N-intein does not contain the first 42 amino acids of the polypeptide as defined in SEQ ID NO: 6.

[0246] In another specific embodiment, the modified NrdJ1 N-inpeptide is a fragment of SEQ ID NO: 7, the fragment comprising or consisting of 25 to 100 amino acids, preferably 30 to 60 amino acids, more preferably 40 to 50 amino acids. The fragment of SEQ ID NO: 7 contains at least one mutation that reduces the affinity between the modified NrdJ1 N-inpeptide and the NrdJ1 C-inpeptide.

[0247] In one specific embodiment, the modified NrdJ1 N-intein does not contain the first 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65 amino acids of the polypeptide as defined in SEQ ID NO: 7.

[0248] In another specific embodiment, the modified NrdJ1 N-inteptide does not contain the last 5, last 10, last 15, or last 20 amino acids of the polypeptide as defined in SEQ ID NO: 7.

[0249] The removal of amino acids at the N-terminus of the NrdJ1 N-inpeptide (i.e., the first amino acid of the polypeptide as defined in SEQ ID NO: 7) and at the C-terminus of the NrdJ1 N-inpeptide (i.e., the last amino acid of the polypeptide as defined in SEQ ID NO: 7) can be combined. In one specific embodiment, the modified NrdJ1 N-inpeptide does not contain the first 5, first 10, first 15, first 20, first 25, first 30, first 35, first 40, first 45, first 50, first 55, or first 60 amino acids of the polypeptide as defined in SEQ ID NO: 7, and does not contain the last 5, last 10, last 15, or last 20 amino acids of the polypeptide as defined in SEQ ID NO: 7.

[0250] In a more specific embodiment, the modified NrdJ1 N-intein does not contain the first 5, first 10, first 15, first 20, first 25, first 30, first 35, first 40, first 45, first 50, or first 55 amino acids of the polypeptide as defined in SEQ ID NO: 7, and does not contain the last 5, last 10, last 15, or last 20 amino acids of the polypeptide as defined in SEQ ID NO: 7.

[0251] In a preferred embodiment, the modified NrdJ1 N-inteptide does not contain the first 56 amino acids of the polypeptide as defined in SEQ ID NO: 7.

[0252] In another specific embodiment, the modified GP41.8 N-inpeptide is a fragment of SEQ ID NO: 8, the fragment comprising or consisting of 19 to 84 amino acids, preferably 30 to 60 amino acids, more preferably 40 to 50 amino acids. The fragment of SEQ ID NO: 8 contains at least one mutation that reduces the affinity between the modified GP41.8 N-inpeptide and the GP41.8 C-inpeptide.

[0253] In one specific embodiment, the modified GP41.8 N-intein does not contain the first 5, first 10, first 15, first 20, first 25, first 30, first 35, first 40, first 45, first 50, first 55, first 60, or first 65 amino acids of the polypeptide as defined in SEQ ID NO: 8.

[0254] In another specific embodiment, the modified GP41.8 N-inteptide does not contain the last 5 amino acids, the last 10 amino acids, the last 15 amino acids, or the last 20 amino acids of the polypeptide as defined in SEQ ID NO:8.

[0255] The removal of amino acids at the N-terminus of the GP41.8 N-inpeptide (i.e., the first amino acid of the polypeptide as defined in SEQ ID NO: 8) and at the C-terminus of the GP41.8 N-inpeptide (i.e., the last amino acid of the polypeptide as defined in SEQ ID NO: 8) can be combined. In one specific embodiment, the modified GP41.8 N-inpeptide does not contain the first 5, first 10, first 15, first 20, first 25, first 30, first 35, first 40, first 45, or first 50 amino acids of the polypeptide as defined in SEQ ID NO: 8, and does not contain the last 5, last 10, last 15, or last 20 amino acids of the polypeptide as defined in SEQ ID NO: 8.

[0256] In a more specific embodiment, the modified GP41.8 N-intein does not contain the first 5, first 10, first 15, first 20, first 25, first 30, first 35, or first 40 amino acids of the polypeptide as defined in SEQ ID NO: 8, and does not contain the last 5, last 10, last 15, or last 20 amino acids of the polypeptide as defined in SEQ ID NO: 8.

[0257] In a preferred embodiment, the modified GP41.8 N-inteptide does not contain the first 40 amino acids of the polypeptide as defined in SEQ ID NO: 8.

[0258] A modified N-inpeptide is an N-inpeptide that does not contain the native sequence of an N-inpeptide protein. Specifically, the modified N-inpeptide contains at least one mutation that reduces the affinity between the modified N-inpeptide and the corresponding C-inpeptide.

[0259] A mutation is a change in the nucleotide sequence of DNA (or in some cases RNA), or a change in the resulting amino acid sequence of a protein. Mutations can occur naturally or can be introduced artificially. In one specific embodiment, the at least one mutation in the modified N-integument that reduces the affinity between the modified N-integument and the corresponding C-integument is a substitution, insertion, or deletion.

[0260] It should be understood that the mutations present in the modified N-inpeptides that destabilize the binding between the modified N-inpeptides and the corresponding C-inpeptides are present only in those N-inpeptide variants containing the mutated sites, and not in those variants where the mutated sites are eliminated due to truncation.

[0261] The mutation in the modified N-inpeptide reduces the affinity between the modified N-inpeptide and the corresponding C-inpeptide. In one specific embodiment, the affinity between the modified N-inpeptide and the C-inpeptide is 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% lower than the affinity between the N-inpeptide and the corresponding C-inpeptide.

[0262] In a preferred embodiment, the affinity between the modified N-inte and the C-inte is 40% to 60% lower than the affinity between the N-inte and the corresponding C-inte.

[0263] In one specific embodiment, the split C-inteptide comprises the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof, and the modified N-inteptide is a modified GP41.1 N-inteptide comprising at least one mutation that reduces the affinity between the modified GP41.1 N-inteptide and the GP41.1 C-inteptide.

[0264] In one specific embodiment, the modified GP41.1 N-inpeptide contains 1 to 4 mutations in the fragment of sequence SEQ ID NO: 5. In a more specific embodiment, the modified GP41.1 N-inpeptide contains 2 mutations in the fragment of sequence SEQ ID NO: 5.

[0265] In one specific embodiment, the modified GP41.1 N-intein contains at least one mutation that replaces a charged amino acid with a neutral amino acid. In a more specific embodiment, the modified GP41.1 N-intein contains at least one mutation that replaces a positively charged amino acid with a neutral amino acid.

[0266] Therefore, in another specific embodiment, the modified N-intein includes at least one mutation that alters the net charge of the modified N-intein fragment compared to the wild-type N-intein. Specifically, the modified GP41.1 N-intein includes at least one mutation that alters the net charge of the modified GP41.1 N-intein fragment compared to the wild-type GP41.1 N-intein.

[0267] In one specific embodiment, the modified GP41.1 N-intein contains at least one mutation at a lysine (K) residue. In a more specific embodiment, the modified GP41.1 N-intein contains at least one mutation that replaces a lysine (K) residue with an alanine (A) residue.

[0268] In a preferred embodiment, the modified GP41.1 N-inpeptide contains mutations at lysine residues at positions 45 and 48 of SEQ ID NO: 5. In a more preferred embodiment, the modified GP41.1 N-inpeptide contains mutations K45A and K48A.

[0269] In one specific embodiment, the modified GP41.1 N-inpeptide comprises the sequence of SEQ ID NO: 31.

[0270] The above-defined mutation applies only when the modified GP41.1 N-inpeptide contains a fragment of SEQ ID NO: 5 containing a mutated position (e.g., K45 and K48).

[0271] In another specific embodiment, the split C-inteptide comprises the sequence of SEQ ID NO: 2 or a functionally equivalent variant thereof, and the modified N-inteptide is a modified IMPDH N-inteptide comprising at least one mutation that reduces the affinity between the modified IMPDH N-inteptide and the IMPDH C-inteptide.

[0272] In one specific embodiment, the modified IMPDH N-inpeptide contains 1 to 4 mutations in the fragment of sequence SEQ ID NO: 6. In a more specific embodiment, the modified IMPDH N-inpeptide contains 2 mutations in the fragment of sequence SEQ ID NO: 6.

[0273] In one specific embodiment, the modified IMPDH N-inte contains at least one mutation that replaces a charged amino acid with a neutral amino acid. In a more specific embodiment, the modified IMPDH N-inte contains at least one mutation that replaces a negatively charged amino acid with a neutral amino acid.

[0274] Therefore, in another specific embodiment, the modified N-intein comprises at least one mutation that alters the net charge of the modified N-intein fragment compared to the wild-type N-intein. Specifically, the modified IMPDH N-intein comprises at least one mutation that alters the net charge of the modified IMPDH N-intein fragment compared to the wild-type IMPDH N-intein.

[0275] In a more specific embodiment, the modified IMPDH N-intein contains at least one mutation at a glutamic acid (E) residue. In a more specific embodiment, the modified IMPDH N-intein contains at least one mutation replacing a glutamic acid (E) residue with an alanine (A) residue.

[0276] In a preferred embodiment, the modified IMPDH N-intein contains mutations at the 46th and 47th glutamic acid residues of SEQ ID NO: 6. In a more preferred embodiment, the modified IMPDH N-intein contains mutations E46A and E47A.

[0277] In one specific embodiment, the modified IMPDH N-inteptide comprises the sequence of SEQ ID NO: 32.

[0278] The above-defined mutation applies only when the modified IMPDH N-inpeptide contains a fragment of SEQ ID NO: 6 containing a mutated position (e.g., E46 and E47).

[0279] In another specific embodiment, the split C-inteptide comprises the sequence of SEQ ID NO: 3 or a functionally equivalent variant thereof, and the modified N-inteptide is a modified NrdJ1 N-inteptide comprising at least one mutation that reduces the affinity between the modified NrdJ1 N-inteptide and the NrdJ1 C-inteptide.

[0280] In one specific embodiment, the modified NrdJ1 N-inpeptide contains 1 to 4 mutations in the fragment of sequence SEQ ID NO: 7. In a more specific embodiment, the modified NrdJ1 N-inpeptide contains 2 mutations in the fragment of sequence SEQ ID NO: 7.

[0281] In one specific embodiment, the modified NrdJ1 N-intein contains at least one mutation that replaces a charged amino acid with a neutral amino acid. In a more specific embodiment, the modified NrdJ1 N-intein contains at least one mutation that replaces a negatively charged amino acid with a neutral amino acid.

[0282] Therefore, in another specific embodiment, the modified N-intein includes at least one mutation that alters the net charge of the modified N-intein fragment compared to the wild-type N-intein. Specifically, the modified NrdJ1 N-intein includes at least one mutation that alters the net charge of the modified NrdJ1 N-intein fragment compared to the wild-type NrdJ1 N-intein.

[0283] In a more specific embodiment, the modified NrdJ1 N-intein contains at least one mutation at a glutamic acid (E) residue. In a more specific embodiment, the modified NrdJ1 N-intein contains at least one mutation that replaces a glutamic acid (E) residue with an alanine (A) residue.

[0284] In a preferred embodiment, the modified NrdJ1 N-inpeptide contains mutations at the 62nd and 64th glutamic acid residues of SEQ ID NO: 7. In a more preferred embodiment, the modified NrdJ1 N-inpeptide contains mutations E62A and E64A.

[0285] In one specific embodiment, the modified NrdJ1 N-inteptide comprises the sequence of SEQ ID NO: 33.

[0286] The above-defined mutation applies only when the modified NrdJ1 N-inpeptide contains a fragment of SEQ ID NO: 7 containing a mutated position (e.g., E62 and E64).

[0287] In another specific embodiment, the split C-inteptide comprises the sequence of SEQ ID NO: 4 or a functionally equivalent variant thereof, and the modified N-inteptide is a modified GP41.8 N-inteptide comprising at least one mutation that reduces the affinity between the modified GP41.8 N-inteptide and the GP41.8 C-inteptide.

[0288] In one specific embodiment, the modified GP41.8 N-inpeptide contains 1 to 4 mutations in the fragment of sequence SEQ ID NO: 8. In a more specific embodiment, the modified GP41.8 N-inpeptide contains 2 mutations in the fragment of sequence SEQ ID NO: 8.

[0289] In one specific embodiment, the modified GP41.8 N-intein contains at least one mutation that replaces a charged amino acid with a neutral amino acid. In a more specific embodiment, the modified GP41.8 N-intein contains at least one mutation that replaces a positively charged amino group with a neutral amino acid.

[0290] Therefore, in another specific embodiment, the modified N-intein comprises at least one mutation that alters the net charge of the modified N-intein fragment compared to the wild-type N-intein. Specifically, the modified GP41.8 N-intein comprises at least one mutation that alters the net charge of the modified GP41.8 N-intein fragment compared to the wild-type GP41.8 N-intein.

[0291] In a more specific embodiment, the modified GP41.8 N-intein contains at least one mutation at a lysine (K) residue. In a more specific embodiment, the modified GP41.8 N-intein contains at least one mutation that replaces a lysine (K) residue with an alanine (A) residue.

[0292] In a preferred embodiment, the modified GP41.8 N-inpeptide contains mutations at lysine residues at positions 52 and 55 of SEQ ID NO: 8. In a more preferred embodiment, the modified GP41.8 N-inpeptide contains mutations K52A and K55A.

[0293] In one specific embodiment, the modified GP41.8 N-inpeptide comprises the sequence of SEQ ID NO: 34.

[0294] The above-defined mutation applies only when the modified GP41.8 N-inpeptide contains a fragment of SEQ ID NO: 8 containing a mutated position (e.g., K52 and K55).

[0295] In another specific embodiment, the first polynucleotide of the present invention further comprises:

[0296] a. The junction region between the modified N-integument and the cleaved C-integument, and / or

[0297] b. The protease cleavage site between the modified N-inte and the cleaved C-inte.

[0298] In one specific embodiment, the linker region between the modified N-integument and the split C-integument contains a sequence of 1 to 200 amino acids (preferably 1 to 100 amino acids).

[0299] The term "protease cleavage site" refers to a specific amino acid sequence within a protein or polypeptide chain that is recognized and cleaved by a protease. Proteases (also known as proteolytic enzymes or peptidases) catalyze the hydrolysis of peptide bonds at these sites, resulting in the protein breaking down into smaller fragments.

[0300] In one specific embodiment, the protease cleavage site is recognized and cleaved by a protease selected from the following: furin, heparanase, calpain, matrix metalloprotease (MMP), cathepsin, serine protease, cysteine ​​protease, aspartic protease, ADAMs and ADAMTS proteins, aminopeptidase, particle-associated serine protease, and caspase.

[0301] Some examples of matrix metalloproteinases (MMPs) include, but are not limited to, MMP-2, MMP-9, MMP-1, MMP-7, MMP-3, and MMP-14.

[0302] Some examples of cathepsins include, but are not limited to, cathepsin B, cathepsin D, and cathepsin L.

[0303] Examples of serine proteases include, but are not limited to, urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), matriptase, kallikrein, elastase, prolyl endopeptidase (PREP), and thymus-specific serine protease (TSSP).

[0304] Some examples of cysteine ​​proteases include, but are not limited to, podin, cathepsin C, cathepsin W, and cathepsin L.

[0305] Examples of aspartic proteases include, but are not limited to, pepsin-like proteases.

[0306] Some examples of ADAMS and ADAMTS proteins include, but are not limited to, ADAM10, ADAM17, and ADAMTS1.

[0307] Some examples of aminopeptidases include, but are not limited to, aminopeptidase N (CD13) and leucine aminopeptidase.

[0308] Some examples of particle-associated serine proteases include, but are not limited to, granzyme A, granzyme B, granzyme K, and granzyme M.

[0309] Some examples of caspases include, but are not limited to, caspase 8, caspase 3 and caspase 9.

[0310] In a preferred embodiment, the protease cleavage site is a furin protease cleavage site.

[0311] The first carrier of the present invention

[0312] In another aspect, the present invention relates to a carrier, hereinafter referred to as the first carrier of the present invention, which comprises the first polynucleotide of the present invention.

[0313] The term "vector" refers to a construct capable of delivering and optionally expressing one or more target genes or sequences within a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granulocytes or phage vectors, DNA or RNA expression vectors bound to cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells (e.g., production cells). Vectors can be stable and self-replicating. An "expression vector" is a vector capable of directing the expression of a gene operatively associated with it.

[0314] Expression vectors are reproducible DNA constructs containing a synthetic or cDNA-derived DNA fragment encoding a fusion protein, operatively linked to a suitable transcriptional or translational regulatory element. This transcriptional or translational regulatory element can be derived from, for example, mammalian, microbial, viral, or insect genes. A transcription unit typically comprises: (1) one or more genetic elements that play a regulatory role in gene expression, such as a transcription promoter or enhancer; (2) a structural or coding sequence transcribed into mRNA and translated into a protein; and (3) suitable transcription and translation initiation and termination sequences. Such regulatory elements may include operator sequences for controlling transcription. The ability to replicate in the host (typically conferred by the origin of replication) and selection genes for facilitating transformant recognition may be additionally incorporated. DNA regions are operatively linked when they are functionally related to each other. For example, if the DNA for the signal peptide is expressed in a precursor form that participates in peptide secretion, then the DNA is operatively linked to the DNA for the peptide; if the promoter controls the transcription of the coding sequence, then the promoter is operatively linked to the coding sequence; or if the location of the ribosome binding site allows translation, then the ribosome binding site is operatively linked to the coding sequence.

[0315] The choice of expression control sequences and expression vectors will depend on the choice of host. A variety of expression host / vector combinations can be used. Expression vectors suitable for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Expression vectors suitable for bacterial hosts include known bacterial plasmids, such as plasmids from *Escherichia coli*, including pCR1, pBR322, pMB9, and their derivatives, as well as plasmids with broader host ranges, such as M13 and filamentous single-stranded DNA bacteriophages.

[0316] The vector may contain at least one promoter. A “promoter” is a DNA segment that controls the expression of a coding sequence or functional RNA. Typically, the coding region is located at the 3' end of the promoter. A promoter may be derived entirely from a natural gene, or may consist of different elements derived from different promoters found in nature, or may even contain synthetic DNA segments. Those skilled in the art will understand that different promoters can direct gene expression in different tissues or cell types, or at different developmental stages, or in response to different environmental or physiological conditions. Promoters that enable gene expression in most cell types most of the time are generally referred to as “constitutive promoters.” It is further recognized that DNA segments of different lengths may have the same promoter activity because the exact boundaries of the regulatory sequence are not fully determined in most cases. A promoter is typically bounded at its 3' end by the transcription start site and extends upstream (in the 5' direction) to contain the minimum number of bases or elements required to initiate transcription at detectable levels above background. Inside the promoter, the transcription start site (e.g., conveniently identified by mapping with nuclease S1) and the protein-binding domain (consensus sequence) responsible for binding RNA polymerase will be found.

[0317] The choice of expression vector and promoter that ultimately allows for operative linking to the polynucleotide encoding the fusion protein depends directly on the desired functional characteristics, such as the location and timing of protein expression, and the host cell to be transformed.

[0318] The first host cell of the present invention

[0319] Therefore, in another aspect, the present invention relates to a host cell, hereinafter referred to as the first host cell of the present invention, which comprises the first polynucleotide of the present invention, the first vector of the present invention, or a fusion protein encoded by the first polynucleotide of the present invention.

[0320] As used herein, the term "host cell" refers to a cell in which the nucleic acid of the present invention (e.g., the first polynucleotide or first vector according to the present invention) has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. The term "host cell" as used refers not only to the specific object cell but also to the offspring or potential offspring of such cells. Such offspring may not be entirely identical to the parent cell due to mutations or environmental influences in subsequent generations, but are still included within the scope of the terminology used herein. The host cell can be any prokaryotic cell (e.g., *Escherichia coli*) or eukaryotic cell (e.g., yeast, plant cells, or animal cells).

[0321] In one specific embodiment, the first host cell of the present invention is a human cell. In a more specific embodiment, the first host cell of the present invention is selected from: T cells, NK cells, induced pluripotent stem cells (iPSCs), derived NK cells, pro-T cells, B cells, and macrophages.

[0322] In another specific embodiment, the first host cell of the present invention is obtained through viral transduction.

[0323] "Viral transduction" refers to the process of introducing exogenous DNA into cells through viruses or viral vectors.

[0324] In a more specific embodiment, the first host cell of the present invention is obtained by viral transduction with lentivirus or by gene knock-in.

[0325] "Gene knock-in" refers to a genetic engineering method that involves replacing DNA sequence information one-to-one at a gene locus, or inserting sequence information that is not present at that locus. In a preferred embodiment, the gene knock-in is performed using a CRISPR-Cas9 system.

[0326] The first fusion protein of the present invention

[0327] In another aspect, the present invention relates to a fusion protein encoded by a first polynucleotide of the present invention, hereinafter referred to as the first fusion protein of the present invention.

[0328] The term "fusion protein" has been defined above, and this definition applies to the first fusion protein of the present invention.

[0329] In one specific embodiment, the first fusion protein of the present invention further comprises a detection tag. In another specific embodiment, the first fusion protein of the present invention comprises one or more detection tags.

[0330] In a more specific embodiment, the first fusion protein of the present invention comprises two detection tags.

[0331] The term "tag" refers to a polypeptide that facilitates the detection, isolation, and / or purification of a protein. Typically, the tag sequence is located in a portion of the target protein that does not adversely affect its function. In one specific embodiment, the detection tag is selected from: c-myc tags, HA tags, FLAG tags, Strep tags, His tags, alfa tags, V5 tags, Spot tags, and NE tags.

[0332] The second fusion protein of the present invention

[0333] In another aspect, the present invention relates to a fusion protein, hereinafter referred to as the second fusion protein of the present invention, comprising, from the N-terminus to the C-terminus:

[0334] i) Heterologous peptides (N-exopeptides) and

[0335] ii) Split N-intein comprising a sequence selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 or a functionally equivalent variant thereof.

[0336] The terms “peptide” and “fusion protein” have been defined or explained above, and these definitions apply to the second fusion protein of the present invention.

[0337] As used herein, the term "heterologous" refers to a vector, plasmid, or host cell element originating from a source other than an endogenous one. Thus, for example, a heterologous sequence (e.g., a polynucleotide or polypeptide sequence) can be a sequence derived from a different gene or plasmid of the same host, from a different host cell line, or from an organism of a different taxonomic group (e.g., a different kingdom, phylum, class, order, family, genus, or species, or any subgroup within any of these taxa). The term "heterologous" is also used synonymously with the term "exogenous" herein.

[0338] When a heteropeptide forms part of a fusion protein comprising a split N-inteptide or a functionally equivalent variant thereof as defined in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, the heteropeptide is also referred to as an N-expeptide.

[0339] "Split N-inpeptide" refers to the N-terminal domain of an inpeptide. The "N-terminal domain of an inpeptide" is an inpeptide sequence containing an N-terminal amino acid sequence that is functional for trans-splicing and / or N-terminal self-cleavage. The N-terminal domain of an inpeptide can be spliced ​​out during trans-splicing.

[0340] The second fusion protein of the present invention comprises ii) a split N-inteptide, said split N-inteptide comprising a sequence selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 or a functionally equivalent variant thereof.

[0341] This refers to the N-terminal domain of the GP41.1 cleavage inteptide.

[0342] This refers to the N-terminal domain of the IMPDH1 cleavage peptide.

[0343] This refers to the N-terminal domain of the NrdJ1 cleavage peptide.

[0344] This refers to the N-terminal domain of the GP41.8 cleavage inteptide.

[0345] The term “functionally equivalent variant of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8” refers to all sequences obtained by modifying, inserting and / or deleting one or more amino acids of the above sequences, provided that the function of the integument is substantially preserved. Suitable assays for determining the integument activity of functionally equivalent variants are described in the patent application published in WO2013045632A1.

[0346] Preferably, variants of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 are (i) polypeptides in which one or more amino acid residues are replaced by conserved or non-conserved amino acid residues (preferably conserved amino acid residues), and such replaced amino acids may or may not be encoded by the genetic code; (ii) polypeptides containing one or more modified amino acid residues, such modified amino acid residues being, for example, residues modified by substituent bonding; (iii) polypeptides generated by variable processing of similar mRNAs; (iv) polypeptide fragments; and / or (v) polypeptides generated by fusing the polypeptides defined in (i) to (iii) with another polypeptide (e.g., a secretory leader sequence or a sequence for purification (e.g., a His tag) or a sequence for detection (e.g., an Sv5 epitope tag)). The fragments include polypeptides generated by proteolytic cleavage (including multisite proteolysis) of the original sequence. The variants may be post-translationally modified or chemically modified. Such variants will be apparent to those skilled in the art.

[0347] Those skilled in the art will recognize that the value of nucleotide sequence identity can be appropriately adjusted by taking into account codon degeneracy, conserved amino acid substitutions, and reading frame positioning to determine the corresponding sequence identity of the two nucleotide sequences encoding the polypeptide of the present invention.

[0348] This invention relates to functionally equivalent variants of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8; and whose amino acid sequences differ from a given sequence by one or more amino acids, the difference being the result of one or more conserved amino acid substitutions. It is known in the art that one or more amino acids in a polypeptide sequence can be replaced by at least one other amino acid having a similar charge and polarity, such that the substitution results in a silencing change in the modified polypeptide, which does not alter the function of the modified polypeptide relative to the function of the unmodified sequence. This invention relates to any polypeptide sequence that differs from the sequence given in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 by one or more amino acids (due to conserved or non-conserved substitutions, and / or due to sequence insertions or deletions), provided that the further provided polypeptide sequence has the same or similar intein N-terminal domain as SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

[0349] The N-terminal domain of an inteptide may contain one or more of the N1, N2, N3, and / or N4 motifs of the HINT (Hog / Intein) family. Therefore, for example, the N-terminal domain of an inteptide may contain both N1 and N3 motifs.

[0350] In some embodiments, the functionally equivalent variants of the split N-integral peptide are characterized by containing an N1 box (A box) sequence. The N1 box is a non-strictly conserved sequence. The N1 box may contain, for example, the sequence... Where X is any amino acid, h is a hydrophobic amino acid, s is a small amino acid, c is a charged amino acid, p is a polar amino acid, and l is a large amino acid. In some embodiments, the N-terminal domain of the contained peptide includes a sequence (SEQ ID NO: 16), where X1 is C; X2 is L, F, or V; X3 is S, T, V, or A; X4 is L, P, G, or Y; X5 is D, E, K, or G; X6 is T or A; X7 is E, Q, L, M, K, or T; X8 is I or V; X9 is L, Q, V, N, K, D, or T; X 10 For T, I, or V; X 11 V, P, Q, N, E, K, or L; X 12 It is E, Q, G, N, Y, I, or E; and X 13 The value can be Y, G, K, P, or D. In some embodiments, the N-terminal domain of the in-cell peptide comprises a sequence... (SEQ ID NO: 17), where X1 is C; X2 is L, F, or V; X3 is S, T, V, or A; X4 is L, P, or G; X5 is D, K, or G; X6 is T or A; X7 is Q, L, M, K, or T; X8 is I or V; X9 is Q, V, N, K, D, or T; X 10 For T, I, or V; X 11 For P, Q, N, E, K, or L; X 12 It is E, Q, G, N, Y, I, or E; and X 13 It can be G, K, P, or D.

[0351] Based on their chemical properties, amino acids can be grouped as follows: (i) charged (D, E, K, R, H), (ii) acidic (D, E), (iii) basic (K, R, H), (iv) small (V, C, S, T, P, G, D, A), (v) polar (N, Q, S, T), (vi) large (E, Q, R, K, H, Y, W, F, M, L, I), (vii) hydrophobic (V, I, L, M, F, Y, W, A) and (viii) nucleophilic (S, T, C).

[0352] In some embodiments, the N-terminal domain of the inteptide is about 50 to about 150 amino acids. In some embodiments, the N-terminal domain of the inteptide is about 60 to about 140 amino acids. In some embodiments, the N-terminal domain of the inteptide is about 75 to about 125 amino acids. In some embodiments, the N-terminal domain of the inteptide is about 70 to about 80, about 80 to about 90, about 90 to about 100, about 100 to about 110, or about 110 to about 120 amino acids.

[0353] The first amino acid in the N-terminal domain of an inpeptide is highly conserved and important for protein splicing. Therefore, in some embodiments, the first amino acid in the N-terminal domain is cysteine. In some embodiments, the first amino acid in the N-terminal domain is serine. In some embodiments, the first amino acid in the N-terminal domain is an amino acid other than serine or cysteine. For example, the first amino acid in the N-terminal domain could be alanine.

[0354] Functionally equivalent variants of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 also include sequences having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequences of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0355] In some implementations, a functionally equivalent variant of SEQ ID NO: 5 is a sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 5.

[0356] In some implementations, a functionally equivalent variant of SEQ ID NO: 6 is a sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 6.

[0357] In some implementations, a functionally equivalent variant of SEQ ID NO: 7 is a sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 7.

[0358] In some implementations, a functionally equivalent variant of SEQ ID NO: 8 is a sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 8.

[0359] In some embodiments, the N-terminal domain of the inteptide contains at least about 10, at least about 20, at least about 30, at least about 40, or at least about 50 amino acids selected from the sequences of SEQ ID NO: 5, 6, 7, and 8. In some embodiments, the N-terminal domain of the inteptide contains at least about 10, at least about 20, at least about 30, at least about 40, or at least about 50 consecutive amino acids selected from the sequences of SEQ ID NO: 5, 6, 7, and 8. In some embodiments, the N-terminal domain of the inteptide contains a deletion of no more than about 5, about 10, about 15, about 20, or about 25 amino acids selected from the sequences of SEQ ID NO: 5, 6, 7, and 8. In some embodiments, the N-terminal domain of the inteptide contains a deletion of no more than about 5, about 10, about 15, about 20, or about 25 consecutive amino acids selected from the sequences of SEQ ID NO: 5, 6, 7, and 8.

[0360] In a preferred embodiment, the functionally equivalent variants of SEQ ID NO: 5, 6, 7 or 8 have at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the corresponding sequence SEQ ID NO: 5, 6, 7 or 8, and said sequence identity is determined over the entire length of the sequence SEQ ID NO: 5, 6, 7 or 8.

[0361] In one specific embodiment, the polypeptide is directly linked to the split N-integral peptide via a peptide bond or via a peptide linker.

[0362] The terms “peptide bond” and “connector” have been defined or explained above, and these definitions apply to the second fusion protein of the present invention.

[0363] In some embodiments, the linker is a peptide containing 1 to 25 amino acid residues, 1 to 20 amino acid residues, 2 to 15 amino acid residues, 3 to 10 amino acid residues, 3 to 7 amino acid residues, 4 to 25 amino acid residues, 4 to 20 amino acid residues, 4 to 15 amino acid residues, 4 to 10 amino acid residues, 5 to 25 amino acid residues, 5 to 20 amino acid residues, 5 to 15 amino acid residues, or 5 to 10 amino acid residues.

[0364] Exemplary linkers include glycine- and serine-rich linkers, such as (GGP)n, (GGGGS)n (SEQ ID NO: 30), or (GGGS)n (SEQ ID NO: 19), where n is 1 to 5. The most commonly used flexible linkers have sequences consisting primarily of Gly and Ser residue segments (“GS” linkers). The length of the GS linker can be optimized by adjusting the copy number “n” to achieve proper separation of functional domains or to maintain necessary interdomain interactions.

[0365] In some embodiments, the first amino acid of the linker is serine, cysteine, or threonine. In some embodiments, the first, second, third, fourth, and / or fifth amino acids of the linker are serine, cysteine, or threonine.

[0366] The second fusion protein of the present invention comprises i) a polypeptide. The polypeptide specifically binds to the target molecule.

[0367] The term "target molecule" refers to a key molecule (e.g., carbohydrates, proteins, and nucleic acids) that is directed to and / or bound to by certain other entities (e.g., endogenous ligands or drugs), thereby causing an alteration in the behavior or function of the key molecule. In one specific embodiment, the target molecule is selected from: target antigens, lectins, and epitopes.

[0368] "Lectin" refers to carbohydrate-binding proteins that are highly specific to glycosyl groups that are part of other molecules, thus causing aggregation of specific cells or precipitation of glycoconjugates and polysaccharides.

[0369] An epitope is a part of an antigen that is recognized by the immune system (especially by antibodies, B cells, or T cells).

[0370] In this invention, "antigen" or "target antigen" refers to any type of molecule capable of binding to a specific antibody or T-cell receptor. Antigens can be proteins, peptides (amino acid chains), polysaccharides (monosaccharide chains), lipids, or nucleic acids. Antigens are recognized by antigen receptors (including antibodies and T-cell receptors). In most cases, antibodies are antigen-specific, meaning that an antibody can only react with and bind to one specific antigen; however, in some cases, antibodies can cross-react and bind to more than one antigen.

[0371] In a more specific embodiment, the target molecule is a target antigen. In a preferred embodiment, the target antigen is selected from: CD19, CD20, CD22, GD2, CD133, EGFR, GPC3, CEA, MUC1, mesothelin, IL-13R, PSMA, ROR1, CAIX, CD1a, CCR9, and Her2.

[0372] In some implementations, the polypeptide that binds specifically to the target molecule, preferably to the target antigen, is an "antigen-binding domain".

[0373] According to the present invention, the terms "antigen-binding domain," "antigen-binding fragment," or "antibody fragment" are used interchangeably and refer to any oligopeptide or polypeptide capable of binding to a target antigen. It may contain an antibody fragment, which refers to at least a portion of a complete antibody or a recombinant variant thereof, such as the antigenic variable region of a complete antibody, which is sufficient to allow the antibody fragment to recognize and specifically bind to the target. In some embodiments, the antigen-binding domain comprises at least a VH region and a VL region. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, ScFv antibody fragments, and linear antibodies. In the context of the present invention, the antigen-binding domain or antibody fragment comprises at least one VH region and one VL region, but it may comprise two VL regions and two VH regions. Thus, for example, in one embodiment, the antigen-binding domain is ScFv, and therefore it will comprise only one VL region and one VH region. In another embodiment, the antigen-binding domain is a Fab fragment, which in this case will contain one VL region and a VH region (Fab or Fab') or two VH regions and two VL regions (Fab2 or F(ab')2).

[0374] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains (VH and VL) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs) or complementarity-determining regions (CDRs). A single VH or VL domain is sufficient to confer antigen-binding specificity.

[0375] As used herein, the terms “hypervariant region,” “HVR,” “complementarity-determining region,” or “CDR” refer to regions within the variable domain of an antibody that are sequence-hypervariant and / or form structurally defined loops (“hypervariant loops”). Typically, a natural four-chain antibody contains six CDRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Therefore, the CDRs determine the protein’s affinity (approximately binding strength) and specificity for a particular antigen. The CDRs of each pair of chains are aligned through the frame region to acquire the function of binding to a specific epitope. Thus, both the heavy chain variable chain and the light chain variable chain are characterized by three CDRs: VH-CDR1, VH-CDR2, VH-CDR3 and VL-CDR1, VL-CDR2, VL-CDR3, respectively.

[0376] "Frame" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. The variable domain FR is usually composed of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences in VH (or VL) usually appear in the following order: FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

[0377] In one specific implementation, i) the polypeptide that binds to the target molecule, preferably specifically to the target antigen, is selected from: single-chain variable fragments (scFv), single-domain antibodies, Ig heavy or light chains, peptides, and ligands.

[0378] As used herein, “single-chain variable fragment (ScFv)” refers to a single-chain polypeptide derived from an antibody and retaining its ability to bind antigens. Examples of ScFv include antibody polypeptides formed using recombinant DNA technology, wherein the variable (Fv) regions of immunoglobulin heavy chain (VH chain) and light chain (VL chain) segments are linked by spacer sequences. Various methods for preparing ScFv are known, including those described in U.S. Patent No. 4,694,778; Nature, Vol. 334, p. 54454 (1989); and Science, Vol. 242, pp. 1038-1041 (1988).

[0379] "Single-domain antibody" is a peptide chain about 110 amino acids long that contains a variable domain (VH) of heavy chain antibody or ordinary IgG.

[0380] The term "ligand" refers to any molecule or atom that can reversibly bind to a protein. In particular, it refers to a molecule that can interact with and bind to a target molecule, preferably a target antigen.

[0381] In another specific embodiment, the second fusion protein of the present invention further comprises a detection tag and / or a solubilization tag. In some embodiments, the second fusion protein of the present invention comprises a detection tag. In other embodiments, the second fusion protein of the present invention comprises a solubilization tag. In still other embodiments, the second fusion protein of the present invention comprises both a detection tag and a solubilization tag.

[0382] The term "detection tag" has been defined or explained above, and this definition applies to the second fusion protein of the present invention. In one specific embodiment, the detection tag is selected from: c-myc tag, HA tag, FLAG tag, Strep tag, His tag, alfa tag, V5 tag, Spot tag, and NE tag.

[0383] The term "solubilizing tag" refers to a class of peptide sequences that readily fold into soluble proteins; when fused with recombinant proteins, they typically enhance solubility. Any soubilizing tag can be used in this invention. In one specific embodiment, the solubilizing tag is selected from: Fasciola hepatica 8-kDa antigen (Fh8), maltose-binding protein (MBP), N-utilization substance (NusA), thioredoxin (Trx), microubiquitin modifier (SUMO), glutathione-S-transferase (GST), solubility-enhancer peptide sequences (SET), IgG domain B1 (GB1) of protein G, IgG repeat domain ZZ (ZZ) of protein A, mutant dehalogenase (HaloTag), solubility eNhancing Ubiquitous Tag (SNUT), seventeen kilodalton protein (Skp), and phage T7 protein kinase. The proteins involved include kinase (T7PK), E. coli secreted protein A (EspA), monomeric phage T7 0.3 protein (Orc protein) (Mocr), E. coli trypsin inhibitor (Ecotin), calcium-binding protein (CaBP), stress-responsive arsenate reductase (ArsC), the N-terminal fragment of translation initiation factor IF2 (IF2-domain I), the N-terminal fragment of translation initiation factor IF2 (expressivity tag (part of IF2-domain I)), stress-responsive proteins (RpoA, SlyD, Tsf, RpoS, PotD, Crr), and E. coli acidic proteins (msyB, yjgD, rpoD).

[0384] In a more specific embodiment, the solubilizing tag is selected from: phage λ head protein D (gpD), small ubiquitin-like modification (SUMO), maltose-binding protein (MBP), glutathione S-transferase (GST), N-utilization substance (NusA), thioredoxin A, and liver fluke 8-kDa antigen (Fh8 tag).

[0385] The second polynucleotide of the present invention

[0386] In another aspect, the present invention relates to a polynucleotide encoding a second fusion protein of the present invention, hereinafter referred to as the second polynucleotide of the present invention.

[0387] The term "polynucleotide" has been defined above, and that definition applies to the second polynucleotide of this invention.

[0388] In one specific embodiment, the second polynucleotide of the present invention further comprises a nucleotide sequence encoding a signal peptide, wherein the signal peptide is within a frame and located at the N-terminus of a polypeptide that specifically binds to a target molecule, and the signal peptide allows the fusion protein to be secreted from the cell.

[0389] As used herein, the term "signal peptide" refers to a relatively short peptide, typically 5 to 40 amino acid residues, that guides synthesized proteins into the secretory pathway. Signal peptides usually contain a series of hydrophobic amino acids that form a secondary α-helix. In addition, many peptides contain a series of positively charged amino acids that can help the protein adopt a topological structure suitable for its translocation. Signal peptides typically have a motif at their carboxyl terminus for recognition by peptidases, which hydrolyze the signal peptide to produce the free signal peptide and the mature protein.

[0390] Any signal peptide that allows the fusion protein to be secreted from the cell can be used in this invention.

[0391] The second carrier of the present invention

[0392] In another aspect, the present invention relates to a carrier, hereinafter referred to as a second carrier of the present invention, which comprises a second polynucleotide of the present invention.

[0393] The term "carrier" has been defined or explained above, and that definition applies to the second carrier of the present invention.

[0394] The choice of expression control sequences and expression vectors will depend on the choice of host. A variety of expression host / vector combinations can be used. Expression vectors suitable for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Expression vectors suitable for bacterial hosts include known bacterial plasmids, such as plasmids from *E. coli*, including pCR1, pBR322, pMB9 and their derivatives, and plasmids with a broader host range, such as M13 and filamentous single-stranded DNA bacteriophages.

[0395] The second host cell of the present invention

[0396] In another aspect, the present invention relates to a host cell, hereinafter referred to as the second host cell of the present invention, which contains the second polynucleotide of the present invention or the second vector of the present invention.

[0397] The term "host cell" has been defined or explained above, and this definition applies to the second host cell of the present invention.

[0398] Suitable host cells include, for example, plants (e.g., tomato, tobacco, Arabidopsis, alfalfa), mammalian cells (e.g., CHO, COS, and 293T cells), filamentous fungi (e.g., *Tricoderma resei* and *Aspergillus sp.*), and insect cells. Examples of suitable mammalian host cell lines include the monkey kidney cell line COS-7 described by Gluzman (Cell 23:175, 1981), as well as other cell lines capable of expressing suitable vectors, including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa, and BHK cell lines. A review of baculovirus systems for producing heterologous proteins in insect cells can be found in Luckow and Summers, Bio / Technology 6:47 (1988).

[0399] The chimeric receptor of the present invention

[0400] In another aspect, the present invention relates to a chimeric receptor, hereinafter referred to as the chimeric receptor of the present invention, comprising, from the N-terminus to the C-terminus:

[0401] i) An extracellular domain comprising a polypeptide that specifically binds to a target molecule, or a polypeptide capable of forming an oligomer in the presence of one or more additional subunits, wherein said oligomer binds specifically to the target molecule.

[0402] ii) The linker region, whose sequence is the result of protein splicing.

[0403] iii) Transmembrane domains, and

[0404] iv) Intracellular signal transduction domains from the receptor,

[0405] The protein splicing reaction occurs between two complementary parts of the splitting inteptide, and the splitting inteptide is selected from: Gp41.1 splitting inteptide, IMPDH splitting inteptide, NrdJ1 splitting inteptide and Gp41.8 splitting inteptide.

[0406] The term "chimeric receptor" refers to a receptor protein that has been modified to give cells a new ability to target specific target molecules.

[0407] The chimeric receptor of the present invention comprises i) an extracellular domain containing a polypeptide that specifically binds to a target molecule, or a polypeptide capable of forming an oligomer in the presence of one or more additional subunits, wherein the oligomer specifically binds to the target molecule.

[0408] The term "target molecule" has been defined or explained above, and this definition applies to the chimeric receptor of the present invention. In one specific embodiment, the target molecule is selected from: target antigens, lectins, and epitopes. In a more specific embodiment, the target molecule is a target antigen.

[0409] The terms “antigen” or “target antigen” have been defined or explained above, and this definition applies to the chimeric receptor of the present invention.

[0410] In one specific implementation, the target antigen is selected from: CD19, CD20, CD22, GD2, CD133, EGFR, GPC3, CEA, MUC1, mesothelin, IL-13R, PSMA, ROR1, CAIX, CD1a, CCR9, and Her2.

[0411] In some embodiments, when the chimeric receptor comprises an extracellular domain containing a polypeptide that specifically binds to a target molecule, the polypeptide is selected from: single-chain variable fragments (scFv), single-domain antibodies, lectins, epitopes, Ig heavy or light chains, peptides, and ligands.

[0412] In other embodiments, when the extracellular domain of the chimeric receptor of the present invention is a polypeptide capable of forming oligomers in the presence of one or more additional subunits, the polypeptide is a heavy or light chain of an immunoglobulin.

[0413] The terms “peptide,” “single-chain variable fragment (scFv),” “single-domain antibody,” “lectin,” “epitope,” “heavy chain and light chain,” and “ligand” have been defined or explained above, and these definitions apply to the chimeric receptor of the present invention.

[0414] The term "oligomer" refers to a molecule composed of several repeating units, which may be derived, either physically or conceptually, from smaller molecules, i.e., monomers. If these units are identical, a homooligomer is obtained; otherwise, a heterooligomer can be used. In one specific embodiment, the polypeptide can form a heterooligomer in the presence of one or more additional subunits.

[0415] In one specific embodiment, when the extracellular domain of the chimeric receptor of the present invention is a polypeptide capable of forming an oligomer in the presence of one or more additional subunits, the oligomer is an immunoglobulin.

[0416] The term "immunoglobulin" refers to a heterodimeric protein composed of two heavy chains and two light chains. Functionally, it can be divided into a variable (V) domain that binds antigens and a constant (C) domain that specifies effector functions (such as complement activation or binding to Fc receptors). The variable domains are generated through a series of complex gene rearrangement events and undergo high-frequency somatic mutations to achieve affinity maturation after exposure to antigens. Each V domain can be divided into three sequence-variable regions, called complementation-determining regions or CDRs, and four relatively constant regions, called frame regions or FRs. The three CDRs of the H chain pair with the three CDRs of the L chain to form antigen-binding sites, as described in the classical definition. The heavy chain C domain has five main classes. Each class defines the isotypes of IgM, IgG, IgA, IgD, and IgE. IgG can be divided into four subclasses: IgG1, IgG2, IgG3, and IgG4, each with its own biological characteristics; and similarly, IgA can be divided into IgA1 and IgA2. The constant domains of the H chain can be switched to alter effector function while maintaining antigen specificity.

[0417] In a more specific embodiment, the immunoglobulin is IgA, IgG, IgM, IgE, or IgD. In a more specific embodiment, the immunoglobulin is IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgM, IgE, or IgD.

[0418] In some embodiments, when the extracellular domain of the chimeric receptor of the present invention is a polypeptide capable of forming an oligomer in the presence of one or more additional subunits, the oligomer is a bispecific or trispecific antibody that binds to more than one antigen.

[0419] Bispecific antibodies (BsAbs) are antibodies with two binding sites targeting two different antigens or two different epitopes on the same antigen. BsAbs come in various forms, ranging from relatively small proteins consisting of only two linked antigen-binding fragments to large immunoglobulin G (IgG)-like molecules linked with additional domains. In one specific embodiment, the bispecific antibody is an IgG-like or non-IgG-like bispecific antibody. IgG-like bispecific antibodies retain the structure of a traditional monoclonal antibody (mAb) with two Fab arms and an Fc region, but the two Fab sites bind different antigens. The most common type is called a trifunctional antibody because it has three distinct binding sites on the antibody: the two Fab regions and the Fc region. Each heavy chain and light chain pair originates from a unique mAb. The Fc region, composed of the two heavy chains, forms the third binding site. Non-IgG-like bispecific antibodies completely lack the Fc region. These include chemically linked Fabs (consisting only of Fab regions), as well as various types of bivalent and trivalent single-chain variable fragments (ScFvs). There are also fusion proteins that mimic the variable domains of two antibodies. The most developed of these newer forms is the bi-specific T-cell engager (BiTE).

[0420] Trispecific antibodies (TsAbs) are antibodies with three targets, meaning they have three binding sites targeting three different antigens or three different epitopes on the same antigen. Examples of trispecific antibodies include, but are not limited to, tri-specific killer cell engagers (TriKEs). TriKEs are combinations of a single-chain Fv targeting CD16 with two tumor-associated antigens. These molecules directly trigger NK cell activation via CD16, enhancing NK cell cytolytic activity and cytokine production against multiple tumor cell antigen targets.

[0421] As used in this article, the "chimeric antigen receptor (CAR)," also known as the chimeric T-cell receptor, T-body, artificial T-cell receptor, and chimeric immune receptor (CIR), is a modified receptor that can be selectively and specifically transplanted into immune effector cells. In a classic CAR, the specificity of a monoclonal antibody is transplanted into T cells. Therefore, a CAR is a fusion protein that contains at least an extracellular domain or antigen-binding domain capable of binding to the target antigen, a transmembrane domain, and at least one intracellular signaling domain derived from the antigen receptor.

[0422] The term "antigen-binding domain" has been defined above, and this definition applies to the chimeric receptor of the present invention.

[0423] In one specific embodiment, the chimeric receptor further includes a hinge domain or hinge region between the binding molecule and the transmembrane domain.

[0424] In a more specific embodiment, the CAR of the present invention further includes a hinge domain between the antigen-binding domain and the transmembrane domain.

[0425] As used herein, "hinge domain," "hinge region," or "spacer region" refers to an amino acid region that allows the binding portion to separate from and become flexible on the T cell membrane. The length of the flexible hinge also allows for better binding to relatively inaccessible epitopes; for example, longer hinge domains may achieve optimal binding. Those skilled in the art will be able to determine the appropriate hinge for a given CAR target.

[0426] In one specific embodiment, the hinge region is selected from: IgG1 hinge region, IgG2 hinge region, IgG4 hinge region, FcγR hinge region, CD3ε hinge region, CD3ζ hinge region, CD8α hinge region, CD4 hinge region, CD28 hinge region, CD7 hinge region, and CD19 hinge region. The length of the hinge domain can be from about 10 amino acids to about 200 amino acids, preferably 30 to 150 amino acids, and more preferably 40 to 125 amino acids.

[0427] Exemplary spacer regions include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 18), and (GGGS)n (SEQ ID NO: 19), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured and can therefore be used as neutral linkers between components. Glycine polymers can be used; the phi-psi space accessible to glycine is even significantly larger than that of alanine and is much less restricted than residues with longer side chains. Exemplary spacer regions may contain amino acid sequences, including but not limited to GGSG (SEQ ID NO: 20), GGSGG (SEQ ID NO: 21), GGSG (SEQ ID NO: 22), GGSGG (SEQ ID NO: 23), GGGSG (SEQ ID NO: 24), GSSSG (SEQ ID NO: 25), etc.

[0428] The chimeric receptor of the present invention includes ii) a linker region whose sequence is the result of a protein splicing reaction.

[0429] The "protein splicing reaction" or "integrity reaction" refers to the self-removal process of integrins. Integrins are internal protein components that detach themselves from the host protein and catalyze the linking of flanking sequences (exopeptides) via peptide bonds. Integrin detachment is a post-translational process and does not require coenzymes or cofactors.

[0430] In some embodiments, an intein reaction (e.g., cleavage, splicing, cyclization) can be initiated by contacting a fusion protein containing the N-terminal domain of an intein with a fusion protein containing the C-terminal domain of an intein. In other embodiments, an intein reaction can be initiated by changing the conditions (e.g., temperature or pH) of incubating a cleavage intein or a combination of cleavage inteins. In some embodiments, C-terminal cleavage is initiated by a change in pH or temperature.

[0431] In some embodiments, the intepitide reaction is initiated by contacting the fusion protein with DTT or other strong nucleophiles. In some embodiments, DTT is used to enhance the reaction. In some embodiments, N-terminal cleavage is initiated by a strong nucleophile (e.g., DTT).

[0432] Another way to induce protein splicing or cleavage is through contact with peptides or peptide mimics that activate splicing or cleavage. Yet another way to induce protein splicing or cleavage is through the removal of peptides or peptide mimics that block or inhibit splicing or cleavage.

[0433] In one specific embodiment, the linker region generated by the protein splicing reaction contains 1 to 8 amino acids. In a more specific embodiment, the linker region generated by the protein splicing reaction contains 1 to 6 amino acids. In an even more specific embodiment, the linker region generated by the protein splicing reaction contains 6 amino acids.

[0434] In one specific embodiment, the linker region resulting from the protein splicing reaction of Gp41.1, IMPDH, NrdJ1, or Gp41.8 splitting the peptide contains a Ser(S), Thr(T), or Cys(C) residue. In a more specific embodiment, the Ser(S), Thr(T), or Cys(C) residue is located at the first position of the linker region.

[0435] In another specific embodiment, the adapter region generated by the protein splicing reaction of the Gp41.1 cleavage of the peptide contains the sequence SGYSIC (SEQ ID NO: 26) or SGYSIS (SEQ ID NO: 35).

[0436] In another specific embodiment, the adapter region generated by the protein splicing reaction of the IMPDH-internal peptide contains the sequence GGGSIC (SEQ ID NO: 27).

[0437] In another specific embodiment, the adapter region generated by the protein splicing reaction of NrdJ1 splitting the peptide contains the sequence NPCSEI (SEQ ID NO: 28).

[0438] In another specific embodiment, the adapter region generated by the protein splicing reaction of the Gp41.8 cleavage of the peptide contains the sequence LNRSAV (SEQ ID NO: 29).

[0439] The chimeric receptor of the present invention includes iii) a transmembrane domain.

[0440] The term "transmembrane domain" has been defined above, and this definition applies to the chimeric receptor of the present invention. In one specific embodiment, the transmembrane domain is selected from the transmembrane domains of CD28, CD8α, 4-1BB, CD4, CTLA4, CD27, or CD3ζ.

[0441] The chimeric receptor of the present invention includes (iv) an intracellular signal transduction domain derived from the receptor.

[0442] The term "intracellular signal transduction domain" has been defined or explained above, and this definition applies to the chimeric receptor of the present invention. In one specific embodiment, the intracellular signal transduction domain is an antigen receptor signal transduction domain.

[0443] In a more specific embodiment, the antigen receptor signal transduction domain is selected from CD3ζ, CD28, 4-1BB, NKG2D, DAP10, OX40, CD70, CD27, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP12 antigen receptor signal transduction domain, or a combination thereof.

[0444] The third host cell of the present invention

[0445] In another aspect, the present invention relates to a host cell comprising the chimeric receptor of the present invention, hereinafter referred to as the third host cell of the present invention.

[0446] The term "host cell" has been defined or explained above, and this definition applies to the third host cell of this invention.

[0447] In one specific implementation, the third host cell of the present invention is a human cell.

[0448] In a more specific embodiment, the third host cell of the present invention is selected from: T cells, NK cells, NK cells derived from induced pluripotent stem cells (iPSCs), proto-T cells, B cells, and macrophages.

[0449] The first composition or kit of the present invention

[0450] In another aspect, the present invention relates to a composition or kit comprising a first component and a second component, wherein:

[0451] i) The first component is the first polynucleotide of the present invention, the first vector of the present invention, the first host cell of the present invention, or the first fusion protein of the present invention, and

[0452] ii) The second component is the second fusion protein of the present invention or the second polynucleotide of the present invention, wherein

[0453] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0454] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0455] - The cleaved C-inteptide comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the cleaved N- comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0456] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0457] Variations of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 have been defined above, and these definitions apply to the first composition or kit of the present invention.

[0458] The proportions of the components in the composition are suitable for the efficient processing of the fusion protein. Suitable proportions of the first component to the second component include, but are not limited to, the following (first component: second component) ratios: 1:1, 1:2, 2:1, 1:3, 3:1, 1:4, 4:1, 1:5, 5:1, 1:10, 10:1, 1:15, 15:1, 1:20, 20:1, 1:30, 30:1, 1:40, 40:1, 1:50, 50:1, 1:75, 75:1, 1:100, 100:1, 1:1000, 1000:1, 1:10000, 10000:1.

[0459] The first and second methods of the present invention for generating chimeric antigen receptors

[0460] In another aspect, the present invention relates to an in vitro method for generating cells comprising chimeric receptors, hereinafter referred to as the first method of the invention, wherein the method comprises:

[0461] i) Providing cells comprising: a first polynucleotide of the present invention, a polypeptide encoded by said polynucleotide, or a first vector of the present invention, wherein the provision is carried out under conditions suitable for expression of a fusion protein encoded by said polynucleotide in said cells, and

[0462] ii) Contact the cells from step i) with the second fusion protein of the present invention.

[0463] The contact step ii) is performed under conditions that allow a splicing reaction to occur between a cleaved C-intein that forms a portion of the fusion protein produced in step (i) and a cleaved N-intein that forms a portion of the second fusion protein of the present invention, and wherein...

[0464] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0465] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0466] - The cleaved C-inteptide comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the cleaved N- comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0467] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0468] The term "in vitro method" refers to the generation of cells containing chimeric receptors outside the subject.

[0469] In one specific embodiment, the chimeric receptor is a chimeric antigen receptor. The term "chimeric antigen receptor" has been defined or explained above, and this definition applies to the first method of the present invention.

[0470] The first step of the first method of the present invention includes i) providing cells comprising: a first polynucleotide of the present invention, a polypeptide encoded by said polynucleotide, or a first carrier of the present invention, said provision being performed under conditions suitable for expression of a fusion protein encoded by said polynucleotide in said cells.

[0471] Conditions suitable for the expression of the fusion protein encoded by the first polynucleotide of the present invention in the cells are well known to those skilled in the art. Conditions suitable for the expression of the polynucleotide in host cells include those that allow optimal growth of the host cells and those that allow protein expression in the host cells. The culture conditions are generally different for each type of host cell. However, these conditions are known to those skilled in the art and are readily determined. Similarly, the duration of maintenance may vary depending on the host cells and the amount of fusion protein desired to be prepared. Again, these conditions are well known and can be readily determined in specific circumstances.

[0472] The second step of the first method of the present invention includes ii) contacting the cells of step i) with the second fusion protein of the present invention, wherein this contacting step ii) is carried out under conditions that allow a splicing reaction to occur between a cleaving C-integument that forms a portion of the fusion protein produced in step (i) and a cleaving N-integument that forms a portion of the second fusion protein of the present invention, wherein

[0473] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0474] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0475] - The cleaved C-inteptide comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the cleaved N- comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0476] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0477] Suitable conditions for allowing a splicing reaction to occur between the cleaved C-intein, a portion of the fusion protein produced in step (i), and the cleaved N-intein, a portion of the second fusion protein of the present invention, are well known to those skilled in the art. The terms “protein splicing reaction” or “intein reaction” have been defined or explained above, and this definition applies to the first method of the present invention.

[0478] As described above, in some embodiments, an intein reaction (e.g., cleavage, splicing, cyclization) can be initiated by contacting a fusion protein containing the N-terminal domain of an intein with a fusion protein containing the C-terminal domain of an intein. In other embodiments, an intein reaction can be initiated by changing the conditions (e.g., temperature or pH) of incubating a cleavage intein or a combination of cleavage inteins. In some embodiments, C-terminal cleavage is initiated by a change in pH or temperature.

[0479] In some embodiments, the intepitide reaction is initiated by contacting the fusion protein with DTT or other strong nucleophiles. In some embodiments, DTT is used to enhance the reaction. In some embodiments, N-terminal cleavage is initiated by a strong nucleophile (e.g., DTT).

[0480] Another way to induce protein splicing or cleavage is through contact with peptides or peptide mimics that activate splicing or cleavage. Yet another way to induce protein splicing or cleavage is through the removal of peptides or peptide mimics that block or inhibit splicing or cleavage.

[0481] Variations of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 have been defined above, and these definitions apply to the first method of the present invention.

[0482] In one specific implementation, the cell is a human cell.

[0483] In a more specific implementation, the cells are selected from: T cells, NK cells, NK cells derived from induced pluripotent stem cells (iPSCs), proto-T cells, B cells, and macrophages.

[0484] In another specific implementation, the cells used in step (i) are obtained from the patient.

[0485] In another aspect, the present invention relates to an in vitro method for generating cells comprising chimeric receptors, hereinafter referred to as a second method of the invention, wherein the method comprises:

[0486] i) Provide cells containing the following:

[0487] a. The first polynucleotide of the present invention, the first vector of the present invention, or a polypeptide encoded by said polynucleotide, wherein said polynucleotide further comprises a nucleotide sequence encoding a signal peptide, wherein said signal peptide is within a frame and located at the N-terminus of said cleaved C-inteptide, and said signal peptide allows said fusion protein to insert into a membrane, and

[0488] b. The second polynucleotide of the present invention, the second vector of the present invention, or the second fusion protein of the present invention, wherein the fusion protein encoded by said polynucleotide or said fusion protein further comprises a nucleotide sequence encoding a signal peptide, wherein said signal peptide is within a frame and located at the N-terminus of a polypeptide that specifically binds to a target molecule, and said signal peptide allows said fusion protein to be secreted from said cell, and

[0489] ii) The cells are maintained under conditions suitable for the expression of the two fusion proteins in the cells, suitable for the insertion of the fusion protein encoded by the first polynucleotide of the present invention into the membrane, suitable for the secretion of the fusion protein encoded by the second polynucleotide of the present invention, and suitable for splicing reactions between the cleavage of C-inte and the cleavage of N-inte, wherein...

[0490] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0491] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0492] - The cleaved C-inteptide comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the cleaved N- comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0493] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0494] The term "in vitro" has been defined or explained above, and that definition applies to the second method of the present invention.

[0495] The first step of the second method of the present invention includes i) providing a cell comprising: a first polynucleotide of the present invention, a first carrier of the present invention, or a polypeptide encoded by the polynucleotide, wherein the polynucleotide further comprises a nucleotide sequence encoding a signal peptide, wherein the signal peptide is within a frame and located at the N-terminus of the cleaving C-inteptide, and the signal peptide allows the fusion protein to insert into a membrane; and b) a second polynucleotide of the present invention, a second carrier of the present invention, or a second fusion protein of the present invention, wherein the fusion protein encoded by the polynucleotide or the fusion protein further comprises a nucleotide sequence encoding a signal peptide, wherein the signal peptide is within a frame and located at the N-terminus of a polypeptide that specifically binds to a target molecule, and the signal peptide allows the fusion protein to be secreted from the cell.

[0496] The “signal peptide” used in this invention has been defined or explained above, and these definitions apply to the second method of this invention. Any signal peptide that allows the insertion of a fusion protein encoded by the first polynucleotide of this invention into the membrane can be used in this invention. Some examples of signal peptides are CD8α, IL-2, GM-CSF receptor (GM-CSF) α chain, mouse Ig-κ (IgK), mouse IgK V-III region (IgKVIII), human IgKVIII, CD33, human tissue plasminogen activator (TPA), and naturally occurring secreted alkaline phosphatase (SEAP).

[0497] The signal peptide at the N-terminus of the polypeptide, which is within the frame and specifically binds to the target molecule, must allow the fusion protein to be secreted from the cell. Any signal peptide that allows the fusion protein to be secreted from the cell may be used in this invention.

[0498] The second step of the second method of the present invention includes ii) maintaining the cell under conditions suitable for the expression of the two fusion proteins in the cell, suitable for the insertion of the fusion protein encoded by the first polynucleotide of the present invention into the membrane, suitable for the secretion of the fusion protein encoded by the second polynucleotide of the present invention, and suitable for splicing reactions between the cleavage of C-inte and the cleavage of N-inte, wherein...

[0499] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0500] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0501] - The cleaved C-inteptide comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the cleaved N- comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0502] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0503] Conditions suitable for the expression of the two fusion proteins encoded by the said polynucleotides in cells are well known to those skilled in the art. Conditions suitable for the expression of the polynucleotides in host cells include those that allow optimal growth of the host cells and those that allow protein expression in the host cells. The culture conditions are generally different for each type of host cell. However, these conditions are known to those skilled in the art and are readily determined. Similarly, the duration of maintenance may vary depending on the host cells and the amount of fusion protein desired to be prepared. Again, these conditions are well known and can be readily determined in specific cases.

[0504] Conditions suitable for the insertion of the fusion protein encoded by the first polynucleotide of the present invention into the membrane and for the secretion of the fusion protein encoded by the second polynucleotide of the present invention are well known to those skilled in the art.

[0505] In one specific implementation, when the chimeric receptor is an oligochimeric receptor, the cell provided in (i) further comprises (c) an additional polypeptide encoding another element of the oligomer.

[0506] In a more specific embodiment, the oligomer is an immunoglobulin. In a more specific embodiment, the immunoglobulin is IgA, IgG, IgM, IgE, or IgD. In a more specific embodiment, the immunoglobulin is IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgM, IgE, or IgD.

[0507] In another specific embodiment, the fusion protein is a heavy or light chain of an immunoglobulin, and therefore the additional polypeptide (c) encodes the corresponding heavy or light chain of the immunoglobulin.

[0508] The terms “oligomer,” “immunoglobulin,” “heavy chain,” and “light chain” have been defined or explained above, and these definitions apply to the second method of the present invention.

[0509] In some embodiments, the protein splicing reaction between the protein encoded by the first polynucleotide of the present invention and the fusion protein encoded by the second polynucleotide of the present invention is intracellular and occurs in the secretory pathway of the cell that produces both polypeptides prior to the secretion of the second polypeptide. In other embodiments, the protein splicing reaction occurs after the second polypeptide is secreted and takes place in the extracellular space.

[0510] The conditions that allow protein splicing or peptide-integrating reactions have been defined or explained in the first method of the present invention, and these conditions are applicable to the second method of the present invention.

[0511] Variations of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 have been defined above, and these definitions apply to the second method of the present invention.

[0512] In one specific implementation, the cell is a human cell.

[0513] In a more specific implementation, the cells are selected from: T cells, NK cells, NK cells derived from induced pluripotent stem cells (iPSCs), proto-T cells, B cells, and macrophages.

[0514] In another specific implementation, the cells used in step (i) are obtained from the patient.

[0515] The first modified cell of the present invention

[0516] In another aspect, the present invention relates to cells, hereinafter referred to as the first modified cell of the present invention, which comprises a chimeric receptor obtained by the first or second method of the present invention.

[0517] Medical use

[0518] In another aspect, the present invention relates to a third host cell or a first modified cell of the present invention for treating diseases requiring a cell-mediated immune response against a target molecule.

[0519] In one specific implementation, the target molecule is a target antigen.

[0520] In one specific aspect, the present invention relates to a third host cell for treating diseases requiring a cell-mediated immune response against a target molecule, preferably against a target antigen.

[0521] In another specific aspect, the present invention relates to a first cell for treating diseases requiring a cell-mediated immune response targeting a target molecule, preferably a target antigen.

[0522] The term "treatment" is used to refer to the application of the third host cell of the present invention or the cells of the present invention before or after the appearance of clinical signs in order to control the progression of disease.

[0523] In another aspect, the present invention relates to a second fusion protein, a second polynucleotide, a second vector, or a second host cell of the present invention for treating diseases requiring a cell-mediated immune response against a target molecule, wherein the patient to be treated is characterized by containing cells comprising: a first polynucleotide, a first vector, or a fusion protein encoded by the first polynucleotide of the present invention, wherein...

[0524] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0525] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0526] - The cleaved C-inteptide comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the cleaved N- comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0527] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0528] In another aspect, the present invention relates to a first polynucleotide, a first vector, a first host cell, or a first fusion protein of the present invention for treating diseases requiring a cell-mediated immune response against a target molecule, wherein the patient to be treated is characterized by containing cells comprising: a second fusion protein, a second polynucleotide, a second vector, or a second host cell of the present invention, wherein...

[0529] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0530] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0531] - The cleaved C-inteptide comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the cleaved N- comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0532] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0533] In one specific implementation, the target molecule is a target antigen.

[0534] The term "treatment" is also used to refer to the administration of the second fusion protein of the present invention, the second polynucleotide of the present invention, the second carrier of the present invention, or the second host cell of the present invention to a patient before or after the appearance of clinical signs, wherein the patient is characterized by having cells comprising: the first polynucleotide of the present invention, the first carrier of the present invention, or the fusion protein encoded by the first polynucleotide of the present invention.

[0535] The term "treatment" is also used to refer to the administration of the first polynucleotide of the present invention, the first carrier of the present invention, the first host cell of the present invention, or the first fusion protein of the present invention to a patient before or after the appearance of clinical signs, in order to control the progression of disease, wherein the patient is characterized by having cells comprising: the second fusion protein of the present invention, the second polynucleotide of the present invention, the second carrier of the present invention, or the second host cell of the present invention.

[0536] As used herein, the terms "subject" or "patient" include any animal that suffers from a disease requiring a cell-mediated immune response against a target molecule, preferably against a target antigen, or is at risk of suffering from a disease requiring a cell-mediated immune response against a target molecule, or exhibits symptoms of a disease requiring a cell-mediated immune response against a target molecule. Suitable subjects (patients) include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals, and domestic animals or pets (e.g., cats or dogs). Non-human primates are included, with human patients being preferred. Preferably, the subject is a mammal, more preferably a human.

[0537] Controlling disease progression is understood as a beneficial or desired clinical outcome, including but not limited to: symptom relief, shortened disease duration, pathological stability (partially avoiding further damage), delayed disease progression, improved pathological condition, and remission (partial and complete remission). Controlling disease progression also involves prolonged survival compared to expected survival without treatment. In this invention, the disease is one that requires a cell-mediated immune response against a target molecule. Cell-mediated immunity, or cellular immunity, is an immune response that does not involve antibodies. Instead, cell-mediated immunity involves the activation of phagocytes, antigen-specific cytotoxic T lymphocytes, and the release of various cytokines in response to antigens.

[0538] In one specific implementation, the diseases requiring a cell-mediated immune response targeting a target molecule, preferably a target antigen, are selected from: cancer, viral infections, bacterial infections, parasitic infections, and autoimmune diseases.

[0539] The term "cancer" refers to a disease characterized by uncontrolled cell division (or increased survival or resistance to apoptosis); the ability of said cells to invade other adjacent tissues (invasion); or to spread through lymphatic vessels and blood vessels to other areas of the body where the cells would not normally be present (metastasis). Tumors are classified as benign or malignant based on whether they can spread through invasion and metastasis: benign tumors are tumors that cannot spread through invasion or metastasis, i.e., they grow only locally; while malignant tumors are tumors that can spread through invasion and metastasis.

[0540] In one implementation, cancer includes, but is not limited to, leukemia (e.g., acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), hairy cell leukemia, polycythemia vera, lymphoma (e.g., Hodgkin's disease or non-Hodgkin's disease), AIDS-related leukemia, Waldenstrom's macroglobulinemia. Macroglobulinemia, multiple myeloma, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, mendotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, Kaposi's sarcoma). Sarcoma, colon cancer, pancreatic cancer, breast cancer, biliary tract cancer, esophageal cancer, ovarian cancer, prostate cancer, oral cancer (including squamous cell carcinoma), basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary gland carcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, teratoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, intraepithelial neoplasia (including Bowen's disease and Paget's disease) Diseases including glioma, glioma, astrocytoma, glioblastoma multiforme (GBM, also known as glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0541] In some implementations, the cancer is a glioma, astrocytoma, glioblastoma multiforme (GBM, also known as glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, Schwannoma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, or retinoblastoma.

[0542] In some embodiments, the cancer is acoustic neuroma, astrocytoma (e.g., Grade I – pilocytic astrocytoma, Grade II – low-grade astrocytoma, Grade III – anaplastic astrocytoma, or Grade IV – glioblastoma (GBM)), chordoma, CNS lymphoma, craniopharyngioma, brainstem glioma, ependymoma, mixed glioma, optic nerve glioma, subependymal tumor, medulloblastoma, meningioma, metastatic brain tumor, oligodendroglioma, pituitary tumor, primitive neuroectodermal (PNET) tumor, or Schwannoma. In some embodiments, the cancer is a type more common in children than in adults, such as brainstem glioma, craniopharyngioma, ependymoma, juvenile pilocytic astrocytoma (JPA), medulloblastoma, optic nerve glioma, pineal tumor, primitive neuroectodermal tumor (PNET), or rhabdomyosarcoma.

[0543] In another implementation, the cancers include, but are not limited to, mesothelioma, hepatobiliary cancer (liver cancer and bile duct cancer), bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma of the skin or eye, ovarian cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, gastrointestinal cancer (stomach cancer, colorectal cancer, and duodenal cancer), uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine system cancers, thyroid cancer, parathyroid cancer, and kidney cancer. Adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, testicular cancer, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, non-Hodgkin's lymphoma, spinal axis tumor, brainstem glioma, pituitary adenoma, adrenocortical carcinoma, gallbladder cancer, multiple myeloma, bile duct carcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the above cancers.

[0544] In some embodiments, the cancer is selected from hepatocellular carcinoma, ovarian cancer, epithelial ovarian cancer, or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer; hepatocholangiocarcinoma; soft tissue and synovial sarcoma; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing sarcoma; anaplastic thyroid cancer; adrenocortical adenoma; pancreatic cancer; pancreatic ductal carcinoma or pancreatic adenocarcinoma; gastrointestinal / stomach (GIST) cancer; lymphoma; squamous cell carcinoma of the head and neck (SCCHN); salivary gland cancer; glioma or brain cancer; neurofibromatosis-1 associated malignant peripheral nerve sheath. (Medullary tumor, MPNST); Waldenström macroglobulinemia; or medulloblastoma.

[0545] In some implementations, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial cancer, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), cholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, anaplastic thyroid carcinoma, adrenocortical adenoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1-associated malignant peripheral nerve sheath tumor (MPNST), Waldenström macroglobulinemia, or medulloblastoma.

[0546] In some implementations, the cancer is a solid tumor, such as a sarcoma, carcinoma, or lymphoma. Solid tumors typically comprise an abnormal mass of tissue that usually does not include cysts or fluid-filled areas. In some implementations, the cancer is selected from renal cell carcinoma or kidney cancer; hepatocellular carcinoma (HCC) or hepatoblastoma or liver cancer; melanoma; breast cancer; colorectal carcinoma or colorectal cancer; colon cancer; rectal cancer; anal cancer; lung cancer, such as non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC); ovarian cancer, epithelial ovarian cancer, ovarian cancer. Carcinoma or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer; hepatobiliary ductal carcinoma; soft tissue and bone synovial sarcoma; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing sarcoma; anaplastic thyroid carcinoma; adrenocortical carcinoma; pancreatic cancer; pancreatic ductal carcinoma or pancreatic adenocarcinoma; gastrointestinal / stomach (GIST) cancer; lymphoma; head and neck squamous cell carcinoma (SCCHN); salivary gland carcinoma; glioma or brain cancer; neurofibromatosis-1 associated malignant peripheral nerve schwannoma (MPNST); Waldenström macroglobulinemia; or medulloblastoma.

[0547] In some implementations, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial carcinoma, ovarian carcinoma, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), cholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, anaplastic thyroid carcinoma, adrenocortical carcinoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1-associated malignant peripheral nerve sheath tumor (MPNST), Waldenström macroglobulinemia, or medulloblastoma.

[0548] In one implementation, the cancer is a primary tumor. As used herein, the term "primary tumor" means a tumor that originates at its location or organ and has not metastasized to that location from another location.

[0549] In another embodiment, the cancer is a metastasis. In the context of this invention, "metastasis" is understood as the spread of cancer from its originating organ to different organs. This usually occurs via the blood or lymphatic system. When cancer cells spread and form new tumors, the latter are called secondary or metastatic tumors. The cancer cells that form secondary tumors are similar to those of the original tumor.

[0550] In a preferred embodiment, the cancer is selected from: leukemia, lymphoma, multiple myeloma, neuroblastoma, prostate cancer, acute myeloid leukemia (AML), pancreatic cancer, ovarian cancer, glioblastoma, sarcoma, breast cancer, colorectal cancer, renal cell carcinoma, and melanoma.

[0551] In one specific embodiment, the cancer is leukemia, preferably B-cell acute lymphoblastic leukemia. In a more specific embodiment, the antigen targeted by the chimeric receptor is CD19.

[0552] In another specific embodiment, the cancer is lymphoma, preferably non-Hodgkin's lymphoma. In a more specific embodiment, the antigen targeted by the chimeric receptor is CD19.

[0553] In another specific embodiment, the cancer is multiple myeloma. In a more specific embodiment, the antigen targeted by the chimeric receptor is B-cell maturation antigen (BCMA).

[0554] In another specific embodiment, the cancer is neuroblastoma. In a more specific embodiment, the antigen targeted by the chimeric receptor is disialotetrahexosylganglioside GD2.

[0555] In another specific embodiment, the cancer is prostate cancer. In a more specific embodiment, the antigen targeted by the chimeric receptor is prostate-specific membrane antigen (PSMA).

[0556] In another specific embodiment, the cancer is acute myeloid leukemia. In a more specific embodiment, the antigen targeted by the chimeric receptor is CD33.

[0557] In another specific embodiment, the cancer is pancreatic cancer. In a more specific embodiment, the antigen targeted by the chimeric receptor is mucin 1 (MUC1).

[0558] In another specific embodiment, the cancer is ovarian cancer. In a more specific embodiment, the antigen targeted by the chimeric receptor is folate receptor α.

[0559] In another specific embodiment, the cancer is glioblastoma. In a more specific embodiment, the antigen targeted by the chimeric receptor is epidermal growth factor receptor variant III (EGFRvIII).

[0560] In another specific embodiment, the cancer is a sarcoma. In a more specific embodiment, the antigen targeted by the chimeric receptor is NY-ESO-1.

[0561] In another specific embodiment, the cancer is breast cancer. In a more specific embodiment, the antigen targeted by the chimeric receptor is HER2.

[0562] In another specific embodiment, the cancer is colorectal cancer. In a more specific embodiment, the antigen targeted by the chimeric receptor is carcinoembryonic antigen (CEA).

[0563] In another specific embodiment, the cancer is renal cell carcinoma. In a more specific embodiment, the antigen targeted by the chimeric receptor is carbonic anhydrase IX (CAIX).

[0564] In another specific embodiment, the cancer is melanoma. In a more specific embodiment, the antigen targeted by the chimeric receptor is melanoma-associated antigen (MAGE).

[0565] In another specific implementation, the disease requiring a cell-mediated immune response targeting a target molecule, preferably a target antigen, is an infection.

[0566] Infection is the process by which pathogens invade and multiply in tissues, and the host tissues respond to the infectious agent and its toxins. Infectious diseases, also known as contagious diseases or infectious diseases, are diseases caused by infection. Infections can be caused by a variety of pathogens, the most common being bacteria, viruses, and parasites.

[0567] In a more specific implementation, the disease requiring a cell-mediated immune response targeting a target molecule, preferably a target antigen, is a viral infection.

[0568] A viral infection is an infection caused by a pathogenic virus. Viral diseases (or viral infections) occur when an organism is invaded by a pathogenic virus, and infectious viral particles (virus particles) attach to and enter susceptible cells. Viral infections can be caused by HIV, rhinovirus, lyssaviruses such as rabies virus, Ebola virus, or Severe acute respiratory syndrome coronavirus 2.

[0569] In another, more specific embodiment, the disease requiring a cell-mediated immune response targeting a target molecule, preferably a target antigen, is a bacterial infection.

[0570] "Bacterial infection" is an infection caused by pathogenic bacteria. Pathogenic bacteria are bacteria that can cause disease. Bacterial infections can be caused by Mycobacterium tuberculosis, Staphylococcus aureus, Escherichia coli, Clostridium botulinum, or Salmonella spp.

[0571] In another, more specific embodiment, the disease requiring a cell-mediated immune response targeting a target molecule, preferably a target antigen, is a parasitic infection.

[0572] A "parasitic infection" is an infection caused by a parasite. A parasite is an organism that obtains nutrients from its host and causes harm to the host. Some examples of parasitic infections are malaria, toxoplasmosis, and babesia.

[0573] In another specific implementation, the disease requiring a cell-mediated immune response targeting a target molecule, preferably a target antigen, is an autoimmune disease.

[0574] The terms “autoimmune disease,” “disease associated with immune dysfunction / dysregulation,” or “immunoinflammatory disease” are used throughout the manual to refer to pathological conditions in which a patient’s immune system is triggered by either self-antigens (autoimmunity) or foreign antigens (immune dysfunction / dysregulation or immunoinflammatory disease). Autoimmunity is present to some degree in everyone. It is usually harmless and may be a universal phenomenon in vertebrate life. However, autoimmunity can be the cause of a wide range of human diseases (called autoimmune diseases). Therefore, an autoimmune disease is defined as a progression from benign autoimmunity to pathogenic autoimmunity. This progression is determined by both genetic influences and environmental triggers. The concept of viewing autoimmunity as the actual cause (rather than a consequence or harmless accompaniment) of human disease can be used to establish the criteria for defining a disease as an autoimmune disease. Autoimmune diseases or diseases characterized by immune dysfunction or dysregulation (immunoinflammatory diseases) that can be treated by this invention include systemic lupus erythematosus (SLE), lupus nephritis, central nervous system (CNS) lupus, diabetes (type I), asthma, ulcerative colitis, Crohn's disease, Graves' disease, Addison's disease, celiac disease, alopecia areata, arthritis (including rheumatoid arthritis and osteoarthritis), pernicious anemia, and multiple sclerosis, etc.

[0575] Many autoimmune diseases can be treated using i) the cells of the present invention or the third host cells of the present invention; ii) the second polynucleotide of the present invention, the second vector of the present invention, or the second host cells of the present invention; or iii) the first polynucleotide of the present invention, the first vector of the present invention, the first host cells of the present invention, or the first fusion protein of the present invention, including: autoimmune blood diseases, including pernicious anemia, autoimmune hemolytic anemia, aplastic anemia, idiopathic thrombocytopenic purpura, and ankylosing spondylitis; autoimmune diseases of muscle tissue, including polymyositis and dermatomyositis; autoimmune diseases of the ear, including autoimmune hearing loss and Meniere's syndrome; and autoimmune eye diseases, including Mooren's disease, Reiter's syndrome, and Vogt-Koyanagi-Harada disease. Diseases; autoimmune diseases of the kidneys, including glomerulonephritis, IgA nephropathy, and lupus nephritis; type I diabetes; autoimmune skin diseases, including pemphigus (autoimmune bullous diseases), such as pemphigus vulgaris, pemphigus foliaceus, pemphigus erythematosus, bullous pemphigoid, vitiligo, acquired epidermolysis bullosa, psoriasis, and alopecia areata; cardiovascular autoimmune diseases, including autoimmune myocarditis, vasculitis (including Churg-Strauss syndrome, giant cell arteritis, Kawasaki disease, polyarteritis nodosa, Takayasu's arteritis, and Wegener's granulomatosis). granulomatosis); endocrine autoimmune diseases, including Addison's disease, autoimmune hypoparathyroidism, autoimmune hypophysitis, autoimmune oophoritis, autoimmune orchitis, Graves' disease, Hashimoto's thyroiditis, polyglandular autoimmune syndrome type 1 (PAS-1), polyglandular autoimmune syndrome type 2 (PAS-2), and polyglandular autoimmune syndrome type 3 (PAS-3); gastrointestinal autoimmune diseases, including autoimmune hepatitis, primary biliary cirrhosis, inflammatory bowel disease, celiac disease, and Crohn's disease; and neuroautoimmune diseases, including multiple sclerosis, myasthenia gravis, Guillain-Barré syndrome, and chronic inflammatory demyelinating neuropathy.And systemic autoimmune diseases, including systemic lupus erythematosus, antiphospholipid syndrome, autoimmune lymphoproliferative disorders, autoimmune polyendocrine disorders, Behçet's disease, Goodpasture's disease, arthritis (including rheumatoid arthritis, osteoarthritis, and septic arthritis), sarcoidosis, scleroderma, Sjogren's syndrome, and psoriasis.

[0576] In one specific implementation, the autoimmune disease is selected from: celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, alopecia areata, Addison's disease, pernicious anemia, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

[0577] The third and fourth polynucleotides of the present invention

[0578] In another aspect, the present invention relates to a polynucleotide encoding a fusion protein (hereinafter referred to as the fourth polynucleotide of the present invention), said fusion protein comprising, from the N-terminus to the C-terminus:

[0579] (i) A modified N-inpeptide selected from modified GP41.1 N-inpeptide, modified IMPDH N-inpeptide, modified NrdJ1 N-inpeptide, or modified GP41.8 N-inpeptide.

[0580] The modified N-inpeptide contains a fragment of the N-inpeptide protein that prevents trans-splicing in the presence of the corresponding C-inpeptide, and

[0581] The modified N-inpeptide contains at least one mutation that reduces the affinity between the modified N-inpeptide and the corresponding C-inpeptide.

[0582] (ii) A cleaved C-inteptide comprising a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 or a functionally equivalent variant thereof, wherein

[0583] - If the modified N-inpeptide is the modified GP41.1 N-inpeptide, then the cleaved C-inpeptide contains the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof.

[0584] - If the modified N-inpeptide is a modified IMPDH N-inpeptide, then the cleaved C-inpeptide contains the sequence of SEQ ID NO: 2 or a functionally equivalent variant thereof, and the modified N-inpeptide is a modified IMPDH N-inpeptide.

[0585] - If the modified N-inpeptide is a modified NrdJ1 N-inpeptide, then the cleaved C-inpeptide contains the sequence of SEQ ID NO: 3 or a functionally equivalent variant thereof, or

[0586] - If the modified N-inpeptide is the modified GP41.8 N-inpeptide, then the cleaved C-inpeptide contains the sequence of SEQ ID NO: 4 or a functionally equivalent variant thereof, and

[0587] (iii) Heterologous polypeptides (C-exopeptides).

[0588] The terms “polynucleotide,” “polypeptide,” “integrity,” “split integrity,” “split C-integrity,” and “split N-integrity” have been defined or explained above, and these definitions apply to the third polynucleotide of this invention.

[0589] The term “functional equivalent variants of SEQ ID NO: 1 to 4” has been defined or explained above, and these definitions apply to the third polynucleotide of the present invention.

[0590] The term "modified N-inpeptide" has been defined or explained above, and this definition applies to the third polynucleotide of the present invention.

[0591] The modified N-inpeptide contains a fragment of the N-inpeptide protein that prevents trans-splicing in the presence of the corresponding C-inpeptide.

[0592] In one specific implementation, the modified N-integrapeptide does not contain the complete sequence of the N-integrapeptide protein.

[0593] In one embodiment, the modified GP41.1 N-inpeptide comprises the fragment of SEQ ID NO: 5, which prevents trans-splicing in the presence of the GP41.1 C-inpeptide. In another embodiment, the modified GP41.1 N-inpeptide does not comprise the complete sequence of SEQ ID NO: 5.

[0594] In another embodiment, the modified IMPDH1 N-inpeptide includes the fragment of SEQ ID NO: 6, which prevents trans-splicing in the presence of the IMPDH1 C-inpeptide. In one embodiment, the modified IMPDH1 N-inpeptide does not include the complete sequence of SEQ ID NO: 6.

[0595] In another embodiment, the modified NrdJ1 N-inpeptide includes the fragment of SEQ ID NO: 7, which prevents trans-splicing in the presence of the NrdJ1 C-inpeptide. In one embodiment, the modified NrdJ1 N-inpeptide does not include the complete sequence of SEQ ID NO: 7.

[0596] In another embodiment, the modified GP41.8 N-inpeptide includes the fragment of SEQ ID NO: 8, which prevents trans-splicing in the presence of the GP41.8 C-inpeptide. In one embodiment, the modified GP41.8 N-inpeptide does not include the complete sequence of SEQ ID NO: 8.

[0597] The term "fragment" has been defined or explained above, and this definition applies to the third polynucleotide of the present invention. In one specific embodiment, the fragment comprises or consists of 10 to 100 amino acids, preferably 20 to 65 amino acids, more preferably 45 to 60 amino acids.

[0598] In one specific embodiment, the modified GP41.1 N-inpeptide is a fragment of sequence SEQ ID NO: 5, said fragment comprising or consisting of 18 to 83 amino acids, preferably 30 to 60 amino acids, more preferably 40 to 50 amino acids. The fragment of sequence SEQ ID NO: 5 contains at least one mutation that reduces the affinity between the modified GP41.1 N-inpeptide and the GP41.1C-inpeptide.

[0599] In one specific embodiment, the modified GP41.1 N-intein does not contain the first 5, first 10, first 15, first 20, first 25, first 30, first 35, first 40, first 45, first 50, first 55, first 60, or first 65 amino acids of the polypeptide as defined in SEQ ID NO: 5.

[0600] In another specific embodiment, the modified GP41.1 N-intein does not contain the last 5 amino acids, the last 10 amino acids, the last 15 amino acids, or the last 20 amino acids of the polypeptide as defined in SEQ ID NO: 5.

[0601] The elimination of amino acids at the N-terminal position of the GP41.1 N-intein (i.e., the first few amino acids of the polypeptide as defined in SEQ ID NO: 5) and amino acids at the C-terminal position of the GP41.1 N-intein (i.e., the last few amino acids of the polypeptide as defined in SEQ ID NO: 5) can be combined. In one specific embodiment, the modified GP41.1 N-intein does not contain the first 5, first 10, first 15, first 20, first 25, first 30, first 35, first 40, first 45, or first 50 amino acids of the polypeptide as defined in SEQ ID NO: 5, and does not contain the last 5, last 10, last 15, or last 20 amino acids of the polypeptide as defined in SEQ ID NO: 5.

[0602] In a more specific embodiment, the modified GP41.1 N-intein does not contain the first 5, first 10, first 15, first 20, first 25, first 30, first 35, or first 40 amino acids of the polypeptide as defined in SEQ ID NO: 5, and does not contain the last 5, last 10, last 15, or last 20 amino acids of the polypeptide as defined in SEQ ID NO: 5.

[0603] In a preferred embodiment, the modified GP41.1 N-intein does not contain the first 41 amino acids of the polypeptide as defined in SEQ ID NO: 5.

[0604] In another specific embodiment, the modified IMPDH N-intein is the fragment of SEQ ID NO: 6, which comprises or consists of 21 to 96 amino acids, preferably 40 to 70 amino acids, more preferably 50 to 60 amino acids. The fragment of sequence SEQ ID NO: 6 contains at least one mutation that reduces the affinity between the modified IMPDH N-intein and the IMPDH C-intein.

[0605] In one specific embodiment, the modified IMPDH N-intein does not contain the first 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 amino acids of the polypeptide as defined in SEQ ID NO: 6.

[0606] In another specific embodiment, the modified IMPDH N-inteptide does not contain the last 5, last 10, last 15, or last 20 amino acids of the polypeptide as defined in SEQ ID NO: 6.

[0607] The elimination of amino acids at the N-terminal position of the IMPDH N-intein (i.e., the first few amino acids of the polypeptide as defined in SEQ ID NO: 6) and amino acids at the C-terminal position of the IMPDH N-intein (i.e., the last few amino acids of the polypeptide as defined in SEQ ID NO: 6) can be combined. In one specific embodiment, the modified IMPDH N-intein does not contain the first 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids of the polypeptide as defined in SEQ ID NO: 6, and does not contain the last 5, 10, 15, or 20 amino acids of the polypeptide as defined in SEQ ID NO: 6.

[0608] In a more specific embodiment, the modified IMPDH N-intein does not contain the first 5, first 10, first 15, first 20, first 25, first 30, first 35, first or first 40 amino acids of the polypeptide as defined in SEQ ID NO: 6, and does not contain the last 5, last 10, last 15 or last 20 amino acids of the polypeptide as defined in SEQ ID NO: 6.

[0609] In a preferred embodiment, the modified IMPDH N-intein does not contain the first 42 amino acids of the polypeptide as defined in SEQ ID NO: 6.

[0610] In another specific embodiment, the modified NrdJ1 N-intein is the fragment of SEQ ID NO: 7, said fragment comprising or consisting of 25 to 100 amino acids, preferably 30 to 60 amino acids, more preferably 40 to 50 amino acids. The fragment of sequence SEQ ID NO: 7 contains at least one mutation that reduces the affinity between the modified NrdJ1 N-intein and the NrdJ1 C-intein.

[0611] In one specific embodiment, the modified NrdJ1 N-intein does not contain the first 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 amino acids of the polypeptide as defined in SEQ ID NO: 7.

[0612] In another specific embodiment, the modified NrdJ1 N-intein does not contain the last 5, last 10, last 15, or last 20 amino acids of the polypeptide as defined in SEQ ID NO: 7.

[0613] The elimination of amino acids at the N-terminal position of the NrdJ1 N-intein (i.e., the first few amino acids of the polypeptide as defined in SEQ ID NO: 7) and amino acids at the C-terminal position of the NrdJ1 N-intein (i.e., the last few amino acids of the polypeptide as defined in SEQ ID NO: 7) can be combined. In one specific embodiment, the modified NrdJ1 N-intein does not contain the first 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids of the polypeptide as defined in SEQ ID NO: 7, and does not contain the last 5, 10, 15, or 20 amino acids of the polypeptide as defined in SEQ ID NO: 7.

[0614] In a more specific embodiment, the modified NrdJ1 N-intein does not contain the first 5, first 10, first 15, first 20, first 25, first 30, first 35, first 40, first 45, first 50, or first 55 amino acids of the polypeptide as defined in SEQ ID NO: 7, and does not contain the last 5, last 10, last 15, or last 20 amino acids of the polypeptide as defined in SEQ ID NO: 7.

[0615] In a preferred embodiment, the modified NrdJ1 N-intein does not contain the first 56 amino acids of the polypeptide as defined in SEQ ID NO: 7.

[0616] In another specific embodiment, the modified GP41.8 N-inpeptide is the fragment of SEQ ID NO: 8, which comprises or consists of 19 to 84 amino acids, preferably 30 to 60 amino acids, more preferably 40 to 50 amino acids. The fragment of sequence SEQ ID NO: 8 contains at least one mutation that reduces the affinity between the modified GP41.8 N-inpeptide and the GP41.8 C-inpeptide.

[0617] In one specific embodiment, the modified GP41.8 N-intein does not contain the first 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65 amino acids of the polypeptide as defined in SEQ ID NO: 8.

[0618] In another specific embodiment, the modified GP41.8 N-intein does not contain the last 5, last 10, last 15, or last 20 amino acids of the polypeptide as defined in SEQ ID NO: 8.

[0619] The elimination of amino acids at the N-terminal position of the GP41.8 N-intein (i.e., the first few amino acids of the polypeptide as defined in SEQ ID NO: 8) and amino acids at the C-terminal position of the GP41.8 N-intein (i.e., the last few amino acids of the polypeptide as defined in SEQ ID NO: 8) can be combined. In one specific embodiment, the modified GP41.8 N-intein does not contain the first 5, first 10, first 15, first 20, first 25, first 30, first 35, first 40, first 45, or first 50 amino acids of the polypeptide as defined in SEQ ID NO: 8, and does not contain the last 5, last 10, last 15, or last 20 amino acids of the polypeptide as defined in SEQ ID NO: 8.

[0620] In a more specific embodiment, the modified GP41.8 N-intein does not contain the first 5, first 10, first 15, first 20, first 25, first 30, first 35, or first 40 amino acids of the polypeptide as defined in SEQ ID NO: 8, and does not contain the last 5, last 10, last 15, or last 20 amino acids of the polypeptide as defined in SEQ ID NO: 8.

[0621] In a preferred embodiment, the modified GP41.8 N-intein does not contain the first 40 amino acids of the polypeptide as defined in SEQ ID NO: 8.

[0622] A modified N-inpeptide is an N-inpeptide that does not contain the natural sequence of an N-inpeptide protein. In particular, a modified N-inpeptide contains at least one mutation that reduces the affinity between the modified N-inpeptide and the corresponding C-inpeptide.

[0623] Mutations in the modified N-inpeptide reduce the affinity between the modified N-inpeptide and the corresponding C-inpeptide. In one specific embodiment, the affinity between the modified N-inpeptide and the C-inpeptide is 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% lower than the affinity between the N-inpeptide and the corresponding C-inpeptide.

[0624] In a preferred embodiment, the affinity between the modified N-inte and the C-inte is 40% to 60% lower than the affinity between the N-inte and the corresponding C-inte.

[0625] In one specific embodiment, the modified N-intein is a modified GP41.1 N-intein containing at least one mutation that reduces the affinity between the modified GP41.1 N-intein and the GP41.1 C-intein.

[0626] In one specific embodiment, the modified GP41.1 N-inpeptide contains 1 to 4 mutations in the fragment of sequence SEQ ID NO: 5. In a more specific embodiment, the modified GP41.1 N-inpeptide contains 2 mutations in the fragment of sequence SEQ ID NO: 5.

[0627] In one specific embodiment, the modified GP41.1 N-intein comprises at least one mutation in which a charged amino acid is replaced by a neutral amino acid. In a more specific embodiment, the modified GP41.1 N-intein comprises at least one mutation in which a positively charged amino acid is replaced by a neutral amino acid.

[0628] Therefore, in another specific embodiment, the modified N-inpeptide contains at least one mutation that alters the net charge of the modified N-inpeptide fragment compared to the wild-type N-inpeptide. Specifically, the modified GP41.1 N-inpeptide contains at least one mutation that alters the net charge of the modified GP41.1 N-inpeptide fragment compared to the wild-type GP41.1 N-inpeptide.

[0629] In one specific embodiment, the modified GP41.1 N-intein contains at least one mutation at a lysine (K) residue. In a more specific embodiment, the modified GP41.1 N-intein contains at least one mutation replacing a lysine (K) residue with an alanine (A) residue.

[0630] In a preferred embodiment, the modified GP41.1 N-inpeptide contains mutations at lysine residues 45 and 48 of SEQ ID NO: 5. In a more preferred embodiment, the modified GP41.1 N-inpeptide contains mutations K45A and K48A.

[0631] In one specific embodiment, the modified GP41.1 N-inpeptide comprises the sequence of SEQ ID NO: 31.

[0632] The above-defined mutations apply only when the modified GP41.1 N-inpeptide contains a fragment of SEQ ID NO: 5 containing a mutation site (e.g., K45 and K48).

[0633] In another specific embodiment, the modified N-inteptide is a modified IMPDH N-inteptide containing at least one mutation that reduces the affinity between the modified IMPDH N-inteptide and the IMPDH C-inteptide.

[0634] In one specific embodiment, the modified IMPDH N-inpeptide contains 1 to 4 mutations in the fragment of sequence SEQ ID NO: 6. In a more specific embodiment, the modified IMPDH N-inpeptide contains 2 mutations in the fragment of sequence SEQ ID NO: 6.

[0635] In one specific embodiment, the modified IMPDH N-intein comprises at least one mutation in which a charged amino acid is replaced by a neutral amino acid. In a more specific embodiment, the modified IMPDH N-intein comprises at least one mutation in which a negatively charged amino acid is replaced by a neutral amino acid.

[0636] Therefore, in another specific embodiment, the modified N-inpeptide contains at least one mutation that alters the net charge of the modified N-inpeptide fragment compared to the wild-type N-inpeptide. Specifically, the modified IMPDH N-inpeptide contains at least one mutation that alters the net charge of the modified IMPDH N-inpeptide fragment compared to the wild-type IMPDH N-inpeptide.

[0637] In a more specific embodiment, the modified IMPDH N-intein contains at least one mutation at the glutamic acid (E) residue. In a more specific embodiment, the modified IMPDH N-intein contains at least one mutation in which an alanine (A) residue replaces a glutamic acid (E) residue.

[0638] In a preferred embodiment, the modified IMPDH N-inte contains mutations at glutamate residues 46 and 47 of SEQ ID NO: 6. In a more preferred embodiment, the modified IMPDH N-inte contains mutations E46A and E47A.

[0639] In one specific embodiment, the modified IMPDH N-inteptide comprises the sequence of SEQ ID NO: 32.

[0640] The above-defined mutations apply only when the modified IMPDH N-inpeptide contains a fragment of SEQ ID NO: 6 containing a mutation site (e.g., E46 and E47).

[0641] In another specific embodiment, the modified N-inteptide is a modified NrdJ1 N-inteptide containing at least one mutation that reduces the affinity between the modified NrdJ1 N-inteptide and the NrdJ1 C-inteptide.

[0642] In one specific embodiment, the modified NrdJ1 N-inpeptide contains 1 to 4 mutations in the fragment of sequence SEQ ID NO: 7. In a more specific embodiment, the modified NrdJ1 N-inpeptide contains 2 mutations in the fragment of sequence SEQ ID NO: 7.

[0643] In one specific embodiment, the modified NrdJ1 N-intein comprises at least one mutation in which a charged amino acid is replaced by a neutral amino acid. In a more specific embodiment, the modified NrdJ1 N-intein comprises at least one mutation in which a negatively charged amino group is replaced by a neutral amino acid.

[0644] Therefore, in another specific embodiment, the modified N-inpeptide contains at least one mutation that alters the net charge of the modified N-inpeptide fragment compared to the wild-type N-inpeptide. Specifically, the modified NrdJ1 N-inpeptide contains at least one mutation that alters the net charge of the modified NrdJ1 N-inpeptide fragment compared to the wild-type NrdJ1 N-inpeptide.

[0645] In a more specific embodiment, the modified NrdJ1 N-inte contains at least one mutation at the glutamic acid (E) residue. In a more specific embodiment, the modified NrdJ1 N-inte contains at least one mutation in which a glutamic acid (E) residue is replaced by an alanine (A) residue.

[0646] In a preferred embodiment, the modified NrdJ1 N-inpeptide contains mutations at glutamic acid residues 62 and 64 of SEQ ID NO: 7. In a more preferred embodiment, the modified NrdJ1 N-inpeptide contains mutations E62A and E64A.

[0647] In one specific embodiment, the modified NrdJ1 N-inteptide comprises the sequence of SEQ ID NO: 33.

[0648] The above-defined mutations apply only when the modified NrdJ1 N-inpeptide contains a fragment of SEQ ID NO: 7 containing a mutation site (e.g., E62 and E64).

[0649] In another specific embodiment, the modified N-inteptide is a modified GP41.8 N-inteptide containing at least one mutation that reduces the affinity between the modified GP41.8 N-inteptide and the GP41.8 C-inteptide.

[0650] In one specific embodiment, the modified GP41.8 N-inpeptide contains 1 to 4 mutations in the fragment of sequence SEQ ID NO: 8. In a more specific embodiment, the modified GP41.8 N-inpeptide contains 2 mutations in the fragment of sequence SEQ ID NO: 8.

[0651] In one specific embodiment, the modified GP41.8 N-intein comprises at least one mutation in which a charged amino acid is replaced by a neutral amino acid. In a more specific embodiment, the modified GP41.8 N-intein comprises at least one mutation in which a positively charged amino group is replaced by a neutral amino acid.

[0652] Therefore, in another specific embodiment, the modified N-inpeptide contains at least one mutation that alters the net charge of the modified N-inpeptide fragment compared to the wild-type N-inpeptide. Specifically, the modified GP41.8 N-inpeptide contains at least one mutation that alters the net charge of the modified GP41.8 N-inpeptide fragment compared to the wild-type GP41.8 N-inpeptide.

[0653] In one more specific embodiment, the modified GP41.8 N-intein contains at least one mutation at a lysine (K) residue. In another more specific embodiment, the modified GP41.8 N-intein contains at least one mutation replacing a lysine (K) residue with an alanine (A) residue.

[0654] In a preferred embodiment, the modified GP41.8 N-inpeptide contains mutations at lysine residues 52 and 55 of SEQ ID NO: 8. In a more preferred embodiment, the modified GP41.8 N-inpeptide contains mutations K52A and K55A.

[0655] In one specific embodiment, the modified GP41.8 N-inpeptide comprises the sequence of SEQ ID NO: 34.

[0656] The above-defined mutations apply only when the modified GP41.8 N-inpeptide contains a fragment of SEQ ID NO: 8 containing a mutation site (e.g., K52 and K55).

[0657] In another specific embodiment, the third polynucleotide of the present invention further comprises:

[0658] a. (i) the linker region between the modified N-integument and (ii) the cleaved C-integument, and / or

[0659] b. (i) The protease cleavage site between the modified N-inte and (ii) the cleavage site of the C-inte.

[0660] In one specific embodiment, the linker region between the modified N-inte and the split C-inte comprises a sequence of 1 to 200 amino acids, preferably 1 to 100 amino acids.

[0661] The term "protease cleavage site" has been defined or explained above, and this definition applies to the third polynucleotide of the present invention. In one specific embodiment, the protease cleavage site is recognized and cleaved by a selection of proteases, including furin, heparanase, calpain, matrix metalloproteinases (MMPs), cathepsins, serine proteases, cysteine ​​proteases, aspartic proteases, ADAMs and ADAMTS proteins, aminopeptidase, particle-associated serine proteases, and caspases.

[0662] In a preferred embodiment, the protease cleavage site is a furin protease cleavage site.

[0663] The third polynucleotide of the present invention comprises (iii) a heterologous polypeptide (C-exopeptide).

[0664] The term "heterogeneous polypeptide" has been defined or explained above, and this definition applies to the third polynucleotide of the present invention. However, in those cases where the term "heterogeneous polypeptide" is used to refer to a polypeptide appearing in a fusion protein attached to or splitting a C-inte, the heteropeptide may also be referred to as a "C-exopeptide" in order to distinguish it from heteropeptides attached to the N-terminus of an N-inte or N-exopeptide.

[0665] In one specific implementation, the heterologous polypeptide (C-exotropic peptide) comprises, from the N-terminus to the C-terminus:

[0666] a. Transmembrane domains, and

[0667] b. Intracellular signal transduction domains and / or co-stimulatory domains from the receptor.

[0668] When the heteropeptide (iii) (C-exopeptide) includes a transmembrane domain and an intracellular signal transduction domain and / or co-stimulatory domain from the receptor, the term "fourth polynucleotide of the invention" is used to refer to the third polynucleotide of the invention.

[0669] Therefore, the term "fourth polynucleotide of the present invention" refers to a polynucleotide encoding a fusion protein comprising, from the N-terminus to the C-terminus:

[0670] (i) A modified N-inpeptide selected from modified GP41.1 N-inpeptide, modified IMPDH N-inpeptide, modified NrdJ1 N-inpeptide, or modified GP41.8 N-inpeptide.

[0671] The modified N-inpeptide contains a fragment of the N-inpeptide protein, which prevents trans-splicing in the presence of the corresponding C-inpeptide, and

[0672] The modified N-inpeptide contains at least one mutation that reduces the affinity between the modified N-inpeptide and the corresponding C-inpeptide.

[0673] (ii) A cleaved C-inteptide comprising a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 or a functionally equivalent variant thereof, wherein

[0674] - If the modified N-inpeptide is the modified GP41.1 N-inpeptide, then the cleaved C-inpeptide contains the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof.

[0675] - If the modified N-inpeptide is a modified IMPDH N-inpeptide, then the cleaved C-inpeptide contains the sequence of SEQ ID NO: 2 or a functionally equivalent variant thereof, and the modified N-inpeptide is a modified IMPDH N-inpeptide.

[0676] - If the modified N-inpeptide is a modified NrdJ1 N-inpeptide, then the cleaved C-inpeptide contains the sequence of SEQ ID NO: 3 or a functionally equivalent variant thereof, or

[0677] - If the modified N-inpeptide is the modified GP41.8 N-inpeptide, then the cleaved C-inpeptide contains the sequence of SEQ ID NO: 4 or a functionally equivalent variant thereof.

[0678] (iii) Transmembrane domains, and

[0679] (iv) Intracellular signal transduction domains and / or co-stimulatory domains from the receptor.

[0680] In one specific embodiment, the fourth polynucleotide of the present invention further comprises a linker between the split C-integer and the transmembrane domain.

[0681] The term "connector" has been defined or explained above, and that definition applies to the fourth polynucleotide of the present invention.

[0682] In one specific embodiment, the linker between the split C-integer and the transmembrane domain is a hinge region or a flexible linker. The terms "hinge region" and "flexible linker" have been defined or explained above, and these definitions apply to the fourth polynucleotide of this invention.

[0683] In another specific embodiment, the third polynucleotide of the present invention comprises a nucleotide sequence encoding a signal peptide, wherein the signal peptide is within a frame and located at the N-terminus of a modified N-integral peptide, and the signal peptide allows the fusion protein to insert into the membrane.

[0684] The term "signal peptide" has been defined or explained above, and this definition applies to the third polynucleotide of this invention.

[0685] In a more specific implementation, the signal peptide is selected from: CD8α, IL-2, GM-CSF receptor (GM-CSF) α chain, mouse Ig-κ (IgK), mouse IgK V-III region (IgKVIII), human IgKVIII, CD33, human tissue plasminogen activator (TPA), and naturally occurring secretory alkaline phosphatase (SEAP).

[0686] The terms “transmembrane domain”, “intracellular signal transduction domain” and “co-stimulatory domain” have been defined or explained above, and these definitions apply to the fourth polynucleotide of this invention.

[0687] In one specific embodiment, the intracellular signal transduction domain from the receptor is an antigen receptor signal transduction domain, preferably selected from CD3ζ, CD28, 4-1BB, NKG2D, DAP10, OX40, CD70, CD27, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP 12 antigen receptor signal transduction domains, or combinations thereof.

[0688] In another specific embodiment, the transmembrane domain is selected from the transmembrane domains of CD28, CD8α, 4-1BB, CD4, CTLA4, CD27, or CD3ζ.

[0689] In another specific implementation, the transmembrane domain and the intracellular signal transduction domain are directly connected by peptide bonds or by amino acid linkers.

[0690] The third and fourth carriers of the present invention

[0691] In another aspect, the present invention relates to a vector comprising the third polynucleotide of the present invention, hereinafter referred to as the third vector of the present invention.

[0692] If the heteropeptide (C-exopeptide) of the third polynucleotide of the present invention comprises a transmembrane domain, and an intracellular signal transduction domain and / or co-stimulatory domain from the receptor, then the vector comprising the third polynucleotide of the present invention is referred to as the fourth vector of the present invention.

[0693] Therefore, in another aspect, the present invention relates to a carrier comprising the fourth polynucleotide of the present invention, hereinafter referred to as the fourth carrier of the present invention.

[0694] The term "carrier" has been defined or explained above, and this definition applies to the third and fourth carriers of the present invention.

[0695] The fourth and fifth host cells of the present invention

[0696] In another aspect, the present invention relates to a host cell comprising the third polynucleotide of the present invention, the third vector of the present invention, or a fusion protein encoded by the third polynucleotide of the present invention, hereinafter referred to as the fourth host cell of the present invention.

[0697] In another aspect, the present invention relates to a host cell comprising the fourth polynucleotide of the present invention, the fourth vector of the present invention, or a fusion protein encoded by the fourth polynucleotide of the present invention, hereinafter referred to as the fifth host cell of the present invention.

[0698] The term "host cell" has been defined or explained above, and this definition applies to the fourth and fifth host cells of this invention.

[0699] In one specific implementation, cells are obtained via viral transduction.

[0700] In a more specific implementation, cells are obtained by viral transduction using lentiviruses or by gene knock-in. In a more specific implementation, gene knock-in is performed using a CRISPR-Cas9 system.

[0701] In another specific implementation, the host cell is a human cell. In a more specific implementation, the cell is selected from: T cells, NK cells, induced pluripotent stem cells (iPSCs), derived NK cells, proto-T cells, B cells, and macrophages.

[0702] The third and fourth fusion proteins of the present invention

[0703] In another aspect, the present invention relates to a fusion protein encoded by a third polynucleotide of the present invention, hereinafter referred to as the third fusion protein of the present invention.

[0704] In another aspect, the present invention relates to a fusion protein encoded by the fourth polynucleotide of the present invention, hereinafter referred to as the fourth fusion protein of the present invention.

[0705] The term "fusion protein" has been defined or explained above, and this definition applies to the third and fourth fusion proteins of the present invention.

[0706] In one specific implementation, the fusion protein further includes a detection tag. In a more specific implementation, the detection tag is selected from: c-myc tag, HA tag, FLAG-tag, Strep-tag, His-tag, alfa tag, V5 tag, Spot tag, and NE tag.

[0707] The second composition or kit of the present invention

[0708] In another aspect, the present invention relates to a composition or kit comprising a first component and a second component (hereinafter referred to as the second composition or second kit of the present invention), wherein

[0709] i) The first component is the third polynucleotide of the present invention, the third vector of the present invention, the fourth host cell of the present invention, or the third fusion protein of the present invention, and

[0710] ii) The second component is the second fusion protein of the present invention or the second polynucleotide of the present invention, wherein

[0711] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0712] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0713] - The C-integer contains a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the N-integer contains a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0714] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0715] The first component of the second composition of the present invention or the first component of the second kit of the present invention further comprises the third polynucleotide of the present invention, wherein (iii) the heteropeptide (C-exopeptide) comprises a transmembrane domain, and an intracellular signal transduction domain and / or a co-stimulatory domain derived from the receptor.

[0716] Therefore, the first component of the second composition of the present invention or the first component of the second kit of the present invention further comprises the fourth polynucleotide of the present invention, the fourth vector of the present invention, the fifth host cell of the present invention, or the fourth fusion protein of the present invention.

[0717] Therefore, the second composition or second kit of the present invention comprises a first component and a second component, wherein

[0718] i) The first component is the third or fourth polynucleotide of the present invention, the third or fourth vector of the present invention, the fourth or fifth host cell of the present invention, or the third or fourth fusion protein of the present invention, and

[0719] ii) The second component is the second fusion protein of the present invention or the second polynucleotide of the present invention, wherein

[0720] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0721] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0722] - The C-integer contains a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the N-integer contains a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0723] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0724] Variations of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 have been defined above, and these definitions apply to the second composition or kit of the present invention.

[0725] The ratio of the components in the composition is sufficient for efficient processing of the fusion protein. Suitable ratios of the first and second components include, but are not limited to, the following ratios of (first component): (second component): 1:1, 1:2, 2:1, 1:3, 3:1; 1:4, 4:1, 1:5, 5:1, 1:10, 10:1, 1:15, 15:1, 1:20, 20:1, 1:30, 30:1, 1:40, 40:1, 1:50, 50:1, 1:75, 75:1, 1:100, 100:1, 1:1000, 1000:1, 1:10000, 10000:1.

[0726] Another method of the present invention

[0727] In another aspect, the present invention relates to a method for cleaving a heterologous polypeptide (C-exopeptide) from a third fusion protein of the present invention, comprising incubating the fusion protein together with a cleaving N-inopeptide under conditions allowing inopeptide splicing, wherein

[0728] - If the cleaved C-inpeptide forming part of the fusion protein contains a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, then the cleaved N-inpeptide contains a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0729] - If the cleaved C-inpeptide forming part of the fusion protein contains a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, then the cleaved N-inpeptide contains a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0730] - If the cleaved C-inpeptide forming part of the fusion protein contains a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, then the cleaved N-inpeptide contains a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0731] - If the split C-inpeptide forming part of the fusion protein contains a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, then the split N-inpeptide contains a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0732] The methods for cleaving heteropeptides (C-exopeptides) also include methods for cleaving heteropeptides from the fourth fusion protein of the present invention.

[0733] In another aspect, the present invention relates to a method for covalently linking the N-terminus of a first polypeptide to the C-terminus of a second polypeptide, comprising incubating a second composition of the invention or combining components of a second kit of the invention under conditions allowing inteptide splicing, wherein the first polypeptide is a heterologous polypeptide (C-exopeptide) forming part of a third fusion protein of the invention, and the second polypeptide is a heterologous polypeptide (N-exopeptide) forming part of a second fusion protein of the invention.

[0734] In another aspect, the present invention relates to an in vitro method for generating cells containing chimeric receptors (hereinafter referred to as a third method of the invention), wherein the method comprises:

[0735] (i) Providing cells comprising: the fourth polynucleotide of the present invention, a polypeptide encoded by said polynucleotide, or the fourth carrier of the present invention, wherein the provision is carried out under conditions suitable for expression of a fusion protein encoded by said polynucleotide in said cells, and

[0736] (ii) Contact the cells from step i) with the second fusion protein of the present invention.

[0737] Contact step ii) is performed under conditions that allow a splicing reaction to occur between a cleaved C-intein that forms a portion of the fusion protein produced in step (i) and a cleaved N-intein that forms a portion of the second fusion protein of the present invention, and wherein...

[0738] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0739] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0740] - The C-integer contains a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the N-integer contains a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0741] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0742] The term "in vitro" has been defined or explained above, and that definition applies to the third method of the present invention.

[0743] In one specific implementation, the chimeric receptor is a chimeric antigen receptor.

[0744] The first step of the first method of the present invention includes i) providing cells comprising: the fourth polynucleotide of the present invention, a polypeptide encoded by the polynucleotide, or the fourth carrier of the present invention, wherein the provision is performed under conditions suitable for expression of a fusion protein encoded by the polynucleotide in the cells.

[0745] Suitable conditions for the expression of the fusion protein encoded by the fourth polynucleotide of the present invention in cells are well known to those skilled in the art. Suitable conditions for the expression of the polynucleotide in host cells include those that allow optimal growth of the host cells and those that allow protein expression in the host cells. These culture conditions are generally different for each type of host cell. However, these conditions are known to those skilled in the art and can be readily determined. Similarly, the duration of maintenance can vary depending on the host cells and the amount of fusion protein desired to be prepared. Again, these conditions are well known and can be readily determined in specific cases.

[0746] The second step of the third method of the present invention includes ii) contacting the cells of step i) with the second fusion protein of the present invention, wherein the contacting step ii) is carried out under conditions that allow a splicing reaction to occur between the cleaving C-integument that forms part of the fusion protein produced in step (i) and the cleaving N-integument that forms part of the second fusion protein of the present invention, wherein

[0747] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0748] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0749] - The C-integer contains a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the N-integer contains a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0750] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0751] Suitable conditions for allowing a splicing reaction to occur between the cleaved C-intein, a portion of the fusion protein produced in step (i), and the cleaved N-intein, a portion of the second fusion protein of the present invention, are well known to those skilled in the art. The terms “protein splicing reaction” or “intein reaction” have been defined or interpreted above, and this definition applies to the third method of the present invention.

[0752] In some embodiments, intein reactions (e.g., cleavage, joining (splicing), cyclization) can be initiated by contacting a fusion protein containing an N-terminal domain of an intein with a fusion protein containing a C-terminal domain of an intein. In other embodiments, intein reactions can be initiated by altering the conditions (e.g., temperature or pH) in which an intein fusion protein or a combination of intein fusion proteins is incubated. In some embodiments, C-terminal cleavage is initiated by a change in pH or temperature.

[0753] In some embodiments, the integrin reaction is initiated by contacting the fusion protein with DTT or another strong nucleophile. In some embodiments, DTT is used to enhance the reaction. In some embodiments, N-terminal cleavage is initiated by a strong nucleophile (e.g., DTT).

[0754] Another way to induce protein splicing or cleavage is through contact with peptides or peptide mimics that activate splicing or cleavage. Another way to induce protein splicing or cleavage is through the removal of peptides or peptide mimics that block or inhibit splicing or cleavage.

[0755] Variations of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 have been defined above, and these definitions apply to the third method of the present invention.

[0756] In one specific implementation, the cell is a human cell.

[0757] In a more specific implementation, the cells are selected from: T cells, NK cells, NK cells derived from induced pluripotent stem cells (iPSCs), proto-T cells, B cells, and macrophages.

[0758] In another specific implementation, the cells used in step (i) have been obtained from the patient.

[0759] In another aspect, the present invention relates to an in vitro method for generating cells containing chimeric receptors (hereinafter referred to as the fourth method of the invention), wherein the method comprises:

[0760] i) Provide cells containing the following:

[0761] a. The fourth polynucleotide of the present invention, the fourth vector of the present invention, or a polypeptide encoded by said polynucleotide, wherein the polynucleotide further comprises a nucleotide sequence encoding a signal peptide, wherein the signal peptide is within a frame and located at the N-terminus of a modified N-integral peptide, and said signal peptide allows the fusion protein to insert into the membrane, and

[0762] b. The second polynucleotide of the present invention, the second vector of the present invention, or the second fusion protein of the present invention, wherein the fusion protein encoded by the polynucleotide or said fusion protein further comprises a nucleotide sequence encoding a signal peptide, wherein the signal peptide is within a frame and located at the N-terminus of a polypeptide that specifically binds to a target molecule, and said signal peptide allows the fusion protein to be secreted from the cell, and

[0763] ii) Maintaining cells under conditions suitable for the expression of both fusion proteins in the cells, suitable for the insertion of the fusion protein encoded by the fourth polynucleotide of the present invention into the membrane, suitable for the secretion of the fusion protein encoded by the second polynucleotide of the present invention, and suitable for splicing reactions between the cleavage C-inte and the cleavage N-inte, wherein...

[0764] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0765] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0766] - The C-integer contains a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the N-integer contains a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0767] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0768] The first step of the fourth method of the present invention includes i) providing a cell comprising: a fourth polynucleotide of the present invention, a fourth carrier of the present invention, or a polypeptide encoded by said polynucleotide, wherein said polynucleotide further comprises a nucleotide sequence encoding a signal peptide, wherein the signal peptide is within a frame and located at the N-terminus of a modified N-integer, and said signal peptide allows the fusion protein to insert into the membrane; and b) a second polynucleotide of the present invention, a second carrier of the present invention, or a second fusion protein of the present invention, wherein the fusion protein encoded by the polynucleotide or said fusion protein further comprises a nucleotide sequence encoding a signal peptide, wherein said signal peptide is within a frame and located at the N-terminus of a polypeptide that specifically binds to a target molecule, and said signal peptide allows the fusion protein to be secreted from the cell.

[0769] The “signal peptides” used in this invention have been defined or explained above, and these definitions apply to the fourth method of this invention. Examples of signal peptides are CD8α, IL-2, GM-CSF receptor (GM-CSF) α chain, mouse Ig-κ (IgK), mouse IgK V-III region (IgKVIII), human IgKVIII, CD33, human tissue plasminogen activator (TPA), and naturally occurring secretory alkaline phosphatase (SEAP).

[0770] The second step of the fourth method of the present invention includes ii) maintaining cells under conditions suitable for the expression of the two fusion proteins in the cells, suitable for the insertion of the fusion protein encoded by the fourth polynucleotide of the present invention into the membrane, suitable for the secretion of the fusion protein encoded by the second polynucleotide of the present invention, and suitable for splicing reactions between the cleavage of C-inte and the cleavage of N-inte, wherein...

[0771] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0772] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0773] - The C-integer contains a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the N-integer contains a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0774] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0775] Suitable conditions for the expression of two fusion proteins encoded by polynucleotides in cells are well known to those skilled in the art. Suitable conditions for the expression of polynucleotides in host cells include those that allow the host cells to grow optimally and those that allow the proteins to be expressed in the host cells. These culture conditions are generally different for each type of host cell. However, these conditions are known to those skilled in the art and can be readily determined. Similarly, the duration of maintenance can vary depending on the host cells and the amount of fusion protein desired to be prepared. Again, these conditions are well known and can be readily determined in specific cases.

[0776] Suitable conditions for inserting the fusion protein encoded by the fourth polynucleotide of the present invention into the membrane and for secreting the fusion protein encoded by the second polynucleotide of the present invention are known to those skilled in the art.

[0777] In one specific implementation, when the chimeric receptor is an oligochimeric receptor, the cell provided in (i) also contains (c) an additional polypeptide encoding another element of the oligomer.

[0778] In a more specific embodiment, the oligomer is an immunoglobulin. In a more specific embodiment, the immunoglobulin is IgA, IgG, IgM, IgE, or IgD. In a more specific embodiment, the immunoglobulin is IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgM, IgE, or IgD.

[0779] In another specific embodiment, the fusion protein is a heavy or light chain of an immunoglobulin, and then an additional polypeptide (c) encodes the corresponding heavy or light chain of the immunoglobulin.

[0780] The terms “oligomer,” “immunoglobulin,” “heavy chain,” and “light chain” have been defined or explained above, and these definitions apply to the fourth method of the present invention.

[0781] In some embodiments, the protein splicing reaction between the protein encoded by the fourth polynucleotide of the present invention and the fusion protein encoded by the second polynucleotide of the present invention is intracellular and occurs in the secretory pathway of the cell that produces both polypeptides prior to the secretion of the second polypeptide. In other embodiments, the protein splicing reaction occurs once the second polypeptide is secreted and takes place in the extracellular space.

[0782] The conditions that allow protein splicing or peptide-integrating reactions have been defined or explained in the first method of the present invention, and these conditions are applicable to the fourth method of the present invention.

[0783] Variations of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 have been defined above, and these definitions apply to the fourth method of the present invention.

[0784] In one specific implementation, the cell is a human cell.

[0785] In a more specific implementation, the cells are selected from: T cells, NK cells, NK cells derived from induced pluripotent stem cells (iPSCs), proto-T cells, B cells, and macrophages.

[0786] In another specific implementation, the cells used in step (i) have been obtained from the patient.

[0787] The second cell of the present invention

[0788] In another aspect, the present invention relates to cells comprising chimeric receptors obtained by the third or fourth method of the present invention, hereinafter referred to as the second cell of the present invention.

[0789] Medical use

[0790] In another aspect, the present invention relates to a third host cell or a second cell of the present invention for treating diseases requiring a cell-mediated immune response against a target molecule.

[0791] In one specific implementation, the target molecule is the target antigen.

[0792] In another aspect, the present invention relates to a second fusion protein, a second polynucleotide, a second vector, or a second host cell of the present invention for treating diseases requiring a cell-mediated immune response against a target molecule, wherein the patient to be treated is characterized by comprising cells containing a fourth polynucleotide, a fourth vector, or a fusion protein encoded by a fourth polynucleotide of the present invention, wherein...

[0793] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0794] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0795] - The C-integer contains a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the N-integer contains a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0796] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0797] In another aspect, the present invention relates to a fourth polynucleotide, a fourth vector, a fourth host cell, or a fourth fusion protein of the present invention for treating diseases requiring a cell-mediated immune response against a target molecule, wherein the patient to be treated is characterized by comprising cells containing a second fusion protein, a second polynucleotide, a second vector, or a second host cell of the present invention.

[0798] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof.

[0799] - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof.

[0800] - The C-integer contains a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the N-integer contains a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or

[0801] - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

[0802] In one specific implementation, the target molecule is the target antigen.

[0803] The terms “treatment” and “object” have been defined or explained above, and these definitions apply to these medical uses.

[0804] In one specific implementation, the diseases requiring a cell-mediated immune response against a target molecule (preferably a target antigen) are selected from: cancer, viral infections, bacterial infections, parasitic infections, and autoimmune diseases.

[0805] The terms “cancer,” “viral infection,” “bacterial infection,” “parasitic infection,” and “autoimmune disease” have been defined or explained above, and these definitions apply to these medical uses.

[0806] In a preferred embodiment, the cancer is selected from: leukemia, lymphoma, multiple myeloma, neuroblastoma, prostate cancer, acute myeloid leukemia (AML), pancreatic cancer, ovarian cancer, glioblastoma, sarcoma, breast cancer, colorectal cancer, renal cell carcinoma, and melanoma.

[0807] In another preferred embodiment, the autoimmune disease is selected from: celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, alopecia areata, Addison's disease, pernicious anemia, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

[0808] Example

[0809] Materials and methods

[0810] DNA synthesis and cloning of plasmids encoding IntC-CAR (cage-locked and non-cage-locked) and scFv-IntN constructs

[0811] Synthetic DNA fragments encoding the IntC-CAR construct or scFv-IntN construct were obtained from Twist Bioscience as linear constructs. The receptor vector pcDNA3.1 and the synthetic DNA fragments were digested with the appropriate restriction enzyme (New England Biolabs, NEB) in 20 μL of reaction mixture containing 1–5 μg DNA, 1 μL of each restriction enzyme, and the recommended NEB buffer, followed by incubation at 37°C for 2 h. Digestion efficiency was confirmed by agarose gel electrophoresis. The obtained DNA fragments were purified using a Macherey-Nagel NucleoSpin gel and a PCR Clean-Up kit according to the manufacturer's protocol and eluted in 20–30 μL of sterile water.

[0812] For ligation, approximately 50 to 100 ng of digested vector and insert were mixed with a reaction mixture containing 1 μL T4 DNA ligase (400 U / μL) and 2 μL ligation buffer (NEB), resulting in a final volume of 20 μL. The mixture was incubated at room temperature for 1 hour. The ligated DNA was transformed into chemically competent *E. coli* (NEB 5-α) by heat shock, followed by recovery in 250 μL LB broth with shaking at 37°C for 1 hour. The transformed cell plates were inoculated onto LB agar plates containing an appropriate antibiotic (ampicillin for pcDNA3.1) and incubated overnight at 37°C. Colony selection was performed the following day using PCR, restriction digestion, and confirmation by Sanger sequencing.

[0813] HEK293F cells were transiently transfected with IntC-CAR (cage-locked and non-cage-locked) and scFv-IntN constructs.

[0814] 24 hours before transfection, the cells were divided into groups with a density of approximately 1.5 × 10⁻⁶ cells / cm². 6 Cells / ml were cultured overnight at 37°C with shaking at 125 rpm in an 8% CO2 incubator.

[0815] On the day of transfection, cells were counted using a hemocytometer, and a sufficient volume of cell culture was centrifuged (1200 rpm - 400 rcf, 10 minutes at room temperature) to remove any residual depleted growth medium that could reduce transfection efficiency. The resulting cell pellet was resuspended in fresh medium to a final density of 3 × 10⁻⁶ cells / mL. 6 Cells / ml were incubated at 37°C with shaking at 125 rpm in an 8% CO2 incubator.

[0816] The DNA / PEI mixture was prepared as follows: Polyethyleneimine (PEI, 25 kDa linear PEI, Polysciences, Inc., catalog number 23966) was prepared into a stock solution with a concentration of 1 mg / mL in a buffer containing 25 mM HEPES and 150 mM NaCl (pH 7.5). PEI was added to the buffer and vortexed until completely dissolved (this may be done for several minutes). Once completely dissolved, the PEI was sterilely filtered using 0.22 mL of solution.

[0817] For each transfection, add expression plasmid DNA to the cells, so that the final amount is (1.25 μg) / (1 × 10⁻⁶) 6 (cells), and added PEI to make the total amount (3.75 μg) / (1×10) 6 (Number of cells). The final amount of DNA shown is the same and is independent of the number of transfected plasmids.

[0818] Prepare a dilution of the total plasmid DNA from the total plasmid DNA stock to a final concentration of 0.5 μg DNA / μl in growth medium. Briefly vortex the mixture and centrifuge to return the solution to the bottom of the tube. Add the diluted DNA mixture to the cells. Vortex the culture to mix the DNA and place the flask back into the shaker platform of an 8% CO2 incubator at 37°C and 125 rpm for 5 minutes.

[0819] The total amount of PEI (3.75 μg) / (1×10) 6 Dilute the cells in growth medium to a final concentration of 0.5 μg / μl. Briefly vortex the mixture and centrifuge to return the solution to the bottom of the tube. Add the PEI / medium mixture to the cells. Vortex the culture to mix the PEI, and place the flask back on the shaker platform in the incubator.

[0820] After 24 hours, the cells were diluted 1:1 with preheated Freestyle expression medium supplemented with valproic acid (VPA) (Sigma catalog number P4543-100G) and d-glucose. The final concentration of VPA was 3.75 mM and the final concentration of d-glucose was 4 g / L relative to the final volume (i.e., after a 1:2 dilution 24 hours after transfection).

[0821] The culture was returned to an 8% CO2 incubator at 37°C and oscillated at 125 rpm on the orbital platform.

[0822] IMAC purification of scFv-IntN-His fusion compound secreted by HEK293F cells

[0823] The supernatant of transfected cells was collected by centrifugation. 5× IMAC binding buffer (250 mM Tris pH 8, 1.5 M NaCl, 100 mM imidazole) was added to adjust the pH and ionic strength for binding to the Ni column. The resulting mixture was then filtered through a 0.45 μm filter and loaded onto a 5 mL Ni column using an Äkta system pre-equilibrated with IMAC binding buffer (50 mM Tris pH 8, 300 mM NaCl, 20 mM imidazole). After loading and washing for unbound proteins, the scFv-IntN-His fusion was eluted from the column using an imidazole gradient obtained with IMAC elution buffer (50 mM Tris pH 8, 300 mM NaCl, 500 mM imidazole). The protein content of the eluted fractions was analyzed by Coomassie staining with SDS-PAGE. Fractions containing the target protein were pooled and dialyzed thoroughly against PBS. The concentration of the recovered scFv-IntN-His fusion was quantified by spectrophotometry. The final sample was stored at -80°C until use.

[0824] ST purification of scFv-IntN-ST fusion compound secreted by HEK293F cells

[0825] The supernatant of transfected cells was collected by centrifugation. 10× Strep tag binding buffer (1 M Tris pH 9, 2 M NaCl) was added to adjust the pH and ionic strength for binding to a Strep-Tactin® XT column. The resulting mixture was then filtered through a 0.45 μm filter and loaded onto a 5 mL Strep-Tactin® XT column using an Äkta system pre-equilibrated with ST binding buffer (100 mM Tris pH 9, 200 mM NaCl). After loading and washing for unbound proteins, the scFv-IntN-ST fusion was eluted from the column using a biotin gradient obtained with ST elution buffer (100 mM Tris pH 9, 200 mM NaCl, 50 mM biotin). The protein content of the eluted fractions was analyzed by Coomassie staining with SDS-PAGE. Fractions containing the target protein were pooled and dialyzed thoroughly against PBS. The concentration of the recovered scFv-IntN-ST fusion was quantified spectrophotometrically. Store the final sample at -80°C until use.

[0826] In vitro trans-splicing reaction of purified scFv-IntN fusion with model IntC-POI

[0827] The trans-splicing capacity of the purified scFv-IntN fusion was evaluated using a model IntC-POI fusion containing the corresponding IntC. The trans-splicing reaction was performed in PBS. The model IntC-POI fusion was added to the reaction in a 2-fold excess compared to the scFv-IntN fusion. The reaction was carried out at 25°C in the presence and absence of a reducing agent. Trans-splicing was monitored for 4 hours, and aliquots were collected at 1, 2, and 4 hours after the start of the reaction. The reaction progress was analyzed by Coomassie-stained SDS-PAGE.

[0828] The expression of IntC-CAR and cage-type IntC-CAR on the surface of HEK293F cells was analyzed by flow cytometry.

[0829] Cell viability was determined 72 hours after transfection. Based on the obtained values, 500,000 cells from each sample were transferred to Eppendorf tubes for flow cytometry staining. Cells were washed twice with 300 μL of cold cell staining buffer (Biolegend). Cells were resuspended by vortexing and then centrifuged (5 min, 200 g) for further washing. After the final wash, cells were resuspended in 200 μL of cell staining buffer containing antibody at the desired dilution. 6 Cell staining was performed at a cell density of 8.33 × 10⁶ cells / mL. Cells were incubated with antibody in the dark at room temperature and 80 rpm for 45 minutes. After this time, the cells were washed twice with 300 μL of cell staining buffer. After the final wash, the cells were resuspended in 600 μL of cell staining buffer + 1 mM EDTA (EDTA was added to prevent cell aggregation during flow cytometry analysis), resulting in a density of 8.33 × 10⁶ cells / mL. 5 Cell suspensions of 100 cells / mL were prepared. These samples were analyzed by cytometry using a BC Gallios flow cytometer. DAPI (4',6-diamidinyl-2-phenylindole) was added to the samples to label live cells prior to analysis.

[0830] Western Imprint

[0831] Protein expression was analyzed using Western blot. Samples were treated differently depending on the nature of the expressed protein (whether intracellular, secreted, or purified).

[0832] With cellular protein (e.g., IntC-CAR fusion) expression, HEK293F cells transfected with the IntC-CAR construct were harvested 72 hours post-transfection. Cell pellets corresponding to 500 μL of cell culture were resuspended in 250 μL of ice-cold RIPA buffer (NaCl 150 mM, EDTA 5 mM, Tris pH 8.0 50 mM, NP40 1%, Na-DOC 0.5%, SDS 0.1%) and sonicated on ice for 3 × 10 pulses (4 cycles, 40% power). The resulting cell lysates were diluted with 2 × reducing loading buffer, and 35 μL of each sample was loaded onto an SDS-PAGE gel.

[0833] The secreted proteins and samples obtained by chromatography were diluted 1:2 with 2× reducing loading buffer, and 20 μL of each sample was loaded onto an SDS-PAGE.

[0834] After separation by SDS-PAGE, the samples were then transferred to a miniature PVDF membrane (Thermo Fisher Scientific, catalog number IB401001) using a Thermo iBlot 2 dry blot system (catalog number IB21001). Following blotting, the PVDF membrane was probed using one of the following antibodies or probes: α-STREP tag (StrepMAB-Classic HRP, Iba, catalog number 2-1509-001), α-FLAG tag (anti-DYKDDDDK, Merck, catalog number MAB3118), or α-HIS tag (HisProbe). TM -HRP conjugate (Thermo Fisher Scientific, catalog number 15165) and α-CD3ζ antibody (CD3ζ antibody, CellSignaling Technology, catalog number 88083). When needed, secondary antibodies can be used, namely α-rabbit HRP conjugate (HRP-AffiniPure goat anti-rabbit IgG (H+L), Jackson ImmunoResearch, catalog number 111-035-003) or α-mouse-HRP conjugate (HRP-AffiniPure goat anti-mouse IgG (H+L), Jackson ImmunoResearch, catalog number 115-036-072). For each antibody / probe used, follow the conditions recommended by the manufacturer.

[0835] Finally, the membrane was washed three times with TBS-T and 0.1% Tween-20 and developed with ECL (Novex ECL, Thermo Fisher Scientific, WP20005). Imaging was performed using a ChemiDoc XRS+ imaging station (Biorad).

[0836] In vitro trans-splicing reaction between HEK293F cells expressing IntC-CAR (cage-locked or non-cage-locked) constructs and purified scFv-IntN fusion.

[0837] HEK293F cells were transfected using the IntC-CAR construct. Forty-eight hours after transfection, the cell culture was centrifuged and washed with PBS to remove residual culture medium. The washed cells were then incubated at 10... 6 Cells were resuspended in PBS at a density of 10 cells / mL, and 500 μL aliquots of this cell suspension were added to Eppendorf tubes, one tube per reaction condition. Simultaneously, the purified scFv-IntN fusion was reduced using an IntN:DTT ratio of 1:200. For reactions where the scFv-IntN fusion was not reduced, scFv-IntN solutions were prepared by adding PBS instead of the reducing agent to maintain the same IntN concentration in all reactions. The scFv-IntN fusion was incubated with the reducing agent or PBS at room temperature for at least 1 hour. After this time, it was added to the transfected cells according to the different reaction conditions to be tested, and the trans-splicing reaction was incubated at 37°C and 5% CO2 for approximately 4 hours. At the end of incubation, cells were harvested by centrifugation (10 min, 300 g). To analyze in vitro trans-splicing by Western blotting, wash cells twice with PBS to remove unreacted scFv-IntN fusion and store at -20°C for further analysis of the trans-splicing reaction using the same protocol used to analyze IntC-CAR expression. To analyze in vitro trans-splicing by flow cytometry, wash cells twice with 300 μL of cold cell staining buffer and apply the staining protocol.

[0838] Lentiviral generation

[0839] Synthetic DNA fragments encoding the IntC-CAR construct were cloned into a second-generation pCCL-based lentivirus backbone with T2A-green fluorescent protein (GFP) added to the 3' end of the cassette. A pCCL vector expressing GFP alone (MOCK vector) was used as a control. Viral particles expressing IntC-CAR were generated using HEK293T cells with a standard polyethylenimine (PEI) transfection protocol and pseudotyped with vesicular stomatitis virus-G (VSV-G). For each generation, transfection was performed using the following plasmids: pRRE (11 μg), pREV (5.45 μg), pVSV-G (5 μg), and pIntC-CAR (22 μg) per plate, and the viral particles were concentrated by ultracentrifugation.

[0840] IntC-CAR T cell production

[0841] Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coat of healthy volunteers by Ficoll-Hypaque gradient centrifugation. The buffy coats were obtained from the Barcelona Blood and Tissue Bank (BST) and approved by the institutional review board (IRB) (HCB / 2018 / 0030). T cells were activated for 2 days in the presence of interleukin (IL)-7 and IL-15 (10 ng / mL, Mitenyi Biotec) using plate-bound OKT3 and anti-CD28 antibody (BD Biosciences). Subsequently, in RPMI-1640 medium (catalog number 12-702Q, Lonza) supplemented with 2 mmol / L L-glutamine, 10% heat-inactivated fetal bovine serum (FBS), and 1 U / ml penicillin / streptomycin, virus particles expressing IntC-CAR were used to target 5·10 cells at different multiples of infection (MOIs) (10 to 30). 5Activated T cells were challenged. Cell plates were then seeded every 2–3 days, and cell counts were recorded. IntC-CAR surface expression was detected by fluorescence-activated cell sorting (FACS). IntC-CAR expression was confirmed by GFP expression and the use of an anti-His tag antibody (BioLegend, clone J095G46). Simultaneously, T cell memory phenotypes were analyzed by staining for CCR7 (BioLegend, clone G043H7) and CD45RA (BioLegend, clone HI100). T cell memory phenotypes were as follows: central memory, CM (CCR7+CD45RA-); naive / stem central memory, N / SCM (CCR7+CD45RA+); effector memory, EM (CCR7-CD45RA-); and terminally differentiated, TEMRA (CCR7-CD45RA+). T cell activation status (CD25 and CD69 markers) was also monitored 5 days post-infection (dpi).

[0842] In vitro trans-splicing reaction between T cells expressing the IntC-CAR construct and purified scFv-IntN fusion.

[0843] T cells expressing IntC-CAR were washed in PBS 1× at 5–7 dpi and resuspended in complete RPMI-1640 medium. They were then incubated for 2 h with the corresponding scFv-IntN fusion previously reduced with 5 mM DTT. To determine the formation of reconstructed scFv-CAR via trans-splicing, cells were washed and immediately treated with allophycocyanin (APC) AffiniPure. TM Staining with F(ab')2 fragment goat anti-human IgG (H+L) antibody (Jackson Immuno Research, 109-136-88) for cell counting was performed. This antibody recognizes cells that have incorporated scFv. Orthologous scFv-IntN cells, which cannot perform trans-splicing, were used as controls. The trans-splicing efficiency per cell was calculated as follows: % PTS = (%scFv positive / %His tag positive cells) 100%.

[0844] CD1a-specific T-ALL cytotoxicity

[0845] As previously described, T cells obtained from three different donors were transfected with either IMPDH.1-C-CAR or NrdJ.1-C-CAR. At 5 dpi, T cells were subjected to trans-splicing using an αCD1a scFv-IMPDH.1-N fusion produced in HEK293F cells and purified via IMAC. After 2 hours of incubation, the T cells were washed in PBS 1× and resuspended in 100 μL of complete RPMI-1640 medium. Simultaneously, a mixture of GFP-expressing CD1ako MOLT4 and wild-type MOLT4 cells was prepared and stained with Cell Violet tracer (Thermo Fisher Scientific, C34557) for 20 minutes, washed with complete RPMI-1640 medium and PBS 1×, and then... 6 100 μl of MOLT4 cells / ml were resuspended in complete RPMI-1640 medium. Then, 100 μl of MOLT4 cells (10 5 100 μl of IMPDH.1-C-CAR T cells or 100 μl of NrdJ.1-C-CAR T cells trans-spliced ​​with αCD1a scFv-IMPDH.1-N fusion were mixed. The concentrations of IMPDH.1-C-CAR T cells or NrdJ.1-C-CAR T cells were maintained such that the effector cell (GFP+ CAR T cells):target cell (GFP-MOLT4 cells) ratio was 2:1 and 4:1, respectively. After 24 or 48 hours, the cells were centrifuged (1500 rpm, 5 min), the supernatant was stored at -80°C until further processing, and the cells were resuspended in PBS 1×. Cell analysis was then performed by flow cytometry, and IFN-γ in the supernatant was determined by ELISA (Human ELISA SET, BD Biosciences).

[0846] Example 1: Expression, purification, and trans-expression of αCD19 scFv-gp41.1-N fusion produced in HEK293F cells. splicing activity

[0847] HEK293F cells were transfected with a construct encoding the α-CD19 scFv-gp41.1-N-ST fusion. The presence of the peptide fusion in the culture supernatant was analyzed at different time points after cell transfection using an anti-Strep tag antibody. Figure 1 The αCD19 scFv-gp41.1-N-ST inteptide fusion was expressed and showed two bands due to the glycosylation site located in the gp41.1-N sequence. Culture supernatant was collected 168 hours after transfection, and the inteptide fusion was purified by strep-tag affinity chromatography. Figure 1B). The trans-splicing capability of the pure α-CD19 scFv-gp41.1-N-ST fusion was tested using the model IntC fusion (gp41.1-C-Trx construct) generated in E. coli. Figure 1 (C) Two reactants were prepared: one containing 1 mM DTT and the other without DTT. After incubation at room temperature for 1, 2, and 4 hours, aliquots were extracted and loaded onto Coomassie-stained SDS-PAGE gels. The two bands corresponding to the α-CD19 scFv-gp41.1-N-ST peptide fusion disappeared, and only a single, lower molecular weight band corresponding to the splice product appeared in the reaction containing DTT, confirming the trans-splicing capability of the pure α-CD19 scFv-gp41.1-N-ST fusion.

[0848] Example 2: Expression, purification, and treatment of αBCMA scFv-IMPDH.1-N fusion product generated in HEK293F cells. trans-splicing activity

[0849] HEK293F cells were transfected with a construct encoding the α-BCMA scFv-IMPDH.1-N-His fusion compound. The presence of the peptide fusion compound in the culture supernatant was analyzed at different time points after cell transfection using an anti-His tag antibody. Figure 2 A). Bands with molecular weights of 40 kDa to 50 kDa corresponding to the expressed α-BCMA scFv-IMPDH.1-N-His inteptide fusion were observed. Culture supernatant was collected 168 hours after transfection, and the inteptide fusion was purified by IMAC. Figure 2 B). The trans-splicing capability of the pure α-BCMA scFv-IMPDH.1-N-His fusion was tested using the model IntC fusion (ST-SUMO-His-IMPDH.1-C-VHH construct) generated in E. coli. Figure 2(C) 2 μM αBCMAscFv-IMPDH.1-N-His inteptide fusion was mixed with 4 μM ST-SUMO-His-IMPDH.1-C-VHH in 200 μL PBS. Two reactants were prepared, one containing 1 mM DTT and the other without DTT. Aliquots were extracted after incubation at room temperature for 2 and 4 hours. After aliquot extraction for 4 hours, the splice product was purified by negative IMAC to distinguish it from the unreacted αBCMAscFv-IMPDH.1-N-His inteptide fusion (which has a nearly identical molecular weight). Aliquots of flow-through and eluent from negative IMAC were obtained and loaded together with the aliquots extracted during the reaction onto a Coomassie-stained SDS-PAGE gel. Splicing products of the reaction between the αBCMAscFv-IMPDH.1-N-His inteptide fusion and the model ST-SUMO-His-IMPDH.1-C-VHH IntC precursor were observed in the flow-through of a negative IMAC containing DTT, confirming the trans-splicing capability of the purified αBCMAscFv-IMPDH.1-N-His inteptide fusion.

[0850] Example 3: Expression, purification, and reaction of αCD22 scFv-gp41.8-N fusion product generated in HEK293F cells. splicing activity

[0851] HEK293F cells were transfected with a construct encoding the α-CD22 scFv-gp41.8-N-His fusion compound. The presence of the peptide fusion compound in the culture supernatant was analyzed at different time points after cell transfection using an anti-His-tagged antibody. Figure 3 A band with a molecular weight of approximately 40 kDa corresponding to the expressed α-CD22 scFv-gp41.8-N-His inteptide fusion was observed. The culture supernatant was collected 168 hours after transfection, and the inteptide fusion was purified by IMAC. Figure 3 (B). Pure α-CD22 scFv-gp41.8-N-His inteptide fusion showed two bands, indicating a post-translational modification at this sequence. This modification was not N-glycosylation, as the molecular weight of the inteptide fusion remained unchanged after treatment with PNGase F (a glycosidase that removes almost all N-linked oligosaccharides from glycoproteins). The nature of this translational modification remains unknown. The trans-splicing capability of pure α-CD22 scFv-gp41.8-N-His fusion was tested using the model IntC fusion gp41.8-C-LipTla construct generated in E. coli. Figure 3(C). 1 μM αCD22 scFv-gp41.8-N-His peptide fusion was mixed with 2 μM gp41.8-C-LipTla in 200 μL PBS. Two reactants were prepared, one containing 1 mM DTT and the other without DTT. After incubation at room temperature for 1, 2, and 4 hours, aliquots were extracted and loaded onto Coomassie-stained SDS-PAGE gels. The two bands corresponding to the α-CD22 scFv-gp41.8-N-His peptide fusion disappeared, and only in the reaction containing DTT did a single lower molecular weight band corresponding to the splice product appear, confirming the trans-splicing capability of the pure α-CD22 scFv-gp41.8-N-ST fusion.

[0852] Example 4: Expression, purification, and reaction of αCD1a scFv-NrdJ.1-N fusion product generated in HEK293F cells. splicing activity

[0853] HEK293F cells were transfected with a construct encoding the α-CD1a scFv-NrdJ.1-N-His fusion compound. The presence of the peptide fusion compound in the culture supernatant was analyzed at different time points after cell transfection using an anti-His tag antibody. Figure 4 A band with a molecular weight of approximately 40 kDa corresponding to the expressed α-CD1a scFv-NrdJ.1-N-His inteptide fusion was observed. The culture supernatant was collected 168 hours after transfection, and the inteptide fusion was purified by IMAC. Figure 4 (B). The trans-splicing capability of the pure α-CD1ascFv-NrdJ.1-N-His fusion was tested using the model IntC fusion NrdJ.1-C-LipTla construct generated in E. coli. Figure 4 (C) Two reactants were prepared: one containing 1 mM DTT and the other without DTT. After incubation at room temperature for 1, 2, and 4 hours, aliquots were extracted and loaded onto Coomassie-stained SDS-PAGE gels. The bands corresponding to the αCD1a scFv-NrdJ.1-N-His peptide fusion disappeared with reaction time, while bands with molecular weights of 60 to 80 kDa appeared, consistent with the splicing products, confirming the trans-splicing capability of the pure α-CD1ascFv-NrdJ.1-N-ST fusion.

[0854] Example 5: Expression of IntC-CAR fusion in HEK293F cells

[0855] HEK293F cells were transfected with constructs encoding His-IntC-CAR. These constructs contain a signal peptide that guides the nascent protein to the secretory pathway and ensures IntC is located on the cell surface, a corresponding IntC domain (gp41.1-C, IMPDH.1-C, gp41.8-C, or NrdJ.1-C) with a His tag at the N-terminus for detection, a hinge region and transmembrane domain of the lymphocyte surface antigen CD8, and intracellular signal transduction domains 4-1BB and CD3z (…). Figure 5 (A). 72 hours post-transfection, the expression of the His-IntC-CAR construct was analyzed by flow cytometry using an APC-labeled α-His antibody. Figure 5 B). The IntC-CAR fusions with the highest expression levels were those containing the NrdJ.1 inteptide (84% of His+ cells), followed by the CAR fused with the IMPDH.1 inteptide (47% of His+ cells), the CAR fused with the gp41.1 inteptide (32% of His+ cells), and the CAR fused with the gp41.8 inteptide, which had the lowest expression level (13% of His+ cells). The integrity of the expressed IntC-CAR constructs was examined by Western blotting using the α-CD3z antibody. Figure 5 (C). All IntC-CAR fusions showed the expected molecular weight of the IntC-CAR monomer (m), which was 31 to 32 kDa, depending on the intepid. They all also showed small amounts of IntC-CAR dimers and a band of about 20 kDa, which could correspond to degradation. The gp41.8-C-CAR fusion protein had an N-glycosylation site on the intepid sequence, which increased the observed molecular weight (glycom). The IMPDH.1-C-CAR and NrdJ.1-C-CAR fusions had additional bands with higher molecular weights than the monomers, which likely correspond to post-translational modifications other than N-glycosylation.

[0856] Example 6: Expression of cage-locked IntC-CAR fusion in HEK293F cells

[0857] HEK293F cells were transfected with constructs encoding ST-cage-His-IntC-CAR. These constructs contain a signal peptide that guides the nascent protein to the secretory pathway and ensures IntC is located on the cell surface, a Strep tag for detection, a cage sequence consisting of amino acids from the C-terminal portion of the corresponding IntN, a peptide linker, a His tag for detection, the corresponding IntC domain (gp41.1-C, IMPDH.1-C, gp41.8-C, or NrdJ.1-C), the hinge region and transmembrane domain of the lymphocyte surface antigen CD8, and the intracellular signal transduction domains 4-1BB and CD3z (…). Figure 6 (A). 72 hours post-transfection, the expression of the ST-cage-lock structure-His-IntC-CAR construct was analyzed by flow cytometry using APC-labeled α-His antibody. Addition of the cage-lock structure sequence to gp41.1-C-CAR and IMPDH.1-C-CAR resulted in a double-fold increase in IntC-CAR expression levels. The effect on NrdJ.1-C-CAR was less significant, as the addition of the cage-lock structure sequence increased IntC-CAR expression by 20%. The IntC-CAR construct with the highest expression increase resulting from the addition of the cage-lock structure sequence was gp41.8, with an 8.5-fold increase in expression levels. Figure 6 In addition to the increased absolute expression levels, cells expressing the caged IntC-CAR fusion showed higher expression levels per cell compared to cells expressing the same IntC-CAR fusion in the absence of a cage structure. This effect was observed by a shift to higher fluorescence values ​​in flow cytometry analysis of cells expressing the caged IntC-CAR fusion compared to cells expressing the non-cage-type IntC-CAR fusion. Figure 6 C).

[0858] Example 7: Fusion of cells expressing the gp41.1-C-CAR construct with purified α-CD19 scFv-gp41.1-N In vitro trans-splicing reaction between substances

[0859] HEK293F cells were transfected with the gp41.1-C-CAR construct. Cells were harvested 48 hours after transfection, and in vitro trans-splicing reactants were prepared by combining cells expressing the gp41.1-C-CAR construct with an α-CD19 scFv-gp41.1-N inteptide fusion compound. Figure 7(A). After incubation, the trans-splicing reaction was analyzed by flow cytometry using a combination of two antibodies: an APC-labeled α-His antibody for detecting cells expressing the gp41.1-C-CAR construct (because the gp41.1-C-CAR fusion has an N-terminal His tag); and a PE-labeled α-FLAG antibody for detecting cells containing reconstructed α-CD19 scFv-CAR (because α-CD19 scFv has an N-terminal FLAG tag). Results showed that 40% of the cells expressed gp41.1-C-CAR, and 36% of the cells underwent trans-splicing. The combined figures from both graphs indicate a CAR reconstructing efficiency of 90% per cell, meaning that 90% of the cells expressing gp41.1-C-CAR contained α-CD19 scFv-CAR after the trans-splicing reaction. Figure 7 (B). The trans-splicing reaction was also analyzed using PE-labeled CD19 antigen. Following the trans-splicing reaction, cells were expected to recognize and bind the CD19 antigen as the reaction introduces α-CD19 scFv to the cell surface. This analysis showed that 43% of cells bound to the CD19 antigen after the trans-splicing reaction, indicating that all cells expressing gp41.1-C-CAR incorporated α-CD19 scFv, and therefore, for CARs containing gp41.1 inteptides, the CAR remodeling efficiency per cell was close to 100%. Figure 7 Notably, both flow cytometry analyses achieved equivalent CAR remodeling efficiencies. Finally, Western blotting analysis of cells expressing gp41.1-C-CAR before and after trans-splicing with the α-CD19 scFv-gp41.1-N intein fusion was performed using the αCD3z antibody to identify the formation of remodeled α-CD19 scFv-CAR. Bands with molecular weights ranging from 50 kDa to 70 kDa appeared after the reaction, corresponding to the expected molecular weight of the remodeled CAR, which was 52.6 kDa. Figure 7 D).

[0860] Example 8: Fusion of cells expressing the IMPDH.1-C-CAR construct with purified α-BCMA scFv-IMPDH.1-N In vitro trans-splicing reaction between compounds

[0861] HEK293F cells were transfected with two IMPDH.1-C-CAR constructs. The first one has... Figure 7 The structure shown in A, and the second one has a connector between the transmembrane structural domains of IntC and CD8 ( Figure 8(C). Following the trans-splicing reaction, the linker remained in the reconstructed CAR, imparting some flexibility to the structure. Cells were harvested 48 hours after transfection, and an in vitro trans-splicing reaction was prepared by combining cells expressing the IMPDH.1-C-CAR construct with an α-BCMA scFv-IMPDH.1-N inteptide fusion. After incubation, the trans-splicing reaction was analyzed by flow cytometry using a combination of two antibodies: an APC-labeled α-His antibody, used to detect cells expressing the IMPDH.1-C-CAR construct, with or without the linker, as the construct has an N-terminal His tag; and a PE-labeled α-FLAG antibody, used to detect cells containing the reconstructed α-BCMA scFv-CAR, as the α-BCMA scFv has an N-terminal FLAG tag. Results showed that 12% of cells expressed IMPDH.1-C-CAR on the surface. After the trans-splicing reaction, 8% of cells had a FLAG tag on the surface. These two percentages represent a CAR remodeling efficiency of approximately 70% per cell. Adding the linker between IntC and the CD8 transmembrane domain doubled the percentage of cells expressing IMPDH.1-C-CAR on the surface, from 12% to 23%. It also increased the percentage of cells incorporating FLAG-labeled scFv after trans-splicing from 8% to 12%, resulting in a CAR remodeling efficiency of 52% per cell. Figure 8 (A and D). The presence of reconstructed α-BCMA scFv-CAR in cells after trans-splicing was analyzed by Western blotting using αCD3z antibody. Bands with molecular weights ranging from 50 kDa to 70 kDa appeared after the reaction. Figure 8 (B). This molecular weight corresponds to the expected molecular weight of the reconstructed CAR, which is 53.3 kDa.

[0862] Example 9: Cells expressing the cage-lock type gp41.1-C-CAR construct and purified α-CD19 scFv-gp41.1- In vitro trans-splicing reaction between N fusion compounds

[0863] HEK293F cells were transfected with a cage-locked gp41.1-C-CAR construct. Cells were harvested 48 hours post-transfection, and an in vitro trans-splicing reaction was prepared by combining cells expressing the cage-locked gp41.1-C-CAR construct with an α-CD19 scFv-gp41.1-N inteptide fusion. Following incubation, the trans-splicing reaction was analyzed by flow cytometry using a combination of two antibodies: an APC-labeled α-His antibody, used to detect cells expressing the cage-locked gp41.1-C-CAR construct due to its N-terminal His tag; and a PE-labeled α-FLAG antibody, used to detect cells containing the reconstructed α-CD19 scFv-CAR due to its N-terminal FLAG tag. Results showed that 84% of cells expressed the cage-locked gp41.1-C-CAR, and 37% of cells underwent trans-splicing. The combination of the two figures shows that the CAR reconstruction efficiency per cell is 44% ( Figure 9 (A). The trans-splicing reaction was also analyzed using PE-labeled CD19 antigen. Following the trans-splicing reaction, cells were expected to recognize and bind the CD19 antigen as the reaction introduces α-CD19 scFv to the cell surface. Results showed that 32% of cells bound to the CD19 antigen after the trans-splicing reaction, indicating that these cells had incorporated α-CD19 scFv, and therefore, for cage-locked CARs containing the gp41.1 inteptide, the CAR remodeling efficiency per cell was 38% (A). Figure 9 Finally, Western blotting analysis was performed using the αCD3z antibody on cells expressing caged gp41.1-C-CAR before and after trans-splicing with the α-CD19 scFv-gp41.1-N inteptide fusion to identify the formation of reconstructed α-CD19 scFv-CAR. A molecular weight shift corresponding to the caged gp41.1-N-CAR band was observed, consistent with the incorporation of α-CD19 scFv. Figure 9 C).

[0864] Example 10: Cells expressing the cage-lock type IMPDH.1-C-CAR construct and purified α-BCMA scFv- In vitro trans-splicing reaction between IMPDH.1-N fusion compounds

[0865] HEK293F cells were transfected with a cage-locked IMPDH.1-C-CAR construct. Cells were harvested 48 hours post-transfection, and an in vitro trans-splicing reaction was prepared by combining cells expressing the cage-locked IMPDH.1-C-CAR construct with an α-BCMA scFv-IMPDH.1-N inteptide fusion. Following incubation, the trans-splicing reaction was analyzed by flow cytometry using a combination of two antibodies: an APC-labeled α-His antibody, used to detect cells expressing the cage-locked IMPDH.1-C-CAR construct due to its N-terminal His tag; and a PE-labeled α-FLAG antibody, used to detect cells containing the reconstructed α-BCMA scFv-CAR due to its N-terminal FLAG tag. Results showed that 87% of cells expressed the cage-locked IMPDH.1-C-CAR on their surface. After the trans-splicing reaction, 7% of cells had a FLAG tag on their surface. Figure 10 (A). These two percentages represent a CAR remodeling efficiency of 8% per cell. The presence of remodeled α-BCMA scFv-CAR in cells after trans-splicing was analyzed by Western blotting using the αCD3z antibody. A molecular weight shift corresponding to the cage-like IMPDH.1-N-CAR was observed, consistent with the incorporation of α-BCMA scFv. Figure 10 B).

[0866] Example 11: The effect of the presence of cage-locking structures in the IntC-CAR construct on the trans-splicing response

[0867] Cage-locking IntC within the IntC-CAR construct increased protein expression on the surface of transfected HEK293F cells (see [link]). Figure 6 B). It also increased the number of cells expressing high levels of IntC-CAR per cell (see B). Figure 6C). As a result, the number of cells incorporating high levels of scFv after trans-splicing was significantly higher in cells expressing caged IntC-CAR than in cells expressing non-caged IntC-CAR. This comparison was based on the total number of cells that underwent trans-splicing and the percentage of these cells exhibiting high levels of fluorescence, indicating that they contain high levels of scFv-CAR on their surface. Both the number of cells that underwent trans-splicing and the percentage of cells exhibiting high levels of fluorescence were obtained from flow cytometry analysis of the trans-splicing reaction between cells expressing IntC-CAR (caged or non-caged) and purified scFv-IntN fusions. In the case of gp41.1 containing peptides, flow cytometry analysis of the trans-splicing reaction was performed using PE-labeled CD19 antigen. Since the trans-splicing reaction introduces αCD19 scFv to the cell surface, cells that have undergone trans-splicing are then able to bind the labeled antigen. The results showed that cage-locked gp41.1-C-CAR increased the percentage of cells with high levels of αCD19 scFv-CAR on the surface by 10-fold, and increased the total number of cells undergoing trans-splicing from 3% in the non-cage-locked construct to 35% in the cage-locked construct. Figure 11 (A). In the case of IMPDH.1 containing a peptide, flow cytometry analysis of the trans-splicing reaction was performed using a PE-labeled α-FLAG antibody. Since the trans-splicing reaction incorporates a FLAG tag at the N-terminus of the reconstructed αBCMA scFv-CAR, cells that have undergone trans-splicing were detected by th...

Claims

1. A polynucleotide encoding a fusion protein, said fusion protein comprising, from the N-terminus to the C-terminus: i) A modified N-inpeptide, wherein the modified N-inpeptide is selected from modified GP41.1 N-inpeptide, modified IMPDH N-inpeptide, modified NrdJ1 N-inpeptide, or modified GP41.8 N-inpeptide. The modified N-inpeptide comprises a fragment of an N-inpeptide protein that prevents trans-splicing in the presence of the corresponding C-inpeptide. The modified N-inpeptide contains at least one mutation that reduces the affinity between the modified N-inpeptide and the corresponding C-inpeptide; ii) A cleaved C-inteptide comprising a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 or a functionally equivalent variant thereof, wherein - If the modified N-inpeptide is a modified GP41.1 N-inpeptide, then the split C-inpeptide contains the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof. - If the modified N-inpeptide is a modified IMPDH N-inpeptide, then the cleaved C-inpeptide contains the sequence of SEQ ID NO: 2 or a functionally equivalent variant thereof, wherein the modified N-inpeptide is a modified IMPDH N-inpeptide. - If the modified N-inpeptide is a modified NrdJ1 N-inpeptide, then the split C-inpeptide contains the sequence of SEQ ID NO: 3 or a functionally equivalent variant thereof, or - If the modified N-inpeptide is a modified GP41.8 N-inpeptide, then the split C-inpeptide comprises the sequence of SEQ ID NO: 4 or a functionally equivalent variant thereof; and iii) Heterologous polypeptides.

2. The polynucleotide according to claim 1, wherein: - The modified GP41.1 N-inpeptide contains the sequence of SEQ ID NO:

31. - The modified IMPDH N-inpeptide contains the sequence of SEQ ID NO:

32. - The modified NrdJ1 N-inpeptide contains the sequence of SEQ ID NO: 33, or - The modified GP41.8 N-inpeptide contains the sequence of SEQ ID NO:

34.

3. The polynucleotide according to any one of claims 1 or 2, wherein the polynucleotide further comprises: a. The junction region between the modified N-integument and the cleaved C-integument, and / or b. The protease cleavage site between the modified N-inte and the cleaved C-inte.

4. The polynucleotide according to any one of claims 1 to 3, wherein the heteropolypeptide comprises, from the N-terminus to the C-terminus: a. Transmembrane domains, and b. Intracellular signal transduction domains and / or co-stimulatory domains from the receptor.

5. The polynucleotide according to any one of claims 1 to 4, wherein the polynucleotide further comprises a nucleotide sequence encoding a signal peptide, wherein the signal peptide is within the frame and located at the N-terminus of the modified N-integral peptide, and the signal peptide allows the fusion protein to insert into the membrane.

6. The polynucleotide according to claim 5, wherein the signal peptide is selected from: CD8α, IL-2, GM-CSF receptor (GM-CSF) α chain, mouse Ig-κ (IgK), mouse IgK V-III region (IgKVIII), human IgKVIII, CD33, human tissue plasminogen activator (TPA) and natural secretory alkaline phosphatase (SEAP).

7. The polynucleotide according to any one of claims 4 to 6, wherein the intracellular signal transduction domain from the receptor is an antigen receptor signal transduction domain, wherein the antigen receptor signal transduction domain is preferably selected from: CD3ζ, CD28, 4-1BB, NKG2D, DAP10, OX40, CD70, CD27, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP12 antigen receptor signal transduction domain or combinations thereof.

8. The polynucleotide according to any one of claims 4 to 7, wherein the transmembrane domain is selected from the transmembrane domains of CD28, CD8α, 4-1BB, CD4, CTLA4, CD27 or CD3ζ.

9. The polynucleotide of claim 8, wherein the transmembrane domain and the intracellular signal transduction domain are directly linked by peptide bonds or by amino acid linkers.

10. A vector comprising a polynucleotide according to any one of claims 1 to 9.

11. A host cell comprising a polynucleotide according to any one of claims 1 to 9, a vector according to claim 10, or a fusion protein encoded by a polynucleotide according to any one of claims 1 to 9.

12. The host cell of claim 11, wherein the cell is obtained by viral transduction.

13. The host cell of claim 12, wherein the cell is obtained by viral transduction with lentivirus or by gene knock-in.

14. The host cell of claim 13, wherein the gene knock-in is performed using a CRISPR-Cas9 system.

15. The host cell according to any one of claims 11 to 14, wherein the host cell is a human cell.

16. The host cell according to claim 15, wherein the cell is selected from: T cells, NK cells, induced pluripotent stem cells (iPSCs), derived NK cells, proto-T cells, B cells, and macrophages.

17. A fusion protein encoded by any one of claims 1 to 9.

18. The fusion protein of claim 17, wherein the fusion protein further comprises a detection tag.

19. The fusion protein of claim 18, wherein the detection tag is selected from: c-myc tag, HA tag, FLAG tag, Strep tag, His tag, alfa tag, V5 tag, Spot tag, and NE tag.

20. A fusion protein, said fusion protein comprising, from the N-terminus to the C-terminus: i) Heterologous peptides, and ii) Split N-intein comprising a sequence selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 or a functionally equivalent variant thereof.

21. The fusion protein of claim 20, wherein the polypeptide and the split N-include peptide are directly linked by peptide bonds or by peptide linkers.

22. The fusion protein according to any one of claims 20 or 21, wherein the polypeptide is selected from: single-chain variable fragments (scFv), single-domain antibodies, lectins, epitopes, Ig heavy or light chains, peptides, and ligands.

23. The fusion protein according to claim 22, wherein the polypeptide specifically binds to a target molecule, the target molecule being selected from: target antigen, lectin, epitope.

24. The fusion protein of claim 23, wherein the target molecule is a target antigen.

25. The fusion protein according to claim 24, wherein the target antigen is selected from: CD19, CD20, CD22, GD2, CD133, EGFR, GPC3, CEA, MUC1, mesothelin, IL-13R, PSMA, ROR1, CAIX, CD1a, CCR9 and Her2.

26. The fusion protein according to any one of claims 20 to 25, wherein the fusion protein further comprises a detection tag and / or a solubilization tag.

27. The fusion protein of claim 26, wherein the detection tag is selected from: c-myc tag, HA tag, FLAG tag, Strep tag, His tag, alfa tag, V5 tag, Spot tag, and NE tag.

28. The fusion protein of claim 26, wherein the solubilizing tag is selected from: phage λ head protein D (gpD), small ubiquitin-like modifier (SUMO), maltose-binding protein (MBP), glutathione S-transferase (GST), N utilization substance (NusA), thioredoxin A, and liver fluke 8-kDa antigen (Fh8 tag).

29. A polynucleotide encoding a fusion protein according to any one of claims 20 to 28.

30. The polynucleotide of claim 29, further comprising a nucleotide sequence encoding a signal peptide, wherein the signal peptide is within a frame and located at the N-terminus of a polypeptide that specifically binds to a target molecule, and wherein the signal peptide allows the fusion protein to be secreted from the cell.

31. A vector comprising a polynucleotide according to any one of claims 29 or 30.

32. A host cell comprising the polynucleotide of any one of claims 29 or 30 or the vector of claim 31.

33. A chimeric receptor, said chimeric receptor comprising, from the N-terminus to the C-terminus: i) An extracellular domain comprising a polypeptide that specifically binds to a target molecule, or a polypeptide capable of forming an oligomer in the presence of one or more additional subunits, wherein the oligomer specifically binds to the target molecule. ii) The linker region, whose sequence is the result of protein splicing. iii) Transmembrane domains, and iv) Intracellular signal transduction domains from the receptor, The protein splicing reaction occurs between two complementary parts of the splitting inteptide, and the splitting inteptide is selected from Gp41.1 splitting inteptide, IMPDH splitting inteptide, NrdJ1 splitting inteptide and Gp41.8 splitting inteptide.

34. The chimeric receptor of claim 33, wherein when the extracellular domain is a polypeptide capable of forming an oligomer in the presence of one or more additional subunits, the receptor further comprises the one or more additional subunits.

35. The chimeric receptor according to claim 33 or 34, wherein the linker region comprises 1 to 6 amino acids.

36. The chimeric receptor according to any one of claims 33 to 35, wherein when the extracellular domain comprises a polypeptide that specifically binds to a target molecule, the polypeptide is selected from: single-chain variable fragments (scFv), single-domain antibodies, lectins, epitopes, Ig heavy or light chains, peptides, and ligands.

37. The chimeric receptor according to any one of claims 33 to 35, wherein when the extracellular domain is a polypeptide capable of forming oligomers in the presence of one or more additional subunits, the polypeptide is a heavy or light chain of an immunoglobulin.

38. The chimeric receptor according to any one of claims 33 to 35 and 37, wherein when the extracellular domain is a polypeptide capable of forming an oligomer in the presence of one or more additional subunits, the oligomer is an immunoglobulin.

39. The chimeric receptor of claim 38, wherein the immunoglobulin is IgA, IgG, IgM, IgE or IgD.

40. The chimeric receptor according to any one of claims 33 to 39, wherein at least the target molecule is selected from: target antigen, lectin, epitope.

41. The chimeric receptor of claim 40, wherein the target molecule is a target antigen.

42. The chimeric receptor according to claim 41, wherein the target antigen is selected from: CD19, CD20, CD22, GD2, CD133, EGFR, GPC3, CEA, MUC1, mesothelin, IL-13R, PSMA, ROR1, CAIX, CD1a, CCR9 and Her2.

43. The chimeric receptor according to any one of claims 33 to 42, wherein the intracellular signal transduction domain from the receptor is an antigen receptor signal transduction domain.

44. The chimeric receptor according to claim 43, wherein the antigen receptor signal transduction domain is selected from: CD3ζ, CD28, 4-1BB, NKG2D, DAP10, OX40, CD70, CD27, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP12 antigen receptor signal transduction domain, or combinations thereof.

45. The chimeric receptor according to any one of claims 33 to 44, wherein the transmembrane domain is selected from the transmembrane domains of CD28, CD8α, 4-1BB, CD4, CTLA4, CD27 or CD3ζ.

46. ​​A host cell comprising a chimeric receptor according to any one of claims 33 to 45.

47. The host cell of claim 46, wherein the cell is a human cell.

48. The host cell according to claim 47, wherein the cell is selected from: T cells, NK cells, NK cells derived from induced pluripotent stem cells (iPSCs), proto-T cells, B cells, and macrophages.

49. A composition or kit comprising a first component and a second component, wherein i) The first component is a polynucleotide according to any one of claims 1 to 9, a vector according to claim 10, a host cell according to any one of claims 11 to 16, or a fusion protein according to any one of claims 17 to 19, and ii) The second component is a fusion protein according to any one of claims 20 to 28 or a polynucleotide according to any one of claims 29 or 30, wherein - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof. - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof. - The cleaved C-inteptide comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the cleaved N- comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

50. A method for cleaving the heterologous polypeptide from a fusion protein according to claims 17 to 19, the method comprising incubating the fusion protein together with a cleaving N-intein under conditions allowing intein splicing, wherein... - If the cleaved C-inpeptide forming part of the fusion protein comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, then the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof. - If the cleaved C-inpeptide forming part of the fusion protein comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, then the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof. - If the cleaved C-inpeptide forming part of the fusion protein comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, then the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or - If the split C-inpeptide forming part of the fusion protein comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, then the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

51. A method for covalently linking the N-terminus of a first polypeptide to the C-terminus of a second polypeptide, the method comprising incubating the composition of claim 49 or combining the components of the kit of claim 49 under conditions allowing inteptide splicing, wherein the first polypeptide is a heteropeptide forming part of a fusion protein according to any one of claims 17 to 19, and the second polypeptide is a heteropeptide forming part of a fusion protein according to any one of claims 20 to 28.

52. An in vitro method for generating cells containing chimeric receptors, wherein the method comprises: i) Provide cells comprising the following: polynucleotides according to any one of claims 4 to 9; A polypeptide encoded by the said polynucleotide; or a vector according to claim 10, provided that it refers to claims 4 to 9; the provision is carried out under conditions suitable for expression of the fusion protein encoded by the said polynucleotide in the cells, and ii) Contact the cells from step i) with the fusion protein according to any one of claims 20 to 28, The contact step ii) is carried out under conditions that allow a splicing reaction to occur between a cleaved C-intein of a portion of the fusion protein produced in step (i) and a cleaved N-intein of a portion of the fusion protein according to any one of claims 20 to 28, and wherein... - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof. - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof. - The cleaved C-inteptide comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the cleaved N- comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

53. An in vitro method for generating cells containing chimeric receptors, wherein the method comprises: i) Provide cells containing the following: a. The polynucleotide according to any one of claims 4 to 9; The vector according to claim 10, provided that it refers to claims 4 to 9; or a polypeptide encoded by the polynucleotide; wherein the polynucleotide further comprises a nucleotide sequence encoding a signal peptide, wherein the signal peptide is within the frame and located at the N-terminus of the modified N-integral peptide, and the signal peptide allows the fusion protein to insert into the membrane, and b. The polynucleotide of any one of claims 29 or 30, the vector of claim 31, or the fusion protein of any one of claims 20 to 28, wherein the fusion protein encoded by the polynucleotide or the fusion protein further comprises a nucleotide sequence encoding a signal peptide, wherein the signal peptide is within a frame and located at the N-terminus of a polypeptide that specifically binds to a target molecule, and the signal peptide allows the fusion protein to be secreted from the cell. ii) The cells are maintained under conditions suitable for the expression of the two fusion proteins in the cells, suitable for the insertion of the fusion protein encoded by the polynucleotide according to any one of claims 4 to 9 into the membrane, suitable for the secretion of the fusion protein encoded by the polynucleotide according to any one of claims 29 or 30, and suitable for splicing reactions between the cleavage of C-inte and the cleavage of N-inte, wherein... - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof. - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof. - The cleaved C-inteptide comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the cleaved N- comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

54. The method of claim 53, wherein when the chimeric receptor comprises an extracellular domain comprising a polypeptide capable of forming an oligomer in the presence of one or more additional subunits, the cell provided in (i) further comprises an additional polypeptide encoding the one or more additional subunits of the oligomer.

55. The method of claim 54, wherein the oligomer is an immunoglobulin.

56. The method according to claim 53 or 54, wherein the fusion protein is a heavy or light chain of an immunoglobulin, and therefore the additional polypeptide (c) encodes the corresponding heavy or light chain of the immunoglobulin.

57. The method according to any one of claims 55 or 56, wherein the immunoglobulin is IgA, IgG, IgM, IgE or IgD.

58. The method according to claims 52 to 57, wherein the cell is a human cell.

59. The method of claim 58, wherein the cells are selected from: T cells, NK cells, NK cells derived from induced pluripotent stem cells (iPSCs), proto-T cells, B cells, and macrophages.

60. The method according to any one of claims 52 to 59, wherein the cells used in step (i) are obtained from a patient.

61. A cell containing a chimeric receptor, obtained by the method according to any one of claims 52 to 60.

62. The cell according to any one of claims 46 to 48 or 61, for treating diseases requiring a cell-mediated immune response targeting a target molecule, preferably a target antigen.

63. A fusion protein according to any one of claims 20 to 28, a polynucleotide according to any one of claims 29 or 30, a vector according to claim 31, or a host cell according to claim 32, for treating diseases requiring a cell-mediated immune response against a target molecule, preferably against a target antigen, wherein the patient to be treated is characterized by containing cells comprising: a polynucleotide according to any one of claims 4 to 9; a vector according to claim 10, provided that it refers to claims 4 to 9; or a fusion protein encoded by a polynucleotide according to any one of claims 4 to 9; wherein - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof. - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof. - The cleaved C-inteptide comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the cleaved N- comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

64. The polynucleotide according to any one of claims 4 to 9; the vector according to claim 10, wherein all references to claims 4 to 9 are accepted; the host cell according to any one of claims 11 to 16, wherein all references to claims 4 to 9 are accepted; or a fusion protein encoded by the polynucleotide according to any one of claims 4 to 9; wherein it is used to treat diseases requiring a cell-mediated immune response against a target molecule, preferably against a target antigen, wherein the patient to be treated is characterized by containing cells comprising: the fusion protein according to any one of claims 20 to 28, the polynucleotide according to any one of claims 29 or 30, the vector according to claim 31, or the host cell according to claim 32, wherein... - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof. - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof. - The cleaved C-inteptide comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the cleaved N- comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

65. The fusion protein, polynucleotide, vector, or host cell of any of claims 63 or 64, wherein the disease requiring a cell-mediated immune response against a target molecule, preferably against a target antigen, is selected from: cancer, viral infection, bacterial infection, parasitic infection, and autoimmune disease.

66. The fusion protein, polynucleotide, vector, or host cell of claim 65, wherein the cancer is selected from: leukemia, lymphoma, multiple myeloma, neuroblastoma, prostate cancer, acute myeloid leukemia (AML), pancreatic cancer, ovarian cancer, glioblastoma, sarcoma, breast cancer, colorectal cancer, renal cell carcinoma, and melanoma.

67. A polynucleotide encoding a fusion protein, said fusion protein comprising, from the N-terminus to the C-terminus: i) A cleaved C-inteptide comprising a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 or a functionally equivalent variant thereof. ii) Transmembrane structural domains, and iii) Intracellular signal transduction domains and / or co-stimulatory domains from the receptor.

68. The polynucleotide of claim 67, further comprising a nucleotide sequence encoding a signal peptide, wherein the signal peptide is within a frame and located at the N-terminus of the cleaved C-inteptide, and wherein the signal peptide allows the fusion protein to insert into a membrane.

69. The polynucleotide of claim 68, wherein the signal peptide is selected from: CD8α, IL-2, GM-CSF receptor (GM-CSF) α chain, mouse Ig-κ (IgK), mouse IgK V-III region (IgKVIII), human IgKVIII, CD33, human tissue plasminogen activator (TPA) and natural secretory alkaline phosphatase (SEAP).

70. The polynucleotide according to any one of claims 67 to 69, further comprising a linker between the cleaved C-intein and the transmembrane domain.

71. The polynucleotide of claim 70, wherein the linker comprises 40 to 45 amino acids.

72. The polynucleotide according to any one of claims 70 or 71, wherein the linker is a hinge region or a flexible linker, the hinge region being selected from: IgG1 hinge region, IgG2 hinge region, IgG4 hinge region, FcγR hinge region, CD3ε hinge region, CD3ζ hinge region, CD8α hinge region, CD4 hinge region, CD28 hinge region, CD7 hinge region, CD19 hinge region, and the flexible linker is preferably a linker rich in glycine and serine.

73. The polynucleotide according to any one of claims 67 to 72, wherein the intracellular signal transduction domain from the receptor is an antigen receptor signal transduction domain, wherein the antigen receptor signal transduction domain is preferably selected from CD3ζ, CD28, 4-1BB, NKG2D, DAP10, OX40, CD70, CD27, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP12 antigen receptor signal transduction domains or combinations thereof.

74. The polynucleotide according to any one of claims 67 to 73, wherein the transmembrane domain is selected from the transmembrane domains of CD28, CD8α, 4-1BB, CD4, CTLA4, CD27 or CD3ζ.

75. The polynucleotide according to any one of claims 67 to 74, wherein the transmembrane domain and the intracellular signal transduction domain are directly linked by peptide bonds or by amino acid linkers.

76. The polynucleotide according to any one of claims 67 to 75, wherein the fusion protein encoded by said polynucleotide further comprises a modified N-inpeptide. The modified N-intein is located at the N-terminus relative to the cleaved C-intein, wherein... - If the cleaved C-inpeptide contains the sequence of SEQ ID NO: 1 or a functionally equivalent variant thereof, then the modified N-inpeptide is the modified GP41.1 N-inpeptide. - If the cleaved C-inpeptide contains the sequence of SEQ ID NO: 2 or a functionally equivalent variant thereof, then the modified N-inpeptide is a modified IMPDH N-inpeptide. - If the cleaved C-inpeptide contains the sequence of SEQ ID NO: 3 or a functionally equivalent variant thereof, then the modified N-inpeptide is a modified NrdJ1 N-inpeptide, or - If the cleaved C-inpeptide contains the sequence of SEQ ID NO: 4 or a functionally equivalent variant thereof, then the modified N-inpeptide is the modified GP41.8 N-inpeptide. The modified N-inpeptide comprises a fragment of an N-inpeptide protein that prevents trans-splicing in the presence of the corresponding C-inpeptide. The modified N-intein contains at least one mutation that reduces the affinity between the modified N-intein and the corresponding C-intein.

77. The polynucleotide according to claim 76, wherein - The modified GP41.1 N-inpeptide contains the sequence of SEQ ID NO:

31. - The modified IMPDH N-inpeptide contains the sequence of SEQ ID NO:

32. - The modified NrdJ1 N-inpeptide contains the sequence of SEQ ID NO: 33, or - The modified GP41.8 N-inpeptide contains the sequence of SEQ ID NO:

34.

78. The polynucleotide according to any one of claims 76 or 77, wherein the polynucleotide further comprises: a. The junction region between the modified N-integument and the cleaved C-integument, and / or b. The protease cleavage site between the modified N-inte and the cleaved C-inte.

79. A vector comprising the polynucleotide according to any one of claims 67 to 78.

80. A host cell comprising a polynucleotide according to any one of claims 67 to 78, a vector according to claim 79, or a fusion protein encoded by a polynucleotide according to any one of claims 67 to 78.

81. The host cell of claim 80, wherein the cell is obtained by viral transduction.

82. The host cell of claim 81, wherein the cell is obtained by viral transduction with lentivirus or by gene knock-in.

83. The host cell of claim 82, wherein the gene knock-in is performed using a CRISPR-Cas9 system.

84. The host cell according to any one of claims 80 to 83, wherein the host cell is a human cell.

85. The host cell according to claim 84, wherein the cell is selected from: T cells, NK cells, induced pluripotent stem cells (iPSCs), derived NK cells, proto-T cells, B cells, and macrophages.

86. A fusion protein encoded by any one of claims 67 to 78.

87. The fusion protein of claim 86, wherein the fusion protein further comprises a detection tag.

88. The fusion protein of claim 87, wherein the detection tag is selected from: c-myc tag, HA tag, FLAG tag, Strep tag, His tag, alfa tag, V5 tag, Spot tag, and NE tag.

89. A composition or kit comprising a first component and a second component, wherein i) The first component is a polynucleotide according to any one of claims 67 to 78, a vector according to claim 79, a host cell according to any one of claims 80 to 85, or a fusion protein according to any one of claims 86 to 88, and ii) The second component is a fusion protein according to any one of claims 20 to 28 or a polynucleotide according to any one of claims 29 or 30, wherein - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof. - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof. - The cleaved C-inteptide comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the cleaved N- comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

90. An in vitro method for generating cells containing a chimeric receptor, wherein the method comprises: i) Providing cells comprising: a polynucleotide according to any one of claims 67 to 78, a polypeptide encoded by said polynucleotide, or a vector according to claim 79, wherein the provision is carried out under conditions suitable for expression of a fusion protein encoded by said polynucleotide in said cells, and ii) Contact the cells from step i) with the fusion protein according to any one of claims 20 to 28, The contact step ii) is carried out under conditions that allow a splicing reaction to occur between a cleaved C-intein of a portion of the fusion protein produced in step (i) and a cleaved N-intein of a portion of the fusion protein according to any one of claims 20 to 28, and wherein... - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof. - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof. - The cleaved C-inteptide comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the cleaved N- comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

91. An in vitro method for generating cells containing chimeric receptors, wherein the method comprises: i) Provide cells containing the following: a. A polynucleotide according to any one of claims 67 to 78, a vector according to claim 79, or a polypeptide encoded by said polynucleotide, wherein said polynucleotide further comprises a nucleotide sequence encoding a signal peptide, wherein said signal peptide is within a frame and located at the N-terminus of said cleaved C-inteptide, and said signal peptide allows the fusion protein to insert into a membrane, and b. The polynucleotide of any one of claims 29 or 30, the vector of claim 31, or the fusion protein of any one of claims 20 to 28, wherein the fusion protein encoded by the polynucleotide or the fusion protein further comprises a nucleotide sequence encoding a signal peptide, wherein the signal peptide is within a frame and located at the N-terminus of a polypeptide that specifically binds to a target molecule, and the signal peptide allows the fusion protein to be secreted from the cell. ii) The cells are maintained under conditions suitable for the expression of the two fusion proteins in the cells, suitable for the insertion of the fusion protein encoded by the polynucleotide according to any one of claims 67 to 78 into the membrane, suitable for the secretion of the fusion protein encoded by the polynucleotide according to any one of claims 29 or 30, and suitable for splicing between the cleaving C-inteptide and the cleaving N-inteptide, wherein... - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof. - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof. - The cleaved C-inteptide comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the cleaved N- comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

92. The method of claim 91, wherein when the chimeric receptor comprises an extracellular domain comprising a polypeptide capable of forming an oligomer in the presence of one or more additional subunits, the cell provided in (i) further comprises an additional polypeptide encoding the one or more additional subunits of the oligomer.

93. The method of claim 92, wherein the oligomer is an immunoglobulin.

94. The method according to claim 92 or 93, wherein the fusion protein is a heavy or light chain of an immunoglobulin, and therefore the additional polypeptide (c) encodes the corresponding heavy or light chain of the immunoglobulin.

95. The method according to any one of claims 93 or 94, wherein the immunoglobulin is IgA, IgG, IgM, IgE or IgD.

96. The method according to claims 90 to 95, wherein the cell is a human cell.

97. The method of claim 96, wherein the cells are selected from: T cells, NK cells, NK cells derived from induced pluripotent stem cells (iPSCs), proto-T cells, B cells, and macrophages.

98. The method according to any one of claims 90 to 97, wherein the cells used in step (i) are obtained from the patient.

99. A cell containing a chimeric receptor, obtained by the method according to any one of claims 90 to 98.

100. The cell according to any one of claims 46 to 48 or 99, for treating diseases requiring a cell-mediated immune response targeting a target molecule, preferably a target antigen.

101. A fusion protein according to any one of claims 20 to 28, a polynucleotide according to any one of claims 29 or 30, a vector according to claim 31, or a host cell according to claim 32, for treating diseases requiring a cell-mediated immune response against a target molecule, preferably against a target antigen, wherein the patient to be treated is characterized by containing cells comprising: a polynucleotide according to any one of claims 67 to 78, a vector according to claim 79, or a fusion protein encoded by a polynucleotide according to any one of claims 67 to 78, wherein... - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof. - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof. - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

102. The polynucleotide of any one of claims 67 to 78, the vector of claim 79, the host cell of any one of claims 80 to 85, or the fusion protein of any one of claims 86 to 88, for treating diseases requiring a cell-mediated immune response against a target molecule, preferably against a target antigen, wherein the patient to be treated is characterized by containing cells comprising: the fusion protein of any one of claims 20 to 28, the polynucleotide of any one of claims 29 or 30, the vector of claim 31, or the host cell of claim 32, wherein... - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 5 or a variant thereof. - The cleaved C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 2 or a variant thereof, and the cleaved N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 6 or a variant thereof. - The cleaved C-inteptide comprises a polypeptide having the sequence of SEQ ID NO: 3 or a variant thereof, and the cleaved N- comprises a polypeptide having the sequence of SEQ ID NO: 7 or a variant thereof, or - The split C-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof, and the split N-inpeptide comprises a polypeptide having the sequence of SEQ ID NO: 8 or a variant thereof.

103. The fusion protein, polynucleotide, vector, or host cell of any of claims 101 or 102, wherein the disease requiring a cell-mediated immune response against a target molecule, preferably against a target antigen, is selected from: cancer, viral infection, bacterial infection, parasitic infection, and autoimmune disease.

104. The fusion protein, polynucleotide, vector, or host cell of claim 103, wherein the cancer is selected from: leukemia, lymphoma, multiple myeloma, neuroblastoma, prostate cancer, acute myeloid leukemia (AML), pancreatic cancer, ovarian cancer, glioblastoma, sarcoma, breast cancer, colorectal cancer, renal cell carcinoma, and melanoma.

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