Methods and reagents for the generation of chimeric antigen receptors using split inteins

Abstract

The present invention relates to methods and reagents for the generation of Chimeric Antigen Receptors using split inteins as well as to the uses of the Chimeric Antigen Receptors, particularly in diseases requiring a cell-mediated immune response against an antigen of interest. The present invention also relates to caged inteins and methods for the generation of chimeric antigen receptors using these caged inteins.

Description

[0001] METHODS AND REAGENTS FOR THE GENERATION OF CHIMERIC ANTIGEN RECEPTORS USING SPLIT INTEINS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to methods and reagents for the generation of Chimeric Antigen Receptors using split inteins as well as to the uses of the Chimeric Antigen Receptors, particularly in diseases requiring a cell-mediated immune response against an antigen of interest. The present invention also relates to caged inteins and methods for the generation of recombinant proteins, in particular, chimeric antigen receptors using these caged inteins.

[0004] BACKGROUND OF THE INVENTION

[0005] Chimeric antigen receptors (CARs) are used in many clinical applications, including cancer treatment. A chimeric antigen receptor is a recombinant receptor composed of an extracellular antigen binding domain and an intracellular T-cell signalling domain. The use of chimeric antigen receptor (CAR)-modified T cells is an innovative immunotherapeutic approach. CAR cell therapy relies on re-engineering T-cells to express a receptor that allows the cells to recognize targeted cells. Typically, CAR treatment includes collecting T cells from a patient and introducing a chimeric antigen into the collected cells ex vivo, expanding the transfected cells, and then infusing them into a patient.

[0006] Immunotherapy has generated unprecedented expectations in cancer treatment and relies on the immune system as a powerful weapon against cancer. In recent years, adoptive cellular immunotherapy based on chimeric antigen receptors (CARs) has shown great potential. T cells or T lymphocytes constantly look for foreign antigens and discriminate abnormal (cancer or infected cells) from normal cells. Genetically modifying T cells with CARs is the most common approach to design tumor-specific T cells CAR-T cells targeting tumor-associated antigens (TAA) can be infused into patients (called adoptive cell transfer or ACT) representing an efficient immunotherapy approach.

[0007] Recent developments using chimeric antigen receptor (CAR) modified T cell (CART) therapy, which relies on redirecting T cells to a suitable cell-surface molecule on cancer cells, show promising results in harnessing the power of the immune system to treat cancers. Given the ongoing need for improved strategies for targeting diseases such as cancer, new compositions and methods for improving CART therapies are highly desirable.

[0008] SUMMARY OF THE INVENTION

[0009] In a first aspect, the present invention relates to a polynucleotide, hereinafter the first polynucleotide of the invention, encoding a fusion protein comprising from N-terminus to C-terminus: i) a split C-intein comprising a sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 or a functionally equivalent variant thereof, ii) a transmembrane domain, and iii) an intracellular signalling domain from a receptor and / or costimulatory domain.

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

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

[0012] In another aspect, the present invention relates to a fusion protein, hereinafter the first fusion protein of the invention, encoded by the first polynucleotide of the invention.

[0013] In another aspect, the present invention relates to a fusion protein, hereinafter the second fusion protein of the invention, comprising from N-terminus to C-terminus: i) a heterologous polypeptide and ii) a split N-intein comprising a sequence selected from the group consisting SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 or a functionally equivalent variant thereof.

[0014] In another aspect, the present invention relates to a polynucleotide, hereinafter the second polynucleotide of the invention, encoding the second fusion protein of the invention.

[0015] In another aspect, the present invention relates to a vector, hereinafter the second vector of the invention, comprising the second polynucleotide of the invention. In another aspect, the present invention relates to a host cell, hereinafter the second host cell of the invention, comprising the second polynucleotide of the invention or the second vector of the invention.

[0016] In another aspect, the present invention relates to a chimeric receptor, hereinafter the chimeric receptor of the invention, comprising from N-terminus to C-terminus: i) an extracellular domain comprising a polypeptide that specifically binds to target molecule or a polypeptide which can form an oligomer in the presence of one or more additional subunits and wherein the oligomer specifically binds to a target molecule, ii) a linker region, the sequence of which is the result of a protein splicing reaction, iii) a transmembrane domain, and iv) an intracellular signalling domain from a receptor, wherein the protein splicing reaction occurs between two complementary portions of a split intein and wherein the split intein is selected from the group consisting of: Gp41.1 split intein, IMPDH split intein, NrdJ1 split intein and Gp41.8 split intein.

[0017] In another aspect, the present invention relates to a host cell, hereinafter the third host cell of the invention, comprising the chimeric receptor of the invention.

[0018] In another aspect, the present invention relates to a composition or kit-of-parts, hereinafter the first composition or the first kit-of-parts, comprising a first component and a second component wherein i) the first component is the first polynucleotide of the invention, the first vector of the invention, the first host cell of the invention or the first fusion protein of the invention and ii) the second component is the second fusion protein of the invention or the second polynucleotide of the invention, wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof. In another aspect, the present invention relates to an in vitro method, hereinafter the first method of the invention, for producing a cell comprising a chimeric receptor, wherein the method comprises: i) providing a cell comprising the first polynucleotide of the invention, a polypeptide encoded by said polynucleotide or the first vector of the invention under conditions adequate for the expression in the cell of the fusion protein encoded by the polynucleotide, and ii) contacting the cell of step i) with the second fusion protein of the invention, wherein the contacting step ii) is performed under conditions that allow a splicing reaction between the split C-intein forming part of the fusion protein produced in step (i) and the split N-intein forming part of the second fusion proteins of the invention and wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof. In another aspect, the present invention relates to an in vitro method, hereinafter the second method of the invention, for producing a cell comprising a chimeric receptor, wherein the method comprises:

[0019] (i) providing a cell comprising a. the first polynucleotide of the invention, the first vector of the 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 in frame and N-terminal to the split C-intein and which allows the insertion of the fusion protein into the membrane and b. the second polynucleotide of the invention, the second vector of the invention or the second fusion protein of the 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 in frame and N-terminal to the polypeptide that specifically binds to a target molecule and which allows the secretion of the fusion protein from the cell and

[0020] (ii) maintaining the cell under conditions adequate for the expression in the cell of both fusion proteins, for the insertion into the membrane of the fusion protein encoded by the first polynucleotide of the invention, for the secretion of the fusion protein encoded by the second polynucleotide of the invention and for the splicing reaction between the split C-intein and the split N-intein and wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO: 3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

[0021] In another aspect, the present invention relates to a cell, hereinafter the first cell of the invention, comprising a chimeric receptor obtained by the first method or the second method of the invention.

[0022] In another aspect, the present invention relates to the third host cell of the invention or the first cell of the invention for use in the treatment of a disease which requires a cell- mediated immune response against a target molecule, preferably against the antigen of interest.

[0023] In another aspect, the present invention relates to the second fusion protein of the invention, the second polynucleotide of the invention, the second vector of the invention or the second host cell of the invention for use in the treatment of a disease which requires a cell-mediated immune response against a target molecule, preferably against an antigen of interest, wherein the patient to be treated is characterized in that it contains cells comprising the first polynucleotide of the invention, the first vector of the invention or the fusion protein encoded by the first polynucleotide of the invention, wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

[0024] In another aspect, the present invention relates to the first polynucleotide of the invention, the first vector of the invention, the first host cell of the invention or the first fusion protein of the invention for use in the treatment of a disease which requires a cell-mediated immune response against a target molecule, preferably against an antigen of interest, wherein the patient to be treated is characterized in that it contains cells comprising the second fusion protein of the invention, the second polynucleotide of the invention, the second vector of the invention or the second host cell of the invention, wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

[0025] In another aspect, the present invention relates to a polynucleotide, hereinafter the third polynucleotide of the invention, encoding a fusion protein comprising from N-terminus to C-terminus:

[0026] (i) a modified N-intein selected from the group consisting of a modified GP41.1 N-intein, a modified IMPDH N-intein, a modified NrdJ1 N-intein or a modified GP41.8 N-intein, wherein the modified N-intein comprises a fragment of the N-intein protein that prevents trans-splicing from occurring in the presence of the corresponding C-intein and wherein the modified N-intein comprises at least one mutation that decreases the affinity between the modified N-intein and the corresponding C-intein,

[0027] (ii) a split C-intein comprising a sequence selected from the group consisting of 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-intein is a modified GP41.1 N-intein, the split C-intein comprises the sequence of SEQ ID NO: 1 or is a functionally equivalent variant thereof,

[0028] If the modified N-intein is a modified IMPDH N-intein, the split C-intein comprises the sequence of SEQ ID NO: 2 or is a functionally equivalent variant thereof, the modified N-intein is a modified IMPDH N-intein If the modified N-intein is a modified NrdJ1 N-intein, the split C-intein comprises the sequence of SEQ ID NO: 3 or is a functionally equivalent variant thereof or

[0029] If the modified N-intein is a modified GP41.8 N-intein, the split C-intein comprises the sequence of SEQ ID NO: 4 or is a functionally equivalent variant thereof, and

[0030] (iii) a heterologous polypeptide.

[0031] In another aspect, the present invention relates to a vector, hereinafter the third vector of the invention, which comprises the third polynucleotide of the invention.

[0032] In another aspect, the present invention relates to a host cell, hereinafter the fourth host cell of the invention, comprising the third polynucleotide of the invention, the third vector of the invention or the fusion protein encoded by the third polynucleotide of the invention.

[0033] In another aspect, the present invention relates to a fusion protein, hereinafter the third fusion protein of the invention, encoded by the third polynucleotide of the invention.

[0034] In another aspect, the present invention relates to a polynucleotide, hereinafter the fourth polynucleotide of the invention, encoding a fusion protein comprising from N-terminus to C-terminus:

[0035] (i) a modified N-intein selected from the group consisting of a modified GP41.1 N-intein, a modified IMPDH N-intein, a modified NrdJ1 N-intein or a modified GP41.8 N-intein, wherein the modified N-intein comprises a fragment of the N-intein protein that prevents trans-splicing from occurring in the presence of the corresponding C- intein and wherein the modified N-intein comprises at least one mutation that decreases the affinity between the modified N-intein and the corresponding C-intein,

[0036] (ii) a split C-intein comprising a sequence selected from the group consisting of 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-intein is a modified GP41.1 N-intein, the split C-intein comprises the sequence of SEQ ID NO: 1 or is a functionally equivalent variant thereof, - If the modified N-intein is a modified IMPDH N-intein, the split C-intein comprises the sequence of SEQ ID NO: 2 or is a functionally equivalent variant thereof, the modified N-intein is a modified IMPDH N-intein

[0037] - If the modified N-intein is a modified NrdJ1 N-intein, the split C-intein comprises the sequence of SEQ ID NO: 3 or is a functionally equivalent variant thereof or

[0038] - If the modified N-intein is a modified GP41.8 N-intein, the split C-intein comprises the sequence of SEQ ID NO: 4 or is a functionally equivalent variant thereof,

[0039] (iii) a transmembrane domain, and

[0040] (iv) an intracellular signalling domain from a receptor and / or costimulatory domain.

[0041] In another aspect, the present invention relates to a vector, hereinafter the fourth vector of the invention, which comprises the fourth polynucleotide of the invention.

[0042] In another aspect, the present invention relates to a host cell, hereinafter the fifth host cell of the invention, comprising the fourth polynucleotide of the invention, the fourth vector of the invention or the fusion protein encoded by the fourth polynucleotide of the invention.

[0043] In another aspect, the present invention relates to a fusion protein, hereinafter the fourth fusion protein of the invention, encoded by the fourth polynucleotide of the invention.

[0044] In another aspect, the present invention relates to a composition or kit-of-parts, hereinafter the second composition or the second kit-of-parts, comprising a first component and a second component wherein i) the first component is the third polynucleotide or fourth polynucleotide of the invention, the third vector or fourth vector of the invention, the fourth host cell or fifth host cell of the invention or the third fusion protein or fourth fusion protein of the invention and ii) the second component is the second fusion protein of the invention or the second polynucleotide of the invention, wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

[0045] In another aspect, the present invention relates to a method for cleaving the heterologous polypeptide from the third fusion protein of the invention comprising incubating the fusion protein with a split N-intein under conditions that allow intein splicing wherein

[0046] If the split C-intein forming part of the fusion protein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof, then the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof,

[0047] If the split C-intein forming part of the fusion protein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof, the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof,

[0048] If the split C-intein forming part of the fusion protein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof, then the split N- intein comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or

[0049] If the split C-intein forming part of the fusion protein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof, then and the split N- intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

[0050] 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 the second composition of the invention or bringing into association the components of the second kit-of-parts of the invention under conditions allowing intein splicing wherein said first polypeptide is the heterologous polypeptide which forms part of the third fusion protein of the invention and said second polypeptide is the heterologous polypeptide which forms part of the second fusion protein of the invention.

[0051] In another aspect, the present invention relates to an in vitro method, hereinafter the third method of the invention, for producing a cell comprising a chimeric receptor, wherein the method comprises:

[0052] (i) providing a cell comprising the fourth polynucleotide of the invention, a polypeptide encoded by said polynucleotide or the fourth vector of the invention, under conditions adequate for the expression in the cell of the fusion protein encoded by the polynucleotide, and

[0053] (ii) contacting the cell of step i) with the second fusion protein of the invention, wherein the contacting step ii) is performed under conditions that allow a splicing reaction between the split C-intein forming part of the fusion protein produced in step (i) and the split N-intein forming part of the second fusion proteins of the invention and wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO: 1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

[0054] In another aspect, the present invention relates to an in vitro method, hereinafter the fourth method of the invention, for producing a cell comprising a chimeric receptor, wherein the method comprises:

[0055] (i) providing a cell comprising a. the fourth polynucleotide of the invention, the fourth vector of the 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 in frame and N-terminal to the modified N-intein and which allows the insertion of the fusion protein into the membrane and b. the second polynucleotide of the invention, the second vector of the invention or the second fusion protein of the 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 in frame and N-terminal to the polypeptide that specifically binds to a target molecule and which allows the secretion of the fusion protein from the cell and

[0056] (ii) maintaining the cell under conditions adequate for the expression in the cell of both fusion proteins, for the insertion into the membrane of the fusion protein encoded by the fourth polynucleotide of the invention, for the secretion of the fusion protein encoded by the second polynucleotide of the invention and for the splicing reaction between the split C-intein and the split N-intein and wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO: 3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof and the split N-intein 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 cell, hereinafter the second cell of the invention, comprising a chimeric receptor obtained by the third method or the fourth method of the invention.

[0058] In another aspect, the present invention relates to the third host cell of the invention or the second cell of the invention for use in the treatment of a disease which requires a cell-mediated immune response against a target molecule, preferably against the antigen of interest. In another aspect, the present invention relates to the second fusion protein of the invention, the second polynucleotide of the invention, the second vector of the invention or the second host cell of the invention for use in the treatment of a disease which requires a cell-mediated immune response against a target molecule, preferably against an antigen of interest, wherein the patient to be treated is characterized in that it contains cells comprising the fourth polynucleotide of the invention, the fourth vector of the invention, or the fusion protein encoded by the fourth polynucleotide of the invention, wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

[0059] In another aspect, the present invention relates to the fourth polynucleotide of the invention, the fourth vector of the invention, the fifth host cell of the invention, or the fusion protein encoded by the fourth polynucleotide of the invention for use in the treatment of a disease which requires a cell-mediated immune response against a target molecule, preferably against an antigen of interest, wherein the patient to be treated is characterized in that it contains cells comprising the second fusion protein of the invention, the second polynucleotide of the invention, the second vector of the invention or the second host cell of the invention, wherein

[0060] - the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

[0061] BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1. Expression, purification and analysis of trans-splicing activity of an aCD19 scFv-gp41.1-N-ST intein fusion. A) a-Strep tag Western blot analysis of the expression of the aCD19 scFv-gp41.1-N-ST intein fusion in HEK293F cells at 144 and 168 hours post-transfection (hpt). B) Coomassie stained affinity purification analysis of a culture supernatant containing the aCD19 scFv-gp41.1-N-ST intein fusion. The analysis shows the protein content of the chromatographic input (in), flow-through (FT), wash (w) and the recovered elution fractions (11 - 27). C) Coomassie stained analysis of a trans- splicing reaction between the aCD19 scFv-gp41.1-N-ST intein fusion and the gp41.1-C- Trx fusion. Lane description: M, protein molecular weight marker; IN, pure aCD19 scFv- gp41.1-N-ST intein fusion at a concentration equivalent to that initially added to the reaction; IC, pure gp41.1-C-Trx intein fusion at a concentration equivalent to that initially added to the reaction; to, aliquot corresponding to the start of the reaction; 1h - 4h, aliquots extracted from the reaction after 1 h, 2h and 4h of incubation, either with or without DTT. The formation of the splicing product (SP) aCD19 scFv-Trx in the reaction with DTT is shown.

[0063] Figure 2. Expression, purification and analysis of trans-splicing activity of an aBCMA scFv-IMPDH.1-N-His intein fusion. A) a-His tag Western blot analysis of the expression of the aBCMA scFv-IMPDH.1-N-His intein fusion in HEK293F cells at 144 and 168 hours post-transfection (hpt). B) Coomassie stained affinity purification analysis of a culture supernatant containing the aBCMA scFv-IMPDH.1-N-His intein fusion. The analysis shows the protein content of the chromatographic input (in), flow-through (FT), wash (w) and the recovered elution fractions (T13 - T21). C) Coomassie stained analysis of a trans-splicing reaction between the aBCMA scFv-IMPDH.1-N-His intein fusion and the model ST-SUMO-His-IMPDH.1-C-VHH IntC fusion. Lane description: M, protein molecular weight marker; IN, pure aBCMA scFv-IMPDH.1-N-His intein fusion at a concentration equivalent to that initially added to the reaction; IC, pure ST-SUMO-His- IMPDH.1-C-VHH intein fusion at a concentration equivalent to that initially added to the reaction; to, aliquot corresponding to the start of the reaction; 2h and 4h, aliquots extracted from the reaction after 2h and 4h of incubation, respectively; FT and res, aliquots extracted from the flow-through and elution of the negative IMAC procedure, either with or without DTT. The formation of the splicing product (SP) aBCMA scFv-VHH in the reaction with DTT is shown in the flow-through of the negative IMAC of the reaction with DTT.

[0064] Figure 3. Expression, purification and analysis of trans-splicing activity of an aCD22 scFv-gp41.8-N-His intein fusion. A) a-His tag Western blot analysis of the expression of the aCD22 scFv-gp41.8-N-His intein fusion in HEK293F cells at 120, 144 and 168 hours post-transfection (hpt). B) Coomassie stained affinity purification analysis of a culture supernatant containing the aCD22 scFv-gp41.8-N-His intein fusion. The analysis shows the protein content of the chromatographic input (in), flow-through (FT), wash (w) and the recovered elution fractions (T6 - T10). C) Coomassie stained analysis of a trans- splicing reaction between the aCD22 scFv-gp41.8-N-His intein fusion and the gp41.8-C- LipTla fusion. Lane description: M, protein molecular weight marker; IN, pure aCD22 scFv-gp41 ,8-N-His intein fusion at a concentration equivalent to that initially added to the reaction; IC, pure gp41.8-C-LipTla intein fusion at a concentration equivalent to that initially added to the reaction; to, aliquot corresponding to the start of the reaction; 1 h - 4h, aliquots extracted from the reaction after 1 h, 2h and 4h of incubation, either with or without DTT. The formation of the splicing product (SP) aCD22 scFv-LipTla occurs in the reaction with DTT.

[0065] Figure 4. Expression, purification and analysis of trans-splicing activity of an aCD1a scFv-NrJ.1-N-His intein fusion. A) a-His tag Western blot analysis of the expression of the aCD1a scFv-NrdJ.1-N-His intein fusion in HEK293F cells at 120, 144 and 168 hours post-transfection (hpt). B) Coomassie stained affinity purification analysis of a culture supernatant containing the aCD1a scFv-NrdJ.1-N-His intein fusion. The analysis shows the protein content of the chromatographic input (in), flow-through (FT), wash (w) and the recovered elution fractions (T 14 - T22). C) Coomassie stained analysis of a trans- splicing reaction between the aCD1a scFv-NrdJ.1-N-His intein fusion and the NrdJ.1-C- LipTla fusion. Lane description: M, protein molecular weight marker; IN, pure aCD1a scFv-NrdJ.1-N-His intein fusion at a concentration equivalent to that initially added to the reaction; IC, pure NrdJ.1-C-LipTla intein fusion at a concentration equivalent to that initially added to the reaction; to, aliquot corresponding to the start of the reaction; 1 h - 4h, aliquots extracted from the reaction after 1 h, 2h and 4h of incubation, either with or without DTT. The formation of the splicing product (SP) aCD1a scFv-LipTla occurs in the reaction with DTT.

[0066] Figure 5. Expression of IntC-CAR fusions in HEK293F cells. A) Schematic representation of the IntC-CAR constructs. SP: signal peptide; His: 6xHis 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; 4-1 BB and CD3z: intracellular signaling domains. B) Flow cytometry analysis of the expression of the different IntC- CAR constructs with an APC-labelled anti-His antibody. C) Anti-CD3z Western blot analysis of the IntC-CAR fusions integrity of the gp41.1 (G1), IMPDH.1 (11), NrdJ.1 (N1) and gp41.8 (G8) inteins. m: monomer; m + PTM: monomer + post-translational modification; glyco m: glycosylated monomer.

[0067] Figure 6. Expression of caged IntC-CAR fusions in HEK293F cells. A) Schematic representation of the caged IntC-CAR constructs. SP: signal peptide; ST: Strep tag; INTNcage: C-terminal fragment of the corresponding IntN; linker: peptide linker; His: 6xHis 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; 4-1 BB and CD3z: intracellular signaling domains. B) Expression levels of uncaged and caged IntC-CAR constructs obtained by flow cytometry analysis using an APC-labelled a-His antibody. G1 : gp41.1-CAR; 11 : IMPDH.1-C-CAR; N1 : NrdJ.1-C-CAR; G8: gp41.8-C-CAR C) Comparison between the flow cytometry analysis obtained with an APC-labelled a- His antibody of the expression of caged and uncaged IntC-CAR constructs of the gp41.1 , IMPDH.1 , NrdJ.1 and gp41.8 inteins.

[0068] Figure 1. Flow cytometry analysis of a trans-splicing reaction between HEK293F cells expressing the gp41.1-C-CAR construct and the O-CD19 scFv-gp41.1-N intein fusion. A) Schematic representation of a trans-splicing reaction between cells expressing an IntC-CAR on the surface and a scFv-lntN fusion to produce a full-length scFv-CAR. B) Flow cytometry analysis of the trans-splicing reaction between the gp41.1-C-CAR exposed on the surface of transfected HEK293F cells and the O-CD19 scFv-gp41.1-N- ST using an APC-labelled a-His antibody to determine the expression level of the gp41.1 - C-CAR and a PE-labelled a-FLAG antibody to detect cells containing the reconstituted a-CD19 scFv-CAR. C) Flow cytometry analysis of the trans-splicing reaction between the gp41.1 -C-CAR exposed on the surface of transfected HEK293F cells and the a- CD19 scFv-gp41.1-N-ST with a PE-labelled CD19 antigen. D) aCD3z Western blot analysis of cells expressing the gp41.1-C-CAR before (-) and after (+) a trans-splicing reaction with the O-CD19 scFv-gp41.1-N intein fusion.

[0069] Flow cytometry analysis of a trans-splicing reaction between HEK293F cells expressing the IMPDH.1-C-CAR construct and the a-BCMA scFv-IMPDH.1-N intein fusion. A) Flow cytometry analysis of the trans-splicing reaction between the IMPDH.1- C-CAR exposed on the surface of transfected HEK293F cells and the a-BCMA scFv- IMPDH.1-N-His using an APC-labelled a-His antibody to determine the expression level of the IMPDH.1-C-CAR and a PE-labelled a-FLAG antibody to detect cells containing the reconstituted a-BCMA scFv-CAR. B) aCD3z Western blot analysis of cells expressing the IMPDH.1-C-CAR that have undergone a trans-splicing reaction with the a-BCMA scFv-IMPDH.1-N intein fusion. C) Schematic representation of the IMPDH.1-C- linker-CAR construct. SP: signal peptide; His: 6xHis tag; INTC: IntC domain corresponding to the IMPDH.1-C intein; linker: amino acid peptide; CD8-HR: CD8 hinge region; CD8 TM: CD8 transmembrane domain; 4-1 BB and CD3z: intracellular signaling domains. D) Flow cytometry analysis of the trans-splicing reaction between the IMPDH.1-C-linker-CAR exposed on the surface of transfected HEK293F cells and the a- BCMA scFv-IMPDH.1-N-His using an APC-labelled a-His antibody to determine the expression level of the IMPDH.1-C-linker-CAR and a PE-labelled a-FLAG antibody to detect cells containing the reconstituted a-BCMA scFv-linker-CAR.

[0070] Flow cytometry analysis of a trans-splicing reaction between expressing the caged gp41.1-C-CAR construct and the a-CD19 scFv-gp41.1-N intein fusion. A) Flow cytometry analysis of the trans-splicing reaction between the caged gp41.1-C-CAR exposed on the surface of transfected HEK293F cells and the a-CD19 scFv-gp41.1-N-ST using an APC-labelled a-His antibody to determine the expression level of the gp41.1-C-CAR and a PE-labelled a-FLAG antibody to detect cells containing the reconstituted a-CD19 scFv-CAR. B) Flow cytometry analysis of the trans-splicing reaction between the caged gp41.1-C-CAR exposed on the surface of transfected HEK293F cells and the a-CD19 scFv-gp41.1-N-ST with a PE-labelled CD19 antigen. C) aCD3z Western blot analysis of cells expressing the caged gp41.1-C-CAR before (-) and after (+) a trans-splicing reaction with the a-CD19 scFv-gp41.1-N intein fusion.

[0071] 10. Flow cytometry analysis of a trans-splicing reaction between HEK293F cells expressing the caged IMPDH.1-C-CAR construct and the a-BCMA scFv-IMPDH.1-N intein fusion. A) Flow cytometry analysis of the expression of the caged IMPDH.1-C-CAR in transfected HEK293F cells using an APC-labelled a-His antibody, which detects the His tag present in the construct, and flow cytometry analysis of the trans-splicing reaction between the caged IMPDH.1-C-CAR and the a-BCMA scFv-IMPDH.1-N-His using a PE- labelled a-FLAG antibody to detect cells containing the reconstituted a-BCMA scFv- CAR. B) aCD3z Western blot analysis of cells expressing the caged IMPDH.1-C-CAR that has undergone a trans-splicing reaction with the a-BCMA scFv-IMPDH.1-N intein fusion.

[0072] Figure 11. Comparison of the percentage of cells with high levels of reconstituted scFv- CAR on the surface between cells expressing a caged IntC-CAR and cells expressing an uncaged IntC-CAR. A) Comparison of the percentage of cells with high levels of reconstituted aCD19 scFv-CAR between cells expressing the caged gp41.1-C-CAR and cells expressing the uncaged gp41.1-C-CAR. The flow cytometry analysis of the trans- splicing reaction was done with a PE-labelled CD19 antigen. B) Comparison of the percentage of cells with high levels of reconstituted aBCMA scFv-CAR between cells expressing the caged IMPDH.1-C-CAR and cells expressing the uncaged IMPDH.1-C- CAR. The flow cytometry analysis of the trans-splicing reaction was done with a PE- labelled a-FLAG antibody.

[0073] Figure 12. Western blot analysis of the co-transfection of HEK293F cells with plasmids encoding the gp41.1-C-CAR fusion, caged or uncaged, and a scFv-gp41.1-N fusion. A) aCD3z Western blot analysis of cells co-transfected with the gp41.1-C-CAR. B) aCD3z Western blot analysis of cells co-transfected with the caged gp41 ,1-C-CAR. In both gels, lanes 1 and 2 correspond to the co-transfection with the scFv-IMPDH.1-N construct, for which no trans-splicing can occur, and lanes 3 and 4 correspond to the co-transfection with the scFv-gp41 ,1-N construct. The gels show the formation of the reconstituted scFv- CAR 48 and 72 hours after transfection. The lower panel shows the bands corresponding to the precursor IntC-CAR fusions (gp41.1-C-CAR in gel A and caged gp41.1-C-CAR in gel B) and the upper panel shows the trans-splicing product (scFv-CAR in both gels).

[0074] Figure 13. Western blot analysis of the co-transfection of HEK293F cells with plasmids encoding different IntC-CAR fusions, caged or uncaged, and a scFv-lntN fusion. A) aCD3z Western blot analysis of cells co-transfected with the plasmid combinations described in the table shown below the Western blot membrane. The trans-splicing products obtained in the co-transfections are labeled as indicated in the table. B) aFLAG Western blot analysis of cells co-transfected with the plasmid combinations described in the table shown below the Western blot membrane. For each sample, the cell lysate and the growth medium containing the secreted proteins were loaded onto the gel as indicated in the table. C) Flow cytometry analysis of cells co-transfected with the plasmid combination indicated below each graph. Cells were incubated with a combination of two antibodies, an APC-labelled a-His antibody to detect the expression of the gp41.1-C- CAR construct and a PE-labelled a-FLAG antibody to detect the expression of the reconstituted O-CD19 scFv-CAR.

[0075] Figure 14. Western blot analysis of the co-transfection of HEK293F cells with plasmids encoding the IMPDH.1-C-CAR fusion, caged or uncaged, and a scFv-gp41.1-N fusion. A) aCD3z Western blot analysis of cells co-transfected with the IMPDH.1-C-CAR. B) aCD3z Western blot analysis of cells co-transfected with the caged IMPDH.1-C-CAR. In both gels, lanes 1 , 2 and 3 correspond to the co-transfection with the CD19scFv-gp41.1- N construct, for which no trans-splicing can occur; lanes 3, 4 and 5 correspond to the co- transfection with the CD19scFv-IMPDH.1-N construct; lanes 6, 7 and 8 correspond to the co-transfection with the BCMAscFv-IMPDH.1-N construct. The gels show the formation of the reconstituted scFv-CAR 24, 48 and 72 hours after transfection. The lower panel shows the bands corresponding to the precursor IntC-CAR fusions (IMPDH.1-C-CAR in gel A and caged IMPDH.1-C-CAR in gel B) and the upper panel shows the trans-splicing product (scFv-CAR in both gels).

[0076] Figure 15. aCD3z Western blot analysis of the co-transfection of HEK293F cells with plasmids encoding a gp41.1-C-CAR and an aPSMA IgG fused to gp41.1-N at the C- terminus of the heavy chain. Lane 1 corresponds to the co-transfection with the CD19scFv-IMPDH.1-N construct, for which no trans-splicing can occur. Lane 2 corresponds to the co-transfection with the PSMAIgG-HC-gp41.1-N construct, which generates a reconstituted CAR with an aPSMA targeting molecule (PSMA-HC-CAR).

[0077] Figure 16. Integration of IntC-CAR does not affect T cell activation and proliferative status. A) Schematic representation of the IntC-CAR constructs used for T cell transduction. SP: signal peptide; His: 6xHis tag; INTC: IntC domain corresponding to IMPDH.1-C or NrdJ.1-C; CD8-HR: CD8 hinge region; CD8 TM: CD8 transmembrane domain; 4-1 BB and CD3z: intracellular signaling domains; T2A sequence; and GFP: green fluorescent protein. B) Proliferation assay of T cells expressing an IntC-CAR construct. Two different IntC-CAR constructs were analyzed: NrdJ.1-C-CAR and IMPDH.1-CAR. No significant differences in cell proliferation were observed as compared to non-transduced activated T cells (sample number= 3).

[0078] 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 phenotype of NrdJ.1-C-CAR and IMPDH.1-C-CAR T cells. Data from one representative donor are shown. Summary (n = 5) of B) uninfected T cells, C) NrdJ.1-C-CAR T cells, and D) IMPDH.1-C-CAR T cells. NaTve / SCM: CCR7+CD45RA+; central memory: CCR7+CD45RA+; effector memory: CCR7-CD45RA-; and terminally differentiated effector memory or TEMRA: CCR7- CD45RA-.

[0079] Figure 18. Trans-splicinq reaction between IMPDH.1-C-CAR T cells and purified aCD1a scFv-IMPDH.1-N fusion. A) The trans-splicing reaction between IMPDH.1-C-CAR T cells and the aCD1a scFv-IMPDH.1-N fusion is specific for the IMPDH.1 intein pair, because when the aCD1a scFv-IMPDH.1-N fusion is combined with NrdJ.1-C-CAR T cells, these cells don’t incorporate the aCD1a scFv on the surface. B) 72.4% of the activated T cells transduced with the IMPDH.1-C-CAR construct express it on the surface. After the trans- splicing reaction with reduced aCD1a scFv-IMPDH.1-N, 63.2 % of the cells contain the aCD1a scFv on the surface, resulting in a trans-splicing efficiency of 87%. C) The trans- splicing efficiency depends on the T cell donor, but it is always higher than 60%. Summary of three independent T cell donors showing the initial percentage of His+ cells and the percentage of aCD1a scFv+ cells after trans-splicing. Trans-splicing efficiency is calculated as (%His+ / %scFv+) * 100.

[0080] Figure 19. aCD1a scFv-CAR T cells reconstituted by IMPDH.1 intein trans-splicing show cytotoxicity against CD1a+ T-ALL cells. A) Gating strategy for MOLT4 cell death analysis. An equal number of cells from a wild-type CD1a-expressing MOLT4 cell line (a T-ALL cell line), previously stained with Cell Violet tracer, and a GFP-expressing CDIako MOLT4 cell line, also previously stained with Cell Violet tracer, were mixed. GFP expression was used as a surrogate for CD1a negativity. For the analysis of the effect of □CD1a scFv-CAR T cells on the MOLT4 mixture, the GFP signal obtained by flow cytometry (CD1a negativity) after gating on the Cell Violet+ cell population was used. B) Representative example of the results obtained after 24 hours of co-incubation between the MOLT4 mixture and aCD1a scFv-CAR T cells obtained after a trans-splicing reaction between IMPDH.1-C-CAR T cells and a reduced aCD1a scFv-IMPDH.1-N fusion. The star indicates the decrease of GFP-, i.e. CD1a+, MOLT4 cells after incubation. C) Specific cytotoxicity of aCD1a scFv-CAR T cells obtained under different trans-splicing conditions against CD1a+ MOLT4 cells after 24 (left) and 48 h (right). D) IFN-Y secretion at 24 (left) and 48 h (right) obtained under each condition shown in figure C. The graphs in figures C and D summarize the results from three independent donors.

[0081] DETAILED DESCRIPTION OF THE INVENTION First polynucleotide of the invention

[0082] In a first aspect, the present invention relates to a polynucleotide, hereinafter the first polynucleotide of the invention, encoding a fusion protein comprising from N-terminus to C-terminus: i) a split C-intein comprising a sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 or a functionally equivalent variant thereof, ii) a transmembrane domain, and iii) an intracellular signalling domain from a receptor and / or costimulatory domain.

[0083] The term "nucleic acid," "polynucleotide," or "nucleic acid molecule" as used herein refers to a polymeric compound comprised of covalently linked subunits called nucleotides. Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single-stranded or double-stranded. DNA includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA. The polynucleotides may contain deoxyribonucleotides, ribonucleotides, and / or their analogs. Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The term "polynucleotide" includes, for example, single-stranded, double-stranded and triple helical molecules, a gene or gene fragment, 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 a native nucleic acid molecule, a nucleic acid molecule of the present invention may also comprise modified nucleic acid molecules.

[0084] The first polynucleotide of the invention encodes a fusion protein. The term “fusion protein” or “fusion polypeptide” refers to a protein or a polypeptide comprised of at least two polypeptides and optionally a linking sequence to operatively link the two polypeptides into one continuous polypeptide. The two polypeptides linked in a fusion polypeptide are typically derived from two independent sources, and therefore a fusion polypeptide comprises two linked polypeptides not normally found linked in nature. The two polypeptides may be operably attached directly by a peptide bond or may be linked indirectly through a linker described herein or otherwise known in the art.

[0085] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. In a preferred embodiment the polypeptide is exclusively formed by amino acids.

[0086] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Furthermore, the term "amino acid" includes both D- and L-amino acids (stereoisomers). Preferably, the amino acids are L-amino acids.

[0087] The term "natural amino acids" or “naturally occurring amino acids” comprises the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.

[0088] The first polynucleotide of the invention encoding a fusion protein comprises i) a split C- intein comprising a sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 or a functionally equivalent variant thereof.

[0089] The term “intein” refers to a protein element that is capable of self-excising from a host protein and catalyzing ligation of the flanking sequences with a peptide bond.

[0090] The term “split inteins” refers to any intein in which the N-terminal domain of the intein and the C-terminal domain of the intein are not directly linked via a peptide bond. Natural split inteins have been identified in cyanobacteria and archaea, but split inteins can also be created artificially by separating an intein's sequence into two pieces. The split inteins used in the present invention can comprise the six conserved protein-splicing motifs of the HINT (Hog / lntein) family.

[0091] The term “split C-intein” refers to the C-terminal domain of an intein. An "intein C-terminal domain" refers to an intein sequence that comprises a C-terminal amino acid sequence that is functional for trans-splicing reactions and / or C-terminal self-cleavage reactions. The intein C-terminal domain can comprise the C1 and / or C2 motifs of the HINT (Hog / lntein) family. The first polynucleotide of the invention encoding a fusion protein comprises a split C- intein comprising a sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 or a functionally equivalent variant thereof.

[0092] SEQ ID NO: 1 (MMLKKILKIE ELDERELIDI EVSGNHLFYA NDILTHN) refers to the C- terminal domain of the GP41.1 split intein.

[0093] SEQ ID NO: 2 (MKFKLKEITS IETKHYKGKV HDLTVNQDHS YNVRGTWHN) refers to the C-terminal domain of the IMPDH1 split intein.

[0094] SEQ ID NO: 3 (MEAKTYIGKL KSRKIVSNED TYDIQTSTHN FFANDILVHN) refers to the C-terminal domain of the NrdJ1 split intein.

[0095] SEQ ID NO: 4 (MCEIFENEID WDEIASIEYV GVEETIDINV TNDRLFFANG ILTHN) refers to the C-terminal domain of the GP41 .8 split intein.

[0096] The term “functionally equivalent variant of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4” relates to all those sequences which result from the modification, insertion and / or deletion of one or more amino acids, including resulting post- translational modifications such as glycosylation, phosphorylation, hydroxylation, acylation, alkylation, gamma-carboxylation, nucleotide addition, iodination, propionylation, S-glutathionylation, S-nitrosylation, succinylation, sulfation, from the above sequences, provided that the function of the split intein is substantially maintained. Suitable assays for determining the intein activity of functionally equivalent variants are as described in the patent application published as WQ2013045632A1.

[0097] “Post-translational modification” (PTM) refers to the covalent process of changing proteins following protein biosynthesis. PTMs may involve enzymes or occur spontaneously. Proteins are created by ribosomes translating mRNA into polypeptide chains, which may then change to form the mature protein product. Post-translational modifications can occur on the amino acid side chains or at the protein's C- or N- termini. Sites that often undergo post-translational modification are those that have a functional group that can serve as a nucleophile in the reaction: the hydroxyl groups of serine, threonine, and tyrosine; the amine forms of lysine, arginine, and histidine; the thiolate anion of cysteine; the carboxylates of aspartate and glutamate; and the N- and C-termini. In addition, although the amide of asparagine is a weak nucleophile, it can serve as an attachment point for glycans. Rarer modifications can occur at oxidized methionines and at some methylene groups in side chains. Post-translational modification of proteins can be experimentally detected by a variety of techniques, including mass spectrometry, Eastern blotting, and Western blotting.

[0098] 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 substituted by a preserved or non-preserved amino acid residue (preferably a preserved amino acid residue) and such substituted amino acid may be coded or not by the genetic code, (ii) polypeptides in which there is one or more modified amino acid residues, for example, residues modified by substituent bonding, (iii) polypeptides resulting from alternative processing of a similar mRNA, (iv) polypeptide fragments and / or (v) polypeptides resulting from fusion of the polypeptide defined in (i) to (iii) with another polypeptide, such as a secretory leader sequence or a sequence being used for purification (for example, His tag) or for detection (for example, Sv5 epitope tag). The fragments include polypeptides generated through proteolytic cut (including multisite proteolysis) of an original sequence. The variants may be post-translationally or chemically modified. Such variants are supposed to be apparent to those skilled in the art.

[0099] One skilled in the art will recognize that the values of identity of nucleotide sequences can be appropriately adjusted in order to determine the corresponding sequence identity of two nucleotide sequences encoding the polypeptides of the present invention, by taking into account codon degeneracy, conservative amino acid substitutions, and reading frame positioning.

[0100] In the context of the present invention "conservative amino acid changes" and "conservative amino acid substitution" are used synonymously in the invention. "Conservative amino acid substitutions" refers to the interchangeability of residues having similar side chains, and mean substitutions of one or more amino acids in a native amino acid sequence with another amino acid(s) having similar side chains, resulting in a silent change that does not alter function of the protein. Conserved substitutes for an amino acid within a native amino acid sequence can be selected from other members of the group to which the naturally occurring amino acid belongs. For example, a group of amino acids having aliphatic side chains includes glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains includes serine and threonine; a group of amino acids having amide-containing side chains includes asparagine and glutamine; a group of amino acids having aromatic side chains includes phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains includes lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains includes cysteine and methionine. In some embodiments of the invention, preferred conservative amino acids substitutions are: valine-leucine, valineisoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. Thus, the invention refers to functionally equivalents variants of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; and that have an amino acid sequence differing in one or more amino acids with the sequence given as the result of one or more conservative amino acid substitutions. It is well known in the art that one or more amino acids in a polypeptide sequence can be substituted with at least one other amino acid having a similar charge and polarity such that the substitution / s result in a silent change in the modified polypeptide that does not alter its function relative to the function of the non-modified sequence. The invention refers to any polypeptide sequence differing in one or more amino acids, either as a result of conserved or non-conserved substitutions, and / or either as a result of sequence insertions or deletions, relative to the sequence given by SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, as long as said further provided polypeptide sequence has the same or similar of intein C-terminal domains as SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0101] In some embodiments, the functionally equivalent variant of the split C-intein is characterized in that it comprises a C2 box (F box) sequence. The C2 box is a non- strictly conserved sequence. The C2 box can comprise, for example, the sequence XhhDIpVXXpHXFX (SEQ ID NO: 9), wherein X is any amino acid, wherein h is a hydrophobic amino acid and p is a polar amino acid. In some embodiments, the intein C-terminal domain comprises the sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 10), wherein Xi is N, E, L, K, Q, D, P, or R; X2is V, L, or T; X3is Y, I, V, H, or F; X4is D; X5is I or L; X6is G, E, T, Q, or K; X7is V or T; X8is E, S, T, D, N, or K; X9is R, G, D, N, Q, S, or K; X is D, E, N, T, or K; Xu is H, R, S, I, or N; X12 is N, L, S, I, or N; X13 is F, Y, L, or I; and X14 is A, Y, F, N, C, or S. In some embodiments, the intein C- terminal domain comprises the sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 11), wherein Xi is E, L, K, Q, D, P, or R; X2is V, L, or T; X3is Y, I, V, H, or F; X4is

[0102] D; X5is I or L; X6is G, E, T, Q, or K; X7 is V or T; X8is E, S, T, D, N, or K; X9is G, D, N,

[0103] Q, S, or K; X10 is D, E, N, T, or K; X11 is H, R, S, I, or N; X12 is N, L, S, I, or N; X13 is F,

[0104] Y, L, or I; and X14 is A, Y, F, N, C, or S.

[0105] Based on chemical properties of the amino acids, they can be grouped as: (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).

[0106] In some embodiments, the functionally equivalent variant of the split C-intein is characterized in that it comprises part of the C1 box (G box) sequence. The C1 box is a non-strictly conserved sequence. The C1 box can comprise, for example, the sequence hNXIhXHNn (SEQ ID NO: 12), wherein X is any amino acid, wherein h is a hydrophobic amino acid and n is a nucleophilic amino acid. In some embodiments, the intein C- terminal domain comprises the sequence X1X2X3X4X5X6X7X8X9 (SEQ ID NO: 13), wherein Xi 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; Xe is V, I, T, or A; X7 is H or S; Xs is N; and X9 is S, T, or C. In some embodiments, the intein C-terminal domain comprises the sequence X1X2X3X4X5X6X7X8X9 (SEQ ID NO: 14), wherein Xi 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; Xe is V, I, or T; X7 is H; Xs is N; and Xg is S, T, or C. Within the C1 box sequence, the amino acids from Xi to Xs correspond to the intein sequence, and X9 corresponds to the first amino acid of the extein.

[0107] The last two amino acids in an intein C-terminal domain are highly conserved and are important for the protein splicing reaction. Therefore, in some embodiments, the last amino acid in an intein C-terminal domain is an asparagine. In some embodiments, the last amino acid in an intein C-terminal domain is a glutamine. In some embodiments, the penultimate amino acid in an intein C-terminal domain is a histidine. In some embodiments, the last amino acid in an intein C-terminal domain is an amino acid other than asparagine or glutamine. In some embodiments, the penultimate amino acid in an intein C-terminal domain is an amino acid other than histidine. In some embodiments, the penultimate amino acid in an intein C-terminal domain is an amino acid other than serine. For example, the last amino acid and / or penultimate amino acid in an intein C- terminal domain can be an alanine.

[0108] In some embodiments, the intein C-terminal domain is about 10 to about 80 amino acids. In some embodiments, the intein C-terminal domain is about 20 to about 70 amino acids. In some embodiments, the intein C-terminal domain is about 30 to about 60 amino acids. In some embodiments, the intein C-terminal domain 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.

[0109] The terms “identity”, "identical" or “percent identity" in the context of two or more amino acid, or nucleotide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid or nucleotide residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences.

[0110] The percentage of sequence identity may be determined by comparing two optimally aligned sequences over a comparison window. The aligned sequences may be polynucleotide sequences or polypeptide sequences. For optimal alignment of the two sequences, the portion of the polynucleotide or amino acid sequence in the comparison window may comprise insertions or deletions (i.e. , gaps) as compared to the reference sequence (that does not comprise insertions or deletions). The percentage of sequence identity is calculated by determining the number of positions at which the identical nucleotide residues, or the identical amino acid residues, occurs in both compared sequences to yield the number of matched positions, then dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Sequence identity between two polypeptide sequences or two polynucleotide sequences can be determined, for example, by using the Gap program in the WISCONSIN PACKAGE version 10.0-UNIX from Genetics Computer Group, Inc. based on the method of Needleman and Wunsch (J. Mol. Biol. 48:443-453, 1970) using the set of default parameters for pairwise comparison (for amino acid sequence comparison: Gap Creation Penalty=8, Gap Extension Penalty=2; for nucleotide sequence comparison: Gap Creation Penalty=50; Gap Extension Penalty=3), or using the TBLASTN program in the BLAST 2.2.1 software suite (Altschul et al., Nucleic Acids Res. 25:3389-3402), using BLOSUM62 matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. U.S.A. 89:10915- 10919, 1992) and the set of default parameters for pair-wise comparison (gap creation cost=11 , gap extension cost=1).

[0111] Identity can exist over a region of the sequences that is at least about 10, about 20, about 40-60 residues in length or any integral value there between, and can be over a longer region than 60-80 residues, for example, at least about 90-100 residues, and in some embodiments, the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a nucleotide sequence for example. Functionally equivalent variants of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 also include sequences with a sequence identity of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%,

[0112] 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %,

[0113] 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%,

[0114] 97%, 98%, or 99% with the sequences SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 and

[0115] SEQ ID NO: 4, respectively.

[0116] In some embodiments, the functionally equivalent variant of SEQ ID NO: 1 is a sequence with a sequence identity of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,

[0117] 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%,

[0118] 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,

[0119] 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the SEQ ID NO:

[0120] 1.

[0121] In some embodiments, the functionally equivalent variant of SEQ ID NO: 2 is a sequence with a sequence identity of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%,

[0122] 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%,

[0123] 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,

[0124] 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the SEQ ID NO:

[0125] 2.

[0126] In some embodiments, the functionally equivalent variant of SEQ ID NO: 3 is a sequence with a sequence identity of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%,

[0127] 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%,

[0128] 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,

[0129] 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the SEQ ID NO:

[0130] 3.

[0131] In some embodiments, the functionally equivalent variant of SEQ ID NO: 4 is a sequence with a sequence identity of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%,

[0132] 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%,

[0133] 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,

[0134] 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the SEQ ID NO:

[0135] 4.

[0136] In some embodiments, the intein C-terminal domain contains at least about 10, at least about 20, at least about 30, at least about 40, or at least about 50 amino acids of a sequence selected from the group consisting of SEQ ID NO: 1 , 2, 3 and 4. In some embodiments, the intein C-terminal domain 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 of a sequence selected from the group consisting of SEQ ID NO: 1 , 2, 3 and 4. In some embodiments, the intein C-terminal domain contains a deletion of no more than about 5, about 10, about 15, about 20, or about 25 amino acids of a sequence selected from the group consisting of SEQ ID NO: 1 , 2, 3 and 4. In some embodiments, the intein C- terminal domain contains a deletion of no more than about 5, about 10, about 15, about 20, or about 25 consecutive amino acids of a sequence selected from the group consisting of SEQ ID NO: 1 , 2, 3 and 4.

[0137] In an embodiment, the functionally equivalent variant of SEQ ID NO: 1 , 2, 3 or 4 has a sequence identity of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,

[0138] 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%,

[0139] 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,

[0140] 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the corresponding sequence SEQ ID NO, 1 , 2, 3 or 4 and the sequence identity is determined throughout the whole length of the sequence SEQ ID NO: 1 , 2, 3 or 4.

[0141] The first polynucleotide of the invention encoding a fusion protein comprises ii) a transmembrane domain.

[0142] As used herein, "transmembrane domain" (TMD) refers to a membrane-spanning protein domain. The transmembrane domain is the transmembrane domain of a transmembrane protein (e.g., a type I transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. TMDs may consist of one or several alpha-helices or a transmembrane beta barrel. Because the interior of the lipid bilayer is hydrophobic, the amino acid residues in TMDs are often hydrophobic, although proteins such as membrane pumps and ion channels can contain polar residues. A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region). The transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membranebound or transmembrane protein. Non limiting examples or transmembrane domains of particular use in this invention may include at least the transmembrane region(s) of e.g., the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD3 zeta, KIRDS2, 0X40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD 278), 4-1 BB (CD137), GITR, CD40, CTLA4, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1 , VLA1 , CD49a, ITGA4, IA4 CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDGA, CDGA, CD103, ITGAL, CDLa, LFA- 1 , ITGAM, CDIIb, ITGAX, CDIc, 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) , LylOS), SLAM (SLAMF1 , CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, Kp30, NKp46, including NKG2D, and / or a transmembrane domain selected from the transmembrane domain of NKG2C.

[0143] In a particular embodiment, the transmembrane domain is selected from the group consisting of the CD28, the CD8a, the 4-1 BB, the CD4, the CTLA4, the CD27 or the CD3 transmembrane domain.

[0144] The first polynucleotide of the invention encoding a fusion protein comprises iii) an intracellular signalling domain from a receptor and / or costimulatory domain. Thus, in a particular embodiment, the first polynucleotide of the invention encoding a fusion protein comprises iii) an intracellular signalling domain from a receptor. In another particular embodiment, the first polynucleotide of the invention encoding a fusion protein comprises iii) a costimulatory domain.

[0145] “Intracellular signalling domain” or “signalling domain” as used herein refers to the intracellular portion of a molecule and more specifically to any oligopeptide or polypeptide known to function as a domain that transmits a signal to cause activation or inhibition of a biological process in a cell.

[0146] In a particular embodiment, the intracellular signalling domain from a receptor is an intracellular signalling domain from an antigen receptor, i.e. an antigen receptor signalling domain. In a more particular embodiment, the antigen receptor signalling domain is selected from the group consisting of the CD3 , CD28, 4-1 BB, NKG2D, DAP10, 0X40, CD70, CD27, CD5, ICAM-1 , LFA-1 (CD11a / CD18), ICOS (CD278), DAP 12 antigen receptor signalling domain or a combination thereof.

[0147] The antigen receptor signalling domain generates a signal that stimulates the immune effector function of chimeric antigen receptor (CAR)-containing cells, for example, CAR- T cells. The effector function of a T cell, for example, may be cytolytic function or helper activity including the secretion of cytokines. Thus, the intracellular signalling domain may be a portion of a protein which transduces the effector function signal and directs the cell (e.g. T cell) to perform a specialised function.

[0148] Generally, the whole intracellular signalling domain can be used; however, it is appreciated that it is not necessary to use the entire domain, provided that whatever part of the signalling domain that is used is still capable of transducing the effector function signal. It will also be appreciated that variants of such intracellular signalling domains with substantially the same or greater functional capability may also be used. By this we include the meaning that the variants should have substantially the same or greater transduction of the effector functional signal. 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 signalling domain, wherein signal transduction of the unmodified intracellular signalling domain corresponds to 100%. Methods for assessing transduction of effector function signal are well known to those skilled in the art and include, for example, assessing the amounts and / or activity of molecules (e.g. proteins such as cytokines) that are indicative of the transduced signal. Thus, when the signal is the cytolytic function of a T-cell, the methods may involve measurement of one or more cytokines secreted by the T-cell, which cytokines are known to have a cytolytic activity (e.g. I FN gamma). Another means of assessing the cytolytic function is by CFSE staining and counting positive cells by Flow cytometry or by a chromium release assay as is well known in the art.

[0149] Examples of intracellular signalling domains include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.

[0150] Sometimes, signals generated through the TCR alone are generally insufficient for full activation of a T cell and that a secondary and / or costimulatory signal may also be required. Thus, T cell activation can be said to be mediated by two distinct classes of intracellular signalling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signalling domains) and those that act in an antigen- independent manner to provide a secondary or costimulatory signal (secondary intracellular signalling domain, such as a costimulatory domain). Costimulatory domains promote activation of effector functions and may also promote persistence of the effector function and / or survival of the cell.

[0151] A primary intracellular signalling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary intracellular signalling domains that act in a stimulatory manner may contain signalling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs (e.g. 2, 3, 4, 5 or more ITAMs). Thus, the intracellular signalling domain may comprise one or more ITAMs. Examples of ITAM containing primary intracellular signalling domains that are of particular use in the invention include those of CD3 zeta, Fc receptor gamma, Fc receptor beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0152] The “costimulatory signaling domain” refers to a portion of the chimeric receptor comprising the intracellular domain of a costimulatory molecule.

[0153] The term “co-stimulating molecule” refers to a recognizable T-cell binding partner that specifically binds to a co-stimulating ligand, thereby mediating the co-stimulatory response exerted by the T-cell, such as, but not limited to, proliferation. Co-stimulating molecules are cell surface molecules other than antigen-specific receptors or their ligands, which are necessary for an effective immune response. A costimulatory molecule may be a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of immune cells (such as lymphocytes) to an antigen. A costimulatory molecule can be represented in 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 4-1 BB (CD137), 0X40, ICOS, DAP10, CD27, CD28, CDS, CD30, CD137 (4-1 BB), 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 a ligand that specifically binds with CD83, and the like.

[0154] In a particular embodiment, the first polynucleotide of the invention comprises a nucleotide sequence encoding a signal peptide, wherein the signal peptide is in frame and N-terminal to the split C-intein and which allows the insertion of the fusion protein into the membrane. The term “signal peptide” refers to a peptide of a relatively short length, generally between 5 and 40 amino acid residues, directing proteins synthesized in the cell to a specific location, particularly allowing the insertion of the fusion protein encoding by the first polynucleotide of the invention into the membrane.

[0155] Any signal peptide that allows the insertion of the fusion protein encoded by the first polynucleotide of the invention into the membrane may be used in the present invention. In a particular embodiment, the signal peptide is selected from the group consisting of: CD8a, IL-2, GM-CSF receptor (GM-CSF) a chain, murine Ig-kappa (IgK), murine IgK V- III region (IgKVIll), human IgKVIll, CD33, human tissue plasminogen activator (TPA) and native secreted alkaline phosphatase (SEAP).

[0156] In another particular embodiment, the first polynucleotide of the invention further comprises a linker between the split C-intein and the transmembrane domain.

[0157] The term “linker” means a suitable peptide that allows for two or more functional domains joined together in a fusion protein. Linkers can be flexible or rigid linkers. In a preferred embodiment the linker is a flexible linker. “Flexible linker” as it is used herein means that the joined domains require a certain degree of movement or interaction. They are generally composed of small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids. The small size of these amino acids provides flexibility, and allows for mobility of the connecting functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduces the unfavourable interaction between the linker and the protein moieties.

[0158] In certain embodiments, the linker is a peptide containing 1-150 amino acid residues, 1- 90 amino acid residues, 2-85 amino acid residues, 3-80 amino acid residues, 5-75 amino acid residues, 5-70 amino acid residues, 5-65amino acid residues, 10-60amino acid residues, 10-50amino acid residues 30-50 amino acids, 10-150 amino acids, 10-125 amino acids, 50-125 amino acids, 75-125 amino acids. . In a preferred embodiment, the linker between the split C-intein and the transmembrane domain comprises between 40 and 45 amino acids.

[0159] In a particular embodiment, the linker between the split C-intein and the transmembrane domain is a hinge region or a flexible linker. As used herein, “hinge domain”, “hinge region” or “spacer” refers to an amino acid region that allows for separation and flexibility of the split C-intein and the transmembrane domain.

[0160] In a particular embodiment, the hinge region is selected from the group consisting of: IgG 1 hinge region, lgG2 hinge region, lgG4 hinge region, FcyR hinge region, CD3E hinge region, CD3 hinge region, CD8a hinge region, CD4 hinge region, CD28 hinge region, CD7 hinge region and CD19 hinge region.

[0161] Exemplary flexible linkers include glycine and serine-rich linkers, e.g., (GGP)n, (GGGGS)n (SEQ ID NO: 30) or (GGGS)n (SEQ ID NO: 19), where n is 1-5. The most commonly used flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues (“GS” linker). By adjusting the copy number “n”, the length of this GS linker can be optimized to achieve appropriate separation of the functional domains, or to maintain necessary inter-domain interactions. In a preferred embodiment, the linker between the split C-intein and the transmembrane domain is a glycine and serine-rich linker.

[0162] 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 acid of the linker is a serine, cysteine, or threonine.

[0163] In another particular embodiment, the transmembrane domain and the intracellular signalling domain are either directly connected by a peptide bond or by an amino acid linker.

[0164] The term “peptide bond” refers to an amide type of covalent chemical bond linking two consecutive alpha-amino acids from C1 (carbon number one) of one alpha-amino acid and N2 (nitrogen number two) of another, along a peptide or protein chain.

[0165] In a particular embodiment, the fusion protein encoded by the first polynucleotide of the invention further comprises a modified N-intein, wherein the modified N-intein is in N- terminal position with respect to the split C-intein, wherein

[0166] If the split C-intein comprises the sequence of SEQ ID NO: 1 or is a functionally equivalent variant thereof, the modified N-intein is a modified GP41.1 N-intein, If the split C-intein comprises the sequence of SEQ ID NO: 2 or is a functionally equivalent variant thereof, the modified N-intein is a modified IMPDH N-intein, If the split C-intein comprises the sequence of SEQ ID NO: 3 or is a functionally equivalent variant thereof, the modified N-intein is a modified NrdJ1 N-intein or If the split C-intein comprises the sequence of SEQ ID NO: 4 or is a functionally equivalent variant thereof, the modified N-intein is a modified GP41.8 N-intein, wherein the modified N-intein comprises a fragment of the N-intein protein that prevents trans-splicing from occurring in the presence of the corresponding C-intein, and wherein the modified N-intein comprises at least one mutation that decreases the affinity between the modified N-intein and the corresponding C-intein.

[0167] The modified N-intein comprises a fragment of the N-intein protein that prevents trans- splicing from occurring in the presence of the corresponding C-intein.

[0168] In a particular embodiment, the modified N-intein does not comprise the complete sequence of the N-intein protein.

[0169] According to the invention, the complete sequence of the N-intein protein is selected from the inteins GP41.1 N-intein, IMPDH N-intein, NrdJ1 N-intein and GP41.8 N-intein. The sequences of the N-terminal domain of the GP41.1 intein, IMPDH intein, NrdJ1 intein and GP41.8 intein are described in SEQ ID NO: 5, 6, 7 and 8, respectively:

[0170] SEQ ID NO: 5 (CLDLKTQVQT PQGMKEISNI QVGDLVLSNT GYNEVLNVFP KSKKKSYKIT LEDGKEIICS EEHLFPTQTG EMNISGGLKE GMCLYVKE) refers to the N-terminal domain of the GP41.1 split intein.

[0171] SEQ ID NO: 6 (CFVPGTLVNT ENGLKKIEEI KVGDKVFSHT GKLQEVVDTL IFDRDEEIIS INGIDCTKNH EFYVIDKENA NRVNEDNIHL FARWVHAEEL DMKKHLLIEL E) refers to the N-terminal domain of the IMPDH1 split intein.

[0172] SEQ ID NO: 7 (CLVGSSEIIT RNYGKTTIKE VVEIFDNDKN IQVLAFNTHT DNIEWAPIKA AQLTRPNAEL VELEINTLHG VKTIRCTPDH PVYTKNRDYV RADELTDDDE LVVAI) refers to the N-terminal domain of the NrdJ1 split intein.

[0173] SEQ ID NO: 8 (CLSLDTMVVT NGKAIEIRDV KVGDWLESEC GPVQVTEVLP IIKQPVFEIV LKSGKKIRVS ANHKFPTKDG LKTINSGLKV GDFLRSRA) refers to the N- terminal domain of the GP41 .8 split intein.

[0174] In a particular embodiment, if the split C-intein comprises the sequence of SEQ ID NO: 1 or is a functionally equivalent variant thereof, the modified GP41.1 N-intein comprises a fragment of the SEQ ID NO: 5 that prevents trans-splicing from occurring in the presence of the GP41.1 C-intein. In a particular embodiment, the modified GP41.1 N- intein does not comprise the complete sequence of the SEQ ID NO: 5. In another particular embodiment, if the split C-intein comprises the sequence of SEQ ID NO: 2 or is a functionally equivalent variant thereof, the modified IMPDH1 N-intein comprises a fragment of the SEQ ID NO: 6 that prevents trans-splicing from occurring in the presence of the IMPDH1 C-intein. In a particular embodiment, the modified IMPDH1 N-intein does not comprise the complete sequence of SEQ ID NO: 6.

[0175] In another particular embodiment, if the split C-intein comprises the sequence of SEQ ID NO: 3 or is a functionally equivalent variant thereof, the modified NrdJ1 N-intein comprises a fragment of the SEQ ID NO: 7 that prevents trans-splicing from occurring in the presence of the NrdJ1 C-intein. In a particular embodiment, the modified NrdJ1 N- intein does not comprise the complete sequence of SEQ ID NO: 7.

[0176] In another particular embodiment, if the split C-intein comprises the sequence of SEQ ID NO: 4 or is a functionally equivalent variant thereof, the modified GP41.8 N-intein comprises a fragment of the SEQ ID NO: 8 that prevents trans-splicing from occurring in the presence of the GP41.8 C-intein. In a particular embodiment, the modified GP41.8 N-intein does not comprise the complete sequence of SEQ ID NO: 8.

[0177] The term “fragment” refers to a portion or subset of a larger amino acid sequence that retains certain defined characteristics or functionality. In a particular embodiment, the fragment comprises or consists between 10 and 100 amino acids, preferably between 20 and 65 amino acids, more preferably between 45 and 60 amino acids. The truncation of the N-intein protein to obtain the modified N-intein prevents trans-splicing from occurring, i.e. the fragment of the N-intein protein prevents trans-splicing from occurring in the presence of the corresponding C-intein.

[0178] In a particular embodiment, the modified GP41.1 N-intein is a fragment of the sequence SEQ ID NO: 5 that comprises or consists of between 18 and 83 amino acids, preferably between 30 and 60 amino acids, more preferably between 40 and 50 amino acids. The fragment of the sequence SEQ ID NO: 5 comprises at least one mutation that decreases the affinity between the modified GP41.1 N-intein and the GP41.1 C-intein.

[0179] In a particular embodiment, the modified GP41.1 N-intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids, the first 40 amino acids, the first 45 amino acids, the first 50 amino acids, the first 55 amino acids, the first 60 amino acids or the first 65 amino acids of the polypeptide as defined in SEQ ID NO: 5. In another particular embodiment, the modified GP41.1 N-intein does not comprise 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.

[0180] The elimination of the amino acids in the N-terminal position of the GP41.1 N-intein (i.e. first amino acids of the polypeptide as defined in SEQ ID NO: 5) and in the C-terminal position of the GP41.1 N-intein (i.e. last amino acids of the polypeptide as defined in SEQ ID NO: 5) may be combined. In a particular embodiment, the modified GP41.1 N- intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids, the first 40 amino acids, the first 45 amino acids or the first 50 amino acids of the polypeptide as defined in SEQ ID NO: 5 and does not comprise 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.

[0181] In a more particular embodiment, the modified GP41.1 N-intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids or the first 40 amino acids of the polypeptide as defined in SEQ ID NO: 5 and does not comprise 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.

[0182] In a preferred embodiment, the modified GP41.1 N-intein does not comprise the 41 first amino acids of the polypeptide as defined in SEQ ID NO: 5.

[0183] In another particular embodiment, the modified IMPDH N-intein is a fragment of the SEQ ID NO: 6 that comprises or consists between 21 and 96 amino acids, preferably between 40 and 70 amino acids, more preferably between 50 and 60 amino acids. The fragment of the sequence SEQ ID NO: 6 comprises at least one mutation that decreases the affinity between the modified IMPDH N-intein and the IMPDH C-intein.

[0184] In a particular embodiment, the modified IMPDH N-intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids, the first 40 amino acids, the first 45 amino acids, the first 50 amino acids, the first 55 amino acids, the first 60 amino acids or the first 65 amino acids of the polypeptide as defined in SEQ ID NO: 6. In another particular embodiment, the modified IMPDH N-intein does not comprise 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: 6.

[0185] The elimination of the amino acids in the N-terminal position of the IMPDH N-intein (i.e. first amino acids of the polypeptide as defined in SEQ ID NO: 6) and in the C-terminal position of the IMPDH N-intein (i.e. last amino acids of the polypeptide as defined in SEQ ID NO: 6) may be combined. In a particular embodiment, the modified IMPDH N-intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids, the first 40 amino acids, the first 45 amino acids, the first 50 amino acids, the first 55 amino acids or the first 60 amino acids of the polypeptide as defined in SEQ ID NO: 6, and does not comprise 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: 6.

[0186] In a more particular embodiment, the modified IMPDH N-intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids, the first or the first 40 amino acids of the polypeptide as defined in SEQ ID NO: 6 and does not comprise 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: 6.

[0187] In a preferred embodiment, the modified IMPDH N-intein does not comprise the 42 first amino acids of the polypeptide as defined in SEQ ID NO: 6.

[0188] In another particular embodiment, the modified NrdJ1 N-intein is a fragment of the SEQ ID NO: 7 that comprises or consists between 25 and 100 amino acids, preferably between 30 and 60 amino acids, more preferably between 40 and 50 amino acids. The fragment of the sequence SEQ ID NO: 7 comprises at least one mutation that decreases the affinity between the modified NrdJ1 N-intein and the NrdJ1 C-intein.

[0189] In a particular embodiment, the modified NrdJ1 N-intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids, the first 40 amino acids, the first 45 amino acids, the first 50 amino acids, the first 55 amino acids, the first 60 amino acids or the first 65 amino acids of the polypeptide as defined in SEQ ID NO: 7. In another particular embodiment, the modified NrdJ1 N-intein does not comprise 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: 7.

[0190] The elimination of the amino acids in the N-terminal position of the NrdJ1 N-intein (i.e. first amino acids of the polypeptide as defined in SEQ ID NO: 7) and in the C-terminal position of the NrdJ1 N-intein (i.e. last amino acids of the polypeptide as defined in SEQ ID NO: 7) may be combined. In a particular embodiment, the modified NrdJ1 N-intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids, the first 40 amino acids, the first 45 amino acids, the first 50 amino acids, the first 55 amino acids or the first 60 amino acids of the polypeptide as defined in SEQ ID NO: 7, and does not comprise 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: 7.

[0191] In a more particular embodiment, the modified NrdJ1 N-intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids, the first 40 amino acids, the first 45 amino acids, the first 50 amino acids or the first 55 amino acids of the polypeptide as defined in SEQ ID NO: 7 and does not comprise 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: 7.

[0192] In a preferred embodiment, the modified NrdJ1 N-intein does not comprise the 56 first amino acids of the polypeptide as defined in SEQ ID NO: 7.

[0193] In another particular embodiment, the modified GP41 .8 N-intein is a fragment of the SEQ ID NO: 8 that comprises or consists between 19 and 84 amino acids, preferably between 30 and 60 amino acids, more preferably between 40 and 50 amino acids. The fragment of the sequence SEQ ID NO: 8 comprises at least one mutation that decreases the affinity between the modified GP41.8 N-intein and the GP41.8 C-intein.

[0194] In a particular embodiment, the modified GP41.8 N-intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids, the first 40 amino acids, the first 45 amino acids, the first 50 amino acids, the first 55 amino acids, the first 60 amino acids or the first 65 amino acids of the polypeptide as defined in SEQ ID NO: 8.

[0195] In another particular embodiment, the modified GP41.8 N-intein does not comprise 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.

[0196] The elimination of the amino acids in the N-terminal position of the GP41.8 N-intein (i.e. first amino acids of the polypeptide as defined in SEQ ID NO: 8) and in the C-terminal position of the GP41.8 N-intein (i.e. last amino acids of the polypeptide as defined in SEQ ID NO: 8) may be combined. In a particular embodiment, the modified GP41.8 N- intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids, the first 40 amino acids, the first 45 amino acids or the first 50 amino acids of the polypeptide as defined in SEQ ID NO: 8, and does not comprise 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.

[0197] In a more particular embodiment, the modified GP41.8 N-intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids or the first 40 amino acids of the polypeptide as defined in SEQ ID NO: 8 and does not comprise 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.

[0198] In a preferred embodiment, the modified GP41.8 N-intein does not comprise the 40 first amino acids of the polypeptide as defined in SEQ ID NO: 8.

[0199] The modified N- intein refers to an N-intein that does not comprise the native sequence of the N-intein protein. In particular, the modified N-intein comprises at least one mutation that decreases the affinity between the modified N-intein and the corresponding C-intein.

[0200] A mutation is a change in the sequence of nucleotides in DNA (or RNA in some cases) or a change in the resulting amino acid sequence of a protein. Mutations can occur naturally or can be introduced artificially. In a particular embodiment, the at least one mutation of the modified N-intein that decreases the affinity between the modified N- intein and the corresponding C-intein is a substitution, an insertion or a deletion. It will be understood that the mutations present in the modified N-intein and that destabilize the binding between the modified N-intein and the corresponding C-intein exist only in those N-intein variants which contain the positions which are mutated and not in those variants in which the truncation has resulted in the elimination of the positions which contain the mutations.

[0201] The mutation (s) in the modified N-intein decrease the affinity between the modified N- intein and the corresponding C-intein. In a particular embodiment, the affinity between the modified N-intein and the C-intein is 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60% lower than the affinity between the N-intein and the corresponding C-intein.

[0202] In a preferred embodiment the affinity between the modified N-intein and the C-intein is between 40% and 60% lower than the affinity between the N-intein and the corresponding C-intein.

[0203] In a particular embodiment, wherein the split C-intein comprises the sequence of SEQ ID NO: 1 or is a functionally equivalent variant thereof, the modified N-intein is a modified GP41.1 N-intein that comprises at least one mutation that decreases the affinity between the modified GP41.1 N-intein and the GP41.1 C-intein.

[0204] In a particular embodiment, the modified GP41.1 N-intein comprises between 1 and 4 mutations in a fragment of the sequence SEQ ID NO: 5. In a more particular embodiment, the modified GP41.1 N-intein comprises 2 mutations in a fragment of the sequence SEQ ID NO: 5.

[0205] In a particular embodiment, the modified GP41.1 N-intein comprises at least one mutation that replaces a charged amino acid with a neutral amino acid. In a more particular embodiment, the modified GP41.1 N-intein comprises at least one mutation that replaces a positively charged amino acid with a neutral amino acid.

[0206] Therefore, in another particular embodiment, the modified N-intein comprises at least one mutation that changes the net charge of the modified N-intein fragment compared with the wild type N-intein. In particular, the modified GP41.1 N-intein comprises at least one mutation that changes the net charge of the modified GP41.1 N-intein fragment compared with the wild type GP41.1 N-intein.

[0207] In a particular embodiment, the modified GP41.1 N-intein comprises at least one mutation in a Lysine (K) residue. In a more particular embodiment, the modified GP41.1 N-intein comprises at least one mutation that replaces a Lysine (K) residue with an Alanine (A) residue.

[0208] In a preferred embodiment, the modified GP41.1 N-intein comprises a mutation in the lysine residues at positions 45 and 48 of the SEQ ID NO: 5. In a more preferred embodiment, the modified GP41.1 N-intein comprises the mutations K45A and K48A.

[0209] In a particular embodiment, the modified GP41.1 N-intein comprises the sequence of SEQ ID NO: 31.

[0210] The mutations defined above are only to be applied when the modified GP41.1 N-intein comprises a fragment of the sequence SEQ ID NO: 5 comprising the mutated positions (e.g. K45 and K48).

[0211] In another particular embodiment, wherein the split C-intein comprises the sequence of SEQ ID NO: 2 or is a functionally equivalent variant thereof, the modified N-intein is a modified IMPDH N-intein that comprises at least one mutation that decreases the affinity between the modified IMPDH N-intein and the IMPDH C-intein.

[0212] In a particular embodiment, the modified IMPDH N-intein comprises between 1 and 4 mutations in a fragment of the sequence SEQ ID NO: 6. In a more particular embodiment, the modified IMPDH N-intein comprises 2 mutations in a fragment of the sequence SEQ ID NO: 6.

[0213] In a particular embodiment, the modified IMPDH N-intein comprises at least one mutation that replaces a charged amino acid with a neutral amino acid. In a more particular embodiment, the modified IMPDH N-intein comprises at least one mutation that replaces a negatively charged amino acid with a neutral amino acid.

[0214] Therefore, in another particular embodiment, the modified N-intein comprises at least one mutation that changes the net charge of the modified N-intein fragment compared with the wild type N-intein. In particular, the modified IMPDH N-intein comprises at least one mutation that changes the net charge of the modified IMPDH N-intein fragment compared with the wild type IMPDH N-intein.

[0215] In a more particular embodiment, the modified IMPDH N-intein comprises at least one mutation in a glutamic acid (E) residue. In a more particular embodiment, the modified IMPDH N-intein comprises at least one mutation that replaces a glutamic acid (E) residue with an Alanine (A) residue. In a preferred embodiment, the modified IMPDH N-intein comprises a mutation in the glutamic acid residues at positions 46 and 47 of the SEQ ID NO: 6. In a more preferred embodiment, the modified IMPDH N-intein comprises the mutations E46A and E47A.

[0216] In a particular embodiment, the modified IMPDH N-intein comprises the sequence of SEQ ID NO: 32.

[0217] The mutations defined above are only to be applied when the modified IMPDH N-intein comprises a fragment of the sequence SEQ ID NO: 6 comprising the mutated positions (e.g. E46 and E47).

[0218] In another particular embodiment, wherein the split C-intein comprises the sequence of SEQ ID NO: 3 or is a functionally equivalent variant thereof, the modified N-intein is a modified NrdJ1 N-intein that comprises at least one mutation that decreases the affinity between the modified NrdJ1 N-intein and the NrdJ1 C-intein.

[0219] In a particular embodiment, the modified NrdJ1 N-intein comprises between 1 and 4 mutations in a fragment of the sequence SEQ ID NO: 7. In a more particular embodiment, the modified NrdJ1 N-intein comprises 2 mutations in a fragment of the sequence SEQ ID NO: 7.

[0220] In a particular embodiment, the modified NrdJ1 N-intein comprises at least one mutation that replaces a charged amino acid with a neutral amino acid. In a more particular embodiment, the modified NrdJ1 N-intein comprises at least one mutation that replaces a negatively charged amino with a neutral amino acid.

[0221] Therefore, in another particular embodiment, the modified N-intein comprises at least one mutation that changes the net charge of the modified N-intein fragment compared with the wild type N-intein. In particular, the modified NrdJ1 N-intein comprises at least one mutation that changes the net charge of the modified NrdJ1 N-intein fragment compared with the wild type NrdJ1 N-intein.

[0222] In a more particular embodiment, the modified NrdJ1 N-intein comprises at least one mutation in a glutamic acid (E) residue. In a more particular embodiment, the modified NrdJ1 N-intein comprises at least one mutation that replaces a glutamic acid (E) residue with an Alanine (A) residue.

[0223] In a preferred embodiment, the modified NrdJ1 N-intein comprises a mutation in the glutamic acid residues at positions 62 and 64 of the SEQ ID NO: 7. In a more preferred embodiment, the modified NrdJ1 N-intein comprises the mutations E62A and E64A. In a particular embodiment, the modified NrdJ1 N-intein comprises the sequence of SEQ ID NO: 33.

[0224] The mutations defined above are only to be applied when the modified NrdJ1 N-intein comprises a fragment of the sequence SEQ ID NO: 7 comprising the mutated positions (e.g. E62 and E64).

[0225] In another particular embodiment, the split C-intein comprises the sequence of SEQ ID NO: 4 or is a functionally equivalent variant thereof, the modified N-intein is a modified GP41 .8 N-intein that comprises at least one mutation that decreases the affinity between the modified GP41.8 N-intein and the GP41.8 C-intein.

[0226] In a particular embodiment, the modified GP41.8 N-intein comprises between 1 and 4 mutations in a fragment of the sequence SEQ ID NO: 8. In a more particular embodiment, the modified GP41.8 N-intein comprises 2 mutations in a fragment of the sequence SEQ ID NO: 8.

[0227] In a particular embodiment, the modified GP41.8 N-intein comprises at least one mutation that replaces a charged amino acid with a neutral amino acid. In a more particular embodiment, the modified GP41.8 N-intein comprises at least one mutation that replaces a positively charged amino with a neutral amino acid.

[0228] Therefore, in another particular embodiment, the modified N-intein comprises at least one mutation that changes the net charge of the modified N-intein fragment compared with the wild type N-intein. In particular, the modified GP41.8 N-intein comprises at least one mutation that changes the net charge of the modified GP41.8 N-intein fragment compared with the wild type GP41.8 N-intein.

[0229] In a more particular embodiment, the modified GP41.8 N-intein comprises at least one mutation in a Lysine (K) residue. In a more particular embodiment, the modified GP41.8 N-intein comprises at least one mutation that replaces a Lysine (K) residue with an Alanine (A) residue.

[0230] In a preferred embodiment, the modified GP41.8 N-intein comprises a mutation in the lysine residues at positions 52 and 55 of the SEQ ID NO: 8. In a more preferred embodiment, the modified GP41.8 N-intein comprises the mutations K52A and K55A.

[0231] In a particular embodiment, the modified GP41.8 N-intein comprises the sequence of SEQ ID NO: 34. The mutations defined above are only to be applied when the modified GP41.8 N-intein comprises a fragment of the sequence SEQ ID NO: 8 comprising the mutated positions (e.g. K52 and K55).

[0232] In another particular embodiment, the first polynucleotide of the invention further comprises: a. a linker region between the modified N-intein and the split C-intein and / or b. a protease cleavage site between the modified N-intein and the split C- intein.

[0233] In a particular embodiment, the linker region between the modified N-intein and the split C-intein comprises a sequence of between 1 and 200 amino acids, preferably between 1 and 100 amino acids.

[0234] The term “protease cleavage site” refers to a specific sequence of amino acids within a protein or polypeptide chain that is recognized and cleaved by a protease enzyme. Proteases, also known as proteinases or peptidases, catalyse the hydrolysis of peptide bonds at these sites, resulting in the separation of the protein into smaller fragments.

[0235] In a particular embodiment, the protease cleavage site is recognised and cleaved by a protease selected from the group consisting of: a furin, an heparanase, calpain, a matrix metalloprotease (MMP), a cathepsin, a serine protease, a cysteine protease, an aspartic protease, ADAMs and ADAMTS protein, an aminopeptidase, a granule-associated serine protease and a caspase.

[0236] Examples of matrix metalloproteinases (MMPs) include, but are not limited to MMP-2, MMP-9, MMP-1 , MMP-7, MMP-3 and MMP-14.

[0237] Examples of cathepsins include, but are not limited to, cathepsin B, cathepsin D and cathepsin L.

[0238] Examples of serine proteases include, but are not limited to urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), matriptase, kallikreins, elastase, prolyl endopeptidase (PREP) and thymus-specific serine protease (TSSP).

[0239] Examples of cysteine proteases include, but are not limited to, legumain, cathepsin C, cathepsin W and cathepsin L.

[0240] Examples of aspartic proteases include, but are not limited to pepsin-like proteases. Examples of ADAMS and ADAMTS proteins include, but are not limited to, ADAM 10, ADAM 17 and ADAMTS1.

[0241] Examples of aminopeptidases include, but are not limited to aminopeptidase N (CD13) and leucine aminopeptidase.

[0242] Examples of granule-associated serine proteases include, but are not limited to granzyme A, granzyme B, granzyme K and granzyme M.

[0243] Examples of caspases include, but are not limited to caspase 8, caspase 3 and caspase 9.

[0244] In a preferred embodiment, the protease cleavage site is a furin cleavage site.

[0245] First vector of the invention

[0246] In another aspect, the present invention relates to a vector, hereinafter the first vector of the invention, which comprises the first polynucleotide of the invention.

[0247] The term “vector” means a construct, which is capable of delivering, and optionally expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells. The vectors can be stable and can be self-replicating. An "expression vector" is a vector that is capable of directing the expression of genes to which it is operably associated.

[0248] Expression vectors are replicable DNA constructs that have synthetic or cDNA-derived DNA fragments encoding a fusion protein, operatively linked to suitable transcriptional or translational regulatory elements. The transcriptional or translational regulatory elements can be derived from, for example, mammalian, microbial, viral, or insect genes. A transcriptional unit generally comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences. Such regulatory elements can include an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated. DNA regions are operatively linked when they are functionally related to each other. For example, DNA for a signal peptide is operatively linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation.

[0249] The choice of expression control sequence and expression vector will depend upon the choice of host. A wide variety of expression host / vector combinations can be employed. Useful expression vectors for eukaryotic hosts, include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from Escherichia coli, including pCR 1 , pBR322, pMB9 and their derivatives, wider host range plasmids, such as M13 and filamentous singlestranded DNA phages.

[0250] The vectors can comprise at least one promoter. "Promoter" refers to a DNA fragment capable of controlling the expression of a coding sequence or functional RNA. In general, a coding region is located 3' to a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity. A promoter is generally bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

[0251] The choice of which expression vector and ultimately to which promoter a fusion proteinencoding polynucleotide is operatively linked depends directly on the functional properties desired, e.g., the location and timing of protein expression, and the host cell to be transformed.

[0252] First host cell of the invention

[0253] Thus, in another aspect, the present invention relates to a host cell, hereinafter the first host cell of the invention, comprising the first polynucleotide of the invention, the first vector of the invention or the fusion protein encoded by the first polynucleotide of the invention.

[0254] The term “host cell”, as used herein, refers to a cell into which a nucleic acid of the invention, such as the first polynucleotide or the first vector according to the invention, has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. The term “host cell” is used such that it refers not only to the particular subject cell, but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. A host cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell (e.g., yeast, plant cells or animal cells).

[0255] In a particular embodiment, the first host cell of the invention is a human cell. In a more particular embodiment, the first host cell of the invention is selected from the group consisting of: T-cell, NK-cell, induced pluripotent stem cell (iPSC), derived NK cell, Pro- Tcell, B cell and macrophage.

[0256] In another particular embodiment, the first host cell of the invention is obtained by viral transduction.

[0257] “Viral transduction” refers to is process by which foreign DNA is introduced into a cell by a virus or viral vector.

[0258] In a more particular embodiment, the first host cell of the invention is obtained by viral transduction with a lentivirus or by gene knock-in.

[0259] “Gene knock-in” refers to a genetic engineering method that involves the one-for-one substitution of DNA sequence information in a genetic locus or the insertion of sequence information not found within the locus. In a preferred embodiment, the gene knock-in is carried out using a CRISPR-Cas9 system.

[0260] First fusion protein of the invention In another aspect, the present invention relates to a fusion protein, hereinafter the first fusion protein of the invention, encoded by the first polynucleotide of the invention.

[0261] The term “fusion protein” has been defined above, and this definition is applicable to the first fusion protein of the invention.

[0262] In a particular embodiment, the first fusion protein of the invention further comprises a detection tag. In a particular embodiment, the first fusion protein of the invention comprises one or more detection tags.

[0263] In a more particular embodiment, the first fusion protein of the invention comprises two detection tags.

[0264] The term “tag” means a polypeptide useful for making the detection, isolation and / or purification of a protein easier. Generally, said labeling sequence is located in a part of the protein of interest that does not adversely affect the functionality thereof. In a particular embodiment, the detection tag is selected from the group consisting of: c-myc tag, HA tag, FLAG-tag, Strep-tag, His-tag, alfa tag, V5 tag, Spot tag and NE tag.

[0265] Second fusion protein of the invention

[0266] In another aspect, the present invention relates to a fusion protein, hereinafter the second fusion protein of the invention, comprising from N-terminus to C-terminus: i) a heterologous polypeptide (N-extein) and ii) a split N-intein comprising a sequence selected from the group consisting SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 or a functionally equivalent variant thereof.

[0267] The terms “polypeptide” and “fusion protein” have been defined or explained above and these definitions are applicable to the second fusion protein of the invention.

[0268] The term “heterologous” as used herein refers to an element of a vector, plasmid or host cell that is derived from a source other than the endogenous source. Thus, for example, a heterologous sequence (e.g., a polynucleotide sequence or a polypeptide sequence) could be a sequence that is derived from a different gene or plasmid from the same host, from a different strain of host cell, or from an organism of a different taxonomic group (e.g., different kingdom, phylum, class, order, family genus, or species, or any subgroup within one of these classifications). The term "heterologous" is also used synonymously herein with the term "exogenous." In the case wherein the heterologous polypeptide forms part of the fusion protein comprising the split N-intein as defined in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 or a functionally equivalent variant thereof, then this heterologous polypeptide is also referred to as N-extein.

[0269] The “split N-intein” refers to the N-terminal domain of an intein. An "intein N-terminal domain" refers to an intein sequence that comprises an N-terminal amino acid sequence that is functional for trans-splicing reactions and / or N-terminal self-cleavage reactions. An intein N-terminal domain can be spliced out when trans-splicing occurs.

[0270] The second fusion protein of the invention comprises ii) a split N-intein comprising a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 or a functionally equivalent variant thereof.

[0271] SEQ ID NO: 5 (CLDLKTQVQT PQGMKEISNI QVGDLVLSNT GYNEVLNVFP KSKKKSYKIT LEDGKEIICS EEHLFPTQTG EMNISGGLKE GMCLYVKE) refers to the N-terminal domain of the GP41.1 split intein.

[0272] SEQ ID NO: 6 (CFVPGTLVNT ENGLKKIEEI KVGDKVFSHT GKLQEVVDTL IFDRDEEIIS INGIDCTKNH EFYVIDKENA NRVNEDNIHL FARWVHAEEL DMKKHLLIEL E) refers to the N-terminal domain of the IMPDH1 split intein.

[0273] SEQ ID NO: 7 (CLVGSSEIIT RNYGKTTIKE VVEIFDNDKN IQVLAFNTHT DNIEWAPIKA AQLTRPNAEL VELEINTLHG VKTIRCTPDH PVYTKNRDYV RADELTDDDE LVVAI) refers to the N-terminal domain of the NrdJ1 split intein.

[0274] SEQ ID NO: 8 (CLSLDTMVVT NGKAIEIRDV KVGDWLESEC GPVQVTEVLP IIKQPVFEIV LKSGKKIRVS ANHKFPTKDG LKTINSGLKV GDFLRSRA) refers to the N- terminal domain of the GP41.8 split intein.

[0275] The term “functionally equivalent variant of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8” relates to all those sequences which result from the modification, insertion and / or deletion of one or more amino acids from the above sequences, provided that the function of the split intein is substantially maintained. Suitable assays for determining the intein activity of functionally equivalent variants are as described in the patent application published as WQ2013045632A1.

[0276] 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 substituted by a preserved or non-preserved amino acid residue (preferably a preserved amino acid residue) and such substituted amino acid may be coded or not by the genetic code, (ii) polypeptides in which there is one or more modified amino acid residues, for example, residues modified by substituent bonding, (iii) polypeptides resulting from alternative processing of a similar mRNA, (iv) polypeptide fragments and / or (v) polypeptides resulting from fusion of the polypeptide defined in (i) to (iii) with another polypeptide, such as a secretory leader sequence or a sequence being used for purification (for example, His tag) or for detection (for example, Sv5 epitope tag). The fragments include polypeptides generated through proteolytic cut (including multisite proteolysis) of an original sequence. The variants may be post-translationally or chemically modified. Such variants are supposed to be apparent to those skilled in the art.

[0277] One skilled in the art will recognize that the values of identity of nucleotide sequences can be appropriately adjusted in order to determine the corresponding sequence identity of two nucleotide sequences encoding the polypeptides of the present invention, by taking into account codon degeneracy, conservative amino acid substitutions, and reading frame positioning.

[0278] The invention refers to functionally equivalents variants of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8; and that have an amino acid sequence differing in one or more amino acids with the sequence given as the result of one or more conservative amino acid substitutions. It is well known in the art that one or more amino acids in a polypeptide sequence can be substituted with at least one other amino acid having a similar charge and polarity such that the substitution / s result in a silent change in the modified polypeptide that does not alter its function relative to the function of the non-modified sequence. The invention refers to any polypeptide sequence differing in one or more amino acids, either as a result of conserved or non-conserved substitutions, and / or either as a result of sequence insertions or deletions, relative to the sequence given by SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, as long as said further provided polypeptide sequence has the same or similar of intein N-terminal domains as SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.

[0279] The intein N-terminal domain can comprise one or more of N1 , N2, N3, and / or N4 motifs of the HINT (Hog / lntein) family. Thus, for example, an intein N-terminal domain can comprise the N1 and N3 motifs.

[0280] In some embodiments, the functionally equivalent variant of the split N-intein is characterized in that it comprises an N1 box (A box) sequence. The N1 box is a non- strictly conserved sequence. The N1 box can comprise, for example, the sequence ChsXcpIhXTXXG (SEQ ID NO: 15), wherein X is any amino acid, wherein 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 I is a large amino acid. In some embodiments, the intein N-terminal domain comprises the sequence X1X2X3X4X5X6X7X8X9X10X11X12X13 (SEQ ID NO: 16), wherein Xi 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; X6is T or A; X7is E, Q, L, M, K, or T; X8is I or V; X9is L, Q, V, N, K, D, or T; X is T, I, or V; X11 is V, P, Q, N, E, K, or L; X12 is E, Q, G, N, Y, I, or E; and X13 is Y, G, K, P, or D. In some embodiments, the intein N-terminal domain comprises the sequence X1X2X3X4X5X6X7X8X9X10X11X12X13 (SEQ ID NO: 17), wherein Xi is C; X2is L, F, or V; X3is S, T, V, or A; X4 is L, P, or G; X5 is D, K, or G; Xe is T or A; X7 is Q, L, M, K, or T; Xs is I or V; X9is Q, V, N, K, D, or T; X10 is T, I, or V; Xu is P, Q, N, E, K, or L; X12 is E, Q, G, N, Y, I, or E, and X13 is G, K, P, or D.

[0281] Based on chemical properties of the amino acids, they can be grouped as: (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).

[0282] In some embodiments, the intein N-terminal domain is about 50 to about 150 amino acids. In some embodiments, the intein N-terminal domain is about 60 to about 140 amino acids. In some embodiments, the intein N-terminal domain is about 75 to about 125 amino acids. In some embodiments, the intein N-terminal domain 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.

[0283] The first amino acid in an intein N-terminal domain is highly conserved and is important for the protein splicing reaction. Therefore, in some embodiments, the first amino acid in an intein N-terminal domain is a cysteine. In some embodiments, the first amino acid in an intein N-terminal domain is a serine. In some embodiments, the first amino acid in an intein N-terminal domain is an amino acid other than serine or cysteine. For example, the first amino acid in an intein N-terminal domain can be an alanine.

[0284] Functionally equivalent variants of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 also include sequences with a sequence identity of at least 50%, 51 %,

[0285] 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%,

[0286] 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %,

[0287] 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the sequences SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0288] In some embodiments, the functionally equivalent variant of SEQ ID NO: 5 is a sequence with a sequence identity of 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% with the SEQ ID NO:

[0289] 5.

[0290] In some embodiments, the functionally equivalent variant of SEQ ID NO: 6 is a sequence with a sequence identity of 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% with the SEQ ID NO:

[0291] 6.

[0292] In some embodiments, the functionally equivalent variant of SEQ ID NO: 7 is a sequence with a sequence identity of 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% with the SEQ ID NO:

[0293] 7.

[0294] In some embodiments, the functionally equivalent variant of SEQ ID NO: 8 is a sequence with a sequence identity of 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% with the SEQ ID NO:

[0295] 8.

[0296] In some embodiments, the intein N-terminal domain contains at least about 10, at least about 20, at least about 30, at least about 40, or at least about 50 amino acids of a sequence selected from the group consisting of SEQ ID NO: 5, 6, 7 and 8. In some embodiments, the intein N-terminal domain 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 of a sequence selected from the group consisting of SEQ ID NO: 5, 6, 7 and 8. In some embodiments, the intein N-terminal domain contains a deletion of no more than about 5, about 10, about 15, about 20, or about 25 amino acids of a sequence selected from the group consisting of SEQ ID NO: 5, 6, 7 and 8. In some embodiments, the intein N- terminal domain contains a deletion of no more than about 5, about 10, about 15, about 20, or about 25 consecutive amino acids of a sequence selected from the group consisting of SEQ ID NO: 5, 6 , 7 and 8.

[0297] In a preferred embodiment, the functionally equivalent variant of SEQ ID NO: 5, 6, 7 or 8 has a sequence identity of 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% with the corresponding sequence SEQ ID NO, 5, 6, 7 or 8 and the sequence identity is determined throughout the whole length of the sequence SEQ ID NO: 5, 6 , 7 or 8.

[0298] In a particular embodiment, the polypeptide and the split N-intein are either directly connected by a peptide bond or by a peptide linker.

[0299] The terms “peptide bond” and “linker” have been defined or explained above and these definitions are applicable to the second fusion protein of the invention.

[0300] In certain embodiments, the linker is a peptide containing 1-25 amino acid residues, 1- 20 amino acid residues, 2-15 amino acid residues, 3-10 amino acid residues, 3-7 amino acid residues, 4-25 amino acid residues, 4-20 amino acid residues, 4-15 amino acid residues, 4-10 amino acid residues, 5-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues, or 5-10 amino acid residues.

[0301] Exemplary linkers include glycine and serine-rich linkers, e.g., (GGP)n, (GGGGS)n (SEQ ID NO: 30) or (GGGS)n (SEQ ID NO: 19), where n is 1-5. The most commonly used flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues (“GS” linker). By adjusting the copy number “n”, the length of this GS linker can be optimized to achieve appropriate separation of the functional domains, or to maintain necessary inter-domain interactions.

[0302] 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 acid of the linker is a serine, cysteine, or threonine.

[0303] The second fusion protein of the invention comprises a i) polypeptide. The polypeptide specifically binds to a target molecule. The term “target molecule” refers to key molecules (such as carbohydrates, proteins, and nucleic acids) to which some other entity (like an endogenous ligand or a drug) is directed and / or binds, resulting in a change in its behaviour or function. In a particular embodiment, the target molecule is selected from the group consisting of: an antigen of interest, a lectin and an epitope.

[0304] “Lectins” refers to carbohydrate-binding proteins that are highly specific for sugar groups that are part of other molecules, so cause agglutination of particular cells or precipitation of glycoconjugates and polysaccharides.

[0305] “Epitope” refers to the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells.

[0306] “Antigen” or “antigen of interest” in the present invention refers to any type of molecule that can bind to a specific antibody or T-cell receptor. Antigens can be proteins, peptides (amino acid chains), polysaccharides (chains of simple sugars), 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 to and bind one specific antigen; in some instances, however, antibodies may crossreact to bind more than one antigen.

[0307] In a more particular embodiment, the target molecule is an antigen of interest. In a preferred embodiment, the antigen of interest is selected from the group consisting of: CD19, CD20, CD22, GD2, CD133, EGFR, GPC3, CEA, MUC1 , Mesothelin, IL-13R, PSMA, ROR1 , CAIX, CD1a, CCR9 and Her2.

[0308] In some embodiments, the polypeptide that specifically binds to a target molecule, preferably to an antigen of interest, is an “antigen binding domain”.

[0309] According to the present invention, the expressions “antigen-binding domain”, “antigenbinding fragment” or “antibody fragment” are used interchangeably and refer to any oligopeptide or polypeptide that can bind to an antigen of interest. It may comprise an antibody fragment, which refers to at least one portion of an intact antibody, or recombinant variants thereof, for example an antigen variable region of an intact antibody that is sufficient to allow recognition and specific binding of an antibody fragment to a 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. Within the context of the present invention, the antigen-binding domain or antibody fragment comprise at least one VH and one VL regions, but it may comprise two VL regions and two VH regions. Thus, for example, in an embodiment, the antigen-binding domain is a ScFv, and therefore, it will comprise only one VL and one VH regions. In another embodiment, the antigen-binding domain is a Fab fragment, in which case it will comprise one VL and VH (Fab or Fab’) or two VH and two VL regions (Fab2, or F(ab’)2).

[0310] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs) or complementary determining regions (CDRs). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0311] The term “hypervariable region”, “HVR”, “complementarity determining regions” or “CDRs” as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops (“hypervariable loops”). Generally, native four-chain antibodies comprise six CDRs; three in the VH (H1 , H2, H3), and three in the VL (L1 , L2, L3). Thus, CDRs determine the protein's affinity (roughly, bonding strength) and specificity for specific antigens. The CDRs of the two chains of each pair are aligned by the framework regions, acquiring the function of binding a specific epitope. Consequently, both the heavy variable chain and the light variable chain are characterized by three CDRs, respectively VH-CDR1 , VH- CDR2, VH-CDR3 and VL-CDR1 , VL-CDR2, VL-CDR3.

[0312] “Framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1 , FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following order in VH (or VL): FR1-H1 (L1)-FR2-H2(L2)-FR3-H3 (L3)-FR4.

[0313] In a particular embodiment, the i) polypeptide that specifically binds to a target molecule, preferably to an antigen of interest, is selected from the group consisting of: a singlechain variable fragment (scFv), a single domain antibody, an Ig heavy or light chain, a peptide and a ligand.

[0314] As used herein, a "single chain variable fragment (ScFv)" means a single chain polypeptide derived from an antibody which retains the ability to bind to an antigen. An example of the ScFv includes an antibody polypeptide which is formed by a recombinant DNA technique and in which variable (Fv) regions of immunoglobulin heavy chain (VH chain) and light chain (VL chain) fragments are linked via a spacer sequence. Various methods for preparing a ScFv are known, and include methods described in US Patent No. 4694778, Nature, vol. 334, p. 54454 (1989), and Science, vol. 242, pp. 1038-1041 (1988).

[0315] A “single domain antibody” is a peptide chain of about 110 amino acids long, comprising one variable domain (VH) of a heavy-chain antibody, or of a common IgG.

[0316] The term “ligand” refers to any molecule or atom which binds reversibly to a protein. Particularly, refers to a molecule that is capable of interacting with and binding to the target molecule, preferably to an antigen of interest.

[0317] In another particular embodiment, the second fusion protein of the invention further comprises a detection tag and / or a solubilisation tag. In some embodiments, the second fusion protein of the invention comprises a detection tag. In other embodiments, the second fusion protein of the invention comprises a solubilisation tag. In other embodiments, the second fusion protein of the invention comprises a detection tag and a solubilisation tag.

[0318] The term “detection tag” has been defined or explained above, and this definition is applicable to the second fusion protein of the invention. In a particular embodiment, the detection tag is selected from the group consisting of: c-myc tag, HA tag, FLAG-tag, Strep-tag, His tag, alfa tag, V5 tag, Spot tag and NE tag.

[0319] The term “solubilisation tag” refers to a class of peptide sequences that readily fold into soluble proteins; when fused to recombinant proteins, they often enhance solubility. Any solubilisation tag can be used in the present invention. In a particular embodiment, the solubilisation tag is selected from the group consisting of: Fasciola hepatica 8-kDa antigen (Fh8), maltose-binding protein (MBP), N-utilization substance (NusA), Thioredoxin (Trx), small ubiquitin modified (SUMO), glutathione-S-transferase (GST), Solubility-enhancer peptide sequences (SET), IgG domain B1 of Protein G (GB1), IgG repeat domain ZZ of Protein A (ZZ), Mutated dehalogenase (HaloTag), Solubility eNhancing Ubiquitous Tag (SNUT), Seventeen kilodalton protein (Skp), Phage T7 protein kinase (T7PK), E. coli secreted protein A (EspA), Monomeric bacteriophage T7 0.3 protein (Orc protein) (Mocr), E. coli trypsin inhibitor (Ecotin), Calcium-binding protein (CaBP), Stress-responsive arsenate reductase (ArsC), N-terminal fragment of translation initiation factor IF2 (IF2-domain I), 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).

[0320] In a more particular embodiment, the solubilisation tag is selected from the group consisting of: phage bacteriophage lambda head protein D (gpD), small ubiquitin-like modifier) SUMO, maltose binding protein (MBP), glutathione-S-transferase (GST), N- utilization substance (NusA), thioredoxinA and Fasciola hepatica 8-kDa antigen (Fh8 tag).

[0321] Second polynucleotide of the invention

[0322] In another aspect, the present invention relates to a polynucleotide, hereinafter the second polynucleotide of the invention, encoding the second fusion protein of the invention.

[0323] The term “polynucleotide” has been defined above, and this definition is applicable to the second polynucleotide of the invention.

[0324] In a particular embodiment, the second polynucleotide of the invention further comprises a nucleotide sequence encoding a signal peptide, wherein the signal peptide is in frame and N-terminal to the polypeptide that specifically binds to a target molecule and which allows the secretion of the fusion protein from the cell.

[0325] As used herein, the term “signal peptide” refers to a peptide of a relatively short length, generally between 5 and 40 amino acid residues, directing proteins synthesized in the cell towards the secretory pathway. The signal peptide usually contains a series of hydrophobic amino acids adopting a secondary alpha helix structure. Additionally, many peptides include a series of positively-charged amino acids that can contribute to the protein adopting the suitable topology for its translocation. The signal peptide tends to have at its carboxyl end a motif for recognition by a peptidase, which is capable of hydrolyzing the signal peptide giving rise to a free signal peptide and a mature protein.

[0326] Any signal peptide that allows the secretion of the fusion protein from the cell may be used in the present invention.

[0327] Second vector of the invention

[0328] In another aspect, the present invention relates to a vector, hereinafter the second vector of the invention, comprising the second polynucleotide of the invention. The term “vector” has been defined or explained above, and this definition is applicable to the second vector of the invention.

[0329] The choice of expression control sequence and expression vector will depend upon the choice of host. A wide variety of expression host / vector combinations can be employed. Useful expression vectors for eukaryotic hosts, include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from Escherichia coli, including pCR 1 , pBR322, pMB9 and their derivatives, wider host range plasmids, such as M13 and filamentous singlestranded DNA phages.

[0330] Second host cell of the invention

[0331] In another aspect, the present invention relates to a host cell, hereinafter the second host cell of the invention, comprising the second polynucleotide of the invention or the second vector of the invention.

[0332] The term “host cell” has been defined or explained above, and this definition is applicable to the second host cell of the invention.

[0333] Suitable host cells include, by way of 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 COS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175, 1981), and other cell lines capable of expressing an appropriate vector including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell lines. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio / Technology 6:47 (1988).

[0334] Chimeric receptor of the invention

[0335] In another aspect, the present invention relates to a chimeric receptor, hereinafter the chimeric receptor of the invention, comprising from N-terminus to C-terminus: i) an extracellular domain comprising a polypeptide that specifically binds to a target molecule or a polypeptide which can form an oligomer in the presence of one or more additional subunits and wherein the oligomer specifically binds to a target molecule, ii) a linker region, the sequence of which is the result of a protein splicing reaction, iii) a transmembrane domain, and iv) an intracellular signalling domain from a receptor, wherein the protein splicing reaction occurs between two complementary portions of a split intein and wherein the split intein is selected from the group consisting of: Gp41.1 split intein, IMPDH split intein, NrdJ1 split intein and Gp41.8 split intein.

[0336] The term “chimeric receptor” refers to receptor proteins that have been engineered to give cells the new ability to target a specific target molecule.

[0337] The chimeric receptor of the invention comprises i) an extracellular domain comprising a polypeptide that specifically binds to a target molecule or a polypeptide which can form an oligomer in the presence of one or more additional subunits and wherein the oligomer specifically binds to a target molecule.

[0338] The term “target molecule” has been defined or explained above, and this definition is applicable to the chimeric receptor of the invention. In a particular embodiment, the target molecule is selected from the group consisting of: an antigen of interest, a lectin and an epitope. In a more particular embodiment, the target molecule is an antigen of interest.

[0339] The term “antigen” or “antigen of interest” has been defined or explained above, and this definition is applicable to the chimeric receptor of the invention.

[0340] In a particular embodiment, the antigen of interest is selected from the group consisting of: CD19, CD20, CD22, GD2, CD133, EGFR, GPC3, CEA, MUC1 , Mesothelin, IL-13R, PSMA, ROR1 , CAIX, CD1a, CCR9 and Her2.

[0341] In some embodiments, when the chimeric receptor comprises an extracellular domain that comprises a polypeptide that specifically binds to a target molecule, then the polypeptide is selected from the group consisting of: a single chain variable fragment (scFv), a single domain antibody, a lectin, an epitope, an Ig heavy or light chain, a peptide and a ligand.

[0342] In other embodiments, when the extracellular domain of the chimeric receptor of the invention is a polypeptide that can form an oligomer in the presence one or more additional subunits, then the polypeptide is a heavy chain or a light chain of an immunoglobulin. The terms “polypeptide”, “single chain variable fragment (scFv)”, “single domain antibody”, “lectins”, “epitope”, “heavy and light chain” and “ligand” have been defined or explained above, and these definitions are applicable to the chimeric receptor of the invention.

[0343] The term “oligomer” refers to molecule that consists of a few repeating units which could be derived, actually or conceptually, from smaller molecules, monomers. If the units are identical, one has a homo-oligomer; otherwise one may use hetero-oligomer. In a particular embodiment, the polypeptide can form a hetero-oligomer in the presence of one or more additional subunits.

[0344] In a particular embodiment, when the extracellular domain of the chimeric receptor of the invention is a polypeptide that can form an oligomer in the presence of one or more additional subunits, then the oligomer is an immunoglobulin.

[0345] The term “immunoglobulin” refers to a heterodimeric protein composed of two heavy and two light chains. They can be separated functionally into variable (V) domains that binds antigens and constant (C) domains that specify effector functions such as activation of complement or binding to Fc receptors. The variable domains are created by means of a complex series of gene rearrangement events, and can then be subjected to somatic hypermutation after exposure to antigen to allow affinity maturation. Each V domain can be split into three regions of sequence variability, termed the complementarity determining regions, or CDRs, and four regions of relatively constant sequence termed the framework regions, or FRs. The three CDRs of the H chain are paired with the three CDRs of the L chain to form the antigen binding site, as classically defined. There are five main classes of heavy chain C domains. Each class defines the IgM, IgG, IgA, IgD, and IgE isotypes. IgG can be split into four subclasses, lgG1 , lgG2, lgG3, and lgG4, each with its own biologic properties; and IgA can similarly be split into lgA1 and lgA2. The constant domains of the H chain can be switched to allow altered effector function while maintaining antigen specificity.

[0346] In a more particular embodiment, the immunoglobulin is IgA, IgG, IgM, IgE or IgD. In a still more particular embodiment, the immunoglobulin is lgA1 , lgA2, lgG1 , lgG2, lgG3, lgG4, IgM, IgE or IgD.

[0347] In some embodiments, when the extracellular domain of the chimeric receptor of the invention is a polypeptide that can form an oligomer in the presence of one or more additional subunits, then the oligomer is a bispecific antibody or a trispecific antibody that binds to more than one antigen.

[0348] Bispecific antibodies (BsAbs) are antibodies with two binding sites directed at two different antigens or two different epitopes on the same antigen. BsAbs come in many formats, ranging from relatively small proteins, merely consisting of two linked antigenbinding fragments, to large immunoglobulin G (IgG)-like molecules with additional domains attached. In a particular embodiment, the bispecific antibody is a IgG-like or a non-IgG-like bispecific antibody. The IgG-like bispecific antibodies retains the traditional monoclonal antibody (mAb) structure of two Fab arms and one Fc region, except the two Fab sites bind different antigens. The most common types are called trifunctional antibodies, as they have three unique binding sites on the antibody: the two Fab regions, and the Fc region. Each heavy and light chain pair is from a unique mAb. The Fc region made from the two heavy chains forms the third binding site. The non-IgG-like bispecific antibodies lack an Fc region entirely. These include chemically linked Fabs, consisting of only the Fab regions, and various types of bivalent and trivalent single-chain variable fragments (ScFvs). There are also fusion proteins mimicking the variable domains of two antibodies. The furthest developed of these newer formats are the bi-specific T-cell engagers (BiTEs).

[0349] Trispecific antibodies (TsAbs) are antibodies that have three targets, i.e. are antibodies with three binding sites directed at three different antigens or three different epitopes on the same antigen. Examples of trispecific antibodies include, but are not limited to, trispecific killer cell engagers (TriKEs). TriKEs are a combination of a single-chain Fv against CD16 and two tumor associated antigens. These molecules directly trigger NK cell activation through CD 16 amplifying NK cell cytolytic activity and cytokine production against various tumor cell antigen targets

[0350] As used herein, a "chimeric antigen receptor (CAR)" also known as chimeric T cell receptors, a T-body, artificial T cell receptors and chimeric immune receptors (CIR), are engineered receptors, which graft an arbitrary specificity onto an immune effector cell. In a classical CAR, the specificity of a monoclonal antibody is grafted on to a T cell. CARs are therefore fusion proteins which comprise at least, an extracellular domain or antigen binding domain capable of binding to an antigen of interest, a transmembrane domain, and at least one intracellular signalling domain from an antigen receptor.

[0351] The term “antigen-binding domain” has been defined above, and this definition is applicable to the chimeric receptor of the invention. In a particular embodiment, the chimeric receptor further comprises a hinge domain or region between the binding molecule and the transmembrane domain,

[0352] In a more particular embodiment, the CAR of the invention further comprises a hinge domain between the antigen-binding domain and the transmembrane domain.

[0353] As used herein, “hinge domain”, “hinge region” or “spacer” refers to an amino acid region that allows for separation and flexibility of the binding moiety and the T cell membrane. The length of the flexible hinges also allows for better binding to relatively inaccessible epitopes, e.g., longer hinge domains are allow for optimal binding. One skilled in the art will be able to determine the appropriate hinge for the given CAR target.

[0354] In a particular embodiment, the hinge region is selected from the group consisting of: IgG 1 hinge region, lgG2 hinge region, lgG4 hinge region, FcyR hinge region, CD3E hinge region, CD3 hinge region, CD8a hinge region, CD4 hinge region, CD28 hinge region, CD7 hinge region, CD19 hinge region. The hinge domain can have a length of from about 10 amino acids to about 200 amino acids, preferably, between 30 and 150 amino acids, more preferably between 40 and 125 amino acids.

[0355] Exemplary spacers 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 therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains. Exemplary spacers can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 20), GGSGG (SEQ ID NO: 21), GSGSG (SEQ ID NO: 22), GSGGG (SEQ ID NO: 23), GGGSG (SEQ ID NO: 24), GSSSG (SEQ ID NO: 25), and the like.

[0356] The chimeric receptor of the invention comprises ii) a linker region, the sequence of which is the result of a protein splicing reaction.

[0357] “Protein splicing reaction” or “intein reaction” refers to the self-excision process of the inteins.lnteins are internal protein elements that self-excise from their host protein and catalyze ligation of the flanking sequences (exteins) with a peptide bond. Intein excision is a posttranslational process that does not require auxiliary enzymes or cofactors. In some embodiments, an intein reaction (e.g., cleavage, linkage (splicing), cyclization) can be initiated by contacting the fusion protein comprising an intein N-terminal domain with the fusion protein comprising an intein C-terminal domain. In other embodiments, an intein reaction can be initiated by shifting the conditions, e.g., the temperature or pH, in which a split intein fusion protein or combination of split intein fusion proteins is incubated. In some embodiments, a C-terminal cleavage is initiated by a pH or temperature shift.

[0358] In some embodiments, an intein reaction is initiated by contacting fusion proteins with DTT or another strong nucleophile. In some embodiments, DTT is used to enhance a reaction. In some embodiments, an N-terminal cleavage is initiated by a strong nucleophile, e.g., DTT.

[0359] Another manner of inducing protein splicing or cleavage is by contact with a peptide or peptidomimetic agent that activates splicing or cleavage. Another manner of inducing protein splicing or cleavage is by removal of a peptide or peptidomimetic agent that blocks or inhibits splicing or cleavage.

[0360] In a particular embodiment, the linker region resulting from the protein splicing reaction comprises between 1 and 8 amino acids. In a more particular embodiment, the linker region resulting from the protein splicing reaction comprises between 1 and 6 amino acids. In a still more particular embodiment, the linker region resulting from the protein splicing reaction comprises 6 amino acids.

[0361] In a particular embodiment, the linker region resulting from the protein splicing reaction of the Gp41.1 , the IMPDH, the NrdJ1 or the Gp41.8 split intein comprises a Ser (S), a Thr (T) or a Cys (C) residue. In a more particular embodiment, the Ser (S), Thr (T) or Cys (C) is in the first position of the linker region.

[0362] In another particular embodiment, the linker region resulting from the protein splicing reaction of the Gp41.1 split intein comprises the sequence SGYSIC (SEQ ID NO: 26) or SGYSIS (SEQ ID NO: 35).

[0363] In another particular embodiment, the linker region resulting from the protein splicing reaction of the IMPDH split intein comprises the sequence GGGSIC (SEQ ID NO: 27).

[0364] In another particular embodiment, the linker region resulting from the protein splicing reaction of the NrdJ1 split intein comprises the sequence NPCSEI (SEQ ID NO: 28). In another particular embodiment, the linker region resulting from the protein splicing reaction of the Gp41.8 split intein comprises the sequence LNRSAV (SEQ ID NO: 29).

[0365] The chimeric receptor of the invention comprises iii) a transmembrane domain.

[0366] The term “transmembrane domain” has been defined above, and this definition is applicable to the chimeric receptor of the invention. In a particular embodiment, the transmembrane domain is selected from the group consisting of the CD28, the CD8a the 4-1 BB, the CD4, the CTLA4, the CD27 or the CD3 transmembrane domain.

[0367] The chimeric receptor of the invention comprises iv) an intracellular signalling domain from a receptor.

[0368] The term “intracellular signalling domain” has been defined or explained above, and this definition is applicable to the chimeric receptor of the invention. In a particular embodiment, the intracellular signalling domain is an antigen receptor signalling domain.

[0369] In a more particular embodiment, the antigen receptor signalling domain is selected from the group consisting of the CD3 , CD28, 4-1 BB, NKG2D, DAP10, 0X40, CD70, CD27, CD5, ICAM-1 , LFA-1 (CD11a / CD18), ICOS (CD278), DAP 12 antigen receptor signalling domain or a combination thereof.

[0370] Third host cell of the invention

[0371] In another aspect, the present invention relates to a host cell, hereinafter the third host cell of the invention, comprising the chimeric receptor of the invention.

[0372] The term “host cell” has been defined or explained above, and this definition is applicable to the third host cell of the invention.

[0373] In a particular embodiment, the third host cell of the invention is a human cell.

[0374] In a more particular embodiment, the third host cell of the invention is selected from the group consisting of: T cell, NK-cell, induced pluripotent stem cell (iPSC) derived NK cells, Pro-Tcell, B cell, and macrophage.

[0375] First composition or kit-of-parts of the invention

[0376] In another aspect, the present invention relates to a composition or kit-of-parts comprising a first component and a second component wherein i) the first component is the first polynucleotide of the invention, the first vector of the invention, the first host cell of the invention or the first fusion protein of the invention and ii) the second component is the second fusion protein of the invention or the second polynucleotide of the invention, wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

[0377] The variants 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 are applicable to the first composition or kit-of parts of the invention.

[0378] The ratio of the components in the compositions is adequate for an efficient processing of the fusion proteins. Suitable ratios of the first and second components include, without limitation, 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.

[0379] First and second methods of the invention for the generation of chimeric antigen receptors

[0380] In another aspect, the present invention relates to an in vitro method, hereinafter the first method of the invention, for producing a cell comprising a chimeric receptor, wherein the method comprises: i) providing a cell comprising the first polynucleotide of the invention, a polypeptide encoded by said polynucleotide or the first vector of the invention under conditions adequate for the expression in the cell of the fusion protein encoded by the polynucleotide, and ii) contacting the cell of step i) with the second fusion protein of the invention, wherein the contacting step ii) is performed under conditions that allow a splicing reaction between the split C-intein forming part of the fusion protein produced in step (i) and the split N-intein forming part of the second fusion proteins of the invention and wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

[0381] The term “in vitro method” means that the production of a cell comprising a chimeric receptor is carried out outside the subject's body.

[0382] In a particular embodiment, the chimeric receptor is a chimeric antigen receptor. The term “chimeric antigen receptor” has been defined or explained above, and this definition is applicable to the first method of the invention.

[0383] The first step of the first method of the invention comprises i) providing a cell comprising the first polynucleotide of the invention, a polypeptide encoding by said polynucleotide or the first vector of the invention under conditions adequate for the expression in the cell of the fusion protein encoded by the polynucleotide.

[0384] Suitable conditions for the expression in the cell of the fusion protein encoded by the first polynucleotide of the invention are well known for the person skilled in the art. Conditions suitable for the expression of the polynucleotide in the host cell include those conditions that allow the optimal growth of said host cell and those conditions that allow the protein expression in said host cell. Said culture conditions are typically different for each type of host cell. However, those conditions are known by skilled workers and are readily determined. Similarly, the duration of maintenance can differ with the host cells and with the amount of the fusion protein desired to be prepared. Again, those conditions are well known and can be readily determined in specific situations.

[0385] The second step of the first method of the invention comprises ii) contacting the cell of step i) with the second fusion protein of the invention, wherein the contacting step ii) is performed under conditions that allow a splicing reaction between the split C-intein forming part of the fusion protein produced in step (i) and the split N-intein forming part of the second fusion proteins of the invention, wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

[0386] Suitable conditions that allow a splicing reaction between the split C-intein forming part of the fusion protein produced in step (i) and the split N-intein forming part of the second fusion protein of the invention are well known for the person skilled in the art. The terms “protein splicing reaction” or “intein reaction” have been defined or explained above, and this definition is applicable to the first method of the invention.

[0387] As explained above, in some embodiments, an intein reaction (e.g., cleavage, linkage (splicing), cyclization) can be initiated by contacting a fusion protein comprising an intein N-terminal domain and a fusion protein comprising an intein C-terminal domain. In other embodiments, an intein reaction can be initiated by shifting the conditions, e.g., the temperature or pH, in which a split intein fusion protein or combination of split intein fusion proteins is incubated. In some embodiments, a C-terminal cleavage is initiated by a pH or temperature shift. In some embodiments, an intein reaction is initiated by contacting fusion proteins with DTT or another strong nucleophile. In some embodiments, DTT is used to enhance a reaction. In some embodiments, an N-terminal cleavage is initiated by a strong nucleophile, e.g., DTT.

[0388] Another manner of inducing protein splicing or cleavage is by contact with a peptide or peptidomimetic agent that activates splicing or cleavage. Another manner of inducing protein splicing or cleavage is by removal of a peptide or peptidomimetic agent that blocks or inhibits splicing or cleavage.

[0389] The variants 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 are applicable to the first method of the invention.

[0390] In a particular embodiment, the cell is a human cell.

[0391] In a more particular embodiment, the cell is selected from the group consisting of: T cell, NK-cell, induced pluripotent stem cell (iPSC) derived NK cells, Pro-Tcell, B cell and macrophage.

[0392] In another particular embodiment, the cell used in step (i) have been obtained from a patient.

[0393] In another aspect, the present invention relates to an in vitro method, hereinafter the second method of the invention, for producing a cell comprising a chimeric receptor, wherein the method comprises: i) providing a cell comprising a. the first polynucleotide of the invention, the first vector of the 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 in frame and N-terminal to the split C-intein and which allows the insertion of the fusion protein into the membrane and b. the second polynucleotide of the invention, the second vector of the invention or the second fusion protein of the 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 in frame and N-terminal to the polypeptide that specifically binds to a target molecule and which allows the secretion of the fusion protein from the cell and ii) maintaining the cell under conditions adequate for the expression in the cell of both fusion proteins, for the insertion into the membrane of the fusion protein encoded by the first polynucleotide of the invention, for the secretion of the fusion protein encoded by the second polynucleotide of the invention and for the splicing reaction between the split C-intein and the split N-intein and wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO: 3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

[0394] The term “in vitro" has been defined or explained above, and this definition is applicable to the second method of the invention.

[0395] The first step of the second method of the invention comprises i) providing a cell comprising the first polynucleotide of the invention, the first vector of the 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 in frame and N-terminal to the split C-intein and which allows the insertion of the fusion protein into the membrane and b. the second polynucleotide of the invention, the second vector of the invention or the second fusion protein of the 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 in frame and N-terminal to the polypeptide that specifically binds to a target molecule and which allows the secretion of the fusion protein from the cell. The “signal peptides” that can be used in the present invention have been defined or explained above, and these definitions are applicable to the second method of the invention. Any signal peptide that allows the insertion of the fusion protein encoded by the first polynucleotide of the invention into the membrane may be used in the present invention. Examples of signal peptides are CD8a, IL-2, GM-CSF receptor (GM-CSF) a chain, murine Ig-kappa (IgK), murine IgK V-lll region (IgKVIll), human IgKVIll, CD33, human tissue plasminogen activator (TPA) and native secreted alkaline phosphatase (SEAP).

[0396] The signal peptide that is in frame and N-terminal to the polypeptide that specifically binds to a target molecule has to allow the secretion of the fusion protein from the cell. Any signal peptide that allows the secretion of the fusion protein from the cell can be used in the present invention.

[0397] The second step of the second method of the invention comprises ii) maintaining the cell under conditions adequate for the expression in the cell of both fusion proteins, for the insertion into the membrane of the fusion protein encoded by the first polynucleotide of the invention, for the secretion of the fusion protein encoded by the second polynucleotide of the invention and for the splicing reaction between the split C-intein and the split N-intein and wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO: 3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

[0398] Suitable conditions for the expression in the cell of the both fusion proteins encoded by the polynucleotides are well known for the person skilled in the art. Conditions suitable for the expression of a polynucleotide in the host cell include those conditions that allow the optimal growth of said host cell and those conditions that allow the protein expression in said host cell. Said culture conditions are typically different for each type of host cell. However, those conditions are known by skilled workers and are readily determined. Similarly, the duration of maintenance can differ with the host cells and with the amount of the fusion proteins desired to be prepared. Again, those conditions are well known and can readily be determined in specific situations.

[0399] Suitable conditions for the insertion into the membrane of the fusion protein encoded by the first polynucleotide of the invention and for the secretion of the fusion protein encoded by the second polynucleotide of the invention are well known for the person skilled in the art.

[0400] In a particular embodiment, when the chimeric receptor is an oligomeric chimeric receptor, then the cell provided in (i) further comprises (c) an additional polypeptide encoding additional elements of the oligomer.

[0401] In a more particular embodiment, the oligomer is an immunoglobulin. In a still more particular embodiment, the immunoglobulin is IgA, IgG, IgM, IgE or IgD. In a still more particular embodiment, the immunoglobulin is lgA1 , lgA2, lgG1 , lgG2, lgG3, lgG-4, IgM, IgE or IgD.

[0402] In another particular embodiment, the fusion protein is a heavy chain or a light chain of an immunoglobulin, and then the additional polypeptide (c) encodes the corresponding heavy or light chain of the immunoglobulin.

[0403] The terms “oligomer”, “immunoglobulin”, “heavy chain” and “light chain” have been defined or explained above, and these definitions are applicable to the second method of the invention.

[0404] In some embodiments, the protein splicing reaction between the protein encoded by the first polynucleotide of the invention and the fusion protein encoded by the second polynucleotide of the invention is either intracellular and occurs in the secretory pathway of the cell producing both polypeptides, before the secretion of the second polypeptide. In other embodiments the protein splicing reaction occurs once the second polypeptide has been secreted and takes place in the extracellular space.

[0405] The conditions that allow a protein splicing reaction or an intein reaction have been defined or explained in the first method of the invention and these conditions are applicable to the second method of the invention. The variants 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 are applicable to the second method of the invention.

[0406] In a particular embodiment, the cell is a human cell.

[0407] In a more particular embodiment, the cell is selected from the group consisting of: T cell, NK-cell, induced pluripotent stem cell (iPSC) derived NK cells, Pro-Tcell, B cell and macrophage.

[0408] In another particular embodiment, the cell used in step (i) have been obtained from a patient.

[0409] First engineered cell of the invention

[0410] In another aspect, the present invention relates to a cell, hereinafter the first engineered cell of the invention, comprising a chimeric receptor obtained by the first method or the second method of the invention.

[0411] Medical uses

[0412] In another aspect, the present invention relates to the third host cell of the invention or the first engineered cell of the invention for use in the treatment of a disease which requires a cell-mediated immune response against a target molecule.

[0413] In a particular embodiment, the target molecule is an antigen of interest.

[0414] In a particular aspect, the invention relates to the third host cell of the invention for use in the treatment of a disease which requires a cell-mediated immune response against a target molecule, preferably against an antigen of interest.

[0415] In another particular aspect, the invention relates to the first cell of the invention for use in the treatment of a disease which requires a cell-mediated immune response against a target molecule, preferably against an antigen of interest.

[0416] The term “treatment” is used to designate the administration of the third host cell of the invention or the cell of the invention to control the progression of the disease before or after the clinical signs have appeared.

[0417] In another aspect, the present invention relates to the second fusion protein of the invention, the second polynucleotide of the invention, the second vector of the invention or the second host cell of the invention for use in the treatment of a disease which requires a cell-mediated immune response against the target molecule, wherein the patient to be treated is characterized in that it contains cells comprising the first polynucleotide of the invention, the first vector of the invention or the fusion protein encoded by the first polynucleotide of the invention, wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

[0418] In another aspect, the present invention relates to the first polynucleotide of the invention, the first vector of the invention, the first host cell of the invention or the first fusion protein of the invention for use in the treatment of a disease which requires a cell-mediated immune response against the target molecule, wherein the patient to be treated is characterized in that it contains cells comprising the second fusion protein of the invention, the second polynucleotide of the invention, the second vector of the invention or the second host cell of the invention, wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof. In a particular embodiment, the target molecule is an antigen of interest.

[0419] The term “treatment” is also used to designate the administration of the second fusion protein of the invention, the second polynucleotide of the invention, the second vector of the invention or the second host cell of the invention to control the progression of the disease before or after the clinical signs have appeared in a patient that is characterized in that it contains cells comprising the first polynucleotide of the invention, the first vector of the invention or the fusion protein encoded by the first polynucleotide of the invention.

[0420] The term “treatment” is also used to designate the administration of the first polynucleotide of the invention, the first vector of the invention, the first host cell of the invention or the first fusion protein of the invention to control the progression of the disease before or after the clinical signs have appeared in a patient that is characterized in that it contains cells comprising the second fusion protein of the invention, the second polynucleotide of the invention, the second vector of the invention or the second host cell of the invention.

[0421] The term “subject” or “patient” as used herein, includes any animal that has disease which requires a cell-mediated immune response against a target molecule, preferably against an antigen of interest, or is at risk for having a disease which requires a cell- mediated immune response against a target molecule or exhibiting a symptom of a disease which requires a cell-mediated immune response against a target molecule. Suitable subjects (patients) include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as cats or dogs). Nonhuman primates and, preferably, human patients, are included. Preferably, the subject is a mammal, more preferably is a human.

[0422] Control of the progression of the disease is understood as the beneficial or desired clinical results which include but are not limited to: reduction of the symptoms, reduction of the duration of the disease, stabilization of pathological conditions (specifically avoiding additional impairment), delaying the progression of the disease, improving the pathological condition and remission (both partial and complete). The control of the progression of the disease also involves a prolongation of survival in comparison to the expected survival if the treatment was not applied. In the present invention, the disease is a disease which requires a cell-mediated immune response against the target molecule. Cell-mediated immunity or cellular immunity is an immune response that does not involve antibodies. Rather, cell-mediated immunity is the activation of phagocytes, antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen.

[0423] In a particular embodiment, the disease requiring a cell-mediated immune response against a target molecule, preferably against an antigen of interest, is selected from the group consisting of: cancer, a viral infection, a bacterial infection, a parasitic infection and an autoimmune disease.

[0424] The term “cancer” is referred to a disease characterized by uncontrolled cell division (or by an increase of survival or apoptosis resistance), by the ability of said cells to invade other neighbouring tissues (invasion) or by the spread to other areas of the body where the cells are not normally located (metastasis) through the lymphatic and blood vessels. Depending on whether or not tumours can spread by invasion and metastasis, they are classified as being either benign or malignant: benign tumours are tumours that cannot spread by invasion or metastasis, i.e., they only grow locally; whereas malignant tumours are tumours that are capable of spreading by invasion and metastasis.

[0425] Cancer includes, in one embodiment, without limitation, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), hairy cell leukemia, polycythemia vera, lymphoma (e.g., Hodgkin’s disease or non-Hodgkin’s disease), AIDS-associated leukemias, Waldenstrom's macroglobulinemia, multiple myeloma, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, mendotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, Kaposi’s sarcoma, colon carcinoma, 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 adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, teratoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, intraepithelial neoplasms including Bowen’s disease and Paget’s disease, neuroglioma, glioma, astrocytoma, glioblastoma multiforme (GBM, also known as glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

[0426] In some embodiments, the cancer is glioma, astrocytoma, glioblastoma multiforme (GBM, also known as glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, or retinoblastoma.

[0427] 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, brain stem glioma, ependymoma, mixed glioma, optic nerve glioma, subependymoma, medulloblastoma, meningioma, metastatic brain tumor, oligodendroglioma, pituitary tumors, primitive neuroectodermal (PNET) tumor, or schwannoma. In some embodiments, the cancer is a type found more commonly in children than adults, such as brain stem glioma, craniopharyngioma, ependymoma, juvenile pilocytic astrocytoma (JPA), medulloblastoma, optic nerve glioma, pineal tumor, primitive neuroectodermal tumors (PNET), or rhabdoid tumor.

[0428] Cancer includes, in another embodiment, without limitation, mesothelioma, hepatobilliary (hepatic and billiary duct), bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal, and duodenal), uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin’s Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, testicular cancer, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, non-Hodgkins’s lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, adrenocortical cancer, gall bladder cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the foregoing cancers. In some embodiments, the cancer is selected from hepatocellular carcinoma, ovarian cancer, ovarian epithelial cancer, or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (LIPSC); prostate cancer; testicular cancer; gallbladder cancer; hepatocholangiocarcinoma; soft tissue and bone 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 tumors (MPNST); Waldenstrom’s macroglobulinemia; or medulloblastoma.

[0429] In some embodiments, 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 (LIPSC), hepatocholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, anaplastic thyroid cancer, adrenocortical adenoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1 associated malignant peripheral nerve sheath tumors (MPNST), Waldenstrom’s macroglobulinemia, or medulloblastoma.

[0430] In some embodiments, a cancer is a solid tumor, such as a sarcoma, carcinoma, or lymphoma. Solid tumors generally comprise an abnormal mass of tissue that typically does not include cysts or liquid areas. In some embodiments, 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 nonsmall cell lung cancer (NSCLC) or small cell lung cancer (SCLC); ovarian cancer, ovarian epithelial cancer, ovarian carcinoma, or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (LIPSC); prostate cancer; testicular cancer; gallbladder cancer; hepatocholangiocarcinoma; soft tissue and bone synovial sarcoma; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing sarcoma; anaplastic thyroid cancer; adrenocortical carcinoma; 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 tumors (MPNST); Waldenstrom’s macroglobulinemia; or medulloblastoma.

[0431] In some embodiments, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial cancer, ovarian carcinoma, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (LIPSC), hepatocholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, anaplastic thyroid cancer, adrenocortical carcinoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1 associated malignant peripheral nerve sheath tumors (MPNST), Waldenstrom’s macroglobulinemia, or medulloblastoma.

[0432] In an embodiment, the cancer is a primary tumor. The term "primary tumor", as used herein, refers to a tumor that originated in the location or organ in which it is present and did not metastasize to that location from another location.

[0433] In another embodiment, the cancer is a cancer metastasis. In the context of the present invention, "metastasis" is understood as the propagation of a cancer from the organ where it started to a different organ. It generally occurs through the blood or lymphatic system. When the cancer cells spread and form a new tumor, the latter is called a secondary or metastatic tumor. The cancer cells forming the secondary tumor are like those of the original tumor.

[0434] In a preferred embodiment, the cancer is selected from the group consisting of: 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.

[0435] In a particular embodiment, the cancer is a leukemia, preferably B-cell acute lymphoblastic leukemia. In a more particular embodiment, the antigen against which the chimeric receptor is directed is CD19.

[0436] In another particular embodiment, the cancer is a lymphoma, preferably a non-Hodgkin lymphoma. In a more particular embodiment, the antigen against which the chimeric receptor is directed is CD19.

[0437] In another particular embodiment, the cancer is multiple myeloma. In a more particular embodiment, the antigen against which the chimeric receptor is directed is B-cell maturation antigen (BCMA). In another particular embodiment, the cancer is neuroblastoma. In a more particular embodiment, the antigen against which the chimeric receptor is directed is disialoganglioside GD2.

[0438] In another particular embodiment, the cancer is prostate cancer. In a more particular embodiment, the antigen against which the chimeric receptor is directed is prostatespecific membrane antigen (PSMA).

[0439] In another particular embodiment, the cancer is acute myeloid leukemia. In a more particular embodiment, the antigen against which the chimeric receptor is directed is CD33.

[0440] In another particular embodiment, the cancer is pancreatic cancer. In a more particular embodiment, the antigen against which the chimeric receptor is directed is mucin 1 (MUC1).

[0441] In another particular embodiment, the cancer is ovarian cancer. In a more particular embodiment, the antigen against which the chimeric receptor is directed is folate receptor alpha.

[0442] In another particular embodiment, the cancer is glioblastoma. In a more particular embodiment, the antigen against which the chimeric receptor is directed is epidermal growth factor receptor variant III (EGFRvlll).

[0443] In another particular embodiment, the cancer is sarcoma. In a more particular embodiment, the antigen against which the chimeric receptor is directed is NY-ESO-1.

[0444] In another particular embodiment, the cancer is breast cancer. In a more particular embodiment, the antigen against which the chimeric receptor is directed is HER2.

[0445] In another particular embodiment, the cancer is colorectal cancer. In a more particular embodiment, the antigen against which the chimeric receptor is directed is carcinoembryonic antigen (CEA).

[0446] In another particular embodiment, the cancer is renal cell carcinoma. In a more particular embodiment, the antigen against which the chimeric receptor is directed is carbonic anhydrase IX (CAIX).

[0447] In another particular embodiment, the cancer is melanoma. In a more particular embodiment, the antigen against which the chimeric receptor is directed is melanoma- associated antigen (MAGE). In another particular embodiment, the disease requiring a cell-mediated immune response against a target molecule, preferably against an antigen of interest, is an infection.

[0448] An “infection” is the invasion of tissues by pathogens, their multiplication, and the reaction of host tissues to the infectious agent and the toxins they produce. An infectious disease, also known as a transmissible disease or communicable disease, is an illness resulting from an infection. Infections can be caused by a wide range of pathogens, most prominently bacteria, viruses and parasites.

[0449] In a more particular embodiment, the disease requiring a cell-mediated immune response against a target molecule, preferably against an antigen of interest, is a viral infection.

[0450] A “viral infection” is an infection caused by a pathogenic virus. A viral disease (or viral infection) occurs when an organism's body is invaded by pathogenic viruses, and infectious virus particles (virions) attach to and enter susceptible cells. Viral infections can be caused by HIV, Rhinovirus, Lyssaviruses such as Rabies virus, Ebolavirus or Severe acute respiratory syndrome coronavirus 2).

[0451] In another more particular embodiment, the disease requiring a cell-mediated immune response against a target molecule, preferably against an antigen of interest, is a bacterial infection.

[0452] A “bacterial infection” is an infection caused by a 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.

[0453] In another more particular embodiment, the disease requiring a cell-mediated immune response against a target molecule, preferably against an antigen of interest, is a parasitic infection.

[0454] A “parasitic infection” is an infection caused by a parasite. Parasites are organisms which derive sustenance from its host while causing it harm. Examples of parasitic infections are malaria, Toxoplasma, Babesia.

[0455] In another particular embodiment, the disease requiring a cell-mediated immune response against a target molecule, preferably against an antigen of interest, is an autoimmune disease. The term “autoimmune disease”, "disease associated with immune dysfunction / dysregulation" or "immune inflammatory disease" is used throughout the specification to refer to a pathogenic condition in which the patient’s immune system results in disease from a self-antigen (autoimmunity) or a foreign antigen (immune dysfunction / dysregulation or immune inflammatory disease). Autoimmunity is present in everyone to some extent. It is usually harmless and probably a universal phenomenon of vertebrate life. However, autoimmunity can be the cause of a broad spectrum of human illnesses, known as autoimmune diseases. Autoimmune diseases are, thus, defined when the progression from benign autoimmunity to pathogenic autoimmunity occurs. This progression is determined by both genetic influences and environmental triggers. The concept of autoimmunity as the actual cause of human illness (rather than a consequence or harmless accompaniment) can be used to establish criteria that define a disease as an autoimmune disease. Autoimmune diseases or diseases which are characterized as involving immune dysfunction or dysregulation (immune inflammatory disease), which may be treated by the present invention include systemic lupus erythematosus (SLE), lupus nephritis, central nervous system (CNS) lupus, diabetes mellitus (type I), asthma, ulcerative colitis, Crohn’s disease, Grave's disease, Addison's disease, celiac disease, alopecia areata, arthritis, including rheumatoid arthritis and osteoarthritis, pernicious anemia, and multiple sclerosis, among numerous others.

[0456] Numerous autoimmune diseases may be treated using i) the cell of the invention or the third host cell of the invention, ii) the second polynucleotide of the invention, the second vector of the invention or the second host cell of the invention or iii) the first polynucleotide of the invention, the first vector of the invention, the first host cell of the invention or the first fusion protein of the invention, including autoimmune blood diseases, including pernicious anemia, autoimmune hemolytic anemia, aplastic anemia, idiopathic thrombocytopenic purpura, ankylosing spondilitis; autoimmune diseases of the musculature including polymyositis and dermatomyositis, autoimmune diseases of the ear including autoimmune hearing loss and Meniere's syndrome, autoimmune eye diseases, including Mooren's disease, Reiter's syndrome and Vogt-Koyanagi-Harada disease, autoimmune diseases of the kidney including glomerulonephritis, IgA nephropathy, and lupus nephritis; diabetes mellitus (type I); autoimmune skin diseases including pemphigus (autoimmune bullous diseases), such as pemphigus vulgaris, pemphigus foliaceus, pemphigus erythematosus, bullous pemphigoid, vitiligo, epidermolysis bullosa acquisita, psoriasis and alopecia areata; cardiovascular autoimmune diseases, including autoimmune myocarditis, vasculitis including Churg- Strauss syndrome, giant cells arteritis, Kawasaki's disease, polyarteritis nodosa, Takayasu's arteritis and Wegener's granulomatosis; endocrine autoimmune diseases, including Addison's disease, autoimmune hypoparathyroidism, autoimmune hypophysitis, autoimmune oophoritis, autoimmune orchitis, Grave's Disease, Hashimoto's thyroiditis, polyglandular autoimmune syndrome type 1 (PAS-I) polyglandular autoimmune syndrome type 2 (PAS-2), and polyglandular autoimmune syndrome type 3 (PAS-3); autoimmune gastroenteric diseases including autoimmune hepatitis, primary biliary cirrhosis, inflammatory bowel disease, celiac disease, Crohn's disease; autoimmune nervous diseases, including multiple sclerosis, myasthenia gravis, Guillan-Barre syndrome and chronic inflammatory demyelinating neuropathy; and systemic autoimmune diseases including systemic lupus erythematosus, antiphospholid syndrome, autoimmune lymphoproliferative disease, autoimmune polyendocrinopathy, Bechet's disease, Goodpasture's disease, arthrtitis, including rheumatoid arthritis, osteoarthritis and septic arthritis, sarcoidosis, scleroderma and Sjogren's syndrome and psoriasis among others.

[0457] In a particular embodiment, the autoimmune disease is selected from the group consisting of: celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel diseases, multiple sclerosis, alopecia areata, Addison's disease, pernicious anemia, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

[0458] Third and fourth polynucleotides of the invention

[0459] In another aspect, the present invention relates to a polynucleotide, hereinafter the fourth polynucleotide of the invention, encoding a fusion protein comprising from N-terminus to C-terminus:

[0460] (i) a modified N-intein selected from the group consisting of a modified GP41.1 N- intein, a modified IMPDH N-intein, a modified NrdJ1 N-intein or a modified GP41.8 N-intein, wherein the modified N-intein comprises a fragment of the N-intein protein that prevents trans-splicing from occurring in the presence of the corresponding C- intein and wherein the modified N-intein comprises at least one mutation that decreases the affinity between the modified N-intein and the corresponding C-intein,

[0461] (ii) a split C-intein comprising a sequence selected from the group consisting of 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-intein is a modified GP41.1 N-intein, the split C-intein comprises the sequence of SEQ ID NO: 1 or is a functionally equivalent variant thereof,

[0462] If the modified N-intein is a modified IMPDH N-intein, the split C-intein comprises the sequence of SEQ ID NO: 2 or is a functionally equivalent variant thereof, the modified N-intein is a modified IMPDH N-intein

[0463] If the modified N-intein is a modified NrdJ1 N-intein, the split C-intein comprises the sequence of SEQ ID NO: 3 or is a functionally equivalent variant thereof or

[0464] If the modified N-intein is a modified GP41.8 N-intein, the split C-intein comprises the sequence of SEQ ID NO: 4 or is a functionally equivalent variant thereof, and

[0465] (iii) a heterologous polypeptide (C-extein).

[0466] The terms “polynucleotide”, “polypeptide”, “intein”, “split intein”, “split C-intein” and “split N-intein” have been defined or explained above and these definitions are applicable to the third polynucleotide of the invention.

[0467] The terms “functionally equivalent variants of SEQ ID NO: 1-4” has been defined or explained above and these definitions are applicable to the third polynucleotide of the invention.

[0468] The term “modified N-intein” has been defined or explained above and this definition is applicable to the third polynucleotide of the invention.

[0469] The modified N-intein comprises a fragment of the N-intein protein that prevents transsplicing from occurring in the presence of the corresponding C-intein.

[0470] In a particular embodiment, the modified N-intein does not comprise the complete sequence of the N-intein protein.

[0471] In a particular embodiment, the modified GP41.1 N-intein comprises a fragment of the SEQ ID NO: 5 that prevents trans-splicing from occurring in the presence of the GP41.1 C-intein. In a particular embodiment, the modified GP41.1 N-intein does not comprise the complete sequence of the SEQ ID NO: 5.

[0472] In another particular embodiment, the modified IMPDH1 N-intein comprises a fragment of the SEQ ID NO: 6 that prevents trans-splicing from occurring in the presence of the IMPDH1 C-intein. In a particular embodiment, the modified IMPDH1 N-intein does not comprise the complete sequence of SEQ ID NO: 6.

[0473] In another particular embodiment, the modified NrdJ1 N-intein comprises a fragment of the SEQ ID NO: 7 that prevents trans-splicing from occurring in the presence of the NrdJ1 C-intein. In a particular embodiment, the modified NrdJ1 N-intein does not comprise the complete sequence of SEQ ID NO: 7.

[0474] In another particular embodiment, the modified GP41.8 N-intein comprises a fragment of the SEQ ID NO: 8 that prevents trans-splicing from occurring in the presence of the GP41.8 C-intein. In a particular embodiment, the modified GP41.8 N-intein does not comprise the complete sequence of SEQ ID NO: 8.

[0475] The term “fragment” has been defined or explained above and this definition is applicable to the third polynucleotide of the invention. In a particular embodiment the fragment comprises or consists between 10 and 100 amino acids, preferably between 20 and 65 amino acids, more preferably between 45 and 60 amino acids.

[0476] In a particular embodiment, the modified GP41.1 N-intein is a fragment of the sequence SEQ ID NO: 5 that comprises or consists between 18 and 83 amino acids, preferably between 30 and 60 amino acids, more preferably between 40 and 50 amino acids. The fragment of the sequence SEQ ID NO: 5 comprises at least one mutation that decreases the affinity between the modified GP41.1 N-intein and the GP41.1 C-intein.

[0477] In a particular embodiment, the modified GP41.1 N-intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids, the first 40 amino acids, the first 45 amino acids, the first 50 amino acids, the first 55 amino acids, the first 60 amino acids or the first 65 amino acids of the polypeptide as defined in SEQ ID NO: 5.

[0478] In another particular embodiment, the modified GP41.1 N-intein does not comprise 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.

[0479] The elimination of the amino acids in the N-terminal position of the GP41.1 N-intein (i.e. first amino acids of the polypeptide as defined in SEQ ID NO: 5) and in the C-terminal position of the GP41.1 N-intein (i.e. last amino acids of the polypeptide as defined in SEQ ID NO: 5) may be combined. In a particular embodiment, the modified GP41.1 N- intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids, the first 40 amino acids, the first 45 amino acids or the first 50 amino acids of the polypeptide as defined in SEQ ID NO: 5 and does not comprise 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.

[0480] In a more particular embodiment, the modified GP41.1 N-intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids or the first 40 amino acids of the polypeptide as defined in SEQ ID NO: 5 and does not comprise 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.

[0481] In a preferred embodiment, the modified GP41.1 N-intein does not comprise the 41 first amino acids of the polypeptide as defined in SEQ ID NO: 5.

[0482] In another particular embodiment, the modified IMPDH N-intein is a fragment of the SEQ ID NO: 6 that comprises or consists between 21 and 96 amino acids, preferably between 40 and 70 amino acids, more preferably between 50 and 60 amino acids. The fragment of the sequence SEQ ID NO: 6 comprises at least one mutation that decreases the affinity between the modified IMPDH N-intein and the IMPDH C-intein.

[0483] In a particular embodiment, the modified IMPDH N-intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids, the first 40 amino acids, the first 45 amino acids, the first 50 amino acids, the first 55 amino acids, the first 60 amino acids, the first 65 amino acids, the first 70 amino acids, the first 75 amino acids or the first 80 amino acids of the polypeptide as defined in SEQ ID NO: 6.

[0484] In another particular embodiment, the modified IMPDH N-intein does not comprise 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: 6.

[0485] The elimination of the amino acids in the N-terminal position of the IMPDH N-intein (i.e. first amino acids of the polypeptide as defined in SEQ ID NO: 6) and in the C-terminal position of the IMPDH N-intein (i.e. last amino acids of the polypeptide as defined in SEQ ID NO: 6) may be combined. In a particular embodiment, the modified IMPDH N-intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids, the first 40 amino acids, the first 45 amino acids, the first 50 amino acids, the first 55 amino acids or the first 60 amino acids of the polypeptide as defined in SEQ ID NO: 6, and does not comprise 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: 6.

[0486] In a more particular embodiment, the modified IMPDH N-intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids, the first or the first 40 amino acids of the polypeptide as defined in SEQ ID NO: 6 and does not comprise 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: 6.

[0487] In a preferred embodiment, the modified IMPDH N-intein does not comprise the 42 first amino acids of the polypeptide as defined in SEQ ID NO: 6.

[0488] In another particular embodiment, the modified NrdJ1 N-intein is a fragment of the SEQ ID NO: 7 that comprises or consists between 25 and 100 amino acids, preferably between 30 and 60 amino acids, more preferably between 40 and 50 amino acids. The fragment of the sequence SEQ ID NO: 7 comprises at least one mutation that decreases the affinity between the modified NrdJ1 N-intein and the NrdJ1 C-intein.

[0489] In a particular embodiment, the modified NrdJ1 N-intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids, the first 40 amino acids, the first 45 amino acids, the first 50 amino acids, the first 55 amino acids, the first 60 amino acids, the first 65 amino acids, the first 70 amino acids, the first 75 amino acids or the first 80 amino acids of the polypeptide as defined in SEQ ID NO: 7.

[0490] In another particular embodiment, the modified NrdJ1 N-intein does not comprise 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: 7.

[0491] The elimination of the amino acids in the N-terminal position of the NrdJ1 N-intein (i.e. first amino acids of the polypeptide as defined in SEQ ID NO: 7) and in the C-terminal position of the NrdJ1 N-intein (i.e. last amino acids of the polypeptide as defined in SEQ ID NO: 7) may be combined. In a particular embodiment, the modified NrdJ1 N-intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids, the first 40 amino acids, the first 45 amino acids, the first 50 amino acids, the first 55 amino acids or the first 60 amino acids of the polypeptide as defined in SEQ ID NO: 7, and does not comprise 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: 7.

[0492] In a more particular embodiment, the modified NrdJ1 N-intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids, the first 40 amino acids, the first 45 amino acids, the first 50 amino acids or the first 55 amino acids of the polypeptide as defined in SEQ ID NO: 7 and does not comprise 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: 7.

[0493] In a preferred embodiment, the modified NrdJ1 N-intein does not comprise the 56 first amino acids of the polypeptide as defined in SEQ ID NO: 7.

[0494] In another particular embodiment, the modified GP41 .8 N-intein is a fragment of the SEQ ID NO: 8 that comprises or consists between 19 and 84 amino acids, preferably between 30 and 60 amino acids, more preferably between 40 and 50 amino acids. The fragment of the sequence SEQ ID NO: 8 comprises at least one mutation that decreases the affinity between the modified GP41.8 N-intein and the GP41.8 C-intein.

[0495] In a particular embodiment, the modified GP41.8 N-intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids, the first 40 amino acids, the first 45 amino acids, the first 50 amino acids, the first 55 amino acids, the first 60 amino acids or the first 65 amino acids of the polypeptide as defined in SEQ ID NO: 8.

[0496] In another particular embodiment, the modified GP41.8 N-intein does not comprise 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.

[0497] The elimination of the amino acids in the N-terminal position of the GP41.8 N-intein (i.e. first amino acids of the polypeptide as defined in SEQ ID NO: 8) and in the C-terminal position of the GP41.8 N-intein (i.e. last amino acids of the polypeptide as defined in SEQ ID NO: 8) may be combined. In a particular embodiment, the modified GP41.8 N- intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids, the first 40 amino acids, the first 45 amino acids or the first 50 amino acids of the polypeptide as defined in SEQ ID NO: 8, and does not comprise 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.

[0498] In a more particular embodiment, the modified GP41.8 N-intein does not comprise the 5 first amino acids, the first 10 amino acids, the first 15 amino acids, the first 20 amino acids, the first 25 amino acids, the first 30 amino acids, the first 35 amino acids or the first 40 amino acids of the polypeptide as defined in SEQ ID NO: 8 and does not comprise 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.

[0499] In a preferred embodiment, the modified GP41.8 N-intein does not comprise the 40 first amino acids of the polypeptide as defined in SEQ ID NO: 8.

[0500] The modified N- intein refers to an N-intein that does not comprise the native sequence of the N-intein protein. In particular, the modified N-intein comprises at least one mutation that decreases the affinity between the modified N-intein and the corresponding C-intein.

[0501] The mutation (s) in the modified N-intein decrease the affinity between the modified N- intein and the corresponding C-intein. In a particular embodiment, the affinity between the modified N-intein and the C-intein is 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60% lower than the affinity between the N-intein and the corresponding C-intein.

[0502] In a preferred embodiment the affinity between the modified N-intein and the C-intein is between 40% and 60% lower than the affinity between the N-intein and the corresponding C-intein.

[0503] In a particular embodiment, the modified N-intein is a modified GP41.1 N-intein that comprises at least one mutation that decreases the affinity between the modified GP41.1 N-intein and the GP41.1 C-intein.

[0504] In a particular embodiment, the modified GP41.1 N-intein comprises between 1 and 4 mutations in a fragment of the sequence SEQ ID NO: 5. In a more particular embodiment, the modified GP41.1 N-intein comprises 2 mutations in a fragment of the sequence SEQ ID NO: 5. In a particular embodiment, the modified GP41.1 N-intein comprises at least one mutation that replaces a charged amino acid with a neutral amino acid. In a more particular embodiment, the modified GP41.1 N-intein comprises at least one mutation that replaces a positively charged amino acid with a neutral amino acid.

[0505] Therefore, in another particular embodiment, the modified N-intein comprises at least one mutation that changes the net charge of the modified N-intein fragment compared with the wild type N-intein. In particular, the modified GP41.1 N-intein comprises at least one mutation that changes the net charge of the modified GP41.1 N-intein fragment compared with the wild type GP41.1 N-intein.

[0506] In a particular embodiment, the modified GP41.1 N-intein comprises at least one mutation in a Lysine (K) residue. In a more particular embodiment, the modified GP41.1 N-intein comprises at least one mutation that replaces a Lysine (K) residue with an Alanine (A) residue.

[0507] In a preferred embodiment, the modified GP41.1 N-intein comprises a mutation in the lysine residues at positions 45 and 48 of the SEQ ID NO: 5. In a more preferred embodiment, the modified GP41.1 N-intein comprises the mutations K45A and K48A.

[0508] In a particular embodiment, the modified GP41.1 N-intein comprises the sequence of SEQ ID NO: 31.

[0509] The mutations defined above are only to be applied when the modified GP41.1 N-intein comprises a fragment of the sequence SEQ ID NO: 5 comprising the mutated positions (e.g. K45 and K48).

[0510] In another particular embodiment, the modified N-intein is a modified IMPDH N-intein that comprises at least one mutation that decreases the affinity between the modified IMPDH N-intein and the IMPDH C-intein.

[0511] In a particular embodiment, the modified IMPDH N-intein comprises between 1 and 4 mutations in a fragment of the sequence SEQ ID NO: 6. In a more particular embodiment, the modified IMPDH N-intein comprises 2 mutations in a fragment of the sequence SEQ ID NO: 6.

[0512] In a particular embodiment, the modified IMPDH N-intein comprises at least one mutation that replaces a charged amino acid with a neutral amino acid. In a more particular embodiment, the modified IMPDH N-intein comprises at least one mutation that replaces a negatively charged amino acid with a neutral amino acid. Therefore, in another particular embodiment, the modified N-intein comprises at least one mutation that changes the net charge of the modified N-intein fragment compared with the wild type N-intein. In particular, the modified IMPDH N-intein comprises at least one mutation that changes the net charge of the modified IMPDH N-intein fragment compared with the wild type IMPDH N-intein.

[0513] In a more particular embodiment, the modified IMPDH N-intein comprises at least one mutation in a glutamic acid (E) residue. In a more particular embodiment, the modified IMPDH N-intein comprises at least one mutation that replaces a glutamic acid (E) residue with an Alanine (A) residue.

[0514] In a preferred embodiment, the modified IMPDH N-intein comprises a mutation in the glutamic acid residues at positions 46 and 47 of the SEQ ID NO: 6. In a more preferred embodiment, the modified IMPDH N-intein comprises the mutations E46A and E47A.

[0515] In a particular embodiment, the modified IMPDH N-intein comprises the sequence of SEQ ID NO: 32.

[0516] The mutations defined above are only to be applied when the modified IMPDH N-intein comprises a fragment of the sequence SEQ ID NO: 6 comprising the mutated positions (e.g. E46 and E47).

[0517] In another particular embodiment, the modified N-intein is a modified NrdJ1 N-intein that comprises at least one mutation that decreases the affinity between the modified NrdJ1 N-intein and the NrdJ1 C-intein.

[0518] In a particular embodiment, the modified NrdJ1 N-intein comprises between 1 and 4 mutations in a fragment of the sequence SEQ ID NO: 7. In a more particular embodiment, the modified NrdJ1 N-intein comprises 2 mutations in a fragment of the sequence SEQ ID NO: 7.

[0519] In a particular embodiment, the modified NrdJ1 N-intein comprises at least one mutation that replaces a charged amino acid with a neutral amino acid. In a more particular embodiment, the modified NrdJ1 N-intein comprises at least one mutation that replaces a negatively charged amino with a neutral amino acid.

[0520] Therefore, in another particular embodiment, the modified N-intein comprises at least one mutation that changes the net charge of the modified N-intein fragment compared with the wild type N-intein. In particular, the modified NrdJ1 N-intein comprises at least one mutation that changes the net charge of the modified NrdJ1 N-intein fragment compared with the wild type NrdJ1 N-intein.

[0521] In a more particular embodiment, the modified NrdJ1 N-intein comprises at least one mutation in a glutamic acid (E) residue. In a more particular embodiment, the modified NrdJ1 N-intein comprises at least one mutation that replaces a glutamic acid (E) residue with an Alanine (A) residue.

[0522] In a preferred embodiment, the modified NrdJ1 N-intein comprises a mutation in the glutamic acid residues at positions 62 and 64 of the SEQ ID NO: 7. In a more preferred embodiment, the modified NrdJ1 N-intein comprises the mutations E62A and E64A.

[0523] In a particular embodiment, the modified NrdJ1 N-intein comprises the sequence of SEQ ID NO: 33.

[0524] The mutations defined above are only to be applied when the modified NrdJ1 N-intein comprises a fragment of the sequence SEQ ID NO: 7 comprising the mutated positions (e.g. E62 and E64).

[0525] In another particular embodiment, the modified N-intein is a modified GP41.8 N-intein that comprises at least one mutation that decreases the affinity between the modified GP41.8 N-intein and the GP41.8 C-intein.

[0526] In a particular embodiment, the modified GP41.8 N-intein comprises between 1 and 4 mutations in a fragment of the sequence SEQ ID NO: 8. In a more particular embodiment, the modified GP41 .8 N-intein comprises 2 mutations in a fragment of the sequence SEQ ID NO: 8.

[0527] In a particular embodiment, the modified GP41.8 N-intein comprises at least one mutation that replaces a charged amino acid with a neutral amino acid. In a more particular embodiment, the modified GP41.8 N-intein comprises at least one mutation that replaces a positively charged amino with a neutral amino acid.

[0528] Therefore, in another particular embodiment, the modified N-intein comprises at least one mutation that changes the net charge of the modified N-intein fragment compared with the wild type N-intein. In particular, the modified GP41.8 N-intein comprises at least one mutation that changes the net charge of the modified GP41.8 N-intein fragment compared with the wild type GP41.8 N-intein.

[0529] In a more particular embodiment, the modified GP41.8 N-intein comprises at least one mutation in a Lysine (K) residue. In a more particular embodiment, the modified GP41.8 N-intein comprises at least one mutation that replaces a Lysine (K) residue with an Alanine (A) residue.

[0530] In a preferred embodiment, the modified GP41.8 N-intein comprises a mutation in the lysine residues at positions 52 and 55 of the SEQ ID NO: 8. In a more preferred embodiment, the modified GP41.8 N-intein comprises the mutations K52A and K55A.

[0531] In a particular embodiment, the modified GP41.8 N-intein comprises the sequence of SEQ ID NO: 34.

[0532] The mutations defined above are only to be applied when the modified GP41.8 N-intein comprises a fragment of the sequence SEQ ID NO: 8 comprising the mutated positions (e.g. K52 and K55).

[0533] In another particular embodiment, the third polynucleotide of the invention further comprises: a. a linker region between (i) the modified N-intein and (ii) the split C-intein, and / or b. a protease cleavage site between (i) the modified N-intein and (ii) the split C-intein.

[0534] In a particular embodiment, the linker region between the modified N-intein and the split C-intein comprises a sequence between 1 and 200 amino acids, preferably between 1 and 100 amino acids.

[0535] The term “protease cleavage site” has been defined or explained above, and this definition is applicable to the third polynucleotide of the invention. In a particular embodiment, the protease cleavage site is recognised and cleaved by a protease selected from the group consisting of: a furin, an heparanase, calpain, a matrix metalloprotease (MMP), a cathepsin, a serine protease, a cysteine protease, an aspartic protease, ADAMs and ADAMTS protein, an aminopeptidase, a granule-associated serine protease and a caspase.

[0536] In a preferred embodiment, the protease cleavage site is a furin cleavage site.

[0537] The third polynucleotide of the invention comprises (iii) an heterologous polypeptide (C- extein).

[0538] The term "heterologous polypeptide" has been defined or explained above, and this definition is applicable to the third polynucleotide of the invention. However, in those cases in which the term “heterologous polypeptide is used to refer to the polypeptide which appears in the fusion protein attached to the C-terminus or the split C-intein, the heterologous polypeptide can also be referred to as the “C-extein” in order to differentiate it from the heterologous polypeptide which is attached to the N-terminus of the N-intein or N-extein.

[0539] In a particular embodiment, the heterologous polypeptide (C-extein) comprises from N- terminus to C-terminus: a. a transmembrane domain, and b. an intracellular signalling domain from a receptor and / or costimulatory domain.

[0540] The term “fourth polynucleotide of the invention” is used to refer to the third polynucleotide of the invention when the heterologous polypeptide (iii) (C-extein) comprises a transmembrane domain, and an intracellular signalling domain from a receptor and / or costimulatory domain.

[0541] Therefore, the term “fourth polynucleotide of the invention” refers to a polynucleotide encoding a fusion protein comprising from N-terminus to C-terminus:

[0542] (i) a modified N-intein selected from the group consisting of a modified GP41.1 N- intein, a modified IMPDH N-intein, a modified NrdJ1 N-intein or a modified GP41.8 N-intein, wherein the modified N-intein comprises a fragment of the N-intein protein that prevents trans-splicing from occurring in the presence of the corresponding C- intein and wherein the modified N-intein comprises at least one mutation that decreases the affinity between the modified N-intein and the corresponding C-intein,

[0543] (ii) a split C-intein comprising a sequence selected from the group consisting of 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-intein is a modified GP41.1 N-intein, the split C-intein comprises the sequence of SEQ ID NO: 1 or is a functionally equivalent variant thereof,

[0544] If the modified N-intein is a modified IMPDH N-intein, the split C-intein comprises the sequence of SEQ ID NO: 2 or is a functionally equivalent variant thereof, the modified N-intein is a modified IMPDH N-intein

[0545] If the modified N-intein is a modified NrdJ1 N-intein, the split C-intein comprises the sequence of SEQ ID NO: 3 or is a functionally equivalent variant thereof or If the modified N-intein is a modified GP41.8 N-intein, the split C-intein comprises the sequence of SEQ ID NO: 4 or is a functionally equivalent variant thereof,

[0546] (iii) a transmembrane domain, and

[0547] (iv) an intracellular signalling domain from a receptor and / or costimulatory domain.

[0548] In a particular embodiment, the fourth polynucleotide of the invention further comprises a linker between the split C-intein and the transmembrane domain.

[0549] The term “linker” has been defined or explained above and this definition is applicable to the fourth polynucleotide of the invention.

[0550] In a particular embodiment, the linker between the split C-intein 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 are applicable to the fourth polynucleotide of the invention.

[0551] In another particular embodiment, the third polynucleotide of the invention comprises a nucleotide sequence encoding a signal peptide, wherein the signal peptide is in frame and N-terminal to the modified N-intein and which allows the insertion of the fusion protein into the membrane.

[0552] The term “signal peptide” has been defined or explained above, and this definition is applicable to the third polynucleotide of the invention.

[0553] In a more particular embodiment, the signal peptide is selected from the group consisting of: CD8a, IL-2, GM-CSF receptor (GM-CSF) a chain, murine Ig-kappa (IgK), murine IgK V-lll region (IgKVIll), human IgKVIll, CD33, human tissue plasminogen activator (TPA) and native secreted alkaline phosphatase (SEAP).

[0554] The terms “transmembrane domain”, “intracellular signalling domain” and “costimulatory domain” have been defined or explained above and these definitions are applicable to the fourth polynucleotide of the invention.

[0555] In a particular embodiment, the intracellular signalling domain from a receptor is an antigen receptor signalling domain, preferably selected from the group consisting of the CD3 , CD28, 4-1 BB, NKG2D, DAP10, 0X40, CD70, CD27, CD5, ICAM-1 , LFA-1 (CD11a / CD18), ICOS (CD278), DAP 12 antigen receptor signalling domain or a combination thereof. In another particular embodiment, the transmembrane domain is selected from the group consisting of the CD28, the CD8a the 4-1 BB, the CD4, the CTLA4, the CD27 or the CD3 transmembrane domain.

[0556] In another particular embodiment, the transmembrane domain and the intracellular signalling domain are either directly connected by a peptide bond or by an amino acid linker.

[0557] Third and fourth vectors of the invention

[0558] In another aspect, the present invention relates to a vector, hereinafter the third vector of the invention, which comprises the third polynucleotide of the invention.

[0559] If the heterologous polypeptide (C-extein) of the third polynucleotide of the invention comprises a transmembrane domain, and an intracellular signalling domain from a receptor and / or costimulatory domain, the vector which comprises the third polynucleotide of the invention is called fourth vector of the invention.

[0560] Therefore, in another aspect, the present invention relates to a vector, hereinafter the fourth vector of the invention, which comprises the fourth polynucleotide of the invention.

[0561] The term “vector” has been defined or explained above and this definition is applicable to the third and fourth vectors of the invention.

[0562] Fourth and fifth host cells of the invention

[0563] In another aspect, the present invention relates to a host cell, hereinafter the fourth host cell of the invention, comprising the third polynucleotide of the invention, the third vector of the invention or the fusion protein encoded by the third polynucleotide of the invention.

[0564] In another aspect, the present invention relates to a host cell, hereinafter the fifth host cell of the invention, comprising the fourth polynucleotide of the invention, the fourth vector of the invention or the fusion protein encoded by the fourth polynucleotide of the invention.

[0565] The term “host cell” has been defined or explained above, and this definition is applicable to the fourth and the fifth host cells of the invention.

[0566] In a particular embodiment, the cell is obtained by viral transduction. In a more particular embodiment, the cell is obtained by viral transduction with a lentivirus or by gene knock-in. In a still more particular embodiment, the gene knock-in is carried out using a CRISPR-Cas9 system.

[0567] In another particular embodiment, the host cell is a human cell. In a more particular embodiment, the cell is selected from the group consisting of: T-cell, NK-cell, induced pluripotent stem cell (iPSC), derived NK cell, Pro-Tcell, B cell and macrophage.

[0568] Third and fourth fusion proteins of the invention

[0569] In another aspect, the present invention relates to a fusion protein, hereinafter the third fusion protein of the invention, encoded by the third polynucleotide of the invention.

[0570] In another aspect, the present invention relates to a fusion protein, hereinafter the fourth fusion protein of the invention, encoded by the fourth polynucleotide of the invention.

[0571] The term “fusion protein” has been defined or explained above, and this definition is applicable to the third and fourth fusion proteins of the invention.

[0572] In a particular embodiment, the fusion protein further comprises a detection tag. In a more particular embodiment, the detection tag is selected from the group consisting of: c-myc tag, HA tag, FLAG-tag, Strep-tag, His-tag, alfa tag, V5 tag, Spot tag and NE tag.

[0573] Second composition or kit-of-parts of the invention

[0574] In another aspect, the present invention relates to a composition or kit-of-parts, hereinafter the second composition or the second kit-of-parts of the invention, comprising a first component and a second component wherein i) the first component is the third polynucleotide of the invention, the third vector of the invention, the fourth host cell of the invention or the third fusion protein of the invention and ii) the second component is the second fusion protein of the invention or the second polynucleotide of the invention, wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

[0575] The first component of the second composition of the invention or the second-kit-of parts of the invention also includes the third polynucleotide of the invention in which (iii) the heterologous polypeptide (C-extein) comprises a transmembrane domain, and an intracellular signalling domain from a receptor and / or costimulatory domain.

[0576] Therefore, the first component of the second composition of the invention or the second- kit-of parts of the invention also include the fourth polynucleotide of the invention, the fourth vector of the invention, the fifth host cell of the invention or the fourth fusion protein of the invention.

[0577] Therefore, the second composition or the second kit-of-parts of the invention comprises a first component and a second component wherein i) the first component is the third polynucleotide or the fourth polynucleotide of the invention, the third vector or fourth vector of the invention, the fourth host cell or fifth host cell of the invention or the third fusion protein or fourth fusion protein of the invention and ii) the second component is the second fusion protein of the invention or the second polynucleotide of the invention, wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

[0578] The variants 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 are applicable to the second composition or kit-of parts of the invention.

[0579] The ratio of the components in the compositions is adequate for an efficient processing of the fusion proteins. Suitable ratios of the first and second components include, without limitation, 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.

[0580] Additional methods of the invention

[0581] In another aspect, the present invention relates to a method for cleaving a heterologous polypeptide (C-extein) from the third fusion protein of the invention comprising incubating the fusion protein with a split N-intein under conditions allowing intein splicing wherein

[0582] If the split C-intein forming part of the fusion protein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof, then the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof,

[0583] If the split C-intein forming part of the fusion protein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof, the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof,

[0584] If the split C-intein forming part of the fusion protein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof, then the split N- intein comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or

[0585] If the split C-intein forming part of the fusion protein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof, then and the split N- intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

[0586] The method for cleaving a heterologous polypeptide (C-extein) also includes a method for cleaving a heterologous polypeptide from the fourth fusion protein of the invention.

[0587] 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 the second composition of the invention or bringing into association the components of the second kit-of-parts of the invention under conditions allowing intein splicing wherein said first polypeptide is the heterologous polypeptide (C-extein) which forms part of the third fusion protein of the invention and said second polypeptide is the heterologous polypeptide (N-extein) which forms part of the second fusion protein of the invention.

[0588] In another aspect, the present invention relates to an in vitro method, hereinafter the third method of the invention, for producing a cell comprising a chimeric receptor, wherein the method comprises:

[0589] (i) providing a cell comprising the fourth polynucleotide of the invention, a polypeptide encoded by said polynucleotide or the fourth vector of the invention under conditions adequate for the expression in the cell of the fusion protein encoded by the polynucleotide, and

[0590] (ii) contacting the cell of step i) with the second fusion protein of the invention, wherein the contacting step ii) is performed under conditions that allow a splicing reaction between the split C-intein forming part of the fusion protein produced in step (i) and the split N-intein forming part of the second fusion proteins of the invention and wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

[0591] The term “in vitro" has been defined or explained above and this definition is applicable to the third method of the invention.

[0592] In a particular embodiment, the chimeric receptor is a chimeric antigen receptor.

[0593] The first step of the first method of the invention comprises i) providing a cell comprising the fourth polynucleotide of the invention, a polypeptide encoded by said polynucleotide or the fourth vector of the invention under conditions adequate for the expression in the cell of the fusion protein encoded by the polynucleotide.

[0594] Suitable conditions for the expression in the cell of the fusion protein encoded by the fourth polynucleotide of the invention are well known for the person skilled in the art. Conditions suitable for the expression of the polynucleotide in the host cell include those conditions that allow the optimal growth of said host cell and those conditions that allow the protein expression in said host cell. Said culture conditions are typically different for each type of host cell. However, those conditions are known by skilled workers and are readily determined. Similarly, the duration of maintenance can differ with the host cells and with the amount of the fusion protein desired to be prepared. Again, those conditions are well known and can be readily determined in specific situations.

[0595] The second step of the third method of the invention comprises ii) contacting the cell of step i) with the second fusion protein of the invention, wherein the contacting step ii) is performed under conditions that allow a splicing reaction between the split C-intein forming part of the fusion protein produced in step (i) and the split N-intein forming part of the second fusion proteins of the invention, wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

[0596] Suitable conditions that allow a splicing reaction between the split C-intein forming part of the fusion protein produced in step (i) and the split N-intein forming part of the second fusion protein of the invention are well known for the person skilled in the art. The terms “protein splicing reaction” or “intein reaction” have been defined or explained above, and this definition is applicable to the third method of the invention.

[0597] In some embodiments, an intein reaction (e.g., cleavage, linkage (splicing), cyclization) can be initiated by contacting a fusion protein comprising an intein N-terminal domain and a fusion protein comprising an intein C-terminal domain. In other embodiments, an intein reaction can be initiated by shifting the conditions, e.g., the temperature or pH, in which a split intein fusion protein or combination of split intein fusion proteins is incubated. In some embodiments, a C-terminal cleavage is initiated by a pH or temperature shift.

[0598] In some embodiments, an intein reaction is initiated by contacting fusion proteins with DTT or another strong nucleophile. In some embodiments, DTT is used to enhance a reaction. In some embodiments, an N-terminal cleavage is initiated by a strong nucleophile, e.g., DTT.

[0599] Another manner of inducing protein splicing or cleavage is by contact with a peptide or peptidomimetic agent that activates splicing or cleavage. Another manner of inducing protein splicing or cleavage is by removal of a peptide or peptidomimetic agent that blocks or inhibits splicing or cleavage.

[0600] The variants 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 are applicable to the third method of the invention.

[0601] In a particular embodiment, the cell is a human cell.

[0602] In a more particular embodiment, the cell is selected from the group consisting of: T cell, NK-cell, induced pluripotent stem cell (iPSC) derived NK cells, Pro-Tcell, B cell and macrophage.

[0603] In another particular embodiment, the cell used in step (i) have been obtained from a patient. In another aspect, the present invention relates to an in vitro method, hereinafter the fourth method of the invention, for producing a cell comprising a chimeric receptor, wherein the method comprises: i) providing a cell comprising a. the fourth polynucleotide of the invention, the fourth vector of the 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 in frame and N-terminal to the modified N-intein and which allows the insertion of the fusion protein into the membrane and b. the second polynucleotide of the invention, the second vector of the invention or the second fusion protein of the 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 in frame and N-terminal to the polypeptide that specifically binds to a target molecule and which allows the secretion of the fusion protein from the cell and ii) maintaining the cell under conditions adequate for the expression in the cell of both fusion proteins, for the insertion into the membrane of the fusion protein encoded by the fourth polynucleotide of the invention, for the secretion of the fusion protein encoded by the second polynucleotide of the invention and for the splicing reaction between the split C-intein and the split N-intein and wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO: 3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

[0604] The first step of the fourth method of the invention comprises i) providing a cell comprising the fourth polynucleotide of the invention, the fourth vector of the 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 in frame and N-terminal to the modified N-intein and which allows the insertion of the fusion protein into the membrane and b. the second polynucleotide of the invention, the second vector of the invention or the second fusion protein of the 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 in frame and N-terminal to the polypeptide that specifically binds to a target molecule and which allows the secretion of the fusion protein from the cell.

[0605] The “signal peptides” that can be used in the present invention have been defined or explained above, and these definitions are applicable to the fourth method of the invention. Examples of signal peptides are CD8a, IL-2, GM-CSF receptor (GM-CSF) a chain, murine Ig-kappa (IgK), murine IgK V-lll region (IgKVIll), human IgKVIll, CD33, human tissue plasminogen activator (TPA) and native secreted alkaline phosphatase (SEAP).

[0606] The second step of the fourth method of the invention comprises ii) maintaining the cell under conditions adequate for the expression in the cell of both fusion proteins, for the insertion into the membrane of the fusion protein encoded by the fourth polynucleotide of the invention, for the secretion of the fusion protein encoded by the second polynucleotide of the invention and for the splicing reaction between the split C-intein and the split N-intein and wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO: 3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

[0607] Suitable conditions for the expression in the cell of the both fusion proteins encoded by the polynucleotides are well known for the person skilled in the art. Conditions suitable for the expression of a polynucleotide in the host cell include those conditions that allow the optimal growth of said host cell and those conditions that allow the protein expression in said host cell. Said culture conditions are typically different for each type of host cell. However, those conditions are known by skilled workers and are readily determined. Similarly, the duration of maintenance can differ with the host cells and with the amount of the fusion proteins desired to be prepared. Again, those conditions are well known and can readily be determined in specific situations.

[0608] Suitable conditions for the insertion into the membrane of the fusion protein encoded by the fourth polynucleotide of the invention and for the secretion of the fusion protein encoded by the second polynucleotide of the invention are well known for the person skilled in the art.

[0609] In a particular embodiment, when the chimeric receptor is an oligomeric chimeric receptor, then the cell provided in (i) further comprises (c) an additional polypeptide encoding additional elements of the oligomer.

[0610] In a more particular embodiment, the oligomer is an immunoglobulin. In a still more particular embodiment, the immunoglobulin is IgA, IgG, IgM, IgE or IgD. In a still more particular embodiment, the immunoglobulin is lgA1 , lgA2, lgG1 , lgG2, lgG3, lgG4, IgM, IgE or IgD.

[0611] In another particular embodiment, the fusion protein is a heavy chain or a light chain of an immunoglobulin, and then the additional polypeptide (c) encodes the corresponding heavy or light chain of the immunoglobulin.

[0612] The terms “oligomer”, “immunoglobulin”, “heavy chain” and “light chain” have been defined or explained above, and these definitions are applicable to the fourth method of the invention. In some embodiments, the protein splicing reaction between the protein encoded by the fourth polynucleotide of the invention and the fusion protein encoded by the second polynucleotide of the invention is either intracellular and occurs in the secretory pathway of the cell producing both polypeptides, before the secretion of the second polypeptide. In other embodiments the protein splicing reaction occurs once the second polypeptide has been secreted and takes place in the extracellular space.

[0613] The conditions that allow a protein splicing reaction or an intein reaction have been defined or explained in the first method of the invention and these conditions are applicable to the fourth method of the invention.

[0614] The variants 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 are applicable to the fourth method of the invention.

[0615] In a particular embodiment, the cell is a human cell.

[0616] In a more particular embodiment, the cell is selected from the group consisting of: T cell, NK-cell, induced pluripotent stem cell (iPSC) derived NK cells, Pro-Tcell, B cell and macrophage.

[0617] In another particular embodiment, the cell used in step (i) have been obtained from a patient.

[0618] Second cell of the invention

[0619] In another aspect, the present invention relates to a cell, hereinafter the second cell of the invention, comprising a chimeric receptor obtained by the third method or the fourth method of the invention.

[0620] Medical uses

[0621] In another aspect, the present invention relates to the third host cell of the invention or the second cell of the invention for use in the treatment of a disease which requires a cell-mediated immune response against a target molecule.

[0622] In a particular embodiment, the target molecule is an antigen of interest.

[0623] In another aspect, the present invention relates to the second fusion protein of the invention, the second polynucleotide of the invention, the second vector of the invention or the second host cell of the invention for use in the treatment of a disease which requires a cell-mediated immune response against the target molecule, wherein the patient to be treated is characterized in that it contains cells comprising the fourth polynucleotide of the invention, the fourth vector of the invention or the fusion protein encoded by the fourth polynucleotide of the invention, wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

[0624] In another aspect, the present invention relates to the fourth polynucleotide of the invention, the fourth vector of the invention, the fourth host cell of the invention or the fourth fusion protein of the invention for use in the treatment of a disease which requires a cell-mediated immune response against the target molecule, wherein the patient to be treated is characterized in that it contains cells comprising the second fusion protein of the invention, the second polynucleotide of the invention, the second vector of the invention or the second host cell of the invention, wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof. In a particular embodiment, the target molecule is an antigen of interest.

[0625] The terms “treatment” and “subject” have been defined or explained above, and these definition is applicable to these medical uses.

[0626] In a particular embodiment, the disease requiring a cell-mediated immune response against a target molecule, preferably against an antigen of interest, is selected from the group consisting of: cancer, a viral infection, a bacterial infection, a parasitic infection and an autoimmune disease.

[0627] The terms “cancer”, “viral infection”, “bacterial infection”, “parasitic infection” and “autoimmune disease” have been defined or explained above, and these definitions are applicable to these medical uses.

[0628] In a preferred embodiment, the cancer is selected from the group consisting of: 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.

[0629] In another preferred embodiment, the autoimmune disease is selected from the group consisting of: celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel diseases, multiple sclerosis, alopecia areata, Addison's disease, pernicious anemia, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

[0630] EXAMPLES

[0631] Material and methods

[0632] DNA synthesis and cloning of plasmids encoding IntC-CAR (caged and uncaged) and scFv-lntN constructs

[0633] Synthesized DNA fragments encoding the IntC-CAR constructs or the scFv-lntN constructs were obtained as linear constructs from Twist Bioscience. The acceptor vector, pcDNA3.1 , and the synthesized DNA fragments were digested with appropriate restriction enzymes (New England Biolabs, NEB) in a 20 pL reaction containing 1-5 pg of DNA, 1 pL of each restriction enzyme, and the recommended NEB buffer, followed by incubation at 37°C for 2 hours. Digestion efficiency was confirmed by agarose gel electrophoresis. The resulting DNA fragments were purified using the Macherey-Nagel NucleoSpin Gel and PCR Clean-Up Kit, according to the manufacturer’s protocol, and eluted in 20-30 pL of sterile water. For the ligation, approximately 50-100 ng of digested vector was mixed with the insert in a reaction containing 1 pL of T4 DNA ligase (400 U / pL) and 2 pL of ligation buffer (NEB) in a final volume of 20 pL. The mixture was incubated at room temperature for 1 hour. The ligated DNA was transformed into chemically competent E. coli (NEB 5-alpha) via heat shock, followed by recovery in 250 pL of LB broth at 37°C for 1 hour with shaking. Transformed cells were plated onto LB agar plates containing the appropriate antibiotic (ampicillin for pcDNA3.1) and incubated overnight at 37°C. Colony screening was performed the following day using PCR, restriction digestions and confirmed by Sanger sequencing.

[0634] HEK293F cells transient transfection with IntC-CAR (caged and uncaged) and scFv-lntN constructs

[0635] Twenty-four hours prior to transfection, cells have been split to a density of ~1.5 x 106cells / ml and cultured overnight in an 8% CO2 incubator with 125 rpm shaking at 37°C.

[0636] The day of the transfection, cells have been counted with a hemocytometer and the sufficient volume of cell culture has been spun down (1200 rpm - 400 ref, 10' at RT) in order to get rid of any residual exhausted growth medium that could reduce the transfection efficiency. The resulting cell pellet has been resuspended with fresh medium at a final density of 3 x 106cells / ml and incubated in an 8% CO2 incubator with 125 rpm shaking at 37°C.

[0637] The preparation of the DNA / PEI mixtures have been prepared according to the following method: polyethylenimine (PEI, 25 kDa linear PEI, Polysciences, Inc., cat. No. 23966) is prepared as a stock solution at a concentration of 1 mg / ml in a buffer containing 25mM HEPES and 150 mM NaCI (pH 7.5). The PEI is added to the buffer and vortexed until completely dissolved (this can take MANY minutes of vortexing). Once fully dissolved PEI can be sterile filtered using a 0.22.

[0638] For each transfection, add the expression plasmid DNA to the cells in a final amount of (1 ,25 pg) / (1x106cells) and PEI at a total amount of (3,75 pg) / (1x106cells). The indicated final amount of DNA is the same independently of the number of plasmids transfected.

[0639] A dilution of the total amount of plasmid DNA is prepared from the total amount of plasmid DNA stock to a final concentration of 0.5 pg DNA / pl in growth medium. Briefly vortex the mixture and spin to return the solution to the bottom of the tube. Add the diluted DNA mix to the cells. Swirl the culture to mix the DNA and return the flask to shaker platform in the 8% CO2 incubator, at 37°C, 125 rpm, for 5 minutes. The total amount of PEI ((3,75 pg) / (1x106cells)) is diluted at a final concentration of 0.5 pg / pl in growth medium, briefly vortex the mixture and spin to return the solution to the bottom of the tube. Add the PEI / medium mix to the cells. Swirl the culture to mix the PEI, return the flask to shaker platform in the incubator.

[0640] After 24 hours, dilute the cells 1 :1 with pre-warmed Freestyle Expression Medium supplemented with valproic acid (VPA) (Sigma cat. No. P4543-100G) to a final concentration of 3,75 mM and d-glucose, to a final concentration of 4 g / l, related to the final volume (i.e. after dilution 1 :2, at 24 hours post transfection).

[0641] Return the culture to the 8% CO2 incubator at 37°C, with shaking at 125 rpm on the orbital platform.

[0642] IMAC purification of scFv-lntN-His fusions secreted by HEK293F cells

[0643] The supernatant of the transfected cells was collected by centrifugation. 5x IMAC binding buffer (250 mM Tris pH 8, 1.5 M NaCI, 100 mM imidazole) was added to adjust pH and ionic strength for binding to the Ni column. The resulting mixture was then filtered through a 0.45 pm filter and loaded onto a 5 mL Ni column using an Akta system previously equilibrated with IMAC binding buffer (50 mM Tris pH 8, 300 mM NaCI, 20 mM imidazole). After loading and washing of unbound proteins, the scFv-lntN-His fusion was eluted from the column using an imidazole gradient obtained with IMAC elution buffer (50 mM Tris pH 8, 300 mM NaCI, 500 mM imidazole). The protein content of the eluted fractions was analyzed by Coomassie-stained SDS-PAGE. The fractions containing the protein of interest were combined and dialyzed extensively against PBS. The concentration of the recovered scFv-lntN-His fusion was quantified by spectrophotometry. The final sample was stored at -80 °C until use.

[0644] ST purification of scFv-lntN-ST fusions secreted by HEK293F cells

[0645] The supernatant of the transfected cells was collected by centrifugation. 10x Strep Tag binding buffer (1 M Tris pH 9, 2 M NaCI) was added to adjust pH and ionic strength for binding to the Strep-Tactin® XT column. The resulting mixture was then filtered through a 0.45 pm filter and loaded onto a 5 mL Strep-Tactin® XT column using an Akta system previously equilibrated with ST binding buffer (100 mM Tris pH 9, 200 mM NaCI). After loading and washing of unbound proteins, the scFv-lntN-ST fusion was eluted from the column using a biotin gradient obtained with ST elution buffer (100 mM Tris pH 9, 200 mM NaCI, 50 mM biotin). The protein content of the eluted fractions was analyzed by Coomassie-stained SDS-PAGE. The fractions containing the protein of interest were combined and dialyzed extensively against PBS. The concentration of the recovered scFv-lntN-ST fusion was quantified by spectrophotometry. The final sample was stored at -80 °C until use.

[0646] In vitro trans-splicing reaction of a purified scFv-lntN fusion with a model IntC-POl

[0647] The ability of a purified scFv-lntN fusion to trans-splice was assessed using a model IntC-POl fusion containing the corresponding IntC. The trans-splicing reaction was performed in PBS. The model IntC-POl fusion was added to the reaction in a 2-fold excess compared to the scFv-lntN fusion. The reaction was performed at 25°C in the presence and absence of a reducing agent. Trans-splicing was monitored for 4 hours, with aliquots taken at 1 hour, 2 hours and 4 hours after the reaction initiation. The progress of the reaction was analyzed by Coomassie-stained SDS-PAGE.

[0648] Analysis of I ntC-CAR and cage-lntC-CAR expression on the surface of HEK293F cells by flow cytometry

[0649] Cell viability was determined after 72 hours of transfection. According to the values obtained, 500,000 cells for each sample were transferred to an Eppendorf tube to perform the flow cytometry staining reaction. Cells were washed twice with 300 pL of cold Cell Staining Buffer (Biolegend). Washes were performed by resuspending the cells by vortexing, followed by centrifugation (5 min, 200g). After the last wash, the cells were resuspended in 200 pL of Cell Staining Buffer containing the antibodies at the desired dilution. Cell staining was performed at a cell density of 2.5- 106cells I mL. Cells were incubated with the antibodies for 45 minutes in the dark at room temperature and 80 rpm. After this time, the cells were washed twice with 300 pL of Cell Staining Buffer. After the final wash, the cells were resuspended in 600 pL of Cell Staining Buffer + 1 mM EDTA (EDTA was added to prevent cell aggregation during the analysis in the flow cytometer), resulting in a cell suspension with a density of 8.33- 105cells I mL. These samples were analyzed by cytometry using a BC Gallios flow cytometer. DAPI (4',6-diamidino-2- phenylindole) was added to the samples prior to the analysis to label live cells.

[0650] Western blotting

[0651] Protein expression has been analyzed via Wester Blotting. Samples were treated differently depending upon the nature of the expressed proteins, whether intracellular or secreted and purified. In the case of the expression of cellular proteins (e.g. IntC-CAR fusions), HEK293F cells transfected with IntC-CAR constructs were harvested 72 hours after transfection. Cell pellets corresponding to 500 pl of cell culture were resuspended in 250 pl of ice-cold RIPA buffer (NaCI 150 mM, EDTA 5 mM, Tris pH 8.0 50 mM, NP40 1 %, Na-DOC 0,5%, SDS 0,1 %) and sonicated 3 x 10 pulses (4 cycle, 40% power) on ice. The resulting cell lysates were diluted with 2x reducing loading buffer and 35 pl of each sample was loaded onto an SDS-PAGE gel.

[0652] Secreted proteins and samples obtained upon chromatography were diluted 1 :2 with 2x reducing loading buffer and 20 pl loaded onto an SDS-PAGE.

[0653] Upon resolution by SDS-PAGE, samples were subsequently transferred to mini PVDF membranes (Thermo Fisher Scientific, Cat. No. IB401001) using the Thermo iBIot 2 Dry Blotting System (Cat. No. IB21001). Upon blotting, PVDF membranes were probed with one of the following antibodies or probes: a-STREP tag (StrepMAB-Classic HRP, Iba, Cat. No. 2-1509-001), a-FLAG tag (Anti-DYKDDDDK, Merck, Cat. No. MAB3118), a-HIS tag (HisProbe™-HRP Conjugate, Thermo Fisher Scientific, Cat. No. 15165) and a-CD3 antibody (CD3 Antibody, Cell Signaling Technology, Cat. No. 88083). When needed, a secondary antibody has been used, either a-rabbit HRP conjugated (HRP-AffiniPure Goat Anti-rabbit IgG (H+L), Jackson ImmunoResearch, Cat. No. 111-035-003) or a- mouse-HRP conjugated (HRP-AffiniPure Goat Anti-Mouse IgG (H+L), Jackson ImmunoResearch, Cat. No. 115-036-072). For each antibody / probe used, the conditions recommended by the manufacturer were followed.

[0654] Finally, the membrane was washed three times with TBS-T, 0.1% Tween-20 and developed with ECL (Novex ECL, Thermo Fisher Scientific, WP20005). Imaging was performed with a ChemiDoc XRS+ imaging station (Biorad).

[0655] In vitro trans-splicing reaction between HEK293F cells expressing an IntC-CAR (caged or uncaged) construct and a purified scFv-lntN fusion

[0656] HEK293F cells were transfected with an IntC-CAR construct. 48 hours after cell transfection, the cell culture was centrifuged and the cells were washed with PBS to remove residual culture media. The washed cells were then resuspended in PBS at a cell density of 106cells / mL and aliquots of 500 pL of this cell suspension were added to Eppendorf tubes, one for each reaction condition to be tested. In parallel, the purified scFv-lntN fusion was reduced using an lntN:DTT ratio of 1 :200. For reactions where the scFv-lntN fusion is not reduced, a solution of scFv-lntN was prepared by adding PBS instead of the reducing agent to maintain the same IntN concentration in all the reactions. The scFv-lntN fusion was incubated with the reducing agent or PBS for at least 1 h at room temperature. After this time, it was added to the transfected cells according to the difference reaction conditions to be tested and the trans-splicing reactions were incubated at 37°C and 5% CO2 for approximately 4 hours. At the end of the incubation, the cells were harvested by centrifugation (10 min, 300g). If in vitro trans-splicing was to be analyzed by Western blot, cells were washed twice with PBS to remove the unreacted scFv-lntN fusion and were stored at -20°C for further analysis of the trans-splicing reaction according to the same protocol applied for the analysis of IntC-CAR expression. If in vitro trans-splicing was to be analyzed by flow cytometry, cells were washed twice with 300 pL of cold Cell Staining Buffer and the staining protocol was applied.

[0657] Lenti virus production

[0658] Synthesized DNA fragments encoding the IntC-CAR constructs were cloned into the pCCL lentiviral-based second-generation backbone, which adds a T2A-green fluorescence protein (GFP) at the 3’-end of the cassette. The 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 were pseudotyped with vesicular stomatitis virus-G (VSV-G). For each production, transfection was carried out using the following plasmids: pRRE (11 pg), pREV (5.45 pg), pVSV-G (5 pg), and plntC-CAR (22 pg) per plate, and viral particles were concentrated by ultracentrifugation.

[0659] IntC-CAR T cells production

[0660] Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy volunteers by Ficoll-Hypaque gradient centrifugation. Buffy coats were obtained from the Barcelona Blood and Tissue Bank (BST), with institutional review board (IRB) approval (HCB / 2018 / 0030). T cells were activated with plate-bound OKT3 and anti-CD28 antibodies (BD Biosciences) for 2 days in the presence of interleukin (I L)-7 and IL-15 (10 ng / mL, Mitenyi Biotec). Subsequently, 5- 105activated T cells were challenged with viral particles expressing IntC-CAR at various multiplicities of infection (MOI 10 to 30) in RPMI-1640 medium (Catalog no. 12-702Q, Lonza) supplemented with 2 mmol / L L- glutamine, 10 % heat-inactivated fetal bovine serum (FBS), and 1 U / ml of penicillin / streptomycin. Cells were then plated every 2-3 days and cell counts were recorded. Surface expression of IntC-CAR was detected by fluorescence-activated cell sorting (FACS). IntC-CAR expression was confirmed by GFP expression and by using an anti-HisTag antibody (BioLegend, clone J095G46). T cells were simultaneously analyzed for the memory phenotype by staining for CCR7 (BioLegend, clone G043H7) and CD45RA (BioLegend, clone H1100). T cell memory phenotypes are as follows: Central memory, CM (CCR7+CD45RA-); NaTve / 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 at 5 days post infection (dpi).

[0661] In vitro trans-splicing reaction between T cells expressing an IntC-CAR construct and a purified scFv-lntN fusion

[0662] 5 to 7 dpi T cells expressing an IntC-CAR were washed in PBS 1X and were resuspended in complete RPMI-1640 medium. They were then incubated for 2 hours with the corresponding scFv-lntN fusion, previously reduced with 5 mM DTT. To determine the formation of a reconstituted scFv-CAR by trans-splicing, the cells were washed and immediately stained with the allophycocyanin (APC) AffiniPure™ F(ab')2Fragment Goat Anti-Human IgG (H+L) antibody (Jackson Immuno Research, 109-136- 88) for cell cytometry, which is capable of recognizing cells that have incorporated the scFv. An orthologous scFv-lntN, which is incapable of trans-splicing, was used as a control. The trans-splicing efficiency per cell was calculated as follows: % PTS = (%scFv positive / %HisTag positive cells) * 100 %.

[0663] CD1a-specific T-ALL cytotoxicity

[0664] T cells obtained from 3 different donors were transfected with either the IMPDH.1-C-CAR or the NrdJ.1-C-CAR as described previously. At 5 dpi, T cells were subjected to a trans- splicing reaction using an aCD1a scFv-IMPDH.1-N fusion produced in HEK293F cells and purified by IMAC. After 2 hours of incubation, the T cells were washed in PBS1X and resuspended in 100 pl of complete RPMI-1640 medium. Meanwhile, a mixture of GFP-expressing CDIako 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 1X, and resuspended in complete RPMI-1640 medium at 106cells / ml. Then, 100 pl of MOLT4 cells (105cells) were mixed with 100 pl of IMPDH.1-C-CAR T cells or 100 pl of NrdJ.1-C-CAR T cells trans-spliced with the aCD1a scFv-IMPDH.1-N fusion. The concentration of the IMPDH.1-C-CAR T cells or the NrdJ.1-C-CAR T cells was kept so that the effector (GFP + CAR T cells): target (GFP- MOLT4 cells) ratio was 2:1 and 4:1 , respectively. After 24 or 48 h, cells were centrifuged (1500 rpm, 5 min), supernatants were stored at -80°C until processing, and cells were resuspended in PBS 1X. Cells were then analyzed by flow cytometry and IFN-y in the supernatants was determined by ELISA (Human ELISA SET, BD Biosciences).

[0665] EXAMPLE 1 : Expression, purification and trans-splicinq activity of an aCD19 scFv- qp41.1-N fusion produced in HEK293F cells

[0666] HEK293F cells were transfected with a construct encoding an a-CD19 scFv-gp41.1 -IM- ST fusion. The presence of the intein fusion in the culture supernatant was analyzed at different time points after cell transfection using an anti-Strep tag antibody (Figure 1 , A). The aCD19 scFv-gp41.1-N-ST intein fusion was expressed and showed two bands due to a glycosylation site located in the gp41.1-N sequence. The culture supernatant was collected 168 h post-transfection and the intein fusion was purified by strep tag affinity chromatography (Figure 1 , B). The ability of the pure a-CD19 scFv-gp41.1-N-ST fusion to trans-splice was tested using a model IntC fusion produced in E. coli, the gp41.1-C- Trx construct (Figure 1 , C). 2 pM of aCD19 scFv-gp41.1-N-ST intein fusion was mixed with 4 pM gp41.1-C-Trx in 200 pL of PBS. Two reactions were prepared, one with 1 mM DTT and one without DTT. Aliquots were extracted after 1 , 2 and 4 hours of incubation at room temperature and loaded onto an SDS-PAGE gel stained with Coomassie. The two bands corresponding to the a-CD19 scFv-gp41.1-N-ST intein fusion disappeared and a single lower molecular weight band corresponding to the splicing product appeared only in the reaction with DTT, confirming the ability of pure a-CD19 scFv-gp41.1-N-ST fusion to trans-splice.

[0667] EXAMPLE 2: Expression, purification and trans-splicinq activity of an aBCMA scFv- IMPDH.1-N fusion produced in HEK293F cells

[0668] HEK293F cells were transfected with a construct encoding an a-BCMA scFv-IMPDH.1- N-His fusion. The presence of the intein fusion in the culture supernatant was analyzed at different time points after cell transfection using an anti-His tag antibody (Figure 2, A). A band with a molecular weight between 40 kDa and 50 kDa corresponding to the expressed a-BCMA scFv-IMPDH.1-N-His intein fusion was observed. The culture supernatant was collected 168 h post-transfection and the intein fusion was purified by IMAC (Figure 2, B). The ability of the pure a-BCMA scFv-IMPDH.1-N-His fusion to trans- splice was tested using a model IntC fusion produced in E. coli, the ST-SUMO-His- IMPDH.1-C-VHH construct (Figure 2, C). 2 pM of aBCMA scFv-IMPDH.1-N-His intein fusion was mixed with 4 pM of ST-SUMO-His-IMPDH.1-C-VHH in 200 pL of PBS. Two reactions were prepared, one with 1 mM DTT and one without DTT. Aliquots were extracted after 2 and 4 hours of incubation at room temperature. After extraction of the 4-hour aliquot, the splicing product was purified by negative IMAC to distinguish it from the unreacted aBCMA scFv-IMPDH.1-N-His intein fusion, which has almost the same molecular weight. Aliquots of the flow-through and elution of the negative IMAC were obtained and loaded onto an SDS-PAGE gel stained with Coomassie together with the aliquots extracted during the reaction. The splicing product of the reaction between the aBCMA scFv-IMPDH.1-N-His intein fusion and the model ST-SUMO-His-IMPDH.1-C- VHH IntC precursor was observed in the flow-through of the negative IMAC of the reaction with DTT, confirming the ability of the purified aBCMA scFv-IMPDH.1-N-His intein fusion to trans-splice.

[0669] EXAMPLE 3: Expression, purification and trans-splicing activity of an aCD22 scFv- gp41.8-N fusion produced in HEK293F cells

[0670] HEK293F cells were transfected with a construct encoding an a-CD22 scFv-gp41.8-N- His fusion. The presence of the intein fusion in the culture supernatant was analyzed at different time points after cell transfection using an anti-His tag antibody (Figure 3, A). A band of a molecular weight of approximately 40 kDa corresponding to the expressed a- CD22 scFv-gp41.8-N-His intein fusion was observed. The culture supernatant was collected 168 h post-transfection and the intein fusion was purified by IMAC (Figure 3, B). The pure a-CD22 scFv-gp41.8-N-His intein fusion showed two bands, indicating the presence of a post-translational modification on the sequence. This modification is not an N-glycosylation, as the molecular weight of the intein fusion didn’t change 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 ability of the pure a-CD22 scFv-gp41.8-N-His fusion to trans- splice was tested using a model IntC fusion produced in E. coli, the gp41.8-C-LipTla construct (Figure 3, C). 1 pM of aCD22 scFv-gp41.8-N-His intein fusion was mixed with 2 pM gp41.8-C-LipTla in 200 pL of PBS. Two reactions were prepared, one with 1 mM DTT and one without DTT. Aliquots were extracted after 1 , 2 and 4 hours of incubation at room temperature and loaded onto an SDS-PAGE gel stained with Coomassie. The two bands corresponding to the a-CD22 scFv-gp41.8-N-His intein fusion disappeared and a single lower molecular weight band corresponding to the splicing product appeared only in the reaction with DTT, confirming the ability of pure a-CD22 scFv-gp41.8-N-ST fusion to trans-splice.

[0671] EXAMPLE 4: Expression, purification and trans-splicing activity of an aCD1a scFv- NrdJ.1-N fusion produced in HEK293F cells HEK293F cells were transfected with a construct encoding an a-CD1a scFv-NrdJ.1-N- His fusion. The presence of the intein fusion in the culture supernatant was analyzed at different time points after cell transfection using an anti-His tag antibody (Figure 4, A). A band with a molecular weight of approximately 40 kDa corresponding to the expressed a-CD1a scFv-NrdJ.1-N-His intein fusion was observed. The culture supernatant was collected 168 h post-transfection and the intein fusion was purified by IMAC (Figure 4,

[0672] B). The ability of the pure a-CD1a scFv-NrdJ.1-N-His fusion to trans-splice was tested using a model IntC fusion produced in E. coli, the NrdJ.1-C-LipTla construct (Figure 4,

[0673] C). 2 pM of aCD1a scFv-NrdJ.1-N-His intein fusion was mixed with 4 pM NrdJ.1-C-LipTla in 200 pL of PBS. Two reactions were prepared, one with 1 mM DTT and one without DTT. Aliquots were extracted after 1 , 2 and 4 hours of incubation at room temperature and loaded onto an SDS-PAGE gel stained with Coomassie. The band corresponding to the aCD1a scFv-NdJ.1-N-His intein fusion disappeared over the reaction time while a band of a molecular weight between 60 and 80 kDa, consistent with the splicing product, appeared, confirming the ability of pure a-CD1a scFv-NrdJ.1-N-ST fusion to trans-splice.

[0674] EXAMPLE 5: Expression of IntC-CAR fusions in HEK293F cells

[0675] HEK293F cells were transfected with constructs encoding the His-lntC-CARs. These constructs contain a signal peptide that directs the nascent protein to the secretion pathway and ensures that IntC is located on the cell surface, the 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, the hinge region and the transmembrane domain of the lymphocyte surface antigen CD8 and the intracellular signaling domains 4-1 BB and CD3z (Figure 5, A). The expression of the His-lntC-CAR constructs was analyzed 72 h post-transfection by flow cytometry using an APC-labelled a-His antibody (Figure 5, B). The IntC-CAR fusion with higher expression levels was that with the NrdJ.1 intein (84% of His + cells), followed by the CAR fused to the IMPDH.1 intein (47% of His + cells), the CAR fused to the gp41.1 intein (32 % of His + cells) and the CAR fused to the gp41.8 intein, which had the lowest expression levels (13% of His + cells). The integrity of the expressed IntC-CAR constructs was checked by Western blot using an a-CD3z antibody (Figure 5, C). All IntC-CAR fusions showed the expected molecular weight for the IntC-CAR monomer (m), between 31 and 32 kDa, depending on the intein. All of them also showed a small amount of IntC-CAR dimer and a band of about 20 kDa, which could correspond to degradation. The gp41.8-C-CAR fusion has an N-glycosylation site on the intein sequence, which increases the observed molecular weight (glyco m). The IMPDH.1-C- CAR and NrdJ.1 -C-CAR fusions have an additional band with a molecular weight higher than that of the monomer, most likely corresponding to a post-translational modification other than an N-glycosylation.

[0676] EXAMPLE 6: Expression of caged IntC-CAR fusions in HEK293F cells

[0677] HEK293F cells were transfected with constructs encoding the ST-cage-His-lntC-CARs. These constructs contain a signal peptide that directs the nascent protein to the secretion pathway and ensures that IntC is located on the cell surface, a Strep tag for detection, the cage sequence consisting of some amino acids from the C-terminal part 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 the transmembrane domain of the lymphocyte surface antigen CD8 and the intracellular signaling domains 4-1 BB and CD3z (Figure 6, A). Expression of the ST-cage-His-lntC- CAR constructs was analyzed 72 h post-transfection by flow cytometry using an APC- labelled a-His antibody. The addition of the cage sequence to gp41.1-C-CAR and IMPDH.1-C-CAR doubles the expression level of the IntC-CAR fusion. The effect on NrdJ.1-C-CAR is less significant, as the addition of the cage sequence increases the expression of IntC-CAR by 20%. The IntC-CAR construct for which the addition of the cage sequence leads to a higher increase in the expression is that of gp41.8, for which the expression levels increase 8.5-fold (Figure 6, B). In addition to the increase in absolute expression levels, cells expressing a caged IntC-CAR fusion show higher expression levels per cell compared to cells expressing the same IntC-CAR fusion without the cage. This effect can be observed by a shift to higher fluorescence values in the flow cytometry analysis of cells expressing the caged IntC-CAR fusions compared to cells expressing uncaged IntC-CAR fusions (Figure 6, C).

[0678] EXAMPLE 7: In vitro trans-splicinq reaction between cells expressing the qp41.1-C-CAR construct and a purified a-CD19 scFv-qp41.1-N fusion

[0679] HEK293F cells were transfected with the gp41.1-C-CAR construct. Cells were harvested 48 hours after transfection and in vitro trans-splicing reactions were prepared by combining the cells expressing the gp41.1-C-CAR construct with the a-CD19 scFv- gp41.1-N intein fusion (Figure 7, A). After incubation, the trans-splicing reactions were analyzed by flow cytometry using a combination of two antibodies, an APC-labelled a- His antibody to detect cells expressing the gp41.1-C-CAR construct (as the gp41.1-C- CAR fusion has an N-terminal His tag) and a PE-labelled a-FLAG antibody to detect cells containing the reconstituted a-CD19 scFv-CAR (as the a-CD19 scFv has an N-terminal FLAG tag). The results show that 40% of the cells express the gp41.1-C-CAR and that 36% of the cells have undergone trans-splicing. The combination of both figures gives a CAR reconstitution efficiency per cell of 90%, i.e. that 90% of the cells expressing the gp41.1-C-CAR contain the O-CD19 scFv-CAR after the trans-splicing reaction (Figure 7, B). The trans-splicing reactions were also analyzed using a PE-labelled CD19 antigen. Following the trans-splicing reaction, the cells are expected to recognize and bind the CD 19 antigen as the reaction introduces an O-CD19 scFv to the cell surface. This analysis shows that 43% of the cells bind to the CD19 antigen after the trans-splicing reaction, indicating that all the cells expressing the gp41.1-C-CAR have incorporated an O-CD19 scFv and therefore the CAR reconstitution efficiency per cell is close to 100 % for the CAR containing the gp41.1 intein (Figure 7, C). It is noteworthy that both flow cytometry analyses achieve equivalent CAR reconstitution efficiencies. Finally, cells expressing the gp41.1-C-CAR before and after a trans-splicing reaction with the O-CD19 scFv-gp41.1-N intein fusion were analyzed by Western blot using an aCD3z antibody to identify the formation of the reconstituted O-CD19 scFv-CAR. A band with a molecular weight between 50 kDa and 70 kDa appeared after the reaction, corresponding to that expected for the reconstituted CAR, which is of 52.6 kDa (Figure 7, D).

[0680] EXAMPLE 8: In vitro trans-splicing reactions between cells expressing IMPDH.1-C-CAR constructs and a purified a-BCMA scFv-IMPDH.1-N fusion

[0681] HEK293F cells were transfected with two IMPDH.1-C-CAR constructs. The first had the structure shown in Figure 7A, and the second had a linker between IntC and the transmembrane domain of the CD8 (Figure 8, C). This linker remains in the reconstituted CAR after the trans-splicing reaction, giving the structure some flexibility. Cells were harvested 48 hours after transfection and in vitro trans-splicing reactions were prepared by combining the cells expressing the IMPDH.1-C-CAR constructs with the a-BCMA scFv-IMPDH.1-N intein fusion. After incubation, the trans-splicing reactions were analyzed by flow cytometry using a combination of two antibodies, an APC-labelled a- His antibody to detect cells expressing the IMPDH.1-C-CAR constructs, either with or without the linker, as the they have an N-terminal His tag, and a PE-labelled a-FLAG antibody to detect cells containing the reconstituted a-BCMA scFv-CAR, as the a-BCMA scFv has an N-terminal FLAG tag. The results show that 12% of the cells express the IMPDH.1-C-CAR on the surface. After the trans-splicing reaction, 8% of the cells have a FLAG tag on the surface. These two percentages represent a CAR reconstitution efficiency per cell close to 70%. The addition of the linker between IntC and the CD8 transmembrane domain doubles the percentage of cells expressing the IMPDH.1-C- CAR on the surface, from 12% to 23%. It also increases the percentage of cells that incorporate the FLAG-tagged scFv after the trans-splicing reaction, from 8% to 12%, resulting in a CAR reconstitution efficiency per cell of 52% (Figure 8, A and D). The presence of the reconstituted a-BCMA scFv-CAR in the cells after the trans-splicing reaction was analyzed by Western blot using an aCD3z antibody. A band with a molecular weight between 50 kDa and 70 kDa appears after the reaction (Figure 8, B). This molecular weight corresponds to that expected for the reconstituted CAR, which is 53.3 kDa.

[0682] EXAMPLE 9: In vitro trans-splicing reaction between cells expressing the caged gp41.1- C-CAR construct and a purified O-CD19 scFv-gp41.1-N fusion

[0683] HEK293F cells were transfected with the caged gp41.1-C-CAR construct. Cells were harvested 48 hours after transfection and in vitro trans-splicing reactions were prepared by combining the cells expressing the caged gp41.1-C-CAR construct with the O-CD19 scFv-gp41.1-N intein fusion. After incubation, the trans-splicing reactions were analyzed by flow cytometry using a combination of two antibodies, an APC-labelled a-His antibody to detect cells expressing the caged gp41.1-C-CAR construct, as it has an N-terminal His tag, and a PE-labelled a-FLAG antibody to detect cells containing the reconstituted O-CD19 scFv-CAR, as the O-CD19 scFv has an N-terminal FLAG tag. The results show that 84% of the cells express the caged gp41.1-C-CAR and that 37% of the cells have undergone trans-splicing. The combination of both figures gives a CAR reconstitution efficiency per cell of 44% (Figure 9, A). The trans-splicing reactions were also analyzed using a PE-labelled CD19 antigen. Following the trans-splicing reaction, the cells are expected to recognize and bind the CD19 antigen as the reaction introduces an O-CD19 scFv to the cell surface. The results show that 32% of the cells bind to the CD19 antigen after the trans-splicing reaction, meaning that these cells have incorporated an O-CD19 scFv and therefore, the CAR reconstitution efficiency per cell is 38 % for the caged CAR containing the gp41.1 intein (Figure 9, B). Finally, cells expressing the caged gp41.1-C- CAR before and after a trans-splicing reaction with the O-CD19 scFv-gp41.1-N intein fusion were analyzed by Western blot using an aCD3z antibody to identify the formation of the reconstituted O-CD19 scFv-CAR. A molecular weight shift of the bands corresponding to the caged gp41.1-N-CAR is observed, consistent with the incorporation of the O-CD19 scFv (Figure 9, C).

[0684] EXAMPLE 10: In vitro trans-splicing reaction between cells expressing the caged

[0685] IMPDH.1-C-CAR construct and a a-BCMA scFv-IMPDH.1-N fusion HEK293F cells were transfected with the caged IMPDH.1-C-CAR construct. Cells were harvested 48 hours after transfection and in vitro trans-splicing reactions were prepared by combining the cells expressing the caged IMPDH.1-C-CAR construct with the a- BCMA scFv-IMPDH.1-N intein fusion. After incubation, the trans-splicing reactions were analyzed by flow cytometry using a combination of two antibodies, an APC-labelled a- His antibody to detect cells expressing the caged IMPDH.1-C-CAR construct, as it has an N-terminal His tag, and a PE-labelled a-FLAG antibody to detect cells containing the reconstituted a-BCMA scFv-CAR, as the a-BCMA scFv has an N-terminal FLAG tag. The results show that 87% of the cells express the caged IMPDH.1-C-CAR on the surface. After the trans-splicing reaction, 7% of the cells have a FLAG tag on the surface (Figure 10, A). These two percentages represent a CAR reconstitution efficiency per cell of 8%. The presence of the reconstituted a-BCMA scFv-CAR in the cells after the trans- splicing reaction was analyzed by Western blot using an aCD3z antibody. A molecular weight shift of the bands corresponding to the caged IMPDH.1-N-CAR is observed, consistent with the incorporation of the a-BCMA scFv (Figure 10, B).

[0686] EXAMPLE 11 : Effect of the presence of the cage in the IntC-CAR construct on the trans- splicing reaction

[0687] Caging IntC in an IntC-CAR construct increases protein expression on the surface of transfected HEK293F cells (see Figure 6B). It also increases the number of cells expressing high levels of IntC-CAR per cell (see Figure 6C). As a result, the number of cells that incorporate high levels of scFv after the trans-splicing reaction is significantly higher in cells expressing a caged IntC-CAR than in cells expressing an uncaged IntC- CAR. This comparison is made based on the total number of cells that have undergone trans-splicing and the percentage of these cells that show high levels of fluorescence, indicating that they contain high levels of scFv-CAR on the surface. Both the number of cells that have undergone trans-splicing and the percentage of cells with high levels of fluorescence are obtained from a flow-cytometry analysis of a trans-splicing reaction between cells expressing the IntC-CAR, caged or uncaged, and a purified scFv-lntN fusion. In the case of the gp41.1 intein, the flow cytometry analysis of the trans-splicing rea...

Claims

CLAIMS1. A polynucleotide encoding a fusion protein comprising from N-terminus to C- terminus: i) a modified N-intein selected from the group consisting of a modified GP41.1 N-intein, a modified IMPDH N-intein, a modified NrdJ1 N-intein or a modified GP41.8 N-intein, wherein the modified N-intein comprises a fragment of the N-intein protein that prevents trans-splicing from occurring in the presence of the corresponding C-intein, wherein the modified N-intein comprises at least one mutation that decreases the affinity between the modified N-intein and the corresponding C-intein, ii) a split C-intein comprising a sequence selected from the group consisting of 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-intein is a modified GP41.1 N-intein, the split C- intein comprises the sequence of SEQ ID NO: 1 or is a functionally equivalent variant thereof,If the modified N-intein is a modified IMPDH N-intein, the split C-intein comprises the sequence of SEQ ID NO: 2 or is a functionally equivalent variant thereof, the modified N-intein is a modified IMPDH N-inteinIf the modified N-intein is a modified NrdJ1 N-intein, the split C-intein comprises the sequence of SEQ ID NO: 3 or is a functionally equivalent variant thereof orIf the modified N-intein is a modified GP41.8 N-intein, the split C- intein comprises the sequence of SEQ ID NO: 4 or is a functionally equivalent variant thereof, and iii) a heterologous polypeptide.

2. The polynucleotide according to claim 1 , wherein:-the modified GP41.1 N-intein comprises the sequence of SEQ ID NO: 31 ,-the modified IMPDH N-intein comprises the sequence of SEQ ID NO: 32,-the modified NrdJ1 N-intein comprises the sequence of SEQ ID NO: 33 or-the modified GP41.8 N-intein comprises 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. a linker region between the modified N-intein and the split C-intein, and / or b. a protease cleavage site between the modified N-intein and the split C- intein.

4. The polynucleotide according to any one of claims 1 to 3, wherein the heterologous polypeptide comprises from N-terminus to C-terminus: a. a transmembrane domain, and b. an intracellular signalling domain from a receptor and / or costimulatory domain.

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 in frame and N-terminal to the modified N- intein and which allows the insertion of the fusion protein into the membrane.

6. The polynucleotide according to claim 5, wherein the signal peptide is selected from the group consisting of: CD8a, IL-2, GM-CSF receptor (GM-CSF) a chain, murine Ig-kappa (IgK), murine IgK V-lll region (IgKVIl I), human IgKVIll, CD33, human tissue plasminogen activator (TPA) and native secreted alkaline phosphatase (SEAP).

7. The polynucleotide according to any one of claims 4 to 6, wherein the intracellular signalling domain from a receptor is an antigen receptor signalling domain, preferably selected from the group consisting of the CD3 , CD28, 4-1 BB, NKG2D, DAP10, 0X40, CD70, CD27, CD5, ICAM-1 , LFA-1 (CD11a / CD18), ICOS (CD278), DAP 12 antigen receptor signalling domain or a combination thereof.

8. The polynucleotide according to any one of claims 4 to 7, wherein the transmembrane domain is selected from the group consisting of the CD28, the CD8a the 4-1 BB, the CD4, the CTLA4, the CD27 or the CD3 transmembrane domain.

9. The polynucleotide according to claim 8, wherein the transmembrane domain and the intracellular signalling domain are either directly connected by a peptide bond or by an amino acid linker.

10. A vector which comprises the polynucleotide according to any one of claims 1 to 9.

11. A host cell comprising the polynucleotide according to any one of claims 1 to 9, a vector according to claim 10 or the fusion protein encoded by the polynucleotide according to any one of claims 1 to 9.

12. The host cell according to claim 11 , wherein the cell is obtained by viral transduction.

13. The host cell according to claim 12, wherein the cell is obtained by viral transduction with a lentivirus or by gene knock-in.

14. The host cell according to claim 13, wherein the gene knock-in is carried out 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 the group consisting of: T-cell, NK-cell, induced pluripotent stem cell (iPSC), derived NK cell, Pro-Tcell, B cell and macrophage.

17. A fusion protein encoded by the polynucleotide of any of claims 1 to 9.

18. The fusion protein according to claim 17, wherein the fusion protein further comprises a detection tag.

19. The fusion protein according to claim 18, wherein the detection tag is selected from the group consisting of: c-myc tag, HA tag, FLAG-tag, Strep-tag, His-tag, alfa tag, V5 tag, Spot tag and NE tag.

20. A fusion protein comprising from N-terminus to C-terminus:i) a heterologous polypeptide and ii) a split N-intein comprising a sequence selected from the group consisting 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 according to claim 20, wherein the polypeptide and the split N-intein are either directly connected by a peptide bond or by a peptide linker.

22. The fusion protein according to any one of claims 20 or 21 , wherein the polypeptide is selected from the group consisting of a single-chain variable fragment (scFv), a single domain antibody, a lectin, an epitope, an Ig heavy or light chain, a peptide and a ligand.

23. The fusion protein according to claim 22, wherein the polypeptide specifically binds to a target molecule selected from the group consisting of: an antigen of interest, a lectin, an epitope.

24. The fusion protein according to claim 23, wherein the target molecule is an antigen of interest.

25. The fusion protein according to claim 24, wherein the antigen of interest is selected from the group consisting of: 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 solubilisation tag.

27. The fusion protein according to claim 26, wherein the detection tag is selected from the group consisting of: c-myc tag, HA tag, FLAG-tag, Strep-tag, His tag, alfa tag, V5 tag, Spot tag and NE tag.

28. The fusion protein according to claim 26, wherein the solubilisation tag is selected from the group consisting of: phage bacteriophage lambda head protein D (gpD), small ubiquitin-like modifier) SUMO, maltose binding protein (MBP), glutathione- S-transferase (GST), N-utilization substance (NusA), thioredoxinA and Fasciola hepatica 8-kDa antigen (Fh8 tag).

29. A polynucleotide encoding the fusion protein according to any one of claims 20 to 28.

30. The polynucleotide according to claim 29 further comprises a nucleotide sequence encoding a signal peptide, wherein the signal peptide is in frame and N-terminal to the polypeptide that specifically binds to a target molecule and which allows the secretion of the fusion protein from the cell.31 . A vector comprising the polynucleotide according to any of claims 29 or 30.

32. A host cell comprising the polynucleotide according to any of claims 29 or 30 or the vector according to claim 31.

33. A chimeric receptor comprising from N-terminus to C-terminus: i) an extracellular domain comprising a polypeptide that specifically binds to a target molecule or a polypeptide which can form an oligomer in the presence of one or more additional subunits and wherein the oligomer specifically binds to a target molecule, ii) a linker region, the sequence of which is the result of a protein splicing reaction, iii) a transmembrane domain, and iv) an intracellular signalling domain from a receptor, wherein the protein splicing reaction occurs between two complementary portions of a split intein and wherein the split intein is selected from the group consisting of the Gp41.1 split intein, the IMPDH split intein, the NrdJ1 split intein and the Gp41.8 split intein.

34. The chimeric receptor according to claim 33, wherein when the extracellular domain is a polypeptide that can form an oligomer in the presence of one or more additional subunits, then the receptor further contains said one or more additional subunits.

35. The chimeric receptor according to claims 33 or 34 wherein the linker region comprises between 1 and 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, then the polypeptide is selected from the group consisting of: a single chain variable fragment (scFv), a single domain antibody, a lectin, an epitope, an Ig heavy or light chain, a peptide and a ligand.

37. The chimeric receptor according to any one of claims 33 to 35, wherein when the extracellular domain is a polypeptide that can form an oligomer in the presence of one or more additional subunits, then the polypeptide is a heavy chain or a 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 that can form an oligomer in the presence of one or more additional subunits, then the oligomer is an immunoglobulin.

39. The chimeric receptor according to 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 the at least a target molecule is selected from the group consisting of: an antigen of interest, a lectin, an epitope.

41. The chimeric receptor according to claim 40, wherein the target molecule is an antigen of interest.

42. The chimeric receptor according to claim 41 , wherein the antigen of interest is selected from the group consisting of: 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 signalling domain from a receptor is an antigen receptor signalling domain.

44. The chimeric receptor according to claim 43, wherein the antigen receptor signalling domain is selected from the group consisting of the CD3 , CD28, 4- 1 BB, NKG2D, DAP10, 0X40, CD70, CD27, CD5, ICAM-1 , LFA-1 (CD11a / CD18),ICOS (CD278), DAP 12 antigen receptor signalling domain or a combination thereof.

45. The chimeric receptor according to any one of claims 33 to 44, wherein the transmembrane domain is selected from the group consisting of the CD28, the CD8a the 4-1 BB, the CD4, the CTLA4, the CD27 or the CD3 transmembrane domain.

46. A host cell comprising the chimeric receptor according to any one of claims 33 to 45.

47. The host cell according to claim 46, wherein the cell is a human cell.

48. The host cell according to claim 47, wherein the cell is selected from the group consisting of: T cell, NK-cell, induced pluripotent stem cell (iPSC) derived NK cells, Pro-Tcell, B cell and macrophage.

49. A composition or kit-of-parts comprising a first component and a second component wherein i) the first component is the polynucleotide according to any one of claims 1 to 9, the vector according to claim 10, the host cell according to any of claims 11 to 16 or the fusion protein according to any of claims 17 to 19 and ii) the second component is the fusion protein according to any one of claims 20 to 28 or the polynucleotide according to any of claims 29 or 30, wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, orthe split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

50. A method for cleaving the heterologous polypeptide from the fusion protein according to claims 17 to 19 comprising incubating the fusion protein with a split N- intein under conditions allowing intein splicing whereinIf the split C-intein forming part of the fusion protein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof, then the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof,If the split C-intein forming part of the fusion protein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof, the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof,If the split C-intein forming part of the fusion protein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof, then the split N-intein comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, orIf the split C-intein forming part of the fusion protein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof, then and the split N-intein 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 comprising incubating the composition of claim 49 or bringing into association the components of the kit-of-parts of claim 49 under conditions allowing intein splicing wherein said first polypeptide is the heterologous polypeptide which forms part of the fusion protein according to any of claims 17 to 19 and said second polypeptide is the heterologous polypeptide which forms part of the fusion protein according to any of claims 20 to 28.

52. An in vitro method for producing a cell comprising a chimeric receptor, wherein the method comprises: i) providing a cell comprising the polynucleotide according to any one of claims 4 to 9, a polypeptide encoded by said polynucleotide or the vectoraccording to claim 10, as far as it depends from claims 4 to 9, under conditions adequate for the expression in the cell of the fusion protein encoded by the polynucleotide, and ii) contacting the cell of step i) with the fusion protein according to any one of claims 20 to 28, wherein the contacting step ii) is performed under conditions that allow a splicing reaction between the split C-intein forming part of the fusion protein produced in step (i) and the split N-intein forming part of the fusion proteins according to any of claims 20 to 28 and wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

53. An in vitro method for producing a cell comprising a chimeric receptor, wherein the method comprises: i) providing a cell comprising a. the polynucleotide according to any one of claims 4 to 9, the vector according to claim 10, as far as it depends from claims 4 to 9, or a polypeptide encoded by said polynucleotide wherein the polynucleotide further comprises a nucleotide sequence encoding a signal peptide, wherein the signal peptide is in frame and N-terminal to the modified N-intein and which allows the insertion of the fusion protein into the membrane and b. the polynucleotide according to any one of claims 29 or 30, the vector according to claim 31 or the fusion protein according to any of claims 20 to 28 wherein the fusion protein encoded by the polynucleotide orthe fusion protein further comprises a nucleotide sequence encoding a signal peptide, wherein the signal peptide is in frame and N-terminal to the polypeptide that specifically binds to a target molecule and which allows the secretion of the fusion protein from the cell and ii) maintaining the cell under conditions adequate for the expression in the cell of both fusion proteins, for the insertion into the membrane of the fusion protein encoded by the polynucleotide according to any one of claims 4 to 9, for the secretion of the fusion protein encoded by the polynucleotide according to any of claims 29 or 30 and for the splicing reaction between the split C-intein and the split N-intein and wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO: 3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

54. The method according to claim 53, wherein when the chimeric receptor comprises an extracellular domain comprising a polypeptide that can form an oligomer in the presence of one or more additional subunits, then the cell provided in (i) further comprises an additional polypeptide encoding said one or more additional subunits of the oligomer.

55. The method according to claim 54, wherein the oligomer is an immunoglobulin.

56. The method according to claims 53 or 54, wherein the fusion protein is a heavy chain or a light chain of an immunoglobulin, and then the additional polypeptide (c) encodes the corresponding heavy or light chain of the immunoglobulin.

57. The method according to any 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 according to claim 58, wherein the cell is selected from the group consisting of: T cell, NK-cell, induced pluripotent stem cell (iPSC) derived NK cells, Pro-Tcell, B cell and macrophage.

60. The method according to any of claims 52 to 59 wherein the cell used in step (i) have been obtained from a patient.

61. A cell comprising a chimeric receptor obtained by a method according to any of claims 52 to 60.

62. The cell according to any of claims 46 to 48 or 61 for use in the treatment of a disease which requires a cell-mediated immune response against a target molecule, preferably against an antigen of interest.

63. The fusion protein according to any of claims 20 to 28, the polynucleotide according to any of claims 29 or 30, the vector according to claim 31 or the host cell according to claim 32 for use in the treatment of a disease which requires a cell-mediated immune response against a target molecule, preferably against an antigen of interest, wherein the patient to be treated is characterized in that it contains cells comprising the polynucleotide according to any one of claims 4 to 9, the vector according to claim 10, as far as it depends from claims 4 to 9, or the fusion protein encoded by the polynucleotide according to any one of claims 4 to 9, wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof,the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

64. The polynucleotide according to any of claims 4 to 9, the vector according to claim 10, as far as it depends from claims 4 to 9, the host cell according to any of claims 11 to 16, as far as it depends from claims 4 to 9, or the fusion protein encoded by the polynucleotide according to any one of claims 4 to 9 for use in the treatment of a disease which requires a cell-mediated immune response against a target molecule, preferably against an antigen of interest, wherein the patient to be treated is characterized in that it contains cells comprising the fusion protein according to any of claims 20 to 28, the polynucleotide according to any of claims 29 or 30, the vector according to claim 31 or the host cell according to claim 32, wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

65. The fusion protein, polynucleotide, vector or host cell for use according to any of claims 63 or 64 wherein the disease requiring a cell-mediated immune response against a target molecule, preferably against an antigen of interest, is selected from the group consisting of: cancer, a viral infection, a bacterial infection, a parasitic infection and an autoimmune disease.

66. The fusion protein, polynucleotide, vector or host cell for use according to claim 65, wherein the cancer is selected from the group consisting of: 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 comprising from N-terminus to C- terminus: i) a split C-intein comprising a sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 or a functionally equivalent variant thereof, ii) a transmembrane domain, and iii) an intracellular signalling domain from a receptor and / or costimulatory domain.

68. The polynucleotide according to claim 67 further comprising a nucleotide sequence encoding a signal peptide, wherein the signal peptide is in frame and N-terminal to the split C-intein and which allows the insertion of the fusion protein into the membrane.

69. The polynucleotide according to claim 68, wherein the signal peptide is selected from the group consisting of: CD8a, IL-2, GM-CSF receptor (GM-CSF) a chain, murine Ig-kappa (IgK), murine IgK V-lll region (IgKVIl I), human IgKVIll, CD33, human tissue plasminogen activator (TPA) and native secreted alkaline phosphatase (SEAP).

70. The polynucleotide according to any one of claims 67 to 69 further comprising a linker between the split C-intein and the transmembrane domain.

71. The polynucleotide according to claim 70, wherein the linker comprises between 40 and 45 amino acids.

72. The polynucleotide according to any of claims 70 or 71 , wherein the linker is a hinge region selected from the group consisting of: I gG 1 hinge region, lgG2 hinge region, lgG4 hinge region, FcyR hinge region, CD3E hinge region, CD3 hinge region, CD8a hinge region, CD4 hinge region, CD28 hinge region, CD7 hingeregion, CD19 hinge region or a flexible linker, preferably a glycine and serine-rich linker.

73. The polynucleotide according to any one of claims 67 to 72, wherein the intracellular signalling domain from a receptor is an antigen receptor signalling domain, preferably selected from the group consisting of the CD3 , CD28, 4-1 BB, NKG2D, DAP10, 0X40, CD70, CD27, CD5, ICAM-1 , LFA-1 (CD11a / CD18), ICOS (CD278), DAP 12 antigen receptor signalling domain or a combination thereof.

74. The polynucleotide according to any one of claims 67 to 73, wherein the transmembrane domain is selected from the group consisting of the CD28, the CD8a the 4-1 BB, the CD4, the CTLA4, the CD27 or the CD3 transmembrane domain.

75. The polynucleotide according to any one of claims 67 to 74, wherein the transmembrane domain and the intracellular signalling domain are either directly connected by a peptide bond or by an amino acid linker.

76. The polynucleotide according to any one of claims 67 to 75, wherein the fusion protein encoded by the polynucleotide further comprises a modified N-intein wherein the modified N-intein is in N-terminal position with respect to the split C- intein, whereinIf the split C-intein comprises the sequence of SEQ ID NO: 1 or is a functionally equivalent variant thereof, the modified N-intein is a modified GP41.1 N-intein,If the split C-intein comprises the sequence of SEQ ID NO: 2 or is a functionally equivalent variant thereof, the modified N-intein is a modified IMPDH N-intein,If the split C-intein comprises the sequence of SEQ ID NO: 3 or is a functionally equivalent variant thereof, the modified N-intein is a modified NrdJ1 N-intein orIf the split C-intein comprises the sequence of SEQ ID NO: 4 or is a functionally equivalent variant thereof, the modified N-intein is a modified GP41.8 N-intein,wherein the modified N-intein comprises a fragment of the N-intein protein that prevents trans-splicing from occurring in the presence of the corresponding C- intein, wherein the modified N-intein comprises at least one mutation that decreases 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-intein comprises the sequence of SEQ ID NO: 31 , the modified IMPDH N-intein comprises the sequence of SEQ ID NO: 32, the modified NrdJ1 N-intein comprises the sequence of SEQ ID NO: 33, or the modified GP41.8 N-intein comprises 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. a linker region between the modified N-intein and the split C-intein and / or b. a protease cleavage site between the modified N-intein and the split C- intein.

79. A vector which comprises the polynucleotide according to any one of claims 67 to 78.

80. A host cell comprising the polynucleotide according to any one of claims 67 to 78, a vector according to claim 79 or the fusion protein encoded by the polynucleotide according to any one of claims 67 to 78.

81. The host cell according to claim 80, wherein the cell is obtained by viral transduction.

82. The host cell according to claim 81 , wherein the cell is obtained by viral transduction with a lentivirus or by gene knock-in.

83. The host cell according to claim 82, wherein the gene knock-in is carried out 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 the group consisting of: T-cell, NK-cell, induced pluripotent stem cell (iPSC), derived NK cell, Pro-Tcell, B cell and macrophage.

86. A fusion protein encoded by the polynucleotide of any of claims 67 to 78.

87. The fusion protein according to claim 86, wherein the fusion protein further comprises a detection tag.

88. The fusion protein according to claim 87, wherein the detection tag is selected from the group consisting of: 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-of-parts comprising a first component and a second component wherein i) the first component is the polynucleotide according to any one of claims 67 to 78, the vector according to claim 79, the host cell according to any of claims 80 to 85 or the fusion protein according to any of claims 86 to 88 and ii) the second component is the fusion protein according to any one of claims 20 to 28 or the polynucleotide according to any of claims 29 or 30, wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprisesa polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

90. An in vitro method for producing a cell comprising a chimeric receptor, wherein the method comprises: i) providing a cell comprising the polynucleotide according to any one of claims 67 to 78, a polypeptide encoded by said polynucleotide or the vector according to claim 79 under conditions adequate for the expression in the cell of the fusion protein encoded by the polynucleotide, and ii) contacting the cell of step i) with the fusion protein according to any one of claims 20 to 28, wherein the contacting step ii) is performed under conditions that allow a splicing reaction between the split C-intein forming part of the fusion protein produced in step (i) and the split N-intein forming part of the fusion proteins according to any of claims 20 to 28 and wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

91. An in vitro method for producing a cell comprising a chimeric receptor, wherein the method comprises: i) providing a cell comprising a. the polynucleotide according to any one of claims 67 to 78, the vector according to claim 79 or a polypeptide encoded by said polynucleotide wherein the polynucleotide further comprises a nucleotide sequence encoding a signal peptide, wherein the signal peptide is in frame andN-terminal to the split C-intein and which allows the insertion of the fusion protein into the membrane and b. the polynucleotide according to any one of claims 29 or 30, the vector according to claim 31 or the fusion protein according to any 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 in frame and N-terminal to the polypeptide that specifically binds to a target molecule and which allows the secretion of the fusion protein from the cell and ii) maintaining the cell under conditions adequate for the expression in the cell of both fusion proteins, for the insertion into the membrane of the fusion protein encoded by the polynucleotide according to any one of claims 67 to 78, for the secretion of the fusion protein encoded by the polynucleotide according to any of claims 29 or 30 and for the splicing reaction between the split C-intein and the split N-intein and wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO: 3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO: 4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

92. The method according to claim 91 , wherein when the chimeric receptor comprises an extracellular domain comprising a polypeptide that can form an oligomer in the presence of one or more additional subunits, then the cell provided in (i) further comprises an additional polypeptide encoding said one or more additional subunits of the oligomer.

93. The method according to claim 92, wherein the oligomer is an immunoglobulin.

94. The method according to claims 92 or 93, wherein the fusion protein is a heavy chain or a light chain of an immunoglobulin, and then the additional polypeptide (c) encodes the corresponding heavy or light chain of the immunoglobulin.

95. The method according to any 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 according to claim 96, wherein the cell is selected from the group consisting of: T cell, NK-cell, induced pluripotent stem cell (iPSC) derived NK cells, Pro-Tcell, B cell and macrophage.

98. The method according to any of claims 90 to 97 wherein the cell used in step (i) have been obtained from a patient.

99. A cell comprising a chimeric receptor obtained by a method according to any of claims 90 to 98.

100. The cell according to any of claims 46 to 48 or 99 for use in the treatment of a disease which requires a cell-mediated immune response against a target molecule, preferably against an antigen of interest.101 . The fusion protein according to any of claims 20 to 28, the polynucleotide according to any of claims 29 or 30, the vector according to claim 31 or the host cell according to claim 32 for use in the treatment of a disease which requires a cell-mediated immune response against a target molecule, preferably against an antigen of interest, wherein the patient to be treated is characterized in that it contains cells comprising the polynucleotide according to any one of claims 67 to 78, the vector according to claim 79 or the fusion protein encoded by the polynucleotide according to any one of claims 67 to 78, wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof,the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

102. The polynucleotide according to any of claims 67 to 78, the vector according to claim 79, the host cell according to any of claims 80 to 85 or the fusion protein according to any of claims 86 to 88 for use in the treatment of a disease which requires a cell-mediated immune response against a target molecule, preferably against an antigen of interest, wherein the patient to be treated is characterized in that it contains cells comprising the fusion protein according to any of claims 20 to 28, the polynucleotide according to any of claims 29 or 30, the vector according to claim 31 or the host cell according to claim 32, wherein the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:1 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:5 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:2 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of e SEQ ID NO:6 or a variant thereof, the split C-intein comprises a polypeptide having the sequence of the SEQ ID NO:3 or a variant thereof and the split N- comprises a polypeptide having the sequence of the SEQ ID NO:7 or a variant thereof, or the split C-intein comprises a polypeptide having the sequence of SEQ ID NO:4 or a variant thereof and the split N-intein comprises a polypeptide having the sequence of SEQ ID NO:8 or a variant thereof.

103. The fusion protein, polynucleotide, vector or host cell for use according to any of claims 101 or 102 wherein the disease requiring a cell-mediated immune response against a target molecule, preferably against an antigen of interest, is selected from the group consisting of: cancer, a viral infection, a bacterial infection, a parasitic infection and an autoimmune disease.

104. The fusion protein, polynucleotide, vector or host cell for use according to claim 103, wherein the cancer is selected from the group consisting of: 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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