Intein mediated modular cars

Abstract

The present invention refers to a chimeric antigen receptor (CAR) precursor comprising at least two polypeptides that are capable of carrying out a trans-splicing reaction to result into a mature CAR. The present invention also provides mature CARs, allogenic CAR-T cells, and methods of producing thereof.

Description

[0001] INTEIN MEDIATED MODULAR CARS

[0002] TECHNICAL FIELD

[0003] The present invention relates to the field of biomedicine. Particularly, the present invention relates to the field of Chimeric Antigen Receptors (CAR), particularly modular CARs, and uses thereof.

[0004] BACKGROUND ART

[0005] Split inteins are specialized protein elements that have revolutionized our understanding of post-translational modifications. These proteins are divided into two distinct fragments, termed the N-intein and C-intein, which independently associate to catalyze a highly specific process called protein trans-splicing. During this process, the intein excises itself from a precursor protein, seamlessly joining the surrounding protein segments (exteins) into a single, functional polypeptide. This reaction occurs without the need for external energy sources or cofactors, relying solely on the intrinsic enzymatic properties of the split inteins.

[0006] In recent years, split inteins have emerged as powerful tools in therapeutic development. Their ability to precisely assemble proteins at specific sites and under controlled conditions makes them ideal for engineering therapeutic molecules. Split inteins have proven particularly useful in the development of cell and gene therapies, where precise protein engineering is critical. In gene therapy, split inteins can facilitate the assembly of therapeutic proteins from smaller gene segments, enabling the delivery of large or complex genes that exceed the packaging limits of viral vectors like adeno-associated viruses (AAVs). Similarly, in cell therapy, split inteins can be employed to create modular and conditionally active therapeutic proteins, such as engineered receptors or cytokines, directly within patient-derived cells. These capabilities not only expand the range of diseases that can be targeted but also improve the safety and specificity of advanced therapies by ensuring that therapeutic proteins are only functional under desired conditions.

[0007] Immunotherapy and particularly chimeric antigen receptor (CAR) T-cell therapy has shown remarkable efficacy against several cancers. However, despite these advances, several barriers continue to limit the overall effectiveness of this approach, including high production costs, extended manufacturing timelines, safety concerns, and the risk of tumor antigen escape due to selective therapeutic pressure. To address these limitations, innovative CAR-T strategies are being developed.Further, despite significant progress in the field, the therapeutic potential of split inteins in immunotherapy is far from fully realized. Current challenges include improving the efficiency and stability of intein-mediated splicing in complex biological environments and enhancing the specificity of intein interactions to avoid unintended reactions. Addressing these limitations is essential to fully unlock the potential of split inteins in therapeutic settings.

[0008] As the field continues to advance, split inteins hold great promise for transforming the landscape of immunotherapy and beyond. However, ongoing innovation is needed to refine their functionality and broaden their applicability. By overcoming current challenges, researchers can leverage split inteins to develop next-generation therapies that are safer, more effective, and tailored to individual patient needs.

[0009] BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1. Dual targeting CARtein strategy. (A) Schematic representation of CARtein modules before and after split intein-mediated protein splicing. (B) Representation of Jurkat (JKT) triple parameter reporter (TPR) expressing the final anti-BCMA and anti-SLAMF7 CARtein constructs. JKT TPR, Jurkat-TPR cells.

[0011] Figure 2. CARtein modules structures prediction. (A) Folding prediction of anti-BCMA antigen recognition module containing the scFv (Belantamab) fused to the N-terminal part of IMPDH-1 split intein (including the N-intein and N-extein domain) compared to the unmodified anti-BCMA scFv. (B) Structure prediction of anti-SLAMF7 antigen recognition module consisting in the scFv (Elotuzumab) fused to the N-terminal part of IMPDH-1 split intein compared to unattached anti-SLAMF7 scFv. (C) Structure prediction of the extracellular domain of the Signaling CARtein module (SCM) formed by CD28 TM and lgG1 spacer domains followed by the C-terminal part of IMPDH-1 intein (including the C-intein and C-extein domain) compared to the intein-depleted SCM. Relevant residues for split intein-mediated protein splicing are shown.

[0012] Figure 3. CARtein complexes prediction. (A) Docking of the predicted structures of anti-BCMA antigen recognition module and SCM, with key residues for intein-mediated protein splicing shown. (B) Docking of the predicted tridimensional structures of anti-SLAMF7 antigen recognition module and SCM, with highlighted relevant residues for split intein-mediated protein splicing. (C) Complex prediction of post splicing anti-BCMA CARtein bound to BCMA ECD compared to an exteins-depleted CAR. Proximal residues of the scFv to the antigen are highlighted. (D) Complex prediction of post-splicing anti-SLAMF7 CARtein bound to SLAMF7ECD compared to a CAR devoid of residual exteins. Proximal residues of Elotuzumab to the surface antigen are shown.

[0013] Figure 4. CARtein strategy and expression after split intein-mediated protein splicing. (A) Representative illustration of anti-BCMA and anti-SLAMF7 CARtein modules before and after intein-mediated CARtein splicing in the ER. (B) Illustrative representation of the staining strategy performed for the evaluation of SCM and CARtein expression. (C) Flow cytometry histogram overlays of SCM and spliced CARteins expression in transduced Jurkat-TPR cells and (D) SLAMF7 and BCMA expression in MM.1s cells. UTD, untransduced cells. JKT TPR, Jurkat-TPR cells.

[0014] Figure 5. T cell activation signaling assay in CARtein Jurkat-TPR cells co-cultured with MM.1s cells. (A) Schematic illustration of anti-BCMA or anti-SLAMF7 CARtein cells or (B) Dual CARtein cells co-cultured with multiple myeloma target cells. (C) Representative flow cytometry analysis of NFAT and NFKB activity and (D) CD69 upregulation 24h after co-culture in a T:E ratio of 1:1. UTD, untransduced cells. JKT TPR, Jurkat-TPR cells.

[0015] Figure 6. kinetics of anti-BCMA, anti-SLAMF7 and Dual CARtein-TPR cell activation upon MM.1s stimulation. (A) Flow cytometry-based statistical comparison of the mean fluorescence intensity (MFI) of NFAT and NFKB activity reporters and CD69 upregulation at three different T:E ratios after MM.1S co-culture. Statistical analysis between T:E ratios is shown (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (B) NFAT, NFKB and CD69 activation signaling kinetics for three T:E ratios of anti-BCMA, (C) anti-SLAMF7 and (D) Dual CARtein cells upon stimulation. Statistical analysis of CARtein cells in 1 :1 ratio is indicated for each time condition (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Data are ± SEM of three different experimental replicates. UTD, untransduced cells.

[0016] Figure 7. Activation signaling assay of anti-BCMA, anti-SLAMF7 and Dual CARtein-TPR cells co-cultured with k562 cells expressing BCMA, SLAMF7 or both surface proteins. (A) Flow cytometry histogram overlays of BCMA and SLAMF7 expression in transduced k562 cells with either one or both surface antigens. (B) Representative flow cytometry analysis of NFAT and (C) NFKB promoter reporters, as well as (D) CD69 activation marker expression in CARtein-TPR cells 24h after co-culture with transduced k562 cells in a T:E ratio of 1:1. UTD, untransduced cells.

[0017] Figure 8. kinetics of anti-BCMA, anti-SLAMF7 and Dual CARtein-TPR cell activation upon k562 cells expressing BCMA, SLAMF7 or both surface proteins stimulation. (A) Flow cytometry comparison of NFAT and NFKB reporters and CD69 MFI of CARtein-TPR cells co-cultured with k562 cells expressing BCMA, SLAMF7 or both surface proteins. Each dot represents an independent experimental triplicate. Statistical comparison between anti-BCMA, anti-SLAMF7 and Dual CARtein-TPR cells is indicated (* p < 0.05, ** p < 0.01, *** p < 0.001 , **** p < 0.0001). (B) NFAT, (C) NFKB and (D) CD69 activation signaling kinetics for anti-BCMA, anti-SLAMF7 and Dual CARtein-TPR cells upon transduced k562 stimulation. Statistical analysis of Dual CARtein cells is shown for each time condition (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Data are ± SEM of three different experimental replicates. UTD, untransduced cells.

[0018] Figure 9: Description of the CARtein platform and surface expression of CD19 / CD20 on target cells. (A) Schematic illustration of the CARtein system, detailing the respective domains of each construct and how they interact. (B) Schematics of the products of the reaction, by which a functional CAR is generated, together with an intein byproduct. (C) Schematic representation of each scFv-intein construct and (D) how they result when bound to the IntStem CAR. (E) Evaluation, by spectral flow cytometry, of CAR expression in Jurkat-TPR cells and (F) CD19 / CD20 expression in the different target cells.

[0019] Figure 10: Activation of CAR-TPR cells upon stimulation with Raji cells. (A) Illustration of the spectral flow cytometry gating strategy, selecting single live CD3+ cells, corresponding to Jurkat-TPR cells. (B) Dot-plot representation of eGFP (NFAT) and CFP (NFKB) fluorescences 24 and 48h post-stimulation, representing cells responding to both promoters. (C) Histogram representation of CD69 (APC) upregulation upon Raji stimulation.

[0020] Figure 11: Kinetics of CAR activation. (A) Graphical representation, at different times and target: effector ratios, of the MFI corresponding to NFAT-eGFP, (B) NFKB-CFP and (C) anti-CD69-APC. Each symbol represents the mean ± SEM of MFI values from three independent experiments (n=3). Statistical analysis is shown for each condition compared to nonstimulated (NS) cells (*p < 0.05, **p < 0.01, ***p < 0.001). NT, Non-transduced.

[0021] Figure 12: Specific activation of the CARtein platform upon binding of individual antigens. (A) NFAT activity (MFI of eGFP) of each CAR 24 and 48h after stimulation with different K562 cell sublines (1:1 ratio), as well as of (B) NFKB promoter activity (CFP) and (C) CD69 (APC) upregulation. (D) Representative histograms of each parameter at 24 hours after stimulation are shown for each condition. Error bars represent the mean ± SEM of MFI values from three replicates (n=3). Statistical analyses have been performed in comparison with non-stimulated (NS) cells. (*p < 0.05, **p < 0.01, ***p < 0.001). NT, Non-transduced.

[0022] Figure 13: CARtein maturation and expression after split intein-mediated protein splicing. (A) Illustration of the cytometry gating strategy, selecting single live cells. (B) Flow cytometry histogram overlays of SCM and spliced CARtein expressing unpermeabilized Jurkat-TPR cells. UTD, untransduced cells.Figure 14: Activation signalling assay of Scituzumab and 2EF CAR / CARtein cells co-cultured with MCF7 cells expressing Trop-2 surface proteins. Representative flow cytometry analysis of CD69 (A) or promotor reporters NFAT (B), NFKB (C) or AP-1 (D) in CARtein-TPR cells 24h after co-culture with either untransduced (UTD) or CARtein transduced k562 cells in a E:T ratio of 32:1, 8:1. and 2:1.

[0023] Figure 15: Flow cytometry comparison of NFAT, NFKB and AP-1 reporters and CD69 MFI for CARtein-TPR cells co-cultured with MCF7 cells in a E:T ratio of 32:0, 16:1, 8:0, 4:1 and 2:1. Synergy of Sacituzumab and 2EF is also evaluated at 2:1, 1:1 and 1:2 Sacituzumab:2EF ratios.

[0024] Figure 16: Activation kinetics of Sacituzumab or2EF CARtein Jurkat-TPR cell upon co-culture with MCF7 cells for 24h at 16:1 target: efector ratio. CD69, NFAT, NFKB and AP-1 activation kinetics for Sacituzumab conventional CAR cells, 2EF CARtein cells or Sacituzumab CAR cells upon co-culture with TROP2 positive MCF7 cells. Synergy of Sacituzumab and 2EF is also shown at 2:1, 1:1 and 1:2 Sacituzumab:2EF ratios.

[0025] Figure 17: CARtein maturation and expression after split intein-mediated protein splicing in YT cells. (A) Illustration of the cytometry gating strategy, selecting single live cells. (B) Flow cytometry histogram overlays of SCM and spliced CARtein expressing unpermeabilized YT cells. UTD, untransduced cells.

[0026] Figure 18: Cytotoxicity of MCF7 target cells by anti-TROP2 CARtein-YT cells compared with unstransduced (WT) or SCM YT cells. Anti-TROP2 CARtein-YT cells were co-cultured with MCF7 cells at effectortarget (E:T) ratios of 1:5 for 24 h. Specific cytotoxicity was measured using D-luciferin.

[0027] FIG. 19: (A) In vitro imaging of wt or anti-TROP2 CAR-engineered YT cells efficacy against MDA-MB-231 target cells expressing Renilla luciferase as a viability dye. (B) Decrease in viability of MDA-MB-231 target cells as revealed by luciferase activity when co-cultured with TROP2 -CAR YT cells.

[0028] FIG.20: expression of the CAR tested in Example 4. CMS corresponds to the first polypeptide (SEQ ID NO: 6, 7, or 8). The second polypeptide is anti-BCMA CARtein (SEQ ID NO: 13). FIG.21 : Cytotoxicity of YT expressing the corresponding CARteins show in the previous figure against either MM.1S or K562 cells expressing BCMA or wyld type K562 cells as controls. FIG. 22: Statistic of the previous figure.GENERAL DEFINITIONS

[0029] It must be noted that, as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Further, unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention. As used herein, the conjunctive term "and / or" between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by "and / or", a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term "and / or" as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term "and / or."

[0030] As used herein, words of approximation such as, without limitation, "about", "around”, “approximately” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as "about" may vary from the stated value by ±1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15%. Preferably the term “about” means exactly the indicated value (± 0%).

[0031] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or "including" or sometimes when used herein with the term "having". Any of the aforementioned terms (comprising, containing, including, having), whenever used herein in the context of an aspect orembodiment of the present invention may be substituted with the term "consisting of", though less preferred.

[0032] When used herein "consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0033] As used herein the term “modular polypeptide” refers to any polypeptide comprised of subunits that when combined reconstitute a mature polypeptide. Non-limiting examples of such modular polypeptide include extracellular domain-containing, intracellular domain-containing, and transmembrane domain-containing polypeptides, as well as polypeptides comprising two or more of such domains e.g., CARs and CCRs.

[0034] The term “targeting moiety” refers to a substance or molecule or part thereof that is able to bind, preferably specifically bind, to a specific antigen.

[0035] The term “affibody” refers to a protein that is derived from the Z domain of protein A and that been engineered to bind to a specific target.

[0036] The term “antibody” refers to a molecule comprising at least one immunoglobulin domain that binds to, or is immunologically reactive with, a particular target. The term includes whole antibodies and any antigen binding portion or single chains thereof and combinations thereof; for instance, the term “antibody” in particular includes bivalent antibodies and bivalent bispecific antibodies. A typical type of antibody comprises at least two heavy chains (“HC”) and two light chains (“LC”) interconnected by disulfide bonds.

[0037] Each “heavy chain” comprises a “heavy chain variable domain” (abbreviated herein as “VH”) and a “heavy chain constant domain” (abbreviated herein as “CH”). The heavy chain constant domain typically comprises three constant domains, CH1, CH2, and CH3.

[0038] Each “light chain” comprises a “light chain variable domain” (abbreviated herein as “VL”) and a “light chain constant domain” (“CL”). The light chain constant domain (CL) can be of the kappa type or of the lambda type. The VH and VL domains can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (“FW’). The variable domains of the heavy and light chains contain a region that interacts with a binding target, and this region interacting with a binding target is also referred to as an “antigen-binding site” or “antigen binding site” herein. The constant domains of the antibodies can mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classicalcomplement system. Exemplary antibodies of the present disclosure include typical antibodies, but also bivalent fragments and variations thereof such as a F(ab’)2.

[0039] As used herein, the term “antibody” encompasses intact polyclonal antibodies, intact monoclonal antibodies, bivalent antibody fragments (such as F(ab’)2), multispecific antibodies such as bispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and any other modified immunoglobulin molecule comprising an antigen binding site.

[0040] An antibody can be of any five major classes (isotypes) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses thereof (e.g. lgG1, lgG2, lgG3, lgG4, lgA1 and lgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as therapeutic agents or diagnostic agents to form immunoconjugates.

[0041] The term “anticalin” refers to a protein that is derived from the lipocalin and that has been engineered to bind to a specific target.

[0042] The term “antigen-binding fragment” or “Fab” refers to an antibody fragment comprising one constant and one variable domain of each of the heavy and light chain. A Fab fragment may be obtained by digesting an intact monoclonal antibody with papain.

[0043] The term “designed ankyrin repeat proteins” or “DARPin” refers to a protein that is derived from an ankyrin repeat that has been engineered to bind to a specific target.

[0044] The term “fynomer” refers to a protein that is derived from the SH3 domain of human Fyn kinase that has been engineered to bind to a specific target. The term “monobody” refers to a protein that is derived from a fibronectin type II domain that has been engineered to bind to a specific target.

[0045] The term “nanobody” refers to a protein comprising the soluble single antigen-binding V-domain of a heavy chain antibody, preferably a camelid heavy chain antibody.

[0046] The term “single-chain antigen-binding fragment” or “scFab” refers to a fusion protein comprising one variable and one constant domain of the light chain of an antibody attached to one variable and one constant domain of the heavy chain of an antibody, wherein the heavy and light chains are linked together through a short peptide.

[0047] The term “single-chain variable fragment” or “scFv” refers to a fusion protein comprising the variable domains of the heavy chain and light chain of an antibody linked to one another with a peptide linker. The term also includes a disulfide stabilized Fv (dsFv).The term “peptide aptamer” refers to a short, 5-20 amino acid residue sequence that can bind to a specific target.

[0048] The term “repebody” refers to a protein that is derived from a leucine-rich repeat module and that has been engineered to bind to a specific target.

[0049] The term “chimeric antigen receptor” or “CAR” refers to a synthetic receptor that targets T-cells or other effector cells, such as for example NK cells, gamma delta T cells, or others, to a chosen antigen and reprograms T cell function, metabolism and persistence.

[0050] The term “costimulatory signaling domain” refers to a signaling moiety that provides to T cells a signal which, in addition to the primary signal provided by for instance the chain of the TCR / CD3 complex, mediates a T cell response, including, but not limited to, activation, proliferation, differentiation, cytokine secretion, and the like. A co-stimulatory domain can include all or a portion of, but is not limited to, CD27, CD28, 4-1 BB (CD137), 0X40 (CD134), CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83. In some embodiments, the costimulatory signaling domain is an intracellular signaling domain that interacts with other intracellular mediators to mediate a cell response including activation, proliferation, differentiation and cytokine secretion, and the like.

[0051] “Intracellular signaling domain” as used herein refers to all or a portion of one or more domains of a molecule (here the chimeric receptor molecule) that provides for activation of a lymphocyte. Intracellular domains of such molecules mediate a signal by interacting with cellular mediators to result in proliferation, differentiation, activation and other effector functions. Examples of intracellular signaling domains for use in a CAR of the invention include the intracellular sequences of the CD3 chain, and / or co-receptors that act in concert to initiate signal transduction following CAR engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability. T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation and provide a T cell receptor like signal (primary cytoplasmic signaling sequences) and those that act in an antigen- independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as receptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences include those derived from CD3 , FcRy, CD3y, CD35, CD3E, CD5, CD22, CD79a, CD79b, and CD66d.It is herein noted by “oligonucleotide,” as referred herein is meant any short segment of DNA, RNA, or DNA / RNA, including both natural and synthetic nucleotides. As used in this invention, the term "oligonucleotide " includes both oligonucleotides as such, as well as the "oligonucleotide analogues". "Oligonucleotide analogues" are the molecules derived therefrom that incorporate some chemical modification in at least one of the nucleotide units that form them, either in the phosphate group, the pentose or one of the nitrogenous bases. The term “cancer” refers to a group of diseases, which can be defined as any abnormal benign or malignant new growth of tissue that possesses no physiological function and arises from uncontrolled usually rapid cellular proliferation and has the potential to invade or spread to other parts of the body.

[0052] The term "epitope" refers to an antigenic determinant in a molecule, i.e., to the part in a molecule that is recognized by the immune system, for example, that is recognized by a targeting moiety, such as an antibody. An epitope of a protein preferably comprises a continuous or discontinuous portion of said protein and is preferably between 5 and 100, preferably between 5 and 50, more preferably between 8 and 30, most preferably between 10 and 25 amino acids in length, for example, the epitope may be preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.

[0053] “Combination therapy”, “in combination with” or “in conjunction with” as used herein denotes any form of concurrent, parallel, simultaneous, sequential or intermittent treatment with at least two distinct treatment modalities (i.e., compounds, components, targeted agents or therapeutic agents). As such, the terms refer to administration of one treatment modality before, during, or after administration of the other treatment modality to the subject. The modalities in combination can be administered in any order. The therapeutically active modalities are administered together (e.g., simultaneously in the same or separate compositions, formulations or unit dosage forms) or separately (e.g., on the same day or on different days and in any order as according to an appropriate dosing protocol for the separate compositions, formulations or unit dosage forms) in a manner and dosing regimen prescribed by a medical caretaker or according to a regulatory agency. In general, each treatment modality will be administered at a dose and / or on a time schedule determined for that treatment modality. Optionally, three or more modalities may be used in a combination therapy. Additionally, the combination therapies provided herein may be used in conjunction with other types of treatment. For example, other anti-cancer treatment may be selected from the group consisting of chemotherapy, surgery, radiotherapy (radiation) and / or hormone therapy, amongst other treatments associated with the current standard of care for the subject.“Administering” or “administration of” a medicament to a patient (and grammatical equivalents of this phrase) refers to direct administration, which may be administration to a patient by a medical professional or may be self-administration, and / or indirect administration, which may be the act of prescribing a drug. E.g., a physician who instructs a patient to self-administer a medicament or provides a patient with a prescription for a drug is administering the drug to the patient.

[0054] As used herein, the term “effective amount” of an agent, e.g., a therapeutic agent such as a CAR-T, is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. For example, in the context of administering a therapeutic agent that treats T-ALL, an effective amount can reduce the number of cancer cells; reduce the tumor size or burden; inhibit (i.e. , slow to some extent and in a certain embodiment, stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and in a certain embodiment, stop) tumor metastasis; inhibit, to some extent, tumor growth; relieve to some extent one or more of the symptoms associated with the cancer; and / or result in a favorable response such as increased progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), or, in some cases, stable disease (SD), a decrease in progressive disease (PD), a reduced time to progression (TTP) or any combination thereof. The term “effective amount” can be used interchangeably with “effective dose,” “therapeutically effective amount,” or “therapeutically effective dose”.

[0055] The terms “individual”, “patient” or “subject” are used interchangeably in the present application to designate a human being and are not meant to be limiting in any way. The “individual”, “patient” or “subject” can be of any age, sex and physical condition. The term “patient in need thereof” usually refers to a patient who suffers from specific antigen-positive cancer.

[0056] As used herein, "pharmaceutically acceptable carrier" or “pharmaceutically acceptable diluent” means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and, without limiting the scope of the present invention, include: additional buffering agents; preservatives; co-solvents; antioxidants, including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers, such as polyesters; salt-forming counterions, such as sodium, polyhydric sugar alcohols; amino acids, such as alanine, glycine, glutamine,asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactitol, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur containing reducing agents, such as glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [alpha]-monothioglycerol, and sodium thiosulfate; low molecular weight proteins, such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; and hydrophilic polymers, such as polyvinylpyrrolidone. Other pharmaceutically acceptable carriers, excipients, or stabilizers may also be included in a pharmaceutical composition described herein, provided that they do not adversely affect the desired characteristics of the pharmaceutical composition.

[0057] The term “sequence identity” refers to a percentage value obtained when two sequences are compared using a pairwise sequence alignment tool. Sequence identity may be calculated based on local or global aligments. In the present case, the sequence identity is preferably obtained using a local alignment tool, preferably a Basic Local Alignment Search Tool (BLAST) tool.

[0058] The terms “treatment” and “therapy”, as used in the present application, refer to a set of hygienic, pharmacological, surgical and / or physical means used with the intent to cure and / or alleviate a disease and / or symptoms with the goal of remediating the health problem. The terms “treatment” and “therapy” include preventive and curative methods, since both are directed to the maintenance and / or reestablishment of the health of an individual or animal. Regardless of the origin of the symptoms, disease and disability, the administration of a suitable medicament to alleviate and / or cure a health problem should be interpreted as a form of treatment or therapy within the context of this application.

[0059] DESCRIPTION OF THE EMBODIMENTS

[0060] The present application is directed to split intein-based CAR precursors for the generation of modular CARs.

[0061] CARs of the invention

[0062] In a first aspect, the present invention provides a chimeric antigen receptor (CAR) precursor, also named “CAR precursor of the invention”, comprising at least two polypeptides. In the context of the present invention, a “CAR precursor” refers to a system of two or more polypeptides that have the ability to undergo further modifications or reactions to become a single fully functional CAR protein. Preferably, the CAR precursor is provided as a system of two separate, i.e., independent, polypeptides, called herein the “first polypeptide of theinvention” and the “second polypeptide of the invention”, respectively. Each of them will be explained in detail below. The modular CAR system is also called herein “CARtein".

[0063] The CAR precursor of the invention comprises at least a pair of split inteins in its sequence. As used herein, the term “intein” refers to any of a variety of auto-catalytic polypeptide sequences capable of self-excision from a larger polypeptide sequence via polypeptide splicing. As used herein, “polypeptide splicing” refers to the auto-catalytic process by which an intein self-excises from a larger polypeptide and which results in the ligation of the intein-flanking sequences (“exteins”) via a new peptide bond. Inteins exist as bifunctional sequences, where the endonuclease domain is present within the splicing domain allowing for cis polypeptide splicing, as well as “split inteins” which are present in separate polypeptide sequences that can self-associate non-covalently to catalyze trans polypeptide splicing. As used herein, an “orthogonal system” is a network of components (e.g., polypeptides, RNAs, DNAs, or small molecules) that interact with each other to achieve a specific function without impeding or being impeded by the native functions of the host cell. Accordingly, the “orthogonal split-intein” will interact with each other to facilitate trans polypeptide splicing of the exteins to which they are fused without impeding or being impeded by the native functions of the host cell. Orthogonality of inteins derives from specificity of split N terminal and C terminal polypeptide sequences that exhibit high binding affinity and high specificity. This orthogonal intein strategy allows construction of complex, mature, trans-spliced proteins from a set of functional domain-containing proteins. Thus, orthogonal split inteins mediate transsplicing reactions. In the context of the present invention, a trans-splicing reaction involves the first polypeptide of the invention comprising a split intein fused at the C terminus of the N-extein, preceded or not by a protein domain of interest (Pol) and the second polypeptide of the invention comprising the compatible split intein fused at the N terminus of the C-extein, followed or not by a Pol. Then, upon association of the split inteins, trans-splicing of first and second polypeptides of the invention (exteins) is catalyzed, resulting in covalent linkage of the first and second polypeptides via a peptide bond and release of the non- covalently associated inteins as well as any sequence downstream of the N-extein and upstream of the C-extein.

[0064] “Compatible split inteins” are also referred herein to as “orthogonal split inteins” or “complementary split inteins”, and refer to a pair of C terminal and N terminal split inteins and exteins that can mediate a trans-splicing reaction of the two polypeptides of the invention. The use of split inteins enables the generation of functional, mature, trans-spliced polypeptides from a given set of available precursor polypeptides. The trans-spliced polypeptide resulting from the reaction of the split inteins in the CAR precursor of the invention is called herein functional CAR of the invention.By “functional CAR of the invention” is referred herein to as a CAR that has the ability to recognize a target antigen, such as a tumor associated antigen, and activate the immune cell carrying said CAR upon antigen binding. The functional CAR of the invention is generated after the intein splicing reaction carried out by the CAR precursor of the invention, and is a mature intein-mediated modular CAR, and will therefore comprise in its sequence the residual exteins sequences resulted after the splicing. The functional CAR of the invention is also called herein “mature intein-mediated modular CAR” or simply “mature CAR”.

[0065] Therefore, the precursor CAR of the invention comprises in its sequence compatible split inteins that trigger a trans-splicing reaction that give rise to a mature intein-mediated modular CAR.

[0066] As explained above, the CAR precursor of the invention comprises at least two polypeptides, namely the first and the second polypeptide of the invention. In order to produce the mature intein-mediated modular CAR, each polypeptide should comprise at least one split intein and extein, which are orthologs or complementary among them. Further, in order to produce a mature intein-mediated modular CAR, the CAR precursor needs to comprise at least one extracellular domain comprising at least one targeting moiety, a transmembrane domain, an intracellular domain and, optionally, one or several costimulatory domains. In an embodiment, at least one of the polypeptides of the CAR precursor comprises a retention motif, preferably an endoplasmic reticulum (ER) retention motif, so that said polypeptide comprising said retention motif is captured in a place of the cell, until the trans-splicing reaction is triggered. In an embodiment, the CAR precursor of the invention comprises at least two polypeptides of the invention, wherein:

[0067] a. the first polypeptide of the invention comprises in the N to C terminal direction:

[0068] i) a C terminal split intein and extein

[0069] ii) a transmembrane domain, and

[0070] iii) an intracellular signaling domain,

[0071] b. the second polypeptide of the invention comprises in the N to C terminal direction:

[0072] i) an extracellular domain comprising at least one targeting moiety, ii) a N terminal split extein and intein, and

[0073] iii) optionally, an endoplasmic reticulum (ER) retention motif,

[0074] wherein the C terminal split intein and extein of a.i) and the N terminal split extein and intein of b.ii) are complementary or orthogonal split inteins so that they can carry out atrans-splicing reaction between the first and the second polypeptides of the invention, thereby resulting into the mature intein-mediated modular CAR of the invention.

[0075] In other words, the first and the second polypeptides must comprise a pair of split inteins that are capable of carrying out a trans-splicing reaction between both peptides, thereby resulting in the functional CAR of the invention.

[0076] Each of the elements comprised in the first and second polypeptides of the invention are detailed below:

[0077] The first polypeptide of the invention

[0078] In an embodiment, the first polypeptide of the invention comprises at least the following domains, preferably in the N to C terminal direction:

[0079] i) a C terminal split intein and extein

[0080] ii) a transmembrane domain,

[0081] iii) an intracellular signaling domain, and

[0082] iv) optionally, a costimulatory domain.

[0083] In an embodiment, the C terminal split intein and extein of i) comprises, consists, or consists essentially of SEQ ID NO: 1, or a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1. Optionally, the C terminal split intein and extein of i) further comprises other split intein and extein, such as the one comprising or consisting of SEQ ID NO: 3, or a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3.

[0084] In an embodiment, the transmembrane domain of ii) may be derived either from a natural or a synthetic source. When the source is natural, the domain may be derived from any membranebound or transmembrane protein. Transmembrane regions may comprise at least the transmembrane region(s) of the a-, -

[0085]

[0086] chain of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 or ICOS.

[0087] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS or a variant thereof having 95%, preferably 98%, sequence identity. In some embodiments, the transmembrane domain comprises the transmembrane domain of CD8 or a variant thereof, wherein the variant thereof has a 95% sequence identity.In some embodiments, the transmembrane domain comprises the transmembrane domain of CD28 or a variant thereof. Preferably, the transmembrane domain consists or consists essentially of SEQ ID NO: 44, or a sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 44.

[0088] In an embodiment, the transmembrane domain is linked to domain i) by a spacer or linker, wherein the spacer or linker is placed at the N-terminal of the transmembrane domain.

[0089] Thus, in an embodiment, a spacer or a linker is placed between i) and ii) domains of the first polypeptide. By “spacer” is referred herein to a sequence, usually a domain, from another protein. Exemplary spacers are immunoglobulin-derived hinges, or CAR-derived hinges, such as lgGiHinge-lgGiCH2-lgGiCH3, lgG4Hinge, lgG4Hinge-lgG4CH2-lgG4CH3, CD8a, or any combination thereof. In an embodiment, the spacer comprises or consists of SEQ ID NO: 59, 60, 61 or 62, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 59, 60, 61 or 62, respectively.

[0090] By “linker” is referred herein to a sequence of between 5-30, preferably 10-25, amino acid residues. The linker may be predominantly G or S residues, or it may be random residues placed to separate two domains and to provide flexibility to the polypeptide. In an embodiment, the linker comprises or consists of SEQ ID NO: 42, 66 or 43, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 42, 66 or 43 respectively.

[0091] The intracellular signaling domain of iii) provides for the activation of at least one function of the cell expressing the CAR upon binding to the ligand expressed on a cell, preferably tumor cells. In some embodiments, the intracellular signaling domain of iii) contains one or more intracellular signaling domains. In some embodiments, the intracellular signaling domain is a portion of and / or a variant of an intracellular signaling domain that provides for activation of at least one function of the CAR-comprising cell.

[0092] In some embodiments, the intracellular signaling domain of iii) comprises the intracellular domain of CD3 , FcRy, CD3y, CD35, CD3E, CD5, CD22, CD79a, CD79b, CD66b, ora variant thereof, wherein the variant thereof has a 95%, preferably 98%, sequence identity. In some embodiments, the intracellular signaling domain comprises the intracellular domain of CD3 FcRy, CD3y, CD35, CD3E, CD5, CD22, CD79a, CD79b or CD66b.

[0093] In some embodiments, the intracellular signaling domain comprises the intracellular domain of CD3 or a variant thereof. In a preferred embodiment, the intracellular signaling domaincomprises, consists or consists essentially of SEQ ID NO: 45, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 45.

[0094] In an embodiment, the intracellular signal domain is linked to domain ii) by a hinge or linker, wherein the hinge or linker is placed at the N-terminal of the intracellular signal domain. Optionally, at least a costimulatory signaling domain iv) may also be present in the first polypeptide. In some embodiments, the costimulatory signaling domain of iv) comprises the intracellular domain of CD27, CD28, CD137, CD134, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, CD276, ICOS, LAT, Lek, orZAP70, a variant thereof, wherein the variant thereof has a 95%, preferably 98%, sequence identity. In some embodiments, the costimulatory signaling domain of iv) comprises the intracellular domain of CD27, CD28, CD137 (4-1 BB), CD134, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, CD276, ICOS or a variant thereof. Preferably, the costimulatory domain is CD28.

[0095] In an embodiment, the intracellular signal domain is linked to domain iii) by a hinge or linker, wherein the hinge or linker is placed at the N-terminal of the costimulatory signaling domain. As it will be readily apparent to the skilled person, each of the domains described herein (domain i), ii), iii), iv), including leader peptide and linkers) comprised in the first polypeptide can be further separated by further linker sequences, such as by random amino acid residues that provide some space and flexibility between domains. Similarly, tags and other sequences can also be added to facilitate purification or downstream processes. In an embodiment, a tag is added, preferably a His tag that comprises or consists of SEQ ID NO: 41. Preferably, a tag is added in the N-terminal of the first polypeptide, and a different tag is added in the N-terminal of the second polypeptide.

[0096] Additionally, a leader or signal peptide can be placed in the N terminal of the first polypeptide, upstream the i) C terminal split intein and extein domain. Preferably, the leader peptide is the endogenous or natural leader peptide of CD8a, hlgkVHI, or IgK. Preferably, the leader peptide comprises or consists of SEQ ID NO: 63, 64, or 65, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 63, 64, or 65, respectively.

[0097] Table 1 below shows preferred embodiments and combination of embodiments of the different components of the first polypeptide of the invention:Table 1 :

[0098]

[0099]

[0100] In an embodiment, the first polypeptide comprises, in the N to C terminal direction:

[0101] i) a signal peptide, a C terminal split intein and extein comprising or consisting of SEQ ID NO: 1 , and a spacer sequence or a linker sequence,

[0102] ii) a transmembrane domain comprising or consisting of CD28, and a linker sequence,

[0103] iii) an intracellular signaling domain comprising or consisting of CD3^.

[0104] In an embodiment, the first polypeptide is the “construct A” in Table 1, that comprises, in the N to C terminal direction:

[0105] i) a C terminal split intein and extein comprising or consisting of SEQ ID NO: 1 , followed by a spacer comprising or consisting of SEQ ID NO: 59,

[0106] ii) a transmembrane domain comprising or consisting of SEQ ID NO: 44, and iii) an intracellular signaling domain comprising or consisting of SEQ ID NO: 45, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, 59, 44, or 45, respectively. Preferably, the first polypeptide comprises or consists of SEQ ID NO: 5, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5.In an embodiment, the first polypeptide is the “construct B” in Table 1, that comprises, in the N to C terminal direction:

[0107] i) a C terminal split intein and extein comprising or consisting of SEQ ID NO: 1 , a further split intein comprising or consisting of SEQ ID NO: 3, and a spacer comprising or consisting of SEQ ID NO: 60,

[0108] ii) a transmembrane domain comprising or consisting of SEQ ID NO: 44, and iii) an intracellular signaling domain comprising or consisting of SEQ ID NO: 45, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, 3, 60, 44, or 45, respectively.

[0109] Preferably, the first polypeptide comprises or consists of SEQ ID NO: 6, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6.

[0110] In an embodiment, the first polypeptide is the “construct C” in Table 1, that comprises, in the N to C terminal direction:

[0111] i) a C terminal split intein and extein comprising or consisting of SEQ ID NO: 1 , a further split intein comprising or consisting of SEQ ID NO: 3, and a spacer comprising or consisting of SEQ ID NO: 61,

[0112] ii) a transmembrane domain comprising or consisting of SEQ ID NO: 44, and iii) an intracellular signaling domain comprising or consisting of SEQ ID NO: 45, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, 3, 61, 44, or 45, respectively.

[0113] Preferably, the first polypeptide comprises or consists of SEQ ID NO: 7, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7.

[0114] In an embodiment, the first polypeptide is the “construct D” in Table 1, that comprises, in the N to C terminal direction:

[0115] i) a C terminal split intein and extein comprising or consisting of SEQ ID NO: 1 , a further split intein comprising or consisting of SEQ ID NO: 3, and a spacer comprising or consisting of SEQ ID NO: 62,ii) a transmembrane domain comprising or consisting of SEQ ID NO: 44, and iii) an intracellular signaling domain comprising or consisting of SEQ ID NO: 45, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, 3, 62, 44, or 45, respectively.

[0116] Preferably, the first polypeptide comprises or consists of SEQ ID NO: 8, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 8.

[0117] In an embodiment, the first polypeptide is the “construct E” in Table 1, that comprises, in the N to C terminal direction:

[0118] i) a C terminal split intein and extein comprising or consisting of SEQ ID NO: 1 , a further split intein comprising or consisting of SEQ ID NO: 3, and a linker of about 11 amino acids,

[0119] ii) a transmembrane domain comprising or consisting of SEQ ID NO: 44, and iii) an intracellular signaling domain comprising or consisting of SEQ ID NO: 45, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, 3, 44, or 45, respectively. Preferably, the first polypeptide comprises or consists of SEQ ID NO: 9, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 9.

[0120] In an embodiment, the first polypeptide is the “construct F” in Table 1, that comprises, in the N to C terminal direction:

[0121] i) a C terminal split intein and extein comprising or consisting of SEQ ID NO: 1 , and a linker,

[0122] ii) a transmembrane domain comprising or consisting of SEQ ID NO: 44, and iii) an intracellular signaling domain comprising or consisting of SEQ ID NO: 45, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, 44, or 45, respectively.Preferably, the first polypeptide comprises or consists of SEQ ID NO: 4, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4.

[0123] Preferably, constructs B, C, D, E further comprise a leader peptide in N terminal of domain i), comprising or consisting of SEQ ID NO: 63. Preferably, constructs A and F further comprise a leader peptide in N terminal of domain i), comprising or consisting of SEQ ID NO: 64.

[0124] The second polypeptide of the invention

[0125] In an embodiment, the second polypeptide of the invention comprises at least the following domains, preferably in the N to C terminal direction:

[0126] i) an extracellular domain comprising at least one targeting moiety,

[0127] ii) a N terminal split extein and intein, and

[0128] iii) optionally, a retention motif, preferably an endoplasmic reticulum (ER) retention motif.

[0129] In an embodiment, the extracellular domain of i) comprises at least one, preferably one or two, targeting moiety. Said at least one targeting moiety is preferably an antibody, anticalin, repebody, monobody, scFv, Fab, scFab, affibody, fynomer, DARPin, nanobody, ligand or peptide aptamer that is capable of binding to an antigen, preferably a tumor associated antigen. By “capable of binding to an antigen” is meant that the targeting moiety has binding affinity for an antigen. Preferably, the binding affinity is specific, i.e., the targeting moiety is capable of specifically binding to said antigen (which is its target molecule) in a manner which distinguishes from the binding to non-target molecules, i.e., from other molecules that are not said antigen. Thus, the targeting moiety either does not bind to non-target molecules or exhibits negligible or substantially-reduced (as compared to the target) e. g. background, binding to nontarget molecules. The targeting moiety specifically recognizes the antigen. Specific binding between the targeting moiety and the antigen can be assessed by numerous techniques, such as surface plasmon resonance, flow cytometry, or ELISA assays. A “Ligand” is considered herein any protein that is capable of binding, preferably specifically binding, to an antigen. As an example, a ligand may be a cellular protein, such as a cellular receptor, that is capable of binding, preferably specifically binding, to a virus antigen.

[0130] Preferably, the antigen is BCMA, CD19, CD20, SLAMF7, and / orTROP2 proteins.

[0131] CD19 targeting moiety.

[0132] In an embodiment, the targeting moiety is capable of binding to CD19 antigen and thus is a CD19 targeting moiety.Preferably, the CD19 targeting moiety is an antibody, F(ab’)2, Fab, preferably scFv, or scFab comprising a VL domain and a VH domain, wherein the VL domain comprises, consists, or consists essentially of SEQ ID NO: 46, or a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 46; and wherein the VH domain comprises, consists, or consists essentially of SEQ ID NO: 47, or a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 47. In some embodiments, the CD19 targeting moiety is an antibody, preferably scFv, Fab, or scFab comprising a VL domain and VH domain, wherein the VL domain comprises or consists of SEQ ID NO: 46 and the VH domain comprises or consists of SEQ ID NO: 47.

[0133] CD20 targeting moiety.

[0134] In an embodiment, the targeting moiety is capable of binding to CD20 antigen and thus is a CD20 targeting moiety.

[0135] Preferably, the CD20 targeting moiety is an antibody, F(ab’)2, Fab, preferably scFv, or scFab comprising a VL domain and a VH domain, wherein the VL domain comprises, consists, or consists essentially of SEQ ID NO: 48, or a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 48; and wherein the VH domain comprises, consists, or consists essentially of SEQ ID NO: 49, or a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 49. In some embodiments, the CD20 targeting moiety is an antibody, preferably scFv, Fab, or scFab comprising a VL domain and VH domain, wherein the VL domain comprises or consists of SEQ ID NO: 48 and the VH domain comprises or consists of SEQ ID NO: 49.

[0136] CD20+CD19 targeting moiety.

[0137] In an embodiment, the extracellular domain of i) comprises two targeting moieties, one that is capable of binding to CD20 antigen and another that is capable of binding to CD19 antigen, and thus is a CD20+CD19 targeting moiety.

[0138] Preferably, the CD20+CD19 targeting moiety comprises two antibody, F(ab’)2, Fab, preferably scFv, or scFab, comprising:

[0139] a VL domain and a VH domain, wherein the VL domain comprises, consists, or consists essentially of SEQ ID NO: 48, or a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 48; and wherein the VH domain comprises, consists, or consists essentially of SEQ ID NO: 49, or a sequence with at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 49, and

[0140] a VL domain and a VH domain, wherein the VL domain comprises, consists, or consists essentially of SEQ ID NO: 46, or a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 46; and wherein the VH domain comprises, consists, or consists essentially of SEQ ID NO: 47, or a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 47.

[0141] Preferably, the CD20+CD19 targeting moiety comprises two antibody, F(ab’)2, Fab, preferably scFv, or scFab, comprising:

[0142] a VL domain and a VH domain, wherein the VL domain comprises, consists, or consists essentially of SEQ ID NO: 48, and wherein the VH domain comprises, consists, or consists essentially of SEQ ID NO: 49, and

[0143] a VL domain and a VH domain, wherein the VL domain comprises, consists, or consists essentially of SEQ ID NO: 46 and wherein the VH domain comprises, consists, or consists essentially of SEQ ID NO: 47.

[0144] Preferably, the CD20+CD19 targeting moiety comprises or consists of SEQ ID NO: 50, or a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 50.

[0145] BCMA targeting moiety.

[0146] In an embodiment, the targeting moiety is capable of binding to BCMA antigen and thus is a BCMA targeting moiety.

[0147] Preferably, the BCMA targeting moiety is an antibody, F(ab’)2, Fab, preferably scFv, or scFab comprising a VL domain and a VH domain, wherein the VL domain comprises, consists, or consists essentially of SEQ ID NO: 51, or a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 51; and wherein the VH domain comprises, consists, or consists essentially of SEQ ID NO: 52, or a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 52. In some embodiments, the BCMA targeting moiety is an antibody, preferably scFv, Fab, or scFab comprising a VL domain and VH domain, wherein the VL domain comprises or consists of SEQ ID NO: 51 and the VH domain comprises or consists of SEQ ID NO: 52.SLAMF7 targeting moiety.

[0148] In an embodiment, the targeting moiety is capable of binding to SLAMF7 antigen and thus is a SLAMF7 targeting moiety.

[0149] Preferably, the SLAMF7 targeting moiety is an antibody, F(ab’)2, Fab, preferably scFv, or scFab comprising a VL domain and a VH domain, wherein the VL domain comprises, consists, or consists essentially of SEQ ID NO: 53, or a seguence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% seguence identity to SEQ ID NO: 53; and wherein the VH domain comprises, consists, or consists essentially of SEQ ID NO: 54, or a seguence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% seguence identity to SEQ ID NO: 54. In some embodiments, the SLAMF7 targeting moiety is an antibody, preferably scFv, Fab, or scFab comprising a VL domain and VH domain, wherein the VL domain comprises or consists of SEQ ID NO: 53 and the VH domain comprises or consists of SEQ ID NO: 54.

[0150] TROP2 targeting moiety.

[0151] In an embodiment, the targeting moiety is capable of binding to TROP2 antigen and thus is a TROP2 targeting moiety. Two TROP2 targeting moieties are described herein as preferred TROP2 targeting moieties. For sake of clarity, we named them TROP2.1 and TROP2.2. Preferably, the TROP2 targeting moiety is an antibody, F(ab’)2, Fab, preferably scFv, or scFab comprising a VL domain and a VH domain, wherein the VL domain comprises, consists, or consists essentially of SEQ ID NO: 55, or a seguence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% seguence identity to SEQ ID NO: 55; and wherein the VH domain comprises, consists, or consists essentially of SEQ ID NO: 56, or a seguence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% seguence identity to SEQ ID NO: 56. In some embodiments, the TROP2 targeting moiety is an antibody, preferably scFv, Fab, or scFab comprising a VL domain and VH domain, wherein the VL domain comprises or consists of SEQ ID NO: 55 and the VH domain comprises or consists of SEQ ID NO: 56.

[0152] Preferably, the TROP2 targeting moiety is an antibody, F(ab’)2, Fab, preferably scFv, or scFab comprising a VL domain and a VH domain, wherein the VL domain comprises, consists, or consists essentially of SEQ ID NO: 57, or a seguence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% seguence identity to SEQ ID NO: 57; and wherein the VH domain comprises, consists, or consists essentially of SEQ ID NO: 58, or a seguence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% seguence identity to SEQ ID NO: 58. In some embodiments, the TROP2 targeting moiety is an antibody, preferably scFv, Fab, or scFabcomprising a VL domain and VH domain, wherein the VL domain comprises or consists of SEQ ID NO: 57 and the VH domain comprises or consists of SEQ ID NO: 58.

[0153] In an embodiment, the N terminal split extein and intein of domain ii) is the ortholog intein (i.e., the compatible intein) of the C terminal split intein and extein of the first polypeptide, in domain i), defined above. Preferably, the N terminal split extein and intein of domain ii) comprises, consists, or consists essentially of SEQ ID NO: 2 (N 1 ), or a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2.

[0154] In an embodiment, a retention motif iii) is preferably placed in the second polypeptide so that it is captured at a place of interest in the cell. Preferably, the retention motif if an endoplasmic reticulum (ER) retention motif. Preferably, the ER retention motif of iii) comprises, consists, or consists essentially of SEQ ID NO: 40, or a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 40. It is noted that if the second polypeptide is provided to the subject in need thereof during the length of the therapy, the ER retention motif is not essential since the second polypeptide will be available to react with the first polypeptide without the need of having it kidnapped in the ER of the cell.

[0155] As it will be readily apparent to the skilled person, each of the domains described herein (domain i), ii), iii), including leader peptide and linkers) comprised in the second polypeptide can be further separated by further linker sequences, such as by random amino acid residues that provide some space and flexibility between domains. Preferably, the linker sequence comprises or consists of SEQ ID NO: 42, 43 or 66, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 42, 43 or 66 respectively.

[0156] Additionally, a leader or signal peptide can be placed in the N terminal of the second polypeptide, upstream the extracellular domain i). Preferably, the leader peptide is the endogenous or natural leader peptide of CD8a, hlgkVHI, or IgK. Preferably, the leader peptide comprises or consists of SEQ ID NO: 63, 64, or 65, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 63, 64, or 65, respectively.

[0157] Preferably a spacer selected from CD8a, I gGi or lgG4 Fc region is placed in between domains i) and ii) of the second polypeptide.

[0158] Table 2 below shows preferred embodiments and combination of embodiments of the different components of the second polypeptide of the invention:Table 2:

[0159]

[0160]

[0161]

[0162] In an embodiment, the second polypeptide comprises, in the N to C terminal direction:

[0163] i) an extracellular domain comprising a targeting moiety, wherein the targeting moiety is preferably selected from a BCMA, SLAMF7, CD19, CD20, CD20+CD19, TROP2 .1 and TROP2 .2 targeting moieties,

[0164] ii) a N terminal split extein and intein comprising or consisting of SEQ ID NO: 2, and

[0165] iii) an endoplasmic reticulum (ER) retention motif, preferably comprising or consisting of SEQ ID NO: 40.

[0166] Preferably, the constructs defined in table 2 comprise a spacer sequence, preferably selected from the list consisting of CD8a, IgG 1 or lgG4 Fc region, between domains i) and ii).

[0167] In an embodiment, the second polypeptide is the “construct 1” in Table 2, that comprises, in the N to C terminal direction:

[0168] i) an extracellular domain comprising a BCMA targeting moiety comprising a VL and a VH, wherein the VL comprises or consists of SEQ ID NO: 51, and the VH comprises or consists of SEQ ID NO: 52,

[0169] ii) a N terminal split extein and intein comprising or consisting of SEQ ID NO: 2, and

[0170] iii) an endoplasmic reticulum (ER) retention motif preferably comprising or consisting of SEQ ID NO: 40,

[0171] or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 51, 52, 2, or 40, respectively. Preferably, the first polypeptide comprises or consists of SEQ ID NO: 13, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 13.

[0172] In an embodiment, the second polypeptide is the “construct 2” in Table 2, that comprises, in the N to C terminal direction:

[0173] i) an extracellular domain comprising a SLAMF7 targeting moiety comprising a VL and a VH, wherein the VL comprises or consists of SEQ ID NO: 53, and the VH comprises or consists of SEQ ID NO: 54,

[0174] ii) a N terminal split extein and intein comprising or consisting of SEQ ID NO: 2, andiii) an endoplasmic reticulum (ER) retention motif preferably comprising or consisting of SEQ ID NO: 40,

[0175] or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 53, 54, 2, or 40, respectively. Preferably, the first polypeptide comprises or consists of SEQ ID NO: 14, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 14.

[0176] In an embodiment, the second polypeptide is the “construct 3” in Table 2, that comprises, in the N to C terminal direction:

[0177] i) an extracellular domain comprising a TROP2 .1 targeting moiety comprising a VL and a VH, wherein the VL comprises or consists of SEQ ID NO: 55, and the VH comprises or consists of SEQ ID NO: 56,

[0178] ii) a N terminal split extein and intein comprising or consisting of SEQ ID NO: 2, and

[0179] iii) an endoplasmic reticulum (ER) retention motif preferably comprising or consisting of SEQ ID NO: 40,

[0180] or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 55, 56, 2, or 40, respectively. Preferably, the first polypeptide comprises or consists of SEQ ID NO: 15, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 15.

[0181] In an embodiment, the second polypeptide is the “construct 4” in Table 2, that comprises, in the N to C terminal direction:

[0182] i) an extracellular domain comprising a TROP2 .2 targeting moiety comprising a VL and a VH, wherein the VL comprises or consists of SEQ ID NO: 57, and the VH comprises or consists of SEQ ID NO: 58,

[0183] ii) a N terminal split extein and intein comprising or consisting of SEQ ID NO: 2, and

[0184] iii) an endoplasmic reticulum (ER) retention motif preferably comprising or consisting of SEQ ID NO: 40,or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 57, 58, 2, or 40, respectively. Preferably, the first polypeptide comprises or consists of SEQ ID NO: 16, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 16.

[0185] In an embodiment, the second polypeptide is the “construct 5” in Table 2, that comprises, in the N to C terminal direction:

[0186] i) an extracellular domain comprising a CD19 targeting moiety comprising a VL and a VH, wherein the VL comprises or consists of SEQ ID NO: 46, and the VH comprises or consists of SEQ ID NO: 47,

[0187] ii) a N terminal split extein and intein comprising or consisting of SEQ ID NO: 2, and

[0188] iii) an endoplasmic reticulum (ER) retention motif preferably comprising or consisting of SEQ ID NO: 40,

[0189] or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 46, 47, 2, or 40, respectively. Preferably, the first polypeptide comprises or consists of SEQ ID NO: 10, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 10.

[0190] In an embodiment, the second polypeptide is the “construct 6” in Table 2, that comprises, in the N to C terminal direction:

[0191] i) an extracellular domain comprising a CD20 targeting moiety comprising a VL and a VH, wherein the VL comprises or consists of SEQ ID NO: 48, and the VH comprises or consists of SEQ ID NO: 49,

[0192] ii) a N terminal split extein and intein comprising or consisting of SEQ ID NO: 2, and

[0193] iii) an endoplasmic reticulum (ER) retention motif preferably comprising or consisting of SEQ ID NO: 40,

[0194] or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 48, 49, 2, or 40, respectively.Preferably, the first polypeptide comprises or consists of SEQ ID NO: 11 , or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 11.

[0195] In an embodiment, the second polypeptide is the “construct 7” in Table 2, that comprises, in the N to C terminal direction:

[0196] i) an extracellular domain comprising a CD20+CD19 targeting moieties comprising or consisting of SEQ ID NO: 50,

[0197] ii) a N terminal split extein and intein comprising or consisting of SEQ ID NO: 2, and

[0198] iii) an endoplasmic reticulum (ER) retention motif preferably comprising or consisting of SEQ ID NO: 40,

[0199] or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 50, 2, or 40, respectively. Preferably, the first polypeptide comprises or consists of SEQ ID NO: 12, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 12.

[0200] Preferably, constructs 3 and 4 further comprise a leader peptide in N terminal of domain i), comprising or consisting of SEQ ID NO: 63. Preferably, constructs 1, 2, 5, 6, and 7 further comprise a leader peptide in N terminal of domain i), comprising or consisting of SEQ ID NO: 65.

[0201] Importantly, although preferred split-inteins are those defined by SEQ ID NOs 1, 2, or 3, it is readily apparent that they may be substituted by any split-intein known in the art, as long as they are orthologs or complementary. That is, the sequence per se of the split-inteins is not relevant and may be changed for another split-intein, but the C terminal split intein and extein of the first polypeptide of the invention needs to be complementary to the N terminal split extein and intein included of the second polypeptide of the invention, so that the trans-splicing reaction can occur in the CAR precursor of the invention, resulting in the functional CAR of the invention.

[0202] Similarly, the spacers, leader peptides and linker sequences can also be replaced by other spacers, leader peptides and linker sequences known in the art, with the proviso that their sequence does not impede the trans-splicing reaction to produce a functional CAR. Means to test that the resulting CAR is a functional CAR are known in the art and include cytotoxicity or killing assays to measure the cytotoxicity or killing capability of a cell carrying out said CAR.Preferred CAR precursors of the invention

[0203] The preferred CAR precursors of the invention are those resulting from the combination of the first and second polypeptides depicted in Tables 1 and 2, respectively.

[0204] Preferably, the CAR precursor of the invention comprises:

[0205] a first polypeptide corresponding to construct A in Table 1 , and

[0206] a second polypeptide corresponding to construct 1, 2, 3, or 4 in Table 2.

[0207] Preferably, the CAR precursor of the invention comprises:

[0208] a first polypeptide corresponding to construct B in Table 1 , and

[0209] a second polypeptide corresponding to construct 1, 2, 3, or 4 in Table 2.

[0210] Preferably, the CAR precursor of the invention comprises:

[0211] a first polypeptide corresponding to construct C in Table 1 , and

[0212] a second polypeptide corresponding to construct 1, 2, 3, or 4 in Table 2.

[0213] Preferably, the CAR precursor of the invention comprises:

[0214] a first polypeptide corresponding to construct D in Table 1 , and

[0215] a second polypeptide corresponding to construct 1, 2, 3, or 4 in Table 2.

[0216] Preferably, the CAR precursor of the invention comprises:

[0217] a first polypeptide corresponding to construct E in Table 1 , and

[0218] a second polypeptide corresponding to construct 1, 2, 3, or 4 in Table 2.

[0219] Preferably, the CAR precursor of the invention comprises:

[0220] a first polypeptide corresponding to construct F in Table 1 , and

[0221] a second polypeptide corresponding to construct 5, 6 or 7 in Table 2.

[0222] Preferably, in the CAR precursor of the invention:

[0223] The first polypeptide comprises SEQ ID NO: 4 and the second polypeptide comprises SEQ ID NO: 10, 11 or 12;

[0224] The first polypeptide comprises SEQ ID NO: 5 and the second polypeptide comprises SEQ ID NO: 13, 14, 15, or 16;

[0225] The first polypeptide comprises SEQ ID NO: 6 and the second polypeptide comprises SEQ ID NO: 13, 14, 15, or 16;

[0226] The first polypeptide comprises SEQ ID NO: 7 and the second polypeptide comprises SEQ ID NO: 13, 14, 15, or 16;The first polypeptide comprises SEQ ID NO: 8 and the second polypeptide comprises SEQ ID NO: 13, 14, 15, or 16; or

[0227] The first polypeptide comprises SEQ ID NO: 9 and the second polypeptide comprises SEQ ID NO: 13, 14, 15, or 16.

[0228] Preferred mature intein-mediated modular CAR of the invention

[0229] In an embodiment, the mature intein-mediated modular CAR of the invention is the functional CAR that results from the trans-splicing reaction carried out between the first and the second polynucleotides of the invention defined in any of the embodiments above.

[0230] Preferably, the mature intein-mediated modular CAR of the invention comprises or consists of SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39, or a sequence that has at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39, respectively.

[0231] Vector of the invention

[0232] In a second aspect, the present invention provides a vector, called herein “the vector of the invention” that comprises and / or encodes the CAR precursor of the invention, or one or more of the first or second polypeptides of the invention, and / or the mature intein-mediated modular CAR of the invention as defined in the first aspect or in any of its embodiments. As used herein, the term “vector” refers to any biological and non-biological element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, nanoparticle, liposome, carrier, vehicle, etc., which is capable of transfer or carry the CAR precursor and / or the mature intein-mediated modular CAR of the invention inside a cell. Thus, the term vector includes cloning and expression vehicles, as well as viral vectors, chemical vector, particles vectors, and oligonucleotide vectors.

[0233] Preferably, the vector is an expression vector that encodes the CAR precursor of the invention, and / or the mature intein-mediated modular CAR of the invention as defined in the first aspect or in any of its embodiments. As used herein, the term “expression vector” refers to a recombinant nucleic acid or oligonucleotide sequence, e.g., a recombinant DNA or RNA molecule, containing a desired coding sequence operably linked to appropriate nucleic acid sequences necessary for the expression of the coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Nucleic acid sequences necessary for expression in eukaryotic cells can include, but are not limited to, promoters, enhancers, and termination and polyadenylation signals.Cells of the invention

[0234] In a third aspect, the invention provides a cell or a plurality of cells, also called herein “the cell or cells of the invention”, comprising and / or expressing one or more of the CARs of the invention (including the CAR precursors and the mature intein-mediated modular CARs of the invention), one of more of the first or second polypeptides of the invention, and / or the vectors of the invention as defined in any of the embodiments and aspects above. Preferably, the cell of the invention comprises and / or encodes for a nucleic acid vector as defined in the second aspect of the invention.

[0235] In some embodiments, the cell is an immune cell, preferably a natural killer cell (NK), a NK cell, a T cell, a monocyte, a macrophage, or a dendritic cell.

[0236] Preferably, the cell is a T cell. In some embodiments, the cell is a naive T cell, memory stem T cell or central memory T cell. It is currently thought that these cells are better suited for adaptive immunotherapy. Preferably, the cell is a T cell and comprises and / or expresses one or more of the CARs defined above (also called herein CAR-T cell).

[0237] In some embodiments, the cell is an autologous T cell. The term “autologous cell” refers to a cell obtained from the same patient that is to be treated using any one of the methods of the invention.

[0238] In some embodiments, the cell is an allo-tolerant T cell. The term “allo-tolerant cell” or “allogenic” refers to a cell that has been engineered to decrease the risk of a Graft-versus-host disease response. In some embodiments, this is achieved by genomic editing-mediated deletion of TCR and / or p2-microglobulin and / or CIITA. Allo-tolerant cells are known in the art. In an embodiment, the allogenic or allo-tolerant cell is the one defined in the seventh aspect of the invention, also called herein the allogenic T cell of the invention.

[0239] In some embodiments, the T cell is a CD3-positive (CD3+) T cell. In some embodiments, the T cell is T a CD8+ T cell. In some instances, the cell is a human cell. In some embodiments, the cell is a lymphoid precursor, embryonic stem cell or an induced pluripotent stem cell with the capacity to differentiate into a mature T cell. In some embodiments, the cell is a CD34-positive (CD34+) cell, preferably selected from the list consisting of HPCs or cord blood cells. In some embodiments, the cell is an NK cell line such as YT-cell or NK92 cell line.

[0240] Pharmaceutical composition of the invention

[0241] In a fourth aspect, the invention provides a pharmaceutical composition, also called herein “the pharmaceutical composition of the invention”, comprising:

[0242] - a plurality of cells of the invention,- one or more of the vectors of the invention,

[0243] - the CAR precursor or the mature intein-mediated modular CAR of the invention, and / or

[0244] - one or more of the first and / or the second polypeptides of the invention,

[0245] and a pharmaceutically acceptable carrier or diluent.

[0246] A pharmaceutical composition as described herein may also contain other substances. These substances include, but are not limited to, cryoprotectants, surfactants, anti-oxidants, and stabilizing agents. The term "cryoprotectant" as used herein, includes agents which provide stability to the CAR-Ts against freezing-induced stresses. Non-limiting examples of cryoprotectants include sugars, such as sucrose, glucose, trehalose, mannitol, mannose, and lactose; polymers, such as dextran, hydroxyethyl starch and polyethylene glycol; surfactants, such as polysorbates (e.g., PS-20 or PS-80); and amino acids, such as glycine, arginine, leucine, and serine. A cryoprotectant exhibiting low toxicity in biological systems is generally used.

[0247] In some embodiments, the pharmaceutical composition comprises other substances, such as cancer growth blockers, that improve the therapeutic effect of composition.

[0248] In some embodiments, the cells are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier) in a therapeutically effective amount. Suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized. The infusion medium can be supplemented with human serum albumin, fetal bovine serum or other human serum components.

[0249] Methods of treatment and uses of the present invention

[0250] In a fifth aspect, the invention provides the CAR precursor of the invention, the mature intein-mediated modular CAR of the invention, the first and second polypeptides of the invention, the vector of the invention, the cells of the invention, and the pharmaceutical composition of the invention, as defined in any of the aspects and embodiments above, for use as a medicament or in therapy.

[0251] Preferably, the use involves administrating a subject indeed thereof a CAR precursor of the invention, or a cell or a vector of the invention comprising and / or expressing the same, for the treatment or prevention of an antigen-positive cancer that comprises cells that are positive for the antigen against which the targeting moiety of domain i) of the second polypeptide isdesigned to bind. “An antigen-positive” cancer, is one comprising cells, which have certain antigen present at their cell surface. The term “antigen-positive” also refers to a cancer that produces sufficient levels of said antigen at the surface of cells thereof, such that a CAR-comprising cell of the present invention have a therapeutic effect, mediated by the binding of the CAR to said antigen.

[0252] More preferably, the use is in a method of treating or preventing a CD19, CD20, BCMA, SLAMF7, or TROP2 positive-cancer. More preferably, the use is in a method of treating or preventing a CD19, CD20, BCMA, SLAMF7, orTROP2 positive-cancer, wherein the targeting moiety of the CARs of the invention is directed against CD19, CD20, BCMA, SLAMF7, or TROP2 antigens, respectively. Preferably, the use involves administrating the “preferred CAR precursors of the invention” or the “preferred mature intein-mediated modular CAR of the invention”, as defined above.

[0253] In an embodiment, the CAR precursor of the invention comprises a first polypeptide comprising SEQ ID NO: 4 and a second polypeptide comprising SEQ ID NO: 10 or 12; and the use is in the treatment or prevention of a CD19 positive cancer, preferably B-cell acute lymphoblastic leukemia (B-ALL), B-cell non-Hodgkin lymphoma (B-NHL), and / or Chronic lymphocytic leukemia (CLL). Preferably, the mature intein-mediated modular CAR of the invention comprises SEQ ID NO 17 or 19, and is used in in the treatment or prevention of a CD19 positive cancer, B-cell acute lymphoblastic leukemia (B-ALL), B-cell non-Hodgkin lymphoma (B-NHL), and / or Chronic lymphocytic leukemia (CLL).

[0254] In an embodiment, the CAR precursor of the invention comprises a first polypeptide comprising SEQ ID NO: 4 and a second polypeptide comprising SEQ ID NO: 11 or 12; and the use is in the treatment or prevention of a CD20 positive cancer, B-cell non-Hodgkin lymphoma (B-NHL). Preferably, the mature intein-mediated modular CAR of the invention comprises SEQ ID NO 18 or 19, and is used in in the treatment or prevention of a CD20 positive cancer, preferably B-cell non-Hodgkin lymphoma (B-NHL).

[0255] In an embodiment, the CAR precursor of the invention comprises a first polypeptide comprising SEQ ID NO: 5, 6, 7, 8, or 9 and a second polypeptide comprising SEQ ID NO: 13, and the use is in the treatment or prevention of a BCMA positive cancer, preferably Multiple Myeloma. Preferably, the mature intein-mediated modular CAR of the invention comprises SEQ ID NO 25, 26, 27, 28, or 29 and is used in in the treatment or prevention of a BCMA positive cancer, preferably Multiple Myeloma.

[0256] In an embodiment, the CAR precursor of the invention comprises a first polypeptide comprising SEQ ID NO: 5, 6, 7, 8, or 9 and a second polypeptide comprising SEQ ID NO: 14,and the use is in the treatment or prevention of a SLAMF7 positive cancer, preferably Multiple Myeloma. Preferably, the mature intein-mediated modular CAR of the invention comprises SEQ ID NO 25, 26, 27, 28, or 29 and is used in in the treatment or prevention of a SLAMF7 positive cancer, preferably Multiple Myeloma.

[0257] In an embodiment, the CAR precursor of the invention comprises a first polypeptide comprising SEQ ID NO: 5, 6, 7, 8, or 9 and a second polypeptide comprising SEQ ID NO: 15 or 16, and the use is in the treatment or prevention of a TROP2 positive cancer, preferably triple-negative breast cancer (TNBC). Preferably, the mature intein-mediated modular CAR of the invention comprises SEQ ID NO 30, 21, 32, 33, 34, 35, 36, 37, 38 or 39, and is used in in the treatment or prevention of a TROP2 positive cancer, preferably triple-negative breast cancer (TNBC).

[0258] In another embodiment, the use is in treating or preventing diseases where the immune response is exacerbated or deregulated, such as infectious diseases, autoimmune diseases, or after organ transplant. Particularly, the CAR precursors or the functional CARs of the invention are used to treat said diseases when the targeting moiety of domain i) of the second polypeptide are designed to target or to bind to CD19, CD20, or both.

[0259] In an embodiment, the CAR precursor of the invention, the mature intein-mediated modular CAR of the invention, the first and second polypeptides of the invention, the vector of the invention, the cells of the invention, or the pharmaceutical composition of the invention, as defined in any of the aspects and embodiments above, is administered to a subject in need thereof intramuscularly, intravenously, intraperitoneally, into the bone marrow, into the lymph node, and / or into cerebrospinal fluid. Preferably, the subject in need thereof is a human, most preferably a human having or suffering from a cancer that comprises cells that are positive for the antigen against which the targeting moiety of domain i) of the second polypeptide is designed to bind.

[0260] Additionally, the combination therapies provided herein may be used in conjunction with other types of treatment. For example, other anti-cancer treatment may be selected from the group consisting of chemotherapy, surgery, radiotherapy (radiation) and / or hormone therapy, amongst other treatments associated with the current standard of care for the subject. Hence, the method and uses thereof also include the administration of two (or more) different treatments that are delivered to the subject during the course of the subject’s affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration.This is sometimes referred to herein as “simultaneous” or “concurrent delivery”. In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0261] It is noted that the therapy and methods describe herein comprise the use of the CARs of the invention and precursor thereof in a therapy in which the first polypeptide is preferably constitutively expressed in the cell of the invention, and the second polypeptide is preferably provided to the subject in need thereof depending on the needs of said subjects and the type of cancer to be treated. For instance, the second peptide can be provided for a sufficient amount of time to as to form a mature CAR of the invention capable to kill a type of cancer cell, and then its administration is ceased for a period of time, or it is replaced by another second polypeptide having a different targeting moiety so as to target a different type of cancer cell.

[0262] Kits

[0263] In a sixth aspect, the present invention provides kits and their use according to any of the previous aspects. The materials described above as well as other materials can be packaged together in any suitable combination as a kit useful for performing, or aiding in the performance of, the disclosed method. It is useful if the kit components in a given kit are designed and adapted for use together in the disclosed method.

[0264] The cell of the seventh aspect of the invention

[0265] In a seventh aspect, the present invention provides a cell, preferably a cell from the immune system, comprising:

[0266] the first polypeptide of the invention as defined in the first aspect or any of its embodiments, and

[0267] a recognition domain (RD).As used herein, a “cell from the immune system” or “an immune cell” means any cell whose lineage, phenotype, or physiological role is associated with innate or adaptive immunity in a vertebrate organism. In one embodiment, the cell from the immune system comprising (i) the first polypeptide of the invention as defined in the first aspect or any of its embodiments, and (ii) a recognition domain (RD), is selected from the group consisting of T lymphocytes (CD8+ cytotoxic, CD4+ helper, regulatory T cells, gamma delta T cells, and invariant NKT cells), natural killer (NK) cells, B lymphocytes, dendritic cells (including conventional and plasmacytoid), monocytes, macrophages (including tissue resident macrophages such as microglia, Kupffer cells, Langerhans cells, and alveolar macrophages), neutrophils, eosinophils, basophils, and mast cells. The cell of the seventh aspect may be autologous or allogeneic, primary or derived from a cell line, and is preferably genetically engineered to coexpress the first polypeptide and the RD. In one embodiment, the immune cell is a natural killer (NK) cell comprising the first polypeptide of the invention as defined in the first aspect or any of its embodiments, and a RD.

[0268] Preferably, the immune cell is a T cell. More preferably the cell of the seventh aspect is an allogenic T cell also called herein after “the allogenic T cell of the invention”.

[0269] Preferably, the present invention provides in a seventh aspect a T cell, preferably an allogenic T cell, having reduced, preferably eliminated, expression of the T cell receptor (TCR), and comprising:

[0270] the first polypeptide of the invention as defined in the first aspect or any of its embodiments, and

[0271] a recognition domain (RD).

[0272] Preferably, the allogenic T cell is a modified T cell, or an engineered T cell, that is, a cell that is non-natural, i.e., that does not exist in nature. The allogenic T cell of the invention has reduced graft-versus-host disease and host-versus-graft response thus is an universal T cell. In another embodiment, the allogenic T cell of the invention has a reduced, preferably eliminated, expression of the T cell receptor (TCR). By “reduced TCR expression” is referred herein to as a decrease in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% of TCR expression, as measured by any method known in the art, such as by western blot or flow cytometry. Preferably, the reduction is a statistically significant reduction.

[0273] By “eliminated TCR expression” is referred herein to as a complete lack of TCR expression, as measured by any method known in the art, such as by western blot or flow cytometry. Preferably, the allogenic T cell of the invention completely lacks any TCR expression.Preferably, the allogenic T cell of the invention lacks the one or more genes encoding one or more components of the TCR.

[0274] Preferably, the reduction or elimination of TCR expression is measured as compared to a control cell. In an embodiment, a control cell is a T cell that has not been modified, that is, a natural T cell. Preferably, the control cell is a T cell of the same linage or cell type as the allogenic T cell of the invention. Preferably, the comparison is performed under the same culturing conditions.

[0275] The TCR is a molecule found on the surface of T cells and is responsible for recognizing fragments of antigens as peptides bound to major histocompatibility complex (MHC) molecules being also responsible for allogenic responses. TCR lacking signaling motifs relays on the CD3 complex for signaling and antibodies against CD3 molecules are used in expansion and activation protocols, as they mimic activation elicited by presenting the cells with a specific antigen that is recognized by the TCR in the context of an MHC molecule. For example, using an anti-CD3 antibody or using artificial antigen presenting systems leads to the activation of the TCR and hence the expansion of the T cells. However, the absence of TCR and the concomitant absence of CD3, hampers the re-stimulation needed for maintenance of a generic T cell until a CAR construct is expressed, and thus prevents us from marketing a universal allogenic T cell ready to be harness with target specificity but still not compromised with specific target recognition.

[0276] This problem is solved herein by the allogenic T cells of the invention that have reduced, preferably eliminated, TCR expression and that comprise a recognition domain and the first polypeptide of the invention. Thanks to the presence of the first polypeptide of the invention and a recognition domain, the cell is capable of translating a signal received from the extracellular media of the cell to the intracellular media of the cell allowing activation and expansion.

[0277] Thus, the first polypeptide of the invention, when present in a T cell together with a recognition domain, can replace the TCR of the cell, and thus this TCR can be eliminated from the cell, thereby resulting in an allogenic T cell (the allogenic T cell of the invention) without yet irreversibly compromising with a specific target recognition.

[0278] The RD is any extracellular domain that is used to or that is capable of activating domains ii) and iii) of the first polypeptide. This RD is capable of binding or being recognized by an exogenous molecule, wherein the binding or recognition between the RD and the exogenous molecule triggers the activation of the allogenic T cell of the invention. In other words, the RD is any extracellular domain that is activatable by an exogenous molecule, such as a ligand, an antibody, etc. By “activatable” is referred herein as the generation and transmission of anactivation signal from the RD to a transmembrane domain, and an intracellular signaling domain (and optionally costimulatory domain) of the first polypeptide, thereby activating the proliferation of the allogenic T cell of the invention. Examples of the RD are polypeptides comprising tags or moieties such as FLAG, HA, His, Myc, V5, Xpress, Thrombin, BAD (Biotin Acceptor Domain), Factor Xa, VSVG, SV40 NLS, Protein C, S Tag, OneStrap, SB1, streptag, avitag or any other epitope recognice by an antibody or any sequence recogniced by a ligand. For example, the RD can comprise any of the ScFV defined above under the first aspect of the present invention (for example, the ScFv that binds to CD19), or any other scFv, as long as the allogenic T cells of the invention are contacted with the exogenous molecule (e.g., CD19) to which the ScFv binds and that will lead to activation.

[0279] Hence, when the RD is engaged with the exogenous molecule, the allogenic T cell of the invention generates an activation signal that results in proliferation and expansion of said allogenic T cell. This is possible even when the allogenic T cell of the invention has reduced, preferably eliminated, TCR expression, because the RD and the first polypeptide of the invention are replacing the TCR signaling, providing an allogenic T cell of the invention. Therefore, the allogenic T cell of the invention is characterized by:

[0280] a) having a reduced, preferably eliminated, TCR expression, and

[0281] b) comprising:

[0282] a recognition domain, and

[0283] the first polypeptide of the invention, as defined in the first aspect or any of its embodiments.

[0284] The RD may be part of the to the first polypeptide of the invention, or it can be present or provided as a separate polypeptide. When the RD is comprised in the first polypeptide of the invention it is preferably placed upstream, that is, in the N terminal, of the first polypeptide of the invention, so that it is located in the surface of the cell. Alternatively, the RD can be provided as a different molecule, for instance by expressing the first polypeptide of the invention and the RD in different polypeptides, as long as the RD is fused by means of, e.g., orthogonal split inteins and thus it will become part of a mature polypeptide (CAR or otherwise) present in the cell until said mature polypeptides are replaced by newly synthesized unspliced first polypeptides.

[0285] Therefore, the RD can be:

[0286] present in the N terminal of the first polypeptide of the invention, orpresent in a further polypeptide that is not the first polypeptide of the invention but can carry out a trans-splicing reaction with the first polypeptide thanks to the presence of orthologs inteins in both polypeptides.

[0287] Embodiments where the recognition domains is present in the first polypeptide of the invention, preferably in its N terminal.

[0288] In an embodiment, the allogenic T cell of the invention comprises a polypeptide that in turn comprises, preferably in the N to C terminal direction:

[0289] a recognition domain, and

[0290] the first polypeptide of the invention as defined in the first aspect or any of its embodiments.

[0291] In other words, the first polypeptide of the invention comprised in the allogenic T cell of the invention comprises, preferably in the N to C terminal direction:

[0292] i) a recognition domain and a C terminal split intein and extein,

[0293] ii) a transmembrane domain, and

[0294] iii) an intracellular signaling domain,

[0295] Domains ii) and iii) have been defined in the first aspect of the invention and all of the embodiments included therein also apply here. The region of domain i) referring to a C terminal split intein and extein has also been defined in the first aspect of the invention and all of the embodiments included therein also apply here.

[0296] Since the RD is connected via peptide bonds to the first polypeptide, once the RD is bound or recognized by the exogenous molecule, the signal will be transmitted to the other domains of the first polypeptide, leading to the activation of the allogenic T cell of the invention. Preferably, the RD is comprised in the first polypeptide of the invention, and the allogenic T cell of the invention constitutively expressed said polypeptide including the RD. By “constitutive expression” is referred to the continuous and consistent expression of a gene (in this case, a gene encoding for the first polypeptide comprising the RD) at a relatively stable level, regardless of external signals or environmental conditions.

[0297] Embodiments where the recognition domains is present in a polypeptide that is not the first polypeptide of the invention:

[0298] As explained above, the RD can be present in the allogenic T cell of the invention in the form of a polypeptide that is different from the first polypeptide, that is, in the form of a separate polypeptide. In this case, the RD is present in the allogenic T cell of the invention in trans withthe first polypeptide of the invention. The ultimate objective is that the RD is fused with the first polypeptide so that it can respond to interacting activatory molecules and the activation signal is transmitted to the cell. The RD can be fused to the first polypeptide by means of orthogonal split inteins, wherein one intein extein is placed in the first polypeptide, and its orthologs are placed in the separate polypeptide together with the RD domain. Preferably, the trans-splicing reaction is carried out between the C terminal split intein and extein comprised in the first polypeptide of the invention and a N terminal split extein and intein comprised in the same polypeptide where the RD is comprised.

[0299] In this case, the allogenic T cell of the invention having a reduced, preferably eliminated, TCR expression comprises and / or encodes for:

[0300] 1. a first polypeptide which is the first polypeptide of the invention as defined in the first aspect of the invention or any of the embodiments above, and

[0301] 2. a further polypeptide comprising, preferably in the N to C terminal direction,

[0302] a. a recognition domain and

[0303] b. a N terminal split extein and intein,

[0304] wherein the C terminal split intein and extein of the 1) first polypeptide of the invention and the N terminal split extein and intein of the further polypeptide of 2) are orthogonal split inteins so that they can carry out a trans-splicing reaction between both 1) and 2) polypeptides. Preferably, the first polypeptide of the invention is constitutively expressed in the allogenic T cell of the invention, and the further polypeptide of 2) is expressed transiently in the allogenic T cell of the invention. By “transient expression” is referred herein as to the temporary and short-term expression of a gene (in this case, a gene expressing the further polypeptide of 2)) or polypeptide in a cell or organism. Also preferably, the first polypeptide of the invention is constitutively expressed in the allogenic T cell of the invention, and the further polypeptide of 2) is directly administrated to the culture media or the subject in need thereof in a sufficient amount to as to fuse with the first polypeptide.

[0305] It is noted that the polypeptide of 2) comprised the allogenic T cell of the invention is different from the “second polypeptide of the invention” as defined above in the first aspect of the invention or any of its embodiments, as it does not necessarily need to comprise a targeting moiety nor a iii) an endoplasmic reticulum (ER) retention motif as it will not be expressed in the same cell but provided in trans. However, it is mandatory that this 2) further polypeptide comprises the orthogonal split intein and extein of the first polypeptide so that, when it is added to the allogenic T cell of the invention, a trans-splicing reaction between both polypeptides occur, thereby giving rise to a construct that is capable of substituting the TCR of the allogenicT cell of the invention, and thus translate the activation signal from the extracellular media to the intracellular media of the cell.

[0306] As the polypeptide of 2) is preferably added in trans (e.g. in a different molecule), once surface molecules are renewed and replaced by newly synthesis polypeptide 1, said polypeptide 1 will be devoted of targeting domains and thus ready to be harnessed with the second polypeptide of the invention either in transient or by constitutive expression.

[0307] Thus, the allogenic T cell of the invention can still be transformed into an allogenic CAR T cell as defined in the first aspect or any of its embodiments, by adding the second polypeptide of the invention. This will trigger the trans-splicing between the first and second polypeptides of the invention, leading to the generation of the mature CAR of the invention as explained in any of the aspects above. This process is particularly useful in cases where an allogenic T cell of the invention is to be generated by activating and expanding it using the RD and the first polypeptide of the invention, and then said activated and expanded allogenic T cell of the invention is to be transformed an allogenic CAR T cell, as defined in the third aspect or any of its embodiments, comprising the mature CAR of the invention.

[0308] Therefore, an eighth aspect of the invention refers to a method of producing the allogenic T cell of the invention, the method comprising the steps of:

[0309] a) providing a T cell, preferably an engineered T cell, having reduced, preferably eliminated, TCR expression, and

[0310] b) introducing (e.g., transducing, transfecting, or genetically modifying) in the cell of a) a RD and the first polypeptide of the invention, or a nucleic acid encoding them.

[0311] The allogenic T cell of the invention can be used in activation and expansion protocols to generate a population of allogenic T cells of the invention. Thus, in an embodiment, the method of the eight aspect is also a method to generate a population of activated and expanded allogenic T cells of the invention, ready to be eventually harnessed with any targeting moiety the method comprising the steps of

[0312] a) providing a T cell, preferably an engineered T cell, having reduced, preferably eliminated, TCR expression,

[0313] b) introducing (e.g., transducing, transfecting, or genetically modifying) in the cell of a) a RD and the first polypeptide of the invention, or a nucleic acid encoding them, and c) contacting the cell of b) with an exogenous molecule that is capable of binding to the RD, thereby activating and expanding said cell.Once the population of allogenic T cells of the invention is created, they can be further modified to become a CAR T cell. This can be achieved by adding a further step of d) introducing a CAR in the cell obtained from step c).

[0314] Therefore, in an embodiment, the method of the eight aspect is a method of producing the allogenic CAR T cell, the method comprising the steps of:

[0315] a) providing a T cell, preferably an engineered T cell, having reduced, preferably eliminated, TCR expression,

[0316] b) introducing (e.g., transducing, transfecting, or genetically modifying) in the cell of a) a RD and the first polypeptide of the invention, or a nucleic acid encoding them, and c) contacting the cell of b) with an exogenous molecule that is capable of binding to the RD of the allogenic CAR precursor of the invention, thereby activating and expanding said cell, and

[0317] d) introducing in the expanded and activated cell of c) a CAR, or a nucleic acid encoding it, thereby producing an allogenic CAR-T cell.

[0318] In an embodiment, the method is a method for producing the CAR-T cells of the invention, as defined in the third aspect described above, comprising the mature CAR of the invention. This can be achieved by adding a further step d) to the method of the eight aspect, comprising or consisting of adding the second polypeptide of the invention as defined above in the first aspect or any of its embodiments. Said second polypeptide of the invention will conduct a trans-splicing reaction with the first polypeptide already present in the allogenic T cell of the invention, as explained in the first aspect or any of its embodiments.

[0319] Therefore, in an embodiment, the method of the eight aspect is a method of producing the allogenic CAR T cell of the invention, the method comprising the steps of:

[0320] a) providing a T cell, preferably an engineered T cell, having reduced, preferably eliminated, TCR expression,

[0321] b) introducing (e.g., transducing, transfecting, or genetically modifying) in the cell of a) a RD and the first polypeptide of the invention, or a nucleic acid encoding them, and c) contacting the cell of b) with an exogenous molecule that is capable of binding to the RD, thereby activating and expanding said cell, and

[0322] d) introducing in the expanded and activated cell of c) the second polypeptide of the invention, thereby producing a trans-splicing reaction between the first and the second polypeptide of the invention, and providing the mature CAR of the invention.

[0323] Step a) may be performed by any means known in the art to genetically modify T cells to reduce or eliminate the expression of a protein, in this case, the TCR. Said methods include,for instance, CRISPR / Cas based methods. By “providing” is understood herein “generating”, “creating”, “producing”.

[0324] Step b) and d) can be performed by any means known in the art aimed at introducing one or more polypeptides into a cell, or one or more nucleic acid vector encoding thereof. Such methods include: lipofection, cell penetrating peptides, electroporation, viral transduction, calcium phosphatate transfections, polymer mediated transfections, liposomes, DNA vectors such as plasmid transfection, microinjection, or modifying the genome of the cell to comprise a gene encoding for relevant polypeptides. By “Introducing” is understood herein “modifying the cell to introduce” or “delivering into the cell” or “administering to the cell” or “loading the cell with”, or “adding to the cell”. Introducing a polypeptide into a cell also encompasses modifying the cell to express said polypeptide, either transiently or constitutively. Preferably, the first polypeptide of the invention are provided for constitutive expression, e.g., modifying the cell to permanently express them, and the remaining polypeptides (e.g., the RD or the second polypeptide of the invention) are provided for transient expression, e.g., provided in the form of polypeptides that are contacted with the cell or that are introduced in the cell temporarily.

[0325] In an embodiment, step b) comprises introducing in the cell the first polypeptide of the invention comprising in its N terminal the RD. In another embodiment, step b) comprises the substeps of:

[0326] - b.1 ) introducing the first polypeptide of the invention, or a nucleic acid vector encoding thereof, without the RD, and

[0327] - b.2) adding to the cell of b.1) another polypeptide comprising the RD, or a nucleic acid vector encoding thereof, wherein the each of the polypeptides of b.1 and b.2 further comprise orthologs split-inteins, so that a trans-splicing reaction is carried out between both polypeptides, resulting in a first polypeptide of the invention comprising the RD domain in its N terminal.

[0328] In another embodiment, step b) comprises the substeps of:

[0329] - b.1) genetically modifying the cell to constitutively express the first polypeptide of the invention, and

[0330] - b.2) introducing a polypeptide comprising the RD into the cell of b.1), so that the cell transiently comprises the polypeptide comprising the RD, wherein the first polypeptides of the invention (b.1) and the transient polypeptide of b.2 each comprise orthologs split-inteins, so that a trans-splicing reaction is carried out between bothpolypeptides, resulting in a first polypeptide of the invention comprising the RD domain in its N terminal.

[0331] Step c) can be performed by any means known in the art to expand and activate T cells. The exogenous molecule can be placed in the surface of an antigen presenting cell or in the surface of beads, or it can be added in suspension to the culture media of the cells. Step c) thus includes stimulation with exogenous molecules, preferably in the presence of cytokines (such as IL-2, IL-7, IL-15). Step c) is performed for a sufficient amount of time to as provide activated and expanded cells. By “contacting” is understood herein “adding to the cell culture media”.

[0332] In a preferred embodiment, the method of the eight aspect is a method of producing the allogenic CAR T cell of the invention, the method comprising the steps of:

[0333] a) providing a T cell, preferably an engineered T cell, having reduced, preferably eliminated, TCR expression,

[0334] b) introducing (e.g., transducing, transfecting, or genetically modifying) in the cell of a) a RD and the first polypeptide of the invention, or a nucleic acid encoding them, wherein this step comprises the sub-steps of:

[0335] - b.1) genetically modifying the cell to constitutively express the first polypeptide of the invention, and

[0336] - b.2) introducing a polypeptide comprising the RD into the cell of b.1), so that the cell transiently comprises the polypeptide comprising the RD, wherein the first polypeptides of the invention (b.1) and the transient polypeptide of b.2) each comprise orthologs split-inteins, so that a trans-splicing reaction is carried out between both polypeptides, resulting in a first polypeptide of the invention comprising the RD domain in its N terminal,

[0337] c) contacting the cell of b) with an exogenous molecule that is capable of binding to the RD, thereby activating and expanding said cell, and

[0338] d) introducing in the expanded and activated cell of c) the second polypeptide of the invention, thereby producing a trans-splicing reaction between the first and the second polypeptide of the invention, and providing the mature CAR of the invention.

[0339] It is noted that, in this preferred embodiment of the method, the polypeptide of b.2) is transiently introduced in the cell, so that when the allogenic T cell of the invention is activated and expanded after step c), the addition of the polypeptide of step b.2) can be ceased, and the second polypeptide of the invention can thus be introduced (constitutively or transiently) to produce the mature CAR of the invention, which will be allogenic.Thus, in a most preferred embodiment, the method of the eight aspect is a method of producing the allogenic CAR T cell of the invention, the method comprising the steps of: a) providing a T cell, preferably an engineered T cell, having reduced, preferably eliminated, TCR expression,

[0340] b) introducing (e.g., transducing, transfecting, or genetically modifying) in the cell of a) a RD and the first polypeptide of the invention, or a nucleic acid encoding them, wherein this step comprises the sub-steps of:

[0341] - b.1) genetically modifying the cell to constitutively express the first polypeptide of the invention, and

[0342] - b.2) introducing a polypeptide comprising the RD into the cell of b.1), so that the cell transiently comprises the polypeptide comprising the RD, wherein the constitutive first polypeptide of the invention (b.1) and the transient polypeptide of b.2) each comprise orthologs split-inteins, so that a transsplicing reaction is carried out between both polypeptides, resulting in a first polypeptide of the invention comprising the RD domain in its N terminal, c) contacting the cell of b.2) with an exogenous molecule that is capable of binding to the RD, thereby activating and expanding said cell and, once the cells are activated and expanded, removing or washing the first polypeptide of the invention comprising the RD domain in its N terminal generated in step b.2) so that a population of allogenic T cell comprising the first polypeptide of the invention but not the RD is generated, and d) introducing in the expanded and activated cell of c) the second polypeptide of the invention, thereby producing a trans-splicing reaction between the first and the second polypeptide of the invention, and providing the mature CAR of the invention.

[0343] The removal of the first polypeptide of the invention comprising the RD domain in its N terminal generated in step b.2) can be performed by simply stop adding the polypeptide of b.2 so that newly first polypeptides are expressed by the cells. Optionally, the cells can be washed to remove non-fused polypeptides. Thus, once the cells are activated and expanded, the removal of the first polypeptide of the invention with the RD in its N terminal can be removed by simply stopping adding the peptide of step b.2), and waiting until a population of activated and expanded cells lacking said polypeptide, but comprising the first polypeptide of the invention (its expression is constitutive) is generated

[0344] Thus, after he removal of the first polypeptide of the invention comprising the RD domain in its N terminal generated in step b.2), the activated and expanded cells of c) will only comprise the first polypeptide of the invention, as it is expressed constitutively in them (see step b.1)), but this first polypeptide of the invention will no longer be fused to the polypeptide comprisingthe RD, since said polypeptide is not present anymore. Once a population of allogenic T cell comprising the first polypeptide of the invention but not the RD is generated, step d) can be performed.

[0345] It is understood that the resulting CAR-T cell of the invention is identical to that defined above in the third aspect of the invention. Its uses and methods for use are also similar as those defined in the fifth aspect of the invention.

[0346] Similarly, the allogenic T cell of the invention as defined in the seventh aspect of the invention can be used in therapy or in the production of a medicament. Preferably, the use involves in immunotherapy as they are allogenic T cells that can be further modified to comprise CARs against a desired antigen, preferably tumor antigen.

[0347] The following items are thus included in the present invention:

[0348] 1. An allogenic T cell having reduced, preferably eliminated, expression of the T cell receptor (TCR), and comprising:

[0349] a first polypeptide of the invention as defined in the first aspects or any of its embodiments and comprising in the N to C terminal direction:

[0350] i) a C terminal split intein and extein,

[0351] ii) a transmembrane domain, and

[0352] iii) an intracellular signaling domain,

[0353] a recognition domain (RD),

[0354] wherein the RD is either bound to the N terminal of the first polypeptide by a peptide bond or is in a separate polypeptide, and wherein if the RD is in a separate polypeptide it further comprises a N terminal split extein and intein so that a trans-splicing reaction is carried out between said polypeptide and the first polypeptide, resulting in the first polypeptide comprising the RD domain in its N terminal.

[0355] 2. The allogenic T cell according to item 1, wherein the recognition domain is a polypeptide capable of being activated by an exogenous molecule and transmit the activation signal to the ii) transmembrane and iii) intracellular signaling domain of the first polypeptide.

[0356] 3. The allogenic T cell according to any of items 1 or 2, wherein the recognition domain comprises a polypeptide that is selected from the list comprising of FLAG, HA, His, Myc, V5, Xpress, Thrombin, BAD (Biotin Acceptor Domain), Factor Xa, VSVG, SV40 NLS, Protein C, S Tag, OneStrap, SB1 , streptag, avitag or any other epitope recognice by an antibody or any sequence recogniced by a ligand.4. The allogenic T cell according to any of items 1 to 3, for use as a medicament or in therapy.

[0357] 5. The allogenic T cell according to any of items 1 to 3, further comprising a CAR, for use in immunotherapy.

[0358] 6. The allogenic T cell for use according to item 5, wherein the CAR is the mature CAR of the invention as defined in the first aspect or any of its embodiments.

[0359] 7. A method for producing an allogenic T cell, the method comprising the steps of:

[0360] a. providing a T cell, preferably engineered T cell, having reduced, preferably eliminated, TCR expression, and

[0361] b. introducing (e.g., transducing, transfecting, or genetically modifying) in the cell of a) a RD and the first polypeptide of the invention, or a nucleic acid encoding them.

[0362] 8. A method for activating and expanding an allogenic T cell, the method comprising a. providing a T cell, preferably engineered T cell, having reduced, preferably eliminated, TCR expression, and

[0363] b. introducing (e.g., transducing, transfecting, or genetically modifying) in the cell of a) a RD and the first polypeptide of the invention, or a nucleic acid encoding them, and

[0364] c. contacting the cell of b) with an exogenous molecule that is capable of binding to the RD, thereby activating and expanding said cell.

[0365] 9. A method for producing an allogenic CAR-T cell, the method comprising

[0366] a. providing a T cell, preferably engineered T cell, having reduced, preferably eliminated, TCR expression,

[0367] b. introducing (e.g., transducing, transfecting, or genetically modifying) in the cell of a) a RD and the first polypeptide of the invention, or a nucleic acid encoding them, and

[0368] c. contacting the cell of b) with an exogenous molecule that is capable of binding to the RD, thereby activating and expanding said cell, and

[0369] d. introducing in the expanded and activated cell of c) the second polypeptide of the invention, thereby producing a trans-splicing reaction between the first andthe second polypeptide of the invention and providing the mature CAR of the invention.

[0370] 10. A method for producing an allogenic CAR-T cell, the method comprising

[0371] a. providing a T cell, preferably engineered T cell, having reduced, preferably eliminated, TCR expression, and

[0372] b. introducing (e.g., transducing, transfecting, or genetically modifying) in the cell of a) a RD and the first polypeptide of the invention, or a nucleic acid encoding them,

[0373] c. contacting the cell of b) with an exogenous molecule that is capable of binding to the RD, thereby activating and expanding said cell, and

[0374] d. introducing in the expanded and activated cell of c) a CAR, or a nucleic acid encoding it.

[0375] 11. The method according to any of items 7 to 10, wherein step b) comprises the substeps of:

[0376] - b.1) genetically modifying the cell to constitutively express the first polypeptide of the invention, and

[0377] - b.2) introducing a polypeptide comprising the RD into the cell of b.1 ) , so that the cell transiently comprises the polypeptide comprising the RD,

[0378] wherein the constitutive first polypeptide of the invention of b.1) and the transient polypeptide of b.2) each comprise orthologs split-inteins, so that a trans-splicing reaction is carried out between both polypeptides, resulting in a first polypeptide of the invention comprising the RD domain in its N terminal.

[0379] 12. A method for producing an allogenic CAR-T cell, the method comprising:

[0380] a. providing a T cell, preferably engineered T cell, having reduced, preferably eliminated, TCR expression,

[0381] b. introducing (e.g., transducing, transfecting, or genetically modifying) in the cell of a) a RD and the first polypeptide of the invention, or a nucleic acid encoding them, wherein this step comprises the sub-steps of:

[0382] - b.1) genetically modifying the cell to constitutively express the first polypeptide of the invention, and

[0383] - b.2) introducing a polypeptide comprising the RD into the cell of b.1), so that the cell transiently comprises the polypeptide comprising the RD,wherein the constitutive first polypeptide of the invention of b.1) and the transient polypeptide of b.2) each comprise orthologs split-inteins, so that a trans-splicing reaction is carried out between both polypeptides, resulting in a first polypeptide of the invention comprising the RD domain in its N terminal,

[0384] c. contacting the cell of b) with an exogenous molecule that is capable of binding to the RD, thereby activating and expanding said cell and, once the cells are activated and expanded, removing or washing the first polypeptide of the invention comprising the RD domain in its N terminal generated in step b.2) so that a population of allogenic T cell comprising the first polypeptide of the invention but not the RD is generated, and

[0385] d. introducing in the expanded and activated cell of c) a CAR, or a nucleic acid encoding it.

[0386] Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention.

[0387] The following further items are also included in the present invention:

[0388] 1. A chimeric antigen receptor (CAR) precursor comprising at least two polypeptides, wherein:

[0389] a. the first polypeptide comprises in the N to C terminal direction:

[0390] i) a C terminal split intein and extein,

[0391] ii) a transmembrane domain, and

[0392] iii) an intracellular signaling domain,

[0393] b. the second polypeptide comprises in the N to C terminal direction:

[0394] i) an extracellular domain comprising at least one targeting moiety, ii) a N terminal split extein and intein, and

[0395] iii) an endoplasmic reticulum (ER) retention motif,

[0396] wherein the C terminal split intein and extein of a.i) and the N terminal split extein and intein of b.ii) are orthogonal split inteins so that they can carry out a trans-splicing reaction between the first and the second polypeptides, thereby resulting into a mature intein-mediated modular CAR.

[0397] 2. The CAR precursor according to item 1 , wherein the C terminal split intein and extein of a.i) comprises SEQ ID NO: 1, and the N terminal split intein and extein of b.ii) comprises SEQ ID NO: 2.3. The CAR precursor according to any one of items 1 or 2, wherein:

[0398] - the endoplasmic reticulum (ER) retention motif of b.i) comprises SEQ ID NO: 40,

[0399] - a tag is placed in the C- terminal of the first and / or the second polypeptides, and / or

[0400] - one or more spacers or linkers are placed between any of a.i-iii) and / or b.i-iii).

[0401] 4. A mature intein-mediated modular CAR comprising in N to C terminal direction: a. an extracellular domain comprising at least one targeting moiety,

[0402] b. a N and a C exteins,

[0403] c. a transmembrane domain, and

[0404] d. an intracellular signaling domain,

[0405] wherein the N and C exteins are the result of a trans-splicing reaction of orthogonal N and C split inteins.

[0406] 5. The CAR precursor or the mature intein-mediated modular CAR according to any of the previous items, wherein the at least one targeting moiety binds to CD19, CD20, BCMA, SLAMF7, orTROP2 tumor-associated proteins.

[0407] 6. The CAR precursor or the mature intein-mediated modular CAR according to any of the previous items, wherein:

[0408] the transmembrane domain comprises CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, orCD154, the intracellular signaling domain comprises CD3 , FcRy, CD3y, CD35, CD3E, CD5, CD22, CD79a, CD79b, or CD66b, and

[0409] optionally, further comprising a costimulatory domain, preferably selected from the list of CD27, CD28, CD137, CD134, CD30, CD40, lymphocyte function- associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, LAT, Lek, ZAP70 or CD276.

[0410] 7. The CAR precursor according to any one of the previous items, wherein:

[0411] The first polypeptide comprises SEQ ID NO: 4 and the second polypeptide comprises SEQ ID NO: 10, 11 or 12;The first polypeptide comprises SEQ ID NO: 5 and the second polypeptide comprises SEQ ID NO: 13, 14, 15, or 16;

[0412] The first polypeptide comprises SEQ ID NO: 6 and the second polypeptide comprises SEQ ID NO: 13, 14, 15, or 16;

[0413] The first polypeptide comprises SEQ ID NO: 7 and the second polypeptide comprises SEQ ID NO: 13, 14, 15, or 16;

[0414] The first polypeptide comprises SEQ ID NO: 8 and the second polypeptide comprises SEQ ID NO: 13, 14, 15, or 16; or

[0415] The first polypeptide comprises SEQ ID NO: 9 and the second polypeptide comprises SEQ ID NO: 13, 14, 15, or 16.

[0416] e intein-mediated modular CAR comprising or consisting of:

[0417] SEQ ID NO: 17: mature intein-mediated modular CAR CD19;

[0418] SEQ ID NO: 18: mature intein-mediated modular CAR CD20;

[0419] SEQ ID NO: 19: mature intein-mediated modular DUAL CAR CD20 / CD19; SEQ ID NO: 20: mature intein-mediated modular CAR BCMA-stemA;

[0420] SEQ ID NO: 21: mature intein-mediated modular CAR BCMA-stemB;

[0421] SEQ ID NO: 22: mature intein-mediated modular CAR BCMA-stemC;

[0422] SEQ ID NO: 23: mature intein-mediated modular CAR BCMA-stemD;

[0423] SEQ ID NO: 24: mature intein-mediated modular CAR BCMA-stemE;

[0424] SEQ ID NO: 25: mature intein-mediated modular CAR SLAMF7-stemA;

[0425] SEQ ID NO: 26: mature intein-mediated modular CAR SLAMF7-stemB;

[0426] SEQ ID NO: 27: mature intein-mediated modular CAR SLAMF7-stemC; SEQ ID NO: 28: mature intein-mediated modular CAR SLAMF7-stemD; SEQ ID NO: 29: mature intein-mediated modular CAR SLAMF7-stemE;

[0427] SEQ ID NO: 30: mature intein-mediated modular CAR TROP2 ,1-stemA; SEQ ID NO: 31 : mature intein-mediated modular CAR TROP2 ,1-stemB; SEQ ID NO: 32: mature intein-mediated modular CAR TROP2 ,1-stemC; SEQ ID NO: 33: mature intein-mediated modular CAR TROP2 ,1-stemD; SEQ ID NO: 34: mature intein-mediated modular CAR TROP2 ,1-stemE; SEQ ID NO: 35: mature intein-mediated modular CAR TROP2 ,2-stemA; SEQ ID NO: 36: mature intein-mediated modular CAR TROP2 ,2-stemB; SEQ ID NO: 37: mature intein-mediated modular CAR TROP2 ,2-stemC; SEQ ID NO: 38: mature intein-mediated modular CAR TROP2 ,2-stemD; and SEQ ID NO: 39: mature intein-mediated modular CAR TROP2 ,2-stemE.A cell comprising a polynucleotide encoding for the CAR precursor or the mature intein-mediated modular CAR as defined in any of the previous items.

[0428] An allogenic T cell having reduced, preferably eliminated, expression of the T cell receptor (TCR), and comprising:

[0429] a first polypeptide comprising in the N to C terminal direction:

[0430] i) a C terminal split intein and extein,

[0431] ii) a transmembrane domain, and

[0432] iii) an intracellular signaling domain,

[0433] a recognition domain (RD),

[0434] wherein the RD is a polypeptide capable of being activated by an exogenous molecule and transmit the activation signal to the ii) transmembrane and iii) intracellular signaling domain of the first polypeptide,

[0435] wherein the RD is either bound to the N terminal of the first polypeptide by a peptide bond or is in a separate polypeptide, and wherein if the RD is in a separate polypeptide it further comprises a N terminal split extein and intein so that a trans-splicing reaction is carried out between said polypeptide and the first polypeptide, resulting in the first polypeptide comprising the RD domain in its N terminal.

[0436] The CAR precursor, the mature intein-mediated modular CAR, or the cell as defined in any of itemsl to 10, for use as a medicament or in therapy.

[0437] The CAR precursor, the mature intein-mediated modular CAR, or cell as defined in any of items 1 to 10, for use in the treatment or prevention of cancer.

[0438] The CAR precursor, the mature intein-mediated modular CAR, or cell as defined in any of items 1 to 9, wherein the targeting moiety comprised in the extracellular domain binds to CD19 and / or CD20, for use as immunosuppressors, preferably in the treatment or prevention of auto-immune or infectious diseases or to avoid immune reactions after surgery or organ transplants.

[0439] A method for producing an allogenic CAR-T cell, the method comprising

[0440] a. providing a T cell, preferably engineered T cell, having reduced, preferably eliminated, TCR expression,

[0441] b. introducing in the cell of a):- a first polypeptide comprising in the N to C terminal direction:

[0442] i) a C terminal split intein and extein,

[0443] ii) a transmembrane domain, and

[0444] iii) an intracellular signaling domain,

[0445] - a recognition domain (RD),

[0446] wherein the RD is a polypeptide capable of being activated by an exogenous molecule and transmit the activation signal to the ii) transmembrane and iii) intracellular signaling domain of the first polypeptide,

[0447] wherein the RD is either bound to the N terminal of the first polypeptide by a peptide bond or is in a separate polypeptide, and wherein if the RD is in a separate polypeptide it further comprises a N terminal split extein and intein so that a trans-splicing reaction is carried out between said polypeptide and the first polypeptide, resulting in the first polypeptide comprising the RD domain in its N terminal.

[0448] c. contacting the cell of b) with an exogenous molecule that is capable of binding to the RD, thereby activating and expanding said cell, and

[0449] d. introducing in the expanded and activated cell of c) a CAR, or a nucleic acid encoding it, preferably the CAR defined in any of items 1 to 8.

[0450] 15. The method according to item 14, wherein step b) comprises the substeps of:

[0451] - b.1) genetically modifying the cell to constitutively express the first polypeptide, and

[0452] - b.2) introducing a polypeptide comprising the RD into the cell of b.1) so that the cell transiently comprises the polypeptide comprising the RD, wherein the RD further comprises a N terminal split extein and intein so that a trans-splicing reaction is carried out between said polypeptide and the first polypeptide constitutively expressed by the cell, resulting in the first polypeptide comprising the RD domain in its N terminal.

[0453] SEQUENCE LISTING

[0454] The following nomenclature has been used in the sequence listing:the different components of the polypeptides are represented below in capital letters and, in order to facilitate its localization, they are depicted by alternating underlying and non-underlying format.

[0455] the symbol () indicates random sequence linkers that are placed in between some of the components of the modules. These are represented in small letter in the sequence, the C1, C2, and N1 split inteins are highlighted in bold in the first and second polypeptide sequences.

[0456] In the mature intein-mediated modular CAR sequences, the leader peptide and the ER retention domain has been removed from the sequence, and the extein “scar” is highlighted in gray.

[0457] SEQ ID NO: 1 C1 terminal split intein and extein:

[0458] MKFKLKEITSIETKHYKGKVHDLTVNQDHSYNVRGTWHNSJC

[0459] SEQ ID NO: 2 N1 terminal split extein and intein:

[0460] GGGCFVPGTLVNTENGLKKIEEIKVGDKVFSHTGKLQEVVDTLIFDRDEEIISINGIDCTKNHE FYVIDKENANRVNEDNIHLFARWVHAEELDMKKHLLIELE

[0461] SEQ ID NO: 3 C2 terminal split intein and extein:

[0462] MMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSSS

[0463] SEQ ID NO: 4 first polypeptide for CAR CD19 / CD20 / CD20+CD19 (construct F in Table 1) MDMRVPAQLLGLLLLWLRGARCdvGGGSGGGSGGGSAWSHPQFEKGGGSGGGSGGSA WSHPQFEKGGGSGGGSGGGLNDIFEAQKI EWH EGGGSGGGSAGGGSGGGGH H H H H Hs sqsprEQKLISEEDLGGGGSGGGGSMKFKLKEITSIETKHYKGKVHDLTVNQDHSYNVRGTV VHNSICspqaepkdvicsmfwvLVVVGGVLACYSLLVTVAFIlFWVRSKRSRGGHSDYMNMTPRR PGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKR RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPR

[0464] Leader (hlqKVIII)-Q-linkerl-Twin Streptaq2-linker-AviTaq-Linker-6HIS-0-myc-linker-lnteinC- ExteinC1-0-CD28

[0465] SEQ ID NO: 5: first polypeptide for CAR BCMA / SLAMF7 / TROP2 (construct A in table 1) MDMRVPAQLLGLLLLWLRGARCdvGGGSGGGSGGGSAWSHPQFEKGGGSGGGSGGSA WSHPQFEKGGGSGGGSGGGLNDIFEAQKI EWH EGGGSGGGSAGGGSGGGGH H H H H Hs sqsprEQKLISEEDLGGGGSGGGGSMKFKLKEITSIETKHYKGKVHDLTVNQDHSYNVRGTV VHNSICspqaEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKmfwvL VVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAA YRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0466] Leader(hlqKVIII)-0-linker1-TwinStreptaa2-linker-AviTag-Linker-6HIS-0-mvc-linker-lnteinC1- ExteinC1-()-lqGi Hinge- lqGiCH2-lgGiCH3-0-CD28

[0467] SEQ ID NO: 6 first polypeptide for CAR BCMA / SLAMF7 / TROP2 (construct B in Table 1) MALPVTALLLPLALLLHAARPEQKLISEEDLdiqfsasqsMKFKLKEITSIETKHYKGKVHDLTVN QDHSYNVRGTWHNSICstMMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSSSasfkpac wESKYGPPCPPCPsIgacdfwyLVVVGGVLACYSLLVTVAFIlFWVRSKRSRGGHSDYMNMTP RRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST ATKDTYDALHMQALPPR

[0468] Leader (CD8a)-mvc-0-inteinC1-ExteinC1-0-inteinC2-ExteinC2-0-lqG4Hinqe-()-CD28-

[0469] SEQ ID NO: 7 first polypeptide for CAR BCMA / SLAMF7 / TROP2 (construct C in Table 1) MALPVTALLLPLALLLHAARPEQKLISEEDLdiqfsasqsMKFKLKEITSIETKHYKGKVHDLTVN QDHSYNVRGTWHNSICstMMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSSSasfkpac wESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGacdfwvLVVVGGVLAC YSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0470] Leader (CD8a)-mvc-

[0471]

[0472] <

[0473] SEQ ID NO: 8 first polypeptide for CAR BCMA / SLAMF7 / TROP2 (construct D in Table 1) MALPVTALLLPLALLLHAARPEQKLISEEDLdiqfsasqsMKFKLKEITSIETKHYKGKVHDLTVN QDHSYNVRGTWHNSICstMMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSSSasfpTT TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDsIgacdfwvLVVVGGVLACYS LLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSA DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0474] Leader (CD8a)-mvc-()-inteinC1 -ExteinC1-Q-inteinC2-ExteinC2-Q- CD8a-Q-CD28-

[0475] SEQ ID NO: 9 first polypeptide for CAR BCMA / SLAMF7 / TROP2 (construct E in Table 1) MALPVTALLLPLALLLHAARPEQKLISEEDLdiqfsasqsMKFKLKEITSIETKHYKGKVHDLTVN QDHSYNVRGTWHNSICstMMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSSSasfkpacdfwvLVVVGGVLACYSLLVTVAFUFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPR DFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0476] Leader (CD8a)-mvc-0-inteinC1-ExteinC1-0-inteinC2-ExteinC2-()-CD28-

[0477] SEQ ID NO: 10 second polypeptide for CAR CD19 (construct 5 in Table 2) METDTLLLWVLLLWVPGSTGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPD GTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTK LEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQ PPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYY GGSYAMDYWGQGTSVTVSSESKYGPPCPPCPGGGCFVPGTLVNTENGLKKIEEIKVGDK VFSHTGKLQEWDTLIFDRDEEIISINGIDCTKNHEFYVIDKENANRVNEDNIHLFARWVHAE ELDMKKHLLIELEGGGGSGGGGSEQKLISEEDLsrppqkmfwvIvHHHHHHHvqqsqqsaaaKDE L

[0478] Leader -VL-Linker-VH-lqG4Hinqe-ExteinN 1 -inteinN1-linker-myc-()-7HIS-0-KDEL

[0479] SEQ ID NO: 11 : second polypeptide for CAR CD20 (construct 6 in Table 2) METDTLLLWVLLLWVPGSTGIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSS PKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKL EIKGSTSGGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWV KQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCAR SNYYGSSYWFFDVWGAGTTVTVSSESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRT PEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRWSVLTVLHQDWLN GKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYT QKSLSLSLGKprpGGGCFVPGTLVNTENGLKKIEElKVGDKVFSHTGKLQEWDTLIFDRDE EIISINGIDCTKNHEFYVIDKENANRVNEDNIHLFARWVHAEELDMKKHLLIELEGGGGSGG GGSEQKLISEEDLsrppqkmfwvIvHHHHHHHvqqsqqsaaaKDEL

[0480] Leader _-Linker-V H-lqG4Hinqe- 1 g G4C H 2-lqG4CH3- () ExteinNI- i n tei n N 1 - linker- my c-Q_-

[0481]

[0482] 7HIS-0-KDEL

[0483] SEQ ID NO: 12: second polypeptide for tandem CAR CD20+CD19 (construct 7 in Table 2) METDTLLLWVLLLWVPGSTGIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSS PKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKL EIKGSTSGGGSGGGSGGGGSSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWV KQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCAR SNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSGGGGSTEVKLQESGPGLV APSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNS KSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGSTSGSGKPGSGE GSTKGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSG VPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITESKYGPPCPPCP GGGCFVPGTLVNTENGLKKIEEIKVGDKVFSHTGKLQEWDTLIFDRDEEIISINGIDCTKNH EFYVIDKENANRVNEDNIHLFARWVHAEELDMKKHLLIELEGGGGSGGGGSEQKLISEEDL

[0484] srppqkmfwvIvHHHHHHHvqqsqqsaaaKDELLeader -V L<CD20)- Linker- VH<CD20)- Linker- VH<CDI 9-Linker-V L<CDI 9)-

[0485]

[0486] lqG4Hinqe-ExteinN1 -inteinN1-linker-myc-()-7HIS-0-KDEL

[0487] SEQ ID NO: 13: second polypeptide for CAR BCMA (construct 1 in Table 2) METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPG KAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTK LEI KGSTSGSGKPGSGEGSTKGQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWM H WV RQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCA RGAIYDGYDVLDNWGQGTLVTVSSGGGCFVPGTLVNTENGLKKIEEIKVGDKVFSHTGKL QEWDTLIFDRDEEIISINGIDCTKNHEFYVIDKENANRVNEDNIHLFARWVHAEELDMKKHL LIELEGGGGSGGGGSEQKLISEEDLsrppqkmfwvIvH H H H H H HvqqsqqsaaaKDEL Leader (lqK)-VL-Linker-VH-ExteinN1-inteinN1-linker-mvc-()-7HIS-0-KDEL

[0488] SEQ ID NO: 14 second polypeptide for CAR SLAMF7 (construct 2 in Table 2) METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPG KVPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSSYPYTFGQGTK VEIGSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVR QAPGKGLEWIGEINPDSSTINYAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDG NYWYFDVWGQGTLVTVSSGGGCFVPGTLVNTENGLKKIEEIKVGDKVFSHTGKLQEWDT LIFDRDEEIISINGIDCTKNHEFYVIDKENANRVNEDNIHLFARWVHAEELDMKKHLLIELEG GGGSGGGGSEQKLISEEDLsrppqkmfwvIvH H H H H H HvqqsqqsaaaKDEL

[0489] Leader (IqK)-V i_-Linker-V H-ExteinN1- i ntei n N 1 - linker- my c-Q-7 H I S-Q- KD E L

[0490] SEQ ID NO: 15: second polypeptide for CAR TROP2 .1 (construct 3 in Table 2) MALPVTALLLPLALLLHAARPEQKLISEEDLDIQLTQSPSSLSASVGDRVSITCKASQDVSIAV AWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITP LTFGAGTKVEIKRGSTSGSG KPGSG EGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTF TNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKAD DTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSGGGCFVPGTLVNTENGLKKIEEIKVGDK VFSHTGKLQEWDTLIFDRDEEIISINGIDCTKNHEFYVIDKENANRVNEDNIHLFARWVHAE ELDMKKHLLIELEGGGGSGGGGSsrppqkmfwvIvH H H H H H HvqqsqqsaaaKDEL Leader (CD8a)- m y c- VL- L i n ke r- VH- Exte i n N 1 -inteinNI- I i n ke r-Q- 7 H I S-Q- K D E L

[0491] SEQ ID NO: 16: second polypeptide for CAR TROP2 .2 (construct 4 in Table 2) MALPVTALLLPLALLLHAARPEQKLISEEDLDIVMTQSPASLAVSLGQRATISCRASQSVSTSS YSYMHWYQQKPGQPPKLLIKYASNLECGVPARFSGSGCGTDFTLNIHPVEEEDSATYYCQH SREI PLTFGAGTKLEI KRGSTSGSG KPGSG EGSTKGDVQLEQFGAELVRPGTSVKMSCKAA GYTFTNYWIGWVKQRPGHGLEWIGDIYPGGGYTNYNEKFKGKATLTADTSSSTAYMQLSSL TFEDFAIYYCARGTGGGDYWGQGTLVTVSSGGGCFVPGTLVNTENGLKKIEEIKVGDKVFSHTGKLQEWDTLIFDRDEEIISINGIDCTKNHEFYVIDKENANRVNEDNIHLFARWVHAEELD MKKHLLIELEGGGGSGGGGSsrppqkmfwvIvH H H H H H HvqqsqqsaaaKDEL

[0492] Leader (CD8a)- m y c- VL- L i n ke r- VH- Exte i n N 1 -inteinNI- I i n ke r-Q- 7 H I S-Q- K D E L

[0493] SEQ ID NO: 17: mature intein-mediated modular CAR CD19 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRF SGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTK GEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYY NSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS ESKYGPPCPPCPGGGSICspqaepkdvicsmfwvLVVVGGVLACYSLLVTVAFIlFWVRSKRSRG GHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELN LGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR VL-Linker-VH-lqG4Hinqe-ExteinN1 -ExteinC1-0-CD28-

[0494] SEQ ID NO: 18: mature intein-mediated modular CAR CD20 IVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFS GSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSGGGSGGGG SSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGD TSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTT VTVSSESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFQSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKprpGGGSICsp qaepkdvicsmfwvLVVVGGVLACYSLLVTVAFUFWVRSKRSRGGHSDYMNMTPRRPGPTRKH YQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR V L-Linker-V H-lqG4Hinqe- 1 gG4C H 2-lqG4CH3- ()- ExteinNI- ExteinCI- 0-CD28-

[0495] SEQ ID NO: 19: mature intein-mediated modular DUAL CAR CD20 / CD19 IVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFS GSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSGGGSGGGG SSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGD TSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTT VTVSSGGGGSGGGGSGGGGSGGGGSTEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYY CAKHYYYGGSYAMDYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSASL GDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISN LEQEDIATYFCQQGNTLPYTFGGGTKLEITESKYGPPCPPCPGGGSICspqaepkdvicsmfwvL VVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAA YRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRVL(CD2o Linker-VHfCD2o Linker-VHfCDi9 Linker-VL(CDi9 lqG4Hinqe-ExteinN1-ExteinC1-O-CD28-

[0496] SEQ ID NO: 20: mature intein-mediated modular CAR BCMA-stemA DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKGSTSGSGKPGSGEGST KGQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGH SDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTL VTVSSGGGSICspqaEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GKmfwvLVVVGGVLACYSLLVTVAFIlFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYA PPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR V L-Linker-V H-ExteinN1-ExteinC1- ()- IqGiHinqe- 1 gG 1 C H 2-lqGiCH3- 0-CD28-

[0497] SEQ ID NO: 21: mature intein-mediated modular CAR BCMA-stemB DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKGSTSGSGKPGSGEGST KGQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGH SDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTL VTVSSGGGSICstMMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSSSasfkpacwESKYG PPCPPCPsIgacdfwvLWVGGVLACYSLLVTVAFIlFWVRSKRSRGGHSDYMNMTPRRPGPT RKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR VL-Linker-VH-ExteinN1-ExteinC1-0-inteinC2-ExteinC2-0-lqG4Hinqe-()-CD28-

[0498] SEQ ID NO: 22: mature intein-mediated modular CAR BCMA-stemC DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKGSTSGSGKPGSGEGST KGQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGH SDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTL VTVSSGGGSICstMMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSSSasfkpacwESKYG PPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGacdfwvLVVVGGVLACYSLLVTV AFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRVL-Linker-VH-ExteinN1-ExteinC1-0-inteinC2-ExteinC2-0-laG4Hinae-laG4CH2-laG4CH3-0- CD28-C

[0499] SEQ ID NO: 23: mature intein-mediated modular CAR BCMA-stemD DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKGSTSGSGKPGSGEGST KGQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGH SDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTL VTVSSGGGSICstMMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSSSasfpTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDsIgacdfwvLVVVGGVLACYSLLVTVAF IIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQ QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL-Linker-VH-ExteinN1-ExteinC1-0-inteinC2-ExteinC2-Q- CD8a-Q-CD28-

[0500] SEQ ID NO: 24: mature intein-mediated modular CAR BCMA-stemE DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKGSTSGSGKPGSGEGST KGQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGH SDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTL VTVSSGGGSICstMMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSSSasfkpacdfwvLVV VGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYR SRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGL YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL-Linker-VH-ExteinN1-ExteinC1-0-inteinC2-ExteinC2-()-CD28-

[0501] SEQ ID NO: 25: mature intein-mediated modular CAR SLAMF7-stemA DIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPGKVPKLLIYWASTRHTGVPDR FSGSGSGTDFTLTISSLQPEDVATYYCQQYSSYPYTFGQGTKVEIGSTSGSGKPGSGEGST KGEVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTI NYAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVTVSS GGGSICspqaEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKmfwv LVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFA AYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR V L-Linker-V H-ExteinN1-ExteinC1- ()- IqGiHinqe- 1 gG 1 C H 2-lqGiCH3- 0-CD28-

[0502] SEQ ID NO: 26: mature intein-mediated modular CAR SLAMF7-stemBDIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPGKVPKLLIYWASTRHTGVPDR FSGSGSGTDFTLTISSLQPEDVATYYCQQYSSYPYTFGQGTKVEIGSTSGSGKPGSGEGST KGEVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTI NYAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVTVSS GGGSICstMMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSSSasfkpacwESKYGPPCP PCPsIgacdfwvLVVVGGVLACYSLLVTVAFIlFWVRSKRSRGGHSDYMNMTPRRPGPTRKHY QPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR VL-Linker-VH-ExteinN1-ExteinC1-0-inteinC2-ExteinC2-0-laG4Hinge-0-CD28-

[0503] SEQ ID NO: 27: mature intein-mediated modular CAR SLAMF7-stemC DIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPGKVPKLLIYWASTRHTGVPDR FSGSGSGTDFTLTISSLQPEDVATYYCQQYSSYPYTFGQGTKVEIGSTSGSGKPGSGEGST KGEVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTI NYAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVTVSS GGGSICstMMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSSSasfkpacwESKYGPPCP PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGacdfwvLVVVGGVLACYSLLVTVAFIIF WVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQG QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL-Linker-VH-ExteinN1-ExteinC1-0-inteinC2-ExteinC2-0-laG4Hinae-laG4CH2-laG4CH3-0- CD28-C

[0504] SEQ ID NO: 28: mature intein-mediated modular CAR SLAMF7-stemD DIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPGKVPKLLIYWASTRHTGVPDR FSGSGSGTDFTLTISSLQPEDVATYYCQQYSSYPYTFGQGTKVEIGSTSGSGKPGSGEGST KGEVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTI NYAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVTVSS GGGSICstMMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSSSasfpTTTPAPRPPTPAPT lASQPLSLRPEACRPAAGGAVHTRGLDFACDsIgacdfwvLVVVGGVLACYSLLVTVAFHFWVR SKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL-Linker-VH-ExteinN1-ExteinC1-0-inteinC2-ExteinC2-Q- CD8a-Q-CD28-

[0505] SEQ ID NO: 29: mature intein-mediated modular CAR SLAMF7-stemE DIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPGKVPKLLIYWASTRHTGVPDR FSGSGSGTDFTLTISSLQPEDVATYYCQQYSSYPYTFGQGTKVEIGSTSGSGKPGSGEGST KGEVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVTVSS GGGSICstMMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSSSasfkpacdfwvLVVVGGVL ACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKF SRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL-Linker-VH-ExteinN1-ExteinC1-0-inteinC2-ExteinC2-()-CD28-

[0506] SEQ ID NO: 30: mature intein-mediated modular CAR TROP2 .1-stemA EQKLISEEDLDIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASY RYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRGSTSGSG KPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKW MGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYF DVWGQGSLVTVSSGGGSICspgaEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIAR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGKmfwvLVVVGGVLACYSLLVTVAFIlFWVRSKRSRGGHSDYMNMTPRRPGPT RKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR

[0507] myc-VL-Linker-VH-ExteinN1 -ExteinC1-0-lgGi Hinge- lgGiCH2-lgGiCH3-0-CD28-

[0508] SEQ ID NO: 31 : mature intein-mediated modular CAR TROP2 .1-stemB EQKLISEEDLDIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASY RYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRGSTSGSG KPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKW MGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYF DVWGQGSLVTVSSGGGSICstMMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSSSasfk gacwESKYGPPCPPCPsIgacdfwvLVVVGGVLACYSLLVTVAFUFWVRSKRSRGGHSDYMNM TPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL STATKDTYDALHMQALPPR

[0509] myc-VL-Linker-VH-ExteinN1-ExteinC1-0-inteinC2-ExteinC2-0-lgG4Hinge-0-CD28-C

[0510] SEQ ID NO: 32: mature intein-mediated modular CAR TROP2 .1-stemC EQKLISEEDLDIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASY RYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRGSTSGSG KPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKW MGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYF DVWGQGSLVTVSSGGGSICstMMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSSSasfk pacwESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGacdfwvLVVVGGVL ACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKF SRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0511] myc-VL-Linker-VH-ExteinN1-ExteinC1-0-inteinC2-ExteinC2-0-lqG4Hinqe-lqG4CH2-lqG4CH3- 0-CD28-

[0512] SEQ ID NO: 33: mature intein-mediated modular CAR TROP2 .1-stemD EQKLISEEDLDIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASY RYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRGSTSGSG KPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKW MGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYF DVWGQGSLVTVSSGGGSICstMMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSSSasf gTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDsIgacdfwvLVVVGGVLA CYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFS RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0513] myc-VL-Linker-VH-ExteinN1 -ExteinC1-Q-inteinC2-ExteinC2-Q- CD8a-Q-CD28-

[0514] SEQ ID NO: 34: mature intein-mediated modular CAR TROP2 .1-stemE EQKLISEEDLDIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASY RYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRGSTSGSG KPGSGEGSTKGQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKW MGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYF DVWGQGSLVTVSSGGGSICstMMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSSSasfk eacdfwvLVVVGGVLACYSLLVTVAFUFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYA PPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR

[0515] myc-VL-Linker-Vn-ExteinNI -ExteinC1-()-inteinC2-ExteinC2-()-CD28-

[0516] SEQ ID NO: 35: mature intein-mediated modular CAR TROP2 .2-stemA EQKLISEEDLDIVMTQSPASLAVSLGQRATISCRASQSVSTSSYSYMHWYQQKPGQPPKLLI KYASNLECGVPARFSGSGCGTDFTLNIHPVEEEDSATYYCQHSREIPLTFGAGTKLEIKRGS TSGSG KPGSG EGSTKGDVQLEQFGAELVRPGTSVKMSCKAAGYTFTNYWIGWVKQRPGH GLEWIGDIYPGGGYTNYNEKFKGKATLTADTSSSTAYMQLSSLTFEDFAIYYCARGTGGGDY WGQGTLVTVSSGGGSICsDqaEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGKmfwvLVVVGGVLACYSLLVTVAFIlFWVRSKRSRGGHSDYMNMTPRRPGPTRK HYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR

[0517] myc-VL-Linker-Vn-ExteinNI -ExteinC1-0-lqGi Hinge- lgGiCH2-lqGiCH3-0-CD28-

[0518] SEQ ID NO: 36: mature intein-mediated modular CAR TROP2 .2-stemB EQKLISEEDLDIVMTQSPASLAVSLGQRATISCRASQSVSTSSYSYMHWYQQKPGQPPKLLI KYASNLECGVPARFSGSGCGTDFTLNIHPVEEEDSATYYCQHSREIPLTFGAGTKLEIKRGS TSGSG KPGSG EGSTKGDVQLEQFGAELVRPGTSVKMSCKAAGYTFTNYWIGWVKQRPGH GLEWIGDIYPGGGYTNYNEKFKGKATLTADTSSSTAYMQLSSLTFEDFAIYYCARGTGGGDY WGQGTLVTVSSGGGSICstMMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSSSasfkpa cwESKYGPPCPPCPsIgacdfwyLWVGGVLACYSLLVTVAFIlFWVRSKRSRGGHSDYMNMTP RRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST ATKDTYDALHMQALPPR

[0519] myc-VL-Linker-VH-ExteinN1-ExteinC1-0-inteinC2-ExteinC2-0-lgG4Hinge-0-CD28-C

[0520] SEQ ID NO: 37: mature intein-mediated modular CAR TROP2 .2-stemC EQKLISEEDLDIVMTQSPASLAVSLGQRATISCRASQSVSTSSYSYMHWYQQKPGQPPKLLI KYASNLECGVPARFSGSGCGTDFTLNIHPVEEEDSATYYCQHSREIPLTFGAGTKLEIKRGS TSGSG KPGSG EGSTKGDVQLEQFGAELVRPGTSVKMSCKAAGYTFTNYWIGWVKQRPGH GLEWIGDIYPGGGYTNYNEKFKGKATLTADTSSSTAYMQLSSLTFEDFAIYYCARGTGGGDY WGQGTLVTVSSGGGSICstMMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSSSasfkpa cwESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGacdfwvLWVGGVLA CYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFS RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0521] myc-Vi.-Linker-VH-ExteinN1-ExteinC1-()-inteinC2-ExteinC2-()-lgG4Hinge-lgG4CH2-lgG4CH3-0-CD28-

[0522] SEQ ID NO: 38: mature intein-mediated modular CAR TROP2 .2-stemD EQKLISEEDLDIVMTQSPASLAVSLGQRATISCRASQSVSTSSYSYMHWYQQKPGQPPKLLI KYASNLECGVPARFSGSGCGTDFTLNIHPVEEEDSATYYCQHSREIPLTFGAGTKLEIKRGS TSGSG KPGSG EGSTKGDVQLEQFGAELVRPGTSVKMSCKAAGYTFTNYWIGWVKQRPGH GLEWIGDIYPGGGYTNYNEKFKGKATLTADTSSSTAYMQLSSLTFEDFAIYYCARGTGGGDY WGQGTLVTVSSGGGSICstMMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSSSasfpTT TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDsIgacdfwvLVVVGGVLACYS LLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSA DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0523] myc-VL-Linker-VH-ExteinN1 -ExteinC1-Q-inteinC2-ExteinC2-Q- CD8a-Q-CD28-SEQ ID NO: 39: mature intein-mediated modular CAR TROP2 .2-stemE EQKLISEEDLDIVMTQSPASLAVSLGQRATISCRASQSVSTSSYSYMHWYQQKPGQPPKLLI KYASNLECGVPARFSGSGCGTDFTLNIHPVEEEDSATYYCQHSREIPLTFGAGTKLEIKRGS TSGSG KPGSG EGSTKGDVQLEQFGAELVRPGTSVKMSCKAAGYTFTNYWIGWVKQRPGH GLEWIGDIYPGGGYTNYNEKFKGKATLTADTSSSTAYMQLSSLTFEDFAIYYCARGTGGGDY WGQGTLVTVSSGGGSICstMMLKKILKIEELDERELIDIEVSGNHLFYANDILTHNSSSasfkpa cdfwvLVVVGGVLACYSLLVTVAFUFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPP RDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP

[0524] R

[0525] myc-VL-Linker-VH-ExteinN1 -ExteinC1-()-inteinC2-ExteinC2-()-CD28-

[0526] SEQ ID NO: 40 ER retention motif: KDEL

[0527] SEQ ID NO: 41 : 7His tag: HHHHHHH

[0528] SEQ ID NO: 42: linker 1 (GGGS)3: GGGSGGGSGGGS

[0529] SEQ ID NO: 43: linker 2 (GGGS)2: GGGSGGGS

[0530] SEQ ID NO: 44 CD28:

[0531] LVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFA AYRS

[0532] SEQ ID NO: 45 CD3^:

[0533] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 46 (CD19, VL):

[0534] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRF SGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT

[0535] SEQ ID NO: 47 (CD19, VH):

[0536] EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYN SALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

[0537] SEQ ID NO: 48 (CD20, VL):IVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFS GSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIK

[0538] SEQ ID NO: 49 (CD20, VH):

[0539] EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTS YNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVT VSS

[0540] SEQ ID NO: 50 (CD20+CD19):

[0541] IVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFS GSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGSTSGGGSGGGSGGGG SSEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGD TSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTT VTVSSGGGGSGGGGSGGGGSGGGGSTEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYY CAKHYYYGGSYAMDYWGQGTSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSASL GDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISN LEQEDIATYFCQQGNTLPYTFGGGTKLEIT

[0542] SEQ ID NO: 51 (BCMA, VL):

[0543] DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIK

[0544] SEQ ID NO: 52 (BCMA, VH):

[0545] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSD TYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTLVTV SS

[0546] SEQ ID NO: 53 (SLAMF7, VL):

[0547] DIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPGKVPKLLIYWASTRHTGVPDR FSGSGSGTDFTLTISSLQPEDVATYYCQQYSSYPYTFGQGTKVEI

[0548] SEQ ID NO: 54 (SLAMF7, VH):

[0549] EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINY APSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVTVSS

[0550] SEQ ID NO: 55 (TROP2 .1, VL):DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRF SGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKR

[0551] SEQ ID NO: 56 (TROP2 .1, VH):

[0552] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEP TYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTV SS

[0553] SEQ ID NO: 57 (TROP2 .2, VL):

[0554] DIVMTQSPASLAVSLGQRATISCRASQSVSTSSYSYMHWYQQKPGQPPKLLIKYASNLECG VPARFSGSGCGTDFTLNIHPVEEEDSATYYCQHSREIPLTFGAGTKLEIKR

[0555] SEQ ID NO: 58 (TROP2 .2, VH):

[0556] DVQLEQFGAELVRPGTSVKMSCKAAGYTFTNYWIGWVKQRPGHGLEWIGDIYPGGGYTNY NEKFKGKATLTADTSSSTAYMQLSSLTFEDFAIYYCARGTGGGDYWGQGTLVTVSS

[0557] SEQ ID NO: 59 IgGiHinge- lgGiCH2-lgGiCH3(A):

[0558] EPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0559] SEQ ID NO: 60 lgG4Hinge (B): ESKYGPPCPPCP

[0560] SEQ ID NO: 61 lgG4Hinge-lgG4CH2-lgG4CH3 (C):

[0561] ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0562] SEQ ID NO: 62 CD8a (D):

[0563] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0564] SEQ ID NO: 63 leader peptide: MALPVTALLLPLALLLHAARP

[0565] SEQ ID NO: 64 leader peptide: MDMRVPAQLLGLLLLWLRGARCSEQ ID NO: 65 Leader peptide: METDTLLLWVLLLWVPGSTG

[0566] SEQ ID NO: 66 linker 3 (GGGS)4: GGGSGGGSGGGSGGGS

[0567] The invention is described below by the following examples, which must be considered as merely illustrative and in no case limiting of the scope of the present invention.

[0568] EXAMPLES EXAMPLE 1: Dual Targeting of BCMA and SLAMF7 with the CARtein System: Chimeric Antigen Receptors with Intein-mediated Splicing Elicit Specific T Cell Activation against Multiple Myeloma

[0569] Materials and methods

[0570] Protein structure prediction and protein docking

[0571] In order to facilitate and simplify the rational design of the CARtein modules, we performed protein structure predictions of these constructs. All protein folding predictions were run with ColabFold open-source software (32). In order to improve the accuracy, the prediction of sequences containing parts of the IMPDH-1 split intein were carried out with the crystal structure of gp41-1 intein (PDB id: 6QAZ) used as template. First, we predicted the tridimensional structure of the extracellular domain of the signaling CARtein module (SCM) containing a CD28 transmembrane (TM) domain and an IgGi spacer followed by the C-terminal part of IMPDH-1 split intein (including the C-intein and C-extein). Then, we predicted the same structure without the C-terminal part of IMPDH-1 with the aim of assessing how much the split intein impacts on the final structure. The same strategy was employed to predict the antigen recognition modules folding. These modules included either an anti-BCMA (Belantamab) or an anti-SLAMF7 (Elotuzumab) scFv fused to the N-terminal region of IMPDH-1. This analysis was also extended to constructs lacking the split intein.

[0572] For the purpose of evaluating whether the partners of IMPDH-1 split inteins in the CARtein modules would have no steric hindrance for performing protein splicing, the antigen recognition modules directed against BCMA or SLAMF7 were docked with the SCM using HADDOCK2.4 modeling platform (Honorato RV, T relief ME, Jimenez-Garcia B, Schaarschmidt J J, Giulini M, Reys V, Koukos PI, Rodrigues JPGLM, Karaca E, van Zundert GCP, et al. The HADDOCK2.4 web server for integrative modeling of biomolecular complexes. Nat Protoc (2024) doi: 10.1038 / s41596-024-01011-0). Moreover, we performed a predictionof the antigen-CARtein complexes after protein splicing with ColabFold, using the structure of CD28 transmembrane domain as template (PDB id: 7VLI5), for comparing the interaction between the scFv and the extracellular domain (ECD) of BCMA (H3BMB5_HUMAN: 1-69aa) or SLAMF7 (SLAF7_HUMAN:23-247aa) without the residual exteins (GGG-SIC). Protein structures were analyzed and rendered with the 3D Protein Imager interface ( Tomasello G, Armenia I, Molla G. The Protein Imager: a full-featured online molecular viewer interface with server-side HQ-rendering capabilities. Bioinformatics (2020) 36:2909-2911. doi: 10.1093 / bioinformatics / btaa009).

[0573] Cell Culture

[0574] Human Embryonic Kidney (HEK Lenti-XTM 293T) (Clontech) cell line was used for lentiviral vector production. A Jurkat T JE6.1 (Jurkat) cell subline known as Jurkat-TPR (Triple Parameter Reporter) expresses eGFP, CFP and mCherry, respectively governed by NFAT, NFKB and AP-1 promoters was established by Prof. Steinberger’s group (Jutz S, Leitner J, Schmetterer K, Doel-Perez I, Majdic O, Grabmeier-Pfistershammer K, Paster W, Huppa JB, Steinberger P. Assessment of costimulation and coinhibition in a triple parameter T cell reporter line: Simultaneous measurement of NF-KB, NFAT and AP-1. Journal of Immunological Methods (2016) 430:10-20. doi: 10.1016 / j.jim.2016.01.007) and previously used by us and others to study CAR T-cell activation signaling. The human multiple myeloma (MM) cell line MM.1S was used as SLAMF7+ / BCMA+ target in activation signaling assays. Lastly, a chronic myelogenous leukemia K562 cell line expressing BCMA and / or SLAMF7 was generated to be used as targets in activation signaling assays. All human cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA).

[0575] Human Embryonic Kidney (HEK Lenti-XTM 293T) (Clontech) as well as Jurkat-TPR cell lines were cultured in DMEM GlutaMax medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% sodium pyruvate, 2 mM L-glutamine, 10 mM HEPES, 50 pM 2-mercaptoethanol (Invitrogen, Carlsbad, CA, USA), 100 units / mL penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA) and 50pg / ml Gentamicin (Gibco) and at 37 °C and 10% CO2. MM.1s cell line was cultured in RPMI 1640 medium (Gibco) equally supplemented at 37 °C and 5% CO2. Genetically modified K562 cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM) (Gibco) supplemented the same way as DMEM GlutaMax and RPMI media at 37 °C and 5% CO2.

[0576] Design of CARtein constructs

[0577] The signaling CARtein module (SCM) contained the same spacer, transmembrane and intracellular domains as our previously reported ACE2-CAR, including a CD3 intracellular domain, a CD28 transmembrane domain mutated for improved membrane expression, aCD28 co-stimulatory domain, and a human IgGi heavy chain spacer spaning hinge, CH2 and CH3 domain, mutated to prevent FC receptor activation. Upstream SCM, the C-terminal IMPDH-1 split intein (including the C-intein and C-extein) was engineered, preceded by a Twin-Strep-tag® (tST) connected through a flexible (GGGGS)2 linker (SEQ ID NO: 43) and a hlgKVIH leader sequence. The SCM construct was synthetized and codon-optimized by GeneArt™ (Thermo Fisher Scientific Inc., Carlsbad, CA, USA) and cloned via a LR Gateway™ reaction into the lentiviral vector pHRSINcPPT CEW, downstream a SFFV promoter.

[0578] Two different antigen recognition modules were generated to interact and bind to the SCM via a peptide bond mediated by split intein protein splicing. Antigen recognition modules contained the complementary N-terminal IMPDH-1 split intein and either an anti-SLAMF7 scFv (Elotuzumab) or anti-BCMA scFv (Belantamab). Both antigen recognition modules were followed by a flexible (GGGGS)2 linker (SEQ ID NO: 43) and an in-frame KDEL sequence, responsible for sequestering these modules in the endoplasmic reticulum (ER) until the interaction with the N-terminal IMPDH-1 with its C-terminal partner in the SCM, occurs ensuing in the mature CAR molecule. These antigen recognition modules were synthetized and codon-optimized by GeneArt™ (Thermo Fisher Scientific Inc., Carlsbad, CA, USA) and cloned via a LR Gateway™ reaction into the expression lentiviral vector pLEX_307 (Addgene plasmid 41392), under the control of an EF-1a promoter.

[0579] Design of SLAMF7 and BCMA Surface antigens

[0580] SLAMF7 and BCMA extracellular and transmembrane domains coding sequences were synthetized by GeneArt™ (Thermo Fisher Scientific Inc., Carlsbad, CA, USA) and then cloned via a LR Gateway™ reaction into the expression lentiviral vector pLEX_307 (Addgene plasmid 41392), governed by an EF-1a promoter.

[0581] Lentiviral vector production

[0582] HEK Lenti-XTM 293T cells were used to produce all lentiviral supernatants. These cells were co-transfected with the corresponding transfer vector, together with plasmids pMD2.G, encoding for the Vesicular Stomatitis Virus G protein (VSVG) and pCMVAR8.91 , encoding for HIV-1 GAG and POL proteins. Polyethylenimine (PEI)-mediated transfection was performed in OptiMEM™ medium (Thermo Fisher Scientific Inc., Carlsbad, CA, USA) according to the method optimized by Tang et al., 2015. Lentiviral supernatants were collected at 48 and 72 h, centrifuged for cell debris removal and concentrated with a lentivirus concentrator solution containing 40% (W / V) PEG-8000 and 1.2M NaCI, according to the 4xLentivirus Concentrator Solution protocol facilitated by the University of Texas M.D. Anderson Cancer Center. The high-titer virus-containing pellets were frozen at -80°C until use and viral titers were determined by evaluating transduction efficiency in Jurkat cells.Cell transduction

[0583] Jurkat-TPR or K562 cells were first cultured for 24h in fresh media, then virus-containing pellets were thawed and resuspended in culture media. 48h after transduction, cells were centrifuged and plated in fresh media. K562 cells (5x105cells / ml) transduced with either BCMA or SLAMF7 expressing lentiviral vectors, were selected by addition of 0.5 .g / ml puromycin Dihydrochloride (Gibco). SLAMF7+ K562 cells were then transduced and re-selected for BCMA ECD expression. Surface antigens expression in K562 cells was analyzed by FACs 72h after selection.

[0584] Jurkat-TPR cells (3x105cells / ml) transduced with lentiviral vectors containing SCM constructs, were selected 48h after transduction by culturing in 40 .g / ml of Blasticidin S HCI (Gibco). SCM expression in Jurkat-TPR transduced cells was evaluated by FACs 72 h after selection. SCM+ Jurkat TPR cells were then transduced by either or both antigen recognition modules directed against BCMA and / or SLAMF7. After 48h, transduced cells were selected through the addition of 0.125ug / ml of puromycin Dihydrochloride (Gibco) to the cell culture media at a final concentration of 3x105cells / ml. CARtein module expression in Jurkat-TPR cells were evaluated by FACs 72h after selection.

[0585] T cell activation signaling assays

[0586] Jurkat-TPR cells expressing the SCM and the final CARtein sequences were co-cultured (105cells / well) with MM.1s target cells at different target to effector (T:E) ratios (1:1, 1:5 and 1:10) in 96-well flat-bottomed plates in a final supplemented DMEM volume of 200ul. Cells were then incubated at 37°C and 10% CO2. T cell activation signaling was measured at three different time points: Oh (unstimulated), 24h and 48h after stimulation. The signal of Jurkat-TPR promoter reporters as well as CD69 upregulation were analyzed for each time point. To further assess CARtein specificity, target K562 cells either expressing BCMA or SLAMF7 ECD, or both (SLAMF7+ / BCMA+ K562) were co-cultured with Jurkat-TPR cells expressing the SCM and the final CARtein sequences (105cells / well) at T:E ratio of 1:1 in 96-well flat-bottomed plates at a final volume of 200ul of supplemented DMEM. Cells were incubated at 37°C and 10% CO2. Jurkat-TPR activation signaling was equally measured at three different time points: Oh (unstimulated), 24h and 48h after stimulation. Signal of NFAT and NFKB promoter reporters as well as CD69 upregulation were analyzed for each measurement. Flow cytometry

[0587] Expression of SCM and anti-BCMAoranti-SLAMF7 post-splicing CARtein sequence in Jurkat-TPR cells were evaluated by staining with Strep-Tactin®XT DY-649 (Iba-Lifesciences, #2-1568-050), Brilliant Violet 421™ anti-human IgG Fc Antibody (Biolegend, San Diego, CA, USA) and Biotinylated Recombinant Protein L Protein, His, Avitag™ (ACROBiosystems, RPL-P81Q7) followed by PE-conjugated streptavidin™ (Thermo Fisher Scientific Inc., Carlsbad, CA, USA). Zombie Violet™ Fixable Viability Kit (Biolegend, San Diego, CA, USA) was used to assess live cells, except for cells stained with Brilliant Violet 421™ anti-human IgG Fc Antibody, for which Zombie NIR™ Fixable Viability Kit (Biolegend, San Diego, CA, USA) was used.

[0588] In order to prevent unwanted binding of antibodies to human FC receptors in K562 and MM.1s cells, they were previously blocked with FcR Blocking Reagent, human (Miltenyi Biotec, Cologne, Germany). Expression of BCMA and SLAMF7 surface antigens in MM.1s cells and transduced K562 cells were evaluated by staining with APC anti-human CD269 (BCMA) Antibody (Biolegend, San Diego, CA, USA) and PE / Cyanine7 anti-human CD319 (CRACC) Antibody (Biolegend, San Diego, CA, USA) respectively. For T cell activation signaling assays analysis, Jurkat-TPR cells were stained with anti-human CD69-APC and CD3-PerCP / Cy5.5 antibodies (Biolegend, San Diego, CA, USA), in order to discriminate CARtein-TPR cells from target cells and evaluate CD69 expression. Cells were also stained with Zombie Violet™ Fixable Viability Kit (Biolegend, San Diego, CA, USA) for live cells determination. Flow cytometry was performed on Cytek® Aurora 5L 16UV-16V-14B-10YG-8R spectral cytometer. Data was collected using SpectroFlo software (Cytek Biosciences) and analysed using FlowJo software V10.1 (TreeStar Inc., Olten, Switzerland).

[0589] Statistical analysis

[0590] Statistical analyses were performed with GraphPad Prism v.9.0.2 (GraphPad, La Jolla, CA, USA). Data are presented as the mean ± standard errors from triplicates. Two-way ANOVA was used for statistical differences comparison among multiple groups followed by a Tukey’s multiple comparison test, p value of less than 0.05 was considered significant, p value significance levels are indicated in the figures (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

[0591] Results

[0592] CARtein structure prediction

[0593] In order to evaluate beforehand whether the integration of split intein modules into mature chimeric antigen receptor would distort protein folding, therefore impairing its functionality, we performed structure and complex predictions with ColabFold software, based on the deep learning model of AlphaFold2 (Mirdita M, Schutze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M. ColabFold: making protein folding accessible to all. Nat Methods (2022) 19:679-682. doi: 10.1038 / s41592-022-01488-1; Jumper J, Evans R, Pritzel A, Green T, Figurnov M, RonnebergerO, Tunyasuvunakool K, Bates R, ZidekA, Potapenko A, etal. Highly accurate protein structure prediction with AlphaFold. Nature (2021) 596:583-589. doi:10.1038 / s41586-021-03819-2). AlphaFold2-based software is capable of performing highly accurate protein structure predictions, and is emerging as a powerful and useful tool in rational protein design, including chimeric antigen receptors development.

[0594] First, we wanted to evaluate whether the integration of IMPDH-1 split intein parts into our CARtein modules would affect their folding and therefore, functionality. We found that structure predictions of anti-BCMA (Belantamab) and anti-SLAMF7 (Elotuzumab) scFvs contained within the antigen recognition modules were sufficiently well-preserved after the addition of the N-terminal part of IMPDH-1 (Figures 2A,B) when comparing it with the unmodified scFv predicted conformation. Furthermore, the N-terminal section of the split intein was observed to be relatively accessible to the split intein partner, and the structural configuration of complementarity-determining region (CDR) loops of both Belantamab and Elotuzumab were comparatively well-preserved. Moreover, we performed a folding prediction of the extracellular and transmembrane domains of the signaling CARtein module (SCM), which comprises a CD28 transmembrane (TM) domain and the IgGi spacer coupled to the C-terminal part of IMPDH-1 split intein ((Figure 2C). The SCM folding conformation in the presence of intein moiety was found to be well conserved when compared with the intein-devoid sequence. In addition, the C-terminal part of IMPDH-1 appeared to be accessible for its split intein partner as well.

[0595] The following step was to analyze whether the IMPDH-1 split intein partner embedded within the CARtein modules were sufficiently accessible for protein splicing. For this purpose, the predicted structure of the antigen recognition modules directed against BCMAorSLAMF7 was docked with the predicted conformation of the SCM using HADDOCK2.4 protein-protein docking platform (Honorato RV, Trellet ME, Jimenez-Garcia B, Schaarschmidt J J, Giulini M, Reys V, Koukos PI, Rodrigues JPGLM, Karaca E, van Zundert GCP, et al. The HADDOCK2.4 web server for integrative modeling of biomolecular complexes. Nat Protoc (2024) doi: 10.1038 / s41596-024-01011-0). It was noted that in both cases, the anti-BCMA and anti-SLAMF7 antigen recognition modules appeared to exhibit an accessible split intein N-terminal region, thereby facilitating engagement with its C-terminal partner contained within the SCM, with no discernible major steric hindrances (Figures 3A,B).

[0596] Following the split intein-mediated protein splicing process, the N-extein (GGG) domain of IMPDH-1, which is embedded within the antigen recognition module, will be covalently linked to the SCM C-extein (SIC) through a peptide bond, resulting in the exclusion of the N-intein and C-intein complex from the final anti-BCMA or SLAMF7 CARtein sequence. Accordingly, to assess the potential influence of residual exteins, encompassing six residues (GGG-SIC), on the spliced anti-BCMA and anti-SLAMF7 CARtein structure on scFv-mediated surfaceantigen recognition, we conducted a prediction of the antigen-CARtein complexes following protein splicing with ColabFold. Structure prediction was performed on the extracellular domain (ECD) of BCMAand SLAMF7, in complex with post-protein spliced CARtein directed against either BCMA or SLAMF7. Additionally, CARtein constructs lacking residual exteins were analyzed. (Figures 3C, D). Comparison of the complex structure prediction between CARtein constructs, in the presence or absence of intervening exteins, suggests that antigen proximal residues in Belantamab and Elotuzumab maintain their relative proximity. Consequently, it can be reasonably deduced that scFv functionality, and therefore CAR activity, should not be adversely affected.

[0597] CARtein system design and establishment of CARtein-TPR cells.

[0598] In order to generate split intein-mediated modular chimeric antigen receptors (CARteins) against BCMA and SLAMF7, three different CARtein modules were designed, including two distinct antigen recognition modules targeting each surface antigen, both contained IMPDH-1 N-terminal portion (encompassing the N-intein-extein), and one signaling CARtein module (SCM), which contained the C-terminal intein (including the C-intein-extein), followed by the IgGi spacer, CD28 transmembrane, and intracellular domains and a CD3 intracellular domain. A Twin-Strep-tag® (tST) sequence was positioned right after the hlgKVHI leader sequence, upstream the C-terminal intein, for easy detection of the unsplit SCM module (Figure 4A).

[0599] The antigen recognition modules comprised the scFv (either Belantamab targeting BCMA or Elotuzumab targeting SLAMF7) followed by the N-terminal part of IMPDH-1 and an in-frame Lys-Asp-Glu-Leu (KDEL) sequence. The objective was to confine these modules within the endoplasmic reticulum (ER) until protein splicing via split inteins occurred, as KDEL receptors (KDELRs) retro-transport KDEL-bearing proteins from the Golgi to the ER. They were designed in such a way that the anti-BCMA and SLAMF7 CARtein constructs following intein-mediated protein splicing, would lose the tST, the KDEL sequence and the split inteins, except for the six residue exteins (GGG-SIC) that are part of the final CARtein sequence. As a consequence of KDEL sequence loss, the spliced CARteins would be able to migrate outside the ER and be expressed on the surface membrane (Figure 4A).

[0600] All three constructs were cloned into lentiviral vectors and then expressed in JKT-TRP to evaluate stimulation mediated activation of Nuclear factor-KB (NFKB) and Nuclear factor of activated T-cells (NFAT), as the synthetic promoters respectively control expression of reporter CFP and eGFP. Jurkat TPR cell line has been previously validated as an efficient platform for the evaluation of CAR functionality. Jurkat-TPR cells were transduced with lentiviral vectors containing the SCM construct, and then selected through the Blasticidin-S resistance gene.SCM expression was evaluated by staining with an anti-IgG antibody recognizing the IgGi spacerand Strep-Tactin®XT- Twin-Strep-tag® (tST). SCM+ Jurkat-TPR cells were transduced with lentiviral vectors bearing antigen recognition modules directed against BCMAorSLAMF7, or both vectors simultaneously (Dual CARtein), and further selected through the Puromycin resistance gene. Since IMPDH-1 reaches its optimal temperature at 37°C, the split intein-mediated protein splicing occurred spontaneously under standard Jurkat cell incubation conditions ( Carvajal-Vallejos P, Pallisse R, Mootz HD, Schmidt SR. Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. J Biol Chem (2012) 287:28686-28696. doi: 10.1074 / jbc.M112.372680).

[0601] In order to analyze SCM, anti-BCMA, anti-SLAMF7 and Dual CARtein expression in Jurkat-TPR cells by flow cytometry, a triple staining strategy was carried out, as illustrated in Figure 4B. Transduced and re-selected cells were stained with an anti-IgG antibody recognizing the IgGi spacer, Strep-Tactin®XT with high affinity fortST and Biotinylated Recombinant Protein L, which recognizes either Belantamab or Elotuzumab VL, followed by PE-conjugated streptavidin™. As evidenced by IgG-staining, the IgGi spacer was highly and consistently expressed among SCM and the different post-protein splicing CARtein cells. As expected, Mean Fluorescence Intensity (MFI) values corresponding to tST staining in SCM+ Jurkat-TPR cells decreased in anti-BCMA, anti-SLAMF7 or Dual CARtein cells, indicating that intein-mediated protein splicing has spontaneously occurred in these cells. Moreover, this is supported by the concomitant increase in protein L MFI in post-splicing CARtein cells, in contrast with single transduced cells that only expressed SCM, and matain high tST and low Protein L staining. Notably, anti-SLAMF7, anti-BCMA and Dual CARtein cells exhibited similar MFI values and flow cytometry histogram profiles for anti-IgG, protein L and Strep-Tactin®XT staining (Figures 4B,C).

[0602] Subsequently, we conducted a flow cytometry analysis to evaluate the expression of BCMA and SLAMF7 surface antigens in the MM.1s multiple myeloma cell line using anti-BCMA and anti-SLAMF7 antibodies for staining. As shown in Figure 4D, MM.1s cells exhibited elevated expression of both tumor antigens.

[0603] Kinetics of CARtein-TPR cells upon SLAMF7 and BCMA stimulation mediated by transduced K562 cells.

[0604] To asses, the specificity of the activation response elicited by CARtein-TPR cells targeting BCMA, SLAMF7 or both surface antigens, K562 cells were transduced with lentiviral vectors expressing BCMA or SLAMF7, and then puromycin selected. After antigen expression and FACS evaluation, SLAMF7+ K562 cells were then transduced and re-selected for BCMA expression. As shown in Figure 7A, transduced K562 cells with BCMA (BCMA+ K562),SLAMF7 (SLAMF7+ K562) or both surface proteins (SLAMF7+ / BCMA+ K562) consistently expressed the corresponding surface antigens.

[0605] Transduced K562 cells were co-cultured with anti-BCMA, anti-SLAMF7 and Dual CARtein-TPR cells for T cell signaling activation analysis at a target to effector ratio of 1:1. CD69 expression, as well as NFAT and NFKB activity of CARtein expressing cells were evaluated at different time points after stimulation (Oh, 24h and 48h) Response of SCM+ cells was evaluated as a control. Untransduced Jurkat-TPR cells (UTD) were used for baseline GFP, CFP or CD69 MFI values. Untransduced K562 cells (SLAMF7- / BCMA- K562) were also cocultured to evaluate K562 mediated non-specific activation. Additionally mock (unstimulated) CARtein cells were also cultured under the same conditions.

[0606] Consistent with previous results, anti-BCMA, anti-SLAMF7 and Dual CARtein-TPR cells elicited NFAT and NFKB activation response directed against their respective targets in transduced K562 cells after 24h (Figures 7B,C). Moreover, CARtein cells exhibited relatively high CD69 activation marker expression (Figure 7D). On the other hand, SCM+ cells did not display CD69 upregulation or SCM-mediated NFAT or NFKB transcriptional activity. Additionally, we did not observe any unspecific activation nor tonic signaling in CARtein-TPR cells or SCM+ cells when co-cultured with untransduced K562 cells. Nonetheless, Dual-CARtein cells exhibited relatively higher GFP and CFP MFI values when co-cultured with SLAMF7+ / BCMA+ K562 cells instead of SLAMF7+ K562 cells.

[0607] For the purpose of comparing activation of either single or dual CARtein when co-cultured with cells expressing BCMA, SLAMF7 or both tumor antigens, we performed a Two-way ANOVA analysis. We did not find statically significant differences when comparing Jurkat-TPR cells stimulated with K562 cells expressing a single antigen or both simultaneously. As illustrated in Figure 8A, anti-BCMA and anti-SLAMF7 CARtein cells exhibited considerable high CD69 and NFAT and NFKB reporters MFI values (**** p < 0.0001) when co-cultured with K562 cells expressing one or both surface proteins. Dual CARtein cells co-cultured with BCMA+ K562 cells elicited a slightly reduced NFAT and NFKB activity, but similar CD69 expression while upon co-culture with SLAMF7+ K562 cells a significant decrease of CD69 (*** p < 0.001), NFAT and NFKB reporters (* p < 0.05) MFI values were observed. Interestingly, in the presence of SLAMF7+ / BCMA+ K562 cells, the stimulation of Dual CARtein-expressing cells resulted in slightly higher NFAT activity than single CARtein cells, although not statistically significant. Dual CARtein cells displayed similar CD69 and NFKB reporter MFI values when compared with CARtein cells targeting only one antigen upon SLAMF7+ / BCMA+ stimulation. Furthermore, no tonic signaling or SCM+ mediated activation response was observed.Regarding CARtein activation kinetics, a two-way ANOVA analysis was conducted in order to compare CARtein mediated cell activation at varying time points following stimulation (Figures 8B-D). CARtein-TPR cells targeting only one or both surface antigens exhibited a pronounced (**** p < 0.0001) GFP and CFP (NFAT and NFKB reporters) upregulation 24h upon stimulation with K562 expressing BCMA, SLAMF7 or both. After 48h, these high GFP and CFP MFI values decreased, although they remained relatively elevated. A similar trend was observed for the CD69 activation marker at 24h and 48h. However, while anti-BCMAand anti-SLAMF7 CARtein cells elicited similar expression patterns, Dual CARtein-TPR cells maintained elevated CD69 expression for 48h.

[0608] Kinetics of T cell activation in CARtein-TPR cells co-cultured with BCMA and SLAMF7 expressing MM.1s cells.

[0609] In order to validate the functionality of cells expressing the different CARtein constructs after split intein-mediated protein splicing, we performed a T cell activation signaling assay, using multiple myeloma cell line MM.1s as a target. We would measure specific CARtein-driven signaling upon BCMA or SLAMF7 stimulation by analyzing expression of CD69 activation marker as well as NFAT and NFKB promoter activity in JKT-TPR cells . For this purpose, we co-cultured BCMA and SLAMF7 expressing multiple myeloma cell line MM.1s, with Jurkat-TPR cells expressing anti-BCMA, anti-SLAMF7 or Dual post-protein splicing CARteins (CARtein-TPR cells) as illustrated in Figures 5A,B. Additionally, Jurkat-TPR cells expressing the SCM (SCM+ cells) were also stimulated, in order to assess any potential SCM-mediated tonic signaling. As a control for baseline eGFP and CFP fluorescence signal MM.1s cells were also co-cultured with untransduced (UTD) Jurkat-TPR cells. Mock (unstimulated) CARtein-TPR cells were also cultured in the same conditions. We then analysed by flow cytometry CD69, NFAT and NFKB activity at different time points (Oh, 24h and 48h) and for different T:E ratios (1:1, 1:5 and 1:10).

[0610] As evidenced in Figures 5C and D, anti-CD69 expression as well as NFAT and NFKB activity considerably increased 24h after MM.1s stimulation in anti-BCMA, anti-SLAMF7 and Dual CARtein-TPR, exhibiting similar activation MFI profiles at 1:1. Moreover, minimal or negligible response was observed in SCM+ cells, as well as in mock samples, which suggests that CAR-mediated activation is strongly specific with little to none tonic activation in CARtein-TPR or SCM+ cells. In addition, the biparametric flow cytometry analysis (Figure S2) revealed that most cells exhibiting CD69 upregulation aligned with those showing higher NFAT or NFKB reporters signals, as well as cells displaying an enhanced NFAT activity with those with higher NFKB MFI values, indicating that most activated cells exhibited a complete signal transduction process.With the aim of comparing anti-BCMA, anti-SLAMF7 and Dual CARtein-mediated T cell activation at different target to effector ratios (1:1, 1:5 and 1:10) upon surface antigen stimulation, we performed a two-way ANOVA analysis. As Figure 6A illustrates, all CARtein cells elicited a strong, specific and sustained NFAT and NFKB-mediated response as well as CD69 upregulation at different target to effector ratios. We found statistically significant differences among T:E ratios for NFAT reporter (GFP) MFI values in CARtein-TPR cells 24h after stimulation. Anti-BCMA CARtein-mediated NFAT activity moderately decreased at 1:5 (* p < 0.05) and 1:10 (** p < 0.01) ratios compared to 1:1 ratio. The same effect was observed in Dual CARtein cells (* p < 0.05), as well as anti-SLAMF7 cells (* p < 0.05) between 1:1 and 1:10 ratios. However, NFAT activity analysis performed after 48h revealed that CARtein cells carrying the same construct exhibited similar MFI values for different T:E ratios. No more significant differences were found when comparing these cells at distinct target to effector ratios. Nonetheless, a delayed NFKB activity seemed to be displayed at 48h in 1:5 and 1:10 ratios when compared to 1 :1 ratio. Notably, SCM+ cells showed none or insignificant activation response, suggesting that no unspecific SCM-driven activation is exhibited by CARtein cells. Interestingly, MFI values for CD69 and NFAT and NFKB reporters exhibited by Dual CARtein cells exceeded those of CARtein cells targeting a single antigen in all T:E ratios and for both, 24h and 48h after co-culture, although the difference is not statically significant. This tendency may be indicative of a synergistic effect in T cell signal transduction when cells expressing these CAR constructs target both antigens.

[0611] To gain further insight into the activation kinetics of these CARtein-TPR cells, a two-way ANOVA analysis was conducted, comparing different time points for a cell expressing a particular intein-mediated CAR (Figures 6B-D). NFAT, NFKB and CD69 MFI values at target to effector 1:1 ratio strongly peak at 24h upon surface antigen stimulation for anti-BCMA, anti-SLAMF7 and Dual CARtein cells (**** p < 0.0001). However, NFAT signal slightly decreases, although no significantly, for all CARtein-expressing cells at 1:1 ratio after 48h. A similar effect can be appreciated for NFKB activity, except that the decrease is more pronounced. Moreover, we can observe a delayed NFAT response for all CARtein-TPR cells at T:E ratios 1:5 and 1:10, since they reach their highest MFI values at 48h (**** p < 0.0001). Interestingly, anti-BCMA CARtein cells at 1:10 ratio and Dual CARtein cells at 1:5 and 1:10 ratios maintained high NFKB MFI values up to 48h, but not in the remaining cases, in which the signal declined. This can be explained by a delayed NFKB activation response in anti-BCMA and Dual CARtein cells at lower target ratios. Regarding CD69 expression, 24h after co-culture, all CARtein-TPR cells exhibit high CD69 MFI values at different T:E ratios. Nonetheless, cells co-cultured at 1 :1 ratio display a slightly higher, although no significant, CD69 expression. CD69 activation marker MFI values remained elevated 48h after stimulation, even though a downward trend isappreciated. NFAT, NFKB and CD69 MFI values remained at background levels for SCM+ cells at any T:E ratio or time point.

[0612] EXAMPLE 2: Intein-Based Modular CAR Platform for Specific CD19 / CD20 CoTargeting

[0613] Materials and methods

[0614] Construction of CARtein, CD19 and CD20 expression plasmids

[0615] The versatile intein signaling module (IntStem CAR) to which different single-chain variable fragments (scFv) can be bound was codon-optimized and synthesized by GeneArt™ (Thermo Fisher Scientific Inc., Carlsbad, CA, USA) with sequences encoding from 5’ to 3’: a hlgKVI 11 leader sequence, the Twin-Strep-tag® (TST) peptide, a flexible interchain linker (GGGS)3 (SEQ ID NO: 42), a IMPDH-1 C-intein-extein domain in frame with a CD28 transmembrane and cytoplasmic domain followed by a TCR-£ domains. The IntStem CAR coding sequence was cloned into the lentiviral shuttle vector pHR'SINcPPT CEWvia a LR Gateway™ reaction, thus allowing its expression under a SFFV promoter.

[0616] Three different scFv-intein constructs (GeneArt™) were synthesized to serve as binding partners for the IntStem CAR module, based on the anti CD19, anti-CD-20 and tandem anti CD19-CD20 scFvs, with a IgK lieder. CDS for anti-CD19 scFv (VL-VH) was followed in frame by sequences coding for an lgG4 hinge domain, N-intein IMPDH.1-N, a flexible (GGGS)2 linker (SEQ ID NO: 43) c-myc and 6His tags and a (G2S)2A3-KDEL sequence that allows scFv to be sequestered in the endoplasmic reticulum until transplicing with the IntStem module occurs. The anti-CD20 scFv construct was engineered like anti CD19 but instead of the lgG4 hinge domain a longer spacer including hinge-CFL-CHs was used, as anti-CD20 CARs require a longer extracellular domain due to the position of CD20 target close to the tumor membrane. In addition, a CD20(VL-VH)-(GGGS)4-CD19(VH-VL) tandem scFv was also engineered with just the hinge spacer as CD19 itself correctly separates antiCD20 from the membrane to reach the shorter CD20 antigen.

[0617] For expression of the human CD20 antigen, a lentiviral expression plasmid encoding for human CD20 (pJRH-1328 LV EF1a-CD20 IRES-EGFP; Addgene plasmid no. 201918), which also encodes for a bleomycin resistance gene, was directly packaged into lentiviral vectors. In the case of CD19, to express both CD19 and CD20 under resistance to different drugs, we generated an expression plasmid by cloning the CD 19 coding sequence from a previously established plasmid (pJRH-1363 LV EF1a-CD19 IRES-EGFP; Addgene plasmid no. 201919)into pLEX_307, [a gift from David Root (Addgene plasmid # 41392; http: / / n2t.net / addgene:41392; RRID:Addgene_41392)], which allows for a strong CD19 expression under puromycin resistance. Both Addgene #201918 (http: / / n2t.net / addgene:201918; RRID:Addgene_201918) and #201919 (http: / / n2t.net / addgene:201918; RRID:Addgene_201919) plasmids were gifts from Jennifer Doudna.

[0618] Cell Lines and Cultures

[0619] All human cell lines were from American Type Culture Collection (ATCC, Manassas, VA). To study T cell activation, a cell subline derived from Jurkat T JE6.1 (Jurkat) was used. This cell subline, known as Jurkat-TPR (Prof. Steinberger, Medical University of Vienna), expresses the fluorescent proteins CFP, eGFP and mCherry respectively governed by NFKB, NFAT and AP-1 promoters, and has previously been used to assess CAR-T cell activity (1 ,2). The human B-lymphoma cell line Raji were used as CD19+CD20+target cells. In addition, K562 cells, a chronic myelogenous leukemia cell line, were employed as parental cells to generate different single-antigen sublines. Lastly, human Embryonic Kidney (HEK Lenti-XTM 293T) cells were used for lentiviral production. All cells were maintained at 37°C and 10% CO2 in standard DMEM GlutaMAX media supplemented with 10% (v / v) FBS (Gibco), 10 mM HEPES, 1% (v / v) sodium pyruvate, 100 units / mL penicillin-streptomycin, 2 mM L-glutamine and 50 pM 2-mercaptoethanol (Invitrogen, Carlsbad, CA, USA), except for K562 cells, which were maintained in Iscove's Modified Dulbecco's Medium (IMDM), with the same supplementation as for DMEM.

[0620] Lentiviral Vector Generation

[0621] Lentiviral supernatants were generated by transient cotransfection of HEK Lenti-XTM 293T cells with the different vector plasmids, pCMVAR8.91 and pMD2.G (VSVG), as previously described (1). Cells were added to collagen-coated 100mm plates in supplemented DMEM, 24h prior to transfection. The following day, cells were transfected in OptiMEM™ medium (Thermo Fisher Scientific Inc., Carlsbad, CA, USA) with 20pg of vector plasmid, 13pg of pCMVAR8.91 and 2.3pg of pMD2.G by polyethylenimine (PEI)-mediated transfection (3). Supernatants were collected 48 and 72 hours after transfection and cells were removed by centrifugation. Lentiviruses were concentrated using the Lenti-X™ concentrator (Clontech) and lentiviral pellets were frozen at -80°C until use.

[0622] Cell Transduction

[0623] A parental IntStem CAR-TPR cell line was generated by transducing 3 105TPR cells in a 48-well plate with the corresponding lentiviral pellet, and 40mg / mL Blasticidin S HCI (ThermoFisher Scientific Inc.) was added 48h post-transduction. IntStem CAR expression was assessed by staining with Strep-Tactin®XT DY-649 (Iba-Lifesciences, #2-1568-050). Once this cell subline was stablished, 3 105IntStem CAR-TPR cell alliquots were left un-transduced or transduced with either anti-CD19-intein (CD19 CAR), anti-CD20-intein (CD20 CAR), tandem scFv-intein (Tandem CAR) or both anti-CD19-intein and anti-CD20-intein (Dual CAR).

[0624] 0,25 .g / mL Puromycin (Thermo Fisher Scientific Inc., Carlsbad, CA, USA) was added 48h after transduction. The efficacy of the intein reaction between IntStem CAR and different scFv-intein partners was assessed by detecting the loss of TST expression with Strep-Tactin®XT DY-649, as well as the presence of the IgG spacer domain by staining with biotinylated goat anti-human IgG antibody (heavy chain) (antibodies-online, Aachen, Germany). The presence of scFv was also assessed by staining with biotinylated protein-L (ACROBiosystems). Both Protein-L and anti-human IgG were subsequently stained with PE-conjugated streptavidin™ (Thermo Fisher Scientific Inc., Carlsbad, CA, USA).

[0625] K562 cells were transduced with lentiviral particles encoding for CD19 or CD20 to obtain individual cell sublines expressing individual antigens. 48h after transduction, 0.25pgg / mL Puromycin was added to cells transduced with CD19, and 100pg / mL Bleomycin (Thermo Fisher Scientific Inc.) to those with CD20. In addition, CD19+CD20+K562 (2Ag-K562) cells were obtained by co-transduction of both antigens and subsequent selection with both Puromycin and Bleomycin. CD19 and CD20 expression in all K562 sublines and in Raji cells was validated by labeling with human anti CD19-PE (Beckman Coulter, Inc., CA, USA) and CD20-APC (Biolegend, San Diego, CA, USA) monoclonal antibodies previously treated with FcR blocking reagent (Miltenyi Biotec) for 15 min at room temperature. Data acquisition was performed on a Cytek® Aurora 5L 16UV-16V-14B-10YG-8R spectral cytometer (Cytek, CA, USA).

[0626] T Cell Activation Assay by Spectral Flow Cytometry

[0627] Effector CAR-T cells were cocultured with Raji cells in 96-well plates at target: effector (T:E) ratios of 1:1, 1:5 and 1:10 in 5% CO2 and 37°C for 24h and 48h. In addition, to test the specificity of each scFv towards its cognate target, effector cells were co-cultured with WT, CD19+, CD20+or 2Ag K562 cells at target: effector (T:E) ratios of 1 :1 in 5% CO2 and 37°C for 24h and 48h. In each case, cells were first counted and centrifuged to replace the consumed medium with fresh supplemented DMEM.

[0628] For each measurement, cells were washed twice in ice-cold PBS and treated with FcR blocking reagent (Miltenyi Biotec) for 15 min at room temperature according to themanufacturer's procedure. Cells were then washed and labeled with anti-human CD69-APC and CD3-PerCP / Cy5.5 antibodies (Biolegend). To exclude dead cells and debris from analysis, they were stained with Zombie NIR™ Fixable Viability Kit (Biolegend) for 15 minutes at room temperature. Data was acquired on a Cytek® Aurora 5L 16UV-16V-14B-10YG-8R spectral cytometer, and the analyses were performed in FlowJo software V10.1 (TreeStar Inc., Olten, Switzerland).

[0629] Statistical Analysis

[0630] All statistical analyses and corresponding graphs were generated using GraphPad Prism 5.0 (GraphPad, La Jolla, CA, USA). For comparisons between more than two groups, a two-way ANOVA was used and Bonferroni's post hoc test was performed to ensure the reliability of multiple comparisons. To compare differences between two groups, a two-tailed unpaired Student's t-test was performed. Values are expressed as the mean of triplicates ± SD, with significance levels marked in the figures (*p < 0.05; **p < 0.01; ***p < 0.001).

[0631] Results

[0632] Development of the CD19-CD20 bispecific CARtein system

[0633] To generate an inert intein signaling CAR module (IntStem), we used a second-generation CD28 / CAR (1) with a Twin-Strep-tag® (TST) for easy identification of unspliced receptors. To enable protein splicing with the different scFv (recognition modules), we included a C-intein in the IntStem CAR, while each scFv includes the orthogonal N-intein. These scFv were expressed in CAR-TPR cells already expressing IntStem, to allow for the transplicing reaction to occur in the endoplasmic reticulum, where scFv are retained due to a KDEL sequence to prevent scFv secretion of unspliced modules (Figure 9A). As a result, CARs gain functionality due to the incorporation of the recognition moiety, leaving a small leftover between the scFv spacer and the IntStem CAR transmembrane domain (TMD). As they react above the TMD, it loses its TST peptide, which is carried away by an irrelevant intein peptide by-product (Figure 9B).

[0634] The above-mentioned design allows as to compare not only anti-CD19 or anti-CD20 recognition modules but also anti-CD19-CD20 tandem CARs onto the same IntStem. As it has been previously shown that the optimal spacer length in CD19 and CD20 CARs differ due to the differential separation of their cognate antigens from the tumor cell membrane, this approach also allows us to customize recognition modules of different lengths that will assemble onto a common IntStem. Since the CD19 antigen is known to reach farther from themembrane than CD20, which is a multiloop transmembrane protein, it was proposed that CD20-CARs, require a longer spacer between the TMD and the scFv, for which we have chosen lgG4 hinge-CH2-CH3. This Fc domain was mutated to prevent interactions with Fc receptors, thus reducing off-target activation hazards. On the other hand a shorter spacer version was used for the anti-CD19 recognition module, including only the lgG4 hinge region. In the case of tandem scFv, the relative position of the antigen was also considered, whereby the CD20-scFv was placed at the N-terminus and the CD19-scFv closer to the hinge-N-intein, both joined by a flexible linker (Figure 9C, D). Efficacy of the reaction was evaluated by loss of the TST peptide, which was expected to be replaced by the different scFv modules. As seen in Figure 9E, scFv-intein coexpression effectively removes TST from IntStem CARs, whereas scFv can be detected by Protein-L on Tandem, Dual and CD20 CAR expressing cells. The chosen CD19-scFv sequence, in contrast, is known to be only mildly detectable by Protein-L. Moreover, human IgG from the anti-CD20-intein construct was also detected on both CD20 and Dual CAR expressing cells. To evaluate the specificity of each recognition module toward its cognate target, K562 cells were transduced with either antigen (CD19 or CD20) or both. Therefore, four different sublines were tested: K562-H / / 7d- Type (WT) (CD19-CD20'), K562-CD19 (CD19+CD20-), K562-CD20 (CD19’ CD20+) and K562-2Ag (CD19+CD20+). Expression of these antigens on target cell surface, as well as in Raji cells (CD19+CD20+), was assessed by spectral flow cytometry (Figure 9F).

[0635] Evaluation of the specificity of CARteins against individual antigens

[0636] To analyze the specificity of this platform, we studied how each CAR-TPR would respond to K562 cells expressing either CD19, CD20, or both (Figure 12). CD69 upregulation, as well as NFAT (eGFP) and NFKB (CFP) promoter activity, were assessed by spectral flow cytometry. Cells expressing CARs including specific scFv were activated upon binding to its cognate antigen (***p<0.001 for each parameter, at each time), whereas remaining unstimulated for the other antigens. Activation of single-scFv CARteins (CD19- and CD20-CARs), elicited high NFAT and NFKB promoter activity and CD69 upregulation in the presence of K562-CD19 or K562-CD20, respectively (***p<0.001), and both are similarly stimulated with K562-2Ag (CD19+CD20+). Activation of Dual CARs is lower to that of Tandem CARs when co-cultured with single antigen K562 cells, but both are similarly activated by K562-2Ag. This suggests that the strength of our platform lies not only in scenarios where one of the antigens has been downregulated but also when both target antigens are expressed. In such cases, dual CARs act synergistically when bound to both antigens, resulting in stronger activation, as observed with Raji cells. In contrast, tandem CARs exhibit similar promoter activity and CD69 upregulation regardless of the presence of CD19, CD20, or both. Furthermore, as when co-cultured with Raji cells, IntStem CAR-TPR cells do not respond to any K562 cell subline, nor to K562-WT cells, reinforcing their suitability as an inert binding module.

[0637] CARtein activation upon targeting Raji cells

[0638] Once the response against individual antigens was evaluated, we aimed to test the functionality of the CARtein platform by targeting Raji cells, which have been widely used as a CD19+CD20+Burkitt lymphoma cell line model (4). Cells were co-cultured at 1:1, 1:5 and 1:10 targeteffector ratios and activation was analyzed after 24 and 48 hours. Effector cells were distinguished from Raji cells by gating single, live CD3+cells (Figure 10A). CD69 upregulation, as well as NFAT (eGFP) and NFKB (CFP) promoter activity, were assessed by spectral flow cytometry. As seen in Figure 10, Raji cells elicit a basal activation of the NFKB promoter and a subtle upregulation of CD69, but it is negligible when compared to that elicited by CAR-specific stimulation. Interestingly, no NFAT activity is seen in non-transduced (NT) or IntStem CAR TPR cells in the presence of Raji cells, yet being highly increased in CAR expressing cells. As expected, the four different effector CAR cell sublines (CD19, CD20, Dual and Tandem CARs) are activated when co-cultured with Raji target cells, manifested by a predominance of the NFAT+NFkB+responder population and upregulation of CD69 in >90% of cells.

[0639] Two-way ANOVA analysis showed that these increases were statistically significant compared with unstimulated cells (Figure 11). High promoter activation, as well as CD69 upregulation, are evident 24h after stimulation with Raji cells, although the peak of activation occurs at 48h, being higher than at 24h in terms of NFAT and NFKB promoter activation (**p<0.01), but not for CD69, whose levels in activated cells were already topped 24h post-activation. Interestingly, this upward trend is more noticeable at lower target: effector (T:E) ratios, being higher at 1:10 and 1:5 ratios. However, no overall differences were observed between the different T:E ratios, despite the fact that stimulation strength tends to increase slightly with higher ratios.

[0640] Of note, IntStem CARs do not respond to Raji stimulation in the absence of any complementary scFv-intein binding partners, thus demonstrating its inert nature. In unstimulated IntStem CAR-TPR cells, only a weak increase in basal CD69 expression is observed. However, the behavior of these cells is similar to that of non-transduced TPR cells when co-cultured with Raji cells, with null NFAT activity but subtle nonspecific NFKB activation and CD69 upregulation so it cannot be explained just by IntStem mediated tonic signals.EXAMPLE 3: Intein-Based Modular CAR Platform for TROP2 Co-targeting in T and NK cells

[0641] Materials and Methods

[0642] Cell Lines and Cultures

[0643] All human cell lines were from the American Type Culture Collection (ATCC, Manassas, VA). Both the NK-like lymphoblastic leukemia / lymphoma cell line YT and the non-Hodgkin's lymphoma NK-92 cell line were cultured at 37°C and 10% CO2 in standard RPMI 1640 medium supplemented with 10% FBS, 2mM L-glutamine, 1X NEAA, and 100 units / mL penicillinstreptomycin (Gibco). For NK-92 cells, 100U / mL rhlL-2 (NIH HIV Reagent Program, now transitioned to BEI Resources) was added. MDA-MB-231, a human triple negative luminal breast cancer cell line and MCF7, a human luminal ER-positive breast cancer cell line, both expressing moderate levels of TROP2 were used as TROP2 positive target and human Embryonic Kidney (HEK Lenti-XTM 293T) cells were used for packaging of lentiviral vectors. Cells were maintained at 37°C and 7% CO2 in standard DMEM GlutaMAX media supplemented with 10% (v / v) FBS (Gibco), 10 mM HEPES, 1% (v / v) sodium pyruvate, 100 units / mL penicillin-streptomycin, 2 mM L-glutamine and 50 pM 2-mercaptoethanol (Invitrogen, Carlsbad, CA, USA).

[0644] Isolation of PBMCs

[0645] Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats obtained from three different healthy donors. These samples were provided by the tissue and cell transfusion center of Seville through the Hospital Universitario Virgen del Rocio-lnstituto de Biomedicina de Sevilla Biobank (Andalusian Public Health System Biobank and Plataforma ISCIII Biomodelos y Biobancos (PISCIIIBB) PT23 / 00134). All samples were obtained upon signature of an informed consent and following approval by the Coordinating Committee for Bioethics in Biomedical Research of Andalusia (CCEIBA). They were then diluted 1:1 with phosphate-buffered saline (PBS) and carefully layered over a Ficoll-Paque (Eurobio Scientific) density gradient, as indicated by the manufacturer. After centrifugation at 400g for 40 minutes at room temperature, the PBMC layer was collected, washed twice with PBS, and resuspended in RPMI 1640 medium with the same supplementation as for both NK-92 and YT cells.

[0646] Construction of CARtein, anti-TROP2 expression plasmids

[0647] Signaling modules (IntStem CARs) to which different single-chain variable fragments (scFv) can be bound, were codon-optimized and synthesized by GeneArt™ (Thermo Fisher Scientific Inc., Carlsbad, CA, USA) with sequences encoding from 5’ to 3’: signal peptide-tag(s) IMPDH-1 C-intein-extein domain-Spacer- CD28 transmembrane and cytoplasmic domains followed by a TCR- domain.

[0648] Four IntStem CAR versions were engineered with different spacers. Specifically, IgG Hinge-CH2-CH3 from I gGi (option A) or lgG4 (option C), just lgG4 Hinge (option B) or CD8a (option D) or just the C2 intein (option E). The results shown in this example, however, are performed with a CAR comprising spacer as defined in SEQ ID NO: 8 (Option D).

[0649] Signal peptides were either hl gicVI 11 for option A or CD8a for option B through D.

[0650] Tags were either Twin-Strep-tag® (TST) peptide-AviTag-6HIS-myc for option A or just the myc tag for option B through E.

[0651] IntStem CAR coding sequence were cloned into the lentiviral shuttle vector pHR'SINcPPT CEWvia a LR Gateway™ reaction, thus allowing its expression under a SFFV promoter. Two different scFv-intein constructs (GeneArt™) were synthesized to serve as binding partners for the IntStem CAR module, one based on the sacituzumab® sequence (SEQ ID NO: 15) and the other, partially based on the 2EF antibody (SEQ ID NO: 16) described in DQI:10.3390 / cancers15143721 and WQ2010089782 with a CD8a signal peptide followed by a myc tag and the CDS for either anti-TROP2 scFv (VL-VH) that in turn were followed in frame by sequences coding for an N-intein IMPDH.1-N, a 7His tag and a KDEL sequence that allows scFv to be sequestered in the endoplasmic reticulum until transplicing with the IntStem module occurs.

[0652] Lentiviral Vector Generation and Cell transduction

[0653] For lentiviral vectors production, HEK 293T cells were seeded in 100mm collagen-coated plates in complete DMEM medium and grown to approximately 70-80% confluency. The following day, cells were transfected in OptiMEM™ medium (Thermo Fisher Scientific Inc., Carlsbad, CA, USA) with 20 pg of vector plasmid, 13 pg of pCMVAR8.91, and 2.3 pg of pMD2.G using polyethylenimine (PEI)-mediated transfection (3). The mixture was incubated for 15 minutes at room temperature before being added dropwise to the cells. After 1.5 hours, the medium was replaced with fresh DMEM containing 10% fetal bovine serum. Lentiviral vector-containing supernatants were collected 48 and 72 hours post-transfection, centrifuged to remove residual cells, and concentrated using the Lenti-X™ concentrator (Clontech). Lentiviral pellets were stored at -80°C until use.

[0654] 0.3 106YT cells were seeded in 48-well plates in 500pL of fresh, complete RPMI medium. To enhance transduction, the protocol was fine-tuned and adapted from previous studies in which YT cells were transduced (2,5,6). Prior to the addition of lentiviral particles, 10pg / mL protaminesulfate and 5pL / mL LentiBOOT (Sirion Biotech) were added to the cell culture. The cells were then spinoculated together with the lentiviral vectors at 800xg at 32°C for 90 minutes. Afterward, the plate was incubated for 48 hours at 37°C and 10% CO2 prior to the addition of 40 ptg / ml Blasticidin S HCI (Gibco).

[0655] Evaluation of YT phenotype

[0656] YT cell phenotype was first studied and compared to that of primary NK and NKT cells. A battery of antibodies, detailed in Table 3, was used. NK and NKT cells were gated based on their CD56+CD3- and CD56+CD3+ phenotypes, respectively. Due to overlapping fluorochromes, several distinct antibody mixes were prepared. Labeling was performed in triplicate for statistical analysis.

[0657] Antibody Conjugated Reference and source

[0658] CCR4 Alexa Fluor®647 TG6 / CCR4 clone, Biolegend

[0659] CCR6 Alexa Fluor®647 TG7 / CCR6 clone, Biolegend

[0660] CCR7 APC G043H7 clone, Biolegend

[0661] CD2 Biotin T Cell Activation / Expansion Kit, Miltenyi CD3 PerCP / Cyanine5.5 SK7 clone, Biolegend

[0662] CD8 StarBright Violet 440 Bio-Rad antibodies

[0663] CD11c PE BU15 clone, Biolegend

[0664] CD14 PerCP HCD14 clone, Biolegend

[0665] CD16 PC7 3G8 clone, Beckman Coulter CD19 ECD HD237 clone, Beckman Coulter CD25 APC M-A251 clone, Biolegend

[0666] CD27 Biotin M-T271 clone, Biolegend

[0667] CD28 Biotin T Cell Activation / Expansion Kit, Miltenyi CD56 Brilliant Violet 421 5.1H11 clone, Biolegend

[0668] CD62L APC / Cyanine7 W21031N clone, Biolegend

[0669] CD69 APC FN50 clone, Biolegend

[0670] CD95 APC VI C-64, BD Biosciences

[0671] CD127 PE / Dazzle™ 594 A019D5 clone, Biolegend

[0672] CD152 (CTLA-4) PerCP / Cyanine5.5 BNI3 clone, Biolegend

[0673] HLA-DR Pacific Blue lmmu-357 clone, Beckman Coulter CD223 (LAG-3) Brilliant Violet 511 11C3C65 clone, Biolegend

[0674] CD279 (PD-1) FITC A17188B clone, Biolegend

[0675] CD319 (SLAMF7) Pe / Cyanine7 162.1 clone, Biolegend

[0676] CD366 (TIM-3) Pe / Cyanine7 F38-2E2 clone, Biolegend

[0677] Table 3 | Table of primary antibodies. All antibodies used to phenotype YT, pNK and pNK-T were either directly conjugated with fluorochrome or biotinylated.

[0678] Cells were pre-treated with a human FcR blocking reagent (Miltenyi Biotec) for 15 minutes at room temperature, then washed and incubated with the appropriate antibody mix for 1 hour at 4°C in the dark. Regarding the labeling of CD2, CD27, and CD28, an additional step was required. After 1 hour of labeling, the cells were washed twice and labeled with PE-conjugated streptavidin™ (Thermo Fisher Scientific Inc.) in PBS + 2% Bovine Serum Albumin (BSA) for 15 minutes on ice and in the dark. After immunostaining, cells were washed and labeled with the Zombie NIR™ Fixable Viability Kit (Biolegend). Data acquisition was conducted using a Cytek® Aurora 5L 16UV-16V-14B-10YG-8R spectral cytometer (Cytek, CA, USA).Viability / cytotoxicity assay

[0679] In order to assess cytotoxic potential of the anti-TROP2 CARtein as defined in SEQ ID NO: 33, anti-TROP2 CAR+ or untransduced YT cells were co-cultured with MDA-MB-231 or MCF7 target cells expressing Renilla or Firefly luciferase (MDA-LUC) at 1:1 E:T ratio (0.3-106cells each) in complete RPMI medium. 24 hours after, 40pg / mL Renilla luciferase substrate (ViviRen™ In Vivo Renilla Luciferase Substrate, Promega, Madison, Wl) was added to each well and after 15 minutes at 37°C, the plate was placed in an I VIS Spectrum in vivo imaging system or a Synergy MX Biotek luminometer. Additional control wells of media and MM1.s cells alone were included to determine minimum and maximum luminescence, respectively. Cytotoxicity activity of anti-TROP2 CARtein-YT cells was measured by adding 150ug / ml of D-Luciferin sodium salt monohydrate (Thermo Scientific) to media and measuring Luminescence 1 minute later in the BioTek Synergy HTX Multi-mode Microplate Reader (Agilent, RRID:SCR_019749). The percentage of lysis was calculated with the following formula: 100 - [(Luminescence sample / Luminescence MM.1S alone) x 100%].

[0680] Statistical Analysis

[0681] Data from spectral flow cytometry were analyzed with FlowJo v10.8.1 software (TreeStar Inc., Olten, Switzerland). Statistical analyses was performed using GraphPad Prism v10.0 (GraphPad, La Jolla, CA, USA). Two-way ANOVA was used for comparisons between more than two groups, followed by Bonferroni's post hoc test to ensure the reliability of multiple comparisons. Two-tailed unpaired Student's t-tests were performed to compare differences between two groups. Values are represented as the mean of triplicates ± SD, with significance levels marked in the figures (*p<0.05; ** <0.01; ***p<0.001).

[0682] Results

[0683] Validation of the anti-TROP2 bispecific CARtein system in Jurkat T cells

[0684] To generate split intein-mediated modular chimeric antigen receptors (CARteins) targeting TROP2, two distinct antigen-recognition CARtein modules were designed (corresponding to SEQ ID NOs: 33 and 38), each based on an anti-TROP2 scFv. One scFv reproduced the sacituzumab® sequence (corresponding to SEQ ID NO: 15), whereas the second was partially derived from the 2EF antibody described in DQI:10.3390 / cancers15143721 and WQ2010089782 (corresponding to SEQ ID NO: 16). In both cases, the constructs included an N-terminal CD8a signal peptide followed by a myc tag and the coding sequence for the corresponding anti-TROP2 scFv (VL-VH), which in turn was fused in frame to an N-intein IMPDH-1-N segment, a 7His tag, and a C-terminal Lys-Asp-Glu-Leu (KDEL) retention sequence. These antigen-recognition modules were intended to remain confined to the endoplasmic reticulum until intein-mediated protein splicing with the universal signalingCARtein module occurred, thereby releasing the mature TROP2-CARtein to the cell surface in an analogous manner to the previously described BCMA / SLAMF7 or CD19 / 20 system. JKT-TPR cells expressing the signaling module were transduced with lentiviral vectors encoding the anti-TROP2 ARM modules, whereas control cells were transfected either with a full-length conventional CAR (positive control) or, with the SCM module alone, or left untransduced as negative controls. The efficiency of the trans-splicing reaction was assessed by flow cytometry using protein L (binding to the variable regions present in the ARM module), recombinant biotinylated TROP2, an anti-Withlow linker antibody (recognizing the linker between variable regions), and an anti-myc antibody (targeting the N-terminal tag of the TROP2 scFvs). As shown in Figure 13, only cells co-expressing the ARM modules or the full-length conventional CAR stained positive for protein L, TROP2 antigen, anti-Withlow linker, and anti-myc, whereas untransduced cells and cells expressing only the SCM module remained negative for all markers. Expression of 2EF CARtein was initially lower (35%) and were immunomagnetic enriched by means of protein L binding.

[0685] Activation signaling assay of Sacituzumab or 2EF CARtein cells co-cultured with MCF7 Trop-2+ cells

[0686] Once successful trans-splicing was confirmed, we next assessed the functionality of the TROP2-directed CARtein platform by co-culturing CARtein-expressing Jurkat-TPR cells with MCF7 cells. Cells were co-cultured at 32:1, 8:1. and 2:1 target: effector ratios and activation was analyzed after 24 hours. Effector cells were identified as single, live CD3+ events, and CD69 upregulation together with NFAT (eGFP), NFKB (CFP) orAP1 promoter activity were quantified by spectral flow cytometry. As shown in Figures 14 and 15, MCF7 cells did not induce promoter activity or CD69 upregulation in untransduced or SCM expressing JKT-TPR cells while inducing robust NFAT, NFKB and AP1 promoter activation and CD69 upregulation in cells expressing anti-TROP2 CARteins based either on the sacituzumab-derived scFv or on the E2F-derived scFv, with both constructs driving the emergence of a distinct NFAT+NFKB+AP+CD69+ responder population. Interestingly Sacituzumab based CARtein induced a slightly stronger stimulation than the conventional CAR.

[0687] In the 2EF experiments, the smaller population of CARtein JKT-TPR cells responding to MCF7 can be attributed to the fact that only 35% of the JKT-TPR cells expressed the CARtein 2EF construct. This limited expression level directly reduces the proportion of cells capable of eliciting a targeted immune response against MCF7, resulting in fewer observable responders compared to Sacituzumab CARtein with higher CARtein expression efficiency. Consequently, the overall efficacy appears diminished, highlighting the need for selection of CARtein positive cells to achieve broader cellular coverage.Kinetics of T cell activation in Jurkat-TPR cells expressing anti-TROP2 CARtein cocultured with TROP2 expressing MCF7 cells.

[0688] To validate the functionality of cells expressing anti-TROP2 CARtein constructs, including both sacituzumab-based and 2EF designs, we monitored NFAT, NFKB and AP1 promoter activity together with CD69 upregulation in JKT-TPR cells at 0, 24 and 48 h. For this purpose, TROP2-expressing target cells were co-cultured with JKT-TPR cells transduced with either sacituzumab- or 2EF derived anti-TROP2 CARteins (CARtein-TPR cells), compared with a conventional Sacituzumab based CAR, while untransduced JKT-TPR cells and mock CARtein-TPR cells were included as negative controls to define baseline reporter and CD69 levels. In Figure 16, flow cytometry analysis showed that sacituzumab CARtein-TPR cells mounted a strong, antigen-dependent increase in NFAT, NFKB and AP1 reporter activation, accompanied by robust CD69 upregulation, that picked at 24 h. 2EF anti-TROP2 CARtein-TPR cells displayed a clearly detectable but quantitatively lower activation profile across all readouts, which we attribute to the fact that this construct was expressed in only 35% of JKT-TPR cells in these experiments, thereby limiting the fraction of CARtein-positive cells capable of engaging TROP2 and triggering downstream signalling. Altogether, these data indicate that anti-TROP2 CARtein designs can efficiently activate NFAT, NFKB and AP1 pathways and induce CD69 expression upon target recognition, and further highlight that the attenuated response observed for 2EF CARteins is primarily driven by reduced construct expression rather than by an intrinsically weaker signalling capacity.

[0689] Validation of the anti-TROP2 bispecific CARtein system in NK YT cells

[0690] To confirm that split intein-mediated assembly of anti-TROP2 CARteins was not restricted to T cells, we next evaluated the same Sacituzumab- ARM construct in the NK cell line YT expressing the universal signaling CARtein module. YT cells were transduced with lentiviral vectors encoding anti-TROP2 ARM, left untransduced, or transduced only with the SCM module, as negative controls. CARtein positive cells were also compared with cells expressing a conventional Sacituzumab based CAR construct. As in JKT-TPR cells, efficient trans-splicing in YT cells was demonstrated by flow cytometry, using protein L, recombinant biotinylated TROP2, and anti-Withlow linker antibody to detect surface expression of the mature TROP2 CARtein (Figure 17). Only YT cells co-expressing the signaling module and sacituzumab-ARMs as well as those expressing the conventional sacituzumab CAR, stained positive for all three markers, whereas untransduced cells and SCM-only cells remained negative, indicating that split intein-mediated CARtein reconstitution proceeds with comparable efficiency in YT and Jurkat backgrounds.

[0691] To extend our functional characterization of split intein-assembled anti-TROP2 CARteins beyond reporter readouts, we next evaluated whether sacituzumab-based CARtein-YT cellscould mediate antigen-specific killing of TROP2-expressing tumor cells. Anti-TROP2 CARtein-redirected YT cells were co-cultured with MCF7 target cells at an effectortarget ratio of 1:5 for 24 h, and specific cytotoxicity was quantified by D-luciferin-based bioluminescence. As shown in Figure 18, untransduced (WT) and SCM-only YT cells displayed a relatively high basal lysis of about 30%, consistent with the intrinsic cytotoxic background of YT cells, whereas anti-TROP2 CARtein-YT cells reached approximately 60% lysis of MCF7 cells. Thus, after accounting for the elevated baseline, sacituzumab CARtein expression still conferred a clear, antigen-dependent increase in cytotoxicity, indicating that CARtein-redirected YT cells can mediate robust and specific killing of TROP2-positive MCF7 cells. Interestingly, sacituzumab CARtein-mediated lysis of MCF7 cells was slightly higher than that achieved with a conventional sacituzumab CAR under the same assay conditions. This modest but consistent increase in cytotoxicity suggests that split intein-reconstituted CARteins can match, and potentially enhance, the effector function of standard full-length CARs despite their modular architecture. These data further support the suitability of the CARtein platform for redirecting cytotoxic lymphocytes against TROP2-positive targets.

[0692] Anti-TROP2 CAR-NK cytotoxicity against MDA-LUC cells

[0693] To analyze anti-TROP2 CAR functionality, YT cells were transduced with the corresponding CARs (SEQ ID NO: 15 + SEQ ID NO: 8) and co-cultured with TROP2 positive tumor cell MDA-LUC cells at 1 :1 E:T ratio in complete RPMI medium. Their cytotoxicity was compared to that of WT YT cells. As shown in Figure 19, although coculturing MDA cell with wt YT cells already showed a decrease in viability, MDA cells co-cultures with anti-TROP2 CAR expressing YT cells showed further decreased their viability indicating a specific CAR mediated tumor lysis effect.

[0694] EXAMPLE 4: Intein-Based Modular CAR Platform for BCMA

[0695] Further to the previous examples using anti-BCMA targeting moiety, more constructs were generated. The materials and methods are as described above. In this case, the sequences used were:

[0696]

[0697] As shown in fig. 20, 21 and 22, the CAR showed cytotoxicity against different cell lines, thereby validating the potential of the platform described in the present invention.

Claims

CLAIMS1. A chimeric antigen receptor (CAR) precursor comprising at least two polypeptides, wherein:The first polypeptide comprises SEQ ID NO: 5 and the second polypeptide comprises SEQ ID NO: 13, 14;The first polypeptide comprises SEQ ID NO: 6, 7 or 8 and the second polypeptide comprises SEQ ID NO: 13;The first polypeptide comprises SEQ ID NO: 4 and the second polypeptide comprises SEQ ID NO: 10, 11 or 12; orThe first polypeptide comprises SEQ ID NO: 8 and the second polypeptide comprises SEQ ID NO: 15 or 16.

2. A mature intein-mediated modular CAR comprising or consisting of:SEQ ID NO: 20: mature intein-mediated modular CAR BCMA-stemA;SEQ ID NO: 21: mature intein-mediated modular CAR BCMA-stemB;SEQ ID NO: 22: mature intein-mediated modular CAR BCMA-stemC;SEQ ID NO: 23: mature intein-mediated modular CAR BCMA-stemD;SEQ ID NO: 25: mature intein-mediated modular CAR SLAMF7-stemA;SEQ ID NO: 17: mature intein-mediated modular CAR CD19;SEQ ID NO: 18: mature intein-mediated modular CAR CD20;SEQ ID NO: 19: mature intein-mediated modular DUAL CAR CD20 / CD19; SEQ ID NO: 33: mature intein-mediated modular CAR TROP2.1-stemD; and SEQ ID NO: 38: mature intein-mediated modular CAR TROP2.2-stemD.

3. A cell comprising a polynucleotide encoding for the CAR precursor or the mature intein-mediated modular CAR as defined in any of the previous claims.

4. An allogenic T cell having eliminated expression of the T cell receptor (TCR), and comprising:a first polypeptide comprising in the N to C terminal direction:i) a C terminal split intein and extein,ii) a transmembrane domain, andiii) an intracellular signaling domain,a recognition domain (RD),wherein the RD is a polypeptide capable of being activated by an exogenous molecule and transmit the activation signal to the ii) transmembrane and iii) intracellular signaling domain of the first polypeptide,wherein the RD is either bound to the N terminal of the first polypeptide by a peptide bond or is in a separate polypeptide, and wherein if the RD is in a separate polypeptide it further comprises a N terminal split extein and intein so that a trans-splicing reaction is carried out between said polypeptide and the first polypeptide, resulting in the first polypeptide comprising the RD domain in its N terminal.

5. The allogenic T cell according to claim 4, wherein the first polypeptide comprises SEQ ID NO: 4, 5, 6, 7 and 8.

6. The CAR precursor, the mature intein-mediated modular CAR, or the cell as defined in any of claims 1 to 5, for use as a medicament or in therapy.

7. The CAR precursor, the mature intein-mediated modular CAR, or cell as defined in any of claims 1 to 5, for use in the treatment or prevention of cancer.

8. The CAR precursor, the mature intein-mediated modular CAR, or the cell as defined in any of claims 1 to 3, for use in the treatment or prevention of cancer, wherein: the targeting moiety comprised in the extracellular domain binds to BCMA and the use is in the treatment or prevention of a BCMA positive cancer;the targeting moiety comprised in the extracellular domain binds to SLAMF7 and the use is in the treatment or prevention of a SLAMF7 positive cancer;the targeting moiety comprised in the extracellular domain binds to CD19 and the use is in the treatment or prevention of a CD 19 positive cancer;the targeting moiety comprised in the extracellular domain binds to CD20 and the use is in the treatment or prevention of a CD20 positive cancer;the targeting moiety comprised in the extracellular domain binds to CD20 and CD19 and the use is in the treatment or prevention of a CD20 and / or CD19 positive cancer; orthe targeting moiety comprised in the extracellular domain binds to TROP2 and the use is in the treatment or prevention of a TROP2 positive cancer.

9. The CAR precursor, the mature intein-mediated modular CAR, or cell as defined in any of claims 1 to 3, wherein the targeting moiety comprised in the extracellular domain binds to CD19 and / or CD20, for use as immunosuppressors, preferably in thetreatment or prevention of auto-immune or infectious diseases or to avoid immune reactions after surgery or organ transplants.

10. A method for producing an allogenic CAR-T cell, the method comprisinga. providing a T cell, preferably engineered T cell, having eliminated TCR expression,b. introducing in the cell of a):- a first polypeptide comprising in the N to C terminal direction:i) a C terminal split intein and extein,ii) a transmembrane domain, andiii) an intracellular signaling domain,- a recognition domain (RD),wherein the RD is a polypeptide capable of being activated by an exogenous molecule and transmit the activation signal to the ii) transmembrane and iii) intracellular signaling domain of the first polypeptide,wherein the RD is either bound to the N terminal of the first polypeptide by a peptide bond or is in a separate polypeptide, and wherein if the RD is in a separate polypeptide it further comprises a N terminal split extein and intein so that a trans-splicing reaction is carried out between said polypeptide and the first polypeptide, resulting in the first polypeptide comprising the RD domain in its N terminal.c. contacting the cell of b) with an exogenous molecule that is capable of binding to the RD, thereby activating and expanding said cell, andd. introducing in the expanded and activated cell of c) a CAR, or a nucleic acid encoding it, preferably the CAR defined in any of claims 1 to 2.

11. The method according to claim 10, wherein step b) comprises the substeps of:- b.1) genetically modifying the cell to constitutively express the first polypeptide, and- b.2) introducing a polypeptide comprising the RD into the cell of b.1) so that the cell transiently comprises the polypeptide comprising the RD, wherein the RD further comprises a N terminal split extein and intein so that a trans-splicing reaction is carried out between said polypeptide and the first polypeptide constitutively expressed by the cell, resulting in the first polypeptide comprising the RD domain in its N terminal.

12. The method according to any one of claims 10 or 11, wherein the first polypeptide comprises SEQ ID NO: 4, 5, 6, 7 and 8.

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