A chimeric transmembrane receptor comprising at least one t-cell immunoreceptor with IG and ITIM domains (TIGIT) polypeptide region, t-cells expressing the chimeric human tigit switch receptor, vectors with nucleic acids encoding for the tigit receptor, kits for preparing the t-cells, as well as corresponding pharmaceutical compositions and methods for treating a patient having a disease and for increasing cytotoxicity of a t-cell in adoptive cell therapy

Abstract

The present invention inter alia relates to a chimeric transmembrane receptor comprising a polypeptide, wherein the polypeptide comprises at least one T cell immunoreceptor with Ig and ITIM domains (TIGIT) polypeptide region comprising a TIGIT extracellular ligand binding domain; further wherein the polypeptide comprises at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises a transmembrane polypeptide region of CD2, CD40, HVEM, or CD30, and wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2, CD40, HVEM, or CD30, or wherein the transmembrane domain is from TIGIT and further wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2 or CD28. The invention also relates to corresponding nucleic acids, vectors and T-cells comprising or expressing the chimeric receptors, to a pharmaceutical composition comprising the T-cells, and to methods for preparing a T-cell for immunotherapy and for treating a disease, respectively, wherein the chimeric transmembrane receptor is used.

Description

A CHIMERIC TRANSMEMBRANE RECEPTOR COMPRISING AT LEAST ONE T- CELL IMMUNORECEPTOR WITH IG AND ITIM DOMAINS (TIGIT) POLYPEPTIDE REGION, T-CELLS EXPRESSING THE CHIMERIC HUMAN TIGIT SWITCH RECEPTOR, VECTORS WITH NUCLEIC ACIDS ENCODING FOR THE TIGIT RECEPTOR, KITS FOR PREPARING THE T-CELLS, AS WELL AS CORRESPONDING PHARMACEUTICAL COMPOSITIONS AND METHODS FOR TREATING A PATIENT HAVING A DISEASE AND FOR INCREASING CYTOTOXICITY OF A T-CELL IN ADOPTIVE CELL THERAPYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority of the European Patent application No. 24163210.8, filed March 13, 2024, the disclosure of which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0002] The present invention relates to a chimeric transmembrane receptor comprising a polypeptide, wherein the polypeptide comprises at least one T-cell immunoreceptor with Ig and ITIM domains (TIGIT) - polypeptide region comprising a TIGIT extracellular ligand binding domain, and further wherein the polypeptide comprises at least one non-TIGIT polypeptide region. The present invention further relates to an isolated nucleic acid comprising a nuclear acid sequence encoding for the chimeric transmembrane receptor, a vector comprising the nucleic acid, and to (isolated) T-cells expressing the chimeric transmembrane receptor and / or comprising the vector or nucleic acid encoding for the chimeric transmembrane receptor. The invention further relates to a kit for preparing the (isolated) T-cell of the present invention, as well as to a pharmaceutical composition comprising the T-cells. The invention also relates to a method for preparing a T-cell for immunotherapy, and to methods for treating a patient having a disease comprising administering the pharmaceutical composition, and / or for increasing cytotoxicity of a T-cell in adoptive cell therapy, comprising introducing the vector into the T-cell.BACKGROUND OF THE INVENTION

[0003] T-cells are known to be important mediators of adaptive cell-mediated immune responses. Adoptive T-cell therapy (ACT) with T-cells expressing native or transgenic ap-T-cell receptors (TCRs) is a promising treatment for cancer, as TCRs cover a wide range of potential target antigens [Chandran and Klebanoff, 2019], Native TCR specificities have successfully been exploited for ACT with tumor infiltrating lymphocytes (TILs) for melanoma [Dafni et al., 2019] and other tumors [Chandran and Klebanoff, 2019], or with virus-specific T-cells (VSTs) for viral-associated malignancies [Leung and Heslop, 2019], Transgenic TCR-based ACT allows the genetic redirection of T-cell specificity in a highly specific and reproducible manner, and has produced promising results in melanoma and several solid tumors [Robbins et al., 2015], multiple myeloma (MM) [Rapoport et al., 2015], viral-associated malignancies [Doran et al., 2019] and acute myeloid leukemia (AML) [ Chapuis et al., 2019], Another promising option in ACT is the treatment with chimeric antigen receptor (CAR)-T-cells, which has produced remarkable clinical responses with certain subsets of B cell leukemia or lymphoma [Sterner and Sterner, 2019], Promising results have also been reported with multiple myeloma.

[0004] T-cell antigen recognition and subsequent T-cell activation is known to depend on the interaction between the T-cell receptor (TCR) and peptide-major histocompatibility complex (pMHC) molecules [Davis and Bjdrkman, 1988], In particular, the CD8 co-receptor plays a major role in CD8 T-cell activation, and the CD4 co-receptor stabilizes the interaction between the TCR on CD4 T-cells and the MHC class II molecule on antigen-presenting cells (APCs). Recently, it has been reported that in adoptive therapy experiments, the efficacy of high avidity CD4 T-cells in providing protective tumor immunity was similar to the therapeutic efficacy seen with CD8 T-cells. Specifically, it has been described that a Co-transfer of Class I TCR- and CD8 coding genes generated high avidity CD4 T-cells [Xue et al., 2013],

[0005] Furthermore, in order to induce an effective immune response, in addition to antigen, T-cells need to receive positive signals. It is known that co-signaling molecules have a crucial role in regulating T-cell activation, subset differentiation, effector function and survival. For example, CD28 is constitutively expressed on naive CD4 and CD8 T- cells and has been shown to act as positive co-stimulatory molecule. CD28 engagement in the immunological synapse decreases the amount of antigen necessary to elicit T-cell activation [Kamphorst et al., 2015],

[0006] In addition to CD28, during the last years, many other costimulatory molecules have been identified. Most co-signaling molecules are members of the immunoglobulin superfamily (IgSF) and tumor necrosis factor receptor superfamily (TNFRSF). For example, TNFRSF co-signaling receptors with co-stimulatory function include HVEM (herpesvirus entry mediator), death receptor 3 (DR3; also known as TNFRSF25), CD40 (also known as TNFRSF5) and lymphotoxin-p receptor (LTBR; also known as TNFRSF3) [Chen and Flies, 2013], Furthermore, all receptors of the type-V, or divergent, family — including 4-1 BB (also known as CD137 or TNFRSF9), 0X40 (also known as TNFSF4), CD27 (also known as TNFRSF7), glucocorticoid-induced TNFR- related protein (GITR; also known as TNFRSF18) and CD30 (also known as TNFRSF8) — also function primarily as co-stimulatory molecules [Croft et al., 2012], For example, IgSF co-signaling receptors with co-stimulatory function include - in addition to CD 28, e.g. the co-stimulatory receptor inducible T-cell co-stimulator (ICOS), CD226, CRTAM, TIM 1 , CD2, SLAM, CD 84, Ly9, and CRACC [Chen and Flies, 2013],

[0007] Furthermore, there are also other receptor families that may play a role in T- cell co-stimulation. For example, although Toll like receptors (TLRs) are highly expressed by innate immune cells, particularly antigen-presenting cells, the very first report of a human TLR also described its expression and function within T-cells. By acting directly on T-cells, TLR agonists can enhance cytokine production by activated T-cells, increase T- cell sensitivity to T-cell receptor stimulation, promote long-lived T-cell memory, and reduce the suppressive activity of regulatory T-cells.

[0008] Despite the progress made during recent years in developing specific ACT’S targeting tumor cell specific antigen genes, a number of challenges of ACT’S such as tumor heterogeneity, antigen escape, T-cell trafficking and an immunosuppressive tumor microenvironment remain to be addressed. For example, solid tumors can effectively evade the immune response, including the promising T cell therapies, through the expression of various inhibitory molecules that can hinder the function of T cells. For example, while, above, receptors of the immunoglobulin superfamily (IgSF) and tumor necrosis factor receptor superfamily (TNFRSF) have been mentioned that are known to have co-stimulatory function, there are also family members which are known to be bound by the inhibitory molecules - e.g. in the immunosuppressive tumor microenvironment - and to transmit the inhibitory effect to the T-cell. Within the IgSG, for example, PD1 and T cell immunoreceptor with Ig and ITIM domains (TIGIT) and TIM-3 have been described to transmit inhibitory signals coming from solid tumors that may inhibit activation, and / or promote exhaustion of T-cells.

[0009] In order to address the issue of the inhibitory effects to the T-cell described above, chimeric switch receptors (i.e. chimeric receptors comprising the extracellular domain of an inhibitory receptor and the cytoplasmic domain of an activating receptor) have been created to reverse the outcomes of their original signaling pathways in order to confer immune cells with the ability to overcome the immunosuppressive tumor microenvironment and to allow them to have greater in vivo persistence.

[0010] Recently, it has been described that two specific TIGIT switch receptors could be coexpressed in T-cells together with an artificial CAR / TCR chimeric receptor [WO 2023 / 215725], In particular, in this international patent application, the two constructs tested for co-expression relate to respective fusions of two specific co-stimulatory domains, respectively, i.e. the co-stimulatory domains of 4-1 BB and CD2, to a TIGIT extracellular domain. However, in view of the diversity of immunosuppressive tumor microenvironments, and in light of the huge number of involved actors which may both positively and negatively regulate the suppressive activity of T-cells in adoptive cell therapy, it still remains a challenging task to provide effective T-cells exhibiting sufficient cytotoxicity and / or cytokine secretion, in particular in the immunosuppressive tumor microenvironment.

[0011] Accordingly, it is an object of the invention to provide for an improvement with respect to the above inconveniences.SUMMARY OF THE INVENTION

[0012] This object is inter alia accomplished by the chimeric transmembrane receptor, the (isolated) nucleic acid, the vectors, the (isolated) T-cells, the pharmaceutical compositions, the kits and the methods, having the features of the respective independent claims.

[0013] In a first aspect, the invention provides a chimeric transmembrane receptor comprising a polypeptide, wherein the polypeptide comprises at least one T cell immunoreceptor with Ig and ITIM domains (TIGIT) polypeptide region having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a TIGIT wildtype polypeptide as set forth in SEQ ID No. 1 , wherein the TIGIT polypeptide region comprises a TIGIT extracellular ligand binding domain; further wherein the polypeptide comprises at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises a transmembrane polypeptide region of CD2, CD40, HVEM, or CD30 , and wherein the at least one non-TIGIT polypeptide regioncomprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2, CD40, HVEM, or CD30.

[0014] In a second aspect, the invention provides a chimeric transmembrane receptor comprising a polypeptide, wherein the polypeptide comprises at least one TIGIT polypeptide region, wherein the TIGIT polypeptide region comprises a TIGIT extracellular ligand binding domain; further wherein the polypeptide comprises at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises a transmembrane polypeptide region of CD2, CD40, HVEM, or CD30, and wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2, CD40, HVEM, or CD30.

[0015] The inventors have found that a chimeric TIGIT-receptor, including the costimulatory domain of CD2, CD40, HVEM, or CD30, and further comprising a transmembrane polypeptide region of CD2, CD40, HVEM, or CD30, together with a TIGIT extracellular ligand binding domain, thus being engineered for use as a switch receptor, is able to turn negative signals e.g. present in a tumor microenvironment into positive signals for T-cell activation. Advantageously, if expressed in T-cells, the chimeric transmembrane receptors comprising the co-stimulatory domains as herein described which are linked to the at least one TIGIT polypeptide region that is still capable to bind to its natural ligand, are conveying resistance to the T-cell to the immunosuppressive tumor microenvironment. The T-cells expressing the chimeric transmembrane receptor as herein provided exhibit less TCR-T exhaustion and depletion through apoptosis, and show stimulated TCR-T proliferation and functional activity.

[0016] Since the chimeric TIGIT switch receptors as herein provided lack the cytoplasmic inhibitory motif / domain / region of the wildtype TIGIT receptor, the binding of a natural ligand to the TIGIT switch receptor, e.g. in tumor microenvironment, does no longer lead to e.g. inhibition of activation, promotion of exhaustion and / or induction of apoptosis of the T-cell expressing the chimeric TIGIT switch receptor, but - instead - even costimulates the T-cell, thereby enhancing its cytotoxic effect.

[0017] In a third aspect, the invention provides a (isolated) nucleic acid encoding for the chimeric transmembrane receptor as herein provided.

[0018] In a fourth aspect, the invention provides a vector comprising a nucleic acid comprising a nuclear acid sequence encoding for a chimeric TIGIT switch receptor as herein provided.

[0019] In some embodiments, in addition to comprising the nucleic acid sequence encoding for the chimeric TIGIT receptor, the vector may further comprise the nucleic acidsequence encoding for an engineered T-cell receptor. According to a further embodiment, the vector may additionally comprise a nucleic acid encoding for a CD8 Co-receptor, such as a wildtype CD8 Co-receptor or a chimeric CD8 Co-receptor.

[0020] For example, overexpression of a chimeric human TIGIT switch receptor as herein described in the T-cells as herein provided, e.g. alongside an engineered transgenic ap-T-cell receptor TCR and, in some embodiments, for example, alongside a CD8 Co-receptor, may offer several advantages and expands the therapeutic potential of this approach.

[0021] The optional, additional provision of the CD8 Co-receptor together with the chimeric TIGIT switch receptor and e.g. together with the engineered T-cell receptor in the T-cell of the present invention may e.g. allow for the efficient incorporation of CD4 T cells into TCR-T-cell therapy, such that it becomes possible to harness their unique properties to augment the antitumor immune response.

[0022] For example, CD4 T cells possess the ability to regulate the function of other immune cells, such as CD8 cytotoxic T-cells, dendritic cells, macrophages and B cells, by providing vital signals through the secretion of cytokines and direct cell-cell interactions. This known helper function may be crucial for enhancing the persistence and potency of TCR-T-cells within the tumour microenvironment.

[0023] In a fifth aspect, the invention provides an isolated T-cell, the T-cell comprising the vector or the nucleic acid according to the present invention.

[0024] In a sixth aspect, the invention provides an isolated T-cell, the T-cell being introduced with, such as transfected (e.g. electroporated), transduced or transformed with the vector or the nucleic acid according to the present invention.

[0025] In a seventh aspect, the invention provides an isolated T-cell, the T-cell being treated, such as transfected (e.g. electroporated), transduced or transformed to express the chimeric TIGIT receptor as herein described. According to some embodiments, the T- cell may be treated, such as transfected, transduced or transformed to express the chimeric TIGIT receptor as herein described together with an engineered T-cell receptor.

[0026] In an eighth aspect, the invention provides a kit comprising means to prepare the isolated and / or engineered T-cells according to the present invention.

[0027] In a ninth aspect, the invention provides a pharmaceutical composition comprising the isolated T-cell according to the present invention.

[0028] In a tenth aspect, the invention provides a method for preparing a T-cell for immunotherapy, comprisingisolating T-cells from a human subject, introducing the vector or the nucleic acid, as herein provided, into the T-cell, and expanding the T-cells in which the vector or nucleic acid has been introduced.

[0029] In an eleventh aspect, the invention provides a pharmaceutical composition comprising T-cells expressing the chimeric TIGIT receptor as herein described. According to some embodiments, the composition may comprise T-cells expressing the chimeric TIGIT receptor as herein described, and further expressing an engineered T-cell receptor.

[0030] In a twelfth aspect, the invention provides a method for treating a patient having a disease, comprising administering to the patient the pharmaceutical composition according to the present invention.

[0031] In a thirteenth aspect, the invention provides a method for treating a patient having a disease, comprising introducing in vivo the nucleic acid as herein described, or the vector as herein provided into a T-cell of the patient.

[0032] In a fourteenth aspect, the invention provides a method for increasing cytotoxicity of a T-cell in adoptive cell therapy, comprising - introducing a vector into the T- cell, wherein the vector comprises a nucleic acid encoding for a chimeric TIGIT-receptor as herein described, and, for example, further encoding an engineered T-cell receptor.

[0033] In a fifteenth aspect, the invention provides an isolated T-cell wherein the T- cell expresses a chimeric transmembrane receptor comprising a polypeptide, wherein said polypeptide comprises at least one T cell immunoreceptor with Ig and ITIM domains (TIGIT) polypeptide region having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a TIGIT wildtype polypeptide as set forth in SEQ ID No. 1 , wherein the TIGIT - polypeptide region comprises a TIGIT extracellular ligand binding domain, and further wherein the TIGIT polypeptide region comprises a TIGIT transmembrane polypeptide domain or region; further wherein the polypeptide comprises at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2, or CD28; wherein the T-cell further expresses a recombinant T-cell receptor.

[0034] In a sixteenth aspect, the invention provides an isolated T-cell wherein the T- cell expresses a chimeric transmembrane receptor comprising a polypeptide, wherein said polypeptide comprises at least one T cell immunoreceptor with Ig and ITIM domains (TIGIT) polypeptide region comprising a TIGIT extracellular ligand bindingdomain, and further wherein the TIGIT polypeptide region comprises a TIGIT transmembrane polypeptide domain or region; further wherein the polypeptide comprises at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2, or CD28; wherein the T-cell further expresses a recombinant T-cell receptor.

[0035] It has been found by the inventors that the T-cells comprising both an engineered T-cell receptor, and a chimeric TIGIT receptor including the costimulatory domain of CD2 or CD28, comprising a TIGIT extracellular ligand binding domain, and further comprising a TIGIT transmembrane polypeptide domain or region, thus being engineered for use as a switch receptor, are able to turn negative signals e.g. present in a tumor microenvironment into positive signals for T-cell activation. Advantageously, the T-cells as provided according to the fifteenth or sixteenth aspect of the invention, by expressing both an engineered T-cell receptor and specifically engineered recombinant chimeric TIGIT receptors, linking the co-stimulatory domains of CD2 or CD28 to at least one TIGIT polypeptide region that is still capable to bind to its natural ligand and to a TIGIT transmembrane polypeptide domain or region, is having resistance to the immunosuppressive tumor microenvironment. The T-cells exhibit less TCR-T exhaustion and depletion through apoptosis, and show stimulated TCR-T proliferation and functional activity. The binding of a natural ligand to the TIGIT switch receptor, e.g. in tumor microenvironment, does no longer lead to e.g. inhibition of activation, promotion of exhaustion and / or induction of apoptosis of the T-cell expressing the chimeric TIGIT switch receptor, but - instead - even costimulates the T-cell, thereby enhancing its cytotoxic effect.

[0036] In further aspects, this invention therefore provides T-cells comprising the chimeric TIGIT receptors according to the fifteenth or sixteenth aspect, and it is herewith contemplated that the chimeric TIGIT receptors according to the fifteenth or sixteenth aspect may also be used in the compositions, kits and methods of the present invention.

[0037] All aspects of the invention provide the above described advantages and improvements related to the provision of- a chimeric transmembrane receptor comprising a polypeptide, wherein the polypeptide comprises at least one T cell immunoreceptor with Ig and ITIM domains (TIGIT) polypeptide region (having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a TIGIT wildtype polypeptide as set forth in SEQ ID No. 1), wherein the TIGIT polypeptide region comprises a TIGIT extracellular ligand binding domain; further wherein the polypeptide comprises at least one non-TIGITpolypeptide region, wherein the at least one non-TIGIT polypeptide region comprises a transmembrane polypeptide region of CD2, CD40, HVEM, or CD30 , and wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2, CD40, HVEM, or CD30, or the provision of- T-cells comprising both an engineered / recombinant T-cell receptor and a chimeric transmembrane receptor comprising a polypeptide, wherein said polypeptide comprises at least one T cell immunoreceptor with Ig and ITIM domains (TIGIT) polypeptide region (having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a TIGIT wildtype polypeptide as set forth in SEQ ID No. 1), wherein the TIGIT polypeptide region comprises a TIGIT extracellular ligand binding domain, and further wherein the TIGIT polypeptide region comprises a TIGIT transmembrane polypeptide domain or region; further wherein the polypeptide comprises at least one non- TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2, or CD28; respectively.The switch receptors and T-cells herein provided are capable of turning negative signals (with respect to e.g. the T-cell’s activation status and / or cytotoxic capacity) into positive signals.BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the drawings, in which:

[0039] Fig. 1 shows a graphical representation of results from a flow cytometric analysis of T-cells transduced with chimeric TIGIT receptor polypeptides. For better clarity, Fig. 1 is depicted on four pages (drawing sheets 1 / 8 - 4 / 8).

[0040] Fig. 2 shows a graphical representation of results from an in-vitro T-cell killing assay of T-cells according to the invention transduced with an engineered T-cell receptor and a chimeric TIGIT receptor polypeptide in CorL23-A2-NLR cells overexpressing TIGIT ligands CD155- and Nectin4. For better clarity, Fig. 2 is depicted on four pages (drawing sheets 5 / 8 - 8 / 8).DETAILED DESCRIPTION OF THE INVENTION

[0041] As explained above, in a first aspect, the invention is directed to a chimeric transmembrane receptor comprising a polypeptide, wherein the polypeptide comprises at least one T cell immunoreceptor with Ig and ITIM domains (TIGIT) polypeptide region having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a TIGIT wildtype polypeptide as set forth in SEQ ID No. 1 , wherein the TIGIT polypeptide region comprises a TIGIT extracellular ligand binding domain; further wherein the polypeptide comprises at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises a transmembrane polypeptide region of CD2, CD40, HVEM, or CD30, and wherein the at least one non- TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2, CD40, HVEM, or CD30.

[0042] In another aspect, the invention provides a chimeric transmembrane receptor comprising a polypeptide, wherein the polypeptide comprises at least one TIGIT polypeptide region, wherein the TIGIT polypeptide region comprises a TIGIT extracellular ligand binding domain; further wherein the polypeptide comprises at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises a transmembrane polypeptide region of CD2, CD40, HVEM, or CD30, and wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2, CD40, HVEM, or CD30.

[0043] According to an embodiment, the TIGIT polypeptide region may be a human TIGIT polypeptide region from a human wildtype TIGIT receptor.

[0044] It is understood that the expression “human wildtype TIGIT receptor” relates to a protein having an amino acid sequence according to UniProtKB database entry No. Q495A1 ■ TIGIT_HUMAN, as set forth e.g. in SEQ ID No. 1.

[0045] As used herein, the term “TIGIT polypeptide region” refers to a polypeptide containing at least a functional portion (e.g., an extracellular ligand binding domain) of a wild-type TIGIT protein or a variant thereof, such as a variant that has at least 60% sequence identity (e.g., at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the amino acid sequence of SEQ ID NO: 1 and that retains the ability to bind an endogenous TIGIT ligand. Similarly, it is to be understood that the terms "TIGIT immunoglobulin variable domain", "TIGIT co-stimulatory cytoplasmic polypeptide domain", "TIGIT homodimerization motif", and the like, refer to a portion of wild-type TIGIT comprising the corresponding protein domain (i.e., the immunoglobulin variable domain, co-stimulatory cytoplasmic polypeptide domain, or homodimerization motif, respectively,of wild-type TIGIT) or a sequence variant thereof, such as a sequence variant recited herein.

[0046] As used herein, the term “non-TIGIT polypeptide domains, regions, or motifs” refers to a polypeptide domain, region, and motif, respectively, that is neither obtained from wild-type TIGIT nor is a functional variant thereof. Examples of non-TIGIT polypeptide domains, regions, or motifs are those that are obtained from, e.g., CD2, CD40, HVEM, CD28 or CD30, as well as sequence variants thereof, such as sequence variants recited herein. Other Examples of non-TIGIT polypeptide domains, regions, or motifs are e.g. those that may be obtained from other costimulatory molecules, as well as sequence variants thereof.

[0047] For example, the extracellular ligand binding domain may comprise a TIGIT immunoglobulin variable (IgV) domain, wherein the TIGIT IgV domain may have at least 85% sequence identity to the amino acid sequence of SEQ ID No. 2. For example, the at least one TIGIT IgV domain may have at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID No. 2.

[0048] The term "sequence identity" or "identity" as used in the present invention means the percentage of pair-wise identical residues, following homology alignment of a sequence of a polypeptide and or nucleic acid of the present invention with a sequence in question, with respect to the number of residues in the longer of these two sequences.

[0049] The percentage of sequence homology or sequence identity can, for example, be determined herein using the program BLASTP, version blastp 2.2.5 (November 16, 2002; cf. Altschul, S. F. et al. (1997) Nucl. Acids Res. 25, 3389-3402). The percentage of homology is based on the alignment of the entire polypeptide sequences (matrix: BLOSIIM 62; gap costs: 11.1 ; cutoff value set to 10-3) including the respective sequences. It is calculated as the percentage of numbers of "positives" (homologous amino acids) indicated as result in the BLASTP program output divided by the total number of amino acids selected by the program for the alignment.

[0050] It is noted in this context that it has been found here for the first time that the chimeric TIGIT receptor comprising both a functional extracellular TIGIT receptor ligand binding domain and at least one non-TIGIT co-stimulatory cytoplasmic polypeptidedomain, region or motif of CD2, CD40, HVEM, or CD30, as well as a non-TIGIT transmembrane polypeptide region of CD2, CD40, HVEM, or CD30, is able to turn negative signals (e.g. present in a tumor microenvironment) into positive signals for T-cell activation. Advantageously, replacing e.g. at least the cytoplasmic “ITIM” motif of wildtype TIGIT (amino acids 229-243 of SEQ ID No. 1), which is characterized by its ability to initiate an inhibition mechanism within cells, or replacing e.g. the complete cytoplasmic domain of TIGIT (amino acids 163- 244) of SEQ ID No. 1) by a at least one non-TIGIT co-stimulatory cytoplasmic polypeptide domain, region or motif of CD2, CD40, HVEM, or CD30, and, furthermore, replacing the transmembrane polypeptide region of TIGIT (amino acids 142- 162 of SEQ ID No. 1) by the transmembrane polypeptide region of CD2, CD40, HVEM, or CD30, enables a T-cell comprising the chimeric TIGIT receptor and e.g. an engineered T- cell receptor to bypass the inhibitory effects of TIGIT ligands expressed by the tumor microenvironment, thus creating resistance to tumor-mediated immune suppression. Secondly, the chimeric TIGIT receptors as herein provided may act as a molecular switch, redirecting the signaling pathways triggered by TIGIT engagement with a TIGIT ligand. Instead of inducing inhibition, the fusion of the TIGIT receptor ligand binding domain to a co-stimulatory domain of CD2, CD40, HVEM, or CD30, as well as to a transmembrane domain of CD2, CD40, HVEM, or CD30 alters the intracellular signaling events, promoting T cell activation, persistence and enhanced anti-tumor responses. By introducing a T-cell as herein provided comprising an engineered chimeric TIGIT receptor together with e.g. an engineered T-cell receptor into T cell therapy, the expression of TIGIT ligands by solid tumors is rendered ineffective in hindering T cell function. This innovative approach empowers the T cells as herein provided to resist the immune evasion mechanisms deployed by solid tumors, enabling them to better recognize and eliminate tumor cells.

[0051] According to an embodiment, the extracellular ligand binding domain of the chimeric TIGIT receptor may be functional in binding at least one TIGIT ligand or any other protein / polypeptide having the ability of binding to the wildtype TIGIT receptor ligand binding domain.

[0052] For example, the at least one TIGIT ligand may comprise at least one of the TIGIT ligands CD155 (PVR) and / or CD112 (PVRL2, nectin-2).

[0053] It is contemplated that the chimeric transmembrane receptor may further comprise an extracellular homodimerization motif, such as a TIGIT homodimerization motif (amino acids 32-42 of SEQ ID No. 1). For example, the extracellular homodimerization motif as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 3. It is herewith envisaged that the extracellular homodimerizationmotif as included in the chimeric transmembrane receptor as herein provided may have one or more conservative amino acid substitutions relative to the extracellular homodimerization motif of the wildtype TIGIT receptor. In particular, all amino acid substitutions that maintain the functional activity of the wildtype extracellular homodimerization motif are envisaged. For example, the TIGIT homodimerization motif may have at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with an amino acid sequence as set forth in SEQ ID No: 3.

[0054] Thus, it is contemplated that the chimeric TIGIT receptor polypeptide as herein provided may comprise, in addition to comprising the TIGIT ligand binding domain, and in addition to comprising the transmembrane domain and at least one costimulatory domain CD2, CD40, HVEM or CD30, further domain regions / motif regions / binding site regions from a wildtype human TIGIT receptor in every conceivable combination to establish a functional chimeric TIGIT receptor polypeptide. “Functional” chimeric TIGIT receptor in this context relates to a chimeric TIGIT receptor that is capable of redirecting the signaling pathways triggered by TIGIT receptor engagement with at least one TIGIT ligand such that - instead of inducing inhibitory pathways in the T-cell - binding of the at least one TIGIT ligand promotes T-cell activation, persistence and enhanced anti-tumor responses of the T-cell expressing a chimeric transmembrane receptor as herein provided. For example, an optional test for functionality of a chimeric transmembrane receptor may be an in-vitro T-cell killing assay as described e.g. by Kalbasi, A., Siurala, M., Su, L.L. et al. “Potentiating adoptive cell therapy using synthetic IL-9 receptors”. Nature 607, 360- 365 (2022) using cells expressing a TIGIT ligand. Thus, the expression “every conceivable combination” of TIGIT receptor polypeptide regions as described above is meant to exclude a combination with wildtype human TIGIT receptor domains / regions / motifs being inhibitory. For example, the chimeric TIGIT receptor polypeptide as herein provided may lack the “ITIM” motif of wildtype TIGIT (amino acids 229-234 of SEQ ID No. 1), or may merely comprise an altered “ITIM” motif which no longer functions in promoting inhibitory pathways of the T-cell, e.g. due to mutations which abolish any inhibitory promoting functionality of the “ITIM” motif.

[0055] According to an embodiment, the at least one TIGIT polypeptide region has at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity with the functional polypeptide domain or a functional polypeptide motif of a wildtype human TIGIT receptor (e.g. Seq ID No. 1). According to an embodiment, the at least one TIGIT polypeptide region may have one or more conservative amino acid substitutions relative to the amino acid sequence of the wildtype TIGIT receptor.

[0056] For example, the at least one TIGIT polypeptide region may have at least 70%, or at least 71 %, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81 %, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with the functional polypeptide domain or a functional polypeptide motif of a wildtype human TIGIT receptor (e.g. Seq ID No. 1).

[0057] The (human) TIGIT polypeptide region comprising a TIGIT extracellular ligand binding domain may have -in general- a sufficient portion of the human wildtype TIGIT extracellular ligand binding domain to be functional in binding at least one TIGIT ligand. For example, said at least one TIGIT polypeptide region having at least 60% sequence identity with TIGIT ligand binding domain of a human wildtype TIGIT receptor may comprise the complete or a considerable part of the human wildtype TIGIT ligand binding domain and / or all amino acids at respective amino acid positions of the wildtype TIGIT receptor that are necessary and sufficient for binding of at least one ligand to the TIGIT receptor. For example, variants of wildtype TIGIT as herein described may comprise an extracellular domain that preserves the ability to bind at least one TIGIT ligand. Suitable methods (such as e.g. surface plasmon resonance assay) for determining said functional ability are known to the skilled person.

[0058] According to an embodiment, the TIGIT polypeptide region may comprise a complete wildtype TIGIT receptor extracellular domain. For example, the expression “wildtype TIGIT receptor extracellular domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 22-141 of UniProtKB database entry No. Q495A1 ■ TIGIT_HUMAN, as set forth e.g. in SEQ ID No. 1. For example, the wildtype TIGIT receptor extracellular domain as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 4. It is herewith envisaged thatthe TIGIT receptor extracellular domain as included in the chimeric transmembrane receptor as herein provided may have one or more conservative amino acid substitutions relative to the extracellular domain of the wildtype TIGIT receptor. In particular, all amino acid substitutions that maintain the functional activity of the wildtype TIGIT extracellular domain are envisaged.

[0059] The expressions “domain region”, “binding site region”, “motif region” as used herein are understood to relate to e.g. a region of the chimeric transmembrane receptor polypeptide which is necessary and / or sufficient for a biological function of the chimeric receptor, or to a region of the chimeric TIGIT receptor which is defined e.g. by a localization with respect to a cell, or to a structurally defined unit of the chimeric TIGIT receptor polypeptide.

[0060] The chimeric transmembrane receptor polypeptide may be a single-chain polypeptide.

[0061] It is envisaged that the chimeric transmembrane receptor as herein provided may comprise- a transmembrane region from CD2 and a costimulatory cytoplasmic polypeptide domain, region or motif from CD2, or- a transmembrane region from CD40 and a costimulatory cytoplasmic polypeptide domain, region or motif from CD40, or- a transmembrane region from HVEM and a costimulatory cytoplasmic polypeptide domain, region or motif from HVEM, or- a transmembrane region from CD30 and a costimulatory cytoplasmic polypeptide domain, region or motif from CD30.

[0062] According to an embodiment, the at least one non-TIGIT polypeptide region may comprise a transmembrane polypeptide region of CD2. The transmembrane domain of wildtype CD2 as referred to herein may relate to a polypeptide comprising amino acid sequence 210-235 of UniProtKB database entry P06729 ■ CD2_HUMAN, as set forth e.g. in SEQ ID No. 5. For example, the transmembrane domain of wildtype CD2 as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 6. It is herewith envisaged that the transmembrane domain as included in the chimeric transmembrane receptor as herein provided may have one or more conservative amino acid substitutions relative to the transmembrane domain of the wildtype CD2 receptor. In particular, all amino acid substitutions that maintain the functional activity of the wildtype transmembrane domain are envisaged.

[0063] For example, the non TIGIT transmembrane polypeptide region may have at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81 %, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 6.

[0064] According to an embodiment, the at least one non-TIGIT polypeptide region may comprise a transmembrane polypeptide region of CD40. The transmembrane domain of wildtype CD40 as referred to herein may relate to a polypeptide comprising amino acid sequence 194-215 of UniProtKB database entry No. P25942- TNR5_HUMAN, as set forth e.g. in SEQ ID No. 7. For example, the transmembrane domain of wildtype CD40 as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 8. It is herewith envisaged that the transmembrane domain as included in the chimeric transmembrane receptor as herein provided may have one or more conservative amino acid substitutions relative to the transmembrane domain of the wildtype CD40 receptor. In particular, all amino acid substitutions that maintain the functional activity of the wildtype transmembrane domain are envisaged.

[0065] For example, the non TIGIT transmembrane polypeptide region may have at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81 %, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 8.

[0066] According to an embodiment, the at least one non-TIGIT polypeptide region may comprise a transmembrane polypeptide region of HVEM. The transmembrane domain of wildtype HVEM as referred to herein may relate to a polypeptide comprising amino acid sequence 203-223 of UniProtKB database entry No. Q92956- TNR14_HUMAN, as set forth e.g. in SEQ ID No. 9. For example, the transmembrane domain of wildtype HVEM as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 10. It is herewith envisaged that the transmembrane domain as included in the chimeric transmembrane receptor as hereinprovided may have one or more conservative amino acid substitutions relative to the transmembrane domain of the wildtype HVEM receptor. In particular, all amino acid substitutions that maintain the functional activity of the wildtype transmembrane domain are envisaged. For example, the non TIGIT transmembrane polypeptide region may have at least 70%, or at least 71 %, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81 %, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 10.

[0067] According to an embodiment, the at least one non-TIGIT polypeptide region may comprise a transmembrane polypeptide region of CD30. The transmembrane domain of wildtype CD30 as referred to herein may relate to a polypeptide comprising amino acid sequence 386-406 of UniProtKB database entry P28908 ■ TNR8_HUMAN, as set forth e.g. in SEQ ID No. 11. For example, the transmembrane domain of wildtype CD30 as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 12. It is herewith envisaged that the transmembrane domain as included in the chimeric transmembrane receptor as herein provided may have one or more conservative amino acid substitutions relative to the transmembrane domain of the wildtype CD30 receptor. In particular, all amino acid substitutions that maintain the functional activity of the wildtype transmembrane domain are envisaged.

[0068] For example, the non TIGIT transmembrane polypeptide region may have at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81 %, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 12.

[0069] According to some embodiments, the transmembrane domain of the chimeric transmembrane receptor of the T-cell as herein provided may be from other proteins which comprise a transmembrane domain, except TIGIT. For example, the transmembrane domain may be from a further co-stimulatory molecule known in the art. In principle, anytransmembrane domain which is functional and allows surface detectable expression of the chimeric transmembrane receptor is herewith envisaged.

[0070] The chimeric transmembrane receptor as herein provided may further comprise at least one linker region. This may be e.g. a polypeptide linker region. Such linker(s) may be included e.g. between functional domains / regions / motifs of the chimeric transmembrane receptor. It may be a linker region naturally occurring e.g. in wildtype TIGIT receptor, or e.g. in co-stimulatory proteins, e.g. in costimulatory proteins from which the costimulatory domain of the receptor is derived. For example, polypeptide linker regions may be included between the transmembrane domain and the ligand binding domain, and / or between the transmembrane domain and the IgV domain of the chimeric TIGIT receptor, and / or between the transmembrane domain and the at least one intracellular co-stimulatory domain, and / or between individual co-stimulatory domains (in embodiments comprising more than one co-stimulatory domains).

[0071] Such linker region may comprise 1-100 amino acids, or e.g. 1-80 amino acids, or e.g. 1-50 amino acids, or e.g. 5-100 amino acids.

[0072] According to an embodiment, a linker region of the chimeric transmembrane receptor as herein provided may comprise the amino acid sequence as set forth in SEQ ID No. 13 (GGGS)n or as set forth in Seq ID No. 14 (GGGGS)n, wherein n is between 0 and 20, or wherein n is between 0 and 10, or where n is between 0 and 5, or where n is between 3 and 5.

[0073] However, in principle, each (polypeptide) linker known in the art is herewith envisaged as being potentially included in the chimeric transmembrane switch receptor of the present invention.

[0074] Turning now to the co-stimulatory domain, in accordance with the present invention, the chimeric transmembrane receptor polypeptide as herein provided may comprise at least one costimulatory cytoplasmic polypeptide domain or cytoplasmic polypeptide motif of CD2, CD40, HVEM, or CD30. The authors have found for the first time that cytoplasmic co-stimulatory domains of CD2, CD40, HVEM, or CD30, together with a transmembrane polypeptide region of CD2, CD40, HVEM, or CD30, may be fused to the at least one TIGIT polypeptide region comprising a functional TIGIT receptor extracellular ligand binding domain in order to generate a functional chimeric TIGIT switch receptor capable of redirecting the signaling pathways triggered by TIGIT receptor engagement with at least one TIGIT ligand such that - instead of inducing inhibitory pathways in the cell - binding of the at least one TIGIT ligand promotes T-cell activation, persistence and enhanced anti-tumor responses of a T-cell expressing the chimeric transmembranereceptors as herein provided, for example when the T-cell at the same time comprises / expresses e.g. an engineered T-cell receptor.

[0075] According to an embodiment, the chimeric TIGIT receptor as herein provided may comprise e.g. at least one complete cytoplasmic domain of CD2, CD40, HVEM, or CD30.

[0076] According to some embodiments, the at least one cytoplasmic polypeptide domain or cytoplasmic polypeptide motif selected from the group consisting of CD2, CD40, HVEM, and CD30 may have an amino acid sequence having at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81 %, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with the respective functional polypeptide domain or a functional polypeptide motif of a wildtype human CD2, CD40, HVEM, or CD30, respectively.

[0077] According to an embodiment, the chimeric transmembrane receptor as herein provided may be able to sustain or enhance cytotoxicity and / or cytokine secretion of a T-cell upon binding a TIGIT ligand.

[0078] According to an embodiment, the chimeric transmembrane receptor as herein provided may be capable of increasing resistance of T-cells to TIGIT ligand expressing cancer cells.

[0079] It is contemplated that the chimeric TIGIT receptor as herein provided may comprise any functional combination of co-stimulatory cytoplasmic polypeptide domain(s) motif(s) and / or region(s) of the cytoplasmic polypeptide domain, region or motif selected from the group consisting of CD2, CD40, HVEM, and CD30.

[0080] According to an embodiment, the chimeric transmembrane receptor as herein provided may comprise at least one cytoplasmic polypeptide domain, region or motif of wildtype CD40. For example, the expression “wildtype human CD40 cytoplasmic domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 216- 277 of UniProtKB database entry No. P25942- TNR5_HUMAN, as set forth e.g. in SEQ ID No. 7. It is herewith envisaged that the cytoplasmic polypeptide domain, region or motif of wildtype CD40 as included in the chimeric transmembrane receptor as herein provided may have one or more conservative amino acid substitutions relative to the amino acid sequence as set forth in SEQ ID No. 15. In particular, all amino acid substitutions thatmaintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype CD40 are envisaged.

[0081] In embodiments wherein a costimulatory region, motif or domain of CD 40 is included in the chimeric transmembrane receptor as herein provided, for example, the polypeptide of the chimeric transmembrane receptor may have an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID NO: 16.

[0082] According to an embodiment, the chimeric transmembrane receptor as herein provided may comprise at least one cytoplasmic polypeptide domain, region or motif of HVEM. For example, a cytoplasmic polypeptide region of HVEM may comprise the complete cytoplasmic domain of wildtype human HVEM. In other embodiments, the cytoplasmic polypeptide region of HVEM included in the chimeric transmembrane receptor may comprise at least one functional, co-stimulatory motif / domain / region of the complete wildtype human HVEM cytoplasmic domain. For example, the expression “wildtype human HVEM cytoplasmic domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 224-283 of UniProtKB database entry No. Q92956- TNR14_HUMAN, as set forth e.g. in SEQ ID No. 9. It is herewith envisaged that the cytoplasmic polypeptide domain, region or motif of wildtype HVEM as included in the chimeric transmembrane receptor as herein provided may have one or more conservative amino acid substitutions relative to the amino acid sequence as set forth in SEQ ID No. 17. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype HVEM are envisaged.

[0083] In embodiments wherein a costimulatory region, motif or domain of HVEM is included in the chimeric transmembrane receptor, for example, the polypeptide of the chimeric transmembrane receptor may have an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID NO: 18.

[0084] According to an embodiment, the chimeric transmembrane receptor as herein provided may comprise at least one cytoplasmic polypeptide domain, region or motif of CD30. For example, a cytoplasmic polypeptide region of CD30 may comprise the complete cytoplasmic domain of wildtype human CD30. In other embodiments, the cytoplasmic polypeptide region of CD30 included in the chimeric transmembrane receptor may comprise at least one functional, co-stimulatory motif / domain / region of the complete wildtype human CD30 cytoplasmic domain. According to some embodiments, the polypeptide comprises a truncated cytoplasmic domain of CD30, optionally wherein the truncated cytoplasmic domain of CD30 comprises or consists of at least one CD30 TRAF binding motif. For example, the expression “wildtype human CD30 cytoplasmic domain”as referred to herein may relate to a polypeptide comprising amino acid sequence 407- 595 of UniProtKB database entry P28908 ■ TNR8_HUMAN, as set forth e.g. in SEQ ID No. 11. It is herewith envisaged that the cytoplasmic polypeptide domain, region or motif of wildtype CD30 as included in the chimeric transmembrane receptor as herein provided may have one or more conservative amino acid substitutions relative to the amino acid sequence as set forth in SEQ ID No. 19. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype CD30 are envisaged.

[0085] In embodiments wherein a costimulatory region, motif or domain of CD30 is included in the chimeric transmembrane receptor, for example, the polypeptide of the chimeric transmembrane receptor may have an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID NO.: 20.

[0086] According to an embodiment, the chimeric transmembrane receptor as herein provided may comprise at least one cytoplasmic polypeptide domain, region or motif of CD2. For example, a cytoplasmic polypeptide region of CD2 may comprise the complete cytoplasmic domain of wildtype human CD2. In other embodiments, the cytoplasmic polypeptide region of CD2 included in the chimeric transmembrane receptor may comprise at least one functional, co-stimulatory motif / domain / region of the complete wildtype human CD2 cytoplasmic domain. For example, the expression “wildtype human CD2 cytoplasmic domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 236-351 of UniProtKB database entry P06729 ■ CD2_HUMAN, as set forth e.g. in SEQ ID No. 5. It is herewith envisaged that the cytoplasmic polypeptide domain, region or motif of wildtype CD2 as included in the chimeric transmembrane receptor of the T-cell as herein provided may have one or more conservative amino acid substitutions relative to the amino acid sequence as set forth in SEQ ID No. 21 . In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype CD2 are envisaged.

[0087] In embodiments wherein a costimulatory region, motif or domain of CD2 is included in the chimeric transmembrane receptor, for example, the polypeptide of the chimeric transmembrane receptor may have an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID NO.: 22.

[0088] According to some embodiments, a chimeric transmembrane receptor as herein provided may e.g. comprise a polypeptide having an amino acid sequence with at least 85% or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or atleast 96% or at least 97%, or at least 98%, or at least 99%, or 100% identity to the amino acids as set forth in any one of the SEQ ID No’s selected from the group consisting of SEQ ID No. 16, 18, 20, and 22. According to some embodiments, a chimeric transmembrane receptor as herein provided may have one or more conservative amino acid substitutions relative to an amino acid sequence as set forth in any one of the SEQ ID No’s selected from the group consisting of SEQ ID No. 16, 18, 20, and 22.

[0089] In a third aspect, the invention provides a (isolated) nucleic acid encoding for the chimeric transmembrane receptor as herein provided.

[0090] In a fourth aspect, the invention provides a vector comprising a nucleic acid comprising a nuclear acid sequence encoding for a chimeric transmembrane switch receptor (chimeric TIGIT switch receptor) as herein provided.

[0091] The term “polynucleotide” or “nucleic acid” as used herein comprises a sequence of polyribonucleotides and polydeoxribonucleotides, e.g. modified or unmodified RNA or DNA, each in single-stranded and / or double-stranded form linear or circular, or mixtures thereof, including hybrid molecules. The nucleic acids according to this invention thus comprise DNA (such as dsDNA, ssDNA, cDNA), RNA (such as dsRNA, ssRNA, mRNA ivtRNA), combinations thereof or derivatives (such as RNA) thereof.

[0092] A polynucleotide may comprise a conventional phosphodiester bond or a non- conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (RNA)). The polynucleotides of the invention may also contain one or more modified bases, such as, for example, tritylated bases and unusual bases such as inosine. Other modifications, including chemical, enzymatic, or metabolic modifications, are also conceivable, as long as a binding molecule of the invention can be expressed from the polynucleotide. The polynucleotide may be provided in isolated form as defined elsewhere herein. A polynucleotide may include regulatory sequences such as transcription control elements (including promoters, enhancers, operators, repressors, and transcription termination signals), ribosome binding site, introns, or the like.

[0093] For example, the present invention provides a polynucleotide comprising or consisting of a nucleic acid that is at least about 80 %, about 85 %, about 90 %, about 91 %, about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 %, or 100 % identical to a reference polynucleotide sequence selected from the group consisting of sequences as depicted in SEQ ID NOs: 23 - 26.

[0094] The polynucleotides described above may or may not comprise additional or altered nucleotide sequences encoding e.g., altered amino acid residues. Thepolynucleotides may further encode fusion polypeptides, fragments, variants and other derivatives of the chimeric transmembrane receptors described herein.

[0095] The nucleic acid sequences of the vectors of the present invention may be codon-optimized for optimal expression in the desired host T-cell, e.g. a human lymphocyte; or for expression in bacterial, yeast or insect T-cells that are particularly envisaged for the expression of a soluble TCR of the invention. Codon-optimization refers to the exchange in a sequence of interest of codons that are generally rare in highly expressed genes of a given species by codons that are generally frequent in highly expressed genes of such species, such codons encoding the same amino acids as the codons that are being exchanged. Selection of optimum codons thus depends on codon usage of the host genome and the presence of several desirable and undesirable sequence motifs.

[0096] A “vector” as understood herein relates to a nucleic acid molecule used as a vehicle to transfer (foreign) genetic material into a host T-cell where it can for instance be replicated and / or expressed.

[0097] The vector may be a viral vector or a non-viral vector.

[0098] Viral vectors may be selected from adenoviruses, poxviruses, alphaviruses, arenaviruses, flaviruses, rhabdoviruses, retroviruses, lentiviruses, herpesviruses, paramyxoviruses, picornaviruses, and combinations thereof. Viruses used for transfection of T-cells may include naturally occurring viruses as well as artificial viruses. Viruses may be either an enveloped or non-enveloped virus. Parvoviruses (such as AAVs) are examples of non-enveloped viruses. The viruses may be enveloped viruses. The viruses used for transfection of T-cells may be retroviruses and in particular lentiviruses. Viral envelope proteins that can promote viral infection of eukaryotic cells may comprise HIV- 1 derived lentiviral vectors (LVs) pseudotyped with envelope glycoproteins (GPs) from the vesicular stomatitis virus (VSV-G), the modified feline endogenous retrovirus (RD114TR), and the modified gibbon ape leukemia virus (GALVTR). These envelope proteins can efficiently promote entry of other viruses, such as parvoviruses, including adeno- associated viruses (AAV), thereby demonstrating their broad efficiency. For example, other viral envelop proteins may be used including Moloney murine leukemia virus (MLV) 4070 env (such as described in Merten et aL, J. Virol.79:834-840, 2005; the content of which is incorporated herein by reference), RD114 env, chimeric envelope protein RD114pro or RDpro (which is an RD114-HIV chimera that was constructed by replacing the R peptide cleavage sequence of RD114 with the HIV-1 matrix / capsid (MA / CA) cleavage sequence, such as described in Bell et al. Experimental Biology and Medicine2010; 235: 1269-1276; the content of which is incorporated herein by reference), baculovirus GP64 env (such as described in Wang et al. J. Virol. 81 :10869-10878, 2007; the content of which is incorporated herein by reference), or GALV env (such as described in Merten et al., J. Virol. 79:834-840, 2005; the content of which is incorporated herein by reference), or derivatives thereof.

[0099] Non-viral vectors may comprise a naked nucleic acid such as naked plasmid DNA, cationic lipids, synthetic polycationic polymers, dendrimers, synthetic peptides such as cell-penetrating peptides (CPP’s), p-1 ,3- glucans, or combinations thereof

[0100] In particular, the term “vector” as used herein encompasses, without limitation, plasmids, viral vectors (including retroviral vectors, lentiviral vectors, adenoviral vectors, vaccinia virus vectors, polyoma virus vectors, and adenovirus-associated vectors (AAV)), phages, phagemids, cosmids and artificial chromosomes (including BACs and YACs). The vector itself is generally a nucleotide sequence, commonly a DNA sequence that comprises an insert (transgene) and a larger sequence that serves as the “backbone” of the vector. Engineered vectors typically comprise an origin for autonomous replication in the host-cells (if stable expression of the polynucleotide is desired), selection markers, and restriction enzyme cleavage sites (e.g. a multiple cloning site, MCS). The vector may additionally comprise promoters, genetic markers, reporter genes, targeting sequences, other regulatory elements, and / or protein purification tags. As known to those skilled in the art, large numbers of suitable vectors are known to those of skill in the art and many are commercially available.

[0101] In some embodiments, the vector may further comprise a nucleic acid encoding a chimeric antigen receptor (CAR).

[0102] In an embodiment, the vector may further comprise a nucleic acid encoding a T-cell receptor comprising a TCRa chain and a TCRp chain. For example, the engineered T-cell receptor may be a recombinant / engineered T-cell receptor.

[0103] In a further embodiment, the vector may further comprise a nucleic acid encoding for a CD8 Co-receptor. For example, the CD 8 Co-receptor may be a wildtype CD 8 Co-receptor. It is contemplated that the nucleic acid may encode e.g. a CD8a and a CD8p Co-receptor. An advantage of incorporation of CD8 co-receptor into the vector is the resulting option of achieving a coordinated CD4+ and CD8+ TCR-T cell response in adoptive cell therapy which broadens and deepens clinical responses. Alternatively, the CD8 Co-receptor may be a chimeric CD8 Co-receptor with CD8 Co-receptor functionality.

[0104] It is understood that the expression human wildtype CD8a Co-receptor relates to a protein having an amino acid sequence according to UniProtKB database entry No. P01732 ■ CD8A_HUMAN, as set forth e.g. in SEQ ID No. 27. It is further understood that the expression human wildtype CD8p Co-receptor relates to a protein having an amino acid sequence according to UniProtKB database entry No. P10966 ■ CD8B_HUMAN, as set forth e.g. in SEQ ID No. 28.

[0105] A chimeric human CD8 Co-receptor polypeptide as referred to herein may comprise a single chain polypeptide comprising both an CD8a IG-like domain region together with an CD8p IG-like domain region, and may be able to maintain the function of an CD8a IG-like domain region and an CD8p IG-like domain region being present on individual, separate polypeptides in a wildtype CD8a co-receptor.

[0106] Thus, the provision of T-cells as herein provided comprising the chimeric transmembrane receptor, the engineered T-cell receptor and, in addition, a chimeric CD8 Co-receptor therefore exhibit enhanced T-cell activation, proliferation, cytokine production, and cytotoxicity, ultimately improving the therapeutic efficacy of TCR-T-cell therapy.

[0107] In some embodiments, the vector may further include one or more multicistronic element(s) and the multicistronic element(s) may be positioned, for example, between any two nucleic acid sequences encoding for the chimeric transmembrane receptor, and the optional TCRa, TCRp, and CD8 Coreceptor. In some embodiments, the multicistronic element(s) may include a sequence encoding a ribosome skip element selected from among a T2A, a P2A, a E2A or a F2A or an internal ribosome entry site (IRES).

[0108] As used herein, the term “self-cleaving 2A peptide” refers to relatively short peptides (of the order of 20 amino acids long, depending on the virus of origin) acting co- translationally, by preventing the formation of a normal peptide bond between the glycine and last proline, resulting in the ribosome skipping to the next codon, and the nascent peptide cleaving between the Gly and Pro. After cleavage, the short 2A peptide remains fused to the C-terminus of the 'upstream’ protein, while the proline is added to the N- terminus of the 'downstream’ protein. Self-cleaving 2A peptide may be selected from porcine teschovirus-1 (P2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), foot- and-mouth disease virus (F2A), or any combination thereof. By adding the linker sequences (GSG or SGSG [SEQ ID No.: 48]) before the selfcleaving 2A sequence, this may enable efficient synthesis of biologically active proteins, e.g., TCRs and chimeric transmembrane receptors as described herein.

[0109] Turning now to a further aspect, there is also provided an (isolated) T-cell, the T-cell comprising a nucleic acid encoding for the chimeric human transmembrane receptor of the present invention.

[0110] In accordance with an embodiment, the T-cell may further comprise a nucleic acid encoding for an engineered T-cell receptor or a CAR.

[0111] It is to be noted that in the context of this invention, the expressions “engineered T-cell receptor” and “recombinant T-cell receptor”, respectively, are to be distinguished from a “CAR” T-cell receptor. For example, unlike chimeric antigen receptors (CARs), engineered / recombinant TCRs recognize HLA-presented peptides derived from proteins of all cellular compartments. Furthermore, the expressions “engineered T-cell receptor” and “recombinant T-cell receptor”, respectively, are understood to embrace TCRs that are not naturally expressed by the recited T cell (e.g., TCRs that are exogenous to the T cell and that are introduced into the T cell genome by way of a genetic engineering technique described herein, and / or e.g. by mRNA based transient expression).

[0112] According to an embodiment, the T-cell may further comprise a nucleic acid encoding for a recombinant CD8 Co-receptor.

[0113] According to an embodiment, the one or more nucleic acid may have been stably integrated into the genome of the T-cell, e.g. by targeted knock-in utilizing e.g. CRISPR / Cas9.

[0114] In accordance with another aspect, a T-cell may express the chimeric transmembrane receptor as herein described.

[0115] For example, the T-cell may further express an engineered T-cell receptor or a CAR.

[0116] The T-cell may, in some embodiments, further express a CD8 co-receptor such as a wildtype CD8 co-receptor or a chimeric CD8 co-receptor. The chimeric CD8 co- receptor may be a chimeric receptor having functionality of a wildtype CD8 Co-receptor. For example, the chimeric CD8 co-receptor may have the same MHC-complex binding functionality as a wildtype CD8 Co-receptor.

[0117] For example, the T-cell may be a CD4 T-cell, and further the CD4 T-cell may additionally expresses a recombinant human CD8 co-receptor, such as e.g. a CD8a and CD8p receptor, or a chimeric CD8 co-receptor.

[0118] According to some embodiments, the vector and / or the nucleic acid as herein described may have been introduced into the T-cell.

[0119] The (isolated) T-cells may be generated using various methods, including those recognized in the literature. For example, a polynucleotide encoding an expressioncassette that comprises a tumor recognition, or another type of recognition moiety, and that also encodes for a chimeric transmembrane receptor as herein described and, optionally, the engineered T-cell receptor or a CAR and, optionally, a CD8 Co-receptor may be stably introduced into the T-cell by a transposon / transposase system or a viralbased gene transfer system, such as a lentiviral or a retroviral system, or another suitable method, such as transfection, electroporation, transduction, lipofection, calcium phosphate (CaPCll), nanoengineered substances, such as Ormosil, mRNA-based therapy, viral delivery methods, including adenoviruses, retroviruses, lentiviruses, adeno-associated viruses, or another suitable method. It is envisaged that T-cells may be generated by in vivo introduction of nucleic acid in T-cells, e.g. by using DNA or mRNA, e.g. by using nanoparticles such as lipid nanoparticles.

[0120] The T-cells may be transfected by means known in the art including lipofection (liposome-based transfection), electroporation, calcium phosphate transfection, biolistic particle delivery (e.g., gene guns), microinjection, or combinations thereof. Various methods of transfecting cells are known in the art. See, e.g., Sambrook & Russell (Eds.) Molecular Cloning: A Laboratory Manual (3rd Ed.) Volumes 1-3 (2001) Cold Spring Harbor Laboratory Press; Ramamoorth & Narvekar “Non Viral Vectors in Gene Therapy- An Overview.” JCIinDiagn Res. (2015) 9(1): GE01-GE06.

[0121] According to an embodiment, the cell may be an p T-cell, y8 T-cell, and / or a natural killer T-cell.

[0122] For example, the ap T-cell may be a CD4 T-cell, or the ap T-cell may be a CD8 T-cell, or the y8 T-cell may comprise e.g. a Vy1 chain or a Vy2 chain, or may be e.g. a Vy9V82+ T-cell.

[0123] It is envisaged that the T-cell may express a chimeric transmembrane receptor as herein described. For example, the T-cell may further express an engineered T-cell receptor or a CAR. In embodiments, the T-cell may be a CD 4 T-cell that further expresses a CD8 Co-receptor, e.g. both CD8a and CD8p Co-receptor, or any engineered protein exhibiting CD8 Co-receptor functionality.

[0124] According to an embodiment, the T-cells may further express an engineered T-cell receptor. Engineered T-cells of the present disclosure can be used to treat a subject in need of treatment for a condition, for example, a cancer described herein. The T-cells may be ap T-cells or y8 T-cells that express the chimeric transmembrane receptor polypeptide as described herein, and furthermore an engineered TCR. Optionally, the T- cells may further express a CD8 Co-receptor such as a wildtype or chimeric CD8 co-receptor. T-cells described herein may be used to treat a cancer, including solid tumors and hematologic malignancies. For example, “hot” tumors or “cold” tumors may be treated by the T-cells herewith provided.

[0125] For example, the engineered T-cell receptor as herein described may specifically bind a MAGE antigen family member, such as MAGE-A1 or MAGE-A4, or wherein the engineered T-cell receptor may specifically bind an antigen selected from the group consisting of a PRAME antigen, a NY-ESO-1 antigen, a GP100 antigen, an AFP antigen, a Col6A3 antigen, an HPV-16 antigen, a WT1 antigen, an HA1 antigen, an HA2 antigen, a mutated KRAS antigen, a mutated NRAS antigen, a mutated HRAS antigen, a mutated TP53 antigen, and an EGFR antigen.

[0126] In this context, it is noted that the expression “mutated” with respect to specific tumor antigens as herein used relates to well-known mutations within the epitope region of the respective protein, polypeptide or peptide that has been correlated with expression in a human cancer.

[0127] According to an embodiment, The T-cells described herein may also be used to treat an infectious disease. The T-cells described herein may be used to treat an infectious disease; an infectious disease may be caused a virus. The T-cells described herein may be used to treat an immune disease, such as an autoimmune disease. The T- cells may be ap T-cells or y8 T-cells that express a chimeric transmembrane receptor as described herein, and optionally an engineered TCR, and optionally a CD8 Co-receptor such as a wildtype or chimeric CD8 co-receptor.

[0128] It is contemplated that the T-cell may be derived from a healthy subject, or the T-cell may be derived from a patient suffering from a disease.

[0129] In some embodiments, the T-cell may be derived from an induced pluripotent stem cell (iPSCs).

[0130] According to a fifteenth aspect, there is provided an isolated T-cell wherein the T-cell expresses a chimeric transmembrane receptor comprising a polypeptide, wherein said polypeptide comprises at least one T cell immunoreceptor with Ig and ITIM domains (TIGIT) polypeptide region having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a TIGIT wildtype polypeptide as set forth in SEQ ID No. 1 , wherein the TIGIT polypeptide region comprises a TIGIT extracellular ligand binding domain, and further wherein the TIGIT polypeptide region comprises a TIGIT transmembrane polypeptide domain or region; further wherein the polypeptide comprises at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmicpolypeptide domain, region or motif of CD2 or CD28, wherein the T-cell further expresses a recombinant T-cell receptor.

[0131] According to a sixteenth aspect, there is provided an isolated T-cell wherein the T-cell expresses a chimeric transmembrane receptor comprising a polypeptide, wherein said polypeptide comprises at least one T cell immunoreceptor with Ig and ITIM domains (TIGIT) polypeptide region comprising a TIGIT extracellular ligand binding domain, and further wherein the TIGIT polypeptide region comprises a TIGIT transmembrane polypeptide domain or region; further wherein the polypeptide comprises at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2 or CD28, wherein the T-cell further expresses a recombinant T-cell receptor.

[0132] The inventors have found that T-cells comprising a chimeric TIGIT-receptor including the costimulatory domain of CD2 or CD28, and further comprising a transmembrane polypeptide region from TIGIT, as well as a TIGIT extracellular ligand binding domain, thus being engineered for use as a switch receptor, is able to turn negative signals e.g. present in a tumor microenvironment into positive signals for T-cell activation. The inventors could show for the first time that, if expressed in T-cells which further express an engineered T-cell receptor, the chimeric transmembrane receptors comprising the at least one co-stimulatory domain of CD2 or CD28 which are linked to the TIGIT transmembrane domain and to the at least one TIGIT polypeptide region that is still capable to bind to its natural ligand, the chimeric transmembrane receptors are conveying resistance to the T-cell to the immunosuppressive tumor microenvironment. The T-cells according to the fifteenth or sixteenth aspect of the invention exhibit less TCR-T exhaustion and depletion through apoptosis, and show stimulated TCR-T proliferation and functional activity.

[0133] With respect to the TIGIT polypeptide region of the chimeric transmembrane receptor expressed by the T-cells according to the fifteenth or sixteenth aspect, each and every embodiment relating to the possible TIGIT polypeptide regions / sequences that have been described herein according to the first aspect with respect to the chimeric TIGIT- receptor including the costimulatory domain of CD2, CD40, HVEM, or CD30 is also contemplated with respect to the chimeric TIGIT-receptor including the costimulatory domain of the fifteenth or sixteenth aspect.

[0134] Furthermore, the TIGIT polypeptide region of the chimeric receptor of the T- cell according to the fifteenth or sixteenth aspect comprises a TIGIT transmembrane polypeptide domain or region. The transmembrane domain of wildtype TIGIT as referredto herein may relate to a polypeptide comprising amino acid sequence 142-162 of UniProtKB database entry No. Q495A1 ■ TIGIT_HUMAN, as set forth e.g. in SEQ ID No. 1. For example, the transmembrane domain of wildtype TIGIT as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 29. It is herewith envisaged that the transmembrane domain as included in the chimeric transmembrane receptor as herein provided may have one or more conservative amino acid substitutions relative to the transmembrane domain of the wildtype TIGIT receptor. In particular, all amino acid substitutions that maintain the functional activity of the wildtype transmembrane domain are envisaged.

[0135] The chimeric transmembrane receptor of the T-cell in accordance with the fifteenth or sixteenth aspect may further comprise at least one linker region. This may be e.g. a polypeptide linker region. Such linker(s) may be included e.g. between functional domains / regions / motifs of the chimeric transmembrane receptor. It may be a linker region naturally occurring e.g. in wildtype TIGIT receptor, or e.g. in co-stimulatory proteins, e.g. in costimulatory proteins from which the costimulatory domain of the receptor is derived. For example, polypeptide linker regions may be included between the transmembrane domain and the ligand binding domain, and / or between the transmembrane domain and the IgV domain of the chimeric TIGIT receptor, and / or between the transmembrane domain and the at least one intracellular co-stimulatory domain, and / or between individual co-stimulatory domains (in embodiments comprising more than one co-stimulatory domains).

[0136] Such linker region may comprise 1-100 amino acids, or e.g. 1-80 amino acids, or e.g. 1-50 amino acids, or e.g. 5-100 amino acids.

[0137] According to an embodiment, a linker region of the chimeric transmembrane receptor as herein provided may comprise the amino acid sequence as set forth in SEQ ID No.13 (GGGS)n or as set forth in Seq ID No. 14 (GGGGS)n, wherein n is between O and 20, or wherein n is between 0 and 10, or where n is between 0 and 5, or where n is between 3 and 5.

[0138] However, in principle, each (polypeptide) linker known in the art is herewith envisaged as being potentially included in the chimeric transmembrane switch receptor of the T-cells according to the fifteenth or sixteenth aspect.

[0139] Turning now to the co-stimulatory domain, in accordance with the present invention, the chimeric transmembrane receptor polypeptide of the T-cells according to the fifteenth or sixteenth aspect may comprise at least one costimulatory cytoplasmic polypeptide domain or cytoplasmic polypeptide motif of CD2, or CD28. The authors havefound for the first time that cytoplasmic co-stimulatory domains of CD2, or CD28, may be fused to the at least one TIGIT polypeptide region comprising a functional TIGIT receptor extracellular ligand binding domain, and further comprising a functional TIGIT transmembrane polypeptide region, in order to generate a functional chimeric TIGIT switch receptor capable of redirecting the signaling pathways triggered by TIGIT receptor engagement with at least one TIGIT ligand such that - instead of inducing inhibitory pathways in the cell - binding of the at least one TIGIT ligand promotes T-cell activation, persistence and enhanced anti-tumor responses of a T-cell expressing the chimeric transmembrane receptors as herein provided, if the T-cell at the same time comprises / expresses an engineered T-cell receptor.

[0140] According to an embodiment, the T-cells expressing the chimeric TIGIT receptor according to the fifteenth or sixteenth aspect may comprise e.g. at least one complete cytoplasmic domain of CD2 or CD28.

[0141] According to some embodiments,, the at least one cytoplasmic polypeptide domain or cytoplasmic polypeptide motif selected from the group consisting of transmembrane polypeptide region of CD2 or CD28 may have an amino acid sequence having at least 70%, or at least 71 %, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81 %, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with the respective functional polypeptide domain or a functional polypeptide motif of a wildtype human CD2 or CD28, respectively.

[0142] According to an embodiment, T-cell comprising the chimeric transmembrane receptor together with the engineered TOR according to the fifteenth or sixteenth aspect may exhibit sustained or enhanced cytotoxicity and / or cytokine secretion upon binding a TIGIT ligand.

[0143] According to an embodiment, T-cells comprising the chimeric transmembrane receptor in accordance with the fifteenth or sixteenth aspect may be capable of increasing resistance to TIGIT ligand expressing cancer cells.

[0144] It is contemplated that the chimeric TIGIT receptors of the T-cells according to the fifteenth or sixteenth aspect may comprise any functional combination of costimulatory cytoplasmic polypeptide domain(s) motif(s) and / or region(s) of the cytoplasmic polypeptide domain, region or motif selected from the group consisting of CD2 and CD 28.

[0145] According to an embodiment, the chimeric transmembrane receptor of the T- cells according to the fifteenth or sixteenth aspect may comprise at least one cytoplasmic polypeptide domain, region or motif of CD2. For example, a cytoplasmic polypeptide region of CD2 may comprise the complete cytoplasmic domain of wildtype human CD2. In other embodiments, the cytoplasmic polypeptide region of CD2 included in the chimeric transmembrane receptor may comprise at least one functional, co-stimulatory motif / domain / region of the complete wildtype human CD2 cytoplasmic domain. For example, the expression “wildtype human CD2 cytoplasmic domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 236-351 of UniProtKB database entry P06729 ■ CD2_HUMAN, as set forth e.g. in SEQ ID No. 5. It is herewith envisaged that the cytoplasmic polypeptide domain, region or motif of wildtype CD2 as included in the chimeric transmembrane receptor of the T-cell as herein provided may have one or more conservative amino acid substitutions relative to the amino acid sequence as set forth in SEQ ID No. 21. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype CD2 are envisaged.

[0146] In embodiments wherein a costimulatory region, motif or domain of CD2 is included in the chimeric transmembrane receptor, for example, the polypeptide of the chimeric transmembrane receptor may have an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID NO.: 30.

[0147] According to an embodiment, the chimeric transmembrane receptor of the T- cell according to the fifteenth or sixteenth aspect may comprise at least one cytoplasmic polypeptide domain, region or motif of CD28. For example, a cytoplasmic polypeptide region of CD28 may comprise the complete cytoplasmic domain of wildtype human CD28. In other embodiments, the cytoplasmic polypeptide region of CD28 included in the chimeric transmembrane receptor may comprise at least one functional, co-stimulatory motif / domain / region of the complete wildtype human CD28 cytoplasmic domain. For example, the expression “wildtype human CD28 cytoplasmic domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 180-220 of UniProtKB database entry No. P10747- CD28_HUMAN, as set forth e.g. in SEQ ID No. 31. It is herewith envisaged that the cytoplasmic polypeptide domain, region or motif of wildtype CD28 as included in the chimeric transmembrane receptor of the T-cell as herein provided may have one or more conservative amino acid substitutions relative to the amino acid sequence as set forth in SEQ ID No. 32. In particular, all amino acid substitutions thatmaintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype CD28 are envisaged.

[0148] In embodiments wherein a costimulatory region, motif or domain of CD28 is included in the chimeric transmembrane receptor, for example, the polypeptide of the chimeric transmembrane receptor may have an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID NO.: 33.

[0149] According to some embodiments, a chimeric transmembrane receptor as herein provided may e.g. comprise a polypeptide having an amino acid sequence with at least 85% or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% identity to the amino acids as set forth in any one of the SEQ ID No’s selected from the group consisting of SEQ ID No. 30 and 33. According to some embodiments, a chimeric transmembrane receptor as herein provided may have one or more conservative amino acid substitutions relative to an amino acid sequence as set forth in any one of the SEQ ID No’s selected from the group consisting of SEQ ID No. 30 and 33.

[0150] According to an embodiment of the fifteenth or sixteenth aspect, the cell may be an p T-cell, y8 T-cell, and / or a natural killer T-cell.

[0151] For example, the ap T-cell may be a CD4 T-cell, or the ap T-cell may be a CD8 T-cell, or the y8 T-cell may comprise e.g. a Vy1 chain or a Vy2 chain, or may be e.g. a Vy9V82+ T-cell.

[0152] It is envisaged that the T-cell may express a chimeric transmembrane receptor as herein described. For example, the T-cell may further express an engineered T-cell receptor or a CAR. In embodiments, the T-cell may be a CD 4 T-cell that further expresses a CD8 Co-receptor, e.g. both CD8a and CD8p Co-receptor, or any engineered protein exhibiting CD8 Co-receptor functionality.

[0153] According to the fifteenth or sixteenth aspect, the T-cells further express an engineered T-cell receptor. Engineered T-cells of the present disclosure can be used to treat a subject in need of treatment for a condition, for example, a cancer described herein. The T-cells may be ap T-cells or y8 T-cells that express the chimeric transmembrane receptor polypeptide as described herein, and furthermore an engineered TCR. Optionally, the T-cells may further express a CD8 Co-receptor such as a wildtype or chimeric CD8 co- receptor. T-cells described herein may be used to treat a cancer, including solid tumorsand hematologic malignancies. For example, “hot” tumors or “cold” tumors may be treated by the T-cells herewith provided.

[0154] For example, the engineered T-cell receptor as herein described may specifically bind a MAGE antigen family member, such as MAGE-A1 or MAGE-A4, or wherein the engineered T-cell receptor may specifically bind an antigen selected from the group consisting of a PRAME antigen, a NY-ESO-1 antigen, a GP100 antigen, an AFP antigen, a Col6A3 antigen, an HPV-16 antigen, a WT1 antigen, an HA1 antigen, an HA2 antigen, a mutated KRAS antigen, a mutated NRAS antigen, a mutated HRAS antigen, a mutated TP53 antigen, and an EGFR antigen.

[0155] In this context, it is noted that the expression “mutated” with respect to specific tumor antigens as herein used relates to well-known mutations within the epitope region of the respective protein, polypeptide or peptide that has been correlated with expression in a human cancer.

[0156] According to an embodiment of the fifteenth or sixteenth aspect, the T-cells described herein may also be used to treat an infectious disease. The T-cells described herein may be used to treat an infectious disease; an infectious disease may be caused a virus. The T-cells described herein may be used to treat an immune disease, such as an autoimmune disease. The T-cells may be ap T-cells or y8 T-cells that express a chimeric transmembrane receptor as described herein, and an engineered TCR, and optionally a CD8 Co-receptor such as a wildtype or chimeric CD8 co-receptor.

[0157] It is contemplated that the T-cell of the fifteenth or sixteenth aspect may be derived from a healthy subject, or the T-cell may be derived from a patient suffering from a disease.

[0158] In some embodiments of the fifteenth or sixteenth aspect, the T-cell may be derived from an induced pluripotent stem cell (iPSCs).

[0159] According to another aspect, it is herewith provided a kit comprising means to prepare the T-cells as herein provided.

[0160] According to a further aspect, this invention relates to a pharmaceutical composition comprising the T-cell provided by the present invention.

[0161] It is herewith contemplated that the pharmaceutical composition may further comprise an adjuvant, excipient, buffer, diluent, carrier, stabilizer or combination thereof.

[0162] According to a further aspect, there is provided a pharmaceutical composition comprising T-cells which express the chimeric transmembrane receptor as herein described. For example, the T-cells may further express an engineered T-cell receptor or a CAR.

[0163] According to an embodiment, the pharmaceutical composition may further comprise CD4 T-cells expressing said chimeric transmembrane receptor, expressing an engineered T-cell receptor, and further expressing a recombinant CD8 Co-receptor, such as e.g. a CD8a receptor and a CD8p receptor, or a chimeric CD8 receptor.

[0164] The pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers. Any pharmaceutically acceptable carrier can be used, as long as the carrier does not impact the viability of the T-cells to be administered is suitable for the chosen route of administration of the pharmaceutical composition. The pharmaceutical acceptable carrier may be a physiological saline solution, optionally with components such as human serum albumin that can improve the viability of the T-cells that express the chimeric transmembrane receptor. It is also possible that the chimeric transmembrane receptor expressing T-cells are stored, after their manufacture, in frozen form, for example at a temperature of between -20°C and -80 °C. In this case, the pharmaceutical composition may contain cryo-protectants that have been added to protect the cells from being damaged by the freezing process. Examples of cryoprotectants that may be used here for the freezing of the pharmaceutical composition containing transduced T-cells include glycerol, DMSO. These cryoprotectants can be used together with crystalloid solutions such as commercially available HypoThermosol® or PlasmaLyte- A solution which are both approved for infusion and are available in pharmaceutical grade. Other possible media that can be used as carrier in the pharmaceutical composition are media of the “CryoStor family”, commercially available animal protein-free defined cryopreservation media from Biolife Solutions such as CyroStor2 (CS2, an optimized freeze media pre-formulated with 2% DMSO), CyroStor5 (CS5, an optimized freeze media pre-formulated with 5% DMSO), or CyroStorlO (CS10, an optimized freeze media preformulated with 10% DMSO).

[0165] Turning to a further aspect, a method for preparing a T-cell for immunotherapy is provided, comprising isolating T-cells from a human subject, introducing the vector as herein provided, or introducing the nucleic acid as herein provided into the T-cell, and expanding the transduced T-cells.

[0166] For example, the method may comprise transforming, transfecting or transducing the isolated T-cells with the vector.

[0167] In accordance with a further aspect, there is also provided a method for treating a patient having a disease, comprising administering to the patient the pharmaceutical composition according to the present invention.

[0168] In accordance with a further aspect, there is provided a method for treating a patient having a disease, comprising introducing in vivo the vector as herein disclosed into a T-cell of the patient.

[0169] According to an embodiment, the nucleic acid may be a DNA or an mRNA.

[0170] For the in vivo introduction, the vector may be - for example - a nonreplicating viral vector.

[0171] According to an embodiment, the nucleic acid may be mRNA, and the mRNA may be in vivo introduced into the T-cell of the patient using nanoparticles, such as lipid nanoparticles.

[0172] In the methods for treating a patient as herewith provided, it is contemplated that the disease may be e.g. an autoimmune disease or a cancer.

[0173] In the methods for treating a patient as herewith provided, for example, a cancer treated by the method may be selected from the group consisting of non-small cell lung cancer, small cell lung cancer, pancreatic cancer, ovarian cancer, melanoma, breast cancer, liver cancer, kidney cancer, esophageal cancer, brain cancer, gastric cancer, Merkel cell carcinoma, leukemia, urinary bladder cancer, uterine cancer, colorectal cancer, gallbladder cancer, bile duct cancer, and prostate cancer.

[0174] For example, the cancer treated may be a solid tumor. In illustrative embodiments of the solid tumor types mentioned above, the lung cancer may be, but is not limited to, non-small cell lung cancer (NSCLC), including squamous cell carcinoma of the lung, adenocarcinoma of the lung, large cell carcinoma of the lung and other histologic types of NSCLC) or small cell lung cancer. In other illustrative examples, the breast cancer may be, but is not limited to, ductal breast cancer, ductal-invasive breast cancer, invasive breast cancer, tubular breast cancer, medullary breast cancer or combinations thereof. In yet other illustrative examples, the gastric cancer may be gastric adenocarcinoma or squamous cell cancer. Turning to sarcoma cancer, the sarcoma cancer may be, but is not limited to, chondrosarcoma cancer, osteosarcoma cancer or combinations thereof. The adenoma cancer may include, but is also not limited to, gastric adenocarcinoma, pancreatic adenocarcinoma or combinations thereof.

[0175] It is contemplated that the cancer cells may express at least one ligand of TIGIT.

[0176] According to a further aspect, as descried above, there is also provided a method for increasing cytotoxicity of a T-cell in adoptive cell therapy, comprising introducing a vector or nucleic acid as herein provided into the T-cell.

[0177] According to an embodiment, the T-cell receptor may be a recombinant T-cell receptor that specifically binds a tumor specific antigen. In some embodiments, only as example, this may be a MAGE antigen such as e.g. a MAGE-A1 antigen.

[0178] The invention will be further illustrated by the following non-limiting Experimental Examples.

[0179] Sequences as used herein are depicted in below Table 1.

[0180] Table 1. Sequences as used herein.Experimental Examples

[0181] Example 1. In-vitro T-cell killing analysis of T-cells according to the present invention transduced with chimeric TIGIT receptor polypeptides according to the present invention and an engineered T-cell receptor

[0182] In order to test the T-cells expressing chimeric TIGIT receptor constructs as described herein together with an engineered T-cell receptor for suitability in adoptive T- cell therapy (ACT), and / or for increasing cytotoxicity of the generated T-cells that express the chimeric TIGIT switch receptors and the engineered T-cell receptor, chimeric TIGIT receptor constructs have been used to transduce CD8 T-cells together with a HLA-I restricted TCR raised against MAGE-A1. Purified transduced T-cells were used in an in-vitro T-cell killing assay with CorL23-A2-NLR cells expressing TIGIT ligands CD155- and Nectin4 for evaluating cytotoxicity of the transduced T-cells.

[0183] 1.1 Materials and MethodsCloning of chimeric human TIGIT receptor constructsChimeric human TIGIT receptor constructs have been generated using standard cloning techniques. Table 2 and Table 3 as presented below summarize the principle structure of the cloned underlying plasmids for chimeric TIGIT receptor constructs created:

[0184] Table 2 and Table 3 as presented below summarize the chimeric TIGIT receptor constructs that have been generated by the inventors in a schematic representation:

[0185] Table 2

[0186] Table 3

[0187] As used in Table 2 and 3, the expression “CYP” relates to the origin of the cytoplasmic domain of the chimeric receptor encoded by the plasmid created by the Inventors. “TM” relates to the origin of the transmembrane domain of the chimeric receptor encoded by the plasmid, and “EC” relates to the origin of the extracellular domain of the chimeric receptor encoded by the plasmid created by the Inventors.

[0188] CD8 Cells Generation

[0189] PBMCs from a healthy donor buffy coat were isolated by density gradient centrifugation with Lymphoprep. Purified polyclonal CD8 T-cells were obtained by positiveselection with anti-CD8+ microbeads. CD3 T-cells were activated using TransAct in presence of IL-7 / IL-15. Two days post activation, CD8 T-cells were separately transduced with either HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) alone, or together with different versions of the TIGIT Switch receptors. The HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) as used herein has been described e.g. in WO 2014 / 118236, which is herewith incorporated by reference in its entirety. In particular, the HLA-I restricted TCR raised against MAGE-A1 as used herein relates to “TCR1367” as described in WO 2014 / 118236. The CDR sequences of the respective a and p chain of “TCR1367” as used herein are further described -for example - in WO 2023 / 083864, which is herewith incorporated by reference in its entirety.

[0190] Transduced CD8 T-cells were further expanded, and at Day 9 the transduced fraction was positively selected using CD34 microbeads. Purified transduced T-cells were cultured for further expansion and were harvested and cryopreserved at Day 10. T-cell characterization was based on transgene expression levels using FACS and killing assay.

[0191] Cell killing assay in CD155- and Nectin4-overexpressing CorL23-A2- NLR cells:The in-vitro T-cell killing assay was performed according to the method described e.g. by Kalbasi, A., Siurala, M., Su, L.L. et al. “Potentiating adoptive cell therapy using synthetic IL-9 receptors”. Nature 607, 360-365 (2022). In particular, the human TCR T-cell repetitive killing assay was conducted using IncuCyte Live Cell Analysis. CD155- and Nectin4- overexpressing CorL23-A2-NLR cells 1x104tumor cells were plated per well in 96-well plates. Transduced human T-cells (transduced with either MAGE_TCR alone, or transduced with MAGE_TCR together with either dominant negative TIGIT receptor or a chimeric TIGIT receptor) were added in triplicates at 1 to 6 E:T ratio.

[0192] Flow Cytometry:

[0193] Extracellular surface staining was performed for 30 minutes at 4°C in flow cytometry FACS buffer (BD Bioscience). The following antibodies were used: from BioLegend: CD8a (clone HIT8a), anti-human TIGIT (clone A15153G); from Invitrogen: CD34 (clone QBEND10), CD34 (clone 4H11); from Miltenyi Biotec: CD8a (clone REA734), from Beckman Coulter: TCRBV3S1 Vp3. PE-conjugated HLA-A*02:01 specific MAGE-A1 MHC tetramer (KVLEYVIKV) (SEQ ID No. 47) (TB-M070-1) was added together with cellsurface staining antibodies. Zombie Yellow™ Fixable Viability Kit was used to discriminate between live and dead cells. The expression of the chimeric TIGIT receptor has been determined for CD8 cells transduced with different chimeric TIGIT receptors as herein provided.

[0194] 1.2. T-cell killing assay analysis

[0195] The Relative cell growth has been observed over time for each transduced T- cell fraction. The results are shown in Fig. 2. In particular, Fig. 2 shows the results of the killing assay with the chimeric TIGIT receptors in CD155- and Nectin4-overexpressing CorL23-A2-NLR cells. “Cancer Cells” relates to a control with no addition of T cells, “TCR only” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR); “TCR+TIGIT DN” relates to a CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a dominant negative version of a TIGIT receptor (a truncated version of the TIGIT receptor which consists of the TIGIT extracellular and transmembrane domains, but which lacks the cytoplasmic domain), “TCR+SwR_TIGIT(EC+TM)-CD2(CYP)” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a chimeric TIGIT receptor comprising a TIGIT extracellular and transmembrane polypeptide region and a CD2 cytoplasmic polypeptide region (pl_977); “TCR-SwR_TIGIT(EC)-CD27(TM+CYP)” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a chimeric TIGIT receptor comprising a TIGIT extracellular and a CD27 transmembrane and cytoplasmic polypeptide region (pl_996); “TCR-SwR_TIGIT(EC+TM)-HVEM(CYP) relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a chimeric TIGIT receptor comprising a TIGIT extracellular and transmembrane polypeptide region and a HVEM cytoplasmic polypeptide region (pl_980); “TCR-SwR_ TIGIT(EC+TM)-CD27(CYP)” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a chimeric TIGIT receptor comprising a TIGIT extracellular and transmembrane polypeptide region and a CD27 cytoplasmic polypeptide region (pl_979); “TCR- SwR_TIGIT(EC+TM)-OX40(CYP)” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a chimeric TIGIT receptor comprising a TIGIT extracellular and transmembrane polypeptide region and a 0X40 cytoplasmic polypeptide region (pl_974); “TCR-SwR_TIGIT(EC+TM)-CD30(CYP)”relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE- A1 (MAGE-A1_TCR) together with a chimeric TIGIT receptor comprising a TIGIT extracellular and transmembrane polypeptide region and a CD30 cytoplasmic polypeptide region (pl_986); “TCR-SwR_TIGIT(EC+TM)-CD40(CYP)” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a chimeric TIGIT receptor comprising a TIGIT extracellular and transmembrane polypeptide region and a CD40 cytoplasmic polypeptide region (pl_978); “TCR- SwR_TIGIT(EC+TM)-ICOS(CYP)” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a chimeric TIGIT receptor comprising a TIGIT extracellular and transmembrane polypeptide region and an ICOS cytoplasmic polypeptide region (pl_971); “TCR-SwR_TIGIT(EC+TM)-41 BB(CYP)” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE- A1 (MAGE-A1_TCR) together with a chimeric TIGIT receptor comprising a TIGIT extracellular and transmembrane polypeptide region and a 41 BB cytoplasmic polypeptide region (pl_973), “TCR-SwR_TIGIT(EC+TM)-CD28(CYP)” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a chimeric TIGIT receptor comprising a TIGIT extracellular and transmembrane polypeptide region and a CD28 cytoplasmic polypeptide region (pl_972), “TCR- SwR_TIGIT(EC)-OX40(TM+CYP)” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a chimeric TIGIT receptor comprising a TIGIT extracellular and a 0X40 transmembrane and cytoplasmic polypeptide region (pl_992); “TCR-SwR_TIGIT(EC)-ICOS(TM+CYP)” relates to CD8 T- cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE- A1_TCR) together with a chimeric TIGIT receptor comprising a TIGIT extracellular and a ICOS transmembrane and cytoplasmic polypeptide region (pl_989); “TCR- SwR_TIGIT(EC)-CD40(TM+CYP)” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a chimeric TIGIT receptor comprising a TIGIT extracellular and a CD40 transmembrane and cytoplasmic polypeptide region (pl_995); “TCR-SwR_TIGIT(EC)-HVEM(TM+CYP)” relates to CD8 T- cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE- A1_TCR) together with a chimeric TIGIT receptor comprising a TIGIT extracellular and a HVEM transmembrane and cytoplasmic polypeptide region (pl_997); “TCR- SwR_TIGIT(EC)-CD2(TM+CYP)” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a chimeric TIGIT receptor comprising a TIGIT extracellular and a CD2 transmembrane and cytoplasmicpolypeptide region (pl_994); “TCR-SwR_TIGIT(EC)-CD30(TM+CYP)” relates to CD8 T- cell fraction transduced with HLA4 restricted TCR raised against MAGE-A1 (MAGE- A1_TCR) together with a chimeric TIGIT receptor comprising a TIGIT extracellular and a CD30 transmembrane and cytoplasmic polypeptide region (pl_1000).

[0196] 1.3 Flow Cytometry Analysis

[0197] CD8 T cells transduced with vectors comprising nucleic acids encoding for different chimeric TIGIT receptors as herein provided have been checked for expression of the chimeric TIGIT receptor. In particular, the respective chimeric TIGIT receptors used in Flow Cytometry Analysis correspond to the respective constructs according to Table 2 and Table 3 as shown above. Furthermore, TIGIT DN and Mock transduces cells have been used as controls. As visible from Fig. 1, the chimeric TIGIT receptors comprising cytoplasmic domains of TLR2 and TLR4, respectively, were not expressed. Furthermore, Fig. 1 shows that the chimeric TIGIT receptor with both transmembrane domain and cytoplasmic domain of CD 28, and the chimeric TIGIT receptor with both transmembrane domain and cytoplasmic domain from 4-1 BB were not expressed. All other chimeric TIGIT receptors as listed in Table 2 and Table 3 show high expression in the transduced CD8 T- cells.

[0198] 1.4 Results

[0199] As visible from Fig. 2, Co-transduction of CD8 T cells with an engineered HLA-I restricted TCR raised against MAGE-A1 together with a chimeric TIGIT receptor comprising a non - TIGIT co-stimulatory cytoplasmic polypeptide domain, motif or region of CD2, CD40, HVEM or CD40, as well as transmembrane polypeptide region of CD2, CD40, HVEM, or CD30, respectively, results in an increased killing activity of the engineered T-cells compared with mock transduced T-cells and / or T-cells transduced with the HLA-I restricted TCR raised against MAGE-A1 only or in combination with double negative TIGIT receptor (TIGIT DN), as visible in CD155- and Nectin4-overexpressing CorL23-A2-NLR cells.

[0200] Furthermore, Co-transduction of CD8 cells with an engineered HLA-I restricted TCR raised against MAGE-A1 together with a chimeric TIGIT receptor comprising a non - TIGIT co-stimulatory cytoplasmic polypeptide domain, motif or region of CD2 or CD28, and comprising a TIGIT transmembrane region, also results in an increased killing activity of the engineered T-cells compared with mock transduced T-cellsand / or T-cells only transduced with the HLA-I restricted TCR raised against MAGE-A1 , as visible in CD155- and Nectin4-overexpressing CorL23-A2-NLR cells.

[0201] Summary and conclusions

[0202] The results described above demonstrate - in principle - suitability of the chimeric TIGIT switch receptor polypeptides as herein provided for improving adoptive cell therapy (ACT). Specifically, it is contemplated that the chimeric TIGIT receptor polypeptides of the present invention, e.g. in combination with an engineered T-cell receptor, may be functional in providing improved resistance to the T-cell in immunosuppressive tumor microenvironment, in preventing T-cell exhaustion and / or depletion through apoptosis; and in stimulating T-cell proliferation and functional activity, such as increased cytotoxicity.

[0203] Thus, it is contemplated that fusion of at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises a transmembrane polypeptide region of CD2, CD40, HVEM, or CD30, and wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2, CD40, HVEM, or CD30 to a TIGIT polypeptide region that comprises the TIGIT ligand binding domain, is able to act like a “switch” receptor, by turning negative signals into positive signals, thereby enhancing cytotoxicity of a T-cell in presence of tumor cells that express at least one TIGIT ligand. Engineered T-cells expressing the chimeric TIGIT receptor polypeptides together with an engineered T-cell receptor as provided herein exhibit an improved killing activity compared to control samples in presence of a TIGIT ligand.

[0204] It is further contemplated that fusion of at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2, or CD28 to a TIGIT polypeptide region that comprises the TIGIT ligand binding domain, and that also comprises a TIGIT transmembrane domain, if co-expressed in a T-cell together with an engineered / recombinant) TCR, is able to act like a “switch” receptor, by turning negative signals into positive signals, thereby enhancing cytotoxicity of a T-cell in presence of tumor cells that express at least one TIGIT ligand. Engineered T-cells expressing the chimeric TIGIT receptor polypeptides together with an engineered T-cell receptor as providedherein exhibit an improved killing activity compared to control samples in presence of a TIGIT ligand.

[0205] It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0206] All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0207] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. Further embodiments of the invention will become apparent from the following claims.

Claims

Claims:What is claimed is:

1. A chimeric transmembrane receptor comprising a polypeptide, wherein the polypeptide comprises at least one T cell immunoreceptor with Ig and ITIM domains (TIGIT) polypeptide region having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a TIGIT wildtype polypeptide as set forth in SEQ ID No. 1 , wherein the TIGIT polypeptide region comprises a TIGIT extracellular ligand binding domain; further wherein the polypeptide comprises at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises a transmembrane polypeptide region of CD2, CD40, HVEM, or CD30, and wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2, CD40, HVEM, or CD30.

2. A chimeric transmembrane receptor comprising a polypeptide, wherein the polypeptide comprises at least one TIGIT polypeptide region comprising a TIGIT extracellular ligand binding domain; further wherein the polypeptide comprises at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises a transmembrane polypeptide region of CD2, CD40, HVEM, or CD30, and wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2, CD40, HVEM, or CD30.

3. The chimeric transmembrane receptor according to claim 1 or 2, wherein the extracellular ligand binding domain is functional in binding at least one TIGIT ligand.

4. The chimeric transmembrane receptor according to any one of the preceding claims, wherein the extracellular ligand binding domain comprises a TIGIT immunoglobulin variable (IgV) domain, wherein the TIGIT IgV domain is having at least 85% sequence identity to the amino acid sequence of SEQ ID No. 2.

5. The chimeric transmembrane receptor according to any one of claims 1 to 4, wherein the polypeptide further comprises an extracellular homodimerization motif, such as a TIGIT homodimerization motif.

6. The chimeric transmembrane receptor according to any one of the preceding claims, wherein said polypeptide is a single-chain polypeptide.

7. The chimeric transmembrane receptor according to any one of claims 1 to 6, wherein the polypeptide comprises a transmembrane region from CD2 and a costimulatory cytoplasmic polypeptide domain, region or motif from CD2.

8. The chimeric transmembrane receptor according to any one of claims 1 to 6, wherein the polypeptide comprises a transmembrane region from CD40 and a costimulatory cytoplasmic polypeptide domain, region or motif from CD40.

9. The chimeric transmembrane receptor according to any one of claims 1 to 6, wherein the polypeptide comprises a transmembrane region from HVEM and a costimulatory cytoplasmic polypeptide domain, region or motif from HVEM.

10. The chimeric transmembrane receptor according to any one of claims 1 to 6, wherein the polypeptide comprises a transmembrane region from CD30 and a costimulatory cytoplasmic polypeptide domain, region or motif from CD30.11 . The chimeric transmembrane receptor according to any one of the preceding claims, wherein the at least one TIGIT polypeptide region comprises a complete TIGIT extracellular domain.

12. The chimeric transmembrane receptor according to any one of claims 1 to 11 , wherein the polypeptide comprises a complete cytoplasmic domain of CD2, CD40, HVEM, or CD30.

13. The chimeric transmembrane receptor to any one of claims 1 to 11 , wherein the polypeptide comprises a truncated cytoplasmic domain of CD30, optionally wherein the truncated cytoplasmic domain of CD30 comprises or consists of at least one CD30 TRAF binding motif.

14. The chimeric transmembrane receptor according to any one of claims 1 to 13, wherein the polypeptide is having an amino acid sequence with at least 85% identity to the amino acid sequence as set forth in any one of the sequences selected from a group consisting of SEQ ID No.: 16, 18, 20 and 22.

15. The chimeric transmembrane receptor according to any one of the preceding claims, wherein the at least one TIGIT polypeptide region has at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity with the functional polypeptide domain, region or motif of a human TIGIT receptor (Seq ID No. 1), and / or wherein the at least one TIGIT polypeptide region is having one ore more conservative amino acid substitutions relative to the functional polypeptide domain, region or motif of the human TIGIT receptor (Seq ID No. 1).

16. The chimeric transmembrane receptor according to anyone of the preceding claims, wherein the chimeric transmembrane receptor is able to sustain or enhancecytotoxicity and / or cytokine secretion of a T-cell upon binding a TIGIT ligand.

17. The chimeric transmembrane receptor according to anyone of the preceding claims, wherein the chimeric transmembrane receptor is capable of increasing resistance of T-cells to TIGIT-ligand expressing cancer cells.

18. An isolated nucleic acid comprising a nuclear acid sequence encoding for the chimeric transmembrane receptor according to any one of the preceding claims.

19. A vector comprising the nucleic acid of claim 18.

20. The vector according to claim 19, wherein the vector is a viral vector or a non-viral vector.

21. The vector according to claim 20, wherein the viral vector is selected from adenoviruses, poxviruses, alphaviruses, arenaviruses, flaviviruses, rhabdoviruses, retroviruses, lentiviruses, herpesviruses, paramyxoviruses, picornaviruses, and combinations thereof.

22. The vector according to claim 20, wherein the non-viral vector comprises a naked nucleic acid, cationic lipids, synthetic polycationic polymers, dendrimers, synthetic peptides such as cell-penetrating peptides (CPP’s), p-1 ,3- glucans, or combinations thereof; optionally wherein the non-viral vector comprises naked plasmid or linear double stranded (ds) DNA.

23. The vector according to any one of claims 19 to 22, wherein the vector further comprises a nucleic acid encoding a chimeric antigen receptor (CAR).

24. The vector according to any one of claims 19 to 22, wherein the vector further comprises a nucleic acid encoding a T-cell receptor comprising a TCR a chain and a TCR p chain.

25. The vector according to claim 24, wherein the T-cell receptor is a recombinant T cell receptor.

26. The vector according to any one of claims 19 to 25, wherein the vector further comprises a nucleic acid encoding for a CD8 Co-receptor.

27. The vector according to claim 26, wherein the nucleic acid encodes a wildtype CD8a and a wildtype CD8p Co-receptor, or wherein the nucleic acid encodes a chimeric receptor with CD8 Co-receptor functionality.

28. An isolated T-cell, wherein the T-cell expresses a chimeric transmembrane receptor according to any one of claims 1 to 17.

29. An isolated T-cell, wherein the T-cell comprises a nucleic acid according to claim 18.

30. The isolated T-cell according to claim 29, wherein the T-cell further comprises a nucleic acid encoding for a recombinant T cell receptor comprising a TCR a chainand a TCR p chain.

31. The isolated T-cell according to claim 30, wherein the recombinant T-cell receptor specifically binds a MAGE antigen family member, such as MAGE-A1 or Mage-A4, or wherein the engineered T-cell receptor specifically binds an antigen selected from the group consisting of a PRAME antigen, a NY-ESO-1 antigen, a GP100 antigen, an AFP antigen, a Col6A3 antigen, an HPV-16 antigen, a WT1 antigen, an HA1 antigen, an HA2 antigen, a mutated KRAS antigen, a mutated NRAS antigen, a mutated HRAS antigen, a mutated TP53 antigen, and an EGFR antigen.

32. The isolated T-cell according to any one of claims 29 to 31 , wherein the T-cell further comprises a nucleic acid encoding for a recombinant CD8 Co-receptor.

33. The T-cell according to any one of claims 29 to 32, wherein the one or more nucleic acid has been stably integrated into the genome of the T-cell by targeted knock-in; optionally wherein CRISPR / Cas9 is used.

34. An isolated T-cell wherein the vector of any one of claims 19 to 27 has been introduced in said T-cell.

35. The T-cell according to any one of claims 28 to 34, wherein the cell is an ap T-cell, a y8 T-cell, and / or a natural killer T-cell.

36. The T-cell of claim 35 wherein the T-cell is a CD4 T-cell, or wherein the ap T- cell is a CD8 T-cell, or wherein the y8 T-cell is a Vc9Vd2+ T-cell, or wherein the y8 T-cell comprises a V81 T-cell.

37. The T-cell of any one of claims 28 to 36, wherein the T-cell is derived from a stem cell, such as from an induced pluripotent stem cell (iPSCs).

38. The T-cell of any one of claims 28 to 36, wherein the T-cell is derived from a healthy subject, or wherein the T-cell is derived from a patient suffering from a disease.

39. An isolated T-cell wherein the T-cell expresses a chimeric transmembrane receptor comprising a polypeptide, wherein said polypeptide comprises at least one T cell immunoreceptor with Ig and ITIM domains (T IGIT) polypeptide region having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a TIGIT wildtype polypeptide as set forth in SEQ ID No. 1 , wherein the TIGIT polypeptide region comprises a TIGIT extracellular ligand binding domain, and further wherein the TIGIT polypeptide region comprises a TIGIT transmembrane polypeptide domain or region; further wherein the polypeptide comprises at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2, or CD28; wherein the T- cell further expresses a recombinant T-cell receptor.

40. An isolated T-cell wherein the T-cell expresses a chimeric transmembrane receptor comprising a polypeptide, wherein said polypeptide comprises at least one T cell immunoreceptor with Ig and ITIM domains (T IGIT) polypeptide region comprising a TIGIT extracellular ligand binding domain, and further wherein the TIGIT polypeptide region comprises a TIGIT transmembrane polypeptide domain or region; further wherein the polypeptide comprises at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2, or CD28; wherein the T-cell further expresses a recombinant T-cell receptor.

41. The isolated T-cell according to claim any one of claims 39 or 40, wherein the extracellular ligand binding domain is functional in binding at least one TIGIT ligand.

42. The isolated T-cell according to any one of claims 39 to 41 , wherein the TIGIT extracellular ligand binding domain comprises a TIGIT immunoglobulin variable (IgV) domain, wherein the TIGIT IgV domain is having at least 85% sequence identity to the amino acid sequence of SEQ ID No. 2.

43. The isolated T-cell according to any one of claims 39 to 42, wherein said polypeptide is a single-chain polypeptide.

44. The isolated T-cell according to any one of claims 39 to 43, wherein the at least one TIGIT polypeptide region comprises a complete TIGIT extracellular domain.

45. The isolated T-cell according to any one of claims 39 to 44, wherein the at least one TIGIT polypeptide region has at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity with the functional polypeptide domain, region or motif of a human TIGIT receptor (Seq ID No. 1), and / or wherein the at least one TIGIT polypeptide region is having one ore more conservative amino acid substitutions relative to the functional polypeptide domain, region or motif of the human TIGIT receptor (Seq ID No. 1).

46. The isolated T-cell according to any one of claims 39 to 45, wherein the least one non-TIGIT co-stimulatory cytoplasmic polypeptide domain, region or motif comprises a complete cytoplasmic domain of CD2.

47. The isolated T-cell according to any one of claims 39 to 45, wherein the least one non-TIGIT co-stimulatory cytoplasmic polypeptide domain, region or motif comprises a complete cytoplasmic domain of CD28.

48. The isolated T-cell according to claim 46, wherein the polypeptide is having anamino acid sequence with at least 85% identity to the amino acid sequence as set forth in SEQ ID No. 30.

49. The isolated T-cell according to claim 47, wherein the polypeptide is having an amino acid sequence with at least 85% identity to the amino acid sequence as set forth in SEQ ID No. 33.

50. The isolated T-cell according to anyone of claims 39 to 49, wherein the chimeric transmembrane is able to sustain or enhance cytotoxicity and / or cytokine secretion of a T-cell upon binding a TIGIT ligand.

51. The isolated T-cell according to any one of claims 39 to 50, wherein the chimeric transmembrane receptor is capable of increasing resistance of T-cells to TIGIT- ligand expressing cancer cells.

52. The isolated T-cell according to any one of claims 39 to 51 , wherein the recombinant T cell receptor comprises a TOR a chain and a TCR p chain.

53. The isolated T-cell according to claim 52, wherein the recombinant T-cell receptor specifically binds a MAGE antigen family member, such as MAGE-A1 or Mage-A4, or wherein the engineered T-cell receptor specifically binds an antigen selected from the group consisting of a PRAME antigen, a NY-ESO-1 antigen, a GP100 antigen, an AFP antigen, a Col6A3 antigen, an HPV-16 antigen, a WT1 antigen, an HA1 antigen, an HA2 antigen, a mutated KRAS antigen, a mutated NRAS antigen, a mutated HRAS antigen, a mutated TP53 antigen, and an EGFR antigen.

54. The isolated T-cell according to any one of claims 39 to 53, wherein the T-cell further comprises a nucleic acid encoding for a recombinant CD8 Co-receptor.

55. The T-cell according to any one of claims 39 to 54, wherein the cell is an ap T-cell, a y8 T-cell, and / or a natural killer T-cell.

56. The T-cell of claim 57, wherein the p T-cell is a CD4 T-cell, or wherein the ap T- cell is a CD8 T-cell, or wherein the y8 T-cell is a Vc9Vd2+ T-cell, or wherein the y8 T-cell comprises a V81 T-cell.

57. The T-cell of any one of claims 39 to 56, wherein the T-cell is derived from a stem cell, such as from an induced pluripotent stem cell (iPSCs), or wherein the T-cell is derived from a healthy subject, or wherein the T-cell is derived from a patient suffering from a disease.

58. A kit comprising means to prepare the T-cell according to any one of claims 28 to 57.

59. A pharmaceutical composition comprising the T-cell of any one of claims 28 to 57.

60. The pharmaceutical composition of claim 59, wherein the composition further comprises an adjuvant, excipient, buffer, diluent, carrier, stabilizer or combinationthereof.

61. The pharmaceutical composition of any one of claims 59 to 60, comprising T-cells expressing the chimeric transmembrane receptor and the recombinant T-cell receptor.

62. The pharmaceutical composition according to claim 61 , further comprising CD4 cells expressing the chimeric transmembrane receptor, the recombinant T-cell receptor and further expressing a recombinant CD8 Co-receptor.

63. A method for preparing a T-cell for immunotherapy, comprising isolating T-cells from a human subject, introducing the vector according to any one of claims 19 to 27, or introducing the nucleic acid according to claim 18 into the T-cell, and expanding the cells.

64. The method according to claim 63, comprising transforming, transfecting or transducing the isolated T-cells with the vector or the nucleic acid.

65. A method for treating a patient having a disease, comprising administering to the patient the composition according to any one of claims 59 to 62.

66. A method for treating a patient having a disease, comprising introducing in vivo the nucleic acid according to claim 18 or the vector according to any one of claims 19 to 27 into a T-cell of the patient.

67. The method according to claim 66, wherein the nucleic acid is a DNA or a mRNA.

68. The method according to claim 66, wherein the vector is a non-replicating viral vector.

69. The method according to claim 66 or 67, wherein the nucleic acid is mRNA, and wherein the mRNA is introduced into the T-cell of the patient using nanoparticles.

70. The method according to any one of claims 65 to 69, wherein the disease is a cancer.

71. The method according to claim 70, wherein the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, pancreatic cancer, ovarian cancer, melanoma, breast cancer, liver cancer, kidney cancer, esophageal cancer, brain cancer, gastric cancer, Merkel cell carcinoma, leukemia, urinary bladder cancer, uterine cancer, colorectal cancer, gallbladder cancer, bile duct cancer, and prostate cancer.

72. The method according to claim 70 or claim 71 , wherein cancer cells express a ligand of TIGIT.

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