Chimeric human CD 95 switch receptor, t-cell expressing said receptor together with an engineered t-cell receptor, respective vectors, kits, pharmaceutical compositions and methods for treating a patient having a disease

Abstract

The present invention inter alia relates to a T-cell expressing a chimeric CD 95 receptor comprising a polypeptide, wherein said polypeptide comprises at least one CD 95-derived polypeptide region having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a human CD 95 wildtype receptor, wherein said human CD 95 polypeptide region comprises a CD 95 extracellular ligand binding domain; further wherein said polypeptide comprises at least one non-CD 95-derived co- stimulatory cytoplasmic polypeptide domain, region or motif of a tumor necrosis factor receptor superfamily protein comprising CD40, CD40L, CD27, HVEM, GITR, CD30, OX40, and / or LTBR, an immunoglobulin superfamily (IgSF) protein, a Toll-like-receptor, and / or an IL6-receptor family protein; wherein the T-cell further expresses an engineered T-cell receptor. The present invention also relates to a vector comprising nucleic acids encoding for the CD 95 receptor and the engineered T-cell receptor, as well as to a kit for preparing the T-cells of the present invention, and a pharmaceutical composition comprising the T-cells. The invention also relates to a method for preparing a T-cell for immunotherapy, and to a method for treating a patient having a disease comprising administering the pharmaceutical composition.

Description

CHIMERIC HUMAN CD 95 SWITCH RECEPTOR, T-CELL EXPRESSING SAID RECEPTOR TOGETHER WITH AN ENGINEERED T-CELL RECEPTOR, RESPECTIVE VECTORS, KITS, PHARMACEUTICAL COMPOSITIONS AND METHODS FOR TREATING A PATIENT HAVING A DISEASECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority of European PatentApplication No. 23193416.7, filed August 25, 2023, the content of which is hereby incorporated by reference it its entirety for all purposes.FIELD OF THE INVENTION

[0002] The present invention relates to an isolated T-cell, wherein the T-cell expresses a chimeric CD 95 receptor comprising a polypeptide, wherein said polypeptide comprises at least one CD 95 polypeptide region having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a wildtype human CD 95 receptor, wherein said human CD 95 polypeptide region comprises a CD 95 extracellular ligand binding domain. The present invention also relates to a vector comprising a nucleic acid encoding the chimeric CD 95 receptor, as well as to isolated T- cells in which the vector has been introduced. 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. The present invention further relates to a chimeric CD 95 receptor comprising a polypeptide, wherein said polypeptide comprises at least one CD 95 polypeptide region having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a wildtype human CD 95 receptor, wherein said human CD 95 polypeptide region comprises a CD 95 extracellular ligand binding domain, further wherein said polypeptide comprises at least one non-CD 95- co-stimulatory cytoplasmic polypeptide domain, region or motif.1BACKGROUND 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 αβ-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-β 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 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. Within the TNFRSF, for example, CD 95 (Fas - receptor) is known to be able to transmit inhibitorysignals when bound by its ligand CD95L (also known as e.g. FASL) that inhibit activation, promote exhaustion and / or induce apoptosis of T-cells.

[0009] Recently, it has been described that fusion of a specific co-stimulatory domain, in particular the co-stimulatory domain of 4-1 BB to the CD 95 receptor could increase pro-survival signaling, proliferation, antitumor function, and altered metabolism in vitro [Oda S.K. et al. , 2020], However, in view of the diversity of immunosuppressive tumor microenvironments, and in light of the huge amount 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, in particular in the immunosuppressive tumor microenvironment.

[0010] Accordingly, it is an object of the invention to provide for an improvement with respect to the above inconveniences.SUMMARY OF THE INVENTION

[0011] This object is inter alia accomplished by the (isolated) T-cells, the vectors, the pharmaceutical compositions, the kits, the methods, and the chimeric CD 95 receptors having the features of the respective independent claims.

[0012] In a first aspect, the invention provides an isolated T-cell, wherein the T-cell expresses a chimeric CD 95 receptor comprising a polypeptide, wherein said polypeptide comprises at least one CD 95 (-derived) polypeptide region having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a human CD 95 receptor as set forth in SEQ ID No. 1 , wherein said CD 95 (-derived) polypeptide region comprises a CD 95 extracellular ligand binding domain; further wherein said polypeptide comprises at least one non-CD 95 (-derived) co-stimulatory cytoplasmic polypeptide domain, region or motif of a tumor necrosis factor receptor superfamily protein comprising CD40, CD40L, CD27, HVEM, GITR, CD30, 0X40, and / or LTBR, an immunoglobulin superfamily (IgSF) protein, a Toll-like-receptor, and / or an IL6-receptor family protein; wherein the T-cell further expresses an engineered T-cell receptor.

[0013] In a second aspect, the invention provides an isolated T-cell, wherein the T- cell expresses a chimeric CD 95 receptor comprising a polypeptide, wherein said polypeptide comprises at least one CD 95 (-derived) polypeptide region comprising a CD 95 extracellular ligand binding domain; further wherein said polypeptide comprises at least one non-CD 95 (-derived) co-stimulatory cytoplasmic polypeptide domain, region or motif of a tumor necrosis factor receptor superfamily protein comprising CD40, CD40L, CD27, HVEM, GITR, CD30, 0X40, and / or LTBR, an immunoglobulin superfamily (IgSF) protein,a Toll-like-receptor, and / or an IL6-receptor family protein; wherein the T-cell further expresses an engineered T-cell receptor.

[0014] The inventors have found that the T-cells comprising both an engineered T- cell receptor and a chimeric CD 95 receptor including the costimulatory domain of CD40, CD40L, CD27, HVEM, GITR, CD30, 0X40, and / or LTBR, an immunoglobulin superfamily (IgSF) protein, a Toll-like-receptor, and / or an IL6-receptor family protein, 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, the T- cells as herein provided, by expressing both an engineered T-cell receptor and specifically engineered recombinant chimeric CD 95 receptors, linking the co-stimulatory domains as herein described to at least one CD 95-derived polypeptide region that is still capable to bind to its natural ligand, 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. Thus, the expression ..region of a tumor necrosis factor receptor superfamily protein” as referred to herein may relate to a protein portion that preserves the ability to propagate co-stimulatory signal transduction of a functional costimulatory tumor necrosis factor receptor superfamily protein analogous to a wildtype tumor necrosis factor receptor superfamily protein. The expression „motif of a tumor necrosis factor receptor superfamily protein” as referred to herein may relate to a protein portion being a recognizable region of protein structure that may be defined by a unique chemical or biological function, and which also preserves the ability to propagate co-stimulatory signal transduction of a functional costimulatory tumor necrosis factor receptor superfamily protein analogous to a wildtype tumor necrosis factor receptor superfamily protein.

[0015] Since the T-cells as herein provided comprise chimeric CD 95 switch receptors which lack the cytoplasmic inhibitory motif / domain / region of the wildtype CD 95 receptor, the binding of CD95L to the CD 95 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 CD 95 switch receptor, but - instead - even costimulates the T-cell, thereby enhancing its cytotoxic effect.

[0016] In a third aspect, the invention provides a vector comprising a nucleic acid comprising a nuclear acid sequence encoding for a chimeric CD 95 receptor comprising a polypeptide, wherein said polypeptide comprises at least one CD 95 (-derived) polypeptide region having at least 60% sequence identity with a polypeptide domain, a polypeptideregion or a polypeptide motif of a human CD 95 receptor as set forth in SEQ ID No. 1 , wherein said human CD 95 polypeptide region comprises a CD 95 extracellular ligand binding domain; further wherein said polypeptide comprises at least one non-CD 95 (- derived) co-stimulatory cytoplasmic polypeptide domain, region or motif of a tumor necrosis factor receptor superfamily protein comprising CD40, CD40L, CD27, HVEM, GITR, CD30, 0X40, and / or LTBR, an immunoglobulin superfamily (IgSF) protein, a Toll- like-receptor, and / or an IL6-receptor family protein; and further comprising a nucleic acid comprising a nuclear acid sequence encoding for an engineered T-cell receptor.

[0017] In a fourth aspect, the invention provides a vector comprising a nucleic acid comprising a nuclear acid sequence encoding for a chimeric CD 95 receptor comprising a polypeptide, wherein said polypeptide comprises at least one CD 95 (-derived) polypeptide region comprising a CD 95 extracellular ligand binding domain; further wherein said polypeptide comprises at least one non-CD 95 (-derived) co-stimulatory cytoplasmic polypeptide domain, region or motif of a tumor necrosis factor receptor superfamily protein comprising CD40, CD40L, CD27, HVEM, GITR, CD30, 0X40, and / or LTBR, an immunoglobulin superfamily (IgSF) protein, a Toll-like-receptor, and / or an IL6-receptor family protein; and further comprising a nucleic acid comprising a nuclear acid sequence encoding for an engineered T-cell receptor.

[0018] In some embodiment, in addition to comprising the nucleic acid sequence encoding for the chimeric CD 95 receptor and the nucleic acid sequence encoding for the engineered T-cell receptor, the vector may further comprise a nucleic acid encoding for a CD8 Co-receptor, such as a wildtype CD8 Co-receptor or a chimeric CD8 Co-receptor.

[0019] For example, overexpression of a chimeric human CD 95 receptor as herein described in the T-cells as herein provided alongside an engineered transgenic αβ-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.

[0020] The optional, additional provision of the CD8 Co-receptor together with the chimeric CD 95 switch receptor and the engineered T-cell receptor in the T-cell of the present invention may e.g. allow for the efficient incorporation of CD4 cells into TCR-T-cell therapy, such that it becomes possible to harness their unique properties to augment the antitumor immune response.

[0021] For example, CD4 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.

[0022] In a fifth aspect, the invention provides an isolated T-cell, the T-cell comprising the vector according to the present invention.

[0023] In a sixth aspect, the invention provides an isolated T-cell, the T-cell being introduced with, such as transfected, transduced or transformed with the vector according to the present invention.

[0024] In a seventh aspect, the invention provides an isolated T-cell, the T-cell being treated, such as transfected, transduced or transformed to express the chimeric CD 95 receptor as herein described together with the engineered T-cell receptor..

[0025] In a eighth aspect, the invention provides a kit comprising means to prepare the isolated and / or engineered T-cells according to the present invention.

[0026] In a ninth aspect, the invention provides a pharmaceutical composition comprising the isolated T-cell according to the present invention.

[0027] In an tenth aspect, the invention provides a method for preparing a T-cell for immunotherapy, comprising isolating T-cells from a human subject, introducing the vector, as herein provided into the T-cell, and expanding the T-cells in which the vector has been introduced.

[0028] In a eleventh aspect, the invention provides a pharmaceutical composition comprising T-cells expressing the chimeric CD 95 receptor as herein described and expressing an engineered T-cell receptor.

[0029] 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.

[0030] In an thirteenth aspect, the invention provides a method for treating a patient having a disease, comprising introducing in vivo the vector as herein provided into a T-cell of the patient.

[0031] 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 CD 95-receptor as herein described, and further encoding an engineered T-cell receptor.

[0032] In a fifteenth aspect, the invention provides a chimeric CD 95 receptor; comprising a polypeptide, wherein said polypeptide comprises at least one CD 95 (- derived) polypeptide region having at least 60% sequence identity with a polypeptidedomain, a polypeptide region or a polypeptide motif of a human CD 95 receptor as set forth in SEQ ID No. 1 , wherein said human CD 95 polypeptide region comprises a CD 95 extracellular ligand binding domain; further wherein said polypeptide comprises at least one non-CD 95 (-derived) co-stimulatory cytoplasmic polypeptide domain, region or motif, wherein said at least one cytoplasmic polypeptide domain or cytoplasmic polypeptide motif is a cytoplasmic polypeptide domain or cytoplasmic polypeptide motif selected from a group consisting of CD40L, CD2, TLR2, TLR4, and IL6R subunit beta.

[0033] In a seventeenth aspect the invention provides a chimeric CD 95 receptor; comprising a polypeptide, wherein said polypeptide comprises at least one CD 95 (- derived) polypeptide region comprising a CD 95 extracellular ligand binding domain; further wherein said polypeptide comprises at least one non-CD 95 (-derived) costimulatory cytoplasmic polypeptide domain, region or motif, wherein said at least one cytoplasmic polypeptide domain or cytoplasmic polypeptide motif is a cytoplasmic polypeptide domain or cytoplasmic polypeptide motif selected from a group consisting of CD40L, CD2, TLR2, TLR4, and IL6R subunit beta.

[0034] It has been found that these chimeric CD 95 receptors are also able to turn negative signals e.g. present in a tumor microenvironment into positive signals for T-cell activation. Advantageously, the chimeric CD 95 receptors as herein provided, by linking the specific co-stimulatory cytoplasmic polypeptide domain / region / motif of CD40L, CD2, TLR2, TLR4, or IL6R subunit beta to at least one CD 95-derived polypeptide region that is still capable to bind to its natural ligand, provides the T-cell with resistance to the immunosuppressive tumor microenvironment, prevents TCR-T exhaustion and depletion through apoptosis, and stimulates TCR-T proliferation and functional activity.

[0035] In further aspects, this invention therefore provides T-cells and vectors comprising the chimeric CD 95 receptors according to the thirteenth aspect, and it is herewith contemplated that the chimeric CD 95 receptors according to the thirteenth aspect may be used in the compositions, kits and methods of the present invention.

[0036] All aspects of the invention provide the above described advantages and improvements related to the provision of T-cells comprising both an engineered T-cell receptor and a chimeric CD 95 receptor comprising a fusion of the specific co-stimulatory cytoplasmic domain of the specific non-CD95 (-derived) polypeptides as herein described, to the human CD 95 receptor polypeptide region comprising a functional CD 95 extracellular ligand binding domain, thereby constituting a switch receptor which is 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

[0037] 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:

[0038] Fig. 1 shows a graphical representation of results from a flow cytometric analysis of T-cells transduced with chimeric CD 95 receptor polypeptides.

[0039] 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 CD 95 receptor polypeptide in HeLa cells.

[0040] Fig. 3 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 chimeric CD 95 receptor polypeptides in NCIH 2030 cells.

[0041] Fig. 4 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, a chimeric CD8 Co-receptor and a chimeric CD 95 receptor polypeptide in HeLa cells.DETAILED DESCRIPTION OF THE INVENTION

[0042] As explained above, in a first aspect the invention is directed to an isolated T- cell, wherein the T-cell expresses a chimeric CD 95 receptor comprising a polypeptide, wherein said polypeptide comprises at least one CD 95 (-derived) polypeptide region having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a wildtype human CD 95 receptor (e.g. SEQ ID No. 1), wherein said CD 95 (-derived) polypeptide region comprises a CD 95 extracellular ligand binding domain; further wherein said polypeptide comprises at least one non-CD 95 (-derived) costimulatory cytoplasmic polypeptide domain, region or motif of a tumor necrosis factor receptor superfamily protein comprising CD40, CD40L, CD27, HVEM, GITR, CD30, 0X40, and / or LTBR, an immunoglobulin superfamily (IgSF) protein, a Toll-like-receptor, and / or an I L6-receptor family protein; wherein the T-cell further expresses an engineered T-cell receptor.

[0043] For example, the T-cell may express a chimeric CD 95 receptor comprising a polypeptide, wherein said polypeptide comprises at least one CD 95 (-derived) polypeptide region having one, two, or three cysteine-rich domains (CRDs) of a human CD 95 receptor, said one, two, or three CRDs selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, wherein said human CD 95 polypeptide region comprises aCD 95 extracellular ligand binding domain; further wherein said polypeptide comprises at least one non-CD 95 (-derived) co-stimulatory cytoplasmic polypeptide domain, region or motif of a tumor necrosis factor receptor superfamily protein comprising CD40, CD40L, CD27, HVEM, GITR, CD30, 0X40, and / or LTBR, an immunoglobulin superfamily (IgSF) protein, a Toll-like-receptor, and / or an IL6-receptor family protein; wherein the T-cell further expresses an engineered T-cell receptor. A particular example for a CRD to be included in the chimeric CD 95 receptor may be the CRD as set forth in SEQ ID NO: 3.

[0044] It is understood that the expression of human wildtype CD 95 receptor relates to a protein having an amino acid sequence according to UniProtKB database entry No. P25445 ■ TNR6_HUMAN, as set forth e.g. in SEQ ID No. 1.

[0045] 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.

[0046] 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.

[0047] It is noted in this context that it has been found here for the first time that the chimeric CD 95 receptor comprising both a functional extracellular CD 95 receptor ligand binding domain and at least one non-CD 95-derived co-stimulatory cytoplasmic polypeptide domain, region or motif of a tumor necrosis factor receptor superfamily protein comprising CD40, CD40L, CD27, HVEM, GITR, CD30, 0X40, and / or LTBR, an immunoglobulin superfamily (IgSF) protein, a Toll-like-receptor, and / or an IL6-receptor family protein, when co-expressed in T-cells together with an engineered T-cell receptor, 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 “death domain” of wildtype CD 95 (amino acids 230-314 of SEQ ID No. 1), which is characterized by its ability to recruite Fas-Associated Death Domain-Containing Protein (FADD) through homotypic interactions, thereby potentially initiating e.g. the implementation of pro- apoptotic signals, or replacing e.g. the complete cytoplasmic domain of CD 95 by a at leastone non-CD 95-derived co-stimulatory cytoplasmic polypeptide domain, region or motif of a tumor necrosis factor receptor superfamily protein comprising CD40, CD40L, CD27, HVEM, GITR, CD30, 0X40, and / or LTBR, an immunoglobulin superfamily (IgSF) protein, a Toll-like-receptor, and / or an I L6-receptor family protein enables the T-cell comprising the chimeric CD 95 receptor and the engineered T-cell receptor to bypass the inhibitory effects of FASL expressed by the tumor microenvironment, thus creating resistance to tumor- mediated immune suppression. Secondly, the T-cells as herein provided thus comprise chimeric CD 95 receptors that may act as a molecular switch, redirecting the signaling pathways triggered by FAS engagement with FASL. Instead of inducing apoptosis and T cell death, the fusion of the CD 95 receptor ligand binding domain to a co-stimulatory domain 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 CD 95 receptor together with an engineered T-cell receptor into T cell therapy, the expression of FASL 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.

[0048] According to an embodiment, the extracellular ligand binding domain of the chimeric CD 95 receptor of the T-cell as herein provided may be functional in binding FAS- ligand (CD95L) or any other protein / polypeptide having the ability of binding to the wildtype CD 95 receptor ligand binding domain. Only for example, e.g. scFv and Fab have been described to have the capacity of binding to the ligand binding domain of wildtype CD 95 receptor.

[0049] According to an embodiment, the at least one CD 95 (-derived) 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% sequence identity with the functional polypeptide domain or a functional polypeptide motif of a wildtype human CD 95 receptor (e.g. Seq ID No. 1). According to an embodiment, the at least one CD 95 (-derived) polypeptide region may have one or more conservative amino acid substitutions relative to the amino acid sequence of the wildtype CD 95 receptor.

[0050] For example, the at least one CD 95 (-derived) 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 %, orat 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 CD 95 receptor (e.g. Seq ID No. 1).

[0051] The human CD 95 polypeptide region comprising a CD 95 extracellular ligand binding domain may have generally a sufficient portion of the human wildtype CD 95 extracellular ligand binding domain to be functional in binding FASL. For example, said at least one CD 95-derived polypeptide region having at least 60% sequence identity with CD 95-derived FASL binding domain of a human wildtype CD 95 receptor may comprise the complete or a considerable part of the human wildtype CD 95 FASL binding domain and / or all amino acids at respective amino acid positions of the wildtype CD 95 receptor that are necessary and sufficient for FASL binding to the CD 95 receptor.

[0052] The expressions “domain region”, “binding site region”, “motif region” as used herein are understood to relate to e.g. a region of the chimeric CD 95 receptor polypeptide which is necessary and / or sufficient for a biological function of the chimeric receptor, or to a region of the chimeric CD 95 receptor which is defined e.g. by a localization with respect to a cell, or to a structurally defined unit of the chimeric CD 95 receptor polypeptide. Furthermore, the expression “cytoplasmic polypeptide domain” and “cytoplasmic polypeptide motif” as used herein may be understood as relating to a region of the chimeric CD 95 receptor which is defined by its localization in the cytoplasm of a cell, and which is necessary and / or sufficient for a biological function of the chimeric receptor.

[0053] The chimeric CD 95 receptor polypeptide may be a single-chain polypeptide.

[0054] According to an embodiment, the CD 95 (-derived) polypeptide region may further comprise at least one, or at least two, or at least three CD 95 (-derived) cysteine- rich domains (CRDs). For example, the at least one CD 95 (-derived) CRD may comprise CRD 1 , CRD 2 and / or CRD 3 of the wildtype CD 95 receptor. CRD 1 of the CD 95 wildtype receptor as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2. CRD 2 of the CD 95 wildtype receptor as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 3. CRD 3 of the CD 95 wildtype receptor as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 4. For example, the CD 95 (- derived) polypeptide region may comprise CRD 1 , CRD 2 and CRD 3 of the wildtype CD 95 receptor. It is herewith envisaged that CRD 1 , 2 and 3 polypeptide regions as included in the chimeric CD 95 receptor of the T-cell as herein provided may have one or more conservative amino acid substitutions relative to the CRD1 , 2, and 3 polypeptide regionsof the wildtype CD 95 receptor. In particular, all amino acid substitutions that maintain the functional activity of the wildtype CRD domains are envisaged. For example, Gary C. Starling et al., 1998, which is incorporated by reference herein in its entirety, discloses a mutagenesis study for identifying amino acid residues contributing to the Fas -FasL interaction. The skilled person is therefore aware of protein portions of CRD 1 , CRD 2 and / or CRD 3 of the wildtype CD 95 receptor and potential amino acid substitutions that maintain functional activity. For example, the CD 95 polypeptide region may comprise CRD 2 of the CD 95 wildtype receptor as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 3 (optionally having one or more conservative amino acid substitutions relative to the CRD 2 polypeptide regions of the wildtype CD 95 receptor).

[0055] According to an embodiment, the at least one CD 95 (-derived) polypeptide region further comprises an extracellular N-terminal pre ligand assembly domain (PLAD) region. The N-Terminal PLAD region of the CD 95 wildtype receptor as referred to herein may relate to a polypeptide comprising amino acid sequence 17-82 of UniProtKB database entry No. P25445 ■ TNR6_HUMAN, as set forth e.g. in SEQ ID No. 1. For example, the N- terminal PLAD region as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 5. It is herewith envisaged that the PLAD region as included in the chimeric CD 95 receptor of the T-cell as herein provided may have one or more conservative amino acid substitutions relative to the PLAD region of the wildtype CD 95 receptor. In particular, all amino acid substitutions that maintain the functional activity of the wildtype PLAD region are envisaged.

[0056] According to another embodiment, the at least one CD 95-derived polypeptide region may further comprise a CD95 wildtype homotypic interaction domain. CD 95 homotypic interaction domain as referred to herein may relate to a polypeptide comprising amino acid sequence 59 - 82 of UniProtKB database entry No. P25445 ■ TNR6_HUMAN, as set forth e.g. in SEQ ID No. 1. For example, the homotypic interaction domain region 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 homotypic interaction domain as included in the chimeric CD 95 receptor of the T-cell as herein provided may have one or more conservative amino acid substitutions relative to the homotypic interaction domain of the wildtype CD 95 receptor. In particular, all amino acid substitutions that maintain the functional activity of the wildtype homotypic interaction domain are envisaged.

[0057] According to another embodiment, the at least one CD 95 (-derived) polypeptide region may comprise a CD-95-derived transmembrane region. Thetransmembrane domain of the CD 95 wildtype receptor as referred to herein may relate to a polypeptide comprising amino acid sequence 174-190 of UniProtKB database entry No. P25445 ■ TNR6_HUMAN, as set forth e.g. in SEQ ID No. 1. For example, the transmembrane domain of wildtype CD 95 as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 7. It is herewith envisaged that the transmembrane domain as included in the chimeric CD 95 receptor of the T-cell as herein provided may have one or more conservative amino acid substitutions relative to the transmembrane domain of the wildtype CD 95 receptor. In particular, all amino acid substitutions that maintain the functional activity of the wildtype transmembrane domain are envisaged.

[0058] Thus, it is contemplated that the chimeric CD 95 receptor polypeptide of the T-cell as herein provided may comprise, in addition to comprising the CD 95 (-derived) ligand binding domain, and in addition to comprising the costimulatory domain of CD40, CD40L, CD27, HVEM, GITR, CD30, 0X40, and / or LTBR, an immunoglobulin superfamily (IgSF) protein, a Toll-like-receptor, and / or an IL6-receptor family protein, further domain regions / motif regions / binding site regions from a wildtype human CD 95 receptor in every conceivable combination to establish a functional chimeric CD 95 receptor polypeptide. “Functional” chimeric CD 95 receptor in this context relates to a chimeric CD 95 receptor that is capable of redirecting the signaling pathways triggered by FAS engagement with FASL such that - instead of inducing apoptosis and T-cell death - binding of FASL promotes T-cell activation, persistence and enhanced anti-tumor responses of the T-cell as herein provided. For example, an optional test for functionality of a chimeric CD 95 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 FASL. Thus, the expression “every conceivable combination” of CD 95 receptor regions as described above is meant to exclude a combination with wildtype human CD 95 receptor domains / regions / motifs being inhibitory and / or promoting apoptosis and T-cell death. For example, the chimeric CD 95 receptor polypeptide of the T-cell as herein provided may lack the “death domain” of wildtype CD 95 (amino acids 230-314 of SEQ ID No. 1), or may merely comprise a altered “death domain” which no longer functions in promoting cell death and / or apoptosis of the T-cell, e.g. due to mutations which abolish any inhibitory and / or apoptosis and / or cell-death promoting functionality of the “death domain”.

[0059] According to an embodiment, the CD 95 (-derived) polypeptide region may comprise a complete wildtype CD 95 receptor extracellular domain. For example, theexpression “wildtype CD 95 receptor extracellular domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 26-173 of UniProtKB database entry No. P25445 ■ TNR6_HUMAN, as set forth e.g. in SEQ ID No. 1. For example, the wildtype CD 95 receptor extracellular domain 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 CD 95 receptor extracellular domain as included in the chimeric CD 95 receptor of the T-cell as herein provided may have one or more conservative amino acid substitutions relative to the extracellular domain of the wildtype CD 95 receptor. In particular, all amino acid substitutions that maintain the functional activity of the wildtype CD 95 extracellular domain are envisaged.

[0060] According to an embodiment, the CD 95-derived polypeptide region may comprise a complete wildtype CD 95 receptor extracellular domain, and an entire CD 95- derived transmembrane domain.

[0061] According to some embodiments, the transmembrane domain of the chimeric CD 95 receptor of the T-cell as herein provided may be derived from other proteins which comprise a transmembrane domain. For example, the transmembrane domain may be derived from a co-stimulatory molecule. In principle, any transmembrane domain which is functional and allows surface detectable expression of the chimeric CD 95 receptor is herewith envisaged.

[0062] The chimeric CD 95 receptor of the T-cell 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 CD 95 receptor. It may be a linker region naturally occurring e.g. in wildtype CD 95 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 a CRD domain of the chimeric CD 95 receptor, and / or between individual CRDs (in embodiments comprising more than one CRD), and / or between the transmembrane domain and the intracellular co-stimulatory domain, and / or between individual co-stimulatory domains (in embodiments comprising more than one co-stimulatory domains).

[0063] 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.

[0064] According to an embodiment, a linker region of the chimeric CD 95 receptor of the T-cell as herein provided may comprise the amino acid sequence as set forth inSEQ ID No. 9 (GGGS)n or as set forth in Seq ID No. 10 (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.

[0065] However, in principle, each (polypeptide) linker known in the art is herewith envisaged as being potentially included in the chimeric CD 95 switch receptor of the present invention.

[0066] Turning now to the co-stimulatory domain, according to an embodiment, the chimeric CD 95 receptor polypeptide of the T-cell as herein provided may comprise at least one cytoplasmic polypeptide domain or cytoplasmic polypeptide motif of CD40, CD40L, CD27, HVEM, GITR, CD30, 0X40, and / or LTBR, an immunoglobulin superfamily (IgSF) protein, a Toll-like-receptor, and / or an IL6-receptor family protein. The authors have found for the first time that cytoplasmic co-stimulatory domains of family members of immunoglobulin superfamily (IgSF) protein, a Toll-like-receptor, and / or an IL6-receptor family protein, or alternatively any one of the TNF-family members CD40, CD40L, CD27, HVEM, GITR, CD30, 0X40, and / or LTBR may be fused to the at least one CD 95-derived polypeptide region comprising a functional CD 95 receptor extracellular ligand binding domain in order to generate a functional chimeric CD 95 switch receptor capable of redirecting the signaling pathways triggered by FAS engagement with FASL such that - instead of inducing apoptosis and T-cell death - binding of FASL promotes T-cell activation, persistence and enhanced anti-tumor responses of the T-cell as herein provided, when the T-cell at the same time comprises / expresses an engineered T-cell receptor. Thus, for example, the expression “T-cell co-stimulatory domain or motif” as used herein may relate to a protein portion which is necessary and / or sufficient to preserve the ability to propagate a co-stimulatory signal in a co-stimulatory molecule / protein as herein described. For example, Hong Ye et al., 1999, which is incorporated herein by reference, discloses - by way of example - TRAF-2 binding sites of diverse TNF-family members that may serve as T-cell co-stimulatory domain or motif in the sense of the present application. The skilled person is further aware of numerous further “T-cell co-stimulatory domain or motif” that have been described in literature. Only for example, an amino acid sequence which is necessary and / or sufficient to preserve the ability to propagate a co- stimulatory signal in a co-stimulatory molecule / protein in the sense of the present invention may therefore e.g. comprise or consist of amino acids 170-177 of SEQ ID No. 28 as herein disclosed.

[0067] According to an embodiment, the chimeric CD 95 receptor of the T-cell as herein provided may comprise e.g. at least one complete cytoplasmic domain of the tumornecrosis factor receptor superfamily members CD40, CD40L, CD27, HVEM, GITR, CD30, 0X40, and / or LTBR, of the member of the immunoglobulin superfamily (IgSF) protein, of the Toll-like-receptor, and / or of the I L6-receptor family protein.

[0068] According to some embodiments,, the at least one cytoplasmic polypeptide domain or cytoplasmic polypeptide motif selected from the group consisting of CD40, CD40L, CD27, ICOS, HVEM, GITR, CD30, CD2, 0X40, LTBR, CD28, TLR2, TLR4, and IL6R subunit beta 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 CD40, CD40L, CD27, ICOS, HVEM, GITR, CD30, CD2, 0X40, LTBR, CD28, TLR2, TLR4, or IL6R subunit beta, respectively.

[0069] According to an embodiment, the chimeric CD 95 receptor polypeptide and the engineered T-cell receptor may be able to enhance cytotoxicity of the T-cell as herein provided.

[0070] According to an embodiment, the chimeric CD 95 receptor polypeptide and the engineered T-cell receptor may be able to enhance activation, proliferation, and / or production of activating cytokines of / in the T-cell as herein provided.

[0071] According to an embodiment, the chimeric CD 95 receptor and the engineered T-cell receptor may be capable of increasing resistance of T-cells as herein provided to CD95L expressing cancer cells.

[0072] It is contemplated that the chimeric CD 95 receptor of the T-cell as herein provided may comprise any functional combination 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 CD40, CD40L, CD27, ICOS, HVEM, 4-1 BB, GITR, CD30, CD2, 0X40, LTBR, CD28, TLR2, TLR4, and IL6R subunit beta.

[0073] According to an embodiment, the chimeric CD 95 receptor of the T-cell 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. 11. It is herewith envisaged that the cytoplasmic polypeptide domain, regionor motif of wildtype CD40 as included in the chimeric CD 95 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. 11. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype CD40 are envisaged.

[0074] In embodiments wherein a costimulatory region, motif or domain of CD40 is included in the chimeric CD 95 receptor of the T-cell as herein provided, for example, the polypeptide of the chimeric CD 95 receptor may have an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID NO: 12.

[0075] According to an embodiment, the chimeric CD 95 receptor of the T-cell as herein provided 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 CD 95 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. 13. It is herewith envisaged that the cytoplasmic polypeptide domain, region or motif of wildtype CD28 as included in the chimeric CD 95 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. 13. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype CD28 are envisaged.

[0076] According to an embodiment, the chimeric CD 95 receptor of the T-cell as herein provided may comprise at least one cytoplasmic polypeptide domain, region or motif of 4-1 BB. For example, a cytoplasmic polypeptide region of 4-1 BB may comprise the complete cytoplasmic domain of wildtype human 4-1 BB. In other embodiments, the cytoplasmic polypeptide region of 4-1 BB included in the chimeric CD 95 receptor may comprise at least one functional, co-stimulatory motif / domain / region of the complete wildtype human 4-1 BB cytoplasmic domain. For example, the expression “wildtype human 4-1 BB cytoplasmic domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 214-255 of UniProtKB database entry No. Q07011 ■ TNR9_HUMAN, as set forth e.g. in SEQ ID No. 14. It is herewith envisaged that the cytoplasmic polypeptide domain, region or motif of wildtype 4-1 BB as included in the chimeric CD 95 receptor ofthe 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. 14. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype 4-1 BB are envisaged.

[0077] In embodiments wherein a costimulatory region, motif or domain of 4-1 BB is included in the chimeric CD 95 receptor as herein provided, for example, the polypeptide of the chimeric CD 95 receptor may have an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID NO: 15.

[0078] According to an embodiment, the chimeric CD 95 receptor of the T-cell as herein provided may comprise at least one cytoplasmic polypeptide domain, region or motif of ICOS. For example, a cytoplasmic polypeptide region of ICOS may comprise the complete cytoplasmic domain of wildtype human ICOS. In other embodiments, the cytoplasmic polypeptide region of ICOS included in the chimeric CD 95 receptor may comprise at least one functional, co-stimulatory motif / domain / region of the complete wildtype human ICOS cytoplasmic domain. For example, the expression “wildtype human ICOS cytoplasmic domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 162-199 of UniProtKB database entry No. Q9Y6W8- ICOS_HUMAN, as set forth e.g. in SEQ ID No. 16. It is herewith envisaged that the cytoplasmic polypeptide domain, region or motif of wildtype ICOS as included in the chimeric CD 95 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. 16. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype ICOS are envisaged.

[0079] In embodiments wherein a costimulatory region, motif or domain of ICOS is included in the chimeric CD 95 receptor, for example, the polypeptide of the chimeric CD 95 receptor may have an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID NO: 17.

[0080] According to an embodiment, the chimeric CD 95 receptor of the T-cell 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 CD 95 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 comprisingamino acid sequence 224-283 of UniProtKB database entry No. Q92956- TNR14_HUMAN, as set forth e.g. in SEQ ID No. 18. It is herewith envisaged that the cytoplasmic polypeptide domain, region or motif of wildtype HVEM as included in the chimeric CD 95 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. 18. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype HVEM are envisaged.

[0081] In embodiments wherein a costimulatory region, motif or domain of HVEM is included in the chimeric CD 95 receptor, for example, the polypeptide of the chimeric CD 95 receptor may have an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID NO: 19.

[0082] According to an embodiment, the chimeric CD 95 receptor of the T-cell as herein provided may comprise at least one cytoplasmic polypeptide domain, region or motif of 0X40. For example, a cytoplasmic polypeptide region of 0X40 may comprise the complete cytoplasmic domain of wildtype human 0X40. In other embodiments, the cytoplasmic polypeptide region of 0X40 included in the chimeric CD 95 receptor may comprise at least one functional, co-stimulatory motif / domain / region of the complete wildtype human 0X40 cytoplasmic domain. For example, the expression “wildtype human 0X40 cytoplasmic domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 236-277 of UniProtKB database entry No. P43489- TNR4_HUMAN, as set forth e.g. in SEQ ID No. 20. It is herewith envisaged that the cytoplasmic polypeptide domain, region or motif of wildtype 0X40 as included in the chimeric CD 95 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. 20. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype 0X40 are envisaged.

[0083] In embodiments wherein a costimulatory region, motif or domain of OX 40 is included in the chimeric CD 95 receptor, for example, the polypeptide of the chimeric CD 95 receptor may have an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID NO: 21.

[0084] According to an embodiment, the chimeric CD 95 receptor of the T-cell as herein provided may comprise at least one cytoplasmic polypeptide domain, region or motif of CD27. For example, a cytoplasmic polypeptide region of CD27 may comprise the complete cytoplasmic domain of wildtype human CD27. In other embodiments, thecytoplasmic polypeptide region of CD27 included in the chimeric CD 95 receptor may comprise at least one functional, co-stimulatory motif / domain / region of the complete wildtype human CD27 cytoplasmic domain. For example, the expression “wildtype human CD27 cytoplasmic domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 213-260 of UniProtKB database entry No. P26842- CD27_HUMAN, as set forth e.g. in SEQ ID No. 22. It is herewith envisaged that the cytoplasmic polypeptide domain, region or motif of wildtype CD27 as included in the chimeric CD 95 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. 22. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype CD27 are envisaged.

[0085] In embodiments wherein a costimulatory region, motif or domain of CD27 is included in the chimeric CD 95 receptor, for example, the polypeptide of the chimeric CD 95 receptor may have an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID NO: 23.

[0086] According to an embodiment, the chimeric CD 95 receptor of the T-cell as herein provided may comprise at least one cytoplasmic polypeptide domain, region or motif of CD40L. For example, a cytoplasmic polypeptide region of CD40L may comprise the complete cytoplasmic domain of wildtype human CD40L. In other embodiments, the cytoplasmic polypeptide region of CD40L included in the chimeric CD 95 receptor may comprise at least one functional, co-stimulatory motif / domain / region of the complete wildtype human CD 40L cytoplasmic domain. For example, the expression “wildtype human CD40L cytoplasmic domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 1-22 of UniProtKB database entry P29965- CD40L_HUMAN, as set forth e.g. in SEQ ID No. 24. It is herewith envisaged that the cytoplasmic polypeptide domain, region or motif of wildtype CD40L as included in the chimeric CD 95 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. 24. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype CD40L are envisaged.

[0087] In embodiments wherein a costimulatory region, motif or domain of CD40L is included in the chimeric CD 95 receptor, for example, the polypeptide of the chimeric CD 95 receptor may have an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID NO: 25.

[0088] According to an embodiment, the chimeric CD 95 receptor of the T-cell as herein provided may comprise at least one cytoplasmic polypeptide domain, region or motif of GITR. For example, a cytoplasmic polypeptide region of GITR may comprise the complete cytoplasmic domain of wildtype human GITR. In other embodiments, the cytoplasmic polypeptide region of GITR included in the chimeric CD 95 receptor may comprise at least one functional, co-stimulatory motif / domain / region of the complete wildtype human GITR cytoplasmic domain. For example, the expression “wildtype human GITR cytoplasmic domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 184-241 of UniProtKB database entry Q9Y5LI5 ■ TNR18_HUMAN, as set forth e.g. in SEQ ID No. 26. It is herewith envisaged that the cytoplasmic polypeptide domain, region or motif of wildtype GITR as included in the chimeric CD 95 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. 26. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype GITR are envisaged.

[0089] In embodiments wherein a costimulatory region, motif or domain of GITR is included in the chimeric CD 95 receptor, for example, the polypeptide of the chimeric CD 95 receptor may have an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID NO: 27.

[0090] According to an embodiment, the chimeric CD 95 receptor of the T-cell 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 CD 95 receptor may comprise at least one functional, co-stimulatory motif / domain / region of the complete wildtype human CD30 cytoplasmic domain. 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. 28. It is herewith envisaged that the cytoplasmic polypeptide domain, region or motif of wildtype CD30 as included in the chimeric CD 95 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. 28. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype CD30 are envisaged.

[0091] In embodiments wherein a costimulatory region, motif or domain of CD30 is included in the chimeric CD 95 receptor, for example, the polypeptide of the chimeric CD 95 receptor may have an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID NO: 29.

[0092] According to an embodiment, the chimeric CD 95 receptor of the T-cell 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 CD 95 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. 30. It is herewith envisaged that the cytoplasmic polypeptide domain, region or motif of wildtype CD2 as included in the chimeric CD 95 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. 30. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype CD2 are envisaged.

[0093] In embodiments wherein a costimulatory region, motif or domain of CD2 is included in the chimeric CD 95 receptor, for example, the polypeptide of the chimeric CD 95 receptor may have an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID NO.: 31.

[0094] According to an embodiment, the chimeric CD 95 receptor of the T-cell as herein provided may comprise at least one cytoplasmic polypeptide domain, region or motif of LTBR. For example, a cytoplasmic polypeptide region of LTBR may comprise the complete cytoplasmic domain of wildtype human LTBR. In other embodiments, the cytoplasmic polypeptide region of LTBR included in the chimeric CD 95 receptor may comprise at least one functional, co-stimulatory motif / domain / region of the complete wildtype human LTBR cytoplasmic domain. For example, the expression “wildtype human LTBR cytoplasmic domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 249-435 of UniProtKB database entry P36941 ■ TNR3_HUMAN, as set forth e.g. in SEQ ID No. 32. It is herewith envisaged that the cytoplasmic polypeptide domain, region or motif of wildtype LTBR as included in the chimeric CD 95 receptor of the T-cell as herein provided may have one or more conservative amino acid substitutionsrelative to the amino acid sequence as set forth in SEQ ID No. 32. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype LTBR are envisaged.

[0095] According to an embodiment, the chimeric CD 95 receptor of the T-cell as herein provided may comprise at least one cytoplasmic polypeptide domain, region or motif of TLR2. For example, a cytoplasmic polypeptide region of TLR2 may comprise the complete cytoplasmic domain of wildtype human TLR2. In other embodiments, the cytoplasmic polypeptide region of TLR2 included in the chimeric CD 95 receptor may comprise at least one functional, co-stimulatory motif / domain / region of the complete wildtype human TLR2 cytoplasmic domain. For example, the expression “wildtype human TLR2 cytoplasmic domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 610-784 of UniProtKB database entry 060603 ■ TLR2_HUMAN, as set forth e.g. in SEQ ID No. 33. . It is herewith envisaged that the cytoplasmic polypeptide domain, region or motif of wildtype TLR2 as included in the chimeric CD 95 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. 33. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype TLR2 are envisaged.

[0096] According to an embodiment, the chimeric CD 95 receptor of the T-cell as herein provided may comprise at least one cytoplasmic polypeptide domain, region or motif of TLR4. For example, a cytoplasmic polypeptide region of TLR4 may comprise the complete cytoplasmic domain of wildtype human TLR4. In other embodiments, the cytoplasmic polypeptide region of TLR4 included in the chimeric CD 95 receptor may comprise at least one functional, co-stimulatory motif / domain / region of the complete wildtype human TLR4 cytoplasmic domain. For example, the expression “wildtype human TLR4 cytoplasmic domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 653-839 of UniProtKB database entry 000206 ■ TLR4_HUMAN, as set forth e.g. in SEQ ID No. 34. It is herewith envisaged that the cytoplasmic polypeptide domain, region or motif of wildtype TLR4 as included in the chimeric CD 95 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. 34. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype TLR4 are envisaged.

[0097] According to an embodiment, the chimeric CD 95 receptor of the T-cell as herein provided may comprise at least one cytoplasmic polypeptide domain, region ormotif of IL6R subunit beta. For example, a cytoplasmic polypeptide region of IL6R subunit beta may comprise the complete cytoplasmic domain of wildtype human IL6R subunit beta. In other embodiments, the cytoplasmic polypeptide region of IL6R subunit beta included in the chimeric CD 95 receptor may comprise at least one functional, costimulatory motif / domain / region of the complete wildtype human IL6R subunit beta cytoplasmic domain. For example, the expression “wildtype human IL6R subunit beta cytoplasmic domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 642-918 of UniProtKB database entry P40189 ■ IL6RB_HUMAN, as set forth e.g. in SEQ ID No. 35. It is herewith envisaged that the cytoplasmic polypeptide domain, region or motif of wildtype IL6R as included in the chimeric CD 95 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. 35. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype IL6R are envisaged.

[0098] Thus, as described herein, the expression “CD 95 -derived polypeptide region” may be used herein interchangeably with the expression “CD95 polypeptide region” to refer to a (functional) portion of a wildtype CD 95 protein (or variants thereof). With respect to variants of wildtype CD 95, such a variant may have 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 corresponding portion of the amino acid sequence of SEQ ID NO: 1. For example, variants of wildtype CD 95 as herein described may comprise an extracellular domain that preserves the ability to bind FASL. Suitable methods (such as e.g. surface plasmon resonance assay) for determining said functional ability are known to the skilled person.

[0099] According to some embodiments, a chimeric CD 95 receptor of the T-cell 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. 12, 15, 17, 19, 21 , 23, 25, 27, 29, and 31. According to some embodiments, a chimeric CD 95 receptor of the T-cell 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. 12, 15, 17, 19, 21 , 23, 25, 27, 29, and 31.

[0100] In a further aspect, the invention provides a vector comprising a nucleic acid comprising a nuclear acid sequence encoding for a chimeric CD 95 receptor comprising a polypeptide, wherein said polypeptide comprises at least one CD 95 (-derived) polypeptide region having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a human CD 95 receptor as set forth in SEQ ID No. 1 , wherein said human CD 95 polypeptide region comprises a CD 95 extracellular ligand binding domain; further wherein said polypeptide comprises at least one non-CD 95 (- derived) co-stimulatory cytoplasmic polypeptide domain, region or motif of a tumor necrosis factor receptor superfamily protein comprising CD40, CD40L, CD27, HVEM, GITR, CD30, 0X40, and / or LTBR, an immunoglobulin superfamily (IgSF) protein, a Toll- like-receptor, and / or an IL6-receptor family protein; and further comprising a nucleic acid comprising a nuclear acid sequence encoding for an engineered T-cell receptor.

[0101] 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.

[0102] 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.

[0103] 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: 36-45.

[0104] The polynucleotides described above may or may not comprise additional or altered nucleotide sequences encoding e.g., altered amino acid residues. The polynucleotides may further encode fusion polypeptides, fragments, variants and other derivatives of the chimeric CD 95 receptors described herein.

[0105] 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.

[0106] 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.

[0107] The vector may be a viral vector or a non-viral vector.

[0108] 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 Medicine 2010; 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.

[0109] 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.

[0110] 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 T-cell receptor.

[0111] In a further embodiment, the vector may further comprise a nucleic acid encoding for a CD8 Co-receptor. For example, the CD8 Co-receptor may be a wildtype CD8 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. For example, the chimeric CD8 Co- receptor may comprise a polypeptide, wherein said polypeptide comprises at least one CD8a-derived polypeptide region having at least 60% sequence identity with a functional polypeptide domain or a functional polypeptide motif of a human wildtype CD8a Co- receptor (e.g. Seq ID No. 46), wherein said least one CD8a-derived polypeptide region comprises an CD8a-derived IG-like domain region; further wherein said polypeptide comprises at least one CD8p-derived polypeptide region having at least 60% sequence identity with a functional polypeptide domain or a functional polypeptide motif of a humanwildtype CD8p Co-receptor (e.g. Seq ID No. 47), wherein said least one CD8p-derived polypeptide region comprises an CD8p-derived IG-like domain region.

[0112] 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. 46. 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. 47.

[0113] It is contemplated that the chimeric human CD8 Co-receptor polypeptide comprising both a CD8a (-derived) IG-like domain region together with a CD8p (-derived) IG-like domain region is able to maintain the function of a CD8a (-derived) IG-like domain region and a CD8p (-derived) IG-like domain region being present on individual, separate polypeptides in a wildtype CD8αβ co-receptor. Thus, the expression “Ig-like domain” as used herein may - in principle - refer to a polypeptide region that is homologous to the V and / or C domains in immunoglobulin proteins.

[0114] According to an embodiment, the at least one CD8a (-derived) 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% sequence identity with the functional polypeptide domain or a functional polypeptide motif of a wildtype human CD8a Co-receptor (e.g. Seq ID No. 46).

[0115] For example, the at least one CD8a (-derived) 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 CD8a Co- receptor (e.g. Seq ID No. 46).

[0116] According to an embodiment, the at least one CD8p (-derived) 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% sequence identity with the functional polypeptide domain or a functional polypeptide motif of a wildtype human CD8p Co-receptor (Seq ID No. 47).

[0117] For example, the at least one CD8p (-derived) 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%, orat 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 CD8p Coreceptor (e.g. Seq ID No. 47).

[0118] According to an embodiment, the chimeric human CD8 Co-receptor polypeptide is a single-chain polypeptide.

[0119] According to an embodiment, the chimeric CD8 Co-receptor comprises the CD8a (derived) IG-like domain region, the CD8p derived IG-like domain region, a stalk region (typically between the transmembrane region and the IG-like domain region of CD8 receptors), a transmembrane domain region; and an intracellular / cytoplasmic domain region, wherein the intracellular domain region comprises a palmitoylation motif region and a LCK binding site region.

[0120] It is herewith envisaged that the stalk region, the transmembrane region, and the intracellular domain of the chimeric CD 8 Co-receptor may be derived from other proteins or from CD8a or CD8p. Thus, it is contemplated that the chimeric CD8 Co- receptor polypeptide may comprise, in addition to comprising the CD8a (-derived) IG-like domain region and the CD8p (-derived) IG-like domain region, further domain regions / motif regions / binding site regions from one or both of a wildtype human CD8a Co- receptor and / or a wildtype human CD8p Co-receptor, and / or from other proteins in every conceivable combination to establish a functional chimeric CD8 Co-receptor polypeptide.

[0121] For example, according to an embodiment, the chimeric CD8 Co-receptor may comprise a CD8a-derived transmembrane region.

[0122] According to an embodiment, the chimeric CD8 receptor may comprise a CD4 Co-receptor (derived) cytoplasmic region. For example, the chimeric CD8 co-receptor may comprise the entire cytoplasmic region of a wildtype CD4 Co-receptor. For example, the expression “wildtype human CD4 Co-receptor cytoplasmic region” as referred to herein may relate to a polypeptide comprising amino acid sequence 419 - 458 of UniProtKB database entry No. P01730 ■ CD4_HUMAN, as set forth e.g. in SEQ ID No. 48.

[0123] According to an embodiment, the chimeric CD8 Co-receptor may further comprise at least one cytoplasmic polypeptide domain or cytoplasmic polypeptide motif of a tumor necrosis factor receptor superfamily protein, of an immunoglobulin superfamily (IgSF) protein, and / or of and / or of an ITAM-associated receptor.

[0124] The enhanced chimeric CD8 Co-receptor which - in some embodiments - is encoded by the vector of the present invention links an extra co-stimulatory domain to thehuman CD8 Receptor. The co-stimulation associated with such co-stimulatory domain provided by the fused cytoplasmic polypeptide domain or cytoplasmic polypeptide motif of a tumor necrosis factor receptor superfamily protein and / or of an immunoglobulin superfamily (IgSF) protein, and / or of an ITAM-associated receptor complements TCR signaling, leading to a more potent TCR-T-cell product.

[0125] Thus, the provision of T-cells as herein provided comprising the chimeric CD 95 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.

[0126] In an embodiment, the chimeric CD8 Co-receptor comprises a CD4 (-derived) cytoplasmic domain. In this embodiment, the at least one CD8a (-derived) polypeptide region may also further comprise a CD8a (-derived) Transmembrane domain region and the at least one CD8p (-derived) polypeptide region may comprise a CD8p (-derived) stalk domain region. In this embodiment, the CD8a (-derived) IG-like domain region may be located closer to the N-terminal end of the enhanced chimeric CD8 Co-receptor polypeptide than the CD8p (-derived) IG-like domain region. For example, the chimeric CD8 Co-receptor may further comprise a costimulatory CD30 motif, as set forth e.g. in SEQ ID No. 49 as herewith presented. Furthermore, the chimeric CD8 Co-receptor according to this embodiment may comprise the cytoplasmic domain of CD40. For example, in accordance with this embodiment, the polypeptide linker sequence GGGS is inserted between the CD30 costimulatory motif and the CD40 cytoplasmic domain. The CD30 motif may be included at the C-terminal end of the chimeric CD8 Co-receptor. An enhanced chimeric CD8 Co-receptor in accordance with this embodiment 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 SEQ ID NO:50 (pTK-0638).

[0127] In some embodiments, the transgene 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 CD 95 receptor, TCRa, TCRp, and the optional 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).

[0128] 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: 51]) before the selfcleaving 2A sequence, this may enable efficient synthesis of biologically active proteins, e.g., TCRs and chimeric CD 95 receptors as described herein.

[0129] 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 CD 95 receptor of the present invention, wherein the nucleic acid further encodes for an engineered T-cell receptor.

[0130] It is to be noted that in the context of this invention, the expression “engineered T-cell receptor” is to be distinguished from a “CAR” T-cell receptor. For example, unlike chimeric antigen receptors (CARs), engineered TCRs recognize HLA- presented peptides derived from proteins of all cellular compartments. Furthermore, the expression “engineered T-cell receptor” is 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).

[0131] In accordance with another aspect, a T-cell may express the chimeric CD 95 receptor as herein described and the engineered T-cell receptor.

[0132] 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 coreceptor 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.

[0133] 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.

[0134] According to some embodiments, the vector and as herein described may have been introduced into the T-cell.

[0135] 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 the chimeric CD 95 receptors as herein described and the engineered T-cell receptor and, optionally a CD8 Co-receptor may be stably introduced into the T-cell by a transposon / transposase system or a viral-based 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.

[0136] 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.

[0137] According to an embodiment, the cell may be a αβ T-cell, yδ T-cell, and / or a natural killer T-cell.

[0138] For example, the αβ T-cell may be a CD4 T-cell, or the αβ T-cell may be a CD8 T-cell, or the yδ T-cell may comprise e.g. a Vy1 chain or a Vy2 chain, or may be e.g. a Vy9V82+ T-cell.

[0139] It is envisaged that the T-cell may express a chimeric CD 95 receptor as herein described, as well as an engineered T-cell receptor. In embodiments, the T-cell may be a CD 4 T-cell that further expresses the CD 8 Co-receptor, e.g. both CD8a and CD8p Co-receptor, or any engineered protein exhibiting Co-receptor functionality.

[0140] In accordance with the present invention, 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 αβ T-cells or yδ T-cells that express the chimeric CD 95 receptor polypeptide as described herein, and furthermore an engineered TCR. Optionally, the T- cells may 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 andhematologic malignancies. For example, “hot” tumors or “cold” tumors may be treated by the T-cells herewith provided.

[0141] 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.

[0142] 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.

[0143] 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 αβ T-cells or yδ T-cells that express a chimeric CD 95 receptor as described herein, and an engineered TCR, and optionally a CD8 Co-receptor such as a wildtype or chimeric CD8 co-receptor.

[0144] In some embodiments, the T-cell may be derived from an induced pluripotent stem cell (iPSCs).

[0145] According to another aspect, it is herewith provided a kit comprising means to prepare the T-cells described above.

[0146] According to a further aspect, this invention relates to a pharmaceutical composition comprising the T-cell provided by the present invention.

[0147] It is herewith contemplated that the pharmaceutical composition may further comprise an adjuvant, excipient, buffer, diluent, carrier, stabilizer or combination thereof.

[0148] According to a further aspect, there is provided a pharmaceutical composition comprising T-cells which express the chimeric CD 95 receptor as herein described and an engineered T-cell receptor.

[0149] According to an embodiment, the pharmaceutical composition may further comprise CD4 T-cells expressing said chimeric CD 95 receptor, 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.

[0150] 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 CD 95 receptor. It is also possible that the chimeric CD 95 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).

[0151] 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 comprising a nucleic acid encoding for a chimeric CD 95 receptor comprising a polypeptide, wherein said polypeptide comprises at least one CD 95-derived polypeptide region having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a human CD 95 receptor as set forth in SEQ ID No. 1 , wherein said human CD 95 polypeptide region comprises a CD 95 extracellular ligand binding domain; further wherein said polypeptide comprises at least one non-CD 95-derived costimulatory cytoplasmic polypeptide domain, region or motif of atumor necrosis factor receptor superfamily protein comprising CD40, CD40L, CD27, HVEM, GITR, CD30, 0X40, and / or LTBR, an immunoglobulin superfamily (IgSF) protein, a Toll-like- receptor, and / or an I L6-receptor family protein; and further comprising a nucleic acid encoding for an engineered T-cell receptor into the T-cell, and expanding the transduced T-cells.

[0152] For example, the method may comprise transforming, transfecting or transducing the isolated T-cells with the vector.

[0153] 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.

[0154] 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.

[0155] According to an embodiment, the nucleic acid may be a DNA or an mRNA.

[0156] For the in vivo introduction, the vector may be - for example - a nonreplicating viral vector.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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, too ment. In other illustrative examples, thebreast 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.

[0161] 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 into a T-cell, wherein the vector comprises a nucleic acid encoding for an engineered T-cell receptor and further encoding for a chimeric CD 95 receptor comprising a polypeptide, wherein said polypeptide comprises at least one CD 95-derived polypeptide region having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a human CD 95 receptor as set forth in SEQ ID No. 1 , wherein said human CD 95 polypeptide region comprises a CD 95 extracellular ligand binding domain; further wherein said polypeptide comprises at least one non-CD 95-derived co-stimulatory cytoplasmic polypeptide domain, region or motif of a tumor necrosis factor receptor superfamily protein comprising CD40, CD40L, CD27, HVEM, GITR, CD30, 0X40, and / or LTBR, an immunoglobulin superfamily (IgSF) protein, a Toll-like-receptor, and / or an IL6-receptor family protein.

[0162] For example, the nucleic acid may further encode for a recombinant CD8 Coreceptor, such as a wildtype CD8 co-receptor or a chimeric CD 8 co-receptor.

[0163] 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.

[0164] The invention will be further illustrated by the following non-limiting Experimental Examples.

[0165] Sequences as used herein are depicted in below Table 1.

[0166] Table 1. Sequences as used herein.Experimental Examples

[0167] Example 1. In-vitro T-cell killing analysis of T-cells according to the present invention transduced with chimeric CD 95 receptor polypeptides and an engineered T-cell receptor

[0168] In order to test the T-cells expressing chimeric CD 95 receptor constructs together with an engineered T-cell receptor described herein for suitability in adoptive T-cell therapy (ACT), and / or for increasing cytotoxicity of the generated T-cells that express the chimeric CD 95 switch receptors and the engineered T-cell receptor, chimeric CD 95 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 NCI-H2030 FASL-expressing cells and HeLa FASL- expressing cells, respectively, for evaluating cytotoxicity of the transduced T-cells.

[0169] 1.1 Materials and MethodsCloning of chimeric human CD95 receptor constructs and chimeric CD8 Co-receptor constructsChimeric human CD95 receptor constructs as well as chimeric CD8 Co-receptor constructs have been generated using standard cloning techniques. Table 2 as presented below summarizes the cloned underlying plasmids for chimeric CD 95 constructs created:

[0170] Table 2 as presented below summarizes the chimeric CD 95 receptor constructs that have been generated by the inventors in a schematic representation:

[0171] Table 2

[0172] As used in Table 2, the expression “EC” relates to the origin of the extracellular domain of the chimeric receptor. “TM” relates to the origin of the transmembrane domain of the chimeric receptor, and “CYP” relates to the origin of the cytoplasmic domain of the generated construct.

[0173] Furthermore, Table 3 as presented below summarizes further chimeric CD 95 receptor constructs as used for the T-cell killing assay shown in Fig. 4, and chimeric CD8 co-receptor type as used in the T-cell killing assay shown in Fig. 4

[0174] Table 3

[0175] As used in Table 3, the expression “EC” relates to the origin of the extracellular domain of the chimeric receptor. “TM” relates to the origin of the transmembrane domain of the chimeric receptor, and “CYP” relates to the origin of the cytoplasmic domain of the generated construct, and Co-receptor type relates to the type of the CD8-coreceptor that is co-transduced into the T-cells, which is - in all constructs according to Table 3, p638, thus encoding a chimeric CD8 co-receptor with costimulatory domain of CD30 (CD30 motif) and CD40 both fused to the cytoplasmic domain of CD4.

[0176] CD8 Cells Generation

[0177] PBMCs from a healthy donor buffy coat were isolated by density gradient centrifugation with Lymphoprep. Purified polyclonal CD8 T-cells were obtained by negative selection with anti-CD4 microbeads for depleting C4 population. 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 SWITCH receptors. TheH LA-1 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.

[0178] 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.

[0179] Cell killing assay in NCIH2030 cells expressing FASL: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. NCIH2030 1x104tumor cells were plated per well in 96-well plates. Untransduced (mock), or transduced human T-cells (transduced with either MAGE_TCR alone, or transduced with MAGE_TCR together with a chimeric CD 95 receptor) were added in triplicates at 1 to 1 or 1 :2 E:T ratio.

[0180] Cell killing assay in HeLa cells expressing FASL:

[0181] 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. HeLa 1x104tumor cells were plated per well in 96-well plates. Untransduced (mock), or transduced human T-cells (transduced with either MAGE_TCR alone, or transduced with MAGE_TCR together with a chimeric CD 95 receptor) were added in triplicates at 1 to 2 E:T ratio. For second and third stimulation, tumor cells (HeLa 1 x104) were added, respectively, to each well after cancer cells from the previous stimulation are killed, typically 50h to 100h from the beginning of the experiment.

[0182] Long term cell killing assay in NCIH 2030 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). PBMCs from a healthy donor buffy coat were isolated by density gradient centrifugation with Lymphoprep. Purified polyclonal CD8+ T cells were obtainedby positive selection with anti-CD8+ microbeads. CD3+ T cells were activated using TransAct in presence of I L-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 SWITCH receptors. Where indicated (results in Fig. 4), a chimeric CD8 Co-receptor was co-transduced (CoR). The chimeric CD8 Co-receptor used in the experiments is the Chimeric CD-8 Co-receptor polypeptide (pTK-0638) having an amino acid sequence as set forth in SeqID No. 50.

[0183] Flow Cytometry:

[0184] 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); from Invitrogen: CD34 (clone QBEND10), CD34 (clone 4H11); from Miltenyi Biotec: CD8a (clone REA734), CD95 (clone DX2), from Beckman Coulter: TCRBV3S1 Vp3. PE-conjugated HLA-A*02:01 specific MAGE-A1 MHC tetramer (KVLEYVIKV) (SEQ ID NO: 52) (TB-M070-1) was added together with cell surface staining antibodies. Zombie Yellow™ Fixable Viability Kit was used to discriminate between live and dead cells. The expression of the chimeric CD 95 receptor has been determined for CD8 cells transduced with different chimeric CD 95 receptors as herein provided.

[0185] 1.2. T-cell killing assay analysis

[0186] The Relative cell growth has been observed over time for each transduced T- cell fraction. The results are shown in Fig. 2 and Fig.3 and Fig.4 wherein Fig. 2 shows the results of the killing assay with the chimeric CD 95 receptors in HeLa cells, whereas Fig. 3 shows the result of the cell killing assay with the chimeric CD 95 receptors in NCIH2030 cells. “Mock” relates to mock-transduced T-cell fraction, “MAGE_TCR” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR); “MAGE-A1_TCR-SwR_CD40” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a chimeric CD 95 receptor comprising CD 40 cytoplasmic domain; “MAGE-A1_TCR- SwR_CD40L” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a chimeric CD 95 receptor comprising CD 40L cytoplasmic domain; “MAGE-A1_TCR-SwR_CD27” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a chimeric CD 95 receptor comprising CD 27 cytoplasmic domain; “MAGE-A1_TCR- SwRJCOS” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a chimeric CD 95 receptor comprisingICOS cytoplasmic domain; “MAGE-A1_TCR-SwR_HVEM” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a chimeric CD 95 receptor comprising HVEM cytoplasmic domain; “MAGE-A1_TCR- SwR_4-1 BB” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a chimeric CD 95 receptor comprising 4-1 BB cytoplasmic domain; “MAGE-A1_TCR-SwR_GITR” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a chimeric CD 95 receptor comprising GITR cytoplasmic domain; “MAGE-A1_TCR- SwR_CD30” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a chimeric CD 95 receptor comprising CD 30 cytoplasmic domain “MAGE-A1_TCR-SwR_CD 2” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a chimeric CD 95 receptor comprising CD 2 cytoplasmic domain; “MAGE-A1_TCR- SwR_OX40” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a chimeric CD 95 receptor comprising 0X40 cytoplasmic domain, respectively, as herewith provided.

[0187] Fig. 4 shows the results of a long term killing assay, wherein T-cells were transduced with either Mock, or a Mage-A1 TCR (MAGE A1 TCR), or a Mage-A1 TCR with a wildtype CD8 co-receptor (Mage A1 TCR WT_CoR), or a Mage-A1 TCR with a chimeric CD8 co-receptor (Mage A1 TCR ESC_CoR), or a Mage-A1 TCR with a chimeric CD8 co-receptor and a respective one of the chimeric CD 95 receptor constructs as herein described (Mage A1 TCR ESC_CoR FAS CD40, Mage A1 TCR ESC_CoR FAS CD30, Mage A1 TCR ESC_CoR FAS 0X40, Mage A1 TCR ESC_CoR FAS CD2, Mage A1 TCR ESC_CoR FAS CD27, Mage A1 TCR ESC_CoR FAS CD40L, Mage A1 TCR ESC_CoR FAS HVEM, and Mage A1 TCR ESC_CoR FAS ICOS, respectively).

[0188] 1.3 Flow Cytometry Analysis

[0189] CD8 cells transduced with vectors comprising nucleic acids encoding for different chimeric CD 95 receptors as herein provided have been checked for expression of the chimeric CD 95 receptor. As visible from Fig. 1 , only the chimeric CD 95 receptors comprising cytoplasmic domains of 41 BB, GITR and CD40L, respectively, show comparably low percentage of Fas-high population, whereas all other tested chimeric CD 95 receptors show high percentages of Fas high population.

[0190] 1.4 Results

[0191] As visible from Fig. 2 and 3, Co-transduction of CD8 cells with an engineered HLA-I restricted TCR raised against MAGE-A1 together with a chimeric CD 95 receptor,comprising a non - CD95 - derived co-stimulatory cytoplasmic polypeptide domain or motif as herewith provided results in an increased killing activity of the engineered T-cells compared with mock transduced T-cells and / or T-cells only transduced with the HLA-I restricted TCR raised against MAGE-A1 , as visible in both HeLa and NCIH2030 cells.

[0192] As visible from Fig. 4, T-cells co-transduced with an engineered HLA-I restricted TCR raised against MAGE-A1 together with a chimeric CD 95 receptor as herein provided, and together with a chimeric CD8 Co-receptor results in an increased killing activity of the engineered T-cells compared with mock transduced T-cells and / or T-cells only transduced with the HLA-I restricted TCR raised against MAGE-A1 , as visible in NCIH2030 cells.

[0193] Summary and conclusions

[0194] The results described above demonstrate - in principle - suitability of the T- cells comprising both a chimeric CD 95 receptor polypeptides as well as an engineered T- cell receptor as herewith provided for improving adoptive cell therapy (ACT). Specifically, it is contemplated that the chimeric CD 95 receptor polypeptides of the present invention, in combination with the 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.

[0195] Thus, it is contemplated that fusion of at least one cytoplasmic costimulatory motif, costimulatory domain, or costimulatory polypeptide region e.g. of a tumor necrosis factor receptor superfamily protein comprising CD40, CD40L, CD27, HVEM, GITR, CD30, 0X40, and / or LTBR, an immunoglobulin superfamily (IgSF) protein, a Toll-like-receptor, and / or an IL6-receptor family protein to a chimeric CD 95 receptor that comprises the CD 95 ligand binding domain is able to act like a “switch” receptor, when the T-cell further comprises an engineered T-cell receptor, by turning negative signals into positive signals, thereby enhancing cytotoxicity of a T-cell in presence of tumor cells that express the ligand FASL. Engineered T-cells expressing the chimeric CD 95 receptor polypeptides together with an engineered T-cell receptor as provided herein exhibit an improved killing activity compared to control samples in presence of FASL.

[0196] 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.

[0197] 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 eachindividual publication was specifically and individually indicated to be incorporated by reference.

[0198] 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.An isolated T-cell, wherein the T-cell expresses a chimeric CD 95 receptor comprising a polypeptide, wherein said polypeptide comprises at least one CD 95 polypeptide region having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a human CD 95 receptor as set forth in SEQ ID No. 1 , wherein said human CD 95 polypeptide region comprises a CD 95 extracellular ligand binding domain; further wherein said polypeptide comprises at least one non-CD 95 co-stimulatory cytoplasmic polypeptide domain, region or motif of a tumor necrosis factor receptor superfamily protein comprising CD40, CD40L, CD27, HVEM, GITR, CD30, 0X40, and / or LTBR, an immunoglobulin superfamily (IgSF) protein, a Toll-like-receptor, and / or an I L6-receptor family protein; wherein the T-cell further expresses an engineered T-cell receptor.

2. An isolated T-cell, wherein the T-cell expresses a chimeric CD 95 receptor comprising a polypeptide, wherein said polypeptide comprises at least one CD 95 polypeptide region comprising a CD 95 extracellular ligand binding domain; further wherein said polypeptide comprises at least one non-CD 95 co-stimulatory cytoplasmic polypeptide domain, region or motif of a tumor necrosis factor receptor superfamily protein comprising CD40, CD40L, CD27, HVEM, GITR, CD30, 0X40, and / or LTBR, an immunoglobulin superfamily (IgSF) protein, a Toll-like-receptor, and / or an I L6-receptor family protein; wherein the T-cell further expresses an engineered T-cell receptor.

3. The T-cell according to claim 1 or 2, wherein the extracellular ligand binding domain of the chimeric CD 95 receptor is functional in binding FAS-ligand (CD95L) or any other protein / polypeptide having the ability of binding to the wildtype CD 95 receptor ligand binding domain.

4. The T-cell according to any one of claims 1 to 3, wherein said polypeptide is a singlechain polypeptide.

5. The T-cell according to any one of claims 1-4, wherein the CD 95 polypeptide region further comprises at least one, or at least two, or at least three CD 95 cysteine-rich domains (CRDs).

6. The T-cell of any one of the foregoing claims, wherein the at least one CD 95 polypeptide region further comprises an extracellular N-terminal PLAD region.

7. The T-cell of any one of the foregoing claims, wherein the at least one CD 95 polypeptide region further comprises a CD95 homotypic interaction domain.

8. The T-cell according to any one of the foregoing claims, wherein the at least oneCD 95 polypeptide region comprises a CD-95 transmembrane region.

9. The T-cell according to any one of the foregoing claims, wherein said CD 95 polypeptide region comprises a complete CD 95 extracellular domain.

10. The T-cell according to claim 9, wherein the CD 95 polypeptide region comprises the complete CD 95 extracellular domain and a CD 95 transmembrane domain.

11. The T-cell according to any one of claims 1-10, wherein the CD 95 polypeptide region comprises at least one linker region.

12. The T-cell according to any one of the foregoing claims, wherein the polypeptide comprises a complete cytoplasmic domain of said tumor necrosis factor receptor superfamily protein, an immunoglobulin superfamily (IgSF) protein, a Toll-like- receptor, and / or an I L6-receptor family protein.

13. The T-cell according to any one of the foregoing claims, wherein said at least one cytoplasmic polypeptide domain or cytoplasmic polypeptide motif of an immunoglobulin superfamily (IgSF) protein, a Toll-like-receptor, and / or an IL6- receptor family protein, is a cytoplasmic polypeptide domain or cytoplasmicpolypeptide motif selected from a group consisting of ICOS, CD28, TLR2, TLR4, and IL6R subunit beta.

14. The T-cell according to any one of the foregoing claims, wherein the polypeptide is having an amino acid sequence with at least 85%, optionally with at least 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 sequences selected from a group consisting of SEQ ID No.: 12, 15, 17, 19, 21 , 23, 25, 27, 29, and 31.

15. The T-cell according to any one of the foregoing claims, wherein the at least one CD 95polypeptide 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% sequence identity with the functional polypeptide domain or a functional polypeptide motif of a human CD95 receptor (Seq ID No. 1).

16. The T-cell of any one of the foregoing claims, having an enhanced cytotoxicity.

17. The T-cell of any one of the foregoing claims, wherein the T-cell is having an increased resistance to CD95L expressing cancer cells.

18. The T-cell according to any one of the foregoing claims, wherein the T-cell further expresses a CD8 Co-receptor.

19. The T-cell according to claim 18, wherein the T-cell is a CD4 T-cell.

20. The T-cell according to claim 18 or 19, wherein the CD8 Co-receptor is a wildtype CD8 Co-receptor such as CD8a and CD8p Co-receptor.

21. The T-cell according to claim 18 or 19, wherein the CD8 Co-receptor is a chimeric CD8 Co-receptor.

22. The T-cell according to claim 21 , wherein the chimeric CD8 Co-receptor is having an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID No.50.

23. The T-cell according to any one of claims 1-22, wherein the engineered 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.

24. A vector comprising a nucleic acid encoding for a chimeric CD 95 receptor comprising a polypeptide, wherein said polypeptide comprises at least one CD 95 polypeptide region having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a human CD 95 receptor as set forth in SEQ ID No. 1 , wherein said human CD 95 polypeptide region comprises a CD 95 extracellular ligand binding domain; further wherein said polypeptide comprises at least one non-CD 95 co-stimulatory cytoplasmic polypeptide domain, region or motif of a tumor necrosis factor receptor superfamily protein comprising CD40, CD40L, CD27, HVEM, GITR, CD30, 0X40, and / or LTBR, an immunoglobulin superfamily (IgSF) protein, a Toll-like-receptor, and / or an I L6-receptor family protein; wherein the vector further comprises a nucleic acid encoding for an engineered T- cell receptor.

25. A vector comprising a nucleic acid encoding for a chimeric CD 95 receptor comprising a polypeptide, wherein said polypeptide comprises at least one CD 95 polypeptide region comprising a CD 95 extracellular ligand binding domain; further wherein said polypeptide comprises at least one non-CD 95 co-stimulatory cytoplasmic polypeptide domain, region or motif of a tumor necrosis factor receptor superfamily protein comprising CD40, CD40L, CD27, HVEM, GITR, CD30, 0X40,and / or LTBR, an immunoglobulin superfamily (IgSF) protein, a Toll-like-receptor, and / or an I L6-receptor family protein; wherein the vector further comprises a nucleic acid encoding for an engineered T- cell receptor.

26. The vector of any one of claims 24 or 25, wherein the vector is a viral vector or a non-viral vector.

27. The vector of claim 26, wherein the viral vector is selected from adenoviruses, poxviruses, alphaviruses, arenaviruses, flaviruses, rhabdoviruses, retroviruses, lentiviruses, herpesviruses, paramyxoviruses, picornaviruses, and combinations thereof.

28. The vector of any one of claims 24-27, wherein the engineered T cell receptor comprises a TCR a chain and a TCR p chain.

29. The vector according to claim 28, wherein the T cell receptor is a recombinant T cell receptor.

30. The vector according to any one of claims 24-29, wherein the vector further comprises a nucleic acid encoding for a CD8 Co-receptor, such as a wildtype or an engineered CD8 Co-receptor.31 . The vector according to claim 30, wherein the nucleic acid encodes a wildtype CD8 Co-receptor such as a CD8a and a CD8p Co-receptor, or wherein the nucleic acid encodes a chimeric CD8 Co-receptor.

32. The vector according to claim 31 , wherein the nucleic acid encodes for a chimeric CD8 Co-receptor, and further wherein said receptor is having at least 85%, optionally at least 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 SEQ ID No. 50.

33. An isolated T-cell, wherein the vector of any one of claims 24-32 has been introduced in said T-cell.

34. The T-cell of any one of claims 1-23 or 33, wherein the cell is a αβ T-cell, yδ T-cell, and / or a natural killer T-cell.

35. The T-cell of claim 34; wherein the αβ T-cell is a CD4 T-cell, or wherein the αβ T- cell is a CD8 T-cell, or wherein the yδ T-cell is a V / 9V82+ T-cell, or wherein the yδ T-cell comprises a V81 T-cell.

36. The T-cell according to any one of claims 1-23 or 33-35, wherein the T-cell is derived from an induced pluripotent stem cell (iPSCs).

37. A kit comprising means to prepare the T-cell according to any one of claims 1-23 or 33-36.

38. A pharmaceutical composition comprising the T-cell of any one of claims 1-23 or 33-36.

39. The pharmaceutical composition of claim 38, wherein the composition further comprises an adjuvant, excipient, buffer, diluent, carrier, stabilizer or combination thereof.

40. 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 24 - 32 into the T-cell, and expanding the T-cells.

41. The method according to claim 40, comprising transforming, transfecting or transducing the isolated T-cells with the vector.

42. The pharmaceutical composition according to claim 38 or 39, comprising T-cells expressing the chimeric CD 95 receptor and the engineered T-cell receptor.

43. The pharmaceutical composition according to claim 42, further comprising CD4 cells expressing said chimeric CD 95 receptor and further expressing a recombinant CD8 Co-receptor.

44. The pharmaceutical composition according to claim 43, wherein the CD8 Coreceptor is a chimeric CD8 Co-receptor.

45. A method for treating a patient having a disease, comprising administering to the patient the composition of claims 38, 39, 42, 43 or 44.

46. A method for treating a patient having a disease, comprising introducing in vivo the vector according to any one of claims 24-32 into a T-cell of the patient.

47. The method according to claim 46, wherein the vector is DNA or an mRNA.

48. The method according to claim 46, wherein the vector is a non-replicating viral vector.

49. The method according to claim 46 or 47, wherein the vector is mRNA, and wherein the mRNA is introduced into the T-cell of the patient using nanoparticles.

50. The method according to any one of claims 45-49, wherein the disease is a cancer or an autoimmune disease.

51. The method according to claim 50, 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.

52. The method according to claim 50 or 51 , wherein cancer cells express FasL.

53. A method for increasing cytotoxicity of a T-cell in adoptive cell therapy, comprising introducing a vector according to any one of claims 24-32 into a T-cell.

54. The method according to claim 53, wherein the vector encodes for a CD8 Coreceptor, such as a human wildtype CD8 Co-receptor or a chimeric CD8 Coreceptor.

55. The method according to claim 53 or 54, wherein the T-cell receptor is a recombinant T-cell receptor that specifically binds a tumor specific antigen.

56. A chimeric human CD 95 receptor; comprising a polypeptide, wherein said polypeptide comprises at least one CD 95 polypeptide region having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a human CD 95 receptor as set forth in SEQ ID No. 1 , wherein said human CD 95 polypeptide region comprises a CD 95 extracellular ligand binding domain; further wherein said polypeptide comprises at least one non-CD 95 co-stimulatory cytoplasmic polypeptide domain, region or motif, wherein said at least one cytoplasmic polypeptide domain or cytoplasmic polypeptide motif is a cytoplasmic polypeptide domain or cytoplasmic polypeptide motif selected from a group consisting of CD40L, CD2, TLR2, TLR4, and IL6R subunit beta.

57. A chimeric human CD 95 receptor; comprising a polypeptide, wherein said polypeptide comprises at least one CD 95 polypeptide region comprising a CD 95 extracellular ligand binding domain; further wherein said polypeptide comprises at least one non-CD 95 co-stimulatory cytoplasmic polypeptide domain, region or motif, wherein said at least one cytoplasmic polypeptide domain or cytoplasmic polypeptide motif is a cytoplasmic polypeptide domain or cytoplasmic polypeptide motif selected from a group consisting of CD40L, CD2, TLR2, TLR4, and IL6R subunit beta.

58. The chimeric CD 95 receptor of any one of claims 56 or 57, wherein the extracellular ligand binding domain is functional in binding FAS-ligand.

59. The chimeric CD 95 receptor of claim 56, 57 or 58, wherein said polypeptide is a single-chain polypeptide.

60. The chimeric CD 95 receptor according to any one of claims 56-59, wherein the CD 95 polypeptide region further comprises at least one, or at least two, or at least three CD 95 cysteine-rich domains (CRDs).61 . The chimeric CD 95 receptor of any one of claims 56-60, wherein the at least one CD 95 polypeptide region further comprises an extracellular N-terminal PLAD region.

62. The chimeric CD 95 receptor of any one of claims 56-61 , wherein the at least one CD 95 polypeptide region further comprises a CD 95 homotypic interaction domain63. The chimeric CD 95 receptor according to any one of claims 56-62, wherein the at least one CD 95 polypeptide region comprises a CD 95 transmembrane region.

64. The chimeric CD 95 receptor according to any one of claims 56-63, wherein said CD 95 polypeptide region comprises a complete CD 95 extracellular domain.

65. The chimeric CD 95 receptor according to claim 64, wherein the CD 95 polypeptide region comprises the complete CD 95 extracellular domain and a CD 95 transmembrane domain.

66. The chimeric CD 95 receptor according to any one of claims 56-65, wherein the CD 95 polypeptide region comprises at least one linker region.

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