A chimeric human CD 95 switch receptor, t-cells expressing the CD 95 switch receptor, vectors with nucleic acids encoding for the CD 95 receptor, kits for preparing the t-cells, as well as corresponding pharmaceutical compositions and methods for treating a patient having a disease and for increasing cytotoxicity of a t-cell in adoptive cell therapy

Abstract

The present invention inter alia relates to a chimeric human CD 95 receptor; comprising a polypeptide, wherein said polypeptide comprises at least one CD 95 polypeptide region comprising or consisting of a polypeptide domain, a polypeptide region or a polypeptide motif of a human CD 95 receptor as set forth in SEQ ID No. 1, or 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 CD 95 polypeptide region comprises a CD 95 extracellular ligand binding domain, further wherein said polypeptide comprises at least two non-CD 95 cytoplasmic co-stimulatory polypeptide domains, regions or motifs, wherein a first non-CD 95 co-stimulatory region comprises a cytoplasmic costimulatory region of OX40, and wherein a second non-CD 95 co-stimulatory region comprises a cytoplasmic costimulatory region of CD40. The present invention also relates to T-cells expressing said chimeric CD 95 receptor, vectors comprising nucleic acids encoding for the CD 95 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

A CHIMERIC HUMAN CD 95 SWITCH RECEPTOR, T-CELLS EXPRESSING THE CD 95 SWITCH RECEPTOR, VECTORS WITH NUCLEIC ACIDS ENCODING FOR THE CD 95 RECEPTOR, KITS FOR PREPARING THE T-CELLS, AS WELL AS CORRESPONDING PHARMACEUTICAL COMPOSITIONS AND METHODS FOR TREATING A PATIENT HAVING A DISEASE AND FOR INCREASING CYTOTOXICITY OF A T-CELL IN ADOPTIVE CELL THERAPYREFERENCE TO SEQUENCE LISTING

[0001] This application contains a sequence listing in a computer readable form. The entire contents of the XML file is incorporated herein by reference.CROSS-REFERENCES

[0002] The present application claims the benefit of priority of the European Patent Application No. 25156082.7, filed 5 February 2025, the content of which is hereby incorporated by reference in its entirety for all purposes.FIELD OF THE INVENTIONThe present invention relates to a chimeric human CD 95 receptor, comprising a polypeptide, wherein said polypeptide comprises at least one CD 95 polypeptide region comprising or consisting of a polypeptide domain, a polypeptide region or a polypeptide motif of a human CD 95 receptor as set forth in SEQ I D No 1 , or 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 CD 95 polypeptide region comprises a CD 95 extracellular ligand binding domain, and further wherein the chimeric receptor comprises a non-CD 95 cytoplasmic co-stimulatory polypeptide domain, region or motif.. The invention further relates to T-cells expressing the chimeric CD 95 receptor, to nucleic acids encoding the chimeric CD 95 receptor, to vectors 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 tomethods for treating a patient having a disease comprising administering the pharmaceutical composition, and / or for increasing cytotoxicity of a T-cell in adoptive cell therapy, comprising introducing the vector into the T-cell.BACKGROUND OF THE INVENTION

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

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

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

[0006] In addition to CD28, during the last years, many other costimulatory molecules have been identified. Most co-signaling molecules are members of the immunoglobulin superfamily (IgSF) and tumor necrosis factor receptor superfamily (TNFRSF). For example, TNFRSF co-signaling receptors with co-stimulatory function include HVEM (herpesvirus entry mediator), death receptor 3 (DR3; also known as TNFRSF25), CD40 (also known as TNFRSF5) and lymphotoxin-p receptor (LTBR; also known as TNFRSF3) [Chen and Flies, 2013], Furthermore, all receptors of the type-V, or divergent, family — including 4-1 BB (also known as CD137 or TNFRSF9), 0X40 (also known as TNFRSF4), 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 IgSF, for example, PD1 andTIGIT 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 inhibitory signals 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 chimeric CD 95 receptors, the (isolated) T-cells, the vectors, the pharmaceutical compositions, the kits, and the methods having the features of the respective independent claims.

[0012] In a first aspect, the invention provides a chimeric human CD 95 receptor; comprising a polypeptide, wherein said polypeptide comprises at least one CD 95 polypeptide region comprising or consisting of a polypeptide domain, a polypeptide region or a polypeptide motif of a human CD 95 receptor as set forth in SEQ ID No 1, or 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 CD 95 polypeptide region comprises a CD 95 extracellular ligand binding domain; further wherein said polypeptide comprises at least two non-CD 95 cytoplasmic co-stimulatory polypeptide domains, regions or motifs, wherein a first non-CD 95 co-stimulatory region comprises a cytoplasmic costimulatory region of 0X40, and wherein a second non-CD 95 co-stimulatory region comprises a cytoplasmic costimulatory region of CD40.

[0013] Surprisingly, the inventors have found that the specific combination of two non-CD 95 cytoplasmic co-stimulatory polypeptide domains, regions or motifs in a CD 95 switch receptor, wherein a first non-CD 95 co-stimulatory region comprises a cytoplasmiccostimulatory region of 0X40, and wherein a second non-CD 95 co-stimulatory region comprises a cytoplasmic costimulatory region of CD40, synergistically improve desirable effects in T-cells.

[0014] In a second aspect, the invention provides a nucleic acid encoding for the CD 95 switch receptor as herein described.

[0015] 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 as herein described.

[0016] In some embodiment, in addition to comprising the nucleic acid sequence encoding for the chimeric CD 95 receptor, the vector may comprise a nucleic acid sequence encoding for an engineered T-cell receptor, and optionally the vector may further comprise a nucleic acid encoding for a CD8 Co-receptor, such as a wildtype CD8 Coreceptor or a chimeric CD8 Co-receptor.

[0017] In a fourth aspect, the invention provides an isolated T-cell, wherein the T-cell expresses a chimeric CD 95 receptor as herein described. Optionally, the T-cells may further express an engineered T-cell receptor.

[0018] The inventors have found that the T-cells comprising both an engineered T-cell receptor and a chimeric CD 95 receptor including both a costimulatory domain of CD40 and a costimulatory domain of 0X40 are able to turn negative signals e.g. present in a tumor microenvironment into positive signals for T-cell activation. Advantageously, the T-cells as 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.

[0019] 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 co-stimulates the T-cell, thereby enhancing its cytotoxic effect.

[0020] For example, overexpression of a chimeric human CD 95 receptor as herein described in the T-cells as herein provided, e.g. alongside an engineered transgenic op-T-cell receptor TCR and, in some embodiments, for example, alongside a CD8 Coreceptor, may offer several advantages and expands the therapeutic potential of this approach.

[0021] The optional, additional provision of the CD8 Co-receptor together with the chimeric 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.

[0022] 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.

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

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

[0025] In a seventh aspect, the invention provides an isolated T-cell, the T-cell being treated, such as transfected (e.g. electroporated), transduced or transformed to express the chimeric CD 95 receptor as herein described, optionally together with the engineered T-cell receptor.

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

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

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

[0029] In an 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.

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

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

[0032] In a fourteenth aspect, the invention provides a method for increasing cytotoxicity of a T-cell in adoptive cell therapy, comprising - introducing a vector into the T-cell, wherein the vector comprises a nucleic acid encoding for a chimeric CD 95-receptor as herein described, and further encoding an engineered T-cell receptor.

[0033] All aspects of the invention provide the above described advantages and improvements related to the provision of 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

[0034] 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:

[0035] Fig. 1 shows a graphical representation of results from a flow cytometric analysis of T-cells transduced with chimeric CD 95 receptor polypeptides.

[0036] Fig. 2 shows a graphical representation of results from an in-vitro T-cell killing assay of T-cells transduced with an engineered T-cell receptor and a chimeric CD 95 receptor polypeptide in FasL expressing HeLa cells.

[0037] Fig. 3 shows a graphical representation of results from an in-vitro T-cell killing assay of T-cells transduced with an engineered T-cell receptor and chimeric CD 95 receptor polypeptides in FasL expressing NCIH 2030 cells.

[0038] Fig. 4 shows a graphical representation of results from an in-vitro T-cell killing assay of T-cells transduced with an engineered T-cell receptor, a chimeric CD8 Coreceptor and a chimeric CD 95 receptor polypeptide in FasL expressingNCIH 2030 cells.

[0039] Fig. 5 shows a graphical representation of results of an experimental analysis of the expression on cell surface of specific constructs introduced into T-cells.

[0040] Fig. 6 shows a graphical representation of results from an in-vitro T-cell killing assay of T-cells transduced with chimeric CD 95 receptors according to the invention together with a CD8 Coreceptor and an engineered T-cell receptor in NCI-H1073 cells.

[0041] Fig. 7 shows a graphical representation of results from a cytokine secretion assay with T-cells transduced with chimeric CD 95 receptors according to the invention together with together with a CD8 Coreceptor and an engineered T-cell receptor, wherein Fig. 7A shows the secretion level of interleukin 2, Fig. 7B shows the secretion level of interferon , Fig. 7C shows the secretion level of Granzyme A, Fig. 7D shows the secretion level of Granzyme B, and Fig. 7E shows the secretion level of Perforin.DETAILED DESCRIPTION OF THE INVENTION

[0042] As explained above, in a first aspect the invention is directed to a chimeric human CD 95 receptor; comprising a polypeptide, wherein said polypeptide comprises at least one CD 95 polypeptide region comprising or consisting of a polypeptide domain, a polypeptide region or a polypeptide motif of a human CD 95 receptor as set forth in SEQ ID No 1, or 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 CD 95 polypeptide region comprises a CD 95 extracellular ligand binding domain; further wherein said polypeptide comprises at least two non-CD 95 cytoplasmic co-stimulatory polypeptide domains, regions or motifs, wherein a first non-CD 95 co-stimulatory region comprises a cytoplasmic costimulatory region of 0X40, and wherein a second non-CD 95 co-stimulatory region comprises a cytoplasmic costimulatory region of CD40.

[0043] In accordance with an embodiment, the extracellular ligand binding domain of the chimeric CD 95 receptor 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. In the art, numerous suitable assays for testing FAS-ligand binding functionality have been described. Any suitable method may be selected.

[0044] In some embodiments, the polypeptide may be a single-chain polypeptide.

[0045] For example, the chimeric CD 95 receptor may have 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 a CD 95 extracellular ligandbinding domain; further wherein said polypeptide comprises a first non-CD 95 costimulatory region of 0X40, and a second non-CD 95 cytoplasmic costimulatory region of CD40. 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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 a first non-CD 95 co-stimulatory region comprising a cytoplasmic costimulatory region of 0X40 in combination with a second non-CD 95 co-stimulatory region comprising a cytoplasmic costimulatory region of CD40, is - in an even synergistically increased matter - 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 least one non-CD 95-derived co-stimulatory cytoplasmic polypeptide domain as herein provided enables the T-cell comprising the chimeric CD 95 receptor and optionally the engineered T-cell receptor, or e.g. a CAR, to bypass the inhibitory effects of FASL expressed by the tumor microenvironment, thus creating resistance to tumor-mediated immune suppression. Secondly, the chimeric CD95 receptors as herein provided 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 the co-stimulatory domain as herein described 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 as herein provided 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.

[0050] According to an embodiment, the extracellular ligand binding domain of the chimeric CD 95 receptor 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.

[0051] 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.

[0052] 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 %, 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 CD 95 receptor (e.g. Seq ID No. 1).

[0053] 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 atleast 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.

[0054] 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.

[0055] The chimeric CD 95 receptor polypeptide may be a single-chain polypeptide.

[0056] 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 regions of 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 compriseCRD 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).

[0057] According to an embodiment, the at least one CD 95 (-derived) polypeptide region may further comprise 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.

[0058] 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.

[0059] According to another embodiment, the at least one CD 95 (-derived) polypeptide region may comprise a CD-95-derived transmembrane region. The transmembrane 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 tothe 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.

[0060] Thus, it is contemplated that the chimeric CD 95 receptor polypeptide as herein provided may comprise, in addition to comprising the CD 95 (-derived) ligand binding domain, and in addition to comprising the at least two cytoplasmic costimulatory domains of CD40, and 0X40 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 an 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”.

[0061] According to an embodiment, the CD 95 (-derived) polypeptide region may comprise a complete wildtype CD 95 receptor extracellular domain. For example, the expression “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 aminoacid substitutions that maintain the functional activity of the wildtype CD 95 extracellular domain are envisaged.

[0062] 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.

[0063] 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.

[0064] 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).

[0065] 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.

[0066] 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 in SEQ 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.

[0067] 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.

[0068] Turning now to the co-stimulatory domain, according to the present invention, the chimeric CD 95 receptor polypeptide as herein provided comprises at least two non-CD 95 cytoplasmic co-stimulatory polypeptide domains, regions or motifs, wherein a first non-CD 95 co-stimulatory region comprises a cytoplasmic costimulatory region of 0X40, and wherein a second non-CD 95 co-stimulatory region comprises a cytoplasmic costimulatory region of CD40. The authors have found for the first time that the specific combination of co-stimulatory regions / motifs as herein described 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. It is thus envisaged that the terms “cytoplasmic costimulatory region of 0X40” and “cytoplasmic costimulatory region of CD40” as used herein encompass wild-type costimulatory regions and variants (specifically, truncations) thereof.

[0069] According to an embodiment, the chimeric CD 95 receptor as herein provided may comprise e.g. the complete cytoplasmic (costimulatory) region of CD40, and / or the first non-CD 95 co-stimulatory region may comprise the complete cytoplasmic (costimulatory) region of 0X40.

[0070] According to some embodiments,, the at least one cytoplasmic costimulatory polypeptide domain, region or motif of CD40, and and / or of 0X40 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 therespective functional polypeptide domain or a functional polypeptide motif of a wildtype human CD40 and / orOX40, respectively.

[0071] According to an embodiment, the chimeric CD 95 receptor polypeptide may be able to enhance cytotoxicity of the T-cell as herein provided.

[0072] According to an embodiment, the chimeric CD 95 receptor polypeptide may be able to enhance activation, proliferation, and / or production of activating cytokines of / in the T-cell as herein provided.

[0073] According to an embodiment, the chimeric CD 95 receptor may be capable of increasing resistance of T-cells as herein provided to CD95L expressing cancer cells.

[0074] It is contemplated that the chimeric CD 95 receptor of the T-cell as herein provided may comprise a further 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.

[0075] As mentioned above, according to the present invention, the chimeric CD 95 receptor as herein provided comprises 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, region or motif of wildtype CD40 as included in the chimeric CD 95 receptor as herein provided may have one or more conservative amino acid substitutions relative to the amino acid sequence as set forth in SEQ ID No. 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.

[0076] According to an embodiment, the second non-CD 95 co-stimulatory region may comprise at least one TRAF binding motif (e.g. TRAF 6 binding motif and / or TRAF 1 / 2 / 3 binding motif) of CD40. TRAF6 binding motifs and TRAF 1 / 2 / 3 binding motifs have been described in the art (e.g. Park HH, Front. Immunol. 9:1999, 2018). There are also numerous suitable methods known in the art (such as e.g. yeast two hybrid assays or surface plasmon resonance assay) for determining binding of TRAF 6 and / or TRAF1, TRAF2 and / or TRAF3 to a specific binding motif. Thus, “CD40 TRAF 6 binding motif” as referred to herein may relate to any polypeptide region of CD40 which is functional in binding TRAF 6. “CD40 TRAF 1 / 2 / 3 binding motif” as referred to herein may relate to any polypeptide region of CD40 which is functional in binding TRAF 1, TRAF 2 and / or TRAF3.CD40 TRAF binding motif as referred to herein may relate to any polypeptide region of CD40 which is functional in binding TRAF 1, TRAF 2, and / or TRAF3, and / or TRAF 6.

[0077] According to an embodiment, the second non-CD 95 costimulatory region may comprise a CD40 TRAF 6 binding motif.

[0078] According to an embodiment, the second non-CD 95 co-stimulatory region may comprise or may consist of the polypeptide region of human CD40 as set forth in SEQ ID No. 56.

[0079] It is contemplated herein that the second non-CD 95 co-stimulatory region may comprise or may consists of the polypeptide region of human CD40 as set forth in SEQ ID No. 56, or that the second non-CD 95 co-stimulatory region may comprise or consist of a polypeptide region of human CD40 having 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. 56. For example, the second non-CD 95 co-stimulatory region may comprise or may consist of a polypeptide region of human CD40 that may have one or more conservative amino acid substitutions relative to the amino acid sequence as set forth in SEQ ID No.56.

[0080]

[0081] According to an embodiment, the second non-CD 95 co-stimulatory region may comprise a polypeptide region of CD40 including amino acid position 237 of SEQ ID No 54 (wildtype CD40) , and further wherein at said position, the second non-CD 95 co-stimulatory region may be mutated, wherein said mutation may consist of an exchange of an asparagine (N) to an aspartic acid (D). According to an embodiment, the second non-CD 95 co-stimulatory region may comprise a polypeptide region of CD40 including amino acid position 229 of SEQ ID No 54 (wildtype CD40) , and further wherein at said position, the second non-CD 95 co-stimulatory region may be mutated, wherein said mutation may consist of an exchange of a proline (P) to an alanine (A). In particular, all amino acid substitutions that maintain the functional activity of the “TRAF6” motif of wildtype CD40 are envisaged. For example, the “TRAF6” binding motif of the second non-CD 95 co-stimulatory 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% sequenceidentity with the amino acid sequence as set forth in SEQ ID NO: 56. Envisaged herein are also second non-CD 95 co-stimulatory regions that may comprise truncated variants of the polypeptide as set forth in SEQ ID NO: 56. For example, the second non-CD95 costimulatory region may comprise or consist of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive amino acids of the polypeptide sequence as set forth in SEQ ID NO: 56, which retain functionality in TRAF 6 binding.

[0082] According to an example, the chimeric CD 95 receptor 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 4-1BB 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. Q0701T 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 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.

[0083] According to an example, the chimeric CD 95 receptor 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 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 thatmaintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype ICOS are envisaged.

[0084] In examples 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.

[0085] According to an example, 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 comprising amino 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.

[0086] In examples 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.

[0087] As mentioned above, according to the present invention, the chimeric CD 95 receptor as herein provided further comprises at least one cytoplasmic polypeptide domain, region or motif of 0X40. For example, the first non-CD95 cytoplasmic costimulatory region of the chimeric CD 95 receptor 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 sequence236-277 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.

[0088] Thus, it is envisaged that the first non-CD 95 co-stimulatory region may comprise or may consists of the polypeptide region of human 0X40 as set forth in SEQ ID No. 20, or that the first non-CD 95 co-stimulatory region may comprise or consist of a polypeptide region of human 0X40 having 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. 20. For example, the first non-CD 95 co-stimulatory region may comprise or may consist of a polypeptide region of human 0X40 that may have one or more conservative amino acid substitutions relative to the amino acid sequence as set forth in SEQ ID No. 20.

[0089] According to an embodiment, the first non-CD 95 co-stimulatory region may comprise at least one TRAF binding motif of 0X40. TRAF binding motifs of 0X40 have been previously described in the art (e.g. Arch R.H. & Thompson, C.B; 1997). As mentioned above, suitable methods are known in the art (such as e.g. yeast two hybrid assays or surface plasmon resonance assay) for determining binding of TRAFs to a specific binding motif. Thus, “0X40 TRAF binding motif” as referred to herein may relate to any polypeptide region of 0X40 which is functional in binding at least one TRAF.

[0090] According to an embodiment, the first non-CD 95 co-stimulatory region may comprise at least one potential PI3K binding motif / potential ubiquitination site of 0X40. Potential PI3K binding motif / potential ubiquitination site of 0X40 have been previously described in the art (e.g. Croft, M. et al., Immunol Rev. 2009 May ; 229(1)).

[0091] According to an embodiment, the first non-CD 95 co-stimulatory region may comprise a polypeptide region of 0X40 including amino acid positions 266 to 268 of SEQ ID No 55. as set forth herein (human wildtype 0X40), and further wherein at said position, the second non-CD 95 co-stimulatory region is mutated, wherein said mutation consists of an exchange of the wildtype 0X40 amino acids EQA to TEP.

[0092] It is contemplated herein that the first non-CD 95 co-stimulatory region may comprise or may consists of the polypeptide region of human 0X40 as set forth in SEQ ID No. 57, or that the first non-CD 95 co-stimulatory region may comprise or consist of apolypeptide region of human 0X40 having 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. 57. For example, the first non-CD 95 co-stimulatory region may comprise or may consist of a polypeptide region of human 0X40 that may have one or more conservative amino acid substitutions relative to the amino acid sequence as set forth in SEQ ID No. 57.

[0093] According to some embodiments, the second co-stimulatory region (CD40) may be located closer to the C-terminal tail of the polypeptide than the first co-stimulatory region (0X40).

[0094] It is further envisaged that the cytoplasmic non-CD 95 co-stimulatory region may comprise a linker between the first and the second non-CD 95 co-stimulatory regions, optionally wherein said linker comprises or consists of a sequence as set forth in SEQ ID No.53.

[0095] According to an example, 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, the cytoplasmic 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.

[0096] In examples 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.

[0097] According to an example, the chimeric CD 95 receptor of the T-cell as herein provided may comprise at least one cytoplasmic polypeptide domain, region or motif ofCD40L. 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.

[0098] In examples 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.

[0099] According to an example, 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 Q9Y5U5 ■ 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.

[0100] In examples wherein a costimulatory region, motif or domain of GITR is included in the chimeric CD 95 receptor, for example, the polypeptide of the chimeric CD95 receptor may have an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID NO: 27.

[0101] According to an example, 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.

[0102] In examples 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.

[0103] According to an example, 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 thatmaintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype CD2 are envisaged.

[0104] In examples 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.

[0105] 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.

[0106] According to some embodiments, a chimeric CD 95 receptor as herein provided may e.g. comprise a polypeptide having an amino acid sequence with at least 85% or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% identity to the amino acids as set forth in any one of the SEQ ID No’s selected from the group consisting of SEQ ID No. 58 and 59. 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. 58 and 59

[0107] In another aspect, the invention provides an (isolated) nucleic acid comprising a nucleic acid sequence encoding for the chimeric CD 95 receptor as herein described.

[0108] In a further aspect, the invention provides a vector comprising the nucleic acid as herein provided. The nucleic acid may optionally further comprise a nuclear acid sequence encoding for an engineered T-cell receptor.

[0109] 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 inventionthus comprise DNA (such as dsDNA, ssDNA, cDNA), RNA (such as dsRNA, ssRNA, mRNA ivtRNA), combinations thereof or derivatives (such as RNA) thereof.

[0110] 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.

[0111] 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: 60 or 61.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] The vector may be a viral vector or a non-viral vector.

[0116] 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.

[0117] 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 theart, large numbers of suitable vectors are known to those of skill in the art and many are commercially available.

[0118] 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.

[0119] 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 human wildtype 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.

[0120] 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.

[0121] 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 CD8ap 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.

[0122] 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).

[0123] 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 Coreceptor (e.g. Seq ID No. 46).

[0124] 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).

[0125] 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%, 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 CD8p Co-receptor (e.g. Seq ID No. 47).

[0126] According to an embodiment, the chimeric human CD8 Co-receptor polypeptide is a single-chain polypeptide.

[0127] 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 domainregion, wherein the intracellular domain region comprises a palmitoylation motif region and a LCK binding site region.

[0128] 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 Coreceptor 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 Coreceptor 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.

[0129] For example, according to an embodiment, the chimeric CD8 Co-receptor may comprise a CD8a-derived transmembrane region.

[0130] 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.

[0131] 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.

[0132] The chimeric CD8 Co-receptor which - in some embodiments - is encoded by the vector of the present invention links an extra co-stimulatory domain to the human 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.

[0133] 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.

[0134] 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 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 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).

[0135] 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).

[0136] 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.

[0137] 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 optionally further encodes for an engineered T-cell receptor.

[0138] There is further provided an (isolated) T-cell, wherein the T-cell further expresses a chimeric human CD 95 receptor as herein described, and further wherein the T-cell may optionally express an engineered T-cell receptor or e.g. a CAR.

[0139] 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, and / or e.g. by mRNA based transient expression).

[0140] In accordance with a specific aspect, a T-cell may express the chimeric CD 95 receptor as herein described and the engineered T-cell receptor.

[0141] 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.

[0142] 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.

[0143] According to some embodiments, the vector and as herein described may have been introduced into the T-cell.

[0144] The (isolated) T-cells may be generated using various methods, including those recognized in the literature. For example, a polynucleotide encoding an expression cassette 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 ora 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, includingadenoviruses, 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.

[0145] 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.

[0146] According to an embodiment, the cell may be an p T-cell, y8 T-cell, and / or a natural killer T-cell.

[0147] For example, the ap T-cell may be a CD4 T-cell, or the ap T-cell may be a CD8 T-cell, or the y8 T-cell may comprise e.g. a Vy1 chain or a Vy2 chain, or may be e.g. a Vy9V82+ T-cell.

[0148] 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.

[0149] 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 ap T-cells or y8 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 and hematologic malignancies. For example, “hot” tumors or “cold” tumors may be treated by the T-cells herewith provided.

[0150] 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 HA2antigen, a mutated KRAS antigen, a mutated NRAS antigen, a mutated HRAS antigen, a mutated TP53 antigen, and an EGFR antigen.

[0151] 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.

[0152] According to an embodiment, The T-cells described herein may also be used to treat an infectious disease. The T-cells described herein may be used to treat an infectious disease, an infectious disease may be caused a virus. The T-cells described herein may be used to treat an immune disease, such as an autoimmune disease. The T-cells may be ap T-cells or 78 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.

[0153] In some embodiments, the T-cell may be derived from an induced pluripotent stem cell (iPSCs).

[0154] According to an embodiment, the T-as herein provided may have an enhanced cytotoxicity. For example, said enhanced cytotoxicity is relative to a T-cell that does not express said chimeric CD95 co-receptor.

[0155]

[0156] According to another aspect, it is herewith provided a kit comprising means to prepare the T-cells described above.

[0157] According to a further aspect, this invention relates to a pharmaceutical composition comprising the T-cell provided by the present invention.

[0158] It is herewith contemplated that the pharmaceutical composition may further comprise an adjuvant, excipient, buffer, diluent, carrier, stabilizer or combination thereof.

[0159] 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 optionally an engineered T-cell receptor or a CAR.

[0160] 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.

[0161] 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 administeredis 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).

[0162] Turning to a further aspect, a method for preparing a T-cell for immunotherapy is provided, comprisingisolating T-cells from a human subject,introducing the vector comprising a nucleic acid encoding for a chimeric CD 95 receptor as herein provided;and further optionally comprising a nucleic acid encoding for an engineered T-cell receptor or a CAR into the T-cell, and expanding the transduced T-cells.

[0163] For example, the method may comprise transforming, transfecting or transducing the isolated T-cells with the vector.

[0164] 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.

[0165] 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.

[0166] According to an embodiment, the nucleic acid may be a DNA or an mRNA.

[0167] For the in vivo introduction, the vector may be - for example - a nonreplicating viral vector.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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, the breast cancer may be, but is not limited to, ductal breast cancer, ductal-invasive breast cancer, invasive breast cancer, tubular breast cancer, medullary breast cancer or combinations thereof. In yet other illustrative examples, the gastric cancer may be gastric adenocarcinoma or squamous cell cancer. Turning to sarcoma cancer, the sarcoma cancer may be, but is not limited to, chondrosarcoma cancer, osteosarcoma cancer or combinations thereof. The adenoma cancer may include, but is also not limited to, gastric adenocarcinoma, pancreatic adenocarcinoma or combinations thereof.

[0172] 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 a chimeric CD 95 receptor as herein provided, and optionally further comprising a nucleic acid encoding for an engineered T-cell receptor or a CAR.

[0173] 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.

[0174] 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 PRAME antigen.

[0175] The invention will be further illustrated by the following non-limiting Experimental Examples.

[0176] Sequences as used herein are depicted in below Table 1.

[0177] Table 1. Sequences as used herein.SEQ ID Name SequenceNO.1 wildtype MLGIWTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNL human EGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFS CD 95 SKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCT receptor KCEHGI IKECTLTSNTKCKEEGSRSNLGWLCLLLLPIPLIVWVKRKEVQ KTCRKHRKENQGSHESPTLNPETVAINLSDVDLSKYITTIAGVMTLSQV KGFVRKNGVNEAKIDEIKNDNVQDTAEQKVQLLRNWHQLHGKKEAYDTL IKDLKKANLCTLAEKIQTI ILKDITSDSENSNFRNEIQSLV2 CRD 1 QNLEGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVdomain ofthe CD 95wildtypereceptor3 CRD 2 PCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVEINCTRTQNTKC domain ofthe CD 95wildtypereceptor4 CRD 3 RCKPNFFCNSTVCEHCDPCTKCEHGI IKECTLTSNTKCKdomain ofthe CD 95wildtypereceptor5 N-terminal RLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCPP PLAD GERKARDCTVNGDEPDCregion ofCD 95wildtypereceptorSEQ ID Name SequenceNO.6 CD95 CHKPCPPGERKARDCTVNGDEPDCwildtypehomotypicinteractiondomain7 transmem LGWLCLLLLPIPLIVWVbranedomain ofwildtypeCD 958 wildtype QVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCPPGERKARDCT CD 95 VNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVEINCTRTQ receptor NTKCRCKPNFFCNSTVCEHCDPCTKCEHGI IKECTLTSNTKCKEEGSRS extracellul Nar domain9 Amino GGGSacid linker10 Amino GGGGSacid linker11 wildtype KKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQE human DGKESRISVQERQCD40cytoplasmic domain12 ChimericCD 95receptorpl_323 MLGIWTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNL andpl_1190 EGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFS SKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCT KCEHGI IKECTLTSNTKCKEEGSRSNLGWLCLLLLPIPLIVWVKKVAKK PTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESR ISVQERQ13 wildtype RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS humanCD28cytoplasmic domain14 wildtype KRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL human 4- 1BBcytoplasmic domainSEQ ID Name SequenceNO.15 ChimericCD 95receptor MLGIWTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNL pl_320 EGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFS SKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCT KCEHGI IKECTLTSNTKCKEEGSRSNLGWLCLLLLPIPLIVWVKRGRKK LLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL16 wildtype CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLhumanICOScytoplasmic domain17 ChimericCD 95receptor MLGIWTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNL pl_318 EGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFS SKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCT KCEHGI IKECTLTSNTKCKEEGSRSNLGWLCLLLLPIPLIVWVCWLTKK KYSSTVHDPNGEYMFMRAVNTAKKSRLTDVTV18 wildtype CVKRRKPRGDWKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEET human IPSFTGRSPNHHVEMcytoplasmic domain19 ChimericCD 95receptor MLGIWTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNL pl_325 EGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFS SKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCT KCEHGI IKECTLTSNTKCKEEGSRSNLGWLCLLLLPIPLIVWVCVKRRK PRGDWKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTG RSPNH20 wildtype ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI human0X40cytoplasmic domainSEQ ID Name SequenceNO.21 ChimericCD 95receptor MLGIWTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNL pl_321 EGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFS SKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCT KCEHGI IKECTLTSNTKCKEEGSRSNLGWLCLLLLPIPLIVWVALYLLR RDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI22 wildtype QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP humanCD27cytoplasmic domain23 ChimericCD 95receptor MLGIWTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNL pl_324 EGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFS SKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCT KCEHGI IKECTLTSNTKCKEEGSRSNLGWLCLLLLPIPLIVWVQRRKYR SNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP24 wildtype MIETYNQTSPRSAATGLPISMKhumanCD40Lcytoplasmic domain25 ChimericCD 95receptor MLGIWTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNL pl_316 EGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFS SKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCT KCEHGI IKECTLTSNTKCKEEGSRSNLGWLCLLLLPIPLIVWVMIETYN QTSPRSAATGLPISMK26 wildtype QLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEE human KGRLGDLWVGITRcytoplasmic domain27 ChimericCD 95receptor MLGIWTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNL pl_330EGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSEQ ID Name SequenceNO.SKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCT KCEHGI IKECTLTSNTKCKEEGSRSNLGWLCLLLLPIPLIVWVQLGLHI WQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGD LWV28 wildtype CHRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTE human PVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPR CD30 VSTEHTNNKIEKI YIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELE cytoplasm ADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK ic domain29 ChimericCD 95receptor MLGIWTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNL pl_331 EGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFS SKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCT KCEHGI IKECTLTSNTKCKEEGSRSNLGWLCLLLLPIPLIVWVCHRRAC RKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEER GLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHT NNKIEKI YIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPH YPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK30 wildtype KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPP human PPGHRSQAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRV CD2 QPKPPHGAAENSLSPSSNcytoplasmic domain31 ChimericCD 95receptor MLGIWTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNL pl_322 EGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFS SKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCT KCEHGI IKECTLTSNTKCKEEGSRSNLGWLCLLLLPIPLIVWVKRKKQR SRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRS QAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPH GAAENSLSPSSN32 wildtype KSHPSLCRKLGSLLKRRPQGEGPNPVAGSWEPPKAHPYFPDLVQPLLPI human SGDVSPVSTGLPAAPVLEAGVPQQQSPLDLTREPQLEPGEQSQVAHGTN LTBR GIHVTGGSMTITGNIYIYNGPVLGGPPGPGDLPATPEPPYPIPEEGDPGcytoplasm PPGLSTPHQEDGKAWHLAETEHCGATPSNRGPRNQFITHDic domainSEQ ID Name SequenceNO.33 wildtype HRFHGLWYMKMMWAWLQAKRKPRKAPSRNICYDAFVSYSERDAYWVENL human MVQELENFNPPFKLCLHKRDFIPGKWI IDNI IDSIEKSHKTVFVLSENF TLR2 VKSEWCKYELDFSHFRLFDENNDAAILILLEPIEKKAIPQRFCKLRKIM cytoplasm NTKTYLEWPMDEAQREGFWVNLRAAIKSic domain34 wildtype KEYEHLMLLAGCIKYGRGENI YDAEVI YSSQDEDWVRNELVKNLEEGVP human PFQLCLHYRDFIPGVAIAANI IHEGFHKSRKVIVWSQHFIQSRWCIFE TLR4 YEIAQTWQFLSSRAGI IFIVLQKVEKTLLRQQVELYRLLSRNTYLEWED cytoplasm SVLGRHI FWRRLRKALLDGKSWNPEGTVGTGCNWQEATS I ic domain35 wildtype NKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTD human VSWEIEANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRP IL6R SISSSDENESSQNTSSTVQYSTWHSGYRHQVPSVQVFSRSESTQPLLD subunit SEERPEDLQLVDHVDGGDGILPRQQYFKQNCSQHESSPDISHFERSKQV beta SSVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAFGPGTEGQVE cytoplasm RFETVGMEAATDEGMPKSYLPQTVRQGGYMPQic domain36 Nucleicacidsequence ATGCTGGGCATCTGGACCCTCCTACCTCTGGTTCTTACGTCTGTTGCTA encoding GAT T AT C G T C C AAAAG T G T T AAT G C C C AAG T GAG T GAG AT C AAC T C C AA forchimeric GGGAT T GGAAT T GAGGAAGAC T GT TAG TACAGT T GAGAC T CAGAAC T T G CD 95 GAAGGCCTGCATCATGATGGCCAATTCTGCCATAAGCCCTGTCCTCCAG receptorGT GAAAGGAAAGC TAGGGAC T GCACAGT CAAT GGGGAT GAAC GAGAC T Gin pl_323and in CGTGCCCTGCCAAGAAGGGAAGGAGTACACAGACAAAGCCCATTTTTCT pl_1194 T C CAAAT GCAGAAGAT G T AGAT T G T G T GAT GAAGGACAT GGC T T AGAAG T G GAAAT AAAC T G GAG C C G GAG C C AGAAT AC C AAG T G C AGAT G T AAAC C AAACTTTTTTTGTAACTCTACTGTATGTGAACACTGTGACCCTTGCACC AAAT GT GAACAT GGAAT CAT CAAGGAAT GCACAC T CACCAGCAACACCA AGTGCAAAGAGGAAGGATCCAGATCTAACTTGGGGTGGCTTTGTCTTCT TCTTTTGCCAATTCCACTAATTGTTTGGGTGAAAAAGGTGGCCAAGAAG CCAACCAATAAGGCCCCCCACCCCAAGCAGGAACCCCAGGAGATCAATT TTCCCGACGATCTTCCTGGCTCCAACACTGCTGCTCCAGTGCAGGAGAC TTTACATGGATGCCAACCGGTCACCCAGGAGGATGGCAAAGAGAGTCGC AT C T CAGT GCAGGAGAGACAG37 Nucleicacidsequence ATGCTGGGCATCTGGACCCTCCTACCTCTGGTTCTTACGTCTGTTGCTA encoding GAT TAT C G T C C AAAAG T G T T AAT G C C C AAG T GAC T GAC AT C AAC T C CAA forGGGAT T GGAAT T GAGGAAGAC T GT TAG TACAGT T GAGAC T CAGAAC T T GchimericSEQ ID Name SequenceNO.CD 95 GAAGGCCTGCATCATGATGGCCAATTCTGCCATAAGCCCTGTCCTCCAG receptorin pl_316 GT GAAAGGAAAGC TAGGGAC T GCACAGT CAAT GGGGAT GAACCAGAC T G and in CGTGCCCTGCCAAGAAGGGAAGGAGTACACAGACAAAGCCCATTTTTCT pl_1190T C CAAAT GCAGAAGAT G T AGAT T G T G T GAT GAAGGACAT GGC T T AGAAG T G GAAAT AAAC T G GAG C C G GAG C C AGAAT AC C AAG T G C AGAT G T AAAC C AAACTTTTTTTGTAACTCTACTGTATGTGAACACTGTGACCCTTGCACC AAAT GT GAACAT GGAAT CAT CAAGGAAT GCACAC T CACCAGCAACACCA AGTGCAAAGAGGAAGGATCCAGATCTAACTTGGGGTGGCTTTGTCTTCT TCTTTTGCCAATTCCACTAATTGTTTGGGTGATGATCGAAACATACAAC CAAACTTCTCCCCGATCTGCGGCCACTGGACTGCCCATCAGCATGAAA38 Nucleicacidsequence ATGCTGGGCATCTGGACCCTCCTACCTCTGGTTCTTACGTCTGTTGCTA encoding GAT T AT C G T C C AAAAG T G T T AAT G C C C AAG T GAC T GAC AT C AAC T C C AA forchimeric GGGAT T GGAAT T GAGGAAGAC T GT TAG TACAGT T GAGAC T CAGAAC T T G CD 95 GAAGGCCTGCATCATGATGGCCAATTCTGCCATAAGCCCTGTCCTCCAG receptorGT GAAAGGAAAGC TAGGGAC T GCACAGT CAAT GGGGAT GAACCAGAC T Gin pl_324or in CGTGCCCTGCCAAGAAGGGAAGGAGTACACAGACAAAGCCCATTTTTCT pl_1195 T C CAAAT GCAGAAGAT G T AGAT T G T G T GAT GAAGGACAT GGC T T AGAAG T G GAAAT AAAC T G C AC C C G GAC C C AGAAT AC C AAG T G C AGAT G T AAAC C AAACTTTTTTTGTAACTCTACTGTATGTGAACACTGTGACCCTTGCACC AAAT GT GAACAT GGAAT CAT CAAGGAAT GCACAC T CACCAGCAACACCA AGTGCAAAGAGGAAGGATCCAGATCTAACTTGGGGTGGCTTTGTCTTCT TCTTTTGCCAATTCCACTAATTGTTTGGGTGCAACGAAGGAAATATAGA TCAAACAAAGGAGAAAGTCCTGTGGAGCCTGCAGAGCCTTGTCATTACA GCTGCCCCAGGGAGGAGGAGGGCAGCACCATCCCCATCCAGGAGGATTA CCGAAAACCGGAGCCTGCCTGCTCCCCC39 Nucleicacidsequence ATGCTGGGCATCTGGACCCTCCTACCTCTGGTTCTTACGTCTGTTGCTA encoding GAT TAT C G T C C AAAAG T G T T AAT G C C C AAG T GAC T GAC AT C AAC T C CAA forchimeric GGGAT T GGAAT T GAGGAAGAC T GT TAG TACAGT T GAGAC T CAGAAC T T G CD 95 GAAGGCCTGCATCATGATGGCCAATTCTGCCATAAGCCCTGTCCTCCAG receptorGT GAAAGGAAAGC TAGGGAC T GCACAGT CAAT GGGGAT GAACCAGAC T Gin pl_318SEQ ID Name SequenceNO.and in CGTGCCCTGCCAAGAAGGGAAGGAGTACACAGACAAAGCCCATTTTTCT pl_1191T C CAAAT GCAGAAGAT G T AGAT T G T G T GAT GAAGGACAT GGC T T AGAAG T G GAAAT AAAC T G GAG C C G GAG C C AGAAT AC C AAG T G C AGAT G T AAAC C AAACTTTTTTTGTAACTCTACTGTATGTGAACACTGTGACCCTTGCACC AAAT GT GAACAT GGAAT CAT CAAGGAAT GCACAC T CACCAGCAACACCA AGTGCAAAGAGGAAGGATCCAGATCTAACTTGGGGTGGCTTTGTCTTCT TCTTTTGCCAATTCCACTAATTGTTTGGGTGTGCTGGCTCACGAAAAAA AAATACTCTTCTACAGTGCACGACCCAAACGGGGAGTACATGTTTATGC G C G C T G T GAAC AC T G C AAAAAAAT C AAG G T T GAC AGAC G T AAC AG T C40 Nucleicacidsequence ATGCTGGGCATCTGGACCCTCCTACCTCTGGTTCTTACGTCTGTTGCTA encoding GAT T AT C G T C C AAAAG T G T T AAT G C C C AAG T GAC T GAC AT C AAC T C C AA forchimeric GGGAT T GGAAT T GAGGAAGAC T GT TAG TACAGT T GAGAC T CAGAAC T T G CD 95 GAAGGCCTGCATCATGATGGCCAATTCTGCCATAAGCCCTGTCCTCCAG receptorGT GAAAGGAAAGC TAGGGAC T GCACAGT CAAT GGGGAT GAACCAGAC T Gin pl_325and in CGTGCCCTGCCAAGAAGGGAAGGAGTACACAGACAAAGCCCATTTTTCT pl_1196 T C CAAAT GCAGAAGAT G T AGAT T G T G T GAT GAAGGACAT GGC T T AGAAG T G GAAAT AAAC T G C AC C C G GAC C C AGAAT AC C AAG T G C AGAT G T AAAC C AAACTTTTTTTGTAACTCTACTGTATGTGAACACTGTGACCCTTGCACC AAAT GT GAACAT GGAAT CAT CAAGGAAT GCACAC T CACCAGCAACACCA AGTGCAAAGAGGAAGGATCCAGATCTAACTTGGGGTGGCTTTGTCTTCT TCTTTTGCCAATTCCACTAATTGTTTGGGTGTGTGTGAAAAGAAGAAAG CCAAGGGGTGATGTAGTCAAGGTGATCGTCTCCGTCCAGCGGAAAAGAC AGGAGGCAGAAGGTGAGGCCACAGTCATTGAGGCCCTGCAGGCCCCTCC GGACGTCACCACGGTGGCCGTGGAGGAGACAATACCCTCATTCACGGGG AGGAGCCCAAACCAC41 Nucleicacidsequence ATGCTGGGCATCTGGACCCTCCTACCTCTGGTTCTTACGTCTGTTGCTA encoding GAT TAT C G T C C AAAAG T G T T AAT G C C C AAG T GAC T GAC AT C AAC T C CAA forchimeric GGGAT T GGAAT T GAGGAAGAC T GT TAG TACAGT T GAGAC T CAGAAC T T G CD 95 GAAGGCCTGCATCATGATGGCCAATTCTGCCATAAGCCCTGTCCTCCAG receptorGT GAAAGGAAAGC TAGGGAC T GCACAGT CAAT GGGGAT GAACCAGAC T Gin pl_320SEQ ID Name SequenceNO.CGTGCCCTGCCAAGAAGGGAAGGAGTACACAGACAAAGCCCATTTTTCT T C CAAAT GCAGAAGAT G T AGAT T G T G T GAT GAAGGACAT GGC T T AGAAG T G GAAAT AAAC T G GAG C C G GAG C C AGAAT AC C AAG T G C AGAT G T AAAC C AAACTTTTTTTGTAACTCTACTGTATGTGAACACTGTGACCCTTGCACC AAAT GT GAACAT GGAAT CAT CAAGGAAT GCACAC T CACCAGCAACACCA AGTGCAAAGAGGAAGGATCCAGATCTAACTTGGGGTGGCTTTGTCTTCT TCTTTTGCCAATTCCACTAATTGTTTGGGTGAAACGGGGCAGAAAGAAA CTCCTGTATATATT C AAAC AAC C AT T T AT GAGAC C AG TAG AAAC T AC T C AAGAGGAAGAT GGC T GTAGC T GCCGAT T T CCAGAAGAAGAAGAAGGAGG ATGTGAACTG42 Nucleicacidsequence ATGCTGGGCATCTGGACCCTCCTACCTCTGGTTCTTACGTCTGTTGCTA encoding GAT T AT C G T C C AAAAG T G T T AAT G C C C AAG T GAC T GAC AT C AAC T C C AA forchimeric GGGAT T GGAAT T GAGGAAGAC T GT TAG TACAGT T GAGAC T CAGAAC T T G CD 95 GAAGGCCTGCATCATGATGGCCAATTCTGCCATAAGCCCTGTCCTCCAG receptorGT GAAAGGAAAGC TAGGGAC T GCACAGT CAAT GGGGAT GAAC GAGAC T Gin pl_330CGTGCCCTGCCAAGAAGGGAAGGAGTACACAGACAAAGCCCATTTTTCT T C CAAAT GCAGAAGAT G T AGAT T G T G T GAT GAAGGACAT GGC T T AGAAG T G GAAAT AAAC T G GAG C C G GAC C C AGAAT AC C AAG T G C AGAT G T AAAC C AAACTTTTTTTGTAACTCTACTGTATGTGAACACTGTGACCCTTGCACC AAAT GT GAACAT GGAAT CAT CAAGGAAT GCACAC T CACCAGCAACACCA AGTGCAAAGAGGAAGGATCCAGATCTAACTTGGGGTGGCTTTGTCTTCT TCTTTTGCCAATTCCACTAATTGTTTGGGTGCAGCTTGGACTGCACATC TGGCAGCTGAGGAGTCAGTGCATGTGGCCCCGAGAGACCCAGCTGCTGC TGGAGGTGCCGCCGTCGACCGAAGACGCCAGAAGCTGCCAGTTCCCCGA GGAAGAGCGGGGCGAGCGATCGGCAGAGGAGAAGGGGCGGCTGGGAGAC CTGTGGGTG43 Nucleicacidsequence ATGCTGGGCATCTGGACCCTCCTACCTCTGGTTCTTACGTCTGTTGCTA encoding GAT TAT C G T C C AAAAG T G T T AAT G C C C AAG T GAC T GAC AT C AAC T C CAA forchimeric GGGAT T GGAAT T GAGGAAGAC T GT TAG TACAGT T GAGAC T CAGAAC T T G CD 95 GAAGGCCTGCATCATGATGGCCAATTCTGCCATAAGCCCTGTCCTCCAGreceptorSEQ ID Name SequenceNO.in pl_331 GT GAAAGGAAAGC TAGGGAC T GCACAGT CAAT GGGGAT GAACCAGAC T G and inpl_1197 CGTGCCCTGCCAAGAAGGGAAGGAGTACACAGACAAAGCCCATTTTTCT T C CAAAT GCAGAAGAT G T AGAT T G T G T GAT GAAGGACAT GGC T T AGAAG T G GAAAT AAAC T G GAG C C G GAG C C AGAAT AC C AAG T G C AGAT G T AAAC C AAACTTTTTTTGTAACTCTACTGTATGTGAACACTGTGACCCTTGCACC AAAT GT GAACAT GGAAT CAT CAAGGAAT GCACAC T CACCAGCAACACCA AGTGCAAAGAGGAAGGATCCAGATCTAACTTGGGGTGGCTTTGTCTTCT TCTTTTGCCAATTCCACTAATTGTTTGGGTGTGCCACCGGAGGGCCTGC AGGAAGCGAATTCGGCAGAAGCTCCACCTGTGCTACCCGGTCCAGACCT CCCAGCCCAAGCTAGAGCTTGTGGATTCCAGACCCAGGAGGAGCTCAAC GCAGCTGAGGAGTGGTGCGTCGGTGACAGAACCCGTCGCGGAAGAGCGA GGGTTAATGAGCCAGCCACTGATGGAGACCTGCCACAGCGTGGGGGCAG CCTACCTGGAGAGCCTGCCGCTGCAGGATGCCAGCCCGGCCGGGGGCCC CTCGTCCCCCAGGGACCTTCCTGAGCCCCGGGTGTCCACGGAGCACACC AAT AAC AAGAT T GAGAAAAT C TAG AT CAT GAAG G C T GAC AC C G T GAT C G TGGGGACCGTGAAGGCTGAGCTGCCGGAGGGCCGGGGCCTGGCGGGGCC AGCAGAGCCCGAGTTGGAGGAGGAGCTGGAGGCGGACCATACCCCCCAC TACCCCGAGCAGGAGACAGAACCGCCTCTGGGCAGCTGCAGCGATGTCA TGCTCTCAGTGGAAGAGGAAGGGAAAGAAGACCCCTTGCCCACAGCTGC CTCTGGAAAG44 Nucleicacidsequence ATGCTGGGCATCTGGACCCTCCTACCTCTGGTTCTTACGTCTGTTGCTA encoding GAT T AT C G T C C AAAAG T G T T AAT G C C C AAG T GAC T GAC AT C AAC T C C AA forchimeric GGGAT T GGAAT T GAGGAAGAC T GT TAG TACAGT T GAGAC T CAGAAC T T G CD 95 GAAGGCCTGCATCATGATGGCCAATTCTGCCATAAGCCCTGTCCTCCAG receptorGT GAAAGGAAAGC TAGGGAC T GCACAGT CAAT GGGGAT GAACCAGAC T Gin pl_322and in CGTGCCCTGCCAAGAAGGGAAGGAGTACACAGACAAAGCCCATTTTTCT pl_1193 T C CAAAT GCAGAAGAT G T AGAT T G T G T GAT GAAGGACAT GGC T T AGAAG T G GAAAT AAAC T G C AC C C G GAC C C AGAAT AC C AAG T G C AGAT G T AAAC C AAACTTTTTTTGTAACTCTACTGTATGTGAACACTGTGACCCTTGCACC AAAT GT GAACAT GGAAT CAT CAAGGAAT GCACAC T CACCAGCAACACCA AGTGCAAAGAGGAAGGATCCAGATCTAACTTGGGGTGGCTTTGTCTTCTT C T T T T GCCAAT T CCAC TAAT T GT T T GGGT GAAAAGGAAAAAACAGAGGSEQ ID Name SequenceNO.AGTCGGAGAAATGATGAGGAGCTGGAGACAAGAGCCCACAGAGTAGCTA CTGAAGAAAGGGGCCGGAAGCCCCACCAAATTCCAGCTTCAACCCCTCA GAATCCAGCAACTTCCCAACATCCTCCTCCACCACCTGGTCATCGTTCC CAGGCACCTAGTCATCGTCCCCCGCCTCCTGGACACCGTGTTCAGCACC AGCCTCAGAAGAGGCCTCCTGCTCCGTCGGGCACACAAGTTCACCAGCA GAAAGGCCCGCCCCTCCCCAGACCTCGAGTTCAGCCAAAACCTCCCCAT GGGGCAGCAGAAAACTCATTGTCCCCTTCCTCTAAT45 Nucleicacidsequence ATGCTGGGCATCTGGACCCTCCTACCTCTGGTTCTTACGTCTGTTGCTA encoding GAT T AT C G T C C AAAAG T G T T AAT G C C C AAG T GAG T GAG AT C AAC T C C AA forchimeric GGGAT T GGAAT T GAGGAAGAC T GT TAG TACAGT T GAGAC T CAGAAC T T G CD 95 GAAGGCCTGCATCATGATGGCCAATTCTGCCATAAGCCCTGTCCTCCAG receptorGT GAAAGGAAAGC TAGGGAC T GCACAGT CAAT GGGGAT GAAC GAGAC T Gin pl_321and in CGTGCCCTGCCAAGAAGGGAAGGAGTACACAGACAAAGCCCATTTTTCT pl_1192 T C CAAAT GCAGAAGAT G T AGAT T G T G T GAT GAAGGACAT GGC T T AGAAG T G GAAAT AAAC T G GAG C C G GAG C C AGAAT AC C AAG T G C AGAT G T AAAC C AAACTTTTTTTGTAACTCTACTGTATGTGAACACTGTGACCCTTGCACC AAAT GT GAACAT GGAAT CAT CAAGGAAT GCACAC T CACCAGCAACACCA AGTGCAAAGAGGAAGGATCCAGATCTAACTTGGGGTGGCTTTGTCTTCT TCTTTTGCCAATTCCACTAATTGTTTGGGTGGCCCTGTACCTGCTCCGG AGGGACCAGAGGCTGCCCCCCGATGCCCACAAGCCCCCTGGGGGAGGCA GTTTCCGGACCCCCATCCAAGAGGAGCAGGCCGACGCCCACTCCACCCT GGCCAAGATC46 WildtypeCD8a Coreceptor MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSN polypeptid PTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTF eVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPT PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVL LLSLVITLYCNHRNRRRVCKCPRPWKSGDKPSLSARYV47 WildtypeCD8b Coreceptor MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCEAKISLSN polypeptid MRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASReSEQ ID Name SequenceNO.FILNLTSVKPEDSGI YFCMIVGSPELTFGKGTQLSWDFLPTTAQPTKK STLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLLVSLGVAIHLCCR RRRARLRFMKQFYK48 wildtypehumanCD4 CoMNRGVPFRHLLLVLQLALLPAATQGKKWLGKKGDTVELTCTASQKKS I receptor QFHWKNSNQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLI IKN polypeptide LKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLESPP GSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFK IDIWLAFQKASSIVYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERA SSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPLHLTLPQALPQYAGS GNLTLALEAKTGKLHQEVNLWMRATQLQKNLTCEVWGPTSPKLMLSLK LENKEAKVSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWST PVQPMALIVLGGVAGLLLFIGLGI FFCVRCRHRRRQAERMSQIKRLLSE KKTCQCPHRFQKTCSPI49 costimulatory CD30motif EADHTPHYPEQETEPPLGSCSDVMLSVEEEGpolypeptide50 ChimericCD-8 Coreceptor MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSN polypeptid PTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTF e (pTK- 0638) VLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPASGGGSGGGSGGG SGGGSLQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDS HHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIY FCMIVGSPELTFGKGTQLSWDFLPTTAQPTKKSTLKKRVCRLPRPETQ KGPLCSPIYIWAPLAGTCGVLLLSLVITCVRCRHRRRQAERMSQIKRLL SEKKTCQCPHRFQKTCSPISGGGSKKVAKKPTNKAPHPKQEPQEINFPD DLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQSGGGSEADHTPH YPEQETEPPLGSCSDVMLSVEEEG51 LinkerSGSG52 MHCtetramerKVLEYVIKVSEQ ID Name SequenceNO.53 LinkerSGGGS54 CD40humanWildtype MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVS full length DCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSE (UniProtentry no.: TDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCP P25942 ■ VGFFSNVSSAFEKCHPWTSCETKDLWQQAGTNKTDWCGPQDRLRALV TNR5_HU VIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGS MAN NTAAPVQETLHGCQPVTQEDGKESRISVQERQ55 0X40humanWildtype MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPG full length NGMVSRCSRSQNTVCRPCGPGFYNDWSSKPCKPCTWCNLRSGSERKQL (UniProtentry no.: CTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNC P43489 ■ TLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWP TNR4_HU RTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQR MAN LPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI56 CD40cytoplasmic coPTNKAPHPKQEPQEINFPDDLPGSNTstimulatory region(aminoacidsequence222 - 247of SEQ IDNo. 54)57 0X40cytoplasmic coALYLLRRDQRLPPDAHKPPGGGSstimulatory region(aminoacidsequence236 - 258of SEQ IDNo. 55)SEQ ID Name SequenceNO.58 ChimericCD 95receptor MLGIWTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNL pl_2234 EGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFS SKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCT KCEHGI IKECTLTSNTKCKEEGSRSNLGWLCLLLLPIPLIVWVALYLLR RDQRLPPDAHKPPGGGSFRTPIQETEPDAHSTLAKISGGGSPTNKAPHP KQEPQEIDFPDDLPGSNT59 ChimericCD 95receptor MLGIWTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNL pl_2232 EGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFS SKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCT KCEHGI IKECTLTSNTKCKEEGSRSNLGWLCLLLLPIPLIVWVALYLLR RDQRLPPDAHKPPGGGSPTNKAPHPKQEPQEIDFPDDLPGSNT60 Nucleicacidsequence ATGCTCGGGATATGGACACTCCTCCCCCTCGTGCTCACGAGTGTAGCCA of pl_2234 G G T T G T C GAG C AAGAG TGTTAATGCT GAG G T C AC AGAT AT C AAT T C C AA AG G C C T C GAG C T T AGAAAGAC AG T T AC TAG AG T G GAGAC T C AGAAT C T G GAGGGACTGCACCATGACGGGCAATTTTGCCATAAACCCTGCCCGCCTG GTGAGCGAAAAGCAAGGGACTGCACCGTTAATGGGGACGAGCCAGATTG TGTGCCGTGTCAAGAGGGCAAAGAATACACGGATAAGGCTCATTTCTCT TCAAAATGCCGCCGATGTCGACTGTGTGATGAGGGGCATGGGCTTGAAG T G GAGAT AAAT T G C AC T C G GAC AC AGAAT AC C AAG T G T AGAT G T AAG C C TAACTTTTTTTGCAACAGTACAGTATGTGAGCACTGTGACCCCTGTACC AAATGCGAGCATGGGATTATCAAGGAATGTACCCTCACTTCCAATACAA AGTGCAAAGAGGAGGGAAGTAGGTCTAATCTGGGATGGTTGTGTTTGCT CCTGCTTCCGATTCCTTTGATAGTCTGGGTCGCATTGTATCTGCTCCGG AGAGACCAAAGGTTGCCGCCAGACGCGCACAAACCTCCGGGTGGAGGGT C T T T C C G GAC T C C GAT AC AAGAAAC G GAAC CGGATGCTCATTC TAG AT T GGCGAAGATCTCCGGAGGGGGCTCCCCTACTAACAAAGCTCCACATCCC AAAC AG GAAC C AC AAGAAAT TGATTTTCCT GAC GATTTGCCCGGATCTA AT ACT61 Nucleicacidsequence ATGCTTGGAATTTGGACACTGCTCCCCCTTGTTCTGACGTCTGTTGCTC of pl_2232G C C T T AG T AG C AAAAG T G T AAAC G C T GAG G T GAC C GAGAT GAAC T C AAASEQ ID Name SequenceNO.AG GAG T T GAAC T T C G GAAAAC AG T AAC AAC T G T G GAGAC T C AGAAT C T G GAGGGTTTGCACCACGATGGACAGTTCTGCCATAAGCCGTGCCCCCCAG GCGAGCGGAAAGCACGGGACTGCACTGTGAACGGAGATGAACCGGACTG TGTGCCGTGCCAGGAGGGGAAGGAGTATACTGACAAAGCCCATTTCTCT TCTAAGTGCCGCCGCTGCCGCCTCTGCGATGAAGGTCACGGATTGGAGG T T GAAAT AAAC T G T AC C C GAAC T C AGAAC AC T AAAT G T AG G T G C AAG C C GAATTTCTTCTGTAATAGTACTGTGTGTGAGCACTGTGATCCTTGTACG AAGTGCGAGCACGGCATAATTAAGGAATGTACCTTGACGTCTAACACAA AATGTAAGGAGGAGGGTAGTCGCTCCAACCTTGGCTGGCTTTGCCTGCT GCTTCTTCCGATCCCTCTTATAGTTTGGGTAGCGTTGTATCTGCTCCGA CGCGACCAACGACTCCCGCCTGACGCACATAAGCCGCCTGGGGGTGGCA GCCCAACCAATAAGGCTCCTCACCCTAAGCAGGAACCCCAGGAGATAGATTTCCCCGATGATCTGCCAGGCTCTAACACAExperimental Examples

[0178] Example 1. In-vitro T-cell killing analysis of T-cells according to the present invention transduced with chimeric CD 95 receptor polypeptides according to the present invention and an engineered T-cell receptor

[0179] In order to test the T-cells expressing chimeric CD 95 receptor constructs of the present invention 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 HeLa FASL-expressing cells (Fig.2), NCI-H2030 FASL-expressing cells (Fig. 3), NCI-H2030 cells (Fig. 4) and NCI-H1703 FasL expressing cells, (Fig. 6) respectively, for evaluating cytotoxicity of the transduced T-cells.

[0180] 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:

[0181] Table 2 as presented below summarizes the chimeric CD 95 receptor constructs that have been generated by the inventors in a schematic representation:

[0182] Table 2EC TM CYP NameCD95 CD95 CD40 pl_323CD95 CD95 CD40L (CD154) pl_316CD95 CD95 CD27 pl_324CD95 CD95 ICOS (CD278) pl_318CD95 CD95 HVEM (CD270) pl_325CD95 CD95 4-1 BB (CD137) pl_320CD95 CD95 GITR (CD357) pl_330CD95 CD95 CD30 (407 to pl_331595aa)CD95 CD95 CD2 pl_322CD95 CD95 0X40 (CD134) pl_321CD95 CD95 LTBR pl_334CD95 CD95 CD28 pl_319CD95 CD95 TLR2 pl_326 / 7CD95 CD95 TLR4 pl_328 / 9CD95 CD95 IL6R subunit beta pl_333CD95 CD95 0X40 aa236to277 pl_2234_ of wildtype 0X40(includingmutationEQA266-268TEP)—linker SGGGS(SEQ ID No. 53)- CD40 aa222to247of wildtype CD40(includingmutation N237D)CD95 CD95 0X40 aa236-258 Pl_2232- of wildtype0X40 - CD40 aa222to247 of wildtype CD40(includingmutation N237D)

[0183] 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.

[0184] 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 or 6, and chimeric CD8 co-receptor type as used in the T-cell killing assay shown in Fig. 4 or 6, and as -partially - used for the cytokine secretion assay shown in Fig. 7.

[0185] Table 3Plasmid EC TM CYP CoR TypeNpl_ 1190 CD95 CD95 Fas- chimeric CD8 CoCD40L receptorpl_ 1191 CD95 CD95 Fas- chimeric CD8 CoICOS receptorpl_ 1192 CD95 CD95 Fas- chimeric CD8 Co0X40 receptorpl_ 1193 CD95 CD95 Fas- chimeric CD8 CoCD2 receptorpl_1194 CD95 CD95 Fas- chimeric CD8 CoCD40 receptorpl_ 1195 CD95 CD95 Fas- chimeric CD8 CoCD27 receptorpl_ 1196 CD95 CD95 Fas- chimeric CD8 CoHVEM receptorpl_ 1197 CD95 CD95 Fas- chimeric CD8 CoCD30 receptorPl_2234 CD95 CD95 0X40 - chimeric CD8 CoCD40 receptorPl_2232 CD95 CD95 0X40- chimeric CD8 CoCD40 receptor

[0186] 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.

[0187] CD8 Cells Generation

[0188] 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, A TCR raised against PRAME alone (in experiments relating to Fig. 5 to 7) or the respective TCR together with different versions of the SWITCH receptors. The HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) as used herein has been described e.g. in WO 2014 / 118236, which is herewith incorporated by reference in its entirety. In particular, the HLA-I restricted TCR raised against MAGE-A1 as used herein relates to “TCR1367” as described in WO 2014 / 118236. The CDR sequences of the respective a and p chain of “TCR1367” as used herein are further described -for example - in WO 2023 / 083864, which is herewith incorporated by reference in its entirety.

[0189] 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.

[0190] 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.

[0191] Cell killing assay in HeLa cells expressing FASL:

[0192] 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 1x104) 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.

[0193] 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 obtained by 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.

[0194] Long term cell killing assay in NCI-H1703 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 obtained by positive selection with anti-CD8+ microbeads. CD3+ T cells were activated using TransAct in presence of IL-7 / IL-15. Two days post activation, CD8 T cells were separately transduced with either HLA-I restricted TCR raised against PRAME alone, or together with different versions of the SWITCH receptors. Where indicated (results in Fig. 6), a chimeric CD8 Co-receptor was co-transduced (CoR). The chimeric CD8 Co-receptor used in theexperiments is the Chimeric CD8 Co-receptor polypeptide (pTK-0638) having an amino acid sequence as set forth in SeqID No. 50.

[0195]

[0196] Flow Cytometry:

[0197] 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.

[0198] 1.2. T-cell killing assay analysis

[0199] 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, Fig.4 and Fig. 6 wherein Fig. 2 shows the results of the killing assay with the chimeric CD 95 receptors in HeLa cells, whereas Fig. 3-4 show the result of the cell killing assay with the chimeric CD 95 receptors in NCIH2030 cells, and Fig. 6 shows the results of the killing assay with the chimeric CD 95 receptors in NCI-H1703 cells. The arrow in Fig. 6 mark the points of cancer cell additions. In Fig. 2, 3 and 4, “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-SwR_ICOS” 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 ICOS 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 cytoplasmicdomain; “MAGE-A1_TCR-SwR_4-1BB” 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.

[0200] 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).

[0201] Fig. 6 also shows the results of a long term killing assay: “Mock” relates to mock-transduced T-cell fraction, “CD8-C0R” relates to CD8 T-cell fraction transduced with the chimeric CD8 receptor and transduced with HLA-I restricted TCR raised against PRAME, “CD8-CoR_FAS-CD40” relates to CD8 T-cell fraction transduced with transduced with HLA-I restricted TCR raised against PRAME, with the chimeric CD8 receptor, and with the chimeric FAS-CD40 receptor according to pl_323, “CD8-CoR_FASOX40 / CD40” relates to CD8 T-cell fraction transduced with transduced with HLA-I restricted TCR raised against PRAME, with the chimeric CD8 receptor, and with the chimeric FAS-OX40 / CD40 receptor according to pl_2232, and “CD8-CoR_FASOX40 / CD40_3” relates to CD8 T-cell fraction transduced with transduced with HLA-I restricted TCR raised against PRAME, with the chimeric CD8 receptor, and with the chimeric FAS-OX40 / CD40 receptor according to pl_2234.

[0202] 1.3 Flow Cytometry Analysis

[0203] 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.

[0204] 1.4 Cytokine Secretion Assay

[0205] NCI-H1703 cells target cells were seeded in 24-well plates overnight. T-cells expressing either TCR alone or TCR+CD8C0R were added to the target cells at 1:1 Effector to Target ratio the following day. After 48hours of co-culture, supernatants were collected and stored at -80 °C. Cytokines in the cell culture supernatants were quantified using LegendPlex Human CD8 / NK Panel (13-plex) (Cat. 741187, BioLegend) according to the manufacturer’s instructions. All samples in the experiment were measured in triplicates. Briefly, 2x diluted supernatants were incubated with beads coated with capture antibodies specific for IL-2, IFN-y, Granzyme A, Granzyme B, or Perforin, respectively, for 2 h at room temperature on shaker. After incubation, beads were washed and incubated with biotin-labeled detection antibodies for 1 h, followed by a final incubation with streptavidin-PE for 30 min at room temperature on shaker. Beads were washed and resuspended with washing buffer. Beads were analyzed by flow cytometry using a FACS Symphony cytometer. Analysis was performed using the LEGENDplex analysis software v8.0, which distinguishes between the 13 different analytes on basis of bead size and internal dye. In the results as shown in Fig. 7, “Cancer cells” relate to non-transduced cancer cells, “Mock” relate to “mock”-transduced cancer cells, “scCD8-CoR” relate to cancer cells transduced with a chimeric CD8 Co-receptor and a TCR directed against PRAME, “scCD8-CoR_FAS-CD40” relate to cancer cells transduced with a chimeric CD8 Co-receptor, a TCR directed against PRAME, and a chimeric CD 95 receptor with only one CD40 cytoplasmic co-stimulatory domain, “scCD8-CoR_FAS-OX40 / CD40” relate to cancer cells transduced with a chimeric CD8 Co-receptor, a TCR directed against PRAME, and a chimeric CD 95 receptor according to the present invention (pl_2232), and “scCD8-CoR_FAS-OX40 / CD40_3” relate to cancer cells transduced with a chimeric CD8 Co-receptor, a TCR directed against PRAME, and a chimeric CD 95 receptor according to the present invention (pl_2234).

[0206] 1.4 Results

[0207] 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.

[0208] 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, and / or T-cells only transduced with the HLA-I restricted TCR raised against MAGE-A1 together with CD8 Co-receptor, as visible in NCIH2030 cells.

[0209] As visible from Fig. 6, chimeric CD 95 receptors as herein provided comprising a first non-CD 95 cytoplasmic co-stimulatory region from 0X40 and a second non-CD 95 cytoplasmic co-stimulatory region from CD40, when co-transduced with an engineered TCR raised against PRAME 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 PRAME together with the chimeric CD8 Co-receptor. Surprisingly, the effect are even slightly increased compared with a chimeric CD 95 receptor having only one single non-CD 95 cytoplasmic co-stimulatory region from CD40.

[0210] Furthermore, Fig. 7 demonstrates a synergistic effect of the specifically combined presence of a first non-CD 95 cytoplasmic co-stimulatory region from 0X40 and a second non-CD 95 cytoplasmic co-stimulatory region from CD40. Fig. 7a shows increased secretion of IL-2 if a chimeric CD 95 receptor according to the present invention (pl_2234) was co-transduced, compared with the effects of a chimeric CD 95 receptor having only a CD40 cytoplasmic co-stimulatory region. Fig. 7B shows increased secretion of IFN-x if a chimeric CD 95 receptor according to the present invention (pl_2234) or pl_2232) was co-transduced, compared with the effects of a chimeric CD 95 receptor having only a CD40 cytoplasmic co-stimulatory region. Fig. 7C shows increased secretion of Granzyme A if a chimeric CD 95 receptor according to the present invention (pl_2234) or pl_2232) was co-transduced, compared with the effects of a chimeric CD 95 receptor having only a CD40 cytoplasmic co-stimulatory region. Fig. 7D shows increased secretion of Granzyme B if a chimeric CD 95 receptor according to the present invention (pl_2234) or pl_2232) was co-transduced, compared with the effects of a chimeric CD 95 receptorhaving only a CD40 cytoplasmic co-stimulatory region. Fig. 7E shows increased secretion of Perforin if a chimeric CD 95 receptor according to the present invention (pl_2234) or pl_2232) was co-transduced, compared with the effects of a chimeric CD 95 receptor having only a CD40 cytoplasmic co-stimulatory region.

[0211] Summary and conclusions

[0212] The results described above demonstrate - in principle - suitability of chimeric CD 95 receptor polypeptides comprising a first non-CD 95 cytoplasmic co-stimulatory region from 0X40 and a second non-CD 95 cytoplasmic co-stimulatory region from CD40, as well as of T-cells comprising the chimeric CD 95 receptors as herein provided, optionally together with an engineered T-cell receptor for improving adoptive cell therapy (ACT). Specifically, it is contemplated that the chimeric CD 95 receptor polypeptides of the present invention, comprising a first non-CD 95 cytoplasmic co-stimulatory region from 0X40 and a second non-CD 95 cytoplasmic co-stimulatory region from CD40, 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. The chimeric CD 95 receptor polypeptides of the present invention, comprising a first non-CD 95 cytoplasmic co-stimulatory region from 0X40 and a second non-CD 95 cytoplasmic co-stimulatory region from CD40, with respect to several advantageous aspects as herein demonstrated, show synergistically improved functions in comparison with chimeric CD 95 receptor polypeptides having only one single CD40 cytoplasmic co-stimulatory region.

[0213] 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.

[0214] All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0215] 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 thefeatures 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

1. Claims:

1. A chimeric human CD 95 receptor; comprising a polypeptide,wherein said polypeptide comprises at least one CD 95 polypeptide region comprising or consisting of a polypeptide domain, a polypeptide region or a polypeptide motif of a human CD 95 receptor as set forth in SEQ ID No 1, or 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 CD 95 polypeptide region comprises a CD 95 extracellular ligand binding domain;further wherein said polypeptide comprises at least two non-CD 95 cytoplasmic costimulatory polypeptide domains, regions or motifs, wherein a first non-CD 95 costimulatory region comprises a cytoplasmic costimulatory region of 0X40, and wherein a second non-CD 95 co-stimulatory region comprises a cytoplasmic costimulatory region of CD40.

2. The chimeric CD 95 receptor of claim 1 , 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.

3. The chimeric CD 95 receptor of claim 1 or 2, wherein said polypeptide is a singlechain polypeptide.

4. The chimeric CD 95 receptor according to any one of claims 1-3, wherein the CD 95-derived polypeptide region further comprises at least one, or at least two, or at least three CD 95 cysteine-rich domains (CRDs).

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

6. The chimeric CD 95 receptor of any one of the foregoing claims, wherein the at least one CD 95 polypeptide region further comprises a CD95 homotypic interaction domain.

7. The chimeric CD 95 receptor according to any one of the foregoing claims, wherein the at least one CD 95 polypeptide region comprises a CD 95 transmembrane region.

8. The chimeric CD 95 receptor according to any one of the foregoing claims, wherein said CD 95 polypeptide region comprises a complete CD 95 extracellular domain.

9. The chimeric CD 95 receptor according to claim 8, wherein the CD 95 polypeptide region comprises the complete CD 95 extracellular domain and a CD 95 transmembrane domain.

10. The chimeric CD 95 receptor according to any one of claims 1-9, wherein the CD 95 polypeptide region comprises at least one linker region.

11. The chimeric CD 95 receptor according to any one of the foregoing claims, wherein the first non-CD 95 co-stimulatory region comprises the complete cytoplasmic costimulatory region of 0X40, and / or wherein the second non-CD 95 co-stimulatory region comprises the complete cytoplasmic costimulatory region of CD40.

12. The chimeric CD 95 receptor according to any one of the foregoing claims, wherein the second non-CD 95 co-stimulatory region comprises at least one TRAF binding motif of CD40.

13. The chimeric CD95 receptor according to any one of the foregoing claims, wherein second non-CD 95 co-stimulatory region comprises the TRAF binding domain of CD40 as comprised in SEQ ID No 56.

14. The chimeric CD95 receptor according to any one of the foregoing claims, wherein second non-CD 95 co-stimulatory region comprises or consists of the polypeptide region of human CD40 having the amino acid sequence as set forth in SEQ ID No 56.

15. The chimeric CD95 receptor according to any one of the foregoing claims, wherein the second non-CD 95 co-stimulatory region comprises a polypeptide region of CD40 including amino acid position 237 of SEQ ID No. 54, and further wherein at said position, the first non-CD 95 co-stimulatory region is mutated, wherein said mutation consists of an exchange of an asparagine (N) to an aspartic acid (D).

16. The chimeric CD 95 receptor according to any one of the foregoing claims, wherein the first non-CD 95 co-stimulatory region comprises at least one TRAF binding site of 0X40.

17. The chimeric CD 95 receptor according to any one of the foregoing claims, wherein the first non-CD 95 co-stimulatory region comprises at least one potential PI3K binding motif / potential ubiquitination site of 0X40.

18. The chimeric CD 95 receptor according to any one of the foregoing claims, wherein the first non-CD 95 co-stimulatory region comprises a polypeptide region of 0X40 including amino acid positions 266 to 268 of SEQ ID No 55, and further wherein at said position, the second non-CD 95 co-stimulatory region is mutated, wherein said mutation consists of an exchange of the wildtype 0X40 amino acids EQA to TEP.

19. The chimeric CD95 receptor according to any one of the foregoing claims, wherein first non-CD 95 co-stimulatory region comprises or consists of the polypeptide region of human 0X40 as set forth in SEQ ID No 57.

20. The chimeric CD95 receptor according to any one of the claims 1 to 18, wherein first non-CD 95 co-stimulatory region comprises or consists of the polypeptide region of human 0X40 as set forth in SEQ ID No 20.

21. The chimeric CD95 receptor according to any one of the foregoing claims, wherein the second costimulatory region (CD40) is located closer to the C- terminal tail of the polypeptide than the first costimulatory region (0X40).

22. The chimeric CD95 receptor according to any one of the foregoing claims, wherein the cytoplasmic non-CD 95 co-stimulatory region comprises a linker between the first and second non-CD 95 co-stimulatory regions, optionally wherein said linker comprises or consists of a sequence as set forth in SEQ ID No. 53.

23. The chimeric CD 95 receptor according to any one of the foregoing claims, wherein the polypeptide is having an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID No.: 58 or 59.

24. The chimeric CD 95 receptor of any one of the foregoing claims, wherein the at least one CD 95 polypeptide region has at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98% or at least 99%, or 100% sequence identity with a functional polypeptide region, domain or motif of a human CD95 receptor (Seq ID No. 1).

25. The chimeric CD 95 receptor of any one of the foregoing claims, wherein the chimeric Co-receptor is able to enhance cytotoxicity of a T-cell.

26. The chimeric CD 95 receptor of any one of the foregoing claims, wherein the chimeric CD 95 receptor is capable of increasing resistance of T-cells to CD95L expressing cancer cells.

27. An isolated nucleic acid comprising a nuclear acid sequence encoding for the chimeric CD 95 receptor according to any one of claims 1-26.

28. A vector comprising the nucleic acid according to claim 27.

29. The vector of claim 28, wherein the vector is a viral vector or a non-viral vector.

30. The vector of claim 28, wherein the viral vector is selected from adenoviruses, poxviruses, alphaviruses, arenaviruses, flaviruses, rhabdoviruses, retroviruses, lentiviruses, herpesviruses, paramyxoviruses, picornaviruses, and combinations thereof.

31. The vector of any one of claims 28-30, wherein the vector further comprises a nucleic acid encoding a chimeric antigen receptor (CAR).

32. The vector of any one of claims 28-30, wherein the vector further comprises a nucleic acid encoding a T cell receptor comprising a TCR a chain and a TCR p chain.

33. The vector according to claim 32, wherein the T cell receptor is a recombinant T cell receptor.

34. The vector according to any one of claims 28 - 33, wherein the vector further comprises a nucleic acid encoding for a CD8 Co-receptor, such as a wildtype or an engineered CD8 Co-receptor.

35. The vector according to claim 34, wherein the nucleic acid encodes a CD8a and a CD8p Co-receptor, or wherein the nucleic acid encodes a chimeric receptor with CD8 Co-receptor functionality.

36. The vector according to claim 35, 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.

37. An isolated T-cell, wherein the T-cell expresses the chimeric CD 95 receptor according to any one of claims 1 to 26; optionally wherein the T-cell further expresses an engineered T-cell receptor.

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

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

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

41. The T-cell according to claim 40, wherein the T-cell is a CD4 T-cell66-1SHEET INCORPORATED BY REFERENCE (RULE 20.6)42. The T-cell according to claim 40 or 41 , wherein the CD8 Co-receptor is a wildtype CD8 Co-receptor such as CD8a and CD8p Co-receptor.

43. The T-cell according to claim 40 or 41, wherein the CD8 Co-receptor is a chimeric CD8 Co-receptor.

44. The T-cell according to claim 43, 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.

45. The T-cell according to any one of claims 37-44, 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.

46. An isolated T-cell, wherein the vector of any one of claims 28 - 36 has been introduced in said T-cell.

47. The T-cell of any one of claims 37 - 46, wherein the cell is a ap T-cell, y8 T-cell, and / or a natural killer T-cell.

48. The T-cell of claim 47; wherein the p T-cell is a CD4 T-cell, or wherein the ap T- cell is a CD8 T-cell, or wherein the y8 T-cell is a V / 9V82+ T-cell, or wherein the y8 T-cell comprises a V81 T-cell.

49. The T-cell according to any one of claims 37 - 48, wherein the T-cell is derived from an induced pluripotent stem cell (iPSCs).

50. A kit comprising means to prepare the T-cell according to any one of claims 37 - 49.67-1SHEET INCORPORATED BY REFERENCE (RULE 20.6)32. The vector of any one of claims 28-30, wherein the vector further comprises a nucleic acid encoding a T cell receptor comprising a TCR > chain and a TCR □chain.

33. The vector according to claim 32, wherein the T cell receptor is a recombinant T cell receptor.

34. The vector according to any one of claims 28 - 33, wherein the vector further comprises a nucleic acid encoding for a CD8 Co-receptor, such as a wildtype or an engineered CD8 Co-receptor.

35. The vector according to claim 34, wherein the nucleic acid encodes a CD8Q and a CD8Q Co-receptor, or wherein the nucleic acid encodes a chimeric receptor with CD8 Co-receptor functionality.

36. The vector according to claim 35, 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.

37. An isolated T-cell, wherein the T-cell expresses the chimeric CD 95 receptor according to any one of claims 1 to 26; optionally wherein the T-cell further expresses an engineered T-cell receptor.

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

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

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

41. The T-cell according to claim 40, wherein the T-cell is a CD4 T-cell.

42. The T-cell according to claim 40 or 41 , wherein the CD8 Co-receptor is a wildtype CD8 Co-receptor such as CD8D and CD8D Co-receptor.

43. The T-cell according to claim 40 or 41, wherein the CD8 Co-receptor is a chimeric CD8 Co-receptor.

44. The T-cell according to claim 43, 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.

45. The T-cell according to any one of claims 37-44, 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.

46. An isolated T-cell, wherein the vector of any one of claims 28 - 36 has been introduced in said T-cell.

47. The T-cell of any one of claims 37 - 46, wherein the cell is a > > T-cell, > > T-cell, and / or a natural killer T-cell.

48. The T-cell of claim 47; wherein the > > T-cell is a CD4 T-cell, or wherein the > > T- cell is a CD8 T-cell, or wherein the yD T-cell is a VD9VD2+ T-cell, or wherein the yD T-cell comprises a VD1 T-cell.

49. The T-cell according to any one of claims 37 - 48, wherein the T-cell is derived from an induced pluripotent stem cell (iPSCs).

50. A kit comprising means to prepare the T-cell according to any one of claims 37 - 49.

51. A pharmaceutical composition comprising the T-cell of any one of claims 37 - 49.

52. The pharmaceutical composition of claim 51, wherein the composition further comprises an adjuvant, excipient, buffer, diluent, carrier, stabilizer or combination thereof.

53. A method for preparing a T-cell for immunotherapy, comprisingisolating T-cells from a human subject,introducing the vector according to any one of claims 28 - 36 into the T-cell, andexpanding the T-cells.

54. The method according to claim 53, comprising transforming, transfecting or transducing the isolated T-cells with the vector.

55. The pharmaceutical composition according to claim 53 or 54, comprising T-cells expressing the chimeric CD 95 receptor and optionally the engineered T-cell receptor.

56. The pharmaceutical composition according to claim 55, further comprising CD4 cells expressing said chimeric CD 95 receptor and further expressing a recombinant CD8 Co-receptor.

57. The pharmaceutical composition according to claim 56, wherein the CD8 Coreceptor is a chimeric CD8 Co-receptor.

58. A method of treating a patient having a disease, comprising administering to the patient the composition of claims 51, 52, or 55 - 57.

59. A method of treating a patient having a disease, comprising introducing in vivo the vector according to any one of claims 28-36 into a T-cell of the patient.

60. The method according to claim 59, wherein the vector is DNA or an mRNA.

61. The method according to claim 59, wherein the vector is a non-replicating viral vector.

62. The method according to claim 59 or 60, wherein the vector is mRNA, and wherein the mRNA is introduced into the T-cell of the patient using nanoparticles.

63. The method according to any one of claims 58- 62, wherein the disease is a cancer or an autoimmune disease.

64. The method according to claim 63, 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.

65. The method according to claim 63 or 64, wherein cancer cells express FasL.

66. A method of increasing cytotoxicity of a T-cell in adoptive cell therapy, comprising introducing a vector according to any one of claims 28 - 36 into a T-cell.

67. The method according to claim 66, wherein the vector encodes for a CD8 Coreceptor, such as a human wildtype CD8 Co-receptor or a chimeric CD8 Coreceptor.

68. The method according to claim 66 or 67, wherein the T-cell receptor is a recombinant T-cell receptor that specifically binds a tumor specific antigen.

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