A chimeric human receptor, a nucleic acid encoding the chimeric human receptor, corresponding vectors, isolated t-cells transduced with the nucleic acid or corresponding vectors and kits for preparing them, as well as corresponding pharmaceutical compositions and methods for treating a patient having a disease

Abstract

The present invention inter alia relates to a chimeric human receptor, comprising a polypeptide having an extracellular polypeptide region, a transmembrane polypeptide region, and a cytoplasmic polypeptide region, wherein the cytoplasmic polypeptide region comprises at least two TNF receptor associated factor 6 (TRAF 6) binding motifs, and / or wherein said cytoplasmic polypeptide region comprises at least two CD40 TNF receptor associated factor 1 / 2 / 3 (TRAF 1 / 2 / 3) binding motifs. The present invention also relates to a nucleic acid encoding the chimeric receptor, a vector comprising the nuclear acid encoding for the chimeric human receptor, as well as to isolated T-cells being transduced, transformed or transfected with the nucleic acid or with the vector, and to isolated T-cells being transduced, transformed or transfected to express the chimeric receptor, as well as to engineered T-cells expressing the chimeric receptor. The invention further relates to a kit for preparing the isolated T-cells of the present invention, as well as to a pharmaceutical composition comprising the T-cells. The invention also relates to a method for preparing a T-cell for immunotherapy, and to a method for treating a patient having a disease comprising administering the pharmaceutical composition.

Description

New PCT ApplicationApplicant: T-Knife GmbHSWK Ref.: TKN18564PCTDate: 5 February 2026A CHIMERIC HUMAN RECEPTOR, A NUCLEIC ACID ENCODING THE CHIMERIC HUMAN RECEPTOR, CORRESPONDING VECTORS, ISOLATED T-CELLS TRANSDUCED WITH THE NUCLEIC ACID OR CORRESPONDING VECTORS AND KITS FOR PREPARING THEM, AS WELL AS CORRESPONDING PHARMACEUTICAL COMPOSITIONS AND METHODS FOR TREATING A PATIENT HAVING A DISEASEREFERENCE 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. 25156077.7, filed 5 February 2025, the content of which is hereby incorporated by reference in its entirety for all purposes.FIELD OF THE INVENTION

[0003] The present invention relates to a chimeric human receptor, comprising a polypeptide having an extracellular polypeptide region, a transmembrane polypeptide region, and a cytoplasmic polypeptide region. The present invention also relates to a nucleic acid encoding the chimeric human receptor, a vector comprising the nuclear acid encoding for the chimeric human receptor, as well as to isolated T-cells being transduced with the nucleic acid or with the vector, and to isolated T-cells being transduced to express the chimeric human receptor. The invention further relates to a kit for preparing the isolated T-cell of the present invention, as well as to a pharmaceutical composition comprising the T-cells. The invention also relates to a method for preparing a T-cell for immunotherapy, and to a method for treating a patient having a disease comprising administering the pharmaceutical composition.BACKGROUND OF THE INVENTION

[0004] 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 TCRspecificities 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.

[0005] 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 CD8ap co-receptor plays a major role in CD8 T-cell activation by increasing antigen sensitivity [Holler and Kramz, 2003] and by stabilizing the TCR-pMHC class I (pMHCI) interaction at the cell surface [Luescher etal., 1995], The pMHCI-CD8 interaction is central to these functional roles.

[0006] 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) [Miceli etal. 1991], It has been reported that during activation of T-cells, the CD4 receptor can fulfill several roles including an adhesion function, a signaling function as well as enhancement of T-cell sensitivity to antigens [Claeys and Vermeire, 2019],

[0007] In the past, adoptive cell therapy (ACT) largely focused on boosting the quantity and quality of anti-tumor CD8+ cytotoxic T lymphocyte (CTL) responses to generate therapeutic benefits. Only 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],

[0008] 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 engagementin the immunological synapse decreases the amount of antigen necessary to elicit T-cell activation [Kamphorst et al., 2015],

[0009] 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 CD28, 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],

[0010] Furthermore, 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 receptors of the immunoglobulin superfamily (IgSF) and tumor necrosis factor receptor superfamily (TNFRSF) have been mentioned that are known to have co-stimulatory function, there are also family members which are known to be bound by the inhibitory molecules - e.g. in the immunosuppressive tumor microenvironment - and to transmit the inhibitory effect to the T-cell. Within the IgSG, for example, PD1 and T cell immunoreceptor with Ig and ITIM domains (TIGIT) and TIM-3 have been described to transmit inhibitory signals coming from solid tumors that may inhibit activation, and / or promote exhaustion of T-cells.

[0011] In order to address the issue of the inhibitory effects to the T-cell described above, chimeric switch receptors (i.e. chimeric receptors comprising the extracellular domain of an inhibitory receptor and the cytoplasmic domain of an activating receptor) have been created to reverse the outcomes of their original signaling pathways in order toconfer immune cells with the ability to overcome the immunosuppressive tumor microenvironment and to allow them to have greater in vivo persistence.

[0012] For example, PD1 switch receptors have been described, wherein the extracellular domain of PD1 has been fused to the cytoplasmic domain of ICOS or CD28, respectively [WO 2013019615A2], In particular, in this international patent application, the described PD1 switch receptors with ICOS or CD28 co-stimulatory domains, respectively, could be co-expressed with a specific CAR in T-cells. However, in view of the diversity of immunosuppressive tumor microenvironments, and in light of the huge number of involved actors which may both positively and negatively regulate the suppressive activity of T-cells in adoptive cell therapy, it still remains a challenging task to provide effective T-cells exhibiting sufficient cytotoxicity and / or cytokine secretion, in particular in the immunosuppressive tumor microenvironment.

[0013] Thus, in view of the huge number of involved actors, in adoptive cell therapy, it thus remains a challenging task to provide effective T-cells exhibiting sufficient cytotoxicity.

[0014] Furthermore, high transduction / transfection efficiency is essential in adoptive T-cell therapies to efficiently introduce TCR / CAR genes into T lymphocytes. Despite mediating high rates of responses in some clinical trials, this approach can be limited by dysfunctional T-cells if they are present at high frequencies in the starting material from a patient. The fitness of the patient’s T-cells, driven by age, chronic infection, disease burden and cancer treatment, is therefore likely to be a crucial limiting factor of T-cell therapy [Mehta et al. 2021], Transduction / transfection efficiency in adoptive T-cell therapies, among other factors, depends on the fitness of the T-cells. The number of engineered T-cells manufactured is dependent on the proliferative potential, as well as the tranfection / transduction potential of the leukapheresis product and can restrict the dose of engineered T-cells administered. For example, robust T-cell activation and proliferation has been described to be needed for efficient CAR transduction with retroviral vectors and to be beneficial for efficient CAR transduction with lentiviral vectors [Riviere et al., 2000], Thus, transgenic ap-TCR, CAR T-cell receptor and / or Co-receptor integration and therefore expression level can be sub-optimal if the leukapheresis product predominantly contains cells with poor transduction / transfection potential due to poor fitness.

[0015] Accordingly, it is an object of the invention to provide for an improvement with respect to the above tasks and inconveniences.SUMMARY OF THE INVENTION

[0016] This object is inter alia accomplished by the chimeric human receptors, the isolated nucleic acids, the vectors, the isolated T-cells, the pharmaceutical compositions, the kits, and the methods having the features of the respective independent claims.

[0017] In a first aspect, the invention provides a chimeric human receptor, comprising a polypeptide having an extracellular polypeptide region, a transmembrane polypeptide region, and a cytoplasmic polypeptide region, wherein the cytoplasmic polypeptide region comprises at least two TNF receptor associated factor 6 (TRAF 6) binding motifs, and / or wherein said cytoplasmic polypeptide region comprises at least two CD40 TNF receptor associated factor 1 / 2 / 3 (TRAF 1 / 2 / 3) binding motifs.

[0018] The Inventors have found that the specific combination of costimulatory elements as herein described in the cytoplasmic polypeptide region of chimeric human receptors advantageously create an improved costimulatory domain which is able to synergistically increase the costimulatory activity of receptors involved in regulating T-cell activation, subset differentiation, effector function and survival. As demonstrated in the Examples of this application, the novel costimulatory domain as herein described functions e.g. in combination with co-receptors of T-cells such as e.g. the CD8 co-receptor, and also in combination with chimeric switch receptors such as e.g. a PD1 switch receptor.

[0019] In an embodiment of the first aspect, the invention is directed to the chimeric human receptor being a chimeric human CD8 co-receptor; and further wherein the polypeptide further comprises at least one CD8a polypeptide region (or a CD8a polypeptide region having at least 60% sequence identity with a functional polypeptide domain or a functional polypeptide motif of a wildtype human CD8a co-receptor), wherein said at least one CD8a polypeptide region comprises an CD8a IG-like domain region; further wherein said polypeptide comprises at least one CD8p polypeptide region (or a CD8p polypeptide region having at least 60% sequence identity with a functional polypeptide domain or a functional polypeptide motif of a wildtype human CD8p co-receptor), wherein said least one CD8p polypeptide region comprises an CD8p IG-like domain region.

[0020] The enhanced chimeric CD8 co-receptor of the present invention links an extra co-stimulatory domain to the human CD8 co-receptor. The co-stimulation associated with the improved costimulatory domain as herein described leads to a more potent TCR-T-cell product. The chimeric CD8 co-receptor as herewith provided thus enhances T-cell activation, proliferation, cytokine production, and cytotoxicity, ultimately improving the therapeutic efficacy of TCR-T-cell therapy.

[0021] For example, overexpression of the enhanced chimeric human CD8 coreceptor as herein described in T-cells alongside an engineered transgenic ap-T-cell receptor TCR and / or a chimeric antigen receptor, may offer several advantages and expands the therapeutic potential of this approach.

[0022] The provision of the enhanced chimeric CD8 co-receptor of the present invention allows for the efficient incorporation of CD4 T-cells into TCR-T-cell therapy, such that it becomes possible to harness their unique properties to augment the antitumor immune response.

[0023] 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 interactions. This known helper function may be crucial for enhancing the persistence and potency of TCR-T-cells within the tumor microenvironment.

[0024] It is noted that the usage of a small, single chain chimeric CD8 co-receptor as herein described, which incorporates both the CD8a IG-like domain region and the CD8p IG-like domain region, allows the addition of an extra co-stimulatory domain without reaching size limits fore.g. efficient viral vector delivery or knock-in template used in geneediting approach, or e.g. for mRNA packaged in nanoparticles such as lipid nanoparticles that may be used for in vivo therapy.

[0025] In this context, the chimeric human CD8 co-receptor according to the present invention offers advantages in terms of e.g. viral vector packaging efficiency. By utilizing a smaller chimeric CD8 co-receptor polypeptide, the overall size of the therapeutic construct may be reduced, compared with utilization of wildtype human CD8 co-receptor ap heterodimer, including the complete CD8a polypeptide and the complete CD8p polypeptide, and a complete tumor necrosis factor receptor superfamily protein or an immunoglobulin superfamily (IgSF) protein, respectively.

[0026] Thus, the chimeric human CD8 co-receptor according to the present invention enables more efficient packaging of genetic material into the vector, e.g. the viral vector. For example, it has been reported that the efficiency of packaging a delivery construct, also known as the transfer vector, into lentiviral particles varies greatly depending on the size of the transfer vector. Therefore, advantageously, this increase in packaging efficiency of the chimeric human CD8 co-receptor according to the present invention leads to higher yields of transduced / transfected T- cells during the manufacturing process, resulting in higher quantities of therapeutically active cells for infusion during adoptive T-cell therapy.

[0027] In addition to the benefits of size reduction, employing the chimeric CD8 coreceptor according to the present invention in TCR-T-cell therapy also offers the advantage of reducing the number of 2A cleavage sites. The 2A peptide sequence is commonly used in gene expression systems to co-express multiple proteins from a single transcript. However, the total number of 2A cleavage sites within the construct that can be theoretically used is limited to four: P2A (Thosea asigna virus 2A), T2A (FMDV 2A), E2A (Equine rhinitis A virus 2A), and F2A (Foot-and-mouth disease virus 2A). Among these, P2A has shown the highest efficiency, followed by T2A and E2A, while F2A has lower cleavage efficiency. Consequently, it is advisable to avoid using F2A, limiting the practical number of 2A sites to three instead of four. Therefore, utilizing the chimeric enhanced CD8 co-receptor according to the present invention may enable the efficient incorporation of additional elements into the vector construct design.

[0028] Thus, the enhanced chimeric CD8 co-receptor as herein described as embodiment in accordance with the first aspect of the invention significantly improves manufacturability, efficacy, and the overall success of adoptive T-cell therapy. By increasing viral vector packaging efficiency and reducing the number of 2A cleavage sites, higher quantities of functional TCR-T-cells may be manufactured, thereby improving treatment outcomes for various diseases, particularly cancer. By fusion of the extra costimulatory domain, moreover, a more potent TCR-T-cell product is generated.

[0029] In a further embodiment of the first aspect, the invention is directed to the chimeric human receptor being a chimeric costimulatory receptor, wherein the extracellular polypeptide region comprises an extracellular domain of a member of the T umor Necrosis Factor Receptor Superfamily (TNFRSF), such as e.g. the extracellular polypeptide domain of CD 95, or wherein the extracellular polypeptide region comprises an extracellular domain of LAG-3, CTLA4, PD1, TIGIT, or TIM-3.

[0030] The chimeric switch receptors as herein described link an extra co-stimulatory domain to the extracellular ligand binding domain of an inhibitory receptor. The Invnetors have found that the chimeric switch receptor polypeptides of the present invention, comprising the novel costimulatory domain as herein described, e.g. in combination with an engineered T-cell receptor, show improved functionality in providing 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.

[0031] In a second aspect, the invention provides an isolated nucleic acid comprising a nuclear acid sequence encoding for the chimeric human receptor according to the present invention.

[0032] In a third aspect, the invention provides a vector comprising the nucleic acid according to the present invention.

[0033] In a fourth aspect, the invention provides an isolated T-cell, the T-cell being introduced with, such as transfected, tranduced or transformed with the nucleic acid according to the present invention.

[0034] In a fifth aspect, the invention provides an isolated T-cell, the T-cell being introduced with, such as transfected, tranduced or transformed with, the vector according to the present invention (e.g. by electroporation).

[0035] In a sixth aspect, the invention provides an isolated T-cell, the T-cell being treated, such as transfected, transduced or transformed to express the chimeric human receptor according to the present invention (e.g. including the cell being treated by electroporation).

[0036] In a seventh aspect, the invention provides an engineered T-cell, the T-cell expressing the chimeric human receptor of the invention.

[0037] In an eighth aspect, the invention provides a kit comprising means to prepare the isolated T-cell according to the present invention.

[0038] In a ninth aspect, the invention provides a pharmaceutical composition comprising the isolated T-cell according to the present invention.

[0039] In a tenth aspect, the invention provides a method for preparing a T-cell for immunotherapy, comprisingisolating T-cells from a human subject,- transducing, transfecting or transforming the T-cells (e.g. using electroporation) with the nucleic acid according to the present invention or with the vector according to the present invention, and expanding the transduced T-cells.

[0040] In an eleventh aspect, the invention provides a pharmaceutical composition comprising T-cells expressing the chimeric human receptor according to the present invention.

[0041] 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.

[0042] In a thirteenth aspect, the invention provides a method for treating a patient having a disease, comprising introducing in vivo the nucleic acid as herein provided or the vector as herein provided into a T-cell of the patient.

[0043] All aspects of the invention provide the above-described advantages and improvements related to the fusion of the extra co-stimulatory domain as herein described to different kinds of human chimeric receptors as described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0044] 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:

[0045] Figs. 1 A and 1 B show a schematic representation of the CD8ap / CD4 derived basis for the construction of the enhanced chimeric CD8 co-receptor according to the present invention. In Fig. 1A both wildtype CD8 ap heterodimer and wildtype CD4 coreceptors are depicted including the functional domains. In particular, the CD8a coreceptor IG-like domain region, the CD8a stalk region, the CD8a transmembrane domain region, the CD8a LCK binding site, the CD8p co-receptor IG-like domain region, the CD8p stalk region, the CD8p transmembrane domain, the CD8p palmitoylation motif region, the CD4 stalk region, the CD4 transmembrane domain, and the CD4 cytoplasmic domain including CD4 Palmitoylation site and CD4 LCK binding site region are schematically illustrated. In Fig. 1B, the CD8ap / CD4 derived basis for the enhanced chimeric CD8 co-receptor according to the present invention including both the CD8a co-receptor IG-like domain and the CD8p co-receptor IG-like domain regions are illustrated together with optional polypeptide domains / regions / motif sites which may be used for construction of the enhanced chimeric CD8 co-receptor according to the present invention.

[0046] Fig. 2 shows a schematic representation of the enhanced chimeric CD8 coreceptors of the present invention. Functional domains are represented by circles, boxes and rhombuses, respectively. Different gradient fills are representative of the respective wildtype co-receptor functional elements are derived from.

[0047] 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 enhanced chimeric CD8 co-receptor polypeptides, against NCI-H2030 cancer cells.

[0048] Fig. 4 shows the graphical representation of the results according to Fig. 3 in a different scale.

[0049] Fig. 5 shows a graphical representation of results from a long term in-vitro T-cell killing assay of T-cells transduced with an engineered T cell receptor and a wildtype CD8 co-receptor or an enhanced chimeric CD8 co-receptor polypeptide, against NCI-H2030 cancer cells.

[0050] Fig. 6 shows a graphical representation of results from a cytokine secretion assay with an engineered T cell receptor and a wildtype or an enhanced chimeric CD8 co-receptor polypeptide, wherein Fig. 6A shows the secretion level of IFN-g, and Fig 6B shows the secretion level of TNF-a.

[0051] Figs. 7A, 7B and 7C show a graphical representation of results from a cytokine secretion assay with an engineered T cell receptor and a wildtype or an enhanced chimeric CD8 co-receptor polypeptide, wherein Fig. 7A shows the secretion level of Granzyme A, Fig 7B shows the secretion level of Granzyme B, and Fig. 7C shows the secretion level of Granulysine.

[0052] Figs. 8A and 8B shows a graphical representation of results from a cytokine secretion assay with an engineered T cell receptor and a wildtype or an enhanced chimeric CD8 co-receptor polypeptide, wherein Fig. 8A shows the secretion level of IL-4, and Fig 8B shows the secretion level of IL-10.

[0053] Fig. 9 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 enhanced chimeric CD8 co-receptor polypeptides comprising the specifically designed costimulatory cytoplasmic domain according to the invention (constructs pl-1296, pl-1298 and pl-1306), in comparison with T-cells transduced with enhanced chimeric CD8 co-receptor polypeptides pTK-638 and pl_1282, against NCI-H2030 cancer cells..

[0054] Fig. 10 shows a schematic representation of the cytoplasmic organization of functional motifs and linkers between said functional motifs of the enhanced chimeric CD8 co-receptor constructs pl_1296, pl_1298 and pl_1306 of the present invention, in comparison with chimeric CD8 co-receptor constucts pTK-638 and pl_1282.

[0055] Fig. 11 shows a schematic representation of a chimeric PD1 receptor polypeptide comprising the specifically designed costimulatory cytoplasmic domain according to the present invention.

[0056] Fig. 12 shows a graphical representation of results from a an in-vitro T-cell killing assay of T-cells transduced with an engineered T-cell receptor and the chimeric PD1 receptor polypeptides shown in Fig. 11 against PD-L1 -expressing NCI-H1703 cells.DETAILED DESCRIPTION OF THE INVENTION

[0057] As explained above, in a first aspect the invention is directed to a chimeric human receptor, comprising a polypeptide having an extracellular polypeptide region, a transmembrane polypeptide region, and a cytoplasmic polypeptide region, wherein the cytoplasmic polypeptide region comprises at least two TNF receptor associated factor 6 (TRAF 6) binding motifs, and / or wherein said cytoplasmic polypeptide region comprises at least two CD40 TNF receptor associated factor 1 / 2 / 3 (TRAF 1 / 2 / 3) binding motifs.

[0058] As demonstrated in the present application, the specifically designed cytoplasmic costimulatory domain / region as herein provided enables the human chimeric receptor of the present invention to increase (e.g. synergistically) the costimulatory activity of receptors involved in regulating T-cell activation, subset differentiation, effector function and survival.

[0059] According to an embodiment, at least one, or at least two, of the at least two TRAF 6 binding motifs may comprise a CD40 TRAF 6 binding motif.

[0060] According to an embodiment, the at least one cytoplasmic polypeptide region may comprise the at least two TNF receptor associated factor 6 (TRAF 6) binding motifs (such as e.g. two CD40 TRAF6 binding motifs), and may further comprise at least one TNF receptor associated factor 1, 2, 3 (TRAF 1 / 2 / 3) binding motif.

[0061] For example, the at least one TRAF 1 / 2 / 3 binding motif may comprise a CD40 TRAF 1 / 2 / 3 binding motif.

[0062] According to an embodiment, the at least one cytoplasmic polypeptide region may comprise the at least two TNF receptor associated factor 6 (TRAF 6) binding motifs (such as e.g. two CD40 TRAF6 binding motifs) and the least one CD40 TRAF 1 / 2 / 3 binding motifs.

[0063] According to an embodiment, the at least one cytoplasmic polypeptide region may comprise the at least two CD40 TNF receptor associated factor 1 / 2 / 3 (TRAF 1 / 2 / 3) binding motifs and at least one TNF receptor associated factor 6 (TRAF 6) binding motifs (such as at least one CD40 TRAF6 binding motif).

[0064] TRAF 6 binding motifs and TRAF 123 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 TRAF6 and / or TRAF1 , TRAF2 and TRAF3 to a specific binding motif. Thus, “TRAF6 binding” motif as referred to herein may relate toany polypeptide region which is functional in binding TRAF6. “TRAF 1 / 2 / 3” binding motifs as referred to herein may relate to any polypeptide region which is functional in binding TRAF1, TRAF2 and / or TRAF3.

[0065] “CD40 TRAF6 binding motif” as referred to herein relates to the specific polypeptide sequence of human wildtype CD40 which is necessary and sufficient for binding to TRAF6.

[0066] According to an embodiment, the CD40 TRAF6 binding motif may comprise or may consists of an amino acid sequence having at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity with the amino acid sequence as set forth in Seq ID No: 69. For example, the CD40 TRAF6 binding motif of the chimeric receptor may have one ore more conservative amino acid substitutions relative to the corresponding polypeptide domain, region or motif of the wildtype human CD40 comprising amino acid sequence 225 to 243 of UniProtKB database entry No. P25942 ■ TNR5_HUMAN, as set forth e.g. in SEQ ID No.79. According to some embodiments, for example, the “TRAF6” binding motif of wildtype CD40 as included in the chimeric receptor as herein provided may comprise a polypeptide region of CD40 including amino acid position 237 of SEQ ID No 79 (wildtype CD40) , and further wherein, at said position, the TRAF6 binding motif may be mutated, optionally wherein said mutation may consist of an exchange of an asparagine (N) to an aspartic acid (D). According to some embodiments, for example, the “TRAF6” binding motif of wildtype CD40 as included in the chimeric PD1 receptor as herein provided may comprise a polypeptide region of CD40 including amino acid position 229 of SEQ ID No 79 (wildtype CD40) , and further wherein, at said position, the TRAF6 binding motif may be mutated, optionally wherein said mutation may consist of an exchange of an 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. Seq ID No. 69 has herein referred to shows the functional CD40 TRAF6 binding motif including the function-preserving mutation at position 229 of SEQ ID No 79.

[0067] For example, the at least one CD40 TRAF6 binding motif may have at least 70%, or at least 71 %, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81 %, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, orat least 98%, or at least 99%, or 100% sequence identity with the functional polypeptide domain or a functional polypeptide motif of a wildtype human CD40 TRAF6 binding motif (e.g. being comprised in or consisting of a polypeptide having an amino acid sequence as set forth in Seq ID No. 69).

[0068] “CD40 TRAF1 / 2 / 3 binding motif” as referred to herein relates to the specific polypeptide sequence of human wildtype CD40 which is necessary and sufficient for binding to TRAF 1 , TRAF 2 and / or TRAF3.

[0069] According to an embodiment, the CD40 TRAF1 / 2 / 3 binding motif may comprise or may consists of an amino acid sequence having at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity with the amino acid sequence as set forth in Seq ID No: 70. For example, the CD40 TRAF6 binding motif may have one ore more conservative amino acid substitutions relative to the functional polypeptide domain, region or motif of the human CD40 (Seq ID 70).

[0070] For example, the at least one CD40 TRAF1 / 2 / 3 binding motif may have at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81 %, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with the functional polypeptide domain or a functional polypeptide motif of a wildtype human CD40TRAF1 / 2 / 3 binding motif (e.g. being comprised in or consisting of a polypeptide having an amino acid sequence as set forth in Seq ID No. 70).

[0071] According to an embodiment, the cytoplasmic polypeptide region may comprise a complete cytoplasmic domain of CD40.

[0072] According to an embodiment, the complete cytoplasmic domain of CD40 may comprise or may consists of an amino acid sequence having at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity with the amino acid sequence as set forth in Seq ID No: 18. For example, the complete cytoplasmic domain of CD40 may have one ore more conservative amino acid substitutions relative to the functional polypeptide domain of the human wildtype CD40 complete cytoplasmic domain (Seq ID No. 18).

[0073] For example, the complete cytoplasmic domain of CD40 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 of a wildtype human CD40 cytoplasmic domain (e.g. Seq ID No. 18).

[0074] It is unserstood herein that a complete cytoplasmic domain of CD40 comprises at least both the at least one CD40 TRAF1 / 2 / 3 binding motif, and the at least one CD40 TRAF6 binding motif as referred to herein.

[0075] According to an embodiment, the cytoplasmic polypeptide region may comprise a complete cytoplasmic domain of CD40, a CD40 TRAF6 binding motif, and a CD40 TRAF 1 / 2 / 3 binding motif.

[0076] It is contemplated that the cytoplasmic polypeptide region may comprise at least one linker between the at least two TRAF 6 binding motifs, and / or between the at least two CD40 TRAF 1 / 2 / 3 binding motifs, and / or between at least one TRAF 1 / 2 / 3 binding motif and at least one TRAF 6 motif, and / or between the complete CD40 cytoplasmic domain and at least one TRAF6 domain, and / or between the complete CD40 cytoplasmic domain and at least one TRAF1 / 2 / 3 domain.

[0077] For example, the linker may comprise or consist of an amino acid sequence as set forth by SEQ ID No. 58 (GGGGS).

[0078] According to an embodiment, the cytoplasmic polypeptide region may comprise or consist of an amino acid sequence having at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity with the amino acid sequence as set forth in any one of the Seq ID No: 80, 81, 82, or 83.

[0079] According to an embodiment, the chimeric human receptor of the present invention may be a chimeric co-receptor, such as a chimeric CD8 co-receptor.

[0080] For example, the polypeptide may comprise a human wildtype CD8a extracellular polypeptide region and a human wildtype CD8a transmembrane polypeptide region, or a human wildtype CD8p extracellular polypeptide region and a human wildtype CD8p transmembrane polypeptide region.

[0081] According to an embodiment, the chimeric human receptor of the present invention may be a chimeric CD8 co-receptor; wherein further the 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. 1), and wherein said least one CD8a (-derived) polypeptide region comprises an CD8a (-derived) IG-like domain region; and 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. 2), wherein said least one CD8p (-derived) polypeptide region comprises an CD8p (-derived) IG-like domain region

[0082] According to an embodiment, the chimeric human receptor of the present invention may be a chimeric CD8 co-receptor; wherein further the polypeptide comprises at least one CD8a polypeptide region, wherein said at least one CD8a polypeptide region comprises an CD8a IG-like domain region; further wherein said polypeptide comprises at least one CD8p polypeptide region, wherein said least one CD8p polypeptide region comprises an CD8p IG-like domain region.

[0083] It is understood that the expression of 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. 1. It is further understood that the expression of 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. 2.

[0084] 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.

[0085] 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: BLOSLIM 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.

[0086] It is noted in this context that it has been found that the chimeric human CD8 co-receptor polypeptide comprising both an CD8a IG-like domain region together with an CD8p IG-like domain region in combination with the specific costimulatory domain as herein described is able to enhance T-cell activation, proliferation, cytokine production, and cytotoxicity, thereby ultimately improving the therapeutic efficacy of TCR-T-cell therapy. The provision of a chimeric CD8 co-receptor polypeptide comprising both an CD8a IG-like domain region together with an CD8p IG-like domain region in combination with the costimulatory domain as described above allows for advantages in terms of viral vector packaging efficiency, by utilizing a smaller chimeric CD8 co-receptor polypeptide.

[0087] According to an embodiment, the at least one CD8a 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. 1).

[0088] For example, the at least one CD8a polypeptide region may have at least 70%, or at least 71 %, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81 %, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with the functional polypeptide domain or a functional polypeptide motif of a wildtype human CD8a Co-receptor (e.g. Seq ID No. 1).

[0089] According to an embodiment, the at least one CD8p 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. 2).

[0090] For example, the at least one CD8p 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. 2).

[0091] The at least one CD8a polypeptide region having at least 60% sequence identity with CD8a IG-like domain region of a human wildtype CD8a co-receptor may have generally a sufficient portion of the human wildtype CD8a IG-like domain region to be able to bind to MHC. For example, said at least one CD8a polypeptide region having at least 60% sequence identity with CD8a IG-like domain region of a human wildtype CD8a co-receptor may comprise the complete or a considerable part of the human wildtype CD8a IG-like domain region, as set forth e.g. in SEQ ID NO.: 4, but in an embodiment may comprise at least 10, 20, 30, 40, 50, 60, 70, 80, 80, 100, or 110 amino acids of the human wildtype CD8a IG-like domain region. 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 immunoglobuline proteins.

[0092] The at least one CD8p polypeptide region having at least 60% sequence identity with CD8p IG-like domain region of a human wildtype CD8p co-receptor may have generally a sufficient portion of the human wildtype CD8p IG-like domain region to be able to bind to MHC. For example, said at least one CD8p polypeptide region having at least 60% sequence identity with CD8p IG-like domain region of a human wildtype CD8p co-receptor may comprise the complete or a considerable part of the human wildtype CD8p IG-like domain region, as set forth e.g. in SEQ ID NO.: 5, but in an embodiment may comprise at least 10, 20, 30, 40, 50, 60, 70, 80, 80, 100, or 110 amino acids of the human wildtype CD8p IG-like domain region. 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 immunoglobuline proteins.

[0093] According to an embodiment, the chimeric human CD8 co-receptor polypeptide may be a single-chain polypeptide.

[0094] According to an embodiment, the chimeric CD8 co-receptor comprises the CD8a IG-like domain region, the CD8p IG-like domain region, a stalk region, a transmembrane domain region; and an intracellular / cytoplasmic domain region, wherein the intracellular domain region comprises a palmitoylation motif region and a LCK binding site region.

[0095] The expressions “domain region”, “binding site region”, “motif region” as used herein are understood to relate to e.g. a region of the chimeric receptor which is necessary and / or sufficient for a biological function of the chimeric receptor, or to a region of the chimeric receptor (e.g. of the chimeric CD8 co-receptor) which is defined e.g. by a localization with respect to a cell, or to a structurally defined unit of the chimeric 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 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.

[0096] According to an embodiment, the at least one CD8a polypeptide region further comprises at least one polypeptide region selected from the group consisting of a stalk domain region, a transmembrane domain region, and a LCK binding site motif region.

[0097] According to another embodiment, the at least one CD8p polypeptide region further comprises at least one polypeptide region selected from the group consisting of a stalk domain region, a transmembrane domain region, a cytoplasmic region, and a palmitoylation motif region.

[0098] Thus, it is contemplated that the chimeric CD8 co-receptor polypeptide may comprise, in addition to comprising the CD8a IG-like domain region and the CD8p IG-like domain region, further domain regions / motif regions / binding site regions from one or both of a wildtype human CD8a co-receptor and / or a wildtype human CD8p co-receptor in every conceivable combination to establish a functional chimeric CD8 co-receptor polypeptide, together with the specific cytoplasmic costimulatory domain as herein described.

[0099] For example, it has been described that wildtype human CD8a co-receptor comprises a stalk domain region, a transmembrane domain region, and a LCK binding site motif region. Furthermore, it has been reported that the human wildtype CD8p- co-receptor comprises a stalk domain region, a transmembrane domain region, and a palmitoylation motif region (e.g. Wong et al.; 2003).

[0100] For example, the expression “wildtype human CD8a co-receptor stalk domain region” as referred to herein may relate to a polypeptide having an amino acid sequenceas set forth in SEQ ID NO: 6. For example, the expression “wildtype human CD8a coreceptor stalk domain region” as referred to herein may relate to a polypeptide comprising amino acid sequence 136-182 of UniProtKB database entry No. P01732 ■ CD8A_HUMAN, as set forth e.g. in SEQ ID No. 1. Thus, the expression “stalk region” as used herein may refer to a protein portion that preserves the ability to propagate signal transduction of a functional CD8 co-receptor analogous to a wildtype human CD8a co-receptor stalk domain region. For example, the expression “wildtype human CD8a co-receptor transmembrane domain region” as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 7. For example, the expression “wildtype human CD8a co-receptor transmembrane domain region” as referred to herein may relate to a polypeptide comprising amino acid sequence 183-203 of UniProtKB database entry No. P01732 ■ CD8A_HUMAN, as set forth e.g. in SEQ ID No. 1. For example, the expression “wildtype human CD8a co-receptor LCK binding site motif region” as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 8. For example, the expression “wildtype human CD8a co-receptor LCK binding site motif domain region” as referred to herein may relate to a polypeptide comprising amino acid sequence CKCP at amino acid positions 215-218 of UniProtKB database entry No. P01732 ■ CD8A_HUMAN, as set forth e.g. in SEQ ID No. 1. Thus, the expression “LCK binding site” as used herein may refer to a protein portion that preserves the ability to recruite the Src family kinase Lek of a functional CD8 co-receptor.

[0101] For example, the expression “wildtype human CD8p co-receptor stalk domain region” as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 9. For example, the expression “wildtype human CD8p co-receptor stalk domain region” as referred to herein may relate to a polypeptide comprising amino acid sequence 133-170 of UniProtKB database entry No. P10966 ■ CD8B_HUMAN, as set forth e.g. in SEQ ID No. 2. Thus, the expression “stalk region” as used herein may refer to a protein portion that preserves the ability to propagate signal transduction of a functional CD8 co-receptor analogous to a wildtype human CD8p co-receptor stalk domain region. For example, the expression “wildtype human CD8p co-receptor transmembrane domain region” as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 10. For example, the expression “wildtype human CD8p co-receptor transmembrane domain region” as referred to herein may relate to a polypeptide comprising amino acid sequence 171-191 of UniProtKB database entry No. P10966 ■ CD8B_HUMAN, as set forth e.g. in SEQ ID No. 2. For example, expression “thewildtype human CD8p co-receptor palmitoylation motif region” as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 11. For example, the expression “wildtype human CD8p co-receptor palmitoylation motif region” as referred to herein may relate to the two conserved amino acids Cysteine at amino acid positions 194 and 195 with respect to the amino acid sequence of UniProtKB database entry No. P10966 ■ CD8B_HUMAN, as set forth e.g. in SEQ ID No. 2. Thus, the expression “palmitoylation motif region” as used herein may refer to a protein portion that preserves the ability to propagate signal transduction of a functional CD8 co-receptor analogous to a wildtype human CD8p palmitoylation motif region region.

[0102] According to an embodiment, the chimeric CD8 Co-receptor may further comprise at least one CD4 (-derived) polypeptide region having at least 60% sequence identity with a functional polypeptide domain or a functional polypeptide motif of a human CD4 co-receptor.

[0103] It is understood that the expression human wildtype CD4 co-receptor relates to a protein having an amino acid sequence according to UniProtKB database entry No. P01730 ■ CD4_HUMAN, as set forth e.g. in SEQ ID No. 3.

[0104] According to an embodiment, the at least one CD4 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 CD4 Co-receptor (Seq ID No. 3).

[0105] For example, the at least one CD4 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 ora functional polypeptide motif of a wildtype human CD4 co-receptor (Seq ID No.3).

[0106] Thus, it is contemplated that the chimeric CD8 co-receptor polypeptide may comprise, in addition to comprising the CD8a IG-like domain region and the CD8p- IG-like domain region, at least one domain region / motif region / binding site region from a wildtype human CD4 co-receptor. Such at least one CD4 domain region / motif region / binding site region may be combined with further CD4 domain region / motif region / binding site regionand / or further domain regions / motif regions / binding site regions from one or both of a wildtype human CD8a co-receptor and / or a wildtype human CD8p co-receptor in every conceivable combination to establish a functional chimeric CD8 co-receptor polypeptide. Without being bound to theory, it is herewith contemplated that inclusion of a CD4 co-receptor derived functional polypeptide domain / motif region / binding site region into the chimeric CD8 co-receptor may contribute to incorporating CD4 T-cells into TCR-T-cell therapy.

[0107] As described herein, the expressions “CD8a-derived polypeptide region”, “CD8p-derived polypeptide region” and “CD4-derived polypeptide region” may be used herein interchangeably with the expressions “CD8a polypeptide region”, “CD8p polypeptide region” and “CD4 polypeptide region”, respectively, to refer to a (functional) portion of a wildtype CD8a, a wildtype CD8p ora wildtype CD4 protein (or variants thereof), respectively. With respect to variants of wildtype CD8a, wildtype CD8p or wildtype CD4 protein portions, 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, SEQ ID NO: 2 or SEQ ID NO 3, respectively. For example, variants of wildtype CD8a as herein described may comprise an extracellular domain that preserve the ability to bind peptide-MCH class I. Suitable methods (such as e.g. surface plasmon resonance assay) for determining said functional ability are known to the skilled person. For example, variants of wildtype CD8p as herein described may comprise an extracellular domain that preserve the ability to bind peptide-MCH class I. Suitable methods (such as e.g. surface plasmon resonance assay) for determining said functional ability are known to the skilled person. For example, variants of wildtype CD4 protein portions as described herein may comprise a CD4 stalk, a CD4 transmembrane and / or a CD4 cytoplasmic domain that preserve the ability to propagate signal transduction. Suitable methods for determining said functional ability are known to the skilled person (e.g. as described by [Capone M., et al., 2021]).

[0108] The wildtype human CD4 co-receptor polypeptide is known to comprise -among others - a stalk domain region, a transmembrane domain region, and a cytoplasmic region comprising a palmitoylation motif region and a LCK binding site region.

[0109] For example, the expression “wildtype human CD4 co-receptor stalk domain region” as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 12. For example, the expression “wildtype human CD4 co-receptor stalk domain region” as referred to herein may relate to a polypeptide comprising amino acid sequence 375-396 of UniProtKB database entry No. P01730 ■ CD4_HUMAN, as set forth e.g. in SEQ ID No. 3. For example, the expression “wildtype human CD4 coreceptor transmembrane domain region” as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 13. For example, the expression “wildtype human CD4 co-receptor transmembrane domain region” as referred to herein may relate to a polypeptide comprising amino acid sequence 397-418 of UniProtKB database entry No. P01730 ■ CD4_HUMAN, as set forth e.g. in SEQ ID No. 3. For example, the expression “wildtype human CD8a co-receptor LCK binding site motif region” as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 14. For example, the expression “wildtype human CD8a co-receptor LCK binding site motif domain region” as referred to herein may relate to a polypeptide comprising amino acid sequence 442-449 (KKTCQCPH) of UniProtKB database entry No. P01730 ■ CD4_HUMAN, as set forth e.g. in SEQ ID No. 3. For example, the expression “wildtype human CD4 co-receptor palmitoylation motif region” as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 15. For example, the expression “wildtype human CD4 co-receptor palmitoylation motif region” as referred to herein may relate to a polypeptide comprising amino acid sequence 419-422 (CVRC) of UniProtKB database entry No. P01730 ■ CD4_HUMAN, as set forth e.g. in SEQ ID No. 3.

[0110] According to an embodiment, the at least one CD4 polypeptide region comprises at least one polypeptide region selected from the group consisting of a stalk domain region, a transmembrane domain region, a cytoplasmic domain, a palmitoylation motif region, and a LCK binding site region.

[0111] In certain embodiments, the chimeric CD8 co-receptor polypeptide may comprise a CD4 polypeptide region comprising a wildtype human CD4 co-receptor cytoplasmic region including a palmitoylation motif region and a LCK binding site region. For example, the expression “wildtype human CD4 co-receptor cytoplasmic region” as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 16. 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. 3.

[0112] It is contemplated that the chimeric CD8 co-receptor may comprise a stalk domain region, a transmembrane domain region, a palmitoylation motif region, and a LCK binding site region.

[0113] According to an embodiment, the chimeric CD8 co-receptor comprises, in an N-terminal to C-terminal order, at least the following polypeptide regions: a CD8a IG-like domain, a CD8p IG-like domain, a stalk region (e.g. a CD8p stalk region), a transmembrane region (e.g. a CD8a transmembrane region), a palmitoylation motif region and an LCK binding site region (e.g. a complete cytoplasmic domain from CD40), and the specific co-stimulatory domain as herein described. For example, the chimeric CD8 co-receptor may further comprise a signal peptide located N-terminally with respect to the above polypeptide regions.

[0114] According to certain embodiments, the CD8a IG-like domain region may be located closer to the N-terminal end of the chimeric CD8 co-receptor polypeptide than the CD8p IG-like domain region. Alternatively, it is herewith also envisaged that the CD8p IG-like domain region may be located closer to the N-terminal end of the chimeric CD8 Co-receptor polypeptide than the CD8a IG-like domain region.

[0115] In some embodiments, the chimeric CD8 co-receptor polypeptide may further comprise at least one linker region.

[0116] For example, the polypeptide may comprise a linker region between the CD8a IG like domain region and said CD8p IG-like domain region.

[0117] 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.

[0118] According to an embodiment, a linker region of the enhanced chimeric CD8 co-receptor may comprise the amino acid sequence (GGGS)nor (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.

[0119] According to an embodiment, the enhanced chimeric CD8 co-receptor polypeptide may comprise at least one linker region comprising the amino acid sequence (GGGS)n, wherein n is 4.

[0120] According to an embodiment, the enhanced chimeric CD8 co-receptor polypeptide may comprise a linker region between the stalk domain region and the IG-like domain region of the polypeptide.

[0121] According to an embodiment, the enhanced chimeric CD8 co-receptor polypeptide may be able to enhance cytotoxicity of a T-cell.

[0122] According to an embodiment, the enhanced chimeric CD8 co-receptor polypeptide may be able to enhance T-cell activation, proliferation, and / or production of activating cytokines.

[0123] According to an embodiment, the enhanced chimeric CD8 co-receptor polypeptide may be able to decrease production of inhibitory cytokines.

[0124] It is further envisaged that the chimeric CD8 co-receptor may comprise at least one cytoplasmic polypeptide domain, region or motif of CD28, HVEM, DR3, CD40, LTBR, 4-1 BB, 0X40, CD27, GITR, CD30, ICOS, CD226, CRTAM, TIM 1, CD2, SLAM, CD 84, Ly9, and / or CRACC.

[0125] In accordance with an embodiment, the chimeric CD8 co-receptor may comprise an Immunoreceptor tyrosine-based activation motif (ITAM), e.g. ITAM1. The ITAM motif has been reported, for example, to be contained in the cytoplasmic domain of transmembrane adaptor molecules that are associated with and transmit signals from various immunoreceptors [LB Ivashkiv, 2009], For example, the expression ITAM1 motif as referred to herein may relate to a polypeptide comprising the amino acid sequence as set forth in SEQ ID. No. 20. Other ITAM motifs known in the art may also be used.

[0126] It is contemplated that the chimeric CD8 co-receptor may comprise any functional combination of the at least one cytoplasmic polypeptide domain or cytoplasmic polypeptide motif of the tumor necrosis factor receptor superfamily protein, and / or of the immunoglobulin superfamily (IgSF) protein, and / or of the ITAM-associated receptor.

[0127] For example, the ITAM 1 polypeptide sequence as set forth in SEQ ID. No.20 may be combined in a chimeric CD8 co-receptor with a cytoplasmic domain of CD28 or with a cytoplasmic domain of 4-1 BB.

[0128] According to an embodiment, the chimeric CD8 co-receptor 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 CD8 co-receptor may comprise at least one functional, costimulatory 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 No. P28908- TNR8_HUMAN, as set forth e.g. in SEQ ID No.17. As explained before, it is further envisaged that the cytoplasmic polypeptide region or motif of CD40 that may be used in the chimeric CD8 co-receptor may comprise the complete cytoplasmic domain of wildtype human CD40. In other embodiments, thecytoplasmic polypeptide region of CD40 included in the chimeric CD8 co-receptor may comprise at least one functional, co-stimulatory motif / domain / region of the complete wildtype human CD40 cytoplasmic domain which comprises the CD40 TRAF6 and or the CD40 TRAF1 / 2 / 3 motif as referred to herein. 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. 18.

[0129] In an embodiment, a cytoplasmic polypeptide region of CD30 that may be used in the chimeric CD8 co-receptor may comprise the amino acid sequence 553 - 583 of UniProtKB database entry No. P28908- TNR8_HUMAN, as set forth e.g. in SEQ ID No.19 (CD30 motif).

[0130] Cytoplasmic polypeptide domains, regions or motifs with co-stimulatory effect of CD30 and CD40 may be combined in any way in the enhanced chimeric CD8 co-receptor.

[0131] For example, the CD30 motif as set forth in Seq ID NO. 19 and the entire CD 40 cytoplasmic domain may be used in combination in the chimeric CD8 co-receptor. It is envisaged that either the entire CD40 cytoplasmic domain, or the CD30 motif may be located closer to the C-terminal end of the chimeric CD8 co-receptor. Between the CD30 motif region and the CD40 cytoplasmic region, for example, a linker region may be provided. In principle, any known linker region may be used, for example one of the polypeptide linker regions as described elsewhere herein. In an embodiment, the linker region may comprise a GGGS polypeptide sequence.

[0132] According to an embodiment, the chimeric CD8 co-receptor may comprise at least one cytoplasmic polypeptide domain, region or motif of CD28. For example, a cytoplasmic polypeptide region of CD28 may comprise the complete cytoplasmic domain of wildtype human CD28. In other embodiments, the cytoplasmic polypeptide region of CD28 included in the chimeric CD8 co-receptor may comprise at least one functional, co-stimulatory motif / domain / region of the complete wildtype human CD28 cytoplasmic domain. For example, the expression “wildtype human CD28 cytoplasmic domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 180-220 of UniProtKB database entry No. P10747- CD28_HUMAN, as set forth e.g. in SEQ ID No.21.

[0133] According to an embodiment, the chimeric CD8 co-receptor 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 domainof wildetype human 4-1 BB. In other embodiments, the cytoplasmic polypeptide region of 4-1 BB included in the chimeric CD8 co-receptor may comprise at least one functional, costimulatory motif / domain / region of the complete wildtype human 4-1 BB cytoplasmic domain. For example, the expression “wildtype human 4-1 BB cytoplasmic domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 214-255 of UniProtKB database entry No. Q07011 ■ TNR9_HUMAN, as set forth e.g. in SEQ ID No.22.

[0134] According to an embodiment, the chimeric CD8 co-receptor 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 wildetype human ICOS. In other embodiments, the cytoplasmic polypeptide region of ICOS included in the chimeric CD8 co-receptor may comprise at least one functional, costimulatory 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. 23.

[0135] According to an embodiment, the chimeric CD8 co-receptor 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 wildetype human HVEM. In other embodiments, the cytoplasmic polypeptide region of HVEM included in the chimeric CD8 co-receptor may comprise at least one functional, costimulatory 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. 24.

[0136] According to an embodiment, the chimeric CD8 co-receptor may comprise at least one cytoplasmic polypeptide domain, region or motif of 0X40. For example, a cytoplasmic polypeptide region of 0X40 may comprise the complete cytoplasmic domain of wildetype human 0X40. In other embodiments, the cytoplasmic polypeptide region of 0X40 included in the chimeric CD8 Co-receptor may comprise at least one functional, costimulatory motif / domain / region of the complete wildtype human 0X40 cytoplasmic domain. For example, the expression “wildtype human 0X40 cytoplasmic domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 236-277of UniProtKB database entry No. P43489- TNR4_HUMAN, as set forth e.g. in SEQ ID No.25.

[0137] According to an embodiment, the chimeric CD8 co-receptor 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 wildetype human CD27. In other embodiments, the cytoplasmic polypeptide region of CD27 included in the chimeric CD8 co-receptor may comprise at least one functional, costimulatory 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.26.

[0138] Cytoplasmic polypeptide domains, regions or motifs with co-stimulatory effect of e.g. CD28, HVEM, DR3, CD40, LTBR, 4-1 BB, 0X40, CD27, GITR, CD30, (ICOS), CD226, CRTAM, TIM 1, CD2, SLAM, CD 84, Ly9, CRACC; and / or ITAM may be combined in anyway in the enhanced chimeric CD8 co-receptor with the CD8a, CD8p- and, in some embodiments, e.g. CD4 polypeptide regions.

[0139] For example, in accordance with an embodiment, the at least one CD4 polypeptide region of the chimeric CD8 co-receptor further comprises a CD4 derived palmitoylation motif region, and a CD4 LCK binding site region. For example, the at least one CD4 polypeptide comprises the entire wildtype human CD4 co-receptor cytoplasmic region. For example, the at least one CD4 polypeptide region may also further comprise a CD4 Transmembrane domain region, and the stalk domain region may be derived from CD8p.

[0140] For example, in accordance with an embodiment, the at least one CD4-derived polypeptide region of the chimeric CD8 co-receptor further comprises a CD4 derived palmitoylation motif region, and a CD4 LCK binding site region. For example, the at least one CD4 polypeptide comprises the entire wildtype human CD4 co-receptor cytoplasmic region. For example, the at least one CD4 polypeptide region may also further comprise a CD4 Transmembrane domain region, and the stalk domain region may be derived from CD8a.

[0141] For example, according to an embodiment, the at least one CD4-derived polypeptide region of the chimeric CD8 co-receptor further comprises a CD4 derivedpalmitoylation motif region, and a CD4 LCK binding site region. For example, the at least one CD4 polypeptide comprises the entire wildtype human CD4 co-receptor cytoplasmic region. For example, the at least one CD4 polypeptide region may also further comprise a CD4 Transmembrane domain region. According to this embodiment, also the stalk region may be derived from wildtype human CD4 co-receptor. Between the CD 4 stalk region and the CD8p IG-like domain region, a linker may be present. According to this embodiment, also between the CD8p IG-like domain region and the CD8a IG-like domain region, a linker may be present. For example, the linker may have an amino acid sequence or (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.

[0142] For example, according to an embodiment, the at least one CD4 polypeptide region of the chimeric CD8 co-receptor further comprises a CD4 derived palmitoylation motif region, and a CD4 LCK binding site region. For example, the at least one CD4 polypeptide comprises the entire wildtype human CD4 co-receptor cytoplasmic region. In this embodiment, the at least one CD8a polypeptide region may also further comprise a CD8a Transmembrane domain region and e.g. a CD8a derived stalk region.

[0143] For example, according to an embodiment, the at least one CD4 polypeptide region of the chimeric CD8 co-receptor further comprises a CD4 derived palmitoylation motif region, and a CD4 LCK binding site region. For example, the at least one CD4 polypeptide comprises the entire wildtype human CD4 co-receptor cytoplasmic region. In this embodiment, the at least one CD8p polypeptide region may also further comprise a CD8p Transmembrane domain region and a CD8p stalk domain region.

[0144] For example, according to a further embodiment, the at least one CD4 polypeptide region of the chimeric CD8 co-receptor further comprises a CD4 palmitoylation motif region, and a CD4 LCK binding site region. For example, the at least one CD4 polypeptide comprises the entire wildtype human CD4 co-receptor cytoplasmic region. In this embodiment, the at least one CD8a polypeptide region may also further comprise a CD8a Transmembrane domain region and the at least one CD8p polypeptide region may comprise a CD8p stalk domain region.

[0145] For example, according to another embodiment, the at least one CD4 polypeptide region of the chimeric CD8 co-receptor further comprises a CD4 palmitoylation motif region, and a CD4-derived LCK binding site region. For example, the at least one CD4 polypeptide comprises the entire wildtype human CD4 co-receptor cytoplasmic region. For example, the at least one CD4 polypeptide region may also further comprise a CD4 Transmembrane domain region. In this embodiment, the at least one CD8apolypeptide region may also further comprise a CD8a stalk region. In this embodiment, the CD8p IG-like domain region may be located closer to the N-terminal end of the chimeric CD8 Co-receptor polypeptide than the CD8a IG-like domain region.

[0146] For example, according to another embodiment, the at least one CD4-derived polypeptide region of the chimeric CD8 co-receptor further comprises a CD4 derived palmitoylation motif region, and a CD4 LCK binding site region. For example, the at least one CD4 derived polypeptide comprises the entire wildtype human CD4 co-receptor cytoplasmic region. For example, the at least one CD8a polypeptide region may also further comprise a CD8a T ransmembrane domain region and a CD8a derived stalk region. In this embodiment, the CD8p IG-like domain region may be located closer to the N-terminal end of the chimeric CD8 co-receptor polypeptide than the CD8a IG-like domain region.

[0147] It is further envisaged that the chimeric CD8 co-receptor may comprise a modified stalk region. For example, the stalk polypeptide region of the chimeric CD8 co-receptor may comprise a modified CD8a stalk region, a modified CD8p stalk region or a modified CD4 stalk region. For example, a modified CD8a stalk region may comprise a polypeptide region which may be elongated, or which may be shortened in length with respect to the length of the wildtype CD8a stalk region. In other embodiments, the CD8 co-receptor may comprise a modified CD8p stalk region which may be elongated, or which may be shortened in length with respect to the length of the wildtype CD8p stalk region. Also envisaged are embodiments wherein the chimeric CD8 co-receptor may comprise a modified CD4 stalk region which may be elongated, or which may be shortened in length with respect to the length of the wildtype CD4 stalk region.

[0148] For example, according to some embodiments, the chimeric CD8 co-receptor may comprise a CD8a stalk region which may be shortened in length with respect to the length of the wildtype CD8a stalk region. It has been suggested that the length of the stalk region of co-receptors may be relevant for establishing the geometry of corresponding ternary TCR-pMHC-co-receptor complexes (Li et al.; 2013). Without being bound to theory, it is thus contemplated that chimeric CD8 co-receptors with a truncated CD8a stalk region may mimic functionality of e.g. shorter CD4 stalk regions, thereby contributing to incorporating CD4+ T- cells into TCR-T-cell therapy.

[0149] For example, according to an embodiment, the at least one CD4 polypeptide region of the chimeric CD8 co-receptor thus further comprises a CD4 derived palmitoylation motif region, and a CD4 LCK binding site region. For example, the at least one CD4 polypeptide comprises the entire wildtype human CD4 co-receptor cytoplasmic region. Furthermore, the at least one CD4 polypeptide region may also further comprise aCD4 Transmembrane domain region. According to this embodiment, the at least one CD8a polypeptide region of the chimeric CD8 co-receptor further comprises a CD8a stalk region which may be shortened in length with respect to the length of the wildtype CD8a stalk region. In this embodiment, the CD8a IG-like domain region may be located closer to the N-terminal end of the chimeric CD8 co-receptor polypeptide than the CD8p-IG-like domain region.

[0150] For example, according to an embodiment, the at least one CD4 polypeptide region of the chimeric CD8 co-receptor thus further comprises a CD4 derived palmitoylation motif region, and a CD4 LCK binding site region. For example, the at least one CD4 polypeptide comprises the entire wildtype human CD4 co-receptor cytoplasmic region. In this embodiment, the at least one CD8a-derived polypeptide region may also further comprise a CD8a transmembrane domain region, and a CD8a stalk region which may be shortened in length with respect to the length of the wildtype CD8a stalk region. According to this embodiment, the CD8a IG-like domain region may be located closer to the N-terminal end of the chimeric CD8 co-receptor polypeptide than the CD8p IG-like domain region.

[0151] In principle, each of the above-described exemplary embodiments may serve as a co-receptor basis for the enhanced chimeric CD8 co-receptor of the present invention. For example, with each of the combinations of CD8a, CD8p and / or CD4 polypeptide regions, the at least one co-stimulatory cytoplasmic polypeptide region as herein provided may be combined in order to constitute the enhanced chimeric CD8 co-receptor as herewith provided.

[0152] According to an embodiment, the at least one CD4 polypeptide region of the chimeric CD8 co-receptor further comprises a CD4-derived palmitoylation motif region, and a CD4-derived LCK binding site region. For example, the at least one CD4-derived polypeptide comprises the entire wildtype human CD4 co-receptor cytoplasmic region. In this embodiment, the at least one CD8a-derived polypeptide region may also further comprise a CD8a-derived Transmembrane domain region and the at least one CD8p-derived polypeptide region may comprise a CD8p-derived stalk domain region. In this embodiment, the CD8a-derived IG-like domain region may be located closer to the N-terminal end of the enhanced chimeric CD8 Co-receptor polypeptide than the CD8p-derived IG-like domain region. For example, the chimeric CD8 co-receptor may further comprise an Immunoreceptor tyrosine-based activation motif (ITAM), e.g. ITAM1. For example, the ITAM1 costimulatory motif may be located at the C-terminal end of the enhanced chimeric CD8 co-receptor. An enhanced chimeric CD8 co-receptor in accordance with this embodiment may e.g. comprise a polypeptide having an amino acidsequence 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:28 (pTK-0628).

[0153] According to an embodiment, the at least one CD4 polypeptide region of the chimeric CD8 co-receptor further comprises a CD4 palmitoylation motif region, and a CD4 LCK binding site region. For example, the at least one CD4 polypeptide comprises the entire wildtype human CD4 co-receptor cytoplasmic region. In this embodiment, the at least one CD8a-derived polypeptide region may also further comprise a CD8a-derived T ransmembrane domain region and the at least one CD8p-derived polypeptide region may comprise a CD8p stalk domain region. In this embodiment, the CD8a-derived IG-like domain region may be located closer to the N-terminal end of the enhanced chimeric CD8 co-receptor polypeptide than the CD8p-derived IG-like domain region. For example, the chimeric CD8 co-receptor may further comprise an Immunoreceptor tyrosine-based activation motif (ITAM), e.g. ITAM1. Furthermore, the chimeric CD8 co-receptor may comprise the cytoplasmic domain of CD28. For example, the ITAM1 costimulatory motif may be located at the C-terminal end of the enhanced chimeric CD8 co-receptor. An enhanced chimeric CD8 co-receptor in accordance with this embodiment may e.g. comprise a polypeptide having an amino acid sequence with at least 85% or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% identity to the amino acids as set forth in SEQ ID NO:29 (pTK-0629).

[0154] According to an embodiment, the at least one CD4-derived polypeptide region of the chimeric CD8 co-receptor further comprises a CD4-derived palmitoylation motif region, and a CD4-derived LCK binding site region. For example, the at least one CD-4 derived polypeptide comprises the entire wildtype human CD4 co-receptor cytoplasmic region. In this embodiment, the at least one CD8a-derived polypeptide region may also further comprise a CD8a-derived Transmembrane domain region and the at least one CD8p-derived polypeptide region may comprise a CD8p-derived stalk domain region. In this embodiment, the CD8a-derived IG-like domain region may be located closer to the N-terminal end of the enhanced chimeric CD8 co-receptor polypeptide than the CD8p-derived IG-like domain region. For example, the chimeric CD8 co-receptor may further comprise an Immunoreceptor tyrosine-based activation motif (ITAM), e.g. ITAM1.Furthermore, the chimeric CD8 co-receptor may comprise the cytoplasmic domain of 4-1BB. For example, the ITAM1 costimulatory motif may be located at the C-terminal end of the enhanced chimeric CD8 co-receptor. An enhanced chimeric CD8 co-receptor in accordance with this embodiment may e.g. comprise a polypeptide having an amino acid sequence with at least 85% or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% identity to the amino acids as set forth in SEQ ID NO:30 (pTK-0630).

[0155] According to an embodiment, the at least one CD4 polypeptide region of the chimeric CD8 co-receptor further comprises a CD4 palmitoylation motif region, and a CD4 LCK binding site region. For example, the at least one CD-4 derived polypeptide comprises the entire wildtype human CD4 co-receptor cytoplasmic region. In this embodiment, the at least one CD8a-derived polypeptide region may also further comprise a CD8a-derived T ransmembrane domain region and the at least one CD8p-derived polypeptide region may comprise a CD8p-derived stalk domain region. In this embodiment, the CD8a-derived IG-like domain region may be located closer to the N-terminal end of the enhanced chimeric CD8 co-receptor polypeptide than the CD8p-derived IG-like domain region. For example, the chimeric CD8 co-receptor may further comprise the cytoplasmic domain of CD28. An enhanced chimeric CD8 co-receptor in accordance with this embodiment may e.g. comprise a polypeptide having an amino acid sequence with at least 85% or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% identity to the amino acids as set forth in SEQ ID NO:31 (pTK-0631).

[0156] According to an embodiment, the at least one CD4 polypeptide region of the chimeric CD8 co-receptor further comprises a CD4-derived palmitoylation motif region, and a CD4-derived LCK binding site region. For example, the at least one CD-4 derived polypeptide comprises the entire wildtype human CD4 Co-receptor cytoplasmic region. In this embodiment, the at least one CD8a-derived polypeptide region may also further comprise a CD8a-derived Transmembrane domain region and the at least one CD8p-derived polypeptide region may comprise a CD8p-derived stalk domain region. In this embodiment, the CD8a-derived IG-like domain region may be located closer to the N-terminal end of the enhanced chimeric CD8 co-receptor polypeptide than the CD8p-derived IG-like domain region. For example, the chimeric CD8 co-receptor may further comprise the cytoplasmic domain of 4-1 BB. An enhanced chimeric CD8 co-receptor in accordance with this embodiment may e.g. comprise a polypeptide having an amino acidsequence 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:32 (pTK-0632).

[0157] According to an embodiment, the at least one CD4 polypeptide region of the chimeric CD8 co-receptor further comprises a CD4 palmitoylation motif region, and a CD4 LCK binding site region. For example, the at least one CD4 polypeptide comprises the entire wildtype human CD4 co-receptor cytoplasmic region. In this embodiment, the at least one CD8a polypeptide region may also further comprise a CD8a-derived T ransmembrane domain region and the at least one CD8p-derived polypeptide region may comprise a CD8p-derived stalk domain region. In this embodiment, the CD8a-derived IG-like domain region may be located closer to the N-terminal end of the enhanced chimeric CD8 co-receptor polypeptide than the CD8p-derived IG-like domain region. For example, the chimeric CD8 co-receptor may further comprise the cytoplasmic domain of ICOS. An enhanced chimeric CD8 co-receptor in accordance with this embodiment may e.g. comprise a polypeptide having an amino acid sequence with at least 85% or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% identity to the amino acids as set forth in SEQ ID NO:33 (pTK-0633).

[0158] According to an embodiment, the at least one CD4 polypeptide region of the chimeric CD8 co-receptor further comprises a CD4 palmitoylation motif region, and a CD4 LCK binding site region. For example, the at least one CD4- derived polypeptide comprises the entire wildtype human CD4 co-receptor cytoplasmic region. In this embodiment, the at least one CD8a polypeptide region may also further comprise a CD8a-derived T ransmembrane domain region and the at least one CD8p-derived polypeptide region may comprise a CD8p stalk domain region. In this embodiment, the CD8a-derived IG-like domain region may be located closer to the N-terminal end of the enhanced chimeric CD8 Co-receptor polypeptide than the CD8p IG-like domain region. For example, the chimeric CD8 co-receptor may further comprise the cytoplasmic domain of HVEM. An enhanced chimeric CD8 co-receptor in accordance with this embodiment may e.g. comprise a polypeptide having an amino acid sequence with at least 85% or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% identity to the amino acids as set forth in SEQ ID NO:34 (pTK-0634).

[0159] According to an embodiment, the at least one CD4 polypeptide region of the chimeric CD8 co-receptor further comprises a CD4 palmitoylation motif region, and a CD4 LCK binding site region. For example, the at least one CD4 polypeptide comprises the entire wildtype human CD4 co-receptor cytoplasmic region. In this embodiment, the at least one CD8a-derived polypeptide region may also further comprise a CD8a-derived T ransmembrane domain region and the at least one CD8p-derived polypeptide region may comprise a CD8p stalk domain region. In this embodiment, the CD8a IG-like domain region may be located closer to the N-terminal end of the enhanced chimeric CD8 Co-receptor polypeptide than the CD8p-derived IG-like domain region. For example, the chimeric CD8 co-receptor may further comprise the cytoplasmic domain of 0X40. An enhanced chimeric CD8 co-receptor in accordance with this embodiment may e.g. comprise a polypeptide having an amino acid sequence with at least 85% or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% identity to the amino acids as set forth in SEQ ID NO:35 (pTK-0635).

[0160] According to an embodiment, the at least one CD4 polypeptide region of the chimeric CD8 co-receptor further comprises a CD4-derived palmitoylation motif region, and a CD4 LCK binding site region. For example, the at least one CD4 polypeptide comprises the entire wildtype human CD4 Co-receptor cytoplasmic region. In this embodiment, the at least one CD8a polypeptide region may also further comprise a CD8a Transmembrane domain region and the at least one CD8p polypeptide region may comprise a CD8p stalk domain region. In this embodiment, the CD8a-derived IG-like domain region may be located closer to the N-terminal end of the enhanced chimeric CD8 co-receptor polypeptide than the CD8p-derived IG-like domain region. For example, the chimeric CD8 co-receptor may further comprise the cytoplasmic domain of CD27. An enhanced chimeric CD8 co-receptor in accordance with this embodiment may e.g. comprise a polypeptide having an amino acid sequence with at least 85% or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% identity to the amino acids as set forth in SEQ ID NO:36 (pTK-0636).

[0161] According to an embodiment, the at least one CD4 polypeptide region of the chimeric CD8 co-receptor further comprises a CD4-derived palmitoylation motif region, and a CD4 LCK binding site region. For example, the at least one CD4 polypeptide comprises the entire wildtype human CD4 co-receptor cytoplasmic region. In thisembodiment, 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 stalk domain region. In this embodiment, the CD8a-derived IG-like domain region may be located closer to the N-terminal end of the enhanced chimeric CD8 co-receptor polypeptide than the CD8p- 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. 19 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 may be present between the CD30 costimulatory motif and the CD40 cytoplasmic domain. The CD40 cytoplasmic domain may be included at the C-terminal end of the chimeric CD8 co-receptor. An enhanced chimeric CD8 co-receptor in accordance with this embodiment may e.g. comprise a polypeptide having an amino acid sequence with at least 85% or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% identity to the amino acids as set forth in SEQ ID NO:37 (pTK-0637).

[0162] According to an embodiment, the at least one CD4 polypeptide region of the chimeric CD8 co-receptor further comprises a CD4 palmitoylation motif region, and a CD4 LCK binding site region. For example, the at least one CD-4 derived polypeptide comprises the entire wildtype human CD4 co-receptor cytoplasmic region. In this embodiment, the at least one CD8a polypeptide region may also further comprise a CD8a-derived Transmembrane domain region and the at least one CD8p polypeptide region may comprise a CD8p stalk domain region. In this embodiment, the CD8a-derived IG-like domain region may be located closer to the N-terminal end of the enhanced chimeric CD8 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. 19 as herewith presented. Furthermore, the chimeric CD8 co-receptor according to this embodiment may comprise the cytoplasmic domain of CD40. For example, in accordance with this embodiment, the polypeptide linker sequence GGGS is inserted between the CD30 costimulatory motif and the CD40 cytoplasmic domain. The CD30 motif may be included at the C-terminal end of the chimeric CD8 Co-receptor. An enhanced chimeric CD8 co-receptor in accordance with this embodiment may e.g. comprise a polypeptide having an amino acid sequence with at least 85% or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least97%, or at least 98%, or at least 99%, or 100% identity to the amino acids as set forth in SEQ ID NO:38 (pTK-0638).

[0163] According to an embodiment, the at least one CD4 polypeptide region of the chimeric CD8 co-receptor further comprises a CD4 palmitoylation motif region, and a CD4-derived LCK binding site region. For example, the at least one CD4 polypeptide comprises the entire wildtype human CD4 co-receptor cytoplasmic region. In this embodiment, the at least one CD8a polypeptide region may also further comprise a CD8a Transmembrane domain region and the at least one CD8p polypeptide region may comprise a CD8p stalk domain region. In this embodiment, the CD8a IG-like domain region may be located closer to the N-terminal end of the enhanced chimeric CD8 co-receptor polypeptide than the CD8p-derived IG-like domain region. For example, the chimeric CD8 co-receptor may further comprise a costimulatory cytoplasmic domain of CD40. An enhanced chimeric CD8 co-receptor in accordance with this embodiment may e.g. comprise a polypeptide having an amino acid sequence with at least 85% or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% identity to the amino acids as set forth in SEQ ID NO:39 (pTK-0639).

[0164] According to an embodiment, the at least one CD4 polypeptide region of the chimeric CD8 co-receptor further comprises a CD4 palmitoylation motif region, and a CD4 LCK binding site region. For example, the at least one CD4- derived polypeptide comprises the entire wildtype human CD4 co-receptor cytoplasmic region. In this embodiment, the at least one CD8a polypeptide region may also further comprise a CD8a-derived T ransmembrane domain region and the at least one CD8p-derived polypeptide region may comprise a CD8p stalk domain region. In this embodiment, the CD8a-derived IG-like domain region may be located closer to the N-terminal end of the enhanced chimeric CD8 Co-receptor polypeptide than the CD8p-derived IG-like domain region. For example, the chimeric CD8 co-receptor may further comprise a costimulatory CD30 motif, as set forth e.g. in SEQ ID No. 19 as herewith presented. An enhanced chimeric CD8 co-receptor in accordance with this embodiment may e.g. comprise a polypeptide having an amino acid sequence with at least 85% or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% identity to the amino acids as set forth in SEQ ID NQ:40 (pTK-0640).

[0165] According to the present invention, the at least one CD4 polypeptide region of the chimeric CD8 co-receptor further comprises a CD4 palmitoylation motif region, and a CD4 LCK binding site region. For example, the at least one CD4 polypeptide comprisesthe entire wildtype human CD4 co-receptor cytoplasmic region. In this embodiment, the at least one CD8a polypeptide region may also further comprise a CD8a-derived Transmembrane domain region and the at least one CD8p polypeptide region may comprise a CD8p stalk domain region. In this embodiment, the CD8a-derived IG-like domain region may be located closer to the N-terminal end of the enhanced chimeric CD8 co-receptor polypeptide than the CD8p-derived IG-like domain region. At the C-terminal side of this construct, the specific costimulatory region as herein described is fused: For example, a complete cytoplasmic domain of human CD40 is included, followed N-terminally by a further CD40 TRAF6 motif and a CD40 TRAF123 motif. An enhanced chimeric CD8 co-receptor in accordance with this embodiment may e.g. comprise a polypeptide having an amino acid sequence with at least 85% or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% identity to the amino acids as set forth in SEQ ID NO:71 (pTK-1296).

[0166] According to the present invention, the at least one CD4 polypeptide region of the chimeric CD8 co-receptor further comprises a CD4 palmitoylation motif region, and a CD4 LCK binding site region. For example, the at least one CD4 polypeptide comprises the entire wildtype human CD4 Co-receptor cytoplasmic region. In this embodiment, the at least one CD8a polypeptide region may also further comprise a CD8a-derived Transmembrane domain region and the at least one CD8p polypeptide region may comprise a CD8p stalk domain region. In this embodiment, the CD8a-derived IG-like domain region may be located closer to the N-terminal end of the enhanced chimeric CD8 co-receptor polypeptide than the CD8p-derived IG-like domain region. At the C-terminal side of the polypeptide comprising said regions / motifs / domains, the specific costimulatory region as herein described is fused: For example, two CD40 TRAF6 motifs are included, followed N-terminally by a further CD40 TRAF1 / 2 / 3 motif. An enhanced chimeric CD8 co-receptor in accordance with this embodiment may e.g. comprise a polypeptide having an amino acid sequence with at least 85% or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% identity to the amino acids as set forth in SEQ ID NO:73 (pTK-1298).

[0167] According to the present invention, the at least one CD4 polypeptide region of the chimeric CD8 co-receptor further comprises a CD4 palmitoylation motif region, and a CD4 LCK binding site region. For example, the at least one CD4 polypeptide comprises the entire wildtype human CD4 co-receptor cytoplasmic region. In this embodiment, the atleast one CD8a polypeptide region may also further comprise a CD8a-derived Transmembrane domain region and the at least one CD8p polypeptide region may comprise a CD8p stalk domain region. In this embodiment, the CD8a-derived IG-like domain region may be located closer to the N-terminal end of the enhanced chimeric CD8 Co-receptor polypeptide than the CD8p-derived IG-like domain region. At the C-terminal side of the polypeptide comprising said regions / motifs / domains, the specific costimulatory region as herein described is fused: For example, a CD40 TRAF 1 / 2 / 3 motif is at its N-terminus followed by a CD40 TRAF6 motif, which is at its N-terminus followed by a further CD40 TRAF1 / 2 / 3 motif. An enhanced chimeric CD8 co-receptor in accordance with this embodiment may e.g. comprise a polypeptide having an amino acid sequence with at least 85% or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% identity to the amino acids as set forth in SEQ ID NO:75 (pTK-1306).

[0168] According to an embodiment, wherein the chimeric human receptor may be a chimeric costimulatory receptor. In some embodiments, the chimeric human receptor may be a chimeric costimulatory switch receptor.

[0169] According to some embodiments, the extracellular polypeptide region may comprise an extracellular domain of a member of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF). For example, the extracellular polypeptide region may comprise an extracellular domain of CD95.

[0170] It is further envisaged that the extracellular polypeptide region may comprise an extracellular domain of LAG-3, CTLA4, PD1, TIGIT, or TIM-3.

[0171] It is contemplated that the transmembrane polypeptide region may comprise a transmembrane region of CD95, LAG-3, CTLA4, PD1, TIGIT, or TIM-3. In some embodiments, the extracellular polypeptide region and the transmembrane polypeptide region may be of (e.g. derived from) the same receptor. For example, when the extracellular polypeptide region may comprise an extracellular domain of PD1, the transmembrane polypeptide region may comprise a transmembrane region from PD1, etc.

[0172] According to an embodiment, the receptor may be a PD1 receptor.

[0173] In embodiments wherein the receptor is a chimeric PD1 (switch) receptor, the polypeptide may comprise at least one PD1- polypeptide region having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a human PD1 receptor as set forth in SEQ ID No. 63, wherein said PD1 polypeptide region comprises a PD1 extracellular ligand binding domain.

[0174] According to an embodiment, the PD1 polypeptide region may be a human PD1 polypeptide region from a human wildtype PD1 receptor.

[0175] It is understood that the expression “human wildtype PD1 receptor” relates to a protein having an amino acid sequence according to UniProtKB database entry No. Q15116 ■ PDCD1_HUMAN, as set forth e.g. in SEQ ID No. 63.

[0176] As used herein, the term “PD1 polypeptide region” refers to a polypeptide containing at least a functional portion (e.g., an extracellular ligand binding domain) of a wild-type PD1 protein or a variant thereof, such as a variant that has at least 60% sequence identity (e.g., at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the amino acid sequence of SEQ ID NO: 63 and that retains the ability to bind an endogenous PD1 ligand. Similarly, it is to be understood that the terms "PD1 immunoglobulin variable domain," "PD1 ligand binding domain," "PD1 co-stimulatory cytoplasmic polypeptide domain," and the like, refer to a portion of wild-type PD1 comprising the corresponding protein domain (i.e., the immunoglobulin variable domain, ligand binding domain, or co-stimulatory cytoplasmic polypeptide domain, respectively, of wild-type PD1) or a sequence variant thereof, such as a sequence variant recited herein.

[0177] In embodiments wherein the receptor is a chimeric PD1 (switch) receptor, the polypeptide may comprise at least one PD1 polypeptide region comprising a PD1 extracellular ligand binding domain.

[0178] According to some embodiments, the extracellular ligand binding domain may be functional in binding a PD1 ligand.

[0179] According to an embodiment the PD1 extracellular ligand binding domain may comprise a PD1 immunoglobulin variable (IgV) domain, wherein the PD1 IgV domain is having at least 85% sequence identity to the amino acid sequence of SEQ ID No. 64.

[0180] For example, the extracellular ligand binding domain may comprise a PD1 immunoglobulin variable (IgV) domain, wherein the PD1 IgV domain may have at least 85% sequence identity to the amino acid sequence of SEQ ID No. 64 (relating to the IgV domain comprising the amino acid sequence 35 - 145 of SEQ ID No 63). For example, the at least one PD1 IgV domain may have at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or atleast 78%, or at least 79%, or at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID No. 64.

[0181] According to an embodiment, the extracellular ligand binding domain of the chimeric PD1 receptor may be functional in binding at least one PD1 ligand or any other protein / polypeptide having the ability of binding to the wildtype PD1 receptor ligand binding domain.

[0182] For example, the at least one PD1 ligand may comprise at least one of the PD1 ligands PD-L1 and / or PD-L2.

[0183] It is contemplated that the chimeric PD1 receptor polypeptide as herein provided may comprise, in addition to comprising the PD1 ligand binding domain, further domain regions / motif regions / binding site regions from a wildtype human PD1 receptor in every conceivable combination to establish a functional chimeric PD1 receptor polypeptide. “Functional” chimeric PD1 receptor in this context relates to a chimeric PD1 receptor that is capable of redirecting the signaling pathways triggered by PD1 receptor engagement with at least one PD1 ligand such that - instead of inducing inhibitory pathways and / or apoptotic pathways in the T-cell - upon binding of the at least one PD1 ligand promote T-cell activation, persistence and enhance anti-tumor responses of the T-cell expressing a chimeric PD1 receptor as herein provided. For example, an optional test for functionality of a chimeric PD1 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 cancer cells expressing a PD1 ligand. Thus, the expression “every conceivable combination” of PD1 receptor polypeptide regions as described above is meant to exclude a combination with wildtype human PD1 receptor domains / regions / motifs being inhibitory. For example, the chimeric PD1 receptor polypeptide as herein provided may lack the “ITIM” motif of wildtype PD1 (amino acids 221 - 226 of SEQ ID No. 63), and / or may lack the “ITSM” motif of wildtype PD1 (amino acids 247-251 of SEQ ID No. 63), or may merely comprise a altered “ITIM” and / or “ITSM” motif, respectively, which no longer function(s) in promoting inhibitory / apoptotic pathways of the T-cell, e.g. due to mutations which abolish any inhibitory promoting functionality of the “ITIM” and / or “ITSM” motif(s), respectively.

[0184] According to an embodiment, the at least one PD1 polypeptide region has at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least95%, or at least 97%, or 100% sequence identity with the functional polypeptide domain or a functional polypeptide motif of a wildtype human PD1 receptor (e.g. Seq ID No. 63). According to an embodiment, the at least one PD1 polypeptide region may have one or more conservative amino acid substitutions relative to the amino acid sequence of the wildtype PD1 receptor.

[0185] For example, the at least one PD1 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 PD1 receptor (e.g. Seq ID No. 63).

[0186] The (human) PD1 polypeptide region comprising a PD1 extracellular ligand binding domain may have -in general- a sufficient portion of the human wildtype PD1 extracellular ligand binding domain to be functional in binding at least one PD1 ligand. For example, said at least one PD1 polypeptide region having at least 60% sequence identity with PD1 ligand binding domain of a human wildtype PD1 receptor may comprise the complete or a considerable part of the human wildtype PD1 ligand binding domain and / or all amino acids at respective amino acid positions of the wildtype PD1 receptor that are necessary and sufficient for binding of at least one ligand to the PD1 receptor.

[0187] According to an embodiment, the PD1 polypeptide region may comprise a complete wildtype PD1 receptor extracellular domain. For example, the expression “wildtype PD1 receptor extracellular domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 24-170 of UniProtKB database entry No. Q15116 ■ PDCD1_HUMAN, as set forth e.g. in SEQ ID No. 63. For example, the wildtype PD1 receptor extracellular domain as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 65. According to some embodiments, a truncated wildtype PD1 receptor extracellular domain may be used. For example, the PD1 polypeptide region may comprise amino acids 24-164 of UniProtKB database entry No. Q15116 ■ PDCD1_HUMAN, as set forth e.g. in SEQ ID No. 63. It is herewith envisaged that the PD1 receptor extracellular domain as included in the chimeric PD1 receptor as herein provided may have one or more conservative amino acid substitutions relative to the extracellular domain of the wildtype PD1 receptor. In particular, all amino acidsubstitutions that maintain the functional activity of the wildtype PD1 extracellular domain are envisaged.

[0188] According to an embodiment, the PD1 polypeptide region may comprise a signal sequence of the wildtype PD1 receptor. For example, a signal sequence of the PD1 receptor as referred to herein may relate to a polypeptide comprising amino acid sequence 1 - 23 of UniProtKB database entry No. Q15116 ■ PDCD1_HUMAN, as set forth e.g. in SEQ ID No. 63.

[0189] It is contemplated herein that the polypeptide of the chimeric switch receptor provided herein may be a single-chain polypeptide.

[0190] According to an embodiment, the polypeptide may comprise a transmembrane domain from CD30, or CD40.

[0191] According to some embodiments, the transmembrane domain of the chimeric PD1 receptor of the T-cell as herein provided may be from other proteins which comprise a transmembrane domain. For example, the transmembrane domain may be from a further co-stimulatory molecule known in the art. In principle, any transmembrane domain which is functional and allows surface detectable expression of the chimeric PD1 receptor is herewith envisaged.

[0192] It is envisaged that the at least one PD1 polypeptide region may comprise a complete PD1 extracellular domain.

[0193] According to an embodiment, the at least one PD1 polypeptide region has at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity with the functional polypeptide domain, region or motif of a human PD1 receptor (Seq ID No. 63), and / or the at least one PD1 polypeptide region is may have one ore more conservative amino acid substitutions relative to the functional polypeptide domain, region or motif of the human PD1 receptor (Seq ID No. 63).

[0194] According to an embodiment, the PD1 polypeptide region may comprise a PD1 transmembrane polypeptide domain or region. The transmembrane domain of wildtype PD1 as referred to herein may relate to a polypeptide comprising amino acid sequence 171 - 191 of UniProtKB database entry No. Q15116 ■ PDCD1_HUMAN, as set forth e.g. in SEQ ID No. 63. For example, the transmembrane domain of wildtype PD1 as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 66. It is herewith envisaged that the transmembrane domain as included in the chimeric PD1 receptor as herein provided may have one or more conservative amino acid substitutions relative to the transmembrane domain of the wildtype PD1 receptor. Inparticular, all amino acid substitutions that maintain the functional activity of the wildtype transmembrane domain are envisaged.

[0195] The chimeric PD1 receptor as herein provided may further comprise at least one linker region. This may be e.g. a polypeptide linker region. Such linker(s) may be included e.g. between functional domains / regions / motifs of the chimeric transmembrane receptor. It may be a linker region naturally occurring e.g. in wildtype PD1 receptor, or e.g. in co-stimulatory proteins, e.g. in costimulatory proteins from which the costimulatory domain of the receptor is derived. For example, polypeptide linker regions may be included between the transmembrane domain and the ligand binding domain, and / or between the transmembrane domain and the IgV domain of the chimeric PD1 receptor, and / or between the transmembrane domain and the at least one intracellular co-stimulatory domain, and / or between individual co-stimulatory domains (in embodiments comprising more than one co-stimulatory domain).

[0196] 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.

[0197] According to an embodiment, a linker region of the chimeric PD1 receptor as herein provided may comprise the amino acid sequence as set forth in SEQ ID No. 57 (GGGS)n or as set forth in Seq ID No. 58 (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.

[0198] However, in principle, each (polypeptide) linker known in the art is herewith envisaged as being potentially included in the chimeric PD1 switch receptor of the present invention.

[0199] According to an embodiment, the chimeric PD1 receptor as herein provided may be able to sustain or enhance cytotoxicity and / or cytokine secretion of a T-cell upon binding a PD1 ligand.

[0200] According to an embodiment, the chimeric PD1 receptor as herein provided may be capable of increasing resistance of T-cells to PD1 ligand expressing cancer cells.

[0201] According to an embodiment, the chimeric PD1 receptor as herein provided comprises a complete PD1 extracellular domain, a complete PD1 transmembrane domain, and a specific cytoplasmic co-stimulators region as herein described, wherein the cytoplasmic polypeptide region comprises - in an N-terminal to C-terminal order - a complete CD40 cytoplasmic region, a further CD40 TRAF6 motif, and a further CD40 TRAF 1 / 2 / 3 motif. A chimeric PD1 (switch) receptor in accordance with this embodiment may e.g. comprise a polypeptide having an amino acid sequence with at least 85% or atleast 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:77 (pl_2355).

[0202] In a further aspect, the invention provides an isolated nucleic acid comprising a nuclear acid sequence encoding for any one of the receptors according to the present invention.

[0203] The term “polynucleotide” or “nucleic acid” as used herein comprises a sequence of polyribonucleotides and polydeoxribonucleotides, e.g. modified or unmodified RNA or DNA, each in single-stranded and / or double-stranded form linear or circular, or mixtures thereof, including hybrid molecules. The nucleic acids according to this invention thus comprise DNA (such as dsDNA, ssDNA, cDNA), RNA (such as dsRNA, ssRNA, mRNA ivtRNA), combinations thereof or derivatives (such as RNA) thereof.

[0204] 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.

[0205] 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: 72, 74, 76 and 78.

[0206] 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 receptors described herein.

[0207] The nucleic acid sequences 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 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.

[0208] According to a further aspect, the present invention provides a vector comprising the nucleic acid encoding for the receptor described herein.

[0209] 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.

[0210] The vector may be a viral vector or a non-viral vector.

[0211] 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), orGALV 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.

[0212] In particular, the term “vector” as used herein encompasses, without limitation, plasmids, viral vectors (including retroviral vectors, lentiviral vectors, adenoviral vectors, vaccinia virus vectors, polyoma virus vectors, and adenovirus-associated vectors (AAV)), phages, phagemids, cosmids and artificial chromosomes (including BACs and YACs). The vector itself is generally a nucleotide sequence, commonly a DNA sequence that comprises an insert (transgene) and a larger sequence that serves as the “backbone” of the vector. Engineered vectors typically comprise an origin for autonomous replication in the host cells (if stable expression of the polynucleotide is desired), selection markers, and restriction enzyme cleavage sites (e.g. a multiple cloning site, MCS). The vector may additionally comprise promoters, genetic markers, reporter genes, targeting sequences, other regulatory elements, and / or protein purification tags. As known to those skilled in the art, large numbers of suitable vectors are known to those of skill in the art and many are commercially available.

[0213] In an embodiment, the vector may further comprise a nucleic acid encoding a chimeric antigen receptor (CAR).

[0214] 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 T-cell receptor may be a recombinant T-cell receptor.

[0215] 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 of TCRa, TCRp, and the chimeric 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).

[0216] 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 fromporcine 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: 55)) before the self cleaving 2A sequence, this may enable efficient synthesis of biologically active proteins, e.g., TCRs and chimeric CD8 Coreceptors as described herein.

[0217] Turning now to a further aspect, there is also provided an isolated T-cell, the T-cell being transduced, transfected or transformed with a nucleic acid encoding for the chimeric human receptor of the present invention.

[0218] In accordance with another aspect, a T-cell may be transduced, transfected or transformed with a vector comprising a nucleic acid encoding for the chimeric human receptor of the present invention. According to a further aspect, a T-cell may be transduced, transfected or transformed to express the chimeric human receptor of the present invention.

[0219] 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 enhanced chimeric receptor as herein provided may be stably introduced into the T-cell by a transposon / transposase system or a viral-based gene transfer system, such as a lentiviral or a retroviral system, or another suitable method, such as transfection, electroporation, transduction, lipofection, calcium phosphate (CaPCll), nanoengineered substances, such as Ormosil, mRNA-based therapy, viral delivery methods, including adenoviruses, retroviruses, lentiviruses, adeno-associated viruses, or another suitable method. It is envisaged that T-cells may be generated by in vivo introduction of nucleic acid in T-cells, e.g. by using DNA or mRNA, e.g. by using nanoparticles such as lipid nanoparticles.

[0220] 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.

[0221] According to a further aspect, there is provided an engineered T-cell, the T-cell expressing the chimeric receptor as herein provided.

[0222] According to an embodiment, the cell may be an ap T-cell, y8 T-cell, and / or a natural killer T-cell.

[0223] For example, the a 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.

[0224] It is envisaged that the T-cell may express the chimeric receptor as provided by the present invention.

[0225] It is further envisaged that the isolated or engineered T-cell as herein provided may express a heterologous T-cell receptor.

[0226] In certain embodiments, the T-cells may further express an engineered T-cell receptor. Engineered T-cells of the present disclosure can be used to treat a subject in need of treatment for a condition, for example, a cancer described herein. The T-cells may be ap T-cells or y8 T-cells that express the chimeric receptor polypeptide as described herein, and optionally an engineered TCR. 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.

[0227] 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 p T-cells ory8 T-cells that express a chimeric receptor as described herein, and optionally an engineered TCR.

[0228] According to another aspect, it is herewith provided a kit comprising means to prepare the T-cells described above.

[0229] According to a further aspect, this invention relates to a pharmaceutical composition comprising the T-cell provided by the present invention.

[0230] It is herewith contemplated that the pharmaceutical composition may further comprise an adjuvant, excipient, buffer, diluent, carrier, stabilizer or combination thereof.

[0231] According to a further aspect, there is provided a pharmaceutical composition comprising T-cells which express the chimeric receptor of the present invention.

[0232] 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 receptor. It is also possible that the chimeric 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 cryoprotectants 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 pre-formulated with 10% DMSO).

[0233] Turning to a further aspect, a method for preparing a T-cell for immunotherapy is provided, comprisingisolating T-cells from a human subject,- transducing, transforming or transfecting the T-cells (e.g. using electroporation) with the nucleic acid as herewith provided, or with the vector as herewith provided, andexpanding the transduced T-cells.

[0234] 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 of the present invention.

[0235] In accordance with a further aspect, there is provided a method for treating a patient having a disease, comprising introducing in vivo the nucleic acid as herein provided or the vector as herein disclosed into a T-cell of the patient.

[0236] According to an embodiment, the nucleic acid may be a DNA or a mRNA.

[0237] For the in vivo introduction, the vector may be - for example - a nonreplicating viral vector.

[0238] 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.

[0239] 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.

[0240] 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.

[0241] For example, the cancer treated may be a solid tumor. In illustrative embodiments of the solid tumor types mentioned above, the lung cancer may be, but is not limited to, non-small cell lung cancer (NSCLC), including squamous cell carcinoma of the lung, adenocarcinoma of the lung, large cell carcinoma of the lung and other histologic types of NSCLC, or small cell lung cancer. In other illustrative examples, the breast cancer may be, but is not limited to, ductal breast cancer, ductal-invasive breast cancer, invasive breast cancer, tubular breast cancer, medullary breast cancer or combinations thereof. In yet other illustrative examples, the gastric cancer may be gastric adenocarcinoma or squamous cell cancer. Turning to sarcoma cancer, the sarcoma cancer may be, but is not limited to, chondrosarcoma cancer, osteosarcoma cancer or combinations thereof. The adenoma cancer may include, but is also not limited to, gastric adenocarcinoma, pancreatic adenocarcinoma or combinations thereof.

[0242] The invention will be further illustrated by the following non-limiting Experimental Examples.

[0243] Sequences as used herein are depicted in below Table 1.

[0244] Table 1. Sequences as used herein.&Experimental Examples

[0245] Example 1. In-vitro T-cell killing analysis of T-cells transduced with chimeric CD8 Co-receptor polypeptides according to the invention

[0246] In order to test the enhanced chimeric CD8 Co-receptor, constructs described herein for suitability in adoptive T-cell therapy (ACT) with T-cells expressing transgenic ap-T-cell receptors, in particular for testing suitability of the chimeric CD8 co-receptor for incorporating CD4+ T-cells into TCR-T-cell therapy, and / or for increasing cytotoxicity of the generated T-cells that express the chimeric CD8 co-receptors, the chimeric CD8 co-receptor constructs have been used to transduce CD4 T-cells together with a HLA-I restricted TCR raised against MAGE-A1. Purified transduced T-cells were used in an in-vitro T-cell killing assay with NCI-H2030 cells for evaluating cytotoxicity of the transduced T-cells (Figure 3, 4, and 5).

[0247] 1.1 Materials and MethodsCloning of enhanced chimeric human CD8 co-receptor constructsChimeric human CD8 co-receptor constructs have been generated using standard cloning techniques. Table 2 as presented below summarizes the cloned underlying plasmids for chimeric constructs created:

[0248] Table 2

[0249] Table 3 as presented below summarizes the chimeric CD8 co-receptor constructs that have been generated by the Inventors in a different schematic representation:

[0250] Table 3

[0251] As used in Table 3, the expression “signal peptide” relates to the the short peptide region present at the N-terminus of newly synthesized CD8 co-receptors. The expression “MHC binder 1” relates to the CD8a -derived, or, respectively, CD8p-derived IG-like domain region the two IG-like domain regions of the chimeric CD8 co-receptor polypeptide which is located closer to the N-terminal end of the chimeric CD8 co-receptor polypeptide. The expression “linker” in Table 3 indicates if - and what kind of - a linker is present between the two IG-like domain regions of the chimeric CD8 co-receptor polypeptide. The expression “MHC binder 2” relates to the CD8a -derived or, respectively, CD8p-derived IG-like domain region of the two IG-like domain regions of the chimeric CD8 co-receptor polypeptide which is located closer to the C-terminal end of the chimeric CD8 co-receptor polypeptide. The expression “stalk” according to Table 3 relates to the “stalk” region of the chimeric CD8 co-receptor polypeptide. The expression “TM” relates to the transmembrane region of the chimeric CD8 co-receptor polypeptide. The expression “Intra 1” according to Table 3 relates to the intracellular domain of the chimeric CD8 co-receptor polypeptide that is derived from CD8a, CD8p or CD4, respectively. Finally, the expression “Intra 2” relates to the cytoplasmic costimulatory polypeptide region of a tumor necrosis factor receptor superfamily protein, of an immunoglobulin superfamily (IgSF) protein, and / or of an ITAM-associated receptor, respectively, that is included in the respective chimeric CD8 co-receptor. “Intra2” depicts the cytoplasmic domain of the chimeric vo-receptor that is located at the C-terminal end of the chimeric vo-receptor. In particular, Table 3 thus schematically represents from which wildtype vo-receptor polypeptide (CD4, CD8a or CD8p) the respective polypeptide region of the generated chimeric CD8 vo-receptor polypeptide is derived from.

[0252] Table 400253] As used in Table 4, the expression “signal peptide” relates to the the short peptide region present at the N-terminus of newly synthesized CD8 receptors. The expression “MHC binder 1” relates to the CD8a -derived, or, respectively, CD8p-derived IG-like domain region the two IG-like domain regions of the chimeric CD8 co-receptor polypeptide which is located closer to the N-terminal end of the chimeric CD8 co-receptor polypeptide. The expression “linker” in Table 4 indicates if - and what kind of - a linker is present between the two IG-like domain regions of the chimeric CD8 Co-receptor polypeptide. The expression “MHC binder 2” relates to the CD8a -derived or, respectively, CD8p-derived IG-like domain region of the two IG-like domain regions of the chimeric CD8 co-receptor polypeptide which is located closer to the C-terminal end of the chimeric CD8 co-receptor polypeptide. The expression “stalk” according to Table 4 relates to the “stalk” region of the chimeric CD8 co-receptor polypeptide. The expression “TM” relates to the transmembrane region of the chimeric CD8 co-receptor polypeptide. The expression “Intra” according to Table 4 relates to the intracellular domain of the chimeric CD8 co-receptor polypeptide that is derived from CD8a, CD8p or CD4, respectively. Finally, the expressions CoStim 1, Costim 2 and Costim 3 refer to the specific costimulatory region / motif present N-terminally in the respective order, and the expression “linker between costims” relates to linker polypeptide sequences used between the respective costimulatory regions / motifs ( between the costimulatory polypeptide regions “CoStim 1” and “CoStim2” , and between the costimulatory polypeptide regions “CoStim 2” and “CoStim3” of the chimeric receptors, respectively.

[0254] CD4 Cells Generation

[0255] PBMCs from a healthy donor buffy coat were isolated by density gradient centrifugation with Lymphoprep. Purified polyclonal CD4 T-cells were obtained by negative selection with anti-CD8+ microbeads for depleting CD8 population. CD3+ T-cells were activated using CD3 / CD28 antibody-coated micro-beads in presence of IL-7 / IL-15. Two days post activation, CD4 T-cells were separately transduced with either HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) alone, or together with wt-CD8ap coreceptor (wt-CD8), or together with a chimeric CD8 co-receptor without costimulatory polypeptide region of a tumor necrosis factor receptor superfamily protein, aimmunoglobulin superfamily (IgSF) protein, and / or an ITAM-associated receptor (pTK-519), or together with different versions of the enhanced chimeric CD8 co-receptor (pTK 0628 to pTK 0640). 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.

[0256] Transduced CD4 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 11. T-cell characterization was based on transgene expression levels using FACS and killing assay.

[0257] CD8 Cells Generation

[0258] For the experiments shown in Fig. 12, 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 CD4 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 a TCR raised against PRAME alone or the PRAME TCR together with different versions of the SWITCH receptors.

[0259] 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.

[0260] CD4 / CD8 Cell Generation

[0261] PBMCs from a healthy donor buffy coat were isolated by density gradient centrifugation with Lymphoprep. CD3+ T-cells were activated using CD3 / CD28 antibody-coated micro-beads in presence of IL-7 / IL-15. Two days post activation, CD4 T-cells were separately transduced with either HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) alone, or CD4 / CD8 cells were transduced with TCR raised against MAGE-A1 together with wt-CD8 co-receptor (wt-CD8), or together with a chimeric CD8 co-receptor without costimulatory polypeptide region of a tumor necrosis factor receptor superfamily protein, a immunoglobulin superfamily (IgSF) protein, and / or an ITAM-associated receptor(pTK-519), or together with different versions of the enhanced chimeric CD8 co-receptor (pTK 0628 to pTK 0640). 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.

[0262] Transduced CD4 or CD4 / 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 11. T-cell characterization was based on transgene expression levels using FACS and killing assay.

[0263] Cell killing assay: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. NCI-H2030 1x104tumor cells were plated per well in 96-well plates. Untransduced, or transduced human T-cells (transduced with either MAGE_TCR alone, or transduced with MAGE_TCR together with a chimeric CD8 co-receptor without costimulatory polypeptide region of a tumor necrosis factor receptor superfamily protein, a immunoglobulin superfamily (IgSF) protein, and / or an ITAM-associated receptor (pTK 519), or transduced with MAGE_TCR together with a respective one of the enhanced chimeric CD8 co-receptors of the present invention (pTK 0628- 0640)) were added in triplicates at 3 to 1 E:T ratio. For second stimulation, tumor cells (NCI-H2030 1x104) were added to each well after cancer cells from the first stimulation are killed, typically 120h to 170h from the beginning of the experiment.

[0264] Long term Cell killing assay: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. NCI-H2030 1x104tumorcells were plated per well in 96-well plates. Untransduced, or transduced human T-cells (transduced with either MAGE_TCR alone, or MAGE_TCR together with wt-CD8ap coreceptor, or transduced with MAGE_TCR together with the enhanced chimeric CD8 coreceptor pTK 0638 of the present invention) were added in triplicates at 1 to 4 E:T ratio. For second stimulation, tumor cells (NCI-H2030 1*104) were added to each well after cancer cells from the first stimulation are killed, typically 120h to 170h from the beginning of the experiment. In a further experiment which was carried out identically (Fig. 9), untransduced, or transduced human T-cells (transduced with either MAGE_TCR alone, or MAGE_TCR together with wt-CD8ap co-receptor, or transduced with MAGE_TCR together with the enhanced chimeric CD8 co-receptor pTK 0638 of the present invention, or transduced with MAGE_TCR together with the enhanced chimeric CD8 co-receptor pl_1282 of the present invention) were added in triplicates at 1 to 4 E:T ratio.

[0265] Cytokine Secretion Assay

[0266] NCI-H2030 HLA-A2 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, IL-4, I IL-6, L-10, IL-17A, TNF-a, Fas, FasL, IFN-y, Granzyme A, Granzyme B, Perforin, Granulysin 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 re-suspended 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.

[0267]

[0268] Cell killing assay in PD-L1 -overexpressing NCI-H1703 cells:

[0269] 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 TCRT-cell repetitive killing assay was conducted using IncuCyte Live Cell Analysis. Using PDL1 expressing NCI-H1703 cells, 1x104 tumor cells were plated per well in 96-well plates. Transduced human CD8 T-cells (transduced with either a PRAME_TCR alone, or transduced with a PRAME_TCR together with a respective chimeric PD1 receptor) were added in triplicates at 2 to 1 E:T ratio (for the experiments shown by Fig 12).

[0270] 1.2. T-cell killing assay analysis

[0271] The Relative cell growth has been observed over time for each transduced T-cell fraction. The results are shown in Fig 3, Fig 4, Fig 5, Fig.9 and Fig.12, wherein Fig.3 and 4 show the results of the killing assay with the enhanced chimeric CD8 Co-receptors pTK 0628 - pTK 0640) in two different scales, whereas Fig 5 and Fig 9 show the result of a long term cell killing assay using specifically the chimeric CD8 co-receptor pTK 0638 (in Fig 5) and a CD8ap wildtype receptor, or using specifically the chimeric CD8 co-receptor pTK 0638, pl_1282, pl_1296, pl_1298, or pl_1306 (in Fig. 9) and a CD8ap wildtype receptor . “Mock” relates to mock-transduced T-cell fraction, “MAGE_TCR” relates to CD4 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR); “wt-CD8” relates to CD4 / CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with wt-CD8ap coreceptor; “519” relates to the CD4 / CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with the chimeric CD8 co-receptor without costimulatory region (pTK 519), “628” - “640”, and “pl-1282” relate to the CD4 / CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with the enhanced chimeric CD8 co-receptor polypeptides including a costimulatory region of a tumor necrosis factor receptor superfamily protein, a immunoglobulin superfamily (IgSF) protein, and / or an ITAM-associated receptor (“pTK 0628” - “pTK0640” and “pl-1282”, respectively). “pl_1296”, “pl_1298”, or “pl_1306” relate to the CD4 / CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with the enhanced chimeric CD8 co-receptor polypeptides including a costimulary polypeptide region as herein provided. Fig. 12 shows the results of the killing assay with chimeric PD1 receptors in PD-L1 -expressing NCI-H1703 cells, wherein an E:T ratio of 2:1 was used. “PRAME TCR only” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against PRAME (PRAME_TCR); “PRAME TCR + PD1-ICOS” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against PRAME (PRAME_TCR) together with a chimeric PD1 receptor comprising a PD1 extracellular polypeptide region, a PD1 transmembrane domain and anICOS cytoplasmic polypeptide region (pl_2352); “PRAME TCR + PD1-41BB” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against PRAME (PRAME_TCR) together with a chimeric PD1 receptor comprising a PD1 extracellular polypeptide region, a PD1 transmembrane domain and an 4-1 BB cytoplasmic polypeptide region (pl_2354), and “PRAME TCR + PD1-CoStim_1296” relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against PRAME (PRAME_TCR) together with a chimeric PD1 receptor comprising a PD1 extracellular polypeptide region, a PD1 transmembrane domain and a CD40 cytoplasmic polypeptide region, wherein the CD40 cytoplasmic polypeptide region comprises the entire cytoplasmic region of human wildtype CD40 fused to a further CD40 TRAF6 motif region and to a further CD40 TRAF123 motif region (pl_2355).

[0272] 1.3. Results

[0273] As visible from Fig. 3 and 4, Co-transduction of CD4 T-cells with an engineered HLA-I restricted TCR raised against MAGE-A1 together with an enhanced chimeric CD8 co-receptor, comprising a costimulatory motif / costimulatory domain of a tumor necrosis factor receptor superfamily protein, an immunoglobulin superfamily (IgSF) protein, and / or an ITAM-associated receptor as herewith provided results in an increased killing activity of the engineered T-cells compared with mock transduced T-cells and / or T-cells transduced with the HLA-I restricted TCR raised against MAGE-A1. The most effective enhanced chimeric CD8 co-receptors are “pTK 0638” and “pTK 0639”.

[0274] Fig. 5 shows that the enhanced cytotoxic activity if the engineered CD4 T-cells expressing - in addition to a TCR raised against MAGE-A1 - an enhanced chimeric CD8 co-receptor as herewith provided (pTK 0638), is conserved over a long time period. This confirms the finding of the T-cell killing assays shown in Fig. 3 and 4. Fig. 5 further demonstrates that the enhanced cytotoxic effect associated with co-expression of an enhanced chimeric CD8 co-receptor as herewith provided (pTK 0638) together with a TCR raised against MAGE-A1 is stronger than a cytotoxic effect associated with co-expression of a wildtype CD8ap co-receptor together with a TCR raised against MAGE-A1. Fig. 9 also shows the enhanced cytotoxic effect associated with co-expression of the enhanced chimeric CD8 co-receptor pTK 0638. Furthermore, Fig 9 shows that, in principle, the specific combination of cytoplasmic costimulatory motifs as included in the enhanced chimeric CD8 co-receptor pTK 0638 also functions in T-cell killing assays when both the linker region between the cytoplasmic CD4 polypeptide region and the fused cytoplasmic co-stimulatory domains on the one hand, and the linker region between the separate CD40and CD30 polypeptide regions on the other hand, respectively, are longer (comprising 3x SGGGS [Seq ID No.: 61] linker sequence between the cytoplasmic CD4 polypeptide region and the fused cytoplasmic co-stimulatory domains and 2x SGGGS [Seq ID No. 61] and 1x SGGGT [Seq ID No.: 62] linker sequences between the separate CD40 and CD30 polypeptide regions, compared with IxSGGGS [Seq ID No.: 61] linker sequence, respectively, see Fig. 10). Furthermore, Fig. 9 demonstrates that the receptors including the specific costimulatory domain / region as herein described show an enhanced cytotoxic effect associated with Co-expression of the chimeric CD8 co-receptor (“pl_1296”, “pl_1298”, and “pl_1306”). Furthermore, as visible from Fig. 12, an increased killing activity is achieved with an engineered HLA-I restricted TCR raised against PRAME together with a chimeric PD1 receptor comprising the specific costimulatory region as herein described in comparison with PD1 receptors comprising the cytoplasmic domain of ICOS or 41 BB. Thus, highest killing acticity is achieved using a costimulatory region comprising full length wildtype CD40 cytoplasmic domain fused to a CD40 TRAF6 motif and a further CD40 TRAF123 motif as present in construct pl_2355 according to the present invention.

[0275]

[0276] 1.4 Cytokine Secretion Assay AnalysisCytokine secretion has been determined for each transduced T-cell fraction. The results are shown in Fig 6, Fig 7 and Fig 8, wherein Fig. 6 shows secretion of IFN-y and TNF-a, Fig. 7 show the results of the determined secretion of Granzyme A (Fig 7A), Granzyme B (Fig. 7B) and Granulysin (Fig. 7C), and wherein Fig 8 shows the secretion of IL-4 (Fig 8A) and IL-10 (Fig. 8B), respectively. The expression “NCIH2030” relates to the respective cytokine secretion of only cancer cells, “Mock” relates to mock-transduced T-cell fraction, “MAGE-A1 TCR” relates to CD4 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR); “wt-CD8” relates to CD4 / CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with wt-CD8ap coreceptor; “pTK-638” relates to the CD4 / CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with the enhanced chimeric CD8 co-receptor including the costimulatory region of both the cytoplasmic CD40 domain and the cytoplasmic CD30 motif as herewith provided.

[0277] 1.5. Results

[0278] As visible from Fig. 9, Co-transduction of CD4 / CD8 cells with an engineered HLA-I restricted TCR raised against MAGE-A1 together with an enhanced chimeric CD8 co-receptor, comprising the specific co-stimulatory domain as herewith provided results in an increased killing activity of the engineered T-cells compared with mock transducedT-cells and / or T-cells merely transduced with the HLA-I restricted TCR raised against MAGE-A1.

[0279] Fig. 7A, Fig 7B and Fig 7C show that co-expression of the enhanced chimeric CD8 co-receptor pTK-638 together with the MAGE-A1 TCR leads to a strong increase in secretion of Granzyme A, Granzyme B, and Granulysin both in comparison with CD4 T-cells only transduced with the MAGE-A1 TCR, and with CD4 T-cells transduced with the MAGE-A1 TCR together with a CD8ap wildtype co-receptor. This finding demonstrates that co-transduction of CD4 T-cells with a recombinant TCR and an enhanced chimeric co-receptor as herewith provided results in a higher secretion of proteolytic enzymes. Without being bound to theory, it is contemplated that this effect -among others - may contribute to the establishing of the enhanced cytotoxic activity of transduced T-cells associated with the enhanced chimeric CD8 co-receptors as herewith provided.

[0280] Fig. 8 shows that the secretion of both IL-4 and IL-10 is reduced when pTK-638 is co-expressed together with the MAGE-A1 TCR, in comparison with co-expression of a CD8ap wildtype co-receptor together with the MAGE-A1 TCR. Since both IL-4 and IL-10 are known to be anti-inflammatory cytokines, it may be concluded that WT CD8ap co-receptor co-transduced CD4 T-cells secrete more anti-inflammatory cytokines compared to pTK-638 co-transduced CD4 T-cells. Again, without being bound to theory, it is contemplated that this effect - among others - may contribute to the establishing of the enhanced cytotoxic activity of transduced CD4 T-cells associated with the enhanced chimeric CD8 co-receptors as herewith provided.

[0281] Summary and conclusions

[0282] The results described above demonstrate - in principle - suitability of the chimeric receptors as herewith provided in adoptive cell therapy (ACT). Specifically, it is contemplated that the enhanced chimeric CD8 co-receptor polypeptides, comprising the specific costimulatory domain as herein described, may be functional in generating high avidity CD4 / CD8 T-cells following co-transfer with e.g. engineered antigen specific TCRs, e.g. PRAME-specific TCRs. Thus, it is contemplated that the inclusion of the specific cytoplasmic costimulatory domain as herein provided to a chimeric CD8 co-receptor that comprises CD8a-derived polypeptide regions, and / or CD8p-derived polypeptide regions, and / or CD4-derived polypeptide regions, and which in particular comprises both a CD8a-derived IG-like domain region and a CD8p-derived IG-like domain region is able toenhance cytotoxicity of a T-cell. Furthermore, it is contemplated that the reduced size of the functional enhanced chimeric CD8 co-receptor polypeptides as described herein also advantageously addreses the problem of low transduction efficiency in ACTs with T-cells of patients having poor overall fitness. Furthermore, as described above, the reduced size of the enhanced chimeric CD8 co-receptor polypeptides as described herein allows the co-transduction of e.g. the costimulatory polypeptide regions, since the smaller size of the vector (and - as described above - possible omission of at least self-cleaving 2A peptide in comparison two transduction of wildtype CD8ap polypeptides) may allow for cotransduction of the at least one additional polypeptide without negatively affecting transduction efficiency (e.g. due to very large vector sizes). Furthermore, the results demonstrate that the chimeric receptors herein provided are suitable for use as powerful chimeric switch receptors.

[0283] 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.

[0284] 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.

[0285] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso ornegative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. Further embodiments of the invention will become apparent from the following claims.

Claims

Claims:What is claimed is:

1. A chimeric human receptor, comprising a polypeptide having an extracellular polypeptide region, a transmembrane polypeptide region, and a cytoplasmic polypeptide region, wherein the cytoplasmic polypeptide region comprises at least two TNF receptor associated factor 6 (TRAF 6) binding motifs, and / or wherein said cytoplasmic polypeptide region comprises at least two CD40 TNF receptor associated factor 1 / 2 / 3 (TRAF 1 / 2 / 3) binding motifs.

2. A chimeric human receptor, comprising a polypeptide having an extracellular polypeptide region, a transmembrane polypeptide region, and a cytoplasmic polypeptide region, wherein the cytoplasmic polypeptide region comprises at least two peptide fragments each having an amino acid sequence that is at least 85% identical to SEQ ID NO: 69, and / or wherein said cytoplasmic polypeptide region comprises at least two peptide fragments each having an amino acid sequence that is at least 85% identical to SEQ ID NO: 70.”3. The chimeric human receptor according to claim 1 or 2, wherein at least one, or at least two, of the at least two TRAF 6 binding motifs comprise a CD40 TRAF 6 binding motif.

4. The chimeric human receptor according to any one of the foregoing claims, wherein the at least one cytoplasmic polypeptide region comprises the at least two TNF receptor associated factor 6 (TRAF 6) binding motifs, and further wherein the at least one cytoplasmic polypeptide region comprises at least one TNF receptor associated factor 1, 2, 3 (TRAF 1 / 2 / 3) binding motif.

5. The chimeric human receptor according to claim 4, wherein the at least one TRAF 1 / 2 / 3 binding motif comprises a CD40 TRAF 1 / 2 / 3 binding motif.

6. The chimeric human receptor according to claim 4 or 5, wherein the at least one cytoplasmic polypeptide region comprises at least two CD40 TRAF 1 / 2 / 3 binding motifs.

7. The chimeric human receptor according to any one of the foregoing claims, wherein the at least one TRAF 6 binding motif comprises or consists of an amino acid sequence having at least 70%, or at least 75%, or at least 80%, or at least8585%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity with the amino acid sequence as set forth in Seq ID No: 69.

8. The chimeric human receptor according to any one of the foregoing claims, wherein the at least one TRAF 1 / 2 / 3 binding motif comprises or consists of an amino acid sequence having at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity with the amino acid sequence as set forth in Seq ID No: 70.

9. The chimeric human receptor according to any one of the foregoing claims, wherein the cytoplasmic polypeptide region comprises a complete cytoplasmic domain of CD40.

10. The chimeric human receptor according to any one of the foregoing claims, wherein the cytoplasmic region comprises at least one linker between the at least two TNF receptor associated factor 6 (TRAF 6) binding motifs, and / or between the at least two CD40 TNF receptor associated factor 1 / 2 / 3 (TRAF 1 / 2 / 3) binding motifs.

11. The chimeric human receptor according to claim 10, wherein the linker comprises or consists of an amino acid sequence as set forth by SEQ ID No. 58 (GGGGS).

12. The chimeric human receptor according to any one of the foregoing claims, wherein the cytoplasmic polypeptide region comprises or consists of an amino acid sequence having at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity with the amino acid sequence as set forth in Seq ID No: 80, 81, 82, or 83.

13. The chimeric human receptor according to any one of claims 1 to 11 , wherein the cytoplasmic polypeptide region comprises or consists of an amino acid sequence having at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity with the amino acid sequence as set forth in Seq ID No: 83.8614. The chimeric human receptor according to any one of claims 1-13, wherein the chimeric human receptor is a chimeric human CD8 Co-receptor.

15. The chimeric human CD8 co-receptor according to claim 14, wherein the polypeptide comprises a human wildtype CD8a extracellular polypeptide region and a human wildtype CD8a transmembrane polypeptide region, or wherein the polypeptide comprises a human wildtype CD8p extracellular polypeptide region and a human wildtype CD8p transmembrane polypeptide region.

16. The chimeric human CD8 co-receptor according to claim 14,wherein said polypeptide comprises at least one CD8a polypeptide region having at least 60% sequence identity with a functional polypeptide domain or a functional polypeptide motif of a wildtype human CD8a co-receptor, wherein said at least one CD8a polypeptide region comprises a CD8a IG-like domain region;further wherein said polypeptide comprises at least one CD8p polypeptide region having at least 60% sequence identity with a functional polypeptide domain or a functional polypeptide motif of a wildtype human CD8p co-receptor, wherein said at least one CD8p polypeptide region comprises a CD8p IG-like domain region.

17. The chimeric human CD8 co-receptor according to claim 14,wherein said polypeptide comprises at least one CD8a polypeptide region, wherein said at least one CD8a polypeptide region comprises a CD8a IG-like domain region;further wherein said polypeptide comprises at least one CD8p polypeptide region, wherein said at least one CD8p polypeptide region comprises a CD8p IG-like domain region.

18. The chimeric CD8 co-receptor according to any one of claims 14 to 17, wherein the Co-receptor further comprises at least one CD4 polypeptide region having at least 60% sequence identity with a functional polypeptide domain or a functional polypeptide motif of a wildtype human CD4 co-receptor.

19. The chimeric human receptor of any one of the foregoing claims, wherein said polypeptide is a single-chain polypeptide.8720. The chimeric CD8 co-receptor of any one of the claims 16 to 19, wherein the at least one CD8a polypeptide region further comprises at least one polypeptide region selected from the group consisting of a stalk domain region, a transmembrane domain region, and a LCK binding site motif region.

21. The chimeric CD8 co-receptor of any one of claims 16 to 20, wherein the at least one CD8p polypeptide region further comprises at least one polypeptide region selected from the group consisting of a stalk domain region, a transmembrane domain region, and a palmitoylation motif region.

22. The chimeric CD8 co-receptor according to any one of claims 18 to 21 , wherein the at least one CD4 polypeptide region comprises at least one polypeptide region selected from the group consisting of a stalk domain region, a transmembrane domain region, a palmitoylation motif region, and a LCK binding site region.

23. The chimeric CD8 co-receptor according to any one of claims 14 to 22, wherein said polypeptide comprises a stalk domain region, a transmembrane domain region, a palmitoylation motif region, and a LCK binding site region.

24. The chimeric CD8 co-receptor according to any one of claims 16 to 23, wherein the CD8a IG-like domain region is located closer to the N-terminal end of said chimeric CD8 Co-receptor polypeptide than the CD8p IG-like domain region.

25. The chimeric CD8 co-receptor according to any one of claims 16 to 23, wherein the CD8p IG-like domain region is located closer to the N-terminal end of said chimeric CD8 co-receptor polypeptide than the CD8a IG-like domain region.

26. The chimeric CD8 co-receptor according to any one of claims 14 to 25, wherein the polypeptide further comprises at least one linker region.

27. The chimeric CD8 co-receptor according to claim 26, wherein the polypeptide comprises a linker region between said CD8a IG like domain region and said CD8p IG-like domain region8828. The chimeric CD8 co-receptor according to claim 26 or 27, wherein the linker region comprises 1-100 amino acids, or wherein the linker region comprises 1-80 amino acids, or wherein the linker region comprises 1-50 amino acids, or wherein the linker region comprises 5-100 amino acids.

29. The chimeric CD8 co-receptor according to any one of claim 26 to 28, wherein the linker region comprises an amino acid sequence as set forth in SEQ ID No. 57 (GGGS)n or as set forth in SEQ ID No. 58 (GGGGS)n, wherein n is between 1 and 20, or wherein n is between 3 and 5.

30. The chimeric CD8 co-receptor according to claim 29, wherein the linker region comprises the amino acid sequence as set forth in SEQ ID No. 57 (GGGS)n, wherein n is 4.

31. The chimeric CD8 co-receptor of any one of claims 26 to 30, further comprising a linker region between the stalk domain region and the IG-like domain region of the polypeptide.

32. The chimeric CD8 co-receptor of any one of the claims 16 to 31, wherein the at least one CD8a 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 human wildtype CD8a Co-receptor.

33. The chimeric CD8 co-receptor of any one of the claims 16 to 31, wherein the at least one CD8p 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 human wildetype CD8p co-receptor.

34. The chimeric CD8 co-receptor of any one of claims 18 to 31, wherein the at least one CD4 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 identity89with the functional polypeptide domain or a functional polypeptide motif of a human wildetype CD4 co-receptor.

35. The chimeric human receptor of any one of the foregoing claims, wherein the chimeric co-receptor is able to enhance cytotoxicity of a T-cell.

36. The chimeric CD8 co-receptor of any one of claim 18 to 35, wherein the at least one CD4 polypeptide region further comprises a CD4 palmitoylation motif region, and / or a CD4 LCK binding site region.

37. The chimeric CD8 co-receptor according to claim 36; wherein the at least one CD8a polypeptide region further comprises a CD8a transmembrane domain region.

38. The chimeric CD8 co-receptor of claim 37, wherein the at least one CD8p polypeptide region further comprises a CD8p stalk domain region.

39. The chimeric CD8 co-receptor according to any one of claims 16 to 38, wherein said cytoplasmic polypeptide region comprises or consists of an amino acid sequence having 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 amino acid sequence as set forth in Seq ID No: 80, 81, or 82.

40. The chimeric CD8 co-receptor according to claim 39, wherein the polypeptide is having an amino acid sequence with 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 amino acid sequence as set forth in SEQ ID No: 71, 73, or 75.

41. The chimeric human receptor according to any one of claims 1-13, wherein the chimeric human receptor is chimeric costimulatory receptor, optionally wherein the chimeric human receptor is a chimeric costimulatory switch receptor.

42. The chimeric costimulatory receptor according to claim 41 , wherein the extracellular polypeptide region comprises an extracellular domain of a member of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF).9043. The chimeric costimulatory receptor according to claim 42, wherein the extracellular polypeptide region comprises an extracellular domain of CD95.

44. The chimeric costimulatory receptor according to claim 41 , wherein the extracellular polypeptide region comprises an extracellular domain of LAG-3, CTLA4, PD1, TIGIT, or TIM-3.

45. The chimeric costimulatory receptor according to any one of the claims 42 to 44, wherein the transmembrane polypeptide region comprises the transmembrane region of CD95, LAG-3, CTLA4, PD1, TIGIT, or TIM-3, optionally wherein the extracellular polypeptide region and the transmembrane polypeptide region are of the same receptor.

46. The chimeric costimulatory receptor according to claim 44 or 45, wherein the receptor is a PD1 receptor.

47. The chimeric PD1 receptor according to claim 46, wherein the polypeptide comprises at least one PD1- polypeptide region having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a human PD1 receptor as set forth in SEQ ID No. 63, wherein said PD1 polypeptide region comprises a PD1 extracellular ligand binding domain.

48. The chimeric PD1 receptor according to claim 46, wherein said polypeptide comprises at least one PD1 polypeptide region comprising a PD1 extracellular ligand binding domain.

49. The chimeric PD1 receptor according to claim 47 or 48, wherein the extracellular ligand binding domain is functional in binding a PD1 ligand.

50. The chimeric PD1 receptor according to any one of claims 47 to 49, wherein the PD1 extracellular ligand binding domain comprises a PD1 immunoglobulin variable (IgV) domain, wherein the PD1 IgV domain is having at least 85% sequence identity to the amino acid sequence of SEQ ID No. 64.

51. The chimeric PD1 receptor according to any one of claims 47 to 50, wherein said polypeptide is a single-chain polypeptide.

52. The chimeric PD1 receptor according to any one of claims 47 to 51 , wherein the at least one PD1 polypeptide region comprises a PD1 transmembrane region.

53. The chimeric PD1 receptor according to any one of claims 47 to 51, wherein the91polypeptide comprises a transmembrane domain from CD30, or CD40.

54. The chimeric PD1 receptor according to any one of claims 47 to 53, wherein the at least one PD1 polypeptide region comprises a complete PD1 extracellular domain.

55. The chimeric PD1 receptor according to any one of claims 47 to 54, wherein the at least one PD1 polypeptide region has at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or 100% sequence identity with the functional polypeptide domain, region or motif of a human PD1 receptor (Seq ID No. 63), and / or wherein the at least one PD1 polypeptide region is having one or more conservative amino acid substitutions relative to the functional polypeptide domain, region or motif of the human PD1 receptor (Seq ID No. 63).

56. An isolated nucleic acid comprising a nuclear acid sequence encoding for the chimeric human receptor according to any one of claims 1-55.

57. A vector comprising the nucleic acid according to claim 56.

58. The vector of claim 57, wherein the vector is a viral vector or a non-viral vector.

59. The vector of claim 57 or claim 58, wherein the vector is a viral vector.

60. The vector of claim 59, wherein the viral vector is selected from adenoviruses, poxviruses, alphaviruses, arenaviruses, flaviruses, rhabdoviruses, retroviruses, lentiviruses, herpesviruses, paramyxoviruses, picornaviruses, and combinations thereof.

61. The vector of any one of claims 57 to 60, wherein the vector further comprises a nucleic acid encoding a chimeric antigen receptor (CAR).

62. The vector of any one of claims 57 to 60, wherein the vector further comprises a nucleic acid encoding a T-cell receptor comprising a TCR a chain and a TCR p chain.

63. The vector according to claim 62, wherein the T-cell receptor is a recombinant T- cell receptor.9264. An isolated T-cell, the T-cell being transfected, transformed, transduced or electroporated with the nucleic acid according to claim 57.

65. An isolated T-cell, the T-cell being transfected, transformed, transduced or electroporated with the vector of any one of claims 57 to 63.

66. An isolated T-cell, the T-cell being transduced to express the chimeric receptor of any one of the claims 1 - 55.

67. An engineered T-cell, the T-cell expressing the chimeric receptor according to any one of claims 1 - 55.

68. The T-cell according to claim 66 or 67, wherein the T-cell expresses a heterologous T-cell receptor.

69. The T-cell of any one of claims 64 to 68, wherein the cell is a ap T-cell, / _8 T-cell, and / or a natural killer T-cell.

70. The T-cell of claim 69; 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.

71. The T-cell of any one of the claims 64 to 70, wherein the T-cell expresses the chimeric receptor according to any one of claims 1 - 54.

72. The T-cell according to any one of claims 64 - 71, wherein the T-cell is derived from an induced pluripotent stem cell (iPSCs).

73. A kit comprising means to prepare the T-cell according to any one of claims 64 to 72.

74. A pharmaceutical composition comprising the T-cell of any one of claims 64 to 72.

75. The pharmaceutical composition of claim 74, wherein the composition further comprises an adjuvant, excipient, buffer, diluent, carrier, stabilizer or combination thereof.

76. A method for preparing a T-cell for immunotherapy, comprisingisolating T-cells from a human subject,transducing, transfecting, transforming or electroporating the T-cells with the nucleic acid of claim 56 or the vector of any one of claims 57 to 63, and expanding the transfected, transformed or transduced T-cells.

77. A pharmaceutical composition comprising T-cells expressing the chimeric receptor according to any one of claims 1-55.

78. A method for treating a patient having a disease, comprising administering to the patient the composition of any one of claims 74, 75 or 77.

79. A method for treating a patient having a disease, comprising introducing in vivo the nucleic acid according to claim 56 or the vector according to any one of claims 57 to 63 into a T-cell of the patient.

80. The method according to claim 79, wherein the nucleic acid is a DNA or a mRNA.

81. The method according to claim 79, wherein the vector is a non-replicating viral vector.

82. The method according to claim 79 or 80, wherein the nucleic acid is mRNA, and wherein the mRNA is introduced into the T-cell of the patient using nanoparticles.

83. The method according to any one of claims 78 to 82, wherein the disease is a cancer or an autoimmune disease.

84. The method according to claim 83, 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, urinarybladder cancer, uterine cancer, colorectal cancer, gallbladder cancer, bile duct cancer, and prostate cancer.95

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