Co-stimulatory t-cell receptor to treat patient with tumor or immune-related disease

Abstract

The invention relates to a chimeric T-cell receptor (TCR) comprising a human transmembrane domain, a human intracellular domain and a human intracellular CD3ε domain wherein in at least one of the CD3ε domains, an arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 54 of SEQ ID NO:25, is substituted or deleted. The invention further relates to a method of producing a T-cell expressing the chimeric co-stimulatory TCR. The invention further relates to a chimeric T-cell receptor (TCR) comprising a human co-stimulatory domain and a human CD3ε domain. The invention further relates to a method of treating a patient having a tumor or an immune-related disease comprising administering T-cells expressing the chimeric TCR to the patient.

Description

[0001] P136875PC00 Title: Co-stimulatory T-cell receptor to treat patient with tumor or immune-related disease FIELD The invention is in the field of immunotherapy, more specifically cell therapy, more specifically the use of T-cells comprising a chimeric T-cell receptor to treat tumors or immune-related diseases. 1. INTRODUCTION Adoptive T-cell therapy involves the administration of genetically engineered immune cells, such as T-cells, harboring antigen specific T cell receptors (TCR) or chimeric antigen receptors (CAR) and is a promising approach for a successful treatment of cancer or immune-related diseases. CARs and TCRs both can recognize a pre-defined tumor antigen, enabling T-cells to selectively destroy cancer cells expressing this target antigen upon activation of the T-cells. Importantly, CARs recognize extracellular antigens (representing about 25 % of all antigens), whereas TCRs recognize extra- as well as intracellular antigens (representing 100 % of antigens). While the usage of CARs has shown impressive results in leukemia and multiple myeloma with response rates up to 90 %, culminating in the EMA and FDA approval of up to six products and numbers further increasing, their current success in the treatment of solid tumors lags behind that of hematological malignancies (Zhang et al., 2022. Frontiers in Immunology 13: 920669; Hammerl et al., 2018. Trends Immunol 39: 921-936). As such, challenges related to antigen expression heterogeneity, off-tumor / on-target toxicity, CAR T-cell trafficking, expansion and persistence need to be circumvented to successfully treat these cancer types. Especially in solid tumors, the administration of CAR T-cell therapy is limited by the lack of truly tumor-restricted targets leading to unwanted on- target / off-tumor toxicities on normal tissue. On the other hand, T-cells comprising engineered TCRs have demonstrated clinical benefit in patients with multiple myeloma, metastatic melanoma and metastatic synovial sarcoma with response rates varying between 20 % and 80 % (Baulu et al., 2023. Science Advances 9: eadf3700). Indeed, adoptive T-cell therapy using TCRs directed against NY-ESO-1 or MAGE-A4 have shown impressive clinical responses in patients with melanoma or synovial sarcoma (Robbins et al., 2011. J Clin Oncol 29: 917-924; Hong et al., 2023. Nature Medicine 29: 104-114). One major challenge in the field of adoptive T-cell therapy when treating solid tumors, is that in most patients, responses are not durable and tumors progress or recur within one year. Recent understanding of the tumor micro- environment shows that lack of co-stimulation (i.e. tumor cells and tumor- associated endothelial cells generally show down-regulated expression of co- stimulatory ligands) hampers sufficient entrance of T-cells into tumor tissue and sustained activation once they have entered the tumor tissue. Along this line, it has been demonstrated that additional co-stimulation of T-cells, for instance via PD1 antagonistic antibodies or 4-1BB agonistic antibodies is indeed advantageous for their anti-tumor performance. The latter antibodies are standard-of-care for certain solid tumor types, yet they are costly, mediate significant toxicity and do not work for all patients. A second line of evidence favoring the requirement for additional co-stimulation comes from studies with CAR T-cells. Even though CARs have not yet proven successful for the treatment of solid tumors (as mentioned above), those that entered standard-of-care treatments for leukemias harbor co- stimulatory domains and studies have revealed that the choice of co-stimulatory domain does affect durability of anti-tumor responses. A last line of evidence favoring the requirement for additional co-stimulation comes from preclinical studies, where a second receptor that harbors a co-stimulatory domain (generally in addition to a CAR) may enhance the efficacy of anti-leukemic T-cell responses. These multi-receptor gene transfer approaches (where more than a single receptor needs to be introduced to create a clinical T-cell product) may, however, be challenged by technical difficulties that limit translation to a clinical setting. To introduce additional co-stimulation, and not be limited by above- mentioned hurdles, newly designed TCRs with built-in domains of co-stimulatory receptors have been developed that were initially put to the test in immunocompetent mouse models. These new murine TCRs rescue T-cell responses and result in highly durable responses in models of solid tumors in which T-cell responses would otherwise become quickly curtailed due to immune suppression. To introduce a co-stimulatory TCR into a therapeutic product to treat patients with solid tumors, such a new TCR preferably comprises human domains or building blocks. This is not trivial as such co-stimulatory TCRs, prior to becoming a candidate for clinical testing, have demonstrated success in preclinical, often murine, cancer models. These mouse chimeric TCRs, besides presenting potential immunogenicity in human patients, are not rebuilt in a straightforward manner to human chimeric TCRs. In fact, our observations, as exemplified in the current filing, have shown that when replacing murine domains by corresponding human orthologues the resulting chimeric TCR may be severely challenged by compromised surface expression by human immune cells or T-cells. This likely relates to the fact that functional expression of TCR chains, in contrast to CAR chains, is intricately regulated and involves interaction of two TCR chains (alpha and beta) and four CD3 accessory molecules (gamma, delta, epsilon and zeta). Despite such challenge, it is critical to create such new human TCRs as they are more likely to function optimally in human T-cells and are less likely to evoke immune response in human patients that may counteract the durability of these T- cell products (Berger et al., 2006. Blood 107: 2294-2302; Lamers et al., 2011. Blood 117: 72-82). 2. BRIEF DESCRIPTION OF THE INVENTION The present invention describes a chimeric co-stimulatory human T-cell receptor (cTCR) that initiates T-cell co-stimulation only when T-cells are at the right location, for example, within a tumor expressing a target antigen, and yields durable T-cell responses against solid tumors. As such, the co-stimulatory domain that is present in the cTCR ensures enhanced effectiveness without affecting safety of treatment with the T-cell product by off-tumor / on-target activity. The chimeric T-cell receptor (TCR) comprises a human transmembrane domain, a human intracellular domain and a human CD3^ intracellular domain. In embodiments, the chimeric T-cell receptor (TCR) comprises a human transmembrane CD28 domain, a human intracellular ICOS domain and a human CD3^ intracellular domain. Notably, T-cell signaling via an intracellular domain such as ICOS within a tumor results in enhanced T-cell fitness, where these T-cells produce inflammatory cytokines and do not become exhausted upon repeated tumor cell exposure. It was found that expression of human cTCR strictly depends on the substitution of key amino acid residues in the CD3^ tail region, which preserves the chimeric TCR’s ability to limit T-cell exhaustion. This enables the generation and use of chimeric TCRs with different specificities. The invention provides a chimeric T-cell receptor (TCR) comprising an extracellular human or humanized TCRα chain and an extracellular human or humanized TCR^ chain, wherein said TCRα chain and said TCR^ chain form an extracellular antigen binding domain that binds to an antigen. Said chains further comprise, in an N-terminal to C-terminal order, a human transmembrane domain from a co-receptor selected from CD8^, CD8^ or CD4, or a co-stimulatory receptor selected from CD28, ICOS, CD27, 2B4 or NKG2D, a first human intracellular domain from a co-stimulatory receptor selected from ICOS, CD28, 4-1BB, OX40, CD27, CD40L, 2B4 or NKG2D; and a human intracellular CD3ε domain, wherein in at least one of the CD3ε domains, an arginine (R) amino acid residue at position 53, 54, or both positions 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. An example of a human transmembrane domain is provided in SEQ ID NO:16. An example of a human intracellular domain is provided in SEQ ID NO:23. In embodiments, the transmembrane domain is or comprises the transmembrane domain from a CD28 or ICOS. Examples of a transmembrane domain are provided in SEQ ID NOs: 16- 18. In embodiments, the first intracellular domain is or comprises an intracellular domain from a co-stimulatory receptor ICOS, CD28, 4-1BB, OX40, or CD40L. Examples of an intracellular domain are provided in SEQ ID NOs: 20-23. In embodiments, the TCRα chain, the TCR^ chain, or both may comprise a second intracellular domain, either N-terminal or C-terminal of the original first intracellular domain. In embodiments, said second intracellular domain is or comprises an intracellular domain from a co-stimulatory receptor selected from ICOS, CD28, 4-1BB, OX40, CD27, CD40L, 2B4 or NKG2D, whereby the second human intracellular domain may differ from the first human intracellular domain. Examples of a second intracellular domain are provided in each of SEQ ID NOs: 20-23. A preferred transmembrane domain of the TCRα chain and / or the TCR^ chain is or comprises the transmembrane domain from the human co-stimulatory receptor CD28, provided herein as SEQ ID NO:16. A preferred intracellular domain of the TCRα chain and / or the TCR^ chain comprises or is the intracellular domain from the human co-stimulatory receptor ICOS, provided herein as SEQ ID NO:23. A preferred chimeric TCR comprises a human transmembrane domain from the co- stimulatory receptor CD28 and a human intracellular domain from the co- stimulatory receptor ICOS. In embodiments, the TCRα chain and the TCR^ chain both comprise the transmembrane domain from the human co-stimulatory receptor CD28 and an intracellular domain from the human co-stimulatory receptor ICOS. In preferred embodiments, a chimeric TCR according to the invention comprises the transmembrane domains of from the co-stimulatory receptor CD28 in both the TCRα chain and the TCR^ chain, the first intracellular co-stimulatory domains from ICOS in both the TCRα chain and the TCR^ chain, and both the TCRα chain and the TCR^ chain have an arginine (R) amino acid residue at position 54 of SEQ ID NO:25 of the human intracellular CD3ε domain substituted or deleted. In embodiments, the substitution of the arginine amino acid residue at position 53 and / or 54 of SEQ ID NO:25 is a substitution into any other amino acid, preferably an aspartic acid, a glutamic acid, a proline, a serine, a threonine, a cysteine, a tyrosine, a phenylalanine, an alanine, a valine, a leucine, an isoleucine, a methionine, or a glycine. In embodiments, the variable region of the TCR α chain and the variable region of the TCR β chain of a chimeric TCR according to the invention are fused into a single-chain TCR constituting an extracellular antigen binding domain. In a chimeric TCR according to the invention, functional expression of said cTCR on the surface of a cell such as an immune cell is enabled by substitution or deletion of at least one arginine (R) amino acid residue at position 53, 54, or both 53 and 54 of SEQ ID NO:25 of both human intracellular CD3ε domains. In embodiments, the TCRα chain and the TCR^ chain form an extracellular antigen binding domain that specifically binds an epitope from an antigen, preferably a cancer-specific antigen. Said extracellular antigen binding domain preferably binds to an epitope selected from a human cancer germline antigen, such as ROPN1, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C2 or NY-ESO1, a mutated antigen, such as KRAS, PIK3CA or TMPRSS2-ERG mutants and / or a cancer neoantigen, including a tumor frameshift antigen. Examples of variable domains of TCRα and TCRβ that bind an epitope derived from a cancer antigen are provided in SEQ ID NOs: 1-6. In embodiments, the TCRα chain and the TCRβ chain constitute an extracellular binding domain that specifically binds an epitope from a self-antigen of a patient, such as MOG, GAD65, TSHR, tTG, and fibrinogen, or an antigen of a pathogen, preferably an antigen of a virus, such as an antigen from EBV, CMV and HIV. The invention further provides a T-cell, preferably a cytotoxic T lymphocyte (CTL), a CD4 helper cell, a γδ T-cell, a tumor-infiltrating lymphocyte (TIL), a regulatory T-cell, a memory T-cell or a Natural Killer T (NKT) cell, or a Natural Killer (NK) cell, expressing a chimeric TCR according to the invention. The invention further provides a pharmaceutical composition, comprising a T-cell expressing a chimeric TCR according to the invention, or an in vivo gene- delivery system encoding a chimeric TCR according to the invention, and a pharmaceutical acceptable carrier. The invention further provides a method of producing a T-cell according to the invention, the method comprising (a) isolating a T-cell from a patient such as an human individual; (b) modifying the T-cell with a construct encoding a chimeric TCR according to any the invention; (c) enabling expression of the chimeric TCR in the T-cell, whereby said chimeric TCR comprises an extracellular antigen binding domain that binds to an antigen of the patient. The invention further provides a method of treating a patient having a tumor, preferably a solid tumor, the method comprising (a) isolating a T-cell from the patient; (b) providing the T-cell with a chimeric TCR according to the invention, said chimeric TCR comprising an extracellular antigen binding domain that binds to a cancer antigen of the patient; (c) enabling expression of the chimeric TCR in the T-cell, thereby generating a modified T-cell product; and (d) administering the modified T-cell product to the patient. The invention further provides a method of treating a patient having or suffering from an immune related disease, such as an autoimmune disease, the method comprising (a) isolating a T-cell from the patient; (b) providing the T-cell with a chimeric TCR according to the invention, said chimeric TCR comprising an extracellular antigen binding domain that binds to a tissue related antigen or self- antigen of the patient; (c) enabling expression of the chimeric TCR in the T-cell, thereby generating a modified T-cell product; and (d) administering the modified T- cell product to the patient. The invention further provides a method of treating a patient having or suffering from an infection with a pathogen, preferably a virus, the method comprising (a) isolating a T-cell from the patient; (b) providing the T-cell with the chimeric TCR according to the invention, said chimeric TCR comprising an extracellular antigen binding domain that binds to an antigen of the pathogen; (c) enabling expression of the chimeric TCR in the T-cell, thereby generating a modified T-cell product and (d) administering the modified T-cell product to the patient. The invention further provides a method of treating a patient having a tumor, preferably a solid tumor, an immune-related disease, preferably an auto-immune disease, or an infection by a pathogen, preferably a virus, the method comprising (a) providing a T-cell with a chimeric TCR according to the invention, said chimeric TCR comprising an extracellular antigen binding domain that binds to a cancer antigen of the patient, to an antigen of the patient or to an antigen of a pathogen; (b) enabling expression of the chimeric TCR in the T-cell, thereby generating a modified T-cell product; and (c) administering the modified T-cell product to the patient. The invention further provides a method of treating a patient having a tumor, preferably a solid tumor, the method comprising (a) providing an in vivo gene- delivery system encoding a chimeric TCR according to the invention, whereby said chimeric TCR comprises an extracellular antigen binding domain that binds to a cancer antigen of the patient, and (b) providing said in vivo gene-delivery system encoding the chimeric TCR to the patient; whereby the gene delivery system is a viral vector, or a nanoparticle. The invention further provides a method of treating a patient having an immune-related disease, preferably an auto-immune disease, comprising (a) providing an in vivo gene-delivery system encoding a chimeric TCR according to the invention, said chimeric TCR comprising an extracellular antigen binding domain that binds to an antigen of the patient; and (b) providing said in vivo gene- delivery system encoding the chimeric TCR to the patient; whereby the gene delivery system preferably is a viral vector or nanoparticle, enabling the expression of the chimeric TCR by the patient’s T-cells. The invention further provides a method of treating a patient having an infection by a pathogen, preferably a virus, comprising (a) providing an in vivo gene-delivery system encoding a chimeric TCR according to the invention, said chimeric TCR comprising an extracellular antigen binding domain that binds to an antigen of the pathogen; and (b) providing said in vivo gene-delivery system encoding the chimeric TCR to the patient; whereby the gene delivery system preferably is a viral vector or nanoparticle, enabling the expression of the chimeric TCR by the patient’s T-cells. The invention further provides a method of treating a cancer patient according to the invention, the method comprising the administration of the pharmaceutical composition of the invention, optionally in combination with surgery, radiation therapy and / or one or more anti-cancer drugs or a combination thereof. The invention further provides a method of treating a patient suffering from an immune-related disease such as an autoimmune disease, the method comprising the administration of the pharmaceutical composition of the invention, optionally in combination with an immunosuppressant. The invention further provides a method of treating a patient suffering from an infection with a pathogen, the method comprising the administration of the pharmaceutical composition of the invention, optionally in combination with an antibiotic, including an antifungal and / or antiviral agent. 3. BRIEF DESCRIPTION OF THE FIGURES Figure 1. TCR:ICOS is a co-stimulatory TCR that results in durable anti- melanoma efficacy following adoptive transfer. (A) Hypothesis regarding the surplus value of co-stimulatory TCRs for anti-tumor T-cells. (B) Design of murine co-stimulatory TCRs, where extracellular TCR-V^C^ and V^C^ chains targeting human gp100 / A2 were coupled to the transmembrane domain of CD28, followed by the intracellular domains of either CD28, OX40, ICOS or 4-1BB, and the intracellular domain of CD3ε (see Materials and Methods for details regarding TCR constructs). (C) C57BL / 6 HHD mice were transplanted with the B16:HHD- YLEP clone expressing the HLA-A2-restricted gp100 epitope YLEPGPVTA. Prior to adoptive T-cell transfer, mice received busulfan and cyclophosphamide, after which mice were administered 6x106wt TCR, TCR:CD28, TCR:OX40 or TCR:ICOS T-cells. Mice were monitored up to 150 days after T-cell transfer for tumor volume, survival and number of TCR T cells in blood (see Materials and Methods for details). (D) Tumor volume in mm3following T-cell transfer (median+SEM, n=4-6 per group). Mice attaining a tumor volume > 1400 mm3were sacrificed and the volume was left at its peak. (E) Survival of mice following T-cell transfer according to Kaplan-Meier curves. Inter-group comparison was done according to Cox regression. (F) Time to tumor relapse in days following T-cell transfer (median±SEM, n=4-6). Inter-group comparison was done according to Kruskal- Wallis testing. (G) Numbers of CD8+ TCR T-cells per µL blood following T-cell transfer as determined by flow cytometry (median±SEM, n=4-6). Inter-group comparison was done according to linear mixed model. (H) Stacked bars displaying overall anti-tumor response rate of mice treated with different TCR T-cells. Definitions of complete, partial and non-response are given in Materials and Methods section. With * = p<0.05, ** = p<0.01, *** = p<0.001. Figure 2. In vitro performance of co-stimulatory TCRs. (A) Dot plot showing surface expression of the different co-stimulatory TCRs in mouse splenocytes (n=2- 5, mean±SEM). (B) Line graphs displaying IFN^ production of TCR T-cells following stimulation with T2 cells that were pre-loaded with titrated amounts of human gp100 peptide (n=6, mean±SEM). TCR T-cells were co-cultured with peptide-loaded T2 cells for 16 h at 37 °C, with supernatants being assayed with IFN^^ELISA. EC50 values were calculated using graphpad prism. Figure 3. Intra-tumoral CD8+ TCR:ICOS T-cells demonstrate a young and co- stimulatory phenotype and create an inflammatory micro-environment. (A) Experimental design, in which mice were transplanted with B16:HHD-YLEP and pre-treated with chemotherapy as described in legend to Figure 1A, after which mice were administered 7.5x106wt TCR or TCR:ICOS T-cells. Monitoring included frequency assessment of subsets CD8+ TCR T-cells in blood (at 3 weeks after T-cell transfer) and TILs (in regressed tumors) according to maturation and co-signaling status as well as transcriptomics of tumors (see Materials and Methods for details). (B) Left panel: stacked bars corresponding to maturation phenotype of CD8+ TCR T-cells in blood following T-cell transfer as determined by flow cytometry (n=3-8 per group). Maturation status of T-cells was defined as follows: naïve T cells, CD62L+CD44-; central memory T-cells, CD62L+CD44+; and effector memory T- cells, CD62L-CD44+. Middle panel: heatmap corresponding to phenotype of CD8+ TCR T-cells in blood following T-cell transfer as determined by flow cytometry. Co- signaling phenotype of T-cells was determined according to the expression of (combinations of) co-inhibitory (CTLA4, LAG3, PD1, TIM3) or co-stimulatory receptors (CD40L, CD137, ICOS, OX40). The heatmap displays median expressions of single or multiple markers, n=3-8 per group. Statistical significance between TCR:ICOS and wt TCR T-cells according to Kruskal-Wallis testing and highlighted at left-hand side (* if p <0.05). Right panel: phenotype of CD8+ TCR:ICOS T-cell subsets that show differential frequencies when compared to CD8+ wt TCR T-cells. (C) Left and middle panels: stacked bars and heatmap corresponding to phenotype of CD8+ TCR TILs as determined by flow cytometry (n=2-4 per group). Heatmap displayed in color, as grayscale did not allow sufficient discrimination between groups. See for details regarding markers and testing legend to Figure 3B. Right panel: phenotype of CD8+ TCR:ICOS TIL subsets that show differential frequencies when compared to CD8+ wt TCR TILs. D. Left panels: differentially expressed genes (left: heatmaps) and enriched pathways (right: enrichment plots) in regressed tumors following transfer of TCR:ICOS T cells when compared to wt TCR T cells. (E) Gene sets that are enriched in regressed tumors following transfer of TCR:ICOS T-cells when compared to wt TCR T-cells. With * = p<0.05, ** = p<0.01, *** = p<0.001. Figure 4. Relapsing tumors do not reflect changes in regressing tumors. Left panel: heatmap displaying enrichment of hallmarks pathways in relapsed tumors following T-cell transfer. Right panel: Heatmap of enriched immune-related signatures that are associated with immune-escape in both regressed and relapsed tumors from wt TCR and TCR:ICOS T-cell-treated mice. Heatmaps are displayed in color as grayscale did not allow sufficient discrimination between treatment groups. Figure 5. In vitro stimulated TCR:ICOS rT-cells activate the PI3K and the NFκB pathway and produce inflammatory cytokines. (A) Left panel: Graphical representation of TCR:ICOS-YF, where the intracellular ICOS domain is mutated at a single site to prevent docking of and downstream signaling through PI3K. Right panel: Experimental design in which mouse splenocytes that expressed either TCR:ICOS, TCR:ICOS-YF or wt TCR were interrogated for intracellular signaling, cytokine production and ability to preserve fitness upon repeated antigen stimulation (see Materials and Methods for details). (B) Left panel: heatmap corresponding to fold-change in protein phosphorylation in wt TCR, TCR:ICOS and TCR:ICOS-YF T cells upon stimulation. Mouse T-cells were incubated for 30 min at 37°C with pre-coated A2Kb pentamers presenting the cognate epitope, after which cells were lysed and lysates were exposed to a fluorescence-based phosphorylation array covering 73 substrates representing T- cell signalling. In the heatmap, phosphorylated targets were categorized according to different intracellular pathways. Heatmap is displayed in color as grayscale did not allow sufficient discrimination between groups. Right panel: box plot displaying ratio of phosphorylated substrates associated with NFκB pathway. Comparison between groups was performed using Kruskal-Wallis, followed by post-hoc Dunn’s test. (C) Bar graphs displaying cytokine production following stimulation with tumor cells. T-cells were incubated for 16h at 37°C with B16 cells that either expressed the cognate epitope or not, after which supernatants were assayed for the presence of 14 cytokines according to a cytokine bead array. Statistical significance between TCR:ICOS and wt TCR T-cells was tested according to 2-way ANOVA with Sidack’s multiple comparison test. (D) Top plots: representative example of flow cytometric changes in percentage of PD1+, TIM3+ within CD8+ TCR T-cells following repeated stimulations. Bottom plot: change in percentage of CD8+ TCR T-cells co-expressing PD1 and TIM3 following repeated stimulations (±SEM, n=9). Co-culture with B16F10 wt tumor cells not expressing the cognate epitope did not result in changed percentages of this T-cell subset, and these backgrounds were used to correct experimental percentages. Statistical significance between TCR:ICOS and wt TCR T-cells (indicated with *) or TCR:ICOS-YF (indicated with +) was tested according to mixed model analysis. With * or + = p < 0.05, ** or ++= p < 0.01, *** or +++ = p < 0.001. Figure 6. In vitro stimulated TCR:ICOS T-cells activate the PI3K pathway and phosphorylate ERK1 / 2. (A) Bar graph displaying phosphorylation of PI3K following stimulation with tumor cells. T-cells were incubated for 2h at 37°C with B16 cells that either expressed the cognate epitope or not, after which T-cells were stained and analyzed for pPI3K (n=4). Statistical significance between TCR:ICOS and wt TCR T-cells was tested according to ANOVA with Sidack’s multiple comparison test. (B) Validation of T-cell stimulation used for the phosphorylation array, tested by pERK1 / 2 expression. Shortly, wt TCR T-cells were stimulated with either cognate peptide (grey curve) or irrelevant peptide (white curve) for 30 min at 37°C, and were stained for pERK1 / 2. With * = p<0.05. Figure 7. Durable anti-tumor response depends on docking site of TCR:ICOS for PI3K. (A) TCR:ICOS and TCR:ICOS-YF T-cells were studied for their in vivo anti-tumor performance comparable to that described in Figure 3A (see also Materials and Methods for details). Line graph displaying tumor volume in mm3following T-cell transfer (median+SEM, n=10-13 per group). (B) Survival of mice following T-cell transfer according to Kaplan-Meier curves. (C) Time to tumor relapse in days following T-cell transfer (median±SEM, n=10-13) and inter-group comparison according to Kruskal-Wallis testing. (D) Quantity of CD8+ TCR T-cells in numbers per µL blood following T-cell transfer as determined by flow cytometry (median±SEM, n=10-13) and inter-group comparison according to linear mixed model. Figure 8. Human orthologue of mTCR:ICOS is not surface expressed. Top panel: Graphical representation of wt TCR, mouse (m) TCR:ICOS and human (h) TCR:ICOS transgenes, in which mouse domains are written in gray and human building blocks in white. Surface expression in human T-cells is listed below graph, according to % TCR-V^^^ for CD3+ cells (mean, n>10 for each construct). Bottom panel: example of flow cytometric plots displaying expression of the different TCRs. Figure 9. Functional performance of human TCR:ICOS relies on single amino acid substitution in cytosolic tail of the CD3^ domain. (A) Selection and testing of hTCR:ICOS variants (see Materials and Methods and Table 1 for details). (B) Identification of single building blocks that are required for surface expression of mTCR:ICOS. Left panel: cartoon of first series of m / h TCR:ICOS variants. Right plot: surface expression of m / h TCR:ICOS variants according to % TCR-V^^^ (mean±SEM, n=6). (C) Identification of single amino acids of mCD3^ that are required for surface expression of hTCR:ICOS. Left plot: amino acid (aa) alignment between human and mouse CD3ε. Right plot: surface expression of mTCR:ICOS with h / m CD3^ aa substitutes according to % TCR-V^^^ (mean±SEM, n=2-10). Statistical significance between h / m TCR:ICOS variants and hTCR:ICOS according to Student’s t-test. (D) Demonstration of antigen reactivity, sensitivity and resistance against repeated stimulation of hTCR:ICOS-RA.1st plot: bar graph displaying surface expression of gp100 wt TCR and TCR:ICOS-RA according to % TCR-V^^^ (mean±SEM, n=10). Statistical significance according to Sidak-Holm’s. 2nd plot: IFN^ production following stimulation with T2 cells that were pre-loaded with titrated amounts of human gp100 peptide. TCR T-cells were co-cultured with peptide-loaded T2 cells for 16h at 37°C, with supernatants being assayed with IFN^ ELISA (mean±SEM, n=4). EC50 values were calculated using graphpad prism. 3rd plot: bar graph displaying IFN^ production of TCR T-cells following stimulation with BLM cells. TCR T-cells were co-cultured with BLM cells that either expressed the human gp100 antigen or not for 16h at 37°C, after which supernatants were assayed according to ELISA (mean±SEM, n=4). Statistical significance between hTCR:ICOS-RA and wt TCR according to two-way ANOVA. 4th plot: change in percentage of CD8+ TCR T cells co-expressing PD1 and TIM3 following repeated stimulations (mean±SEM n=3). For details see Materials and Methods. Statistical significance between TCR:ICOS and wt TCR T-cells was tested according to 2-way ANOVA, with Sidak’s post-hoc testing. (E) The hTCR:ICOS-RA format is extendable to other TCR specificities. 1st plot: bar graph displaying surface expression of ROPN1 wt TCR and TCR:ICOS-RA according to % TCR-V^^^^^ (mean±SEM, n=4). Statistical significance between hTCR:ICOS-RA and wt TCR according to Sidak-Holm’s. 2nd plot: IFN^ production following stimulation with T2 cells that were pre-loaded with titrated amounts of peptide (mean±SEM, n=4). 3rd plot: bar graph displaying IFN^ production following stimulation with human MM- 231 cells (mean±SEM, n=4).4th panel: change in percentage of CD8+ TCR T-cells co-expressing PD1 and TIM3 following repeated stimulations of 48h (mean±SEM, n=3). Experiments were performed similarly as described in legend to Figure 9D. Figure 10. Chimeric TCR expression in human T cells requires the transmembrane domain of either CD28 or ICOS, but not 4-1BB, OX40 nor CD40L. (A) Diagram depicting gp100 hTCR:ICOS variants, in which the transmembrane domain of CD28 was replaced by the transmembrane domain of other co- stimulatory receptors. (B) Bar chart representing the surface expression of TCRs on T cells after retroviral transduction. Level of expression is corrected for Vß14 expression in mock T cells and depicted relatively to the expression level of the parental hTCR:ICOS including the CD28 transmembrane domain (mean, n=2). Figure 11. Chimeric TCR expression in human T cells is not adversely affected by the intracellular domains of either CD28, CD40L, OX40 or 4-1BB. (A) Diagram depicting gp100 chimeric TCR variants, in which the intracellular domain of ICOS was replaced by the intracellular domain of other co-stimulatory receptors. (B) Bar chart representing the surface expression of TCRs on T cells after retroviral transduction. Level of expression is corrected for Vß14 expression in mock T cells and depicted relatively to the expression level of the parental hTCR:ICOS including the ICOS intracellular domain (mean + SEM, n=4). (C) T cells transduced with different chimeric TCRs were co-cultured overnight with BLM cells either expressing the gp100 target antigen or not. Bar charts represent IFN-γ levels measured in supernatant by ELISA (mean, n=2). Figure 12. The CD3ε R54 mutation is necessary for surface expression of chimeric TCRs in human T cells. (A) Diagram depicting gp100 hTCR:ICOS variants, in which the transmembrane domain of CD28 was replaced by the transmembrane domain of other co-stimulatory receptors and in which wt CD3ε was included, harboring an arginine at position 54. (B) Diagram representing chimeric gp100 TCR variants, in which the intracellular domain of ICOS was replaced by the intracellular domain of other co-stimulatory receptors and in which wt CD3ε was included, harboring an arginine at position 54. (C) Bar chart representing the surface expression of TCRs on T cells after retroviral transduction. Level of expression is corrected for Vß14 expression in mock T cells and depicted relatively to the expression level of the parental hTCR:ICOS including the CD28 transmembrane domain and CD3ε R54A (mean, n=2). (D) Bar chart representing the average surface expression of TCRs on T cells after retroviral transduction. Level of expression is corrected for Vß14 expression in mock T cells and depicted relatively to the expression level of the parental hTCR:ICOS including the ICOS intracellular domain and CD3ε R54A (mean + SEM n=4). Figure 13. hTCR:ICOS is expressed in human T cells when deleting hCD3ε R54 or exchanging it for another amino acid. (A) Diagram depicting ROPN1 hTCR:ICOS variants, in which the arginine at position 54 of the intracellular domain of CD3ε was replaced by all possible single amino acids or deleted. (B) Bar chart representing the surface expression of TCRs on T cells after retroviral transduction. Level of expression is depicted relatively to the expression level of the parental ROPN1 hTCR:ICOS including CD3ε R54A (mean, n=2-4). (C) T cells transduced with different hTCR:ICOS-RA variants were co-cultured overnight with MM-231 cells either expressing the ROPN1 target antigen or not. Bar charts represent IFN-γ levels measured in supernatant by ELISA (mean, n=2). Figure 14. Expression of hTCR:ICOS in human T cells is rescued not only by mutating R54, but also by mutating R53, or by mutating or deleting both R53 and R54. (A) Diagram depicting hTCR:ICOS, in which the arginine at position 53 of CD3ε was mutated to alanine, or both arginines at positions 53 and 54 were mutated to alanine or deleted. (B) Bar chart representing the surface expression of TCRs targeting gp100 (left panel), ROPN1 (middle panel) or NY-ESO1 (right panel) on T cells after retroviral transduction. Level of expression is corrected for Vß14 or Vß13.1 (gp100 or ROPN1 / NY-ESO1, respectively) expression in mock T cells and depicted relatively to the parental hTCR:ICOS including the CD28 transmembrane domain, the ICOS intracellular domain and CD3ε R54A (mean + SEM, n=2-4). 4. DETAILED DESCRIPTION OF THE INVENTION Definitions The terms “tumor” and “cancer”, as used herein, refer to a disease which is characterized by deregulated or abnormal cell growth. In embodiments, these terms specifically refer to a cancerous tumor, which can spread into or invade nearby tissues and other parts of the body, a process called “metastasis”. The term “solid tumor”, as used herein, refers to a primary tumor, which is a localized abnormal mass of tissue, in or near an organ. Said solid tumor may also refer to a metastatic tumor, derived from the primary solid tumor. Said solid tumor is for example colorectal cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, anal cancer, uterine cancer, liver cancer, pancreatic cancer, lung cancer, endometrial cancer, bone cancer, testicular cancer, skin cancer, melanoma, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer and salivary gland cancer. The term ‘immune-related disease’, as used herein, refers to a condition resulting from a response of the adaptive immune system, wherein this system targets and attacks parts of its own body. The term “autoimmune disease”, as used herein, refers to an immune-related disease that results from an erroneous response of adaptive immune cells, specifically B or T cells, that target and attack healthy cells resulting in damage of healthy tissues or organs. Although there are over 80 different autoimmune diseases, most recognized examples include systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes mellitus, Sjögren syndrome, Graves’ disease, multiple sclerosis, psoriasis, inflammatory bowel disease, inflammatory myositis, coeliac disease, scleroderma, myasthenia gravis, pernicious anemia, Guillain Barrè syndrome, and vitiligo. The term “infection”, or “infectious disease”, as is used herein, refers to an immune-related disease that results from the establishment and / or invasion of one or more pathogenic agents, including bacteria, fungi, protozoans, and viruses, in the body of a host. The term infection includes the reaction of host tissues to the infectious agent and the toxins they produce, where the immune response starts with involving innate immune cells, such as granulocytes and myeloid cells, followed by involving adaptive immune cells, such as B and T cells. The term ‘immunotherapy’, as used herein, refers to the application of an immune system component, such as a T-cell, to a patient in need thereof. Said patient may be characterized by an insufficient or inadequate immune response against a tumor. Said immune system component may be retrieved from an individual to be treated, or from a donor, such as an allogeneic donor. Immunotherapy may be used, for example, for the treatment of a tumor, such as a solid tumor. The terms “adoptive immunotherapy”, “cellular adoptive immunotherapy” and “adoptive cell transfer”, as used herein, refer to the use of a recombinant immune cell, such as a T-cell, for the treatment of a tumor, including a solid tumor in a patient. Said patient may be characterized by an insufficient or inadequate immune response. Said adoptive immunotherapy includes, for example, a chimeric antigen receptor T-cell (CAR T-cell) and a T-cell comprising a non-modified T-cell receptor (TCR), or a chimeric TCR (cTCR). The term “immune cell”, as used herein, refers to a cell of hematopoietic origin, such as a lymphocyte, a natural killer cell and a myeloid cell, that plays a role in an immune response. The term “immune response”, as used herein, refers to an integrated response involving white blood cells to an antigen. Said term preferably refers to a cellular immune response or a cellular as well as a humoral immune response. An example of an immune response includes a protective, a preventive, a prophylactic or a therapeutic response against an antigen such as a tumor antigen. The term “T-cell”, as used herein, refers to a lymphocyte that matures in the thymus and participates in a variety of cell-mediated immune reactions. A T-cell comprises a T-cell receptor on the cell surface. Examples of a T-cell include all cell types of immune cells expressing a T-cell receptor or marker (CD3), such as a T- helper cell (i.e. CD4+ T-cell), a Th1 cell, a Th2 cell, a Th17 cell, a Th21 cell, a Th22 cell, a cytotoxic T-cell (i.e. CD8+ T-cell), a natural killer T-cell, a regulatory T-cell (CD4+ CD25+ T cells), and a γ^^T-cell. Most of these cells are effector T cells and can be used therapeutically to destroy cancer cells or autoimmune lymphocytes. The regulatory T cells, however, may suppress effector T cell responses, and as such may limit an immune response, including autoimmune responses, of lymphocytes (Yang et al., 2022. Sci Transl Med 14: eabn1716; Doglio et al., 2024. Nat Comm 15: 2542). The term “recombinant T-cell” refers to a T-cell comprising an exogenous expression construct, such as a construct encoding for a TCR, such as a chimeric TCR. The term “extracellular antigen binding domain”, as used herein in the context of a TCR, refers to an extracellular variable domain of an ^- and^^-TCR chain that each comprises three hypervariable complementarity determining regions (CDRs) that typically bind to a peptide:MHC complex. The term “variable domain”, as used herein, refers to a part of an antigen binding receptor, such as a TCR, that binds to a specific part of an antigen or epitope, as is known to a person skilled in the art. A variable domain is characterized by an immunoglobulin fold comprising two closely packed anti- parallel ^-sheets bridged by a conserved disulfide bond, which is crucial for its functionality. The term “complementary determining region”, abbreviated as “CDR”, as used herein, refers to a part of an antigen binding receptor, such as a TCR, more specifically to a part of a variable domain of an antigen binding receptor. Each variable domain comprises three such regions named CDR1, CDR2 and CDR3, which are interspersed by so called framework regions (FR). The term “humanized”, as used herein in the context of a TCR or chimeric TCR, refers to TCRα and TCRβ gene sequences, especially encoding the variable regions of TCRα and TCRβ, that may include sequences that encode the CDR1-3 and framework regions of the variable domains of the TCRα and TCRβ chains that are non-human, preferably of murine origin. In embodiments, said humanized TCRα and TCRβ gene sequences may include sequences that encode the constant TCRα and TCRβ domains that are of non-human origin, preferably of murine origin. The term “Major Histocompatibility Complex” molecule, abbreviated as “MHC”, as used herein, refers to a molecule that may present an antigenic determinant to a T-cell, thereby activating the corresponding T-cell. Said MHC molecule includes either an MHC class I or II molecule, which present a peptide to a cytotoxic T-cell or to a T-helper cell, respectively. The term “chimeric”, as used herein, refers to a recombinant protein that is created by fusion of two or more genes or part of genes that naturally encode for individual proteins or part of proteins. The term “T-cell receptor”, as used herein, refers to a molecule on the surface of a T-cell that recognizes an antigen, and more specifically a peptide derived from an antigen. Said peptide may be presented by an MHC molecule. An endogenous TCR is a heterodimeric protein, comprising an ^- and^^-TCR chain in complex with CD3 accessory molecules. Said TCR^ chain comprises a variable (V), a joining (J) and a constant (C) gene segment, while said^TCR^ chain comprises a variable (V), diversity (D), joining (J) and a constant (C) gene segment. The term “chimeric T-cell receptor”, abbreviated as “cTCR”, as used herein, refers to a recombinant T-cell receptor comprising customized domains, for example transmembrane and / or intracellular domains. In embodiments, a chimeric T-cell receptor comprises extracellular parts of ^- and^^-TCR chains each comprising an extracellular antigen binding domain, an extracellular TCR constant domain, and further comprising a transmembrane domain, an intracellular domain, and an CD3^ activation domain capable of activating or stimulating an immune cell. A chimeric TCR may be further modified, such as by insertion of one or more additional intracellular domains. The terms “co-stimulatory domain” and “intracellular domain”, as used herein, refer to an intracellular domain that relays a secondary, non-specific activation signal. The CD3^ domain, generally positioned at the C-terminus from the co-stimulatory domain and representing a component of the normal TCR-CD3 complex, relays a primary, antigen-specific signal. The latter signal is often necessary for T-cell effector functioning, whereas the former, co-stimulatory signal is often necessary for T-cell fitness and a prolonged anti-tumor T-cell response. The term “inducible co-stimulator”, abbreviated as “ICOS”, as used herein, refers to a transmembrane protein, more specifically a T-cell specific surface glycoprotein CD278, belonging to the CD28 receptor family and which may provide co-stimulatory signals required for T-cell proliferation and cytokine expression. The intracellular domain of CD278 is present in the intracellular part of CD278 and corresponds to amino acid residues 165-199 of UniProt entry Q9Y6W8. The transmembrane domain is present in the transmembrane region of CD278 corresponding to amino acid residues 141-164 of UniProt entry Q9Y6W8. The term “chimeric T-cell receptor: inducible co-stimulator”, abbreviated as “cTCR:ICOS”, as used herein, refers to a chimeric T-cell receptor comprising an extracellular antigen binding domain, a TCR-constant domain, a transmembrane domain, an activation domain capable of activating or stimulating an immune cell as well as the intracellular domain of ICOS or a domain with 70 – 100 % sequence similarity to said ICOS domain. In embodiments, a cTCR:ICOS may comprise a further intracellular domain. The term “cluster of differentiation 28”, abbreviated as “CD28”, as used herein, refers to a transmembrane protein, more specifically a T-cell specific surface glycoprotein CD28, which provides co-stimulatory signals required for T- cell activation and survival. The intracellular domain is present in the cytoplasmic part of CD28 corresponding to amino acid residues 180-220 of UniProt entry P10747. The transmembrane domain is present in the transmembrane region of CD28 corresponding to amino acid residues 153-179 of UniProt entry P10747. The terms “affinity”, “specifically binds” and “binding affinity”, as used herein, refer to an interaction, i.e., specific binding, between an antigen binding site and its epitope (i.e., being part of an antigen, in general a peptide:MHC complex). While binding to other targets cannot be excluded, it is preferred that binding to other targets occurs with low affinity, preferably at least 10 times lower than the binding affinity of the antigen binding site for its cognate epitope. This apparent binding affinity may be expressed as the equilibrium dissociation constant (KD) between a extracellular antigen binding domain and an epitope. In embodiments, a TCR that binds to a specified target antigen has a KD of ≤ 1 ^M, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM. Examples of a KD include 10-8M or less, from 10-8M to 10-13M or from 10-9M to 10-13M. The term “antigen binding site”, as used herein, refers to a motif of an antigen receptor that is capable of specifically interacting with a specific antigen or a specific group of antigens. An example of such antigen binding site is the variable region of a TCR. The term “antigen”, as used herein, refers to a molecule that comprises an epitope that is recognized by an antigen receptor, such as a T-cell receptor. Said antigen may be a protein which, optionally after processing, induces an immune response, which is specific for the antigen or derived epitope. The terms “epitope” and “antigenic determinant”, as used herein, refer to a site on, a part in, or a fragment of an antigen that may be recognized by an antigen receptor, such as a TCR. In particular, said epitope is recognized by a TCR when presented by an MHC molecule. The term "neoantigen", as used herein, refers to an antigen that is produced by a cancer cell, but is not produced by a healthy cell. In embodiments, this neoantigen is a protein or a specific modification of a protein. Said neoantigen may develop due to a mutation in the sequence of a gene. In embodiments, said neoantigen is a tumor frameshift antigen. The term “autologous”, as is used herein, refers to any material derived from an individual to whom it is later to be re-introduced to. Said term also refers to any material derived from an individual that is modified and to whom it is later to be re-introduced to. The term “allogeneic”, as is used herein, refers to any material derived from a different member of the same species as the individual into which the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more genomic loci of the individuals are not identical. The term “patient”, as used herein, includes reference to an individual who has a tumor, such as a solid tumor. The term “expression cassette”, as used herein, refers to a nucleic acid molecule comprising a coding sequence and a functionally coupled regulatory sequence, such as a promoter, regulating the expression of the coding sequence. The term “coding sequence”, as used herein, refers to a part(s) of a gene(s) that codes for a sequence of a protein, including a chimeric protein. Said coding sequence may or may not comprise introns and exons. In particular, a coding sequence starts with a start codon, such as ATG, and ends with a stop codon, such as TAA. The term “vector”, as used herein, refers to an isolated nucleic acid molecule, such as a plasmid, which can be used to deliver a nucleic acid to the interior of a cell. Numerous vectors are known to a person skilled in the art. The terms “combination” and “combination therapy”, as used herein, refer to the medical treatment of an individual in need thereof by providing modified T- cells as described herein, and an additional tumor / cancer therapy to the individual. Said additional tumor / cancer therapy may comprise more than one additional tumor / cancer therapies. The term “medical treatment”, as used herein, refers to a therapy or collection of therapies with which a patient in need thereof is being treated for a tumor, preferably a solid tumor. In embodiments, said medical treatment may include one or more administrations of a therapy. The term “sequence identity”, as used herein, refers to a sequence with 70 – 100 % identity, such as 75 %, 80 %, 85 %, 90 %, 95 %, or 99 % identity, to another sequence, preferably over its whole length. Such sequence identity indicates a percentage of similarity of nucleic acids or amino acids at their sequence levels and does not necessarily refer to an evolutionary relationship. The term “transduction”, as used herein, refers to an introduction of a nucleic acid molecule into a cell, such as a mammalian cell, typically by a virus or viral vector. In embodiments, said introduction is a stable introduction, meaning that the introduced nucleic acid molecule has been integrated into the genome of the cell. Chimeric T-cell receptor The invention comprises a chimeric T-cell receptor, comprising a human or humanized extracellular antigen binding domain, a human or humanized extracellular TCR-constant domain, a human transmembrane domain, a human intracellular domain and a human intracellular CD3ε domain wherein an arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, the invention provides a chimeric T-cell receptor, comprising extracellular parts of TCRα and TCRβ chains forming an N-terminal human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human or humanized extracellular TCR-constant domain, and a human transmembrane CD28 domain, a human intracellular ICOS domain and a human intracellular CD3ε domain wherein an arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. A chimeric T-cell receptor of a T-cell is designed to recognize a target cell such as a tumor cell, which will activate the T-cell and in turn will elicit an immune response in order to kill the target cell. A human or humanized extracellular antigen binding domain may be designed to recognize and bind an antigen present on a target cell, such as a tumor cell. A transmembrane domain links the extracellular domain with an intracellular domain and thereby anchors the receptor in the membrane of a T-cell. Moreover, a transmembrane domain may influence the expression level of a TCR and its stability and may play a role in signaling or synapse formation. An intracellular domain, when activated as a consequence of for instance binding of a target antigen by a cTCR, in turn initiates downstream signaling pathways generally by phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAM) domains present in (other) intracellular signaling molecules. Upon binding to an antigen, the incorporation of both an extracellular antigen binding domain and an intracellular co-stimulatory domain in one chimeric T-cell receptor will result in T-cell proliferation, the full activation of effector cell functions, such as cytokine release and the killing of a target tumor cell, as well as enhanced T-cell fitness and longevity (Sterner and Sterner, 2021. Blood Cancer Journal 11: 69). A person skilled in the art will appreciate that said human or humanized extracellular antigen binding domain, the human TCR-constant domain, the human transmembrane domain, the human intracellular domain and the human intracellular CD3ε domain wherein an arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted, may function independent from each other. Hence, different human or humanized extracellular antigen binding domains, or variants of each domain, or single-chain combinations of such variants, can be combined with a human TCR-constant domain, a human transmembrane, a human intracellular and a human CD3^^domain into a chimeric T-cell receptor. The domains of a chimeric T-cells receptor, especially the extracellular variable and / or constant regions of the TCRα and TCR^ chains, may be human or humanized, as such domains have less risk for inducing an immunogenic response, and maximally enable functioning of said cTCR in human T-cells or other immune cells. A cTCR comprises a human or humanized extracellular antigen binding domain by the variable domains of the TCRα chain and TCR^ chain. Each of said variable domains comprises three CDRs. Said extracellular binding domain is responsible for the binding of a cTCR to an epitope. CDR sequences to be used with the present ‘cTCR-format’, can be derived from any TCR. To illustrate this ‘plug- and-play’ characteristic, the use of CDR sequences derived from TCRs specific for human gp100, ROPN1 and NY-ESO-1 together with the ‘cTCR-format’ is exemplified herein. A person skilled in the art knows how to select CDR regions targeting a specific antigen, such as by making use of a TCR database (Gowthaman and Pierce, 2019. Bioinformatics 35: 5323-5325) or a CDR prediction tool, such as Kabat, IMGT or Chothia (Abhinandan and Martin, 2008. Mol Immunol 45: 3832- 3839). Said extracellular antigen binding domain preferably is human or humanized. Preferred methods for humanizing an extracellular antigen binding domain are known to a person skilled in the art and include for example grafting of human CDRs (Chen et al., 2018. Immunology 155: 123-136). A cTCR comprises a human transmembrane domain that is N-terminally fused to the C-terminal end of the extracellular part of the TCR-constant domain and C-terminally fused to the N-terminal end of a human intracellular domain, the latter followed by a human CD3^^domain. An example of a transmembrane domain includes a transmembrane domain of a CD28, CD4, CD8α, CD8^, 4ICOS, CD27, 2B4 and NKG2D molecule. CD28 is a T-cell specific surface glycoprotein. The transmembrane domain corresponds to amino acid residues 153-179 of UniProt entry P10747. CD4 is an integral membrane glycoprotein found on most helper T lymphocytes that functions as a coreceptor for a peptide: MHC class II complex. The transmembrane domain of CD4 corresponds to amino acid residues 397-418 of UniProt entry P01730. CD8α is an integral membrane glycoprotein found on most cytotoxic T lymphocytes that functions as a coreceptor for a peptide: MHC class I complex. The transmembrane domain of CD8α corresponds to amino acid residues 183-203 of UniProt entry P01732. CD8^ is an integral membrane glycoprotein found on most cytotoxic T lymphocytes that functions as a coreceptor for a peptide: MHC class I complex. The transmembrane domain of CD8^ corresponds to amino acid residues 177-191 of UniProt entry P10966. ICOS or CD278 is a T-cell specific surface glycoprotein. The transmembrane domain is present in the transmembrane region of CD278 corresponding to amino acid residues 141-161 of UniProt entry Q9Y6W8. CD27, also termed tumor necrosis factor receptor superfamily member 7, is a member of the TNF-receptor superfamily. The transmembrane domain corresponds to amino acid residues 192–212 of UniProt entry P26842. 2B4, also termed CD244, is a cell surface receptor expressed on natural killer cells and some T-cells. The transmembrane domain corresponds to amino acid residues 230–250 of UniProt entry Q9BZW8. NKG2D is a transmembrane protein belonging to the CD94 / NKG2 family of C-type lectin-like receptors. NKG2D is expressed by natural killer (NK) cells, most NK T-cells and subsets of γδ T-cells. In addition, NKG2D is present on the cell surface of all human CD8 T-cells. The transmembrane domain corresponds to amino acid residues 52-72 of UniProt entry P26718. Said transmembrane domain may be a variant of the transmembrane domains of CD28, CD4, CD8α, CD8^, ICOS, CD27, 2B4 and NKG2D molecule as is indicated herein above, that has at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 %, at least 99 % sequence identity to the indicated transmembrane domain, or a part thereof. Besides the examples provided herein above, it will be clear to a person skilled in the art that said transmembrane domain may be a transmembrane domain of any T-cell-derived protein involved in T-cell function, or a variant thereof. A cTCR comprises a human intracellular domain that is N-terminally fused to the C-terminal end of a human transmembrane domain and C-terminally to the N-terminally end of a human intracellular CD3ε domain. A human intracellular domain provides a secondary activation that enhances T-cell expansion, IL-2 production, evasion of T-cell anergy and apoptosis. A preferred human intracellular domain is a human intracellular ICOS domain. Upon ICOS activation, class IA phosphatidylinositol 3-kinase (PI3K) is recruited through the YMFM motif of ICOS, which in turn generates phosphatidylinositol 3,4,5-triphosphate (PIP3), resulting in activation of protein kinase B (Akt) as well as Nuclear Factor of ^B (NF^B) which promote cellular proliferation, production of cytokines and T-cell fitness. The intracellular domain of ICOS is present in the cytoplasmatic part of ICOS and corresponds to amino acid residues 162-199 of UniProt entry Q9Y6W8. An alternative example of an intracellular domain includes an intracellular domain of CD28, 4-1BB, OX40, CD27, CD40L, 2B4 or NKG2D molecule. CD28 is a receptor for CD80 (B7.1) and CD86 (B7.2) proteins and provides co- stimulatory signals required for T-cell activation and survival. The signaling domain corresponds to the cytoplasmic part of CD28, corresponding to amino acid residues 180 – 220 of UniProt entry P10747. This part comprises a YMNM motif, beginning at tyrosine 191, which is important for recruitment of SH2-domain containing proteins, generally kinases, which may subsequently become activated and relay phosphorylation to ITAM motifs present in (other) intracellular signaling molecules. 4-1BB, also termed CD137, or tumor necrosis factor receptor superfamily member 9, is a member of the tumor necrosis factor (TNF) receptor family. The signaling domain corresponds to the cytoplasmic part of CD137, corresponding to amino acid residues 214-255 of Uniprot entry Q07011. OX40, also termed CD134 or tumor necrosis factor receptor superfamily member 4, is a member of the tumor necrosis factor (TNF) receptor family. The signaling domain corresponds to the cytoplasmic part of CD134, corresponding to amino acid residues 236-277 of Uniprot entry P43489. CD27 polypeptide, also termed tumor necrosis factor receptor superfamily member 7, is a member of the TNF-receptor superfamily. The signaling domain corresponds to the cytoplasmic part of CD27, corresponding to amino acid residues 213–260 of Uniprot entry P26842. CD40L polypeptide, also termed tumor necrosis factor ligand superfamily member 5, is a member of the TNF superfamily. The signaling domain corresponds to the cytoplasmic part of CD40L, corresponding to amino acid residues 1-22 of Uniprot entry P29965. 2B4, also termed CD244, is a cell surface receptor expressed on natural killer cells and some T-cells. The signaling domain corresponds to the cytoplasmic part of CD244, corresponding to amino acid residues 251–370 of Uniprot entry Q9BZW8. NKG2D is a transmembrane protein belonging to the CD94 / NKG2 family of C-type lectin-like receptors. NKG2D is expressed by natural killer (NK) cells, most NK T-cells and subsets of γδ T-cells. In addition, NKG2D is present on the cell surface of all human CD8 T-cells. The signaling domain corresponds to the cytoplasmic part of NKG2D, corresponding to amino acid residues 1–51 of Uniprot entry P26718. Said human intracellular domain may be a functional variant of the human intracellular domains of ICOS, CD28, 4-1BB, OX40, CD27, CD40L, 2B4 or NKG2D that has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% sequence identity to the indicated human intracellular domain, or a part thereof. Besides the examples provided herein above, said intracellular domain may be an intracellular domain of any T-cell-derived protein involved in T -cell co- stimulation, or a variant thereof. A cTCR comprises a human intracellular CD3^ domain that is N-terminally fused to the C-terminal end of a human intracellular domain, such as a human intracellular domain. CD3^ refers to the epsilon chain of the T-cell surface glycoprotein CD3. CD3^ comprises an immunoreceptor tyrosine-based activation motif (ITAM). Phosphorylation of this motif results in immediate activation of downstream signaling pathways, such as activation of nuclear factor of activated T- cells (NFAT) transcription factors, resulting in activation of T-cell effector functions, such as IL-2 production and tumor cell killing (Govers et al., 2014. J Immunol 139: 5315-5326). Said intracellular CD3^ domain comprises the amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, at position 54, at both positions 53 and 54, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. Said intracellular CD3ε domain may be a functional variant thereof that has at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 %, at least 99 % sequence identity with SEQ ID NO:25, provided that the R53 and / or R54 is not an arginine. In embodiments, deletion of an arginine (R) amino acid residue at position 53, at position 54, or at both positions 53 and 54, may be accomplished by deletion of the 5 C-terminal amino acid residues, deletion of the 4 C-terminal amino acid residues, deletion of the 3 C-terminal amino acid residues, or deletion of the 2 C-terminal amino acid residues. It is noted that R53 may be included in a putative endoplasmic reticulum (ER) retention signal (Mallabiabarrena et al., 1992. Nature 357: 593-596; Mallabiabarrena et al., 1995. EMBO J 10: 2257-2268), but R54 is not. Said single amino acid substitution or deletion of R53 and / or R54 results in the optimal functional performance of a cTCR according to the invention, including target cell recognition, T-cell mediated killing and limited T-cell exhaustion, and enables the production and use of cTCRs with different specificities. At least one of R53 and R54 may, independently, be substituted by an aspartic acid, a glutamic acid, a proline, a serine, a threonine, a cysteine, a tyrosine, a phenylalanine, an alanine, a valine, a leucine, an isoleucine, a methionine, or a glycine. R53 and R54 are preferably substituted by an amino acid residue comprising a shorter side chain, when compared to arginine, such as a smaller charged or neutral amino acid residue. In embodiments, R53 and R54 are substituted by a non-polar aliphatic amino acid residue. Examples of such as an amino acid substitution include a substitution selected from a group consisting of an alanine, a valine, a leucine, an isoleucine, a methionine, or a glycine. In embodiments, R54 is substituted by an alanine (R54A), but could be replaced by any amino acid, preferably the ones listed above. In embodiments, a chimeric T-cell receptor, may comprise one or more further human intracellular domains. In embodiments, said one or more further human intracellular domains differ from the first intracellular domain. In embodiments, said second or more human intracellular domain may be N-terminally fused to the C-terminal end of a human transmembrane domain and C-terminally fused to the N-terminal end of a first human intracellular domain. In embodiments, said further human intracellular domain may be N-terminally fused to the C-terminal end of a first human intracellular domain and C-terminally fused to the N-terminal end of a human intracellular CD3^ domain. Examples of said further human intracellular domain include the intracellular domains of ICOS, CD28, 4-1BB, OX40, CD27, CD40L, 2B4 or NKG2D, as described herein above. In embodiments, a hinge region, also called spacer, may be present between a human or humanized extracellular TCR-constant domain (which follows the extracellular antigen binding domain) and a human transmembrane domain of a cTCR. Said hinge region may provide flexibility to the human or humanized extracellular antigen binding domain of a cTCR. Said flexibility may help this domain to associate and bind to an epitope. Examples of said hinge region include a CD28 hinge region (corresponding to amino acid residues 114-152 of UniProt entry P10747), a CD8^ hinge region (corresponding to amino acid residues 138-184 of UniProt entry P01732), a CD4 hinge region (corresponding to amino acid residues 200-206 of UniProt entry P01730), an IgG4-CH3 hinge region (corresponding to amino acid residues 99-110 of UniProt entry P01861) and an IgD hinge region (corresponding to amino acid residues 228-264 of UniProt entry P0DOX3) (Qin et al., 2017. J Hematol Oncol 10: 68). In embodiments, a linker may be present between a human or humanized TCR-constant domain (which follows the extracellular antigen binding domain) and a human transmembrane domain of a cTCR, and / or between a human transmembrane domain and a human intracellular domain, such as a human intracellular ICOS domain, and / or between a human intracellular domain and a human intracellular CD3^ domain. Examples of such a linker include an ACPK linker, a GSPK linker, a PK linker and a Gly-Ser linker, for example of the type (Glyx Sery)z such as (Gly4 Ser)3, (Gly4 Ser)7 or (Gly3 Ser2)3 as described in WO 99 / 42077, and the GS30, GS15, GS9 and GS7 linkers described in, for example, WO 06 / 040153 and WO 06 / 12282. In embodiments, a cTCR may comprise a TCRα and TCRβ chain, such as an extracellular TCRα and TCRβ chain, forming a human or humanized extracellular antigen binding domain, each of said TCRα and TCRβ chains further comprising a human TCR constant domain, a human transmembrane CD28 domain, a human intracellular ICOS domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54 of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCRβ chain forming a human or humanized extracellular antigen binding domain, each of said TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD4 domain, a human intracellular ICOS domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54 or both 53 and 54 of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of said TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD8^ domain, a human intracellular ICOS domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCRβ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD8^ domain, a human intracellular ICOS domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane ICOS domain, a human intracellular ICOS domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD27 domain, a human intracellular ICOS domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54,or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane 2B4 domain, a human intracellular ICOS domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane NKG2D domain, a human intracellular ICOS domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD28 domain, a human intracellular CD28 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD4 domain, a human intracellular CD28 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD8^ domain, a human intracellular CD28 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD8^ domain, a human intracellular CD28 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane ICOS domain, a human intracellular CD28 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD27 domain, a human intracellular CD28 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane 2B4 domain, a human intracellular CD28 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 an / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane NKG2D domain, a human intracellular CD28 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD28 domain, a human intracellular 4-1BB domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD4 domain, a human intracellular 4-1BB domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD8^ domain, a human intracellular 4-1BB domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD8^ domain, a human intracellular 4-1BB domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane ICOS domain, a human intracellular 4-1BB domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD27 domain, a human intracellular 4-1BB domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane 2B4 domain, a human intracellular 4-1BB domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane NKG2D domain, a human intracellular 4-1BB domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD28 domain, a human intracellular OX40 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD4 domain, a human intracellular OX40 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD8^ domain, a human intracellular OX40 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD8^ domain, a human intracellular OX40 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane ICOS domain, a human intracellular OX40 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD27 domain, a human intracellular OX40 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane 2B4 domain, a human intracellular OX40 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane NKG2D domain, a human intracellular OX40 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD28 domain, a human intracellular CD27 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD4 domain, a human intracellular CD27 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD8^ domain, a human intracellular CD27 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD8^ domain, a human intracellular CD27 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane ICOS domain, a human intracellular CD27 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD27 domain, a human intracellular CD27 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane 2B4 domain, a human intracellular CD27 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane NKG2D domain, a human intracellular CD27 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD28 domain, a human intracellular CD40L domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD4 domain, a human intracellular CD40L domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD8^ domain, a human intracellular CD40L domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD8^ domain, a human intracellular CD40L domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane ICOS domain, a human intracellular CD40L domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD27 domain, a human intracellular CD40L domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane 2B4 domain, a human intracellular CD40L domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane NKG2D domain, a human intracellular CD40L domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD28 domain, a human intracellular 2B4 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD4 domain, a human intracellular 2B4 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD8^ domain, a human intracellular 2B4 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD8^ domain, a human intracellular 2B4 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane ICOS domain, a human intracellular 2B4 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD27 domain, a human intracellular 2B4 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane 2B4 domain, a human intracellular 2B4 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane NKG2D domain, a human intracellular 2B4 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCRβ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD28 domain, a human intracellular NKG2D domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD4 domain, a human intracellular NKG2D domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD8^ domain, a human intracellular NKG2D domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD8^ domain, a human intracellular NKG2D domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane ICOS domain, a human intracellular NKG2D domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD27 domain, a human intracellular NKG2D domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane 2B4 domain, a human intracellular NKG2D domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane NKG2D domain, a human intracellular NKG2D domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD28 domain, a human intracellular ICOS domain, a human intracellular CD28 domain, and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25 is substituted or deleted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD28 domain, a human intracellular ICOS domain, a human intracellular 4-1BB domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25 is deleted or substituted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD28 domain, a human intracellular ICOS domain, a human intracellular OX40 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25 is deleted or substituted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD28 domain, a human intracellular ICOS domain, a human intracellular CD27 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25 is deleted or substituted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD28 domain, a human intracellular ICOS domain, a human intracellular CD40L domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25 is deleted or substituted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD28 domain, a human intracellular ICOS domain, a human intracellular 2B4 domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25 is deleted or substituted. In embodiments, a cTCR may comprise a TCRα and TCR^ chain forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane CD28 domain, a human intracellular ICOS domain, a human intracellular NKG2D domain and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25 is deleted or substituted. In embodiments, a cTCR may comprise a TCR-variable α domain and a TCR- variable ^ domain that are fused into a single-chain antigen binding site that is followed by a human TCR-constant ^ domain, a human transmembrane domain, a human intracellular domain, and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25 is substituted or deleted. Alternatively, this single chain cTCR contains any (combination) of variants regarding human transmembrane and intracellular domains as mentioned above. Methods to generate single-chain TCR-based antigen binding sites are known in the art. For example, Chung et al., 1994 (Proc Nat Acad Science 91: 12654-12658) and Knies et al., 2016 (Oncotarget 7: 21199–21221) describe the generation of functional ingle- chain T-cell receptors. Antigens An antigen may be present on the surface of a tumor cell and may be targeted by an extracellular antigen binding domain of a cTCR according to the invention. Said tumor is preferably a solid tumor, such as a sarcoma or a carcinoma. Examples of solid tumors include melanoma, breast cancer, lung tumor, pancreatic tumor, colorectal tumor, prostate tumor, testicular tumor, ovarian tumor, glioblastoma, synovial tumor, osteosarcoma, liposarcoma, esophageal tumor, head and neck tumor, bladder tumor, cervical tumor and kidney tumor. A person skilled in the art knows that an extracellular antigen binding domain may recognize and bind part of an antigen, named an epitope, presented by a tumor cell. In embodiments, said antigen is generally an intracellular antigen, but not excluding surface antigens. In embodiments, said antigen is specifically expressed by tumor cells. In embodiments, said epitope is specifically derived from said antigen and presented by MHC on tumor cells. Said extracellular antigen binding domain preferably targets an antigen of a solid tumor, such as a melanoma. Examples of antigens of a melanoma include a glycoprotein 100 (gp100), C-kit / cluster of differentiation 117 (CD117), Melanoma Antigen Gene (MAGE), such as MAGE A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10 and MAGE-A12, rhophilin associated tail protein 1 (ROPN1), rhophilin associated tail protein 1B (ROPN1B), Melanoma antigen recognized by T-cells 1 (MART-1), Intercellular Adhesion Molecule 1 (ICAM1), Chondroitin Sulfate Proteoglycan 4 (CSPG4), Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), Receptor Tyrosine Kinase AXL, Glypican 3 (GPC3), CD147, Programmed death-ligand 1 (PDL1), NKG2D-L or alkaline phosphatase placental-like 2 (ALPPL2). Said antigen binding domain preferably targets an antigen of a solid tumor, such as a breast cancer tumor. Examples of antigens of a breast cancer tumor include rhophilin associated tail protein 1 (ROPN1), rhophilin associated tail protein 1B (ROPN1B), human epidermal growth factor receptor 2 (HER2), Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), epithelial cell adhesion molecule (EpCAM), B7-H3, interleukin 13 receptor subunit a2 (IL13Ra2), GPI- anchored carcinoembryonic antigen (CEA), Intercellular Adhesion Molecule 1 (ICAM1), Chondroitin Sulfate Proteoglycan 4 (CSPG4), CD32A, Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), Receptor Tyrosine Kinase AXL, Folate Hydrolase 1 (FOLH1), Folate Receptor alpha (FRα), CD147, Claudin 18 (CLD18), NKG2D-L or alkaline phosphatase placental-like 2 (ALPPL2). Said extracellular antigen binding domain preferably targets an antigen of a solid tumor, such as a lung tumor. Examples of antigens of a lung tumor include Melanoma Antigen Gene (MAGE), such as MAGE A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10 and MAGE-A12, Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), epithelial cell adhesion molecule (EpCAM), B7-H3, Mesothelin, GPI-anchored carcinoembryonic antigen (CEA), Intercellular Adhesion Molecule 1 (ICAM1), Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), Folate Receptor alpha (FRα), Glypican 3 (GPC3), CD147, Claudin 18 (CLD18), Programmed death-ligand 1 (PDL1), NKG2D-L or alkaline phosphatase placental-like 2 (ALPPL2). Said extracellular antigen binding domain preferably targets an antigen of a solid tumor, such as a pancreatic tumor. Examples of antigens of a pancreatic tumor include Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), Mesothelin, interleukin 13 receptor subunit a2 (IL13Ra2), Receptor Tyrosine Kinase AXL, CD147, NKG2D-L, or alkaline phosphatase placental-like 2 (ALPPL2) or Fibroblast-activation protein (FAP). Said extracellular antigen binding domain preferably targets an antigen of a solid tumor, such as a colorectal tumor. Examples of antigens of a colorectal tumor include Actin-like Protein 8 (ACTL8), Guanylyl cyclase C (GUCY2C), epithelial cell adhesion molecule (EpCAM), GPI-anchored carcinoembryonic antigen (CEA), CD32A, Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), Folate Hydrolase 1 (FOLH1), CD147, Claudin 18 (CLD18), NKG2D-L, or alkaline phosphatase placental-like 2 (ALPPL2) or Fibroblast-activation protein (FAP). Said extracellular antigen binding domain preferably targets an antigen of a solid tumor, such as a prostate tumor. Examples of antigens of a prostate tumor include epithelial cell adhesion molecule (EpCAM), B7-H3, Prostate Stem Cell Antigen (PSCA), Prostate Specific Membrane Antigen (PSMA), Receptor Tyrosine Kinase AXL, NKG2D-L or alkaline phosphatase placental-like 2 (ALPPL2). Said extracellular antigen binding domain preferably targets an antigen of a solid tumor, such as a testicular tumor. Examples of antigens of a testicular tumor include Cancer / Testis Antigen 1 also called New York esophageal squamous cell carcinoma 1 (NY-ESO-1), Cancer / testis antigen 83 (CT83) or alkaline phosphatase placental-like 2 (ALPPL2). Said extracellular antigen binding domain preferably targets an antigen of a solid tumor, such as an ovarian tumor. Examples of antigens of an ovarian tumor include BORIS / CTCFL, Mucin 1, Mucin 16, L1-cell adhesion molecule (L1CAM), epithelial cell adhesion molecule (EpCAM), Mesothelin, Glypican 3 (GPC3), CD147, NKG2D-L, alkaline phosphatase placental-like 2 (ALPPL2), Folate receptor-alpha (FRα) or Fibroblast-activation protein (FAP). Said extracellular antigen binding domain preferably targets an antigen of a solid tumor, such as a glioblastoma. Examples of antigens of a gliobastoma include Cancer / Testis Antigen 45A3, B7-H3, epidermal growth factor receptor (EGFR) and EGFRvIII, interleukin 13 receptor subunit a2 (IL13Ra2), Receptor Tyrosine Kinase AXL, CD147, NKG2D-L or Hepatocyte growth factor (HFG) receptor. In an embodiment, an antigen of a solid tumor that is specifically targeted by an extracellular antigen binding domain, more specifically melanoma or breast cancer, may bind to any epitope from any antigen, but preferably binds to any epitope selected from human cancer germline antigens, such as ROPN1, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C2, NY-ESO1 (Kortleve et al., 2022. Trends Immunol 43: 391-403), mutated antigens, such as KRAS, PIK3CA and TMPRSS2- ERG mutants, and / or tumor neoantigens, such as peptides generated by frameshift from putative non-coding regions of the human genome. Generating an expression cassette for the expression of a chimeric TCR The invention further provides an expression cassette that enables the expression of a cTCR in a T-cell. Said construct preferably comprises a coding sequence for the cTCR and an operationally coupled promoter that is capable of directing transcription of the coding sequence in a T-cell. Said promoter preferably is a constitutive promoter that is active in T-cells, for example a mammalian promoter, such as a hPGK promoter or a synthetic promoter comprising a RPL13a promoter fused to parts of a RPL41 gene (RPBSA promoter), or a viral promoter, such as a EF-1 promoter, preferably derived from a human cytomegalovirus, such as a CMV promoter and derivatives (Deer & Allison, 2004. Biotechnol Prog 20: 880–889; US patent No: 5888809; Rad et al., 2020. PLoS one 15: e0232915). Said coding sequence may contain a sequence encoding for a human or humanized extracellular antigen binding domain, human TCR-constant domains, a human transmembrane domain, a human intracellular domain, such as a human intracellular domain, and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, said coding sequence may comprise a further intracellular domain, such as a second intracellular domain or a second and a third intracellular domain. In embodiments, said coding sequence may comprise a sequence coding a TCRα chain and a TCR^ chain, together forming a human or humanized extracellular antigen binding domain, each of the TCRα and TCRβ chains further comprising a human TCR-constant domain, a human transmembrane domain, such as a human CD28 transmembrane domain, a human intracellular domain(s), such as a human intracellular ICOS domain, and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. In embodiments, said coding sequence comprises a sequence coding for a TCRα chain and a TCR^ chain, each of which is linked to a human transmembrane domain, such as a human CD28 transmembrane domain, a human intracellular domain(s), such as a human intracellular ICOS domain, and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and or 54 of SEQ ID NO:25, is substituted or deleted. These two sequences representing TCRα and TCR^ chains, and together responsible for the extracellular antigen binding domain, can be gene-transduced either via separate constructs or be part of a single construct, in which they are linked together by sequence encoding for a peptide such as a 2A peptide. Said peptide serves as a ribosomal skipping sequence and mediates cleavage of the polypeptide during translation (Liu et al., 2017. Sci Rep 7: 2193). Examples of 2A peptides include a F2A, T2A, E2A and P2A peptide sequence. In an alternative embodiment, the CDR regions of TCRα and TCR^ chains constitute a single chain TCR, which is then followed by a human transmembrane domain, such as a human CD28 transmembrane domain, a human intracellular domain(s), such as a human intracellular ICOS domain, and a human CD3ε domain comprising an amino acid sequence of SEQ ID NO:25 wherein an arginine (R) amino acid residue at position 53, 54, or both 53 and 54, of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted. Said expression cassette and optionally, parts of an expression cassette, may be generated by molecular cloning methods known to a skilled person. Examples of such molecular cloning methods include overlap PCR, Golden Gate cloning, Gateway Cloning, Gibson assembly and Restriction enzyme-based cloning (Green & Sambrook, 2012. Molecular Cloning: A Laboratory Manual). T-cells The invention further describes a recombinant T-cell expressing a cTCR. In embodiments, said recombinant T-cell may be expressing an endogenous TCR on its cell surface. Host T cells in the context of this invention are preferably human T cells, more preferably human CD4+ or CD8+ T cells, and may be autologous or allogeneic host cells, preferably autologous cells. A skilled person knows how to isolate T-cells, using standard laboratory procedures, such as leukapheresis (Wang et al., 2022. J Transl Med 20: 608; Van de Griend, 1984. J Immunol Methods 66: 285-298). Allogenic T-cells may be isolated from a healthy donor’s bone marrow or peripheral blood whereas autologous T-cells may be isolated from a cancer patient’s bone marrow, peripheral blood or pieces of tumor. In embodiments, T-cells may be present in a sample of peripheral blood mononuclear cells (PBMC) from a patient. As is known to a person skilled in the art, an isolated T-cell may be cultured in culture medium, such as RPMI-1640 or DMEM, with added cytokines, generally being common-^ cytokines, such as interleukin (IL-)2, IL-7, IL-15 and / or IL-21, in order to increase the number of T-cells. Producing a T-cell comprising a chimeric T-cell receptor The invention further comprises a method to produce a recombinant T-cell according to the invention, comprising isolating a T-cell from the patient, providing the T-cell with a chimeric TCR according to the invention, and enabling the expression of a chimeric TCR in the recombinant T-cell. Methods for isolating T-cells are described above. Isolated T-cells may subsequently be modified to allow expression of a cTCR according to the invention. An expression construct, such as a vector, as mentioned earlier, may be provided to a T-cell, preferably a viral vector. Said vector may comprise an expression cassette encoding a cTCR according to the invention. Examples of viral vectors include an adeno-associated viral vector, a herpes virus-based vector, such as Epstein-Barr Virus, a lentivirus-based vector, such as a human immunodeficiency virus, a retrovirus-based vector, a vaccinia virus-based vector, a bovine papilloma virus- based vector, or a gamma-retrovirus-based vector, such as a pMP71 vector or a vector based on a Moloney Murine Leukemia Virus (MoMLV), a Spleen-Focus Forming Virus (SFFV), a Myeloproliferative Sarcoma Virus (MPSV), a Murine Stem Cell Virus (MSCV) or a SFG gamma retroviral vector (Rivière et al., 1995. PNAS 92: 6733-6737). Said viral vector comprising a cassette encoding for a cTCR may be introduced in a T-cell through transduction, which is known by a person skilled in the art (Lamers et al., 2006, Cancer Gene Ther, 13: 503-509). As an alternative, said cassette may be directly provided to the T-cell, without a vector such as a viral vector. For example, said expression cassette, preferably as part of a construct, may be introduced into a cell by a transfection method, such as by lipofection, by calcium phosphate co-precipitation, by DEAE dextran and by electroporation. Said construct may be transiently transfected in a T-cell, such as by integration of a non-integrative plasmid, or may be stable integrated into the genome of a T-cell. Stable integration of a construct may be enforced by making use of gene editing tools, such as homologous recombination, transcription activator-like effector nuclease (TALEN), Zinc-finger nuclease (ZFN) or clustered regularly interspaced short palindromic repeats (CRISPR). Said gene editing tools may further be used to insert the expression cassette into a specific locus within the genome (Eyquem et al., 2017. Nature 543: 113-117). Examples of preferred genomic loci that support stable long-term expression of the cTCR and do not interfere with the regulation of endogenous expression of the genes normally present at said loci, include the T-cell receptor α-chain (TRAC) and / or the T-cell receptor ^-chain (TRBC) gene loci, the b2m gene locus, the AAVS1 locus and the PD-1 locus, as is known to a skilled person. In embodiments, said expression cassette may be replacing an endogenous TCR sequence of a T-cell. Said replacement of an endogenous TCR sequence may be performed with a genomic editing tool, such as CRISPR or by homologous recombination, and introducing the cTCR gene into the T-cell receptor α-chain (TRAC) and / or the T-cell receptor ^-chain (TRBC) gene loci. The resulting modified recombinant T-cells comprising a cTCR according to the invention may be grown under conditions similar to those for unmodified T- cells, such as in RPMI-1640 or DMEM culture medium supplemented with cytokines. Recombinant T-cells expressing a cTCR according to the invention may be stimulated to proliferate and expand, for example with a T-cell activator, such as anti-CD3 / CD28 beads, by exposure to antigen-positive cells, by exposure to specific cytokines, such as IL-2, IL-7, IL-15 and / or IL-21 (Lamers et al., 2014. Hum Gene Ther Methods 25: 345–357) or by exposure to a recombinant antigen or an antibody directed to the cTCR. Modified recombinant T-cells expressing a cTCR according to the invention may be collected after expansion of said cells, based on their expressed cTCR. As such, recombinant T-cells that are alive and healthy and are expressing a cTCR according to the invention can be retained and / or enriched. By using antibodies directed to the cTCR, desired recombinant T-cell expressing a cTCR according to the invention may be collected. An example of a method to collect modified recombinant T-cells expressing a cTCR according to the invention, is cell sorting, such as fluorescent-activated Cell Sorting (FACS), magnetic-activated cell sorting (MACS) or buoyancy-activated cell sorting (BACS). A recombinant T-cell expressing a construct encoding for a cTCR that enables the expression of the cTCR according to the invention, may be preserved, for example by cryopreservation or immediately administered to a patient in need thereof. Methods of treatment The invention provides a recombinant T-cell expressing a cTCR as disclosed herein, wherein said recombinant T-cell is for use in therapy. For example, the recombinant T-cell is for use in the treatment of a tumor, preferably a solid tumor, in a patient. In embodiments, a T-cell was isolated from the patient, provided with an expression construct expressing the cTCR, followed by the provision of the recombinant T-cell back to the patient. In preferred embodiments, the chimeric TCR comprises an extracellular antigen binding domain that binds to a cancer antigen of the patient. The invention further provides a recombinant T-cell expressing a cTCR, as disclosed herein, wherein said recombinant T-cell is for use as a medicament, preferably a medicament for treatment of a tumor, preferably a solid tumor. The invention further provides use of a chimeric TCR as disclosed herein, preferably of a recombinant T-cell expressing said chimeric TCR, in the preparation of a medicament for treatment of a tumor, preferably a solid tumor. The invention further provides a pharmaceutical composition comprising a recombinant T-cell expressing a cTCR, as disclosed herein, and a pharmaceutically acceptable excipient. Examples of pharmaceutical acceptable excipients include a stabilizer, a bulking agent, a buffer, a carrier, a diluent, a vehicle, a solubilizer or a binder. A skilled person understands that the selection of an appropriate carrier or a diluent depends on the route of administration and the dosage form, as well as the active ingredient and other factors. Alternatively, an in vivo gene-delivery system encoding a chimeric TCR is applied to create cTCR-expressing T-cells in the patient. A recombinant T-cell expressing a cTCR according to the invention is preferably provided to a patient in need thereof in a therapeutically effective amount. Said therapeutically effective amount is an amount that evokes the desired effect in which the symptoms of the tumor are ameliorated but preferably does not cause severe adverse side effects. Said therapeutically effective amount for a patient having a tumor may depend on size and health of the patient and the nature and extent of the tumor and can be determined by the skilled person in a routine manner, and expectedly will range between 107and 1010cTCR-expressing T-cells per infusion. A recombinant T-cell expressing a cTCR according to the invention, or a pharmaceutical composition according to the present invention, may for example be administered through injection or infusion when the recombinant T-cells according to the invention are present in a liquid such as an aqueous liquid, a suspension or an emulsion. Administration of a recombinant T-cell expressing a cTCR according to the invention may be systemic or local. Example of administration routes of a recombinant T-cell expressing a cTCR according to the invention include parental administration, such as intravenous, intramuscular, intraperitoneal, subcutaneous, intra-arterial, intra-tumoral, or intracerebral administration and enteral administration, such as oral and rectal administration. Alternatively, an in vivo gene-delivery system, such a viral vector encoding a chimeric TCR and / or nanoparticles encapsulating nucleic acids encoding said TCR, in which vectors and / or particles may have tropism for immune cells, such as T-cells, is applied directly to the patient, thereby enabling in-vivo gene delivery of the cTCR cassette into the patient’s T-cells. Examples of nanoparticles include polymeric nanoparticles, such as PEG, chitosan and collagen nanoparticles, inorganic nanoparticles, such as metallic nanoparticles, such as gold and silver nanoparticles, protein-based nanoparticles, such as gelatine and albumin nanoparticles, liposomes, dendrimers, and nanohydrogels (Hamimed et al., 2022. Naunyn Schmiedebergs Arch Pharmacol 395: 769-787)) The invention further provides a method of treating a patient having a tumor, preferably a solid tumor by administration of a pharmaceutical composition according to the invention and is optionally combined with an additional tumor / cancer therapy. Said additional tumor therapy may include surgery, radiation therapy or one or more anti-cancer drugs or a combination thereof, which are known to a skilled person. The recombinant T-cells, as disclosed herein, and additional tumor / cancer therapy may be administered together at the same time point, such as in the form of a single pharmaceutical composition, separately from each other at the same time point, such as in the form of separate pharmaceutical compositions, or separately from each other at one or more different time points. Said one or more further anti-cancer drugs may include chemotherapeutic drugs, such as an alkylating agent, for example a nitrogen mustard, such as bendamustine, chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, and melphalan, a nitrosourea, such as carmustine, lomustine, and streptozocin, an alkyl sulfonate, such as busulfan, a triazine, such as dacarbazine and temozolomide, an ethylenimine, such as altretamine and thiotepa, an antimetabolite, such as 5-fluorouracil, hydroxyurea and methotrexate, an alkaloid, such as taxane and camptothecan, a mitotic inhibitor, such as vinblastine, paclitaxel and etoposide, an antitumor antibiotic, such as anthracycline and chromomycin, and / or a topoisomerase inhibitor, such as camptothecin. Said one or more anti-cancer drugs may include one or more molecules for targeted therapy, immunotherapy or hormone therapy. A molecule for targeted therapy is a molecule that specifically blocks growth of cancer cells by interfering with specific molecules, often kinases, which are necessary for carcinogenesis and tumor growth, such as a tyrosine-kinase inhibitor and a phosphoinositide 3-kinase inhibitor. A molecule for immunotherapy overcomes inhibition of the immune system of the individual to kill cancer-associated tumor cells, and includes for example, an immune checkpoint inhibitor, such as an anti-PD-1, an anti-PD-L1, an anti- CTLA4, a CTLA B7-1 and a CTLA B7-2 chimeric molecule. In addition, a molecule for immunotherapy can be either a molecule or an antibody that directs the immune system to attack tumor cells by targeting antigens displayed on tumor cells, such as antibody drug conjugates. A molecule for hormone therapy is a molecule that blocks or lowers a concentration of one or more specific hormones. This can be performed by either preventing the ability of an individual to produce said specific hormone or by interfering with how a specific hormone behaves in the human body. Some cancers, such as breast, prostate, ovarian and endometrial cancer, require hormone stimulation, such as steroid stimulation, to grow and / or develop. Hormone therapy specifically prevents the growing and division of hormone dependent / sensitive cancer cells. Examples of such hormone therapeutic molecules include a hormone antagonist, such as flutamide, goserelin, mitotane or tamoxifen, and an aromatase inhibitor, such as anastrozole, exemestane or letrozole, or a combination thereof. In embodiments, a chimeric TCR may be used for treatment of a patient having, or suffering from, an immune-related disease such as an autoimmune disease. For this, the chimeric TCR comprises an extracellular antigen binding domain that binds to an antigen of the patient. The invention further provides a recombinant T-cell expressing a cTCR, as disclosed herein, wherein said recombinant T-cell is for use as a medicament, preferably a medicament for treatment of a patient suffering from an immune-related disease such as an autoimmune disease. In embodiments, the TCRα chain and TCRβ chain form an extracellular antigen binding domain that specifically binds an epitope from an antigen derived from a mature tissue or organ (i.e., a self-antigen), where said self-antigen includes, for example, MOG, GAD65, TSHR, tTG, or fibrinogen. Specifically, recent studies have demonstrated that adoptive T cell therapy using gene-engineered T cells expressing a CD19 CAR show clinical effects in B cell-mediated autoimmunity, such as systemic lupus erythematosus, inflammatory myositis and sclerosis (Mûller et al., 2024. New Engl J Med 390: 687-700). The invention further provides a method of treating a patient suffering from an immune-related disease by administration of a pharmaceutical composition according to the invention and is optionally combined with an additional immunosuppressant. Said additional immunosuppressant may be one or more of a corticosteroid, cyclosporine, methotrexate, azathioprine, hydroxychloroquine, sulfasalazine, mycophenolic acid, cyclophosphamide, leflunomide, an interleukin (IL) -1 blocker, an IL-6 blocker, a TNF inhibitor, a Janus-Kinase (JAK) inhibitor, or any combination thereof. In embodiments, a chimeric TCR may be used for treatment of a patient having, or suffering from, an infection such as a bacterial infection, a viral infection, a fungal infection or a parasitic infection. For this, the chimeric TCR comprises an extracellular antigen binding domain that binds to an antigen of the pathogen that is presented by an MHC molecule. The invention further provides a recombinant T-cell expressing a cTCR, as disclosed herein, wherein said recombinant T-cell is for use as a medicament, preferably a medicament for treatment of a patient suffering from an infection. An infectious disease is a transmissible disease that results from an infection, where an infection refers to the tissue invasion by pathogens and the onset of the immune response against such invaded pathogens. Initially, said immune response involves innate immune cells, generally being granulocytes (i.e., neutrophilic, basophilic or eosinophilic granulocytes) and myeloid cells (i.e., monocytes, macrophages and dendritic cells), and is followed by a response involving adaptive immune cells (i.e., B and T cells). Pathogens are mostly bacteria, fungi and viruses. Examples of viruses, where the adaptive immune response involves CD8 T cells, include Epstein-Barr virus (EBV), Cytomegalovirus (CMV) and Human or Simian Immunodeficiency Virus (HIV or SIV). EBV is a gammaherpesvirus (i.e., a double- stranded DNA virus that belongs to the family of herpesviruses) and one of the most common viruses that infects humans. EBV infection may lead to mononucleosis and to the development of tumors, such as nasopharyngeal carcinoma. CMV is a betaherpesvirus that infects humans. Diseases that occur as a result of CMV infection include mononucleosis and pneumonia. HIV is a lentivirus (i.e., a single-stranded RNA virus that belongs to the family of retroviruses) that infect humans. Over time, an HIV infection causes the disease called acquired immunodeficiency syndrome (AIDS), which shows progressive failure of the immune system resulting in opportunistic infections and cancers. Specifically, TCRs against epitopes from HIV, CMV and EBV have already been isolated and are tested in clinical or preclinical settings (Varela-Rohena et al., 2008. Nat Med 14, 1390-1395; Ma et al., 2024. J Immunotherapy Cancer 12: e007735; Zheng et al., 2015. Cancer Immunol Res 3: 1138–1147). The invention further provides a method of treating a patient suffering from an infection by administration of a pharmaceutical composition according to the invention and is optionally combined with an additional antibiotic, including an antifungal and an antiviral agent. Said additional antibiotic may be one or more of penicillin, a cephalosporin, a lincosamide, a macrolide, a fluoroquinolone, a trimethoprim-sulfamethoxazole, nitrofurantoin, a tetracycline, teicoplanin, ivermectin, remdesivir, amphotericin B deoxycholate, nystatin, ketoconazole, fluconazole, chloroquine, artemether-lumefantrine, mefloquine, pyrimethamine, sulfadiazine, or any combination thereof. 5. EXAMPLES Example 1 Materials and Methods Cell culture Primary mouse T-cells were isolated from spleens of HHD mice (Straetemans et al., 2015. Mol Ther 23: 396-406 & Schaft et al., 2003. J Immunol Baltim Md 170: 2186-2194) and were cultured at 1x106cells / mL in RPMI 1640 (Gibco) with 25mM HEPES (Gibco), 10% Fetal Bovine Serum (FBS; Greiner Bio-one), Penicillin / Streptavidin (100 U / mL and 100 µg / mL, respectively, Life Technologies), 2 mM L-glutamine (Life Technologies), 1 % non-essential amino acids (Gibco), 1mM sodium pyruvate (Life Technologies) and 50 µM ß-Mercaptoethanol (VWR) (Complete Mouse Medium), which was supplemented with 180 IU / mL Human Recombinant Interleukin-2 (rhIL-2) (Proleukin; Chiron). Human T-cells were isolated from PBMC from healthy human donors (Sanquin) by centrifugation viaFicoll-Isopaque (density = 1.077 g / cm3; Amersham Pharmacia Biotech) and werecultured at 1x106cells / mL in RPMI 1640 with 25 mM HEPES, 5 % Human Serum (Sanquin), Penicillin / Streptavidin, 2 mM L-glutamine (T-cell medium), which was supplemented with 360 IU / mL rhIL-2. The packaging cells Phoenix-Ampho (ATCC CRL-3213) and 293T (ATCC-3216) were cultured in DMEM (Gibco) with 10 % FBS, 1 % non-essential amino acids, 2 mM L-glutamine and Penicillin / Streptavidin (100 U / mL and 100 µg / mL, respectively) (DMEM Complete). BLM wt (Cellosaurus CVCL_7035), a BLM-gp100 clone (overexpressing the gp100 protein), BL / 6 B16F10 wt (CRL-6475) as well as a B16:HHD-YLEP clone (expressing a fusion protein between the human gp100280-288epitope YLEPGPVTA and the HHD molecule; Straetemans et al., 2015. Mol Ther 23: 396-406 & Schaft et al., 2003. J ImmunolBaltim Md 170: 2186-2194) were cultured in DMEM complete, which in case of theoverexpression clones was supplemented with neomycin (1mg / mL G418, Calbiochem). MDA-MB-231 (MM-231) wt and its clone overexpressing the ROPN1 protein were cultured in RPMI 1640 with 10 % FBS, Penicillin / Streptavidin (100 U / mL and 100 µg / mL, respectively) and 2 mM L-glutamine, which in case of the MM231 clone was supplemented with 2 µg / mL puromycin (Life Technologies). T2 cells were cultured in RPMI 1640 with 10 % FBS, 2 mM L-glutamine and Penicillin / Streptavidin (100 U / mL and 100 µg / mL, respectively). Co-culture experiments were performed in RPMI1640 with 25 mM HEPES, 10 % FBS, 2 mM L-glutamine and Penicillin / Streptavidin (100 U / mL and 100 µg / mL, respectively) (Cytotox medium) unless stated otherwise. Design of murine co-stimulatory TCRs Murine (m)TCRα and β genes specific for the human gp100280-288epitope presented by HLA-A2 (gp100 / A2) were derived from clone CTL-296 (Schaft et al., 2003. J Immunol 170: 2186-2194), murinized and codon-optimized as described earlier (Pouw et al., 2007. J Gene Med 9: 561-570). Subsequently, TCR genes were cloned into the pMP71 vector (Engels et al., 2003. Hum Gene Ther.14:1155-68), in which the TCRα and β chains were separated by an optimized T2A ribosome skipping sequence according to a TCR^-2A-TCR^ format. Murine TCR:CD28 was generated as described in Govers et al., 2014 (J Immunol 193: 5315-5326) with its cassette containing human (h)TCR-V^^coupled to mC^ and hTCR-V^ coupled to C^ (all extracellular domains), and in which each TCR chain was followed by a PK linker, the mCD28 transmembrane, mCD28 intracellular and mCD3^ intracellular domains. Murine OX40, ICOS and 4-1BB intracellular domains were ordered via GeneArt (Life Technologies) and introduced into the TCR:CD28 cassette (replacing the intracellular domain of CD28) via overlap PCR with use of Q5 High Fidelity DNA Polymerase (New England Biolabs). One variant to TCR:ICOS, named TCR:ICOS-YF, was designed by site directed mutagenesis of Y181F in the mICOS domain to prevent downstream signalling of PI3K, as described before by Gigoux et al. (Proc Natl Acad Sci USA 106: 20371-20376). Sequences were obtained and checked using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the ABI sequence analyzer. All mouse cTCRs used in this study are schematically depicted in Figures 1B and 5A. Design of human co-stimulatory TCRs To translate the gp100 / A2 mTCR:ICOS into human hTCR:ICOS, we generated the pMP71 cassette that consisted of gp100 / A2 hTCR-VDJ-C^-T2A- hTCR-VJ-C^ (extracellular domains), and in which each TCR chain was followed by a PK linker, the hCD28 transmembrane, hICOS intracellular and hCD3^ intracellular domains. This hTCR:ICOS construct demonstrated poor surface expression in human T-cells (see Figure 8). To identify which building blocks (or combinations thereof) and / or which boundaries between building blocks determine functional expression of TCR:ICOS in human T-cells, a panel of 49 variants was designed (all produced by GeneArt), which were tested for surface expression in a stepwise approach. An overview of the different constructs, including detailed specifics, is provided in Table 1. In all transduction experiments, the wt TCR was taken along as a reference. A scheme of the study design to select and test hTCR:ICOS variants is shown in Figure 9A. Following selection of hTCR:ICOS-RA, in which the mutation R54A was introduced in the hCD3^ domain, this construct was further tested for antigen recognition, sensitivity and resistance to T-cell exhaustion. To test the applicability of hTCR:ICOS-RA to other TCR specificities, the gp100 / A2 TCR-V^ and V^ domains were exchanged with those of the TCR directed against Ropporin-1 (ROPN1 / A2). The ROPN1 / A2 hTCR:ICOS-RA was tested for surface expression, antigen recognition, sensitivity and resistance to T- cell exhaustion. Gene introduction of wt and co-stimulatory TCRs into T-cells Mouse T-cell transduction was performed as previously described by Pouw and colleagues (Pouw et al., 2007. J Gene Med 9: 561-570). Shortly, 293T and Ph-A packaging cells were transfected with pHit60 and pHit123 helper constructs (Soneoka et al., 1995. Nucleic Acids Res 23: 628-633), and the TCR of interest, using calcium phosphate (Promega Profection Mammalian Transfection system). Mouse splenocytes were isolated, activated for 24h with 0.5µg / mL Concavalin A (Sigma) and exposed to the virus supernatant. Human T-cell transduction was performed as previously described by Lamers and colleagues (Lamers et al., 2002. Cancer Gene Ther 9: 613-623). In this case, 293T and Ph-A packaging cells were transfected with pHit60 and pColtGalV (Weijtens et al., 1998. Gene Ther 5: 1195- 1203) helper constructs, and the TCR of interest, using the procedure described above. Human Peripheral Blood Mononuclear Cells, isolated from buffy coats obtained from Sanquin (Amsterdam, the Netherlands), were activated for 48 h using 10 ng / mL soluble anti-CD3 antibody (OKT3, Thermo Fisher Scientific) before receiving 2 hits with virus supernatant. TCR expression level was determined using flow cytometric detection of the introduced TCR-Vß, corrected for percentage positivity as observed with mock T-cells from the same donor, and displayed as % of positive cells within the CD3+ T-cell population. Adoptive cell therapy BL6 HHD mice expressing the chimeric A2:Kb molecule (Pascolo et al., 1997. J Exp Med 185: 2043-2051) were bred at Erasmus MC and were used for adoptive T-cell experiments as described previously with minor adaptations (Govers et al., 2014. J Immunol 193: 5315-5326). Shortly, mice were transplanted with 0.5x106 B16:HHD-YLEP clones 12 days prior to T-cell transfer at day 0. TCR T-cells (6 to 7.5x106 cells) were transferred intravenously, preceded by intraperitoneal injections of busulphan (16.5µg / kg on days -4 and -3; Sigma) and cyclophosphamide (200 mg / kg on day -2; Sigma). For mock T-cells, the same number of cells was transferred as for wt TCR T-cells. The tumor volume was measured three times per week using a caliper and calculated using the formula 0.4 x (AxB2), where A represents the largest diameter measured, and B2 the perpendicular diameter. Tumor regression was defined as a decrease in tumor volume of at least 30 % after ACT, whereas tumor relapse was defined as increases in tumor volume at 3 consecutive measurements after the initial tumor regression. Anti-tumor responses of mice at the end of experiments were categorized as follows: a complete response as 100 % initial tumor regression with tumor remaining non-palpable; partial response as tumor regression followed by a tumor relapse; and non-response no tumor regression.3-4 mice with regressing tumors 5 days after transfer were sacrificed, and tumors were used for RNA sequencing (following snap freezing and storage at -80 ºC) as well as flow cytometric phenotyping of TILs (following the making of a single cell suspension). Mice were sacrificed when the tumor burden exceeded 1400 mm3. Blood samples were collected from the tail vein at weekly intervals after ACT and used for flow cytometric counting and phenotyping of TCR T-cells. Flow cytometry To quantify circulating TCR+ T-cells in mice, cells were washed once with Phosphate Buffered Saline (PBS; Lonza) and incubated with 10µL of an antibody cocktail for 15 min at RT. For this, the following antibodies were used: anti-TCR- Vß14-FITC (clone CAS1.1.3, Beckman Coulter, diluted 1:2), anti-mCD3-PerCP (clone 145-2C11, BD, diluted 1:10), anti-mCD8-APC (clone 53-6.7, eBioscience, diluted 1:50) and anti-mCD4-BV650 (clone RM4-5, BD, diluted 1:2000). Following incubation, the cells were resuspended in 1% paraformaldehyde (PFA, Brunschwig) containing Flow-Count Fluorospheres (Beckman Coulter). To assess frequencies of T-cell subsets, three panels of antibodies were used (using the same procedure as above). These were: (1) a T-cell maturation panel consisting of: anti-mCD62L (PE, clone MEL-14, eBioscience, diluted 1:20) and anti-mCD44 (APC, clone IM7, BD, diluted 1:20); (2) a T-cell co-stimulation panel consisting of: anti-m4-1BB (mCD137- BV421, clone 1AH2, BD, diluted 1:20), anti-mOX40 (mCD134-APC, clone OX-86, Biolegend, diluted 1:100), anti-mCD40L (PE, clone MR1, eBiosciences, diluted 1:10) and anti-mICOS (mCD278-PE-Cy7, 7E, clone 17G9, eBiosciences, diluted 1:100); and (3) a T-cell co-inhibition panel consisting of: anti-mPD-1 (mCD279-PE-Cy7, clone RMP1-30, Biolegend, diluted 1:100), anti-mTIM3 (mCD366-APC, clone B8.2C12, Biolegend, diluted 1:100), anti-mLAG3 (mCD223-BV421, clone C9B7W, BD, diluted 1:20) and anti-mCTLA-4 (mCD152-PE, clone UC10-4B9, eBiosciences, diluted 1:20). Backbone compounds / antibodies (present in these three panels) were: 7-Amino-Actinomycin D (7-AAD; BD Biosciences, diluted 1:20), anti-mCD14-PerCP (clone rmC5-3, eBiosciences, diluted 1:20), anti-mCD3-BV510 (clone 17A2, BD Biosciences, diluted 1:100), anti-mCD4-BV650 (clone RM4-5, BD, diluted 1:2000), anti-mCD8-APC-Cy7 (clone 53-6.7, BD, diluted 1:1600) and anti-TCRVß14-FITC (clone CAS1.1.3, Beckman Coulter, diluted 1:2). To detect phosphorylated (p)ERK, T-cells were first stained with a fixable viability dye (eFluor 666, eBioscience, diluted 1:1000) for 10 min at RT, after which cells were stimulated (as described under intracellular phosphorylation), fixed for 10 min at 37°C (according to FoxP3 kit, eBioscience), washed and stained with anti-mCD3-BV510 (clone 17A2, BD, diluted 1:100), anti-mCD8-APC-Cy7 (clone 53-6.7, BD, diluted 1:1600), antiTCRVß14-PE (clone CAS1.1.3, Beckman Coulter, diluted 1:3) and anti- pERK1 / 2-FITC (clone 6B8B69, BioLegend, diluted 1:20) for 20 min at 4°C. To detect pPI3K, T-cells were stimulated with tumor cells, fixed for 12 min at 37°C (according to Cytofix kit, BD), permeabilized for 20 min at RT, washed twice and incubated with antibodies for 30 min at RT. The antibody used was pPI3K (PE, clone PI3KY458-1A11, ThermoFisher, diluted 1:20), which was added to the backbone antibodies anti-mCD3-BV510, anti-TCRVß-14-FITC, and anti-mCD8- APC-Cy7. To assess T-cell exhaustion upon repeated stimulations with tumor cells, cells were stained with anti-mCD8, anti-mCD3, anti-TCRVß-14, anti-mPD-1 and anti-mTIM-3 (see details above). To detect TCR surface expression of human T- cells, samples were stained with 7-AAD, anti-hCD3-BV421 (clone SP34-2, BD, diluted 1:200), anti-hCD8-BV650 (clone RPA-T8, BD, diluted 1:1000) and either anti-TCRVß-14-PE (gp100 / A2 TCR; clone CAS1.1.3, Beckman Coulter, diluted 1:3) or anti-TCRVß-13.1-FITC (ROPN1 / A2 TCR; clone IMMU 222, diluted 1:10). Lastly, to assess human T-cell exhaustion upon repeated stimulations, cells were stained with anti-hCD8, anti-hCD3, anti-hTCR, anti-hPD-1(APC-Cy7, clone EH12.2H7, Biolegend, diluted 1:50) and anti-hTIM-3 (APC, clone F38-2E2, Biolegend, diluted 1:50). Following above stainings, samples were washed once again, resuspended in 1% PFA, and data was acquired either with a FACS Celesta, a FACS Fortessa or a FACS Symphony A1. All flow cytometry data was analyzed using FlowJo (Treestar, version 8). Transcriptomics Tumor tissues were disrupted by sonification while kept on ice, and RNA was isolated (Machery Nagel). RNA concentration and quality was measured with TapeStation (Agilent), and only samples with an RNA integrity number of 6 or higher and a quantity of more than 250ng were used for 3’ RNA sequencing (QuantSeq, Illumina, Erasmus MC). Differential gene expression analysis was performed using the DESeq2 package from R, and gene set enrichment was performed with the GSA and fgsea packages. References used in these analyses were a laboratory list of immune-related genes that are associated to immune escape (Hammerl et al., 2020. Clin Cancer Res 26: 505-517), and the mouse Hallmark database (available at “https: / / data.broadinstitute.org / gsea- msigdb / msigdb / release / 2022.1.Mm / ”, mh.all.v2022.1.Mm.symbols.gmt). Intracellular phosphorylation Mouse TCR T-cells expressing either wt TCR or TCR:ICOS (6x104 cells / well) were co-cultured with B16:HHD-YLEP clone or B16F10 wt (2x104 cells / well) in Complete Mouse Medium in a tissue culture treated 96 wells plate at 37°C. After 2h, cells were fixed and stained and assessed for pPI3K (as described under ‘flow cytometry’). To assess the ability of TCR:ICOS to phosphorylate a large array of intracellular substrates, we treated wells from a non-tissue culture 96-wells plate that were streptavidin pre-coated (Pierce) with 50µL of 1mg / mL biotinylated A2:Kb pentamer fused to the gp100 peptide YLEPGPVTA or an irrelevant peptide (FLYTYIAKV, Proimmune, Oxford, UK) for 1h at RT, after which wells were washed three times with PBS + 0.5% Tween-20 (Sigma). Mouse T-cells expressing either gp100 / A2 wt TCR, TCR:ICOS or TCR:ICOS-YF were added to the plates (0.25x106 / well in 50µL Cytotox medium) for 30 min at 37°C, with frequencies of TCR-expressing T-cells normalized towards TCR:ICOS T-cells using mock T-cells. Following pMHC stimulations, plates were placed on ice and T-cells were harvested and lysed with lysis beads, after which proteins were extracted, biotinylated and placed on the antibody array, followed by the addition of a Cy3- Streptavidin dye (GEPA43001, Merck) according to the manufacturer’s instructions (antibody array assay kit KAS02 and T-Cell Receptor Phospho-Antibody Array PTC188, Fullmoon Biosystems). To validate the effect of stimulation, one well from each condition was kept separate for flow cytometric detection of pERK1 / 2 (see above, and Figure 6B). Detection of the Cy3 signal was performed using a Typhoon 5 fluorescent scanner and quantified using Imagequant TL with normalization according to the running ball background subtraction tool (Fiji, ImageJ 1.53t). Downstream analysis was performed using R, where median background of each slide was subtracted from the corresponding experimental values, and replicates (n=5) were averaged. Finally, the phosphorylation ratio after stimulation was calculated using the following formula: (phosphorylated protein 1) / (unphosphorylated protein 1) (phosphorylated protein 2) / (unphosphorylated protein 2) where 1 refers to the stimulated condition and 2 to the unstimulated condition. Release of inflammatory cytokines Mouse T-cells expressing either wt TCR, TCR:ICOS or TCR:ICOS-YF (0.75x106cells / well) were co-cultured with B16:HHD-YLEP clone or B16F10 wt (0.25 x106cells / well) in Complete Mouse Medium in a tissue culture treated 24 wells plate for 16h at 37°C. Mock T-cells served as negative controls. Supernatants were assessed for the presence of cytokines (according to a customized Luminex assay, R&D Systems Minneapolis USA; measuring IFN-γ, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, TNF-α, CCL-5, CCL-20, CXCL-2, MMP-8 and MMP-9). Repeated stimulations with tumor cells Mouse TCR T-cells expressing either wt TCR, TCR:ICOS or TCR:ICOS-YF (0.75x106) were co-cultured with B16:HHD-YLEP clone or B16F10 wt (0.25x106) in Complete Mouse Medium supplemented with 25 IU / mL IL-2 in a 24 wells plate for 16h at 37°C. After this 1st cycle of exposure, T-cells were harvested with 20% of the sample being exposed to flow cytometric detection of T-cell exhaustion, and 80% of the sample being again co-cultured for the 2nd cycle, with frequencies of TCR- expressing T-cells normalized towards TCR:ICOS T cells using mock T-cells. Tumor cells were freshly pre-seeded to ensure an 80% confluency at start of co- culture with T-cells. Experiments were performed over a minimum of 4 cycles. Repeated stimulation with human TCR T-cells were performed in a similar manner, using either BLMgp100 (gp100 / A2 TCR) or MM-231-ROPN1 (ROPN1 / A2 TCR) as target cells, and co-culturing the cells in T-cell medium supplemented with 50 IU / mL IL-2. Mock T-cells served as negative controls. Detection of antigen reactivity and sensitivity of human TCR T-cells To assess antigen reactivity, human TCR T-cells expressing either wt TCR or TCR:ICOS-RA (6x104cells / well) were co-cultured with BLM-gp100 (gp100 / A2 TCR) or MM-231-ROPN1 (ROPN1 / A2 TCR) in Cytotox medium in a tissue culture treated 96 wells plate for 16h at 37°C. BLM wt and MM-231 wt cell lines as well as mock T-cells were used as negative controls. To assess antigen sensitivity, TCR T- cells (6x104cells / well) were co-cultured with T2 cells (2x104cells / well) loaded with cognate peptide (gp100 or ROPN1 peptide) at increasing concentrations (from 0.1 nM to 30µM) in Cytotox medium in a tissue culture treated 96 wells plate for 16h at 37°C. Supernatants were assessed for the presence of IFN^ by enzyme-linked immunosorbent assay (ELISA, Biolegend). Statistical analyses Statistical analysis and data display was performed using either GraphPad Prism (GraphPad Software, La Jolla, CA, version 8.0.2) or Rstudio (version 4.3.1). Statistical tests were considered significant when p < 0.05. Changes over time (in tumor volume, number of T-cells, or frequency of T-cell subsets) were compared according to the linear mixed model, whereas survival was compared according to the Cox regression model. Multiple group comparisons were performed either with ANOVA or, when the requirements were not met, Kruskal-Wallis test. Post-hoc testing was performed either with t-tests (with Sidak’s correction if needed) or Dunns’ test.

[0002] Table 1: Specifics of all hTCR:ICOS variants tested for surface expression. TCR TCR-constant Trans Intra Intra Hinge Linker T2A Remark n° domain membrane cellular cellular CD28 ICOS CD3^ (KNRKA ACPK / 10 minimally murinized* n / a (FWVLV- (CWLTK- var2 - QRRI) GSPK IIFWV) DVTL)

[0003] CD28 ICOS TRAC******+TCRBC** CD3^ (KNRKA 44 n / a (FWVLV- (TKKKY- PK / PK var3 *** 6 mouse AA - QRRI) Sequences in bold are murine sequences. For each domain, the first 5 and last 4 amino acids are listed. Different variants of the T2A sequence were used, namely: var 1 (amino acids): SGSGRSGSGEGRGSLLTCGDVEENPGPR; var 2: RSGSGRSGSGEGRGSLLTCGDVEENPGPRMH; and var 3: GSGEGRGSLLTCGDVEENPGP.

[0004] ***** Amino acid sequence PLKEQPALNDSR was replaced by AYKESNYS in constant region of TCR^ chain. ****** Amino acid sequence RSMDFKSNSAV was replaced by KAMDSKSNGAI in constant region of TCR^ chain. Table 2: Sequences of individual building blocks of co-stimulatory TCR^ and TCR^. SEQ ID Name Sequence NO 1 Human ROPN1 TCRα MMKSLRVLLV ILWLQLSWVW SQQKEVEQNS GPLSVPEGAI ASLNCTYSDR GSQSFFWYRQ variable chain YSGKSPELIM SIYSNGDKED GRFTAQLNKA SQYVSLLIRD SQPSDSATYL CAVNGDSSYK LIFGSGTRLL VRPD 2 Human ROPN1 TCRß MSIGLLCCAA LSLLWAGPVN AGVTQTPKFQ VLKTGQSMTL QCAQDMNHEY MSWYRQDPGM variable chain GLRLIHYSVG AGITDQGEVP NGYNVSRSTT EDFPLRLLSA APSQTSVYFC ASSYSLGDGY TFGSGTRLTV VE 3 Human gp100 TCRα MASIRAVFIF LWLQLDLVNG ENVEQHPSTL SVQEGDSAVI KCTYSDSASN YFPWYKQELG variable chain KRPQLIIDIR SNVGEKKDQR IAVTLNKTAK HFSLHITETQ PEDSAVYFCA ASTSGGTSYG KLTFGQGTIL TVHPN 4 Human gp100 TCRß MGPQLLGYVV LCLLGAGPLE AQVTQNPRYL ITVTGKKLTV TCSQNMNHEY MSWYRQDPGL variable chain GLRQIYYSMN VEVTDKGDVP EGYKVSRKEK RNFPLILESP SPNQTSLYFC ASSLGSSYEQ YFGPGTRLTV TE 5 Human NY-ESO1 METLLGLLIL WLQLQWVSSK QEVTQIPAAL SVPEGENLVL NCSFTDSAIY NLQWFRQDPG TCRα variable chain KGLTSLLLIQ SSQREQTSGR LNASLDKSSG RSTLYIAASQ PGDSATYLCA VRPLYGGSYI PTFGRGTSLI VHPY 6 Human NY-ESO1 MSIGLLCCAA LSLLWAGPVN AGVTQTPKFQ VLKTGQSMTL QCAQDMNHEY MSWYRQDPGM TCRß variable chain GLRLIHYSVA IGITDQGEVP NGYNVSRSTT EDFPLRLLSA APSQTSVYFC ASSYVGNTGE LFFGEGSRLT VLE 7 Human TCRα IQNPDPAVYQ LRDSKSSDKS VCLFTDFDSQ TNVSQSKDSD VYITDKTVLD MRSMDFKSNS extracellular constant AVAWSNKSDF ACANAFNNSI IPEDTFFPSP ESS domain

[0005] Human TCRß DLKNVFPPEV AVFEPSEAEI SHTQKATLVC LATGFYPDHV ELSWWVNGKE VHSGVSTDPQ extracellular constant PLKEQPALND SRYCLSSRLR VSATFWQNPR NHFRCQVQFY GLSENDEWTQ DRAKPVTQIV domain 1 SAEAWGRAD Human TCRß DLKNVFPPEV AVFEPSEAEI SHTQKATLVC LATGFYPDHV ELSWWVNGKE VHSGVSTDPQ extracellular constant PLKEQPALND SRYCLSSRLR VSATFWQNPR NHFRCQVQFY GLSENDEWTQ DRAKPVTQIV domain 2 SAEAWGRAD Human CD8α Hinge TTTPAPRPPT PAPTIASQPL SLRPEACRPA AGGAVHTRGL DFACDIY domain Human IgG4 Hinge ESKYGPPCPS CP domain Linker variant 1 PK Linker variant 2 CPK Linker variant 3 ACPK Linker variant 4 GSPK Human CD28 FWVLVVVGGV LACYSLLVTV AFIIFWV transmembrane domain Human OX40 VAAILGLGLV LGLLGPLAIL LALYLL transmembrane domain Human 4-1BB IISFFLALTS TALLFLLFFL TLRFSVV transmembrane domain Human ICOS FWLPIGCAAF VVVCILGCIL ICWL transmembrane domain Human CD28 RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR S intracellular domain

[0006] Human OX40 RRDQRLPPDA HKPPGGGSFR TPIQEEQADA HSTLAKI intracellular domain Human 4-1BB KRGRKKLLYI FKQPFMRPVQ TTQEEDGCSC RFPEEEEGGC EL intracellular domain Human ICOS TKKKYSSSVH DPNGEYMFMR AVNTAKKSRL TDVTL intracellular domain Human ICOS-YF TKKKYSSSVH DPNGEFMFMR AVNTAKKSRL TDVTL intracellular domain Human CD3^ KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQRRI intracellular domain Human CD3^-RA KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQRAI intracellular domain Human CD40L IFMYLLTVFL ITQMIGSALF AVYL transmembrane domain Human CD40L MIETYNQTSPR SAATGLPISM K intracellular domain Human CD3ε-R54C KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQRCI intracellular domain Human CD3ε-R54D KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQRDI intracellular domain Human CD3ε-R54E KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQREI intracellular domain Human CD3ε-R54F KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQRFI intracellular domain Human CD3ε-R54G KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQRGI intracellular domain Human CD3ε-R54H KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQRHI intracellular domain

[0007] Human CD3ε-R54I KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQRII intracellular domain Human CD3ε-R54K KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQRKI intracellular domain Human CD3ε-R54L KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQRLI intracellular domain Human CD3ε-R54M KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQRMI intracellular domain Human CD3ε-R54N KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQRNI intracellular domain Human CD3ε-R54P KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQRPI intracellular domain Human CD3ε-R54Q KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQRQI intracellular domain Human CD3ε-R54S KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQRSI intracellular domain Human CD3ε-R54T KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQRTI intracellular domain Human CD3ε-R54V KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQRVI intracellular domain Human CD3ε-R54W KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQRWI intracellular domain Human CD3ε-R54Y KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQRYI intracellular domain Human CD3ε-R54de KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQRI intracellular domainl Human CD3ε-R53A KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQARI intracellular domain

[0008] Human CD3ε- KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQAAI R53+R54A 51 intracellular domain Human CD3ε-R53+R54 KNRKAKAKPV TRGAGAGGRQ RGQNKERPPP VPNPDYEPIR KGQRDLYSGL NQI del intracellular 52 domain Table 3. Overview of constructs. TCR no Target Transmembrane Intracellular (co-Intracellular (CD3) stimulatory) 1 gp100 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRAI) 2 gp100 4-1BB (IISF – FSVV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRAI) 3 gp100 CD40L (LYVA – YMFI) ICOS (TKKK– DVTL) CD3ε (KNRK – QRAI) 4 gp100 ICOS (FWLP – ICWL) ICOS (TKKK– DVTL) CD3ε (KNRK – QRAI) 5 gp100 OX40 (VAAI – LYLL) ICOS (TKKK– DVTL) CD3ε (KNRK – QRAI) 6 gp100 CD28 (FWVL-IFWV) CD28 (RSKR - AYRS) CD3ε (KNRK – QRAI) 7 gp100 CD28 (FWVL-IFWV) CD40L (KMSI - TEIM) CD3ε (KNRK – QRAI) 8 gp100 CD28 (FWVL-IFWV) 4-1BB (KRGR - GCEL) CD3ε (KNRK – QRAI) 9 gp100 CD28 (FWVL-IFWV) OX40 (RRDQ - LAKI) CD3ε (KNRK – QRAI) 10gp100 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRRI) 11gp100 4-1BB (IISF – FSVV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRRI) 12gp100 CD40L (LYVA – YMFI) ICOS (TKKK– DVTL) CD3ε (KNRK – QRRI) 13gp100 ICOS (FWLP – ICWL) ICOS (TKKK– DVTL) CD3ε (KNRK – QRRI) 14gp100 OX40 (VAAI – LYLL) ICOS (TKKK– DVTL) CD3ε (KNRK – QRRI) 15gp100 CD28 (FWVL-IFWV) CD28 (RSKR - AYRS) CD3ε (KNRK – QRRI) 16gp100 CD28 (FWVL-IFWV) CD40L (KMSI - TEIM) CD3ε (KNRK – QRRI)

[0009] gp100 CD28 (FWVL-IFWV) 4-1BB (KRGR - GCEL) CD3ε (KNRK – QRRI) gp100 CD28 (FWVL-IFWV) OX40 (RRDQ - LAKI) CD3ε (KNRK – QRRI) ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRAI) ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRRI) ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRCI) ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRDI) ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QREI) ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRFI) ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRGI) ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRHI) ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRII) ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRKI) ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRLI) ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRMI) ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRNI) ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRPI) ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRQI) ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRSI) ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRTI) ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRVI) ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRWI) ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRYI) ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – NQRI) gp100 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QARI) gp100 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QAAI) gp100 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – LNQI)

[0010] ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QARI) ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QAAI) ROPN1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – LNQI)NY-ESO1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRRI)NY-ESO1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QRAI)NY-ESO1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QARI)NY-ESO1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – QAAI)NY-ESO1 CD28 (FWVL-IFWV) ICOS (TKKK– DVTL) CD3ε (KNRK – LNQI)

[0011] Results TCR:ICOS mediates durable anti-melanoma T cell responses In the context of adoptive cell therapy (ACT), the duration of an effective T- cell response is significantly challenged by the immune-suppressive tumor micro- environment, that is generally deficient in providing T-cell co-stimulation. In this study, we hypothesized that co-stimulatory T-cell receptors (TCRs) restore T-cell co-stimulation, as normally provided by co-stimulatory ligands, and rescue T-cell response that would otherwise become curtailed within tumors (schematically represented in Figure 1A). We have previously reported the identification of a two- chain TCR linked to the co-stimulatory molecule CD28 (i.e., TCR:CD28) (Govers et al., 2014. J Immunol 193: 5315-5326). Importantly, this initial study showed that the functional expression of TCR:CD28 in T-cells is governed by a strict format, i.e. the particular type and order of building blocks, and their amino acid boundaries. In fact, the transmembrane domain of CD28 turned out to be required for surface expression, whereas the intracellular domains of both CD28 and CD3ε were required for T-cell function. In the present study, we aimed to further improve the functional performance of TCR T-cells, particularly in relation to the duration of their anti-tumor response. To this end, we designed multiple new co-stimulatory TCRs, all directed against the HLA-A2-restricted epitope gp100280-288, where, besides the intracellular CD28 domain, alternative intracellular domains were incorporated, such as CD134 (OX40), CD137 (4-1BB), and CD278 (ICOS) (illustrated in Figure 1B, and their construction detailed in the Materials and Methods section). All TCR constructs were successfully expressed on the surface of murine T-cells following gene transfer, except for the TCR with an introduced 4- 1BB domain (Figure 2A). The lack of surface expression of the TCR:4-1BB was likely a consequence of its size, and this construct was excluded from further analyses. The surface-expressed co-stimulatory TCRs were tested for antigen reactivity and sensitivity and demonstrated antigen-specific responsiveness, although with a lower avidity than wildtype (wt) TCR (Figure 2B). Subsequently, these TCRs were used in a melanoma mouse model for adoptive cell therapy, where treatment with wt TCR, TCR:CD28 and TCR:OX40 T-cells led to an initial tumor regression, which was, however, quickly followed by tumor relapse (Figure 1C and D). Notably, treatment with TCR:ICOS T-cells was the only treatment to provide significant advantage over wt TCR T-cells regarding durability of anti-tumor response (estimate effect of 500 mm3and p = 0.003 according to linear mixed model). In addition, treatment with TCR:ICOS T-cells led to significantly increased survival, with 60% of the mice surviving at the end of the experiment (150 days after T-cell transfer), compared to 0% for wt TCR and TCR:CD28 and 16% for TCR:OX40 (p = 0.02 according to cox regression analysis, Figure 1E). Furthermore, mice treated with TCR:ICOS T-cells showed a longer median time to relapse than those treated with wt TCR T-cells (140 days for TCR:ICOS vs 22, 32 and 35 days for wt TCR, TCR:OX40 and TCR:CD28, respectively, p = 0.048 according to Kruskal-Wallis test, TCR:ICOS vs wt TCR p = 0.007 according to post-hoc Dunn’s test, Figure 1F). The durable anti-tumor response observed for mice treated with TCR:ICOS T-cells was accompanied by a long-term persistence of circulating CD8+ T-cells expressing the introduced TCR (CD8+ TCR T-cells). Indeed, mice treated with TCR:ICOS T-cells had a median of 109 CD8+ TCR T-cells per microL blood at 100 days post-treatment compared to 77 and 4 for CD8+ TCR:OX40 and wt TCR T- cells (estimate effect of 179 cells / µL and p = 0.0015 according to linear mixed model, Figure 1G). Note that the long-term persistence observed with TCR:OX40 T- cells is due to one mouse. The enhanced persistence of TCR:ICOS T-cells was also observed in the CD4 fraction, albeit to a lesser extent (data not shown). Looking at extent of response (i.e., complete, partial or non), TCR:ICOS T-cell-treated mice had a significantly higher overall anti-tumor response rate than mice treated with wt TCR or other co-stimulatory TCR T-cells (Figure 1H). Collectively, TCR:ICOS T- cells showed an improved anti-melanoma efficacy compared to other co-stimulatory TCRs and wt TCR T-cells, especially in terms of durability of the response. Intra-tumoral CD8+ TCR:ICOS T cells demonstrate a young and co-stimulatory phenotype and create an inflammatory micro-environment To better understand the effect of TCR:ICOS on the durability of anti-tumor response, we determined the phenotype of circulating and tumor infiltrating T- cells, specifically regarding their maturation state and expression of co-signalling receptors (Figure 3A). In blood of mice that were treated with either TCR:ICOS or wt TCR T-cells, we observed that CD8+ TCR T-cells were highly differentiated, with no statistically significant differences between treatment groups (Figure 3B). We observed low fractions of circulating T-cells co-expressing multiple co-inhibitory receptors, whereas we observed high fractions of circulating T-cells co-expressing multiple co-stimulatory receptors following both treatments. For instance, 16% CD8+ TCR:ICOS T cells and 17% CD8+ wt TCR T-cells co- expressing PD1 and TIM3 were detected, whereas 76% CD8+ TCR:ICOS T-cells and 78% CD8+ wt TCR T-cells co-expressing CD40L and 4-1BB were detected. Circulating CD4+ T-cells showed a similar pattern with little to no differences observed between TCR:ICOS T cells and wt TCR T-cells (data not shown). In regressed tumors a higher percentage of CD8+ TCR TILs with a naïve-like or central memory and a significantly lower percentage with an effector memory state following treatment with TCR:ICOS T cells vs wt TCR T cells were observed (31 vs 50% CD62L-CD44+ T cells, p = 0.043 according to Kruskal-Wallis, Figure 3C). Notably, it was demonstrated that TCR:ICOS TILs have a larger fraction of CD8+ T cells expressing either 4-1BB or OX40 compared to wt TCR TILs (33 vs 15% 4- 1BB+ T-cells, p = 0.034; and 6 vs 1% OX40+ T cells, p = 0.034 according to Kruskal- Wallis, Figure 3C right panel). The percentage of cells expressing 4-1BB or OX40 together with a different co-stimulatory molecule was also higher in TCR:ICOS T- cells than in wt TCR T-cells. Taken together, in TILs of mice treated with TCR:ICOS T-cells, less differentiated CD8+ T-cells and a higher fraction of T-cells expressing 4-1BB were detected, which potentially represent fit T-cells and may support our earlier finding of a higher persistence of these cells in blood. The above finding guided us to perform transcriptomic analysis of regressed and relapsed tumors upon treatment with TCR:ICOS T-cells. Analysis of differential gene expression showed that regressed tumors treated with TCR:ICOS T-cells had a distinct expression profile from tumors treated with wt TCR T-cells. Interestingly, most of the differentially expressed genes were immune-related (i.e., Cxcl2, Il-1b or Ccl-5, Figure 3D). With gene set enrichment analysis, we found that the inflammatory response represented the most enriched pathway in regressed tumors from TCR:ICOS T-cells-treated mice (NES=2.5, p.adj=1.2x10-23, Figure 3E). Additionally, the TNF-α signaling via NFκB pathway was also a highly enriched pathway in these tumors (NES=2.2, padj= 1.6 x10-13). These gene expression results indicate that TCR:ICOS T-cells create an inflammatory tumor micro- environment, in part by the activation of the NFκB pathway. Transcriptomics from relapsed tumors did not reveal enrichment of these gene sets, and when assessing the occurrence of immune-related signatures, relapsed tumors showed decreased occurrence of antigen presentation (Figure 4). In vitro stimulated TCR:ICOS T cells activate the NFκB pathway, produce inflammatory cytokines and do not become exhausted upon repeated tumor cell exposure In the next series of experiments, we further explored the signaling pathways and functional performance of TCR:ICOS T-cells using controlled stimulations in vitro. In a first step, we assessed the phosphorylation of recognized targets downstream of ICOS using flow cytometric detection of pPI3K following stimulation with the B16:HHD-YLEP clone (Figure 6A). We showed that the fraction of CD8+T-cells expressing pPI3K upon stimulation was significantly higher in TCR:ICOS T-cells than in wt TCR T-cells (14 vs 2% pPI3K+ cells, p=0.019 according to Sidack’s test). This stimulation effect was not observed with BL / 6 B16F10 wt cells. In an effort to abolish TCR:ICOS’ ability to activate PI3K, and consequently its downstream substrates, we developed a TCR:ICOS-YF mutant (see Materials and Methods for details regarding its construction; Figure 5A). T- cells expressing either wt TCR, TCR:ICOS or TCR:ICOS-YF were stimulated either with gp100 A2Kb:peptide or an irrelevant A2Kb:peptide after which cell lysates were exposed to a kinase micro-array. The occurrence of T-cell phosphorylation was validated by detection of an increase in the fraction of wt TCR T-cells positive for pERK1 / 2 (Figure 6B). Expectedly, stimulated TCR:ICOS T-cells, but not TCR:ICOS-YF nor wt TCR T-cells, demonstrated PI3K activation as exemplified by an accumulated presence of the p85 subunit of PI3K (Figure 5B). Remarkably, stimulation of TCR:ICOS T-cells induced a significant phosphorylation of multiple members of the NFκB family, which was lost in stimulated TCR:ICOS-YF T-cells with levels becoming similar to those found in stimulated wt TCR-T-cells (Figure 5B). Moreover, many of the accumulated phospho-members, such as pIKBε, were specific to the canonical NFκB pathway, whereas accumulated phospho-members in wt TCR T-cells, such as pRelB, mostly belonged to the non-canonical pathway. Furthermore, other pathways were also differentially phosphorylated in TCR:ICOS T-cells, although not to the same extent as the NFκB pathway. Especially early TCR signaling, with proteins such as Lck, appeared to be increasingly activated in stimulated TCR:ICOS T-cells compared to wt TCR T-cells. These findings suggest that activation of PI3K as a consequence of antigen-specific triggering of TCR:ICOS is relayed into the activation of the canonical NFκB pathway. In a second step, we stimulated TCR T-cells with B16 cells, and measured the production of a large number of cytokines. We found that TCR:ICOS T-cells produced significantly more cytokines and chemokines in response to antigen- positive, but not antigen-negative melanoma cells, when compared to either wt TCR or TCR:ICOS-YF T-cells. In particular, secretion of IL-2, IL-6, TNF-α, CCL-5 and CXCL-2 were significantly increased (Figure 5C), whereas for other cytokines atrend towards higher detection was found. It is noteworthy that many of thesecytokines, such as IL-1ß, IL-6, and TNF-α, represent direct downstream targets of the NFκB pathway and take actively part in tissue inflammation (Liu et al., 2017. Signal Transduct Target Ther 2: 17023; Su et al., 2021. Sci Rep 11: 13464). Moreover, some of these mediators, such as IL-1ß, IL-6, CCL-5 and CXCL-2, showed higher gene expression in regressed tumors following treatment with TCR:ICOS T-cells (Figure 3D). In a third and last step, TCR T-cells were repeatedly exposed to antigen- positive melanoma cells and assessed for the expression of PD1 and TIM3 as markers of T-cell exhaustion. We found that the fraction of PD1+, TIM3+CD8+T cells over 3 rounds of stimulation was significantly less for TCR:ICOS when compared to wt TCR T-cells or TCR:ICOS-YF T-cells (7, 25 and 15% for TCR:ICOS, wt TCR and TCR:ICOS-YF T cells, p<0.0001 according to mixed effect analysis, Figure 5D). This difference in expression was not observed when using antigen negative melanoma cells. Taking this part together, TCR:ICOS T-cells activate the PI3K and NFκB pathways, produce inflammatory cytokines and chemo-attractants and are more resistant to the expression of T-cell exhaustion markers upon repeated antigen stimulation. These results support the notion that TCR:ICOS T- cells, once recognizing tumor cells, contribute to an NF^B-driven inflammatory tumor milieu and are able, in line with their co-stimulatory phenotype, to be less prone to enter T-cell exhaustion. Durable anti-tumor response depends on docking site of TCR:ICOS for PI3K To investigate whether PI3K and NF^B pathway activation determines in vivo performance of TCR:ICOS T-cells, we tested the requirement for ICOS to dock PI3K and activate downstream signaling pathways in adoptive T-cell transfer experiments. To this end, TCR:ICOS-YF, being unable to dock PI3K, was tested, together with TCR:ICOS and wt TCR, in the melanoma mouse model (Figure 3A). While mice treated with TCR:ICOS T cells exhibited an effective and durable antitumor response, mice treated with TCR:ICOS-YF T-cells showed a response comparable to that of mice treated with wt TCR T-cells, and displayed a significantly worse survival (30, 20 and 55% of mice were alive at 150 days after treatment with TCR:ICOS-YF, wt TCR or TCR:ICOS respectively, Figure 7A and B). In addition, short median time to relapse was observed in mice that received TCR:ICOS-YF T cells (30 and 26 days for TCR:ICOS-YF and wt TCR, and median time to relapsed not reached at 150 days for TCR:ICOS T cells, p=0.04 according to Kruskal-Wallis Figure 7C). Furthermore, CD8+TCR:ICOS-YF T-cells had a poor persistence in the blood, comparable to that of wt TCR T-cells (estimate of 36 cells / µL and p=0.44 for wt TCR vs TCR:ICOS-YF; estimate of 153 cells / µL and p=0.0033 for wt TCR vs TCR:ICOS according to linear mixed model, Figure 7D). CD4+T-cells had an overall low persistence across treatment groups, but was again lower for TCR:ICOS-YF and wt TCR T-cells compared to TCR:ICOS T-cells (data not shown). In short, the durability of anti-tumor responses as conveyed by TCR:ICOS critically depends on the docking of PI3K to ICOS and activation of its downstream pathways. Functional performance of human TCR:ICOS is governed by a single amino acid substitution in cytosolic tail of the CD3^ domain In a final series of experiments, we set out to design and stringently test TCR:ICOS for its performance in human T-cells. The fully murine gp100 / A2 TCR:ICOS resulted in an average surface expression of 23% in human T-cells, whereas the fully human gp100 / A2 TCR:ICOS using identical orthologous sequences only led to expression in 6.5% of the cells (Figure 8). Although this difference may be unexpected, it likely points to the strict regulation of TCR surface expression, which, in contrast to chimeric antigen receptors (CARs), depends on two chains and accessory molecules. To address this challenge and create a functionally expressed hTCR:ICOS while maintaining its native format, we designed a panel of about 50 different variants to identify which building block (or combinations thereof) and / or which boundaries between building blocks are responsible for a low expression of hTCR:ICOS (Figure 9A). In this stepwise approach, we started by exchanging each individual building block of the mTCR:ICOS (i.e., the extracellular TCR-C, the CD28 transmembrane, the ICOS intracellular and the CD3ε intracellular domains) for their human orthologues (Figure 9B). These TCR:ICOS variants were introduced into human T- cells, and surface expressions were compared to mTCR:ICOS and wt TCR (with on average 3 to 4 transductions per variant). Both the hCD28 and hICOS domains did not impair expression compared to mTCR:ICOS. In contrast, the hCD3ε and hTCR- C domains both led to a significant decrease in expression (12 and 6% of CD3+ cells respectively, compared to 18% for mTCR:ICOS). Re-introduction of the mTCR-C domain in an otherwise fully hTCR:ICOS did not rescue the surface expression of the variant, whereas re-introduction of the mCD3ε domain lifted the expression to a level comparable to that of mTCR:ICOS. In the next step, we continued by exchanging individual amino acids from mCD3^ into hTCR:ICOS. To this end, we selected 5 amino acids that are different between m and hCD3^ and constructed the corresponding single amino acid substituents (Figure 9C). Strikingly, substitution of a single amino acid, namely hCD3ε R54A (corresponding to construct no45 in Table 1), led to expression levels of hTCR:ICOS that exceeded those of mTCR:ICOS and were comparable to those of wt TCR (38 vs 36% for hTCR:ICOS-RA vs wt hTCR). We then set out to validate the in vitro functionality of TCR:ICOS-RA, characterizing its antigen reactivity, sensitivity as well as resistance to T-cell exhaustion (Figure 9D). When stimulating T-cells with human melanoma cells that do or do not express the gp100 antigen, we observed that TCR:ICOS-RA mediated T-cell IFN^ production in an antigen-specific manner. When challenging hTCR:ICOS T-cells with titrated amounts of cognate peptide, we observed that EC50 values (with half-maximal levels of IFN^) as well as amplitudes (the plateau levels of IFN^) were slightly higher than those for wt TCR T-cells (EC50:128 vs 1.6nM; amplitude: 32 vs 10 ng / mL for TCR:ICOS vs wt TCR T-cells). These data are in line with our findings for mTCR:ICOS T-cells (Figure2B) and point out that the RA mutation in itself does not change antigen reactivitynor sensitivity of hTCR:ICOS. When testing the benefit of hTCR:ICOS regarding resistance to the development of T-cell exhaustion upon repeated antigen stimulation, we observed that CD8+ hTCR:ICOS T-cells show significantly lower fractions that co-express PD1 and TIM3 when compared to wt TCR T-cells (p=0.0015 according to two-way ANOVA, Figure 9D). In a final set of experiments, we have put surface expression and in vitro performance of hTCR:ICOS-RA to the test using a second TCR specificity (Figure 9E). To this end, we used ROPN1 / A2 TCR that is directed against Ropporin and is being developed for adoptive T-cell therapy to treat Triple Negative Breast Cancer (TNBC) (Kortleve et al., 2024. Cancer Discovery 14: 2450-2470), and created ROPN1 / A2 hTCR:ICOS-RA (see Materials and Methods for details). With this second TCR:ICOS-RA, we again demonstrated high surface expression and reactivity towards ROPN1-expressing TNBC cell lines. In addition, using ROPN1 peptide titrations, we demonstrated that hTCR:ICOS-RA T-cells have a similar EC50 value and higher amplitude compared to wt TCR T-cells (see Figure 9E for details). Importantly, also for this antigen target, CD8+ hTCR:ICOS-RA T-cells showed lower fractions that co-express PD1 and TIM3 upon multiple rounds of repeated stimulation when compared to wt TCR. Taken together, we have developed a single amino acid variant of hTCR:ICOS that enables functional expression in human T-cells, preserves its benefit regarding resistance towards development of T-cell exhaustion, and which can be extended to multiple TCR specificities. Conclusion To sum up, we have developed an advanced chimeric TCR that incorporates the ICOS domain and yields significantly prolonged anti-tumor T-cell responses in an immune competent melanoma mouse model. Mechanistically, murine TCR:ICOS T-cells, when present in regressing tumors, show a young and co- stimulatory cell state, are responsible for an inflammatory milieu, and make use of the NF^B pathway. These characteristics are recapitulated in vitro, where these T- cells produce inflammatory cytokines and do not become exhausted upon repeated tumor cell exposure. These advantages were shown to be reversed when disrupting ICOS-mediated activation of PI3K, a pathway upstream of NF^B. Notably, functional expression of human TCR:ICOS strictly depends on a single amino acid substitution in the intracellular tail, which preserves the TCR’s ability to limit T- cell exhaustion and enables the incorporation of the ‘ICOS cassette’ into TCRs with different specificities. Example 2 Materials and Methods Cell culture Human T cells were isolated from PBMC from healthy human donors (Sanquin) by centrifugation via Ficoll-Isopaque (density = 1.077 g / cm3; Amersham Pharmacia Biotech) and were cultured at 0.5 x106cells / mL in T cell medium consisting of X-vivo 15 (Lonza) with 2% Human Serum (Sanquin), and supplemented with 110 IU / mL Interleukin-15 (IL-15, Miltenyi Biotec) and 0.1 IU / mL Interleukin-21 (IL-21, Miltenyi Biotec). Packaging cells, BLM clones and MDA-MB-231 (MM-231) clones were cultured as in Example 1. Co-culture experiments with the BLM and MM231 cell lines and T cells were performed in RPMI 1640 with 25mM HEPES (Gibco), 5% Human Serum and Penicillin / Streptavidin (100 U / mL and 100 µg / mL respectively). Design of human chimeric TCRs The human gp100 (h)TCR:ICOS, was put into the pMP71 cassette and consisted of gp100 / A2 hTCRV^-C^-T2A-hTCRV^-C^^^extracellular domains), in which each TCR chain was followed by a PK linker, the hCD28 transmembrane, hICOS intracellular domain and hCD3^ intracellular with an R54A mutation (corresponding to TCR no45 in Table 1). In some cases, the TCRV^^and^V^-domains targeting gp100 / A2 were replaced with those TCRV^^and^V^-domains targeting ROPN1 / A2 (Kortleve et al., 2024, Cancer Discovery 14: 2450-2470) or NY-ESO1 / A2 (clone 1G4, Robbins et al. 2008. J. Immunol 180: 6116-31). To test the surface expression of alternative transmembrane and intracellular domains as well as alternative mutations in CD3ε, a panel of 50 variants was designed (all synthesized by GeneArt). An overview of these constructs, including detailed specifics, is provided in Table 3. In all transduction experiments, the hTCR:ICOS- RA (corresponding to TCR no45 in Table 1) was taken along as a reference. Gene introduction of wt and chimeric TCRs into T cells Human T-cell transduction was performed as previously described (Lamers et al., 2014. Human Gene Ther Methods 25: 345-57; Kortleve et al., 2024. Cancer Discovery 14: 2450-2470). In short, 293T and Ph-A packaging cells were transfected with pHit60 and pColtGalV (Weijtens et al., 1998. Gene Ther 5: 1195-1203) helper constructs, and the TCR of interest. Human PBMCs were activated for 48 h using 30 ng / mL soluble anti-CD3 antibody (OKT3, Miltenyi Biotec) and 30 ng / mL anti- CD28 antibody (15E8, Miltenyi Biotec) before being exposed to virus supernatant on two consecutive days, followed by expansion in the presence of IL-15 and IL-21 (110U / mL and 0.1U / mL, respectively, Miltenyi Biotec). TCR expression level was determined using flow cytometric detection of binding of the peptide:MHC or the expression of the TCR-Vß transgene by the transduced T cells, corrected for background expression levels as observed for mock T cells from the same donor. TCR expression level is displayed as percentage of positive cells within the CD3+ T cell population in comparison to the parental hTCR:ICOS-RA. A new chimeric TCR was considered non-successful in case surface expression with all donors was lower in comparison to the hTCR:ICOS-RA reference. Flow cytometry To detect surface expression of chimeric TCRs in human T cells, transduced cells were washed once with Phosphate Buffered Saline (PBS) and incubated with 10µL of an antibody cocktail for 15 min at room temperature. The following antibodies were used: anti-hCD3-FITC (clone UCHT1-2, eBioscience, diluted 1:30), anti-hCD8-APC (clone SK1, eBioscience, diluted 1:300) and either anti-TCRVß-14- PE (gp100 / A2 TCR; clone CAS1.1.3, Beckman Coulter, diluted 1:3) or anti-TCRVß- 13.1-FITC (ROPN1 / A2 and NY-ESO1 / A2 TCR; clone IMMU 222, diluted 1:10). In some experiments, a peptide:MHC-PE dextramer specific for the ROPN1 epitope FLYTYIAKV (Immudex, diluted 1:40) was used to stain cells for 10 minutes at room temperature prior to the addition of CD3 and CD8 antibodies. Following above stainings, samples were washed once again, resuspended in 1% PFA, and data was acquired with a FACS Symphony A1. All flow cytometry data was analyzed using FlowJo (Treestar, version 8). Detection of antigen reactivity of human T cells expressing chimeric TCRs To assess antigen reactivity, human T cells expressing chimeric TCR (6x104cells / well) were co-cultured with either BLM-gp100 (gp100 / A2 TCR, 2x104cells / well) or MM-231-ROPN1 (ROPN1 / A2 TCR, 2x104cells / well) in a tissue culture treated 96 wells plate for 16h at 37°C. BLM wt and MM-231 wt cell lines as well as mock T cells were used as negative controls. Supernatants were assessed for the presence of IFN-^ (expressed in pg / mL ) by enzyme-linked immunosorbent assay (ELISA MAX Deluxe set Human IFNg, BioLegend). Results Chimeric TCRs show expression in human T cells when harboring transmembrane domains of CD28 or ICOS but not CD40L, OX40 nor 41BB In order to test whether human (h)TCR:ICOS-RA (the end product of Example 1) is expressed in primary human T cells when alternative transmembrane domains are used, we designed hTCR:ICOS-RA variants in which we exchanged the CD28 transmembrane domain for the transmembrane domains of either 4-1BB, CD40L, ICOS or OX40 (Figure 10A, TCRs no1-5 in Table 3). Following transduction, we found that using the 4-1BB, CD40L or OX40 transmembrane domains decreased surface expression of the new chimeric TCRs when compared to the parental hTCR:ICOS-RA (which includes the CD28 transmembrane domain) by more than 50% (Figure 10B). In contrast, usage of the ICOS transmembrane domain led to an increased expression of the chimeric TCR when compared to the parental hTCR:ICOS-RA. These data show that the expression of hTCR:ICOS-RA is strictly dependent on the selection of transmembrane domains of specific co-stimulatory receptors, such as CD28 or ICOS, but not necessarily all co-stimulatory receptors. Chimeric TCRs show expression in human T cells when harboring intracellular domains of ICOS, CD28, CD40L, OX40 or 4-1BB Next, we tested whether chimeric TCRs are expressed in primary human T cells when alternative intracellular domains are used, to which end we exchanged the ICOS intracellular domain from hTCR:ICOS-RA for the intracellular domains of either CD28, 4-1BB, CD40L or OX40, while retaining the R54A mutation in CD3ε (Figure 11A, TCR Nos 6-10 in Table 3). We observed that all these chimeric TCRs were expressed at a similar level to hTCR:ICOS-RA (Figure 11B). Importantly, T cells transduced with these alternative co-stimulatory TCRs showed antigen-specific IFN-γ production upon co-culture with tumor cells (Figure 11C). The above findings suggest that the expression of chimeric TCRs is not strictly regulated by the inclusion of intracellular domains of specific co-stimulatory receptors and accepts, at least according to our data, those of ICOS, CD28, 4-1BB, OX40 or CD40L. Expression of chimeric TCR variants in human T cells depends on mutating hCD3ε R54 Following the inclusion of alternative transmembrane and intracellular domains in the chimeric TCR format, we tested whether the expression of these TCRs in primary human T cells depends on the mutation of the arginine at position 54 in the cytosolic tail of hCD3^. To this end, we compared surface expressions of TCRs comprising alternative transmembrane and intracellular domains with and without CD3ε R54A (Figure 12A and 12B, TCRs Nos 11-18 in Table 3). Our results show that while chimeric TCRs that harbored CD3ε R54A with either CD28 or ICOS transmembrane domain showed robust surface expression, the variants without R54A (i.e., harboring wt CD3ε) showed little to negligible expression in primary human T cells (Figure 12C). In line with these findings, leaving out the R54A mutation drastically reduced surface expression of TCRs harboring intracellular domains of ICOS, 4-1BB, OX40 or CD40L when compared to the same TCRs that do harbor the mutation (Figure 12D). It is noteworthy that the exception to this rule was constituted by the chimeric TCR harboring the intracellular domain of CD28, where the usage of wt CD3ε sequence was not detrimental to the surface expression. In short, mutating the arginine at position 54 in CD3ε is critical for surface expression of chimeric TCRs, except for TCRs harboring the CD28 intracellular domain. Expression of hTCR:ICOS is enabled when deleting hCD3ε R54 or exchanging it for any other amino acid After having demonstrated that the expression of chimeric TCRs in human T cells critically depends on R54A in hCD3^ (see also Example 1, Figures 9D,E), we tested whether deletion or mutation of R54 into any other single amino acid would have the same positive effect on the surface expression of hTCR:ICOS. Along this line, we designed a panel of ROPN1 hTCR:ICOS variants, each harboring a different single point mutation (covering all natural amino acids) or a deletion at position 54 of hCD3ε (Figure 13A, TCRs Nos 19-39 of Table 3). We found that replacing the arginine by another positively charged amino acid (i.e., histidine or lysine), an aromatic amino acid (i.e., tryptophan), or a polar uncharged amino acid (i.e., asparagine) decreased the surface expression compared to hTCR:ICOS-RA. Notably, however, the incorporation of any other amino acids showed equal or improved surface expression compared to hTCR:ICOS-RA. Also, deletion of arginine at position 54 retained the surface expression as was observed with hTCR:ICOS-RA (Figure 13B). A panel of six of these hTCR:ICOS variants (R54C, R54G, R54I, R54L R54M, R54V, R54del) was also tested for functionality by co- culturing transduced T cells with a cell line overexpressing the ROPN1 antigen (Figure 13C). These TCR-transduced T cells showed antigen-specific IFN-γ production, implying functionality of hTCR:ICOS when alternative substitutions or deletions to R54A are applied. Collectively, these results show that hTCR:ICOS is functionally expressed by human T cells as long as the arginine at position 54 of hCD3ε is substituted by aspartic acid, glutamic acid, glycine, alanine, valine, leucine, isoleucine, methionine, proline, serine, threonine, cysteine, tyrosine or phenylalanine, or when R54 is deleted. Expression of hTCR:ICOS is further enabled by mutating R53, or by mutating or deleting both R53 and R54 Given the critical role of arginine at position 54 in hCD3^ regarding the functional expression of hTCR:ICOS, we set out to evaluate the role of the arginine at position 53 towards the surface expression of this TCR. The rationale for this study comes from the recognition that this arginine is part of the endoplasmic reticulum retention (ERR) signal of CD3ε (Mallabiabarrena et al., 1994. EMBO J 14: 2257-2268). To enable the evaluation of R53, we designed and tested new hTCR:ICOS variants in which we mutated the arginine at the respective position into an alanine (Figure 14 A, TCRs Nos 40-50 in Table 3). We observed that TCRs harboring R53A brought the expression to a level comparable to TCRs that harbored CD3ε R54A, again providing a considerably improved surface expression compared to those that contain wt CD3ε (Figure 14B). Moreover, when testing multiple antigen specificities, the R53A variants of gp100 or ROPN1-specific TCR:ICOS showed increased surface expressions (28 and 46 %, respectively), whereas the R53A variant of NYESO1 TCR:ICOS showed a similar surface expression when compared to TCR:ICOS-R54A (82 %). Interestingly, mutating both R53 and R54 appeared to have an additive effect on the surface expression of gp100 and ROPN1 but not NY-ESO1 TCR:ICOS. Furthermore, deleting both R53 and R54 appeared to have the strongest effect on the surface expression of gp100 and ROPN1 but again not NY-ESO1 TCR:ICOS (Figure 14B). In short, the above results show that deleting or mutating arginine at position 53 and / or 54 of CD3ε significantly improves the surface expression of hTCR:ICOS for multiple antigen specificities. Conclusion In this second Example, we have made and assessed 50 different human chimeric TCRs to define the rules governing surface expression of hTCR:ICOS-RA (as the lead product from Example 1). Specifically, expression of chimeric TCRs show a narrow acceptance of transmembrane domains of either CD28 or ICOS, whereas it shows a broad acceptance of intracellular domains such as ICOS, CD28, 4-1BB, OX40 or CD40L. In addition, the arginine at position 54 in the cytosolic CD3^ domain critically enables surface expression of chimeric TCRs, an effect that was maintained when replacing the arginine by all possible single amino acids, although glutamine, histidine, tryptophan, lysine and asparagine were not as efficient as alanine. Moreover, deletions or mutations of the arginines at position 53 and / or 54 are able to yield significant surface expression of hTCR:ICOS with different antigen specificities in human T cells. In total, our results demonstrate that expression of chimeric TCRs is governed by (1) differential stringencies regarding transmembrane or intracellular domains of different co-stimulatory receptors as well as (2) mutations in the cytosolic tail of CD3^ that encompass critical arginine residues at positions 53 and / or 54.These findings, besides elucidating the determinants of optimal expression of human chimeric TCRs, and hTCR:ICOS-RA in particular, are providing a molecular basis for the generation of next-generation TCRs for adoptive therapy to treat patients with tumors, auto- immunity or infections.

Claims

Claims 1. A chimeric T-cell receptor (TCR) comprising: - an extracellular human or humanized TCRα chain and an extracellular human or humanized TCR^ chain, wherein said TCRα chain and said TCR^ chain form an extracellular antigen binding domain that binds to an antigen; and wherein each of said chains further comprises, in an N-terminal to C- terminal order: - a human transmembrane domain from a co-receptor selected from CD8^, CD8^ or CD4, or a co-stimulatory receptor selected from CD28, ICOS, CD27, 2B4 or NKG2D; - a first human intracellular domain from a co-stimulatory receptor selected from ICOS, CD28, 4-1BB, OX40, CD27, CD40L, 2B4 or NKG2D; and - a human intracellular CD3ε domain, wherein in at least one of the CD3ε domains, an arginine (R) amino acid residue at position 53, 54, or both positions 53 and 54 of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted.

2. The chimeric TCR according to claim 1, wherein the transmembrane domain comprises a transmembrane domain from CD28 or ICOS.

3. The chimeric TCR according to claim 1 or claim 2, wherein the transmembrane domain is a transmembrane domain from CD28 or ICOS.

4. The chimeric TCR according to any one of claims 1-3, wherein the first intracellular domain from a co-stimulatory receptor comprises an intracellular domain from a co-stimulatory receptor selected from ICOS, CD28, 4-1BB, OX40, or CD40L, 2B4.

5. The chimeric TCR according to any one of claims 1-4, wherein the first intracellular domain from a co-stimulatory receptor is an intracellular domain from a co-stimulatory receptor selected from ICOS, CD28, 4-1BB, OX40, or CD40L.

6. The chimeric TCR according to any one of claims 1-5, wherein the TCRα chain, the TCR^ chain, or both comprise a further intracellular domain from a co- stimulatory receptor, either N-terminal or C-terminal of the original intracellular domain, wherein the further intracellular domain preferably comprises an intracellular domain from a co-stimulatory receptor selected from ICOS, CD28, 4- 1BB, OX40, CD27, CD40L, 2B4 or NKG2D.

7. The chimeric TCR according to claim 6, wherein the further intracellular domain is an intracellular domain from a co-stimulatory receptor selected from ICOS, CD28, 4-1BB, OX40, CD27, CD40L, 2B4 or NKG2D.

8. The chimeric TCR according to any one of claims 1-7, wherein in both CD3ε domains an arginine (R) amino acid residue at position 53, 54 or both 53 and 54 of SEQ ID NO:25, or an arginine (R) amino acid residue at a position that corresponds to said arginine (R) amino acid residue at position 53 and / or 54 of SEQ ID NO:25, is substituted or deleted.

9. The chimeric TCR according to any one of claims 1-8, wherein the substitution of the arginine amino acid residue at position 53 and / or 54 of SEQ ID NO:25 is a substitution into another amino acid, preferably an aspartic acid, a glutamic acid, a proline, a serine, a threonine, a cysteine, a tyrosine, a phenylalanine, an alanine, a valine, a leucine, an isoleucine, a methionine, or a glycine.

10. The chimeric TCR according to any one of claims 1-9, wherein the transmembrane domain of the TCRα chain and / or the TCR^ chain comprises the human transmembrane domain from the co-stimulatory receptor CD28.

11. The chimeric TCR according to any one of claims 1-10, wherein the transmembrane domain of the TCRα chain and / or the TCR^ chain is the human transmembrane domain from the co-stimulatory receptor CD28.

12. The chimeric TCR according to any one of claims 1-11, wherein the first intracellular domain of the TCRα chain and / or the TCR^ chain comprises the intracellular domain from the co-stimulatory receptor ICOS.

13. The chimeric TCR according to any one of claims 1-12, wherein the first intracellular domain of the TCRα chain and / or the TCR^ chain is the intracellular domain from the co-stimulatory receptor ICOS.

14. The chimeric TCR according to any one of claims 1-13, wherein the transmembrane domains of the TCRα chain and the TCR^ chain are the transmembrane domains from the co-stimulatory receptor CD28, wherein the first intracellular domains of the TCRα chain and the TCR^ chain are the intracellular domains from the co-stimulatory receptor ICOS, and wherein the arginine (R) amino acid residue at position 54 of SEQ ID NO:25 of both human intracellular CD3ε domains is substituted or deleted.

15. The chimeric TCR according to any one of claims 1-14, wherein the variable region of the TCR α chain and the variable region of the TCR- ^ chain are fused into a single-chain TCR constituting an extracellular antigen binding domain.

16. The chimeric TCR according to any one of claims 1-15, wherein the functional expression of said TCR is enabled by substitution or deletion of at least one arginine (R) amino acid residue at position 53, 54, or both 53 and 54 of SEQ ID NO:25 of both human intracellular CD3ε domains.

17. The chimeric TCR according to any one of claims 1-16, wherein the TCRα chain and the TCR^ chain constitute an extracellular antigen binding domain that specifically binds an epitope from a cancer antigen, preferably an epitope selected from a human cancer germline antigen, such as ROPN1, MAGE-A4, MAGE-A6,MAGE-A9, MAGE-C2 or NY-ESO1, a mutated antigen, such as a KRAS, PIK3CA or TMPRSS2-ERG mutant and / or a cancer neoantigen, including a tumor frameshift antigen.

18. The chimeric TCR according to any one of claims 1-17, wherein the TCRα chain and the TCRβ chain constitute an extracellular binding domain that specifically binds an epitope from a self-antigen of a patient, such as MOG, GAD65, TSHR, tTG, and fibrinogen, or an antigen of a pathogen, preferably an antigen of a virus, such as an antigen from EBV, CMV and HIV.

19. A T-cell, preferably a cytotoxic T lymphocyte (CTL), a CD4 helper cell, a γδ T- cell, a tumor-infiltrating lymphocyte (TIL), a regulatory T-cell, a memory T-cell or a Natural Killer T (NKT) cell, or a Natural Killer (NK) cell, expressing a chimeric TCR according to any one of claims 1-18.

20. A pharmaceutical composition, comprising the T-cell according to claim 19 or an in vivo gene-delivery system encoding a chimeric TCR according to any one of claims 1-18, and a pharmaceutical acceptable carrier.

21. A method of producing a T-cell according to claim 19, the method comprising: (a) isolating a T-cell from a human individual; (b) modifying the T-cell with a construct encoding the chimeric TCR according to any one of claims 1-18; and (c) enabling expression of the chimeric TCR in the T-cell.

22. A method of treating a patient having a tumor, preferably a solid tumor, the method comprising: (a) isolating a T-cell from the patient; (b) providing the T-cell with the chimeric TCR according to any one of claims 1-18, said chimeric TCR comprising an extracellular antigen binding domain that binds to a cancer antigen of the patient; (c) enabling expression of the chimeric TCR in the T-cell, thereby generating a modified T-cell product; and(d) administering the modified T-cell product to the patient.

23. A method of treating a patient having an immune-related disease, preferably an autoimmune disease, the method comprising: (a) isolating a T-cell from the patient; (b) providing the T-cell with the chimeric TCR according to any one of claims 1-16 or 18, said chimeric TCR comprising an extracellular antigen binding domain that binds to an antigen of the patient; (c) enabling expression of the chimeric TCR in the T-cell, thereby generating a modified T-cell product; and (d) administering the modified T-cell product to the patient.

24. A method of treating a patient having an infection by a pathogen, preferably a virus, the method comprising: (a) isolating a T-cell from the patient; (b) providing the T-cell with the chimeric TCR according to any one of claims 1-16 or 18, said chimeric TCR comprising an extracellular antigen binding domain that binds to an antigen of the pathogen; (c) enabling expression of the chimeric TCR in the T-cell, thereby generating a modified T-cell product; and (d) administering the modified T-cell product to the patient.

25. A method of treating a patient having a tumor, preferably a solid tumor, an immune-related disease, preferably an auto-immune disease, or an infection by a pathogen, preferably a virus, the method comprising: (a) providing a T-cell with the chimeric TCR according to any one of claims 1- 18, said chimeric TCR comprising an extracellular antigen binding domain that binds to a cancer antigen of the patient, to an antigen of the patient or to an antigen of a pathogen; (b) enabling expression of the chimeric TCR in the T-cell, thereby generating a modified T-cell product; and (c) administering the modified T-cell product to the patient.

26. A method of treating a patient having a tumor, preferably a solid tumor, comprising: (a) providing an in vivo gene-delivery system encoding a chimeric TCR according to any one of claims 1-17, whereby said chimeric TCR comprises an extracellular antigen binding domain that binds to a cancer antigen of the patient, and (b) providing said in vivo gene-delivery system encoding the chimeric TCR to the patient; whereby the gene delivery system preferably is a viral vector or nanoparticle, enabling the expression of the chimeric TCR by the patient’s T- cells.

27. A method of treating a patient having an immune-related disease, preferably an auto-immune disease, comprising: (a) providing an in vivo gene-delivery system encoding a chimeric TCR according to any one of claims 1-16 or 18, said chimeric TCR comprising an extracellular antigen binding domain that binds to an antigen of the patient; and (b) providing said in vivo gene-delivery system encoding the chimeric TCR to the patient; whereby the gene delivery system preferably is a viral vector or nanoparticle, enabling the expression of the chimeric TCR by the patient’s T- cells.

28. A method of treating a patient having an infection by a pathogen, preferably a virus, comprising: (a) providing an in vivo gene-delivery system encoding a chimeric TCR according to any one of claims 1-16 or 18, said chimeric TCR comprising an extracellular antigen binding domain that binds to an antigen of the pathogen; and (b) providing said in vivo gene-delivery system encoding the chimeric TCR to the patient; whereby the gene delivery system preferably is a viral vector or nanoparticle, enabling the expression of the chimeric TCR by the patient’s T- cells.

29. The method of treating a patient according claim 22, 25 or 26, the method comprising the administration of the pharmaceutical composition of claim 20, optionally in combination with surgery, radiation therapy and / or one or more anti- cancer drugs or a combination thereof.

30. The method of treating a patient according to claim 22, the method comprising the administration of the pharmaceutical composition of claim 20, optionally in combination with an immunosuppressant.

31. The method of treating a patient according to claim 23, the method comprising the administration of the pharmaceutical composition of claim 20, optionally in combination with an antibiotic, including an antifungal and / or antiviral agent.

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