WO2024100136A1

WO2024100136A1 - Novel method for obtaining gamma t-cell (or delta t-cell) receptor chains (or gamma delta t-cell receptors) or fragments thereof that mediate an anti-tumour or an anti-infective response

Info

Publication number: WO2024100136A1
Application number: PCT/EP2023/081185
Authority: WO
Inventors: Laurens SAND; Thijs VAN MONTFORT
Original assignee: Gadeta B.V.
Priority date: 2022-11-08
Filing date: 2023-11-08
Publication date: 2024-05-16

Abstract

A novel method for obtaining yT-cell (or delta T-cell) receptor chains (or yδTCRs) or fragments thereof that mediate an anti-tumour or an anti-infective response, and novel yT-cell receptor chains, δT-cell receptor chains, yδTCRs, or fragments thereof, mediating anti-tumour responses or anti-infective responses are provided.

Description

Novel method for obtaining gamma T-cell (or delta T-cell) receptor chains (or gamma delta T-cell receptors) or fragments thereof that mediate an anti-tumour or an anti-infective response

Field of the invention

The present invention relates to a method for identifying yT-cell receptor chains, 5T-cell receptor chains, ydT-cell receptors, or fragments thereof, that mediate an anti-tumour or anti-infective response. It further relates to yT-cell receptor chains, 5T-cell receptor chains, y5T-cell receptors, fragments thereof, and cells comprising or expressing them. The invention is useful for anti-tumour and anti-infective therapeutics.

Background of the invention

Our immune system utilizes different lines of defence to protect us from infections as well as cancer. In order to cover the magnitude of potential invaders and internal threats our adaptive immune system has the possibility to raise up to 10¹⁶ aPTCR combinations as well as 10¹¹ variations in immunoglobulins (Chien YH, et al, 2014. Annu.Rev.lmmunol.).

Among all immune receptor chains, TCR5s have even the highest potential diversity in the CDR3 loop (approximately 10¹⁶ combinations for murine TCR5) owing to the presence of multiple D gene segments (two in mice, three in human, and up to five in cattle) that can join together. Each D gene segment can be read in all three open reading frames, and N nucleotides can be inserted into the junctions of the joining segments. Thus, despite the limited diversity at the VJ junctions of TCR y-chains, the potential diversity generated at the combined CDR3 junctions (approximately 10¹⁸ combinations) is still higher than that of aPTCRs (~10¹⁶) and immunoglobulins (~10¹¹). (Chien YH et al, 2014. Annu Rev. Immunol.). TCR5 and TCRy chains may be particularly useful for immunotherapeutics against cancer and infections, particularly for the development of improved treatments. Accordingly, there is still a need for new yT-cell receptor chains, 5T-cell receptor chains, and yST-cell receptors, that mediate an anti-tumour response. There is still a need for yT-cell receptor chains, 5T-cell receptor chains, and yST-cell receptors, that will mediate an anti- infective response. There is still a need for improved treatments utilizing yT-cell receptor chains, 5T-cell receptor chains, and yST-cell receptors.

Brief description of the figures

Fig. 1A-1C. Amino acid position numbering in a Cy1 constant region (Fig. 1A; SEQ ID NO: 1 ), a Cy2 (Fig. 1 B; SEQ ID NO: 41) constant region, and a C5 constant region (Fig. 1C; SEQ ID NO: 2).

Fig. 2A-2C. TCR expression in apT-cells transduced with wild-type (E57; SEQ ID NO: 96, 99, Fig. 2A, 2C) or mutant ySTCRs comprising a single transmembrane domain mutation in either the TRGC1 (Fig. 2A, 2C; T136L (SEQ ID NO: 3), L143E (SEQ ID NO: 4), L143D (SEQ ID NO: 5), F153A (SEQ ID NO: 8), C158F (SEQ ID NO: 9), or C159F (SEQ ID NO: 10)) or TRDC regions (Fig. 2B (wild-type control is displayed in Fig. 2A); T128L (SEQ ID NO: 11 ), G131V (SEQ ID NO: 12), T139V (SEQ ID NO: 13), or F144L (SEQ ID NO: 14)). apT-cells transduced with lentivirus encoding the wild-type E57 y5TCR or yBTCRs comprising a single mutation in eitherthe TRGC1 or TRDC regions (MOI = 3) were produced as TEGs and were analyzed with FACS flow cytometry. Expression is displayed as density plot and the percentage of yBTCR expression displayed in each quadrant was determined on untransduced apT-cells. Comparison of wild-type and single transmembrane mutants are donor matched. Fig. 3A-3C. TCR expression in a T-cells transduced with wild-type (E57; SEQ ID NO: 96, 99) or mutant ySTCRs comprising two or more transmembrane domain mutations in the TRGC1 and TRDC regions. apT- cells transduced with lentivirus (MOI = 3) encoding the wild-type E57 y6-TCR or ySTCRs comprising two or more mutations in either the TRGC1 or TRDC regions were produced as TEGs and were analyzed with FACS flow cytometry. y5-TCR expression is displayed as density plot and the percentage of ap/yS-TCR expression displayed in each quadrant is shown. The expression profile of 2, 3, 4, or 5 transmembrane mutations are displayed as density plots. All comparisons of the wildtype and transmembrane mutants are donor matched.

Fig. 4A-4D. Competition of the endogenous a TCR with the exogenous ybTCRs for the CD3 complex. aPT- cells transduced with lentivirus (MOI = 3) encoding the wild-type ybTCR (E57; SEQ ID NO: 96, 99) or mutant ySTCRs with single (Fig. 4A), double (Fig. 4B), triple (Fig. 4C) or more (Fig. 4D) mutations in the TRGC1 and/or TRDC regions were produced as TEGs and were analyzed with FACS flow cytometry. The percentage of single positive ySTCR-expressing cells over double positive y5-a TCR-expressing cells from the transduced cell population was determined.

Fig. 5A-5B. Cytolysis of cell lines recognized by the wildtype E57 TCR (SEQ ID NO: 96, 99) and by TEGs expressing the mutant ySTCRs comprising a single transmembrane domain mutation in either the TRGC1 (T136L (SEQ ID NO: 3), F153A (SEQ ID NO: 8), C158F (SEQ ID NO: 9), or C159F (SEQ ID NO: 10)) or TRDC regions (T128L (SEQ ID NO: 11), G131 V (SEQ ID NO: 12), T139V (SEQ ID NO: 13), or F144L (SEQ ID NO: 14)). TEGs were co-cultured for 72 hours with either HT-29 (Fig. 5A) or RKO (Fig. 5B) cells. The percentage of cytolysis of two independent experiments is shown as normalized data +/- SEM.

Fig. 6A-6D. Cytolysis (Fig. 6A-6B) and IFN release (Fig. 6C-6D) by TEGs targeting tumour cell lines recognized by the wildtype E57 TCR (SEQ ID NO: 96, 99) and by TEGs expressing the mutant ySTCRs comprising transmembrane domain mutations in the TRGC1 and TRDC regions. TEGs expressing the wildtype E57 TCR or a mutant ySTCRs were co-cultured for 72 hours with two different Luc-tom transduced colorectal tumour cell lines. The percentage of cytolysis of HT-29 (Fig. 6A) or RKO (Fig. 6B) cells from two independent experiments is shown and data is presented as normalized data +/- SEM. The percentage of release of IFN in the supernatant from TEGs expressing mutant ySTCRs relative to the wild-type E57 TCR after co-culture with HT-29 (Fig. 6C) or RKO (Fig. 6D) cells is shown as normalized data from two experiments +/- SEM.

Fig. 7A-7B. Expression profile of TEGs expressing wild-type clone 4 (SEQ ID NO: 97, 100) or clone 5 ySTCRs (SEQ ID NO: 98, 101) or mutant ySTCRs comprising transmembrane domain mutations in the TRGC1 and TRDC regions. apT-cells transduced with lentivirus (MOI = 3 in the case of clone 4 and mutants thereof; MOI = 5 in the case of clone 5 and mutants thereof) were analyzed with FACS flow cytometry. Expression is displayed as density plot and the percentage of ySTCR expression displayed in each quadrant was determined on untransduced apT-cells. Comparison of expression of wild-type and mutant y5TCRs are donor matched (Fig. 7A). The percentage of single positive ySTCR-expressing cells over double positive yS-apTCR-expressing cells from the transduced cell population was determined. (Fig. 7B).

Fig. 8A-8D. Cytolysis (Fig. 8A-8B) and IFN release (Fig. 8C-8D) by TEGs expressing the mutant y5TCRs comprising transmembrane domain mutations in the TRGC1 and TRDC regions targeting tumour cell lines recognized by the wild-type clone 4 (SEQ ID NO: 97, 100) and clone 5 y5TCRs (SEQ ID NO: 98, 101). TEGs expressing the wild-type clone 4 or 5 ySTCR or a mutant y5TCR were co-cultured for 72 hours with target Luc-Tom transduced MDA-MB-231 tumour cells. The percentage of tumour cytolysis and accumulated IFNy in co-culture supernatant for TEGs expressing the ySTC from clone 4 (Fig. 8A and Fig. 8C) or mutants and TEGs expressing the ySTCR from clone 5 (Fig. 8B and Fig. 8D) or mutants was determined. For clone 5, TEG input was normalized based on percentage of TEG expressing the ySTCR or number of viral integration based on eGFP expression. Data was normalized and plotted in bars +/-SEM.

Fig. 9A-9B. TOR expression by afT-cells transduced with wild-type (SEQ ID NO: 96, 99) or FG-loop mutant E57 with or without modification in the con . a T cells from two donors were transduced with lentivirus (MOI = 3) containing the wild-type E57 y6-TCR with mutated FG-loop (yFG, SEQ ID NO: 22 or PFG, SEQ ID NO: 23) or mutated FG-loop and a-loop (yFG conP; SEQ ID NO: 24, PFG conP; SEQ ID NO: 25) in the TRGC1 . The produced TEGs were subjected to FACS flow cytometry and analyzed for the expression of aPTCR and ySTCR. Expression is displayed as density plot and the percentage of y5-TCR expression displayed in each quadrant was determined on untransduced ap-T cells (Fig. 9A). Comparison of wildtype and mutant are donor matched. The percentage of single positive ySTCR-expressing cells over double positive y5- aPTCR-expressing cells from the transduced cell population was determined. (Fig. 9B).

Fig. 10A-10C. TCR expression by aPT-cells transduced with wild-type clone 4 (SEQ ID NO: 97, 100) (MOI = 3), clone 5 (SEQ ID NO: 98, 101) (MOI = 5), E57 (SEQ ID NO: 96, 99) (MOI = 3) or E57 G5D6 (SEQ ID NO: 120, 121) (MOI = 3) or FG-loop mutants with or without modification in the Conp. MOI between wildtype and mutants for each particular clone was the same. aPT cells from two donors were transduced with lentivirus containing with different y5-TCRs with mutated FG-loop (yFG, SEQ ID NO: 22), a-loop (conp, SEQ NO ID: 21), or mutated FG-loop and a-loop (yFG conP; SEQ ID NO: 24) in the TRGC1 . The produced TEGs were subjected to FACS flow cytometry and analyzed for the expression of aPTCR and, y5TCR. Expression is displayed as density plot and the percentage of yS-TCR expression displayed in each quadrant was determined on untransduced apT-cells (Fig. 10A-10B, representative donor shown). The percentage of single positive expressing yST-cells over yS-apT cells from the transduced cell population (expressing the y5-TCR) was determined for all FG mutants (Fig. 10C). Comparison of wildtype and FG mutant y5-TCRs are donor matched.

Fig. 11A-11B. Cytolysis and IFN release by TEGs expressing y5TCR E57 (SEQ ID NO: 96, 99), mutant yFG (SEQ ID NO: 22), PFG (SEQ ID NO: 23), yFG conp (SEQ ID NO: 24), or PFG conp (SEQ ID NO: 25) against two colorectal tumour cell lines. apT cells transduced with lentivirus containing the E57 y5-TCR with FG-loop mutated TRGC1 were produced as TEGs and were co-cultured for 72 hours with target Luc-Tom transduced HT-29 and RKO tumour cells. The percentage of tumour cytolysis and accumulated IFNy in coculture supernatant targeting HT-29 (Fig. 11 A) or RKO (Fig. 11 B) cells was determined. Data was normalized and plotted in bars +/-SEM of indicated experiments.

Fig. 12A-12B. IFN release by TEGs expressing clone 5 y5-TCR (SEQ ID NO: 98, 101) or clone 5 y5-TCR with mutant yFG conp (SEQ ID NO: 24) against two tumour cell lines. apT cells transduced with lentivirus containing the y5-TCR with FG-mutated TRGC1 were produced as TEGs and were co-cultured for 72 hours with target Luc-Tom transduced MDA-MB-231 and MZ1851 RC tumour cells. The percentage of accumulated IFNy in co-culture supernatant targeting MZ1851RC (Fig. 12A), and MDA-MB-231 (Fig. 12B) was determined. Data was normalized and plotted in bars +/-SEM of independent experiments. Fig 13. TCR expression by apT-cells transduced with wild-type E7 (SEQ ID NO: 96, 99) or mutant E57, having mutations in the constant y-TCR region (SEQ ID NO: 26 (K49A K50A), 27 (K96D N97K), 28 (E79D), 29 (E79A K80A), 30 (K80R), 31 (S81 K), or 32 (K96A N97A)). a T-cells were transduced with lentivirus (MOI = 3) containing wild-type and indicated mutations in the TRGC1 that are within loop structures that are in close proximity with CD3 subunits. The produced TEGs were subjected to FACS flow cytometry and analyzed for the expression of apTCR and, ybTCR. Expression is displayed as density plot and the percentage of yb-TCR expression displayed in each quadrant was determined on untransduced apT-cells. Comparison of wildtype and mutant y5-TCRs are donor matched.

Fig 14A-14B. IFN release by apT-cells expressing E57 (SEQ ID NO: 96, 99) or E57 with mutations in the TRGC1 (SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, or 95) against two colorectal tumour cell lines. aPT cells transduced with lentivirus containing the E57 y5-TCR with mutations in the constant TRGC1 were produced as TEGs and were co-cultured for 72 hours with target Luc-Tom transduced HT-29 and RKO tumour cells. The percentage of accumulated IFNy in co-culture supernatant targeting HT-29 (Fig. 14A) or RKO (Fig. 14B) was determined. Data was normalized and plotted in bars +/-SEM of independent experiments.

Fig 15A-15B. TCR expression by apT-cells transduced with wild-type E57 (SEQ ID NO: 96, 99), cl5 (SEQ ID NO: 102, 103), or mutant E57 or cl5 ySTCRs having mutations in the constant y- and/or 5-TCR region (N66S; SEQ ID NO: 35, N14S; SEQ ID NO: 36). apT-cells were transduced with lentivirus (MOI = 3) for E57 containing wild-type and indicated mutations in the TRGC1 and/or mutations in the TRDC that disrupt glycosylation of a potential N-linked glycosylation site. The number of integration sites for cl5 with or without mutations ranged between 3.5 and 3.9. The produced TEGs were subjected to FACS flow cytometry and analyzed for the expression of apTCR and, ySTCR. Expression is displayed as density plot and the percentage of y5-TCR expression displayed in each quadrant was determined on untransduced apT-cells (Fig. 15A). Comparison of wildtype and mutant y5-TCRs are donor matched. The percentage of single positive y5TCR-expressing cells over double positive yS-apTCR-expressing cells from the transduced cell population was determined (Fig. 15B).

Fig 16. TCR expression by apT-cells transduced with wild-type cl5 ySTCR (SEQ ID NO: 102, 103), or mutant cl5 having mutations in the constant y- (Q58C; SEQ ID NO: 37) and 8-TCR region (V48C; SEQ ID NO: 38). apT cells were transduced with lentivirus (MOI = 4.5) containing wild-type and indicated mutations in the TRGC1 and mutations in the TRDC that allow cysteine bond formation between the constant regions. The produced TEGs were subjected to FACS flow cytometry and analyzed for the expression of apTCR and, ySTCR. Expression is displayed as density plot and the percentage of y5-TCR expression displayed in each quadrant was determined on untransduced apT-cells. Comparison of wildtype and mutant yS-TCRs are donor matched.

Fig 17. Cytolysis by apT-cells expressing cl5 y5TCR (SEQ ID NO: 102, 103) or mutant cl5 with mutations in the TRGC1 and TRDC regions (Q58C; SEQ ID NO: 37), (V48C; SEQ ID NO: 38) against two tumour cell lines. apT-cells transduced with lentivirus containing the cl5 y5-TCR with mutated TRGC1 and TRDC were produced as TEGs and were co-cultured for 72 hours with target HT-29 and MZ1851 RC tumour cells. The percentage of tumour cytolysis (measured with the xCELLigence assay) of TEGs in co-culture with HT-29 and MZ1851 RC was determined. Data was normalized and plotted in bars +/-SEM of independent experiments. Fig. 18. Cytolysis by apT-cells expressing G115 y5TCR (SEQ ID NO: 167, 168) or mutant G115 with mutations in the TRGC1 and TRDC regions (C158F/F144L (SEQ ID NO: 9/ SEQ ID NO: 14), S149A/T139V (SEQ ID NO: 6/ SEQ ID NO: 13), S149A-C158F/T139V (SEQ ID NO: 16/ SEQ ID NO: 13), S149A/T128L- T139V (SEQ ID NO: 6/ SEQ ID NO: 18), S149A-F153A/T128L-T139V (SEQ ID NO: 175/SEQ ID NO: 18), S149A/T139V-F144L (SEQ ID NO: 6/SEQ ID NO: 19), S149A-V151 L/T139V-F144L (SEQ ID NO: 15/ SEQ ID NO: 19), S149A/T128L-T139V-F144L (SEQ ID NO: 6/SEQ ID NO: 20), S149A-V151 L/T128L-T139V- F144L (SEQ ID NO: 15/SEQ ID NO: 20), S149A-C158F/T139V-F144L (SEQ ID NO: 16/SEQ ID NO: 19), and S149A-V151 L-C158F/T139V-F144L (SEQ ID NO: 17/SEQ ID NO: 19)) against Luc-Tom HT-29 cells. aPT-cells transduced with lentiviruses containing the reference or mutated G115 yQ-TCRs were produced as TEGs and were co-cultured for 48 hours at an 1 :1 E:T ratio with target Luc-Tom HT-29 cells in the presence of 10 pM pamidronate (PAM). Cytolysis was measured via luciferase assay. Data was normalized and plotted in bars +/-SEM of independent (triplicate) experiments.

Summary of the invention

In a first aspect, there is provided a method for obtaining a yT-cell receptor chain, a 5T-cell receptor chain, a y5T-cell receptor, or a fragment thereof that mediates an anti-tumour or anti-infective response, said method comprising the steps of:

A) providing a yT -cell receptor chain or fragment thereof, comprising a Cy constant region or a fragment thereof, and a 5T-cell receptor chain or fragment thereof, comprising a C5 constant region or a fragment thereof;

B) introducing an amino acid modification in said Cy constant region or fragment thereof and/or in said C5 constant region or fragment thereof;

C) expressing the yT-cell receptor chain or fragment thereof and the 5T-cell receptor chain or fragment thereof obtained in step B) in an immunoresponsive cell;

D) determining the anti-tumour or anti-infective response of the engineered immunoresponsive cell of step C);

E) obtaining the yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, or fragment thereof that mediates the anti-tumour or anti-infective response, preferably wherein the amino acid modification is selected from an amino acid substitution, deletion, insertion, or a combination thereof.

In some embodiments, the immunoresponsive cell is selected from a T-cell, an a(3T-cell, a y5T-cell, CD4+ T-cell, CD8+ T-cell, an induced pluripotent stem cell derived T-cell, a T-effector cell, a lymphocyte, a B-cell, an NK-cell, an NKT-cell, a myeloid cell, a monocyte, a macrophage, or a neutrophil.

In some embodiments, in step B) at least two, at least three, at least four, or at least five amino acid modifications are introduced in the Cy constant region or fragment thereof and/or in the 05 constant region or fragment thereof.

In some embodiments, in step A) the Cy constant region or fragment thereof comprises or consists of an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1, and/or the C5 constant region or fragment thereof comprises or consists of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, the amino acid modification in step B) is selected from:

B1) an amino acid modification in a position corresponding to a position selected from D1 to V8 of SEQ ID NO: 1 or SEQ ID NO: 39;

B2) an amino acid modification in a position corresponding to a position selected from K49 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39; B3) an amino acid modification in a position corresponding to a position selected from T136 to L161 of SEQ ID NO: 1 or SEQ ID NO: 39;

B4) an amino acid modification in a position corresponding to a position selected from N14 to V48 of SEQ ID NO: 2;

B5) an amino acid modification in a position corresponding to a position selected from L127 to F152 of SEQ ID NO: 2, or;

B6) a combination of any one of B1 ) to B5).

In some embodiments, the amino acid modification in B3), B5), or B6) is an amino acid substitution of a polar or an uncharged amino acid by a hydrophobic amino acid, preferably is a substitution of:

- a S by a G, A, V, L, I, P, F, M, or W;

- a V by a G, A, L, I, P, F, M, or W;

- a F by a G, A, V, L, I, P, M, or W;

- a C by a G, A, V, L, I, P, F, M, or W;

- a T by a G, A, V, L, I, P, F, M, or W, or;

- a combination thereof.

In some embodiments, the amino acid modification in B2), B3), B4), B5), or B6) is an amino acid substitution selected from: i. an amino acid substitution in a position corresponding to position K49 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine; ii. an amino acid substitution in a position corresponding to position K50 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine; ill. an amino acid substitution in a position corresponding to position Q58 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a cysteine; iv. an amino acid substitution in a position corresponding to position N66 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a serine; v. an amino acid substitution in a position corresponding to position K96 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine or an aspartic acid; vi. an amino acid substitution in a position corresponding to position N97 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine or a lysine; vii. an amino acid substitution in a position corresponding to position T136 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a leucine; viii. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine; lx. an amino acid substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a leucine; x. an amino acid substitution in a position corresponding to position F153 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine; xi. an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a phenylalanine; xii. an amino acid substitution in a position corresponding to position C159 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a phenylalanine; xiii. an amino acid substitution in a position corresponding to position K49 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position K50 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine; xiv. an amino acid substitution in a position corresponding to position E79 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position K80 of SEQ ID NO: 1, preferably a substitution by an alanine; xv. an amino acid substitution in a position corresponding to position K96 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine or an aspartic acid, and an amino acid substitution in a position corresponding to position N97 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine or a lysine; xvi. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a leucine; xvii. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a phenylalanine; xviii. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position F153 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine; xix. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, an amino acid substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a leucine, and an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a phenylalanine; xx. an amino acid substitution in a position corresponding to position K49 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, an amino acid substitution in a position corresponding to position K50 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, an amino acid substitution in a position corresponding to position K96 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an aspartic acid, and an amino acid substitution in a position corresponding to position N97 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a lysine; xxi. an amino acid substitution in a position corresponding to position N14 of SEQ ID NO: 2, preferably a substitution by a serine; xxii. an amino acid substitution in a position corresponding to position V48 of SEQ ID NO: 2, preferably a substitution by a cysteine; xxiii. an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, preferably a substitution by a leucine; xxiv. an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, preferably a substitution by a valine; xxv. an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, preferably a substitution by a leucine; xxvi. an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, preferably a substitution by a leucine, and an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, preferably a substitution by a valine; xxvii. an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, preferably a substitution by a valine, and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, preferably a substitution by a leucine; xxviii. an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, preferably a substitution by a leucine, an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, preferably a substitution by a valine, and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, preferably a substitution by a leucine, or; xxix. a combination of any one of i) to xxviii).

In some embodiments, the amino acid modification in B1), B2), or B6) is an amino acid substitution selected from: a1) a substitution of the positions corresponding to positions from D1 to V8 of SEQ ID NO: 1 or SEQ ID NO: 39 by EDLKNVF (SEQ ID NO: 43), DKEDLKNVF (SEQ ID NO: 176), or a variant thereof, preferably by EDLKNVF (SEQ ID NO: 43) or a variant thereof; b1) a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39 by VQFYGLSENDEWTQDRAKPVTQIV (SEQ ID NO: 47), VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50), or a variant thereof, or; c1) a combination of a1) and b1).

In a second aspect, there is provided a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof, wherein said Cy constant region or fragment thereof comprises: a) an amino acid modification in a position corresponding to a position selected from D1 to V8 of SEQ ID NO: 1 or SEQ ID NO: 39; b) an amino acid modification in a position corresponding to a position selected from K49 to Q101 of SEQ ID NO : 1 or SEQ ID NO: 39; c) an amino acid modification in a position corresponding to a position selected from T136 to L161 of SEQ ID NO: 1 or SEQ ID NO: 39, or; d) a combination of any one of a) to c).

In some embodiments, the amino acid modification is an amino acid substitution, and the Cy constant region or fragment thereof comprises:

I. an amino acid substitution in a position corresponding to position K49 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine;

II. an amino acid substitution in a position corresponding to position K50 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine;

III. an amino acid substitution in a position corresponding to position Q58 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a cysteine;

IV. an amino acid substitution in a position corresponding to position N66 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a serine;

V. an amino acid substitution in a position corresponding to position K96 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine or an aspartic acid;

VI. an amino acid substitution in a position corresponding to position N97 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine or a lysine;

VII. an amino acid substitution in a position corresponding to position T136 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a leucine;

VIII. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine;

IX. an amino acid substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a leucine;

X. an amino acid substitution in a position corresponding to position F153 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine; XI. an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a phenylalanine;

XII. an amino acid substitution in a position corresponding to position C159 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a phenylalanine;

XIII. an amino acid substitution in a position corresponding to position K49 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position K50 of SEQ ID NO: 1, preferably a substitution by an alanine;

XIV. an amino acid substitution in a position corresponding to position E79 of SEQ ID NO: 1, preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position K80 of SEQ ID NO: 1 , preferably a substitution by an alanine;

XV. an amino acid substitution in a position corresponding to position K96 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine or an aspartic acid, and an amino acid substitution in a position corresponding to position N97 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine or a lysine;

XVI. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a leucine;

XVII. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a phenylalanine;

XVIII. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position F153 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine;

XIX. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, an amino acid substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a leucine, and an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a phenylalanine;

XX. an amino acid substitution in a position corresponding to position K49 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, an amino acid substitution in a position corresponding to position K50 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, an amino acid substitution in a position corresponding to position K96 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an aspartic acid, and an amino acid substitution in a position corresponding to position N97 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a lysine;

XXI. a substitution of the positions corresponding to positions from D1 to V8 of SEQ ID NO: 1 or SEQ ID NO: 39 by EDLKNVF (SEQ ID NO: 43), DKEDLKNVF (SEQ ID NO: 176), or a variant thereof, preferably by EDLKNVF (SEQ ID NO: 43) or a variant thereof;

XXII. a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39 by VQFYGLSENDEWTQDRAKPVTQIV (SEQ ID NO: 47), VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50), or a variant thereof, or;

XXIII. a combination of any one of I. to XXII.

In some embodiments, the Cy constant region or fragment thereof comprises at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or similarity with SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably with SEQ ID NO: 1 . In some embodiments, the Cy constant region or a fragment thereof comprises or consists of an amino acid sequence represented by SEQ ID NO: 3, 6, 7, 8, 9, 10, 15, 16, 17, 21 , 22, 23 , 24, 25, 26, 27, 29, 32, 35, 37, 95, or 177.

In some embodiments, the yT-cell receptor chain or fragment thereof further comprises a variable region or fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 108, 109, 110, 111, 112, 113, 122, or 150.

In some embodiments, the yT-cell receptor chain or fragment thereof further comprises a CDR3 region comprising, consisting essentially of, or consisting of an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 124, 125, 126, 127, 128, 129, 130, or 152.

In some embodiments, the yT-cell receptor chain or fragment thereof further comprises a CDR3 region comprising: an amino acid modification, preferably an amino acid substitution, at an amino acid position corresponding to position 4 of SEQ ID NO: 124, an amino acid modification, preferably an amino acid substitution, more preferably a substitution by a glutamic acid, at an amino acid position corresponding to position 5 of SEQ ID NO: 124; an amino acid modification, preferably an amino acid substitution, more preferably a substitution by an alanine, at an amino acid position corresponding to position 6 of SEQ ID NO: 124; an amino acid modification, preferably an amino acid substitution, more preferably a substitution by an alanine, serine, or tyrosine, at an amino acid position corresponding to position 7 of SEQ ID NO: 124; an amino acid modification, preferably an amino acid substitution, more preferably a substitution by a phenylalanine, at an amino acid position corresponding to position 8 of SEQ ID NO: 124; an amino acid modification, preferably an amino acid substitution, at an amino acid position corresponding to position 9 of SEQ ID NO: 124; an amino acid modification, preferably an amino acid substitution, at an amino acid position corresponding to position 10 of SEQ ID NO: 124, or; any combination thereof.

In some embodiments, the yT-cell receptor chain or fragment thereof further comprises a CDR3 region comprising a modification at an amino acid position corresponding to a position selected from one or more of positions 4-10 of SEQ ID NO: 124, said modification selected from WDAFYYK, WEAFYYK, WDGYFYK, WDGYYYK, WDGAYYK, or WDGSYYK.

In some embodiments, the yT-cell receptor chain or fragment thereof further comprises a CDR1 region comprising, consisting essentially of, or consisting of amino acid sequence SEQ ID NO: 154, 155, 156, or 157, or of an amino acid sequence comprising one, two, or three amino acid modifications relative to SEQ ID NO: 154, 155, 156, or 157, and a CDR2 region comprising, consisting essentially of, or consisting of amino acid sequence SEQ ID NO: 158, 159, 160, or 161, or of an amino acid sequence comprising one, two, or three amino acid modifications relative to SEQ ID NO: 158, 159, 160, or 161.

In some embodiments, the yT-cell receptor chain or fragment thereof is soluble, and preferably comprises a T-cell- and/or NK-cell binding domain, more preferably a CD3-binding domain.

In some embodiments, the yT-cell receptor chain or fragment thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, or 265.

In a third aspect, there is provided a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof, wherein the C5 constant region or fragment thereof comprises: e) an amino acid modification in a position corresponding to a position selected from N14 to V48 of SEQ ID NO: 2; f) an amino acid modification in a position corresponding to a position selected from L127 to L152 of SEQ ID NO: 2, or; g) a combination of e) and f).

In some embodiments, the amino acid modification is an amino acid substitution, and the C6 constant region or fragment thereof comprises:

XXIV. an amino acid substitution in a position corresponding to position N14 of SEQ ID NO: 2, preferably a substitution by a serine;

XXV. an amino acid substitution in a position corresponding to position V48 of SEQ ID NO: 2, preferably a substitution by a cysteine;

XXVI. an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, preferably a substitution by a leucine;

XXVII. an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, preferably a substitution by a valine;

XXVIll.an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, preferably a substitution by a leucine;

XXIX. an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, preferably a substitution by a leucine, and an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, preferably a substitution by a valine;

XXX. an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, preferably a substitution by a valine, and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, preferably a substitution by a leucine;

XXXI. an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, preferably a substitution by a leucine, an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, preferably a substitution by a valine, and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, preferably a substitution by a leucine, or;

XXXII. a combination of any one of XXIV. to XXXI.

In some embodiments, the C5 constant region or fragment thereof comprises at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or similarity with SEQ ID NO: 2.

In some embodiments, the C5 constant region or fragment thereof comprises or consists of an amino acid sequence represented by SEQ ID NO: 11, 13, 14, 18, 19, 20, 36, or 38.

In some embodiments, the 5T-cell receptor chain or fragment thereof further comprises a variable region or fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 114, 115, 116, 117, 118, 119, 123, or 151. In some embodiments, the 6T-cell receptor chain or fragment thereof further comprises a CDR3 region comprising, consisting essentially of, or consisting of an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 131, 132, 133, 134, 135, 136, 137, or 153.

In some embodiments, the 5T-cell receptor chain or fragment thereof further comprises a CDR3 region comprising:

- an amino acid modification, preferably an amino acid substitution, more preferably a substitution by a leucine, at an amino acid position corresponding to position 7 of SEQ ID NO: 131 ;

- an amino acid modification, preferably an amino acid substitution, more preferably a substitution by a lysine, at an amino acid position corresponding to position 8 of SEQ ID NO: 131;

- an amino acid modification, preferably an amino acid substitution, at an amino acid position corresponding to position 9 of SEQ ID NO: 131 ;

- an amino acid modification, preferably an amino acid substitution, more preferably a substitution by a phenylalanine, at an amino acid position corresponding to position 10 of SEQ ID NO: 131;

- an amino acid modification, preferably an amino acid substitution, at an amino acid position corresponding to position 11 of SEQ ID NO: 131 ;

- an amino acid modification, preferably an amino acid substitution, at an amino acid position corresponding to position 12 of SEQ ID NO: 131 , or;

- any combination thereof.

In some embodiments, the 5T-cell receptor chain or fragment thereof further comprises a CDR3 region comprising a modification at an amino acid position corresponding to a position selected from one or more of positions 7-12 of SEQ ID NO: 131 , said modification selected from IRGFTG, IKGYTG , IKGFTG, LRGFTG, LKGFTG, or LKGYTG.

In some embodiments, the 5T-cell receptor chain or fragment thereof further comprises a CDR1 region comprising, consisting essentially of, or consisting of amino acid sequence SEQ ID NO: 162, 163, or 164, or of an amino acid sequence comprising one, two, or three amino acid modifications relative to SEQ ID NO: 162, 163, or 164, and a CDR2 region comprising, consisting essentially of, or consisting of amino acid sequence EKD, QGS, or SEQ ID NO: 166, or of an amino acid sequence comprising one, two, or three amino acid modifications relative to amino acid sequence EKD, QGS, or SEQ ID NO: 166.

In some embodiments, the 5T-cell receptor chain or fragment thereof is soluble, and preferably comprises a T-cell- and/or NK-cell binding domain, more preferably a CD3-binding domain.

In some embodiments, the 5T cell receptor chain or fragment thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281 , 282, 283, 284, 285, 286, 287, 288, or 289.

In a fourth aspect, there is provided a yST-cell receptor or a fragment thereof, wherein said receptor or fragment thereof comprises:

-a yT-cell receptor chain or fragment thereof of the second aspect;

-a 5T-cell receptor chain or fragment thereof of the third aspect;

-a yT-cell receptor chain or fragment thereof of the second aspect and a 5T-cell receptor chain or fragment thereof of the third aspect. In a fifth aspect, there is provided a chimeric antigen-recognition receptor comprising:

-a Cy constant region of a YT-cell receptor chain or a fragment thereof of the second aspect;

-a C5 constant region of a 5T-cell receptor chain or a fragment thereof of the third aspect;

-a Cy constant region of a yT-cell receptor chain or a fragment thereof of the second aspect and a C5 constant region of a 5T-cell receptor chain or fragment thereof of the third aspect, preferably wherein the chimeric antigen-recognition receptor further comprises an antigen-binding domain from or derived from an antibody, an antibody variable region, an scFv, a single domain antibody, a Fab, a Fab', a F b^, a dimer or a trimer of Fab conjugates, an Fv, a minibody, a diabody, a triabody, a tetrabody, an affibody, an ankyrin protein, an ankyrin repeat, a DARPin, a monobody, a nanobody, an avimer, an adnectin, an anticalin, a fynomer, a Kunitz domain, a knottin, a P-hairpin mimetic, or a combination thereof.

In a sixth aspect, there is provided a nucleic acid molecule encoding a yT-cell receptor chain or a fragment thereof of the second aspect, a 5T-cell receptor chain or a fragment thereof of the third aspect, a Y6T-cell receptor or fragment thereof of the fourth aspect, or a chimeric antigen-recognition receptor of the fifth aspect.

In a seventh aspect, there is provided a cell, preferably an engineered cell, expressing the YT-cell receptor chain or fragment thereof of the second aspect, the 5T-cell receptor chain or fragment thereof of the third aspect, the yST-cell receptor or fragment thereof of the fourth aspect, or the chimeric antigen-recognition receptor of the fifth aspect, or comprising the nucleic acid molecule of the sixth aspect, preferably wherein the cell is selected from a T-cell, an iPSC-derived T-cell, an aPT-cell, a Y^T-cell, or an NK cell, more preferably from a Y6T-cell or aPT-cell, most preferably is an aPT-cell.

In an eighth aspect, there is provided a composition, preferably a pharmaceutical composition, comprising a YT-cell receptor chain or a fragment thereof of the second aspect, a 5T-cell receptor chain or a fragment thereof of the third aspect, a yST-cell receptor or fragment thereof of the fourth aspect, a chimeric antigenrecognition receptor of the fifth aspect, a nucleic acid molecule of the sixth aspect, or a cell of the seventh aspect.

In a ninth aspect, there is provided a YT-cell receptor chain or a fragment thereof of the second aspect, a 5T-cell receptor chain or a fragment thereof of the third aspect, a Y3T-cell receptor or fragment thereof of the fourth aspect, a chimeric antigen-recognition receptor of the fifth aspect, a nucleic acid molecule of the sixth aspect, a cell of the seventh aspect, or a composition of the eighth aspect, for use as a medicament, preferably for use in the treatment, regression, curing, and/or delaying of cancer or an infection.

Description of the invention

Method for identifying vT-cell receptor chains, 5T-cell receptor chains, v5T-cell receptors, or fragments thereof

In the context of the disclosure, the term "fragment” of a YT-cell chain or 5T-cell receptor chain or Y6T-cell receptor may be replaced by the term "part”, the two terms being interchangeable. As used herein, the term "T-cell receptor” may be abbreviated as "TCR”. The term "YT-cell receptor chain” (or YCDR3 region) may be alternatively referred to as "gamma T-cell receptor chain” (or gamma CDR3 region) or "gT-cell receptor chain” (or gCDR3 region). The term ”5T-cell receptor chain” (or 5CDR3 region) may be alternatively referred to as "delta T-cell receptor chain” (or delta CDR3 region) or "dT-cell receptor chain” (or dCDR3 region).

A fragment (or part) of a polypeptide may correspond to at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% of the length of a polypeptide, for example as represented by an amino acid sequence with a specific SEQ ID NO, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the length of the polypeptide. A fragment of a polypeptide may correspond to an extracellular domain of a polypeptide, such as a yT-cell receptor chain, a 5T-cell receptor chain, or a y5T-cell receptor, or a fragment of the extracellular domain, as discussed later herein. A fragment of a polypeptide preferably comprises a constant region of a yT-cell receptor chain, a 5T-cell receptor chain, a yST-cell receptor, or a fragment thereof. A fragment of a polypeptide may correspond to a variable region and a fragment of a constant region of a yT-cell receptor chain, a 5T-cell receptor chain, or a yQT-cell receptor. A fragment of a polypeptide may correspond to a fragment of a variable region and a fragment of a constant region of a yT-cell receptor chain, a 5T-cell receptor chain, or a y5T-cell receptor. A fragment of a polypeptide preferably comprises a CDR3 region of a yT-cell receptor chain, a 5T-cell receptor chain, or a yST-cell receptor or a fragment thereof. A fragment of a polypeptide is preferably a functional fragment or part thereof. Functional in this context means that the fragment preferably exhibits a similar biological activity as the polypeptide it is derived from (corresponding original polypeptide). In the context of the disclosure, a biological activity may for example be the mediation of an anti-tumour or an anti-infective response as explained later herein. A similar anti-tumour or anti-infective response may mean that the fragment of the polypeptide mediates at least 50% of the anti-tumour or anti- infective response, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 110%, or at least 120%, or more, as compared to the polypeptide it is derived from. In preferred embodiments, a fragment of a yT-cell receptor chain, a 5T-cell receptor chain, or a yST-cell receptor described herein is a polypeptide comprising, respectively, a yCDR3 region, a 5CDR3 region, or a yCDR3 and a 5CDR3 region as described later herein and, respectively, a Cy constant region or fragment thereof, a C6 constant region or fragment thereof, or a Cy and a C5 constant region or fragment thereof as described later herein. Preferably, the fragment of the yT-cell receptor chain, 5T-cell receptor chain, or y5T-cell receptor further comprises, respectively, a yCDR1 and a yCDR2 region, a 5CDR1 and a 5CDR2 region, or a yCDR1 , a yCDR2, a 5CDR1, and a 5CDR2 region as described later herein.

A "constant” region with respect to a yT-cell receptor or a 5T-cell receptor chain (or fragment thereof) as used herein corresponds to its general definition in the art. ySTCRs are cell-surface multimeric transmembrane complexes formed by two y and 5 glycoproteins, non-covalently linked to up to six different CD3 subunits. Each of the y and 6 chains of the y5 heterodimer are composed of a variable region, which protrudes into the extracellular milieu and is responsible for the cognate antigen-binding, and by a constant region, which anchors the receptor to the cell membrane and is involved in interactions with CD3 molecules and signal transduction. The modular structure of the y and 5 TCR chains is the result of both somatic rearrangements and splicing of variable (V), diversity (D), joining (J), and constant (C) region-encoding genes, each representing a multigene sub-family, which occurs during the development of T lymphocytes in the thymus. First, the variable region is assembled by the somatic rearrangements of V and J genes in TRG (y chain) loci, and between V, D, and J genes in TRD (5 chain) loci. This event, together with N- insertions between recombined V(D)J, determines the clonal variability of the TCR repertoire. After transcription, the resulting rearranged V-(D)-J region is spliced to the C gene, which encodes the constant region of the receptor. The constant regions are generally conserved. A Cy constant region may be a Cy1 or a Cy2 constant region. A Cy2 constant region as used herein refers to a region encoded by a TRGC2 gene or variant thereof, preferably of human origin. Such a gene and region are known to the skilled person, for example see Uniprot Ref: P03986, SEQ ID NO: 40 and SEQ ID NO: 41 provided herein. A Cy1 constant region as used herein refers to a region encoded by a TRGC1 gene or variant thereof, preferably of human origin. Such a gene and region are known to the skilled person, for example see Uniprot Ref: P0CF51 , SEQ ID NO: 1, and SEQ ID NO: 39 provided herein. A C5 constant region as used herein refers to a region encoded by a TRDC gene or variant thereof, preferably of human origin. Such a gene and region are known to the skilled person, for example see Uniprot Ref: B7Z8K6 and SEQ ID NO: 2 provided herein.

In the human genome, the TRD locus contains three variable genes (TRDV1-3), three diversity genes (TRDD1-3), four joining genes (TRDJ1-4) and one constant TRDC gene, which rearrange to encode a TCR5 chain. The TRG locus that rearranges to encode a TCRy chain contains 14 variable genes, of which only 6 are functional (TRGV2-5, TRGV8, and TRGV9), five joining genes (TRGJ1, J2, JP, JP1, JP2), and two constant genes (TRGC1, TRGC2, as discussed above). The two TRGC genes resulted, with their associated TRGJ genes, from a recent duplication in the locus (see Antonacci et al, 2020, Genes 1(6): 624, incorporated herein by reference in its entirety). However, there are structural differences: TRGJP1 , TRGJ1 , and TRGC1 cross-hybridize to TRGJP2, TRGJ, and TRGC2, respectively, whereas the TRGJP has no equivalent in the TRGJP2-J2-C2 cluster (see Lefranc M.-P et al, 1986, PNAS 83:9596-9600; Brenner MB, 1987, Nature 325: 689-694, both of which incorporated herein by reference in their entireties).

The TRGC genes encode the extracellular region of typically 110 amino acids (C-region), the connecting region (CO), the transmembrane region (TM), and the cytoplasmic region (CY). The TRGC1 gene comprises three exons and typically encodes a C-region of 173 AA (Cy1), whereas the TRGC2 gene comprises four or five exons, owing to the duplication or triplication of a region that includes Exon 2 (EX2, EX2T and/or EX2R) and typically encodes a C-region (Cy2) of 189 or 205 amino acids in length, respectively (see Le Franc M.-P, The T Cell Receptor FactsBook, 2001, Academic Press, MA, USA, incorporated herein by reference in its entirety), with 189 AA considered as the canonical length.

Accordingly, a TRGC2 (Cy2) constant region typically differs from a TRGC1 (Cy1) constant region by having 16-32 extra amino acids in the connecting peptide. Additionally, Exon 2 of the TRGC1 gene typically has a cysteine involved in the interchain disulfide bridge, whereas the cysteine is typically not conserved in Exon 2 of the human TRGC2 gene.

The frequency of y5TCR comprising Cy1 or Cy2 constant regions differs among the different y5T-cell subsets. For example, the constant gamma region of the Vy9V52 TCR expressed by the most abundant y5T lymphocytes in human adult blood is exclusively encoded by TRGC1 gene, while the non-Vy9V52 TCRs tend to express a Cy2 domain encoded by the TRGC2 gene (Casorati et al, 1989, JEM 170(5): 1521-35).

A yT-cell receptor chain or fragment thereof which comprises a Cy2 constant region or a fragment thereof can be distinguished from a chain or fragment thereof which comprises a Cy1 constant region or a fragment thereof based on the structural and sequence differences between Cy2 and Cy1 constant regions discussed above.

The present inventors have surprisingly found that improved yT-cell receptor chains, 5T-cell receptor chains, yST-cell receptors, or fragments thereof, may be obtained by introducing an amino acid modification in their Cy constant region (or fragment thereof) and/or C5 constant region (or fragment thereof). The resulting modified yT-cell receptor chains, 5T-cell receptor chains, y5T-cell receptors, or fragments thereof may mediate an improved anti-tumour or anti-infective response compared to the yT-cell receptor chains, 6T-cell receptor chains, y5T-cell receptors, or fragments thereof, that they originate from. The resulting modified yT-cell receptor chains, 5T-cell receptor chains, yST-cell receptors, or fragments thereof may demonstrate improved expression, preferably surface expression, compared to the yT-cell receptor chains, 5T-cell receptor chains, yST-cell receptors, or fragments thereof, that they originate from, in a cell, such as a T-cell, an aPT-cell, a yST-cell, CD4+ T-cell, CD8+ T-cell, an induced pluripotent stem cell derived T-cell, a T-effectorcell, a lymphocyte, a B-cell, an NK-cell, an NKT-cell, a myeloid cell, a monocyte, a macrophage, or a neutrophil. Such a cell may be an immunoresponsive cell. The inventors’ finding is surprising, as the previously available methods in the art concerning the improvement of yT-cell receptor chains, 5T-cell receptor chains, or yST-cell receptors focus on the variable regions. Modification of the constant regions has hitherto not been explored.

Accordingly, the aspects and embodiments provided herein solve at least some of the problems discussed herein.

Provided herein is a method for obtaining a yT-cell receptor chain or a fragment thereof, a 5T-cell receptor chain or a fragment thereof, or a yST-cell receptor or a fragment thereof, that mediates an anti-tumour or anti-infective response. A yT -cell receptor chain or fragment thereof is understood to comprise a y-constant (Cy) constant region or a fragment thereof. A 5T-cell receptor chain or fragment thereof is understood to comprise a 5-constant (C5) constant region or a fragment thereof. A yST-cell receptor or fragment thereof is understood to comprise a y-constant (Cy) constant region or a fragment thereof, and a 5-constant (C5) constant region or a fragment thereof. In some embodiments, the Cy constant region or fragment thereof is a Cy2 constant region or fragment thereof. In preferred embodiments, the Cy constant region or fragment thereof is a Cy1 constant region or fragment thereof.

A yT-cell receptor chain or fragment thereof is understood to comprise a yCDR3 region. A 5T-cell receptor chain or fragment thereof is understood to comprise a 5CDR3 region. A y5T-cell receptor or fragment thereof is understood to comprise a yCDR3 and a 5CDR3 region.

Accordingly, in an aspect, there is provided a method for obtaining a yT-cell receptor chain, a 5T-cell receptor chain, a y5T-cell receptor, or a fragment thereof that mediates an anti-tumour or anti-infective response, said method comprising the steps of:

E) obtaining the yT-cell receptor chain, 5T-cell receptor chain, y5T-cell receptor, or fragment thereof that mediates the anti-tumour or anti-infective response, preferably wherein the amino acid modification is selected from an amino acid substitution, deletion, insertion, or a combination thereof. Step A)

In step A), a yT-cell receptor chain or fragment thereof, comprising a Cy constant region or a fragment thereof, and a 5T-cell receptor chain or fragment thereof, comprising a C5 constant region or a fragment thereof, are provided. Preferably, the yT-cell receptor chain or fragment thereof and/or 6T-cell receptor chain or fragment thereof are of mammalian, more preferably of human origin. In some embodiments, the Cy constant region or fragment thereof is a Cy2 constant region or fragment thereof. In preferred embodiments, the Cy constant region or fragment thereof is a Cy1 constant region or fragment thereof.

In some embodiments, the provided Cy constant region or fragment thereof comprises or consists of an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 , and/or the provided C6 constant region or fragment thereof comprises or consists of an amino acid sequence represented by SEQ ID NO: 2.

A yT-cell receptor chain or fragment thereof may, for example, be a y2T-, y3T-, y4T-, y5T-, y8T-, y9T-, or y11T-cell receptor chain or fragment thereof. In some embodiments, the yT-cell receptor chain or fragment thereof is a y4T-cell receptor chain or fragment thereof. In some embodiments, the yT-cell receptor chain or fragment thereof is a y9T-cell receptor chain or fragment thereof. In some embodiments, the yT-cell receptor chain or fragment thereof is a y5T-cell receptor chain or fragment thereof. In some embodiments, the yT-cell receptor chain or fragment thereof is a y2T-cell receptor chain or fragment thereof. A 5T-cell receptor chain or fragment thereof may, for example, be a 51T-, 52T-, 53T-, or 55T-cell receptor chain or fragment thereof. In some embodiments, the 6T-cell receptor chain or fragment thereof is a 55T-cell receptor chain or fragment thereof. In some embodiments, the 5T-cell receptor chain or fragment thereof is a 51 T- cell receptor chain or fragment thereof. In some embodiments, the 5T-cell receptor chain or fragment thereof is a 52T-cell receptor chain or fragment thereof.

It is understood that "providing” in step A) is not restricted to the physical provision of an original yT-cell receptor chain or fragment thereof and an original 5T-cell receptor chain or fragment thereof. For example, one may begin the method with an in silica yT-cell receptor chain (or fragment thereof) sequence, to which the amino acid modification of step B) as described later herein is introduced using standard sequence editing software (e.g., CloneManager supplied by Sci-Ed, CO, USA), and the modified yT-cell receptor chain or fragment thereof may then be obtained (for example synthesized by a commercial supplier) and expressed in an immunoresponsive cell in step C). The amino acid modification may be introduced in the amino acid sequence of the yT-cell receptor chain or fragment thereof, or a respective modification in the nucleotide sequence encoding the amino acid sequence may be introduced. In such an embodiment, the original yT-cell receptor chain or fragment thereof is not physically provided, whereas the modified yT-cell receptor chain or fragment thereof is. This is also applicable to the provision of a 5T-cell receptor chain or fragment thereof.

It is further understood that "providing” in step A) also encompasses the provision of an immunoresponsive cell expressing the yT-cell receptor chain or fragment thereof and/or 5T-cell receptor chain or fragment thereof. In such embodiments, the immunoresponsive cell may already express one or both provided chains or fragments thereof, and the introduction of a modification in step B) as described later herein may be done directly in the cell using standard molecular toolbox techniques, for example as described in standard handbooks such as Sambrook and Green, Molecular Cloning. A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press (2012) and Ausubel et al., Current Protocols in Molecular Biology, 3rd edition, John Wiley & Sons Inc (2003), both of which are incorporated herein by reference in their entireties. In some embodiments, an immunoresponsive cell already expresses the provided yT-cell receptor chain or fragment thereof and expression of a modified ST-cell receptor chain or fragment thereof (as described later herein) is introduced to the cell. In some embodiments, an immunoresponsive cell already expresses the provided ST-cell receptor chain or fragment thereof and expression of a modified yT-cell receptor chain or fragment thereof (as described later herein) is introduced to the cell. It is further understood that expression of the provided yT-cell receptor chains, ST-cell receptor chains, or fragments thereof, may be introduced to cells that have not yet differentiated into an immunoresponsive cell, for example stem cells, progenitor cells, precursor cells, or IPSC cells, which can later be differentiated into immunoresponsive cells.

Step B)

In step B), an amino acid modification in the Cy constant region or fragment thereof and/or in the C5 constant region or fragment thereof is introduced. In some embodiments, an amino acid modification in the Cy constant region, preferably Cy1 constant region, or fragment thereof, is introduced. In some embodiments, an amino acid modification in the C5 constant region or fragment thereof is introduced. In some embodiments, an amino acid modification in the Cy constant region, preferably Cy1 constant region, or fragment thereof and the C5 constant region or fragment thereof is introduced.

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 , is introduced. In some embodiments, an amino acid modification in a C5 constant region or fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2 is introduced.

In some embodiments, at least two, at least three, at least four, or at least five amino acid modifications are introduced in the Cy constant region or fragment thereof and/or in the C5 constant region or fragment thereof. In some embodiments, at least two, at least three, at least four, or at least five amino acid modifications are introduced in the Cy constant region or fragment thereof. In some embodiments, at least two, at least three, at least four, or at least five amino acid modifications are introduced in the C5 constant region or fragment thereof.

In some embodiments, at least two amino acid modifications are introduced. In some embodiments, at least three amino acid modifications are introduced. In some embodiments, at least four amino acid modifications are introduced. In some embodiments, at least five amino acid modifications are introduced.

An "amino acid modification” (alternatively referred to herein as a mutation) as described herein refers to a modification resulting in an amino acid sequence being modified (altered). Accordingly, an amino acid modification is to be understood as also encompassing modifications to the nucleotide sequence which encodes an amino acid sequence to be modified, for example the modification of codons translating to amino acids corresponding to specific positions in an amino acid sequence. An amino acid modification is preferably selected from an amino acid substitution, deletion, insertion, or a combination thereof. In some embodiments, an amino acid modification is an amino acid substitution. In some embodiments, an amino acid modification is an amino acid insertion. In some embodiments, an amino acid modification is an amino acid deletion. In some embodiments, at least two, at least three, at least four, or at least five amino acid substitutions are introduced in the Cy constant region or fragment thereof and/or in the C6 constant region or fragment thereof. In some embodiments, at least two, at least three, at least four, or at least five amino acid insertions are introduced in the Cy constant region or fragment thereof and/or in the C5 constant region or fragment thereof. In some embodiments, at least two, at least three, at least four, or at least five amino acid deletions are introduced in the Cy constant region or fragment thereof and/or in the C5 constant region or fragment thereof.

An amino acid modification in a parent amino acid sequence results in a modified amino acid sequence (alternatively referred to herein as mutant or variant or derivative amino acid sequence). Accordingly, a yT- cell receptor chain, 5T-cell receptor chain, y5T-cell receptor, or a fragment thereof, comprising a modified amino acid sequence relative to a parent sequence may be called a variant or mutant or derivative yT-cell receptor chain, ST-cell receptor chain, ybT-cell receptor, or fragment thereof. A parent sequence may be an original sequence or a variant of an original sequence. A parent sequence may be a wild-type sequence. A parent sequence may be a sequence that is not available in nature. A definition of "wild-type” is provided later herein.

An amino acid substitution refers to a sequence modification that replaces an amino acid residue in a parent amino acid sequence by another amino acid (or a nucleotide in a nucleotide sequence comprised by a nucleic acid molecule encoding the amino acid sequence) which results in a modified (variant or mutant or derivative) sequence that has the same number of amino acids. An amino acid substitution may correspond to a substitution by any other amino acid. An amino acid substitution may correspond to a substitution of a polar amino acid by a hydrophobic amino acid. An amino acid substitution may correspond to a substitution of an uncharged amino acid by a hydrophobic amino acid. An amino acid substitution may correspond to a substitution of a S (serine), V (valine), F (phenylalanine), C (cysteine), or T (threonine) by a G (glycine), A (alanine), V (valine), L (leucine), I (isoleucine), P (proline), F (phenylalanine), M (methionine), or W (tryptophan). Preferably, in embodiments wherein a S, V, F, C, and/or T is substituted, a S is substituted by a G, A, V, L, I, P, F, M, or W, a V is substituted by a G, A, L, I, P, F, M, or W, a F is substituted by a G, A, V, L, I, P, M, or W, a C is substituted by a G, A, V, L, I, P, F, M, or W, and a T is substituted by a G, A, V, L, I, P, F, M, or W.

An amino acid substitution may correspond to a substitution of an L-amino acid by a D-amino acid. An amino acid substitution may correspond to a substitution by a non-natural amino acid. An amino acid substitution may be conservative. A definition of "conservative” amino acid substitutions is provided later herein.

In embodiments wherein multiple amino acids are substituted, the amino acids may correspond to consecutive positions in an amino acid sequence, to positions that are not consecutive, or to positions that are spatially apart in an amino acid sequence. The skilled person understands that amino acid modifications in the context of the disclosure may be combined, e.g., an amino acid sequence may comprise an amino acid substitution and an amino acid insertion and/or deletion relative to an amino acid sequence, for example relative to an amino acid sequence having a SEQ ID NO as described herein.

An amino acid modification may be introduced in a specific position in a Cy (such as Cy1 or Cy2) or 06 constant region (or fragments thereof). The location of a Cy1 or Cy2 constant region (or fragment thereof) in a yT-cell receptor chain (or fragment thereof) and of a C6 constant region (or fragment thereof) in a 6T- cell receptor chain (or fragment thereof) described herein may easily be identified as the constant regions of yT-cell receptor chains and 6T-cell receptor chains are generally conserved as described above.

For example, the location of a Cy1 constant region or fragment thereof in a yT-cell receptor chain or fragment thereof in question may be identified by aligning the amino acid sequence of the yT-cell receptor chain or fragment thereof with Uniprot Ref: P0Cf51 , SEQ ID NO: 1, SEQ ID NO: 39, or fragment thereof, using sequence alignment tools described later herein. The specific amino acid position to be modified in the yT-cell receptor chain or fragment thereof in question may then be identified based on its corresponding position in Uniprot Ref: P0Cf51, SEQ ID NO: 1 , SEQ ID NO: 39, or fragment thereof. A Cy1 constant region typically has a length of 173 amino acids. The first amino acid position of a Cy1 constant region is denoted by D1 (i.e., an aspartic acid in position 1) and the last amino acid position is denoted by S173 (i.e., a serine in last position 173), as also shown in Uniprot Ref: P0Cf51 , SEQ ID NO: 1 , and SEQ ID NO: 39.

Similarly, the location of a Cy2 constant region or fragment thereof in a yT-cell receptor chain or fragment thereof in question may be identified by aligning the amino acid sequence of the yT-cell receptor chain or fragment thereof with Uniprot Ref: P03986, SEQ ID NO: 40, SEQ ID NO: 41 , or fragment thereof, and the specific amino acid position to be modified may be identified based on its corresponding position in Uniprot Ref: P03986, SEQ ID NO: 40, SEQ ID NO: 41, or fragment thereof. A Cy2 constant region typically has a length of 189 amino acids. The first amino acid position of a Cy2 constant region with a length of 189 amino acids is denoted by D1 (i.e., an aspartic acid in position 1) and the last position is denoted by S189 (i.e., a serine in last position 189), as also shown in Uniprot Ref: P03986, SEQ ID NO: 40, and SEQ ID NO: 41 .

Similarly, the location of a C5 constant region or fragment thereof in a 5T-cell receptor chain or fragment thereof in question may be identified by aligning the amino acid sequence of the 5T-cell receptor chain or fragment thereof with Uniprot Ref: B7Z8K6 or SEQ ID NO: 2, or fragment thereof, and the specific amino acid position to be modified in the ST-cell receptor chain or fragment thereof in question may be identified based on its corresponding position in Uniprot Ref: B7Z8K6 or SEQ ID NO: 2. A C5 constant region typically has a length of 153 amino acids. The first amino acid position of a C5 constant region is denoted by S1 (i.e., a serine in position 1) and the last amino acid position is denoted by L153 (i.e., a leucine in last position 153), as also shown in Uniprot Ref: B7Z8K6 and SEQ ID NO: 2.

The skilled person utilizing this information may then modify an amino acid corresponding to any specific position of a Cy1 or Cy2 constant region (or fragment thereof) in a yT-cell receptor chain or fragment thereof, and/or of a C5 constant region (or fragment thereof) in a 5T-cell receptor chain or fragment thereof.

In some embodiments, an amino acid modification is introduced in the transmembrane domain of a Cy constant region, preferably a Cy1 or Cy2 region, more preferably a Cy1 region, or fragment thereof. The first amino acid position of the transmembrane domain of a Cy1 constant region is denoted by T136 (i.e., a threonine in position 136) and the last amino acid position is denoted by L161 (i.e., a leucine in position 161), as also shown in Uniprot Ref: P0Cf51 , SEQ ID NO: 1, and SEQ ID NO: 39. The first amino acid position of the transmembrane domain of a Cy2 constant region is denoted by T152 (i.e., a threonine in position 152) and the last amino acid position is denoted by L177 (i.e., a leucine in position 177), as also shown in Uniprot Ref: P03986, SEQ ID NO: 40, and SEQ ID NO: 41 . In some embodiments, an amino acid modification is introduced in the transmembrane domain of a C5 constant region or fragment thereof. The first amino acid position of the transmembrane domain of a C5 constant region is denoted by L127 (i.e., a leucine in position 127) and the last amino acid position is denoted by F152 (i.e., a phenylalanine in position 177), as also shown in Uniprot Ref: B7Z8K6 and SEQ ID NO: 2.

In some embodiments, an amino acid modification is introduced in the FG-loop of a Cy constant region, preferably a Cy1 or Cy2 region, more preferably a Cy1 region, or fragment thereof, and/or to an adjacent amino acid position. Modification of amino acid positions "adjacent” to an FG-loop in the context of the disclosure refers to the amino acids corresponding to up to the three positions immediately preceding and up to the three positions immediately following the FG-loop sequence. The first amino acid position of the FG-loop of a Cy1 constant region is denoted by E93 (i.e., a glutamic acid in position 93) and the last amino acid position is denoted by D100 (i.e., an aspartic acid in position 100), as also shown in Uniprot Ref: P0Cf51, SEQ ID NO: 1, and SEQ ID NO: 39 (ENNKNGVD (SEQ ID NO: 44)). The first amino acid position of the FG-loop of a Cy1 constant region including adjacent amino acids is denoted by V90 (i.e. , a valine in position 90) and the last amino acid position is denoted by Q101 (i.e., a glutamine in position 101), as also shown in Uniprot Ref: P0Cf51, SEQ ID NO: 1 , and SEQ ID NO: 39 (VRHENNKNGVDQ (SEQ ID NO: 48)). The first amino acid position of the FG-loop of a Cy2 constant region is denoted by E93 (i.e., a glutamic acid in position 93) and the last amino acid position is denoted by D100 (i.e., an aspartic acid in position 100), as also shown in Uniprot Ref: P03986, SEQ ID NO: 40, and SEQ ID NO: 41 (ENNKNGID (SEQ ID NO: 45)). The first amino acid position of the FG-loop of a Cy2 constant region including adjacent amino acids is denoted by V90 (i.e., a valine in position 90) and the last amino acid position is denoted by Q101 (i.e., a glutamine in position 101), as also shown in Uniprot Ref: P03986, SEQ ID NO: 40, and SEQ ID NO: 41 (VRHENNKNGIDQ (SEQ ID NO: 49)).

The three amino acids immediately preceding the first amino acid position of the FG loop correspond to the end of the anti-parallel F sheet of a yT-cell receptor chain. The three amino acids immediately after the last amino acid position of the FG loop correspond to the start of the anti-parallel G sheet of a yT-cell receptor chain.

In some embodiments, an amino acid modification is introduced in the a-loop of a Cy constant region, preferably a Cy1 or Cy2 region, more preferably a Cy1 region, or fragment thereof. The first amino acid position of the a-loop of a Cy1 and Cy2 region is denoted by D1 (i.e., an aspartic acid in position 1) and the last amino acid position is denoted by V8 (i.e., a valine in position 8), as also shown in Uniprot Ref: P0Cf51 , Uniprot Ref: P03986, SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41 (DKQLDADV (SEQ ID NO: 42)). In some embodiments, a preferred amino acid modification in the a-loop of a Cy constant region is an amino acid substitution of positions D1 to V8 by EDLKNVF (SEQ ID NO: 43) or by DKEDLKNVF (SEQ ID NO: 176), preferably is an amino acid substitution of positions D1 to V8 by EDLKNVF (SEQ ID NO: 43).

In some embodiments, an amino acid modification is introduced in a CD3-interacting position of a Cy constant region, preferably a Cy1 or Cy2 region, more preferably a Cy1 region, or fragment thereof. Preferred CD3-interacting positions of a Cy1 and a Cy2 constant region are K49, K50, E79, K80, S81, K96, and N97. In some embodiments, an amino acid modification is introduced in CD3-interacting position of a C5 constant region or fragment thereof.

In some embodiments, an amino acid modification is introduced in a glycosylation position of a Cy constant region, preferably a Cy1 or Cy2 region, more preferably a Cy1 region, or fragment thereof. In some embodiments, an amino acid modification is introduced in a glycosylation position of a C5 constant region. Such positions typically comprise an asparagine in a NxT/S motif in the amino acid sequence, to which an N-linked glycan is attached. A preferred glycosylation position of a Cy1 and a Cy2 constant region to be modified is N66. A preferred glycosylation position of a C5 constant region to be modified is N14.

In some embodiments, an amino acid in a position corresponding to a position selected from D1 to V8 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably of SEQ ID NO: 1 , is modified. In some embodiments, an amino acid in a position corresponding to a position selected from K49 to Q101 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably of SEQ ID NO: 1, is modified. Among positions corresponding to a position selected from K49 to Q101 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably of SEQ ID NO: 1 , positions corresponding to a position selected from K49 to E79 and from S81 to G98 are preferred.

In some embodiments, an amino acid in a position corresponding to a position selected from K49 to S81 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably of SEQ ID NO: 1 , is modified. In some embodiments, an amino acid in a position corresponding to a position selected from V90 to Q101 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably of SEQ ID NO: 1, is modified. In some embodiments, an amino acid in a position corresponding to a position selected from E93 to D100 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably of SEQ ID NO: 1 , is modified. In some embodiments, an amino acid in a position corresponding to a position selected from E93 to D100 of SEQ ID NO: 40 or SEQ ID NO: 41 is modified.

In some embodiments, an amino acid in a position corresponding to a position selected from T136 to L161 of SEQ ID NO: 1 orSEQ ID NO: 39, preferably of SEQ ID NO: 1 , is modified. Among positions corresponding to a position selected from T136 to L161 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably of SEQ ID NO: 1 , positions corresponding to a position selected from T136 to Y140 and from Y142 to L161 are preferred.

In some embodiments, an amino acid in a position corresponding to a position selected from T152 to L177 of SEQ ID NO: 40 or SEQ ID NO: 41, is modified. Among positions corresponding to a position selected from T152 to L177 of SEQ ID NO: 40 or SEQ ID NO: 41 , positions corresponding to a position selected from T152 to Y156 and from Y158 to L177 are preferred.

In some embodiments, an amino acid in a position corresponding to a position selected from N14 to V48 of SEQ ID NO: 2 is modified. In some embodiments, an amino acid in a position corresponding to a position selected from L127 to F152 of SEQ ID NO: 2 is modified.

Preferably, the amino acid modification is selected from an amino acid substitution, deletion, insertion, or a combination thereof, more preferably is an amino acid substitution. Combinations of the abovementioned amino acid modifications are also encompassed by the disclosure.

In some embodiments, an amino acid modification introduced in a position of a Cy constant region, preferably a Cy1 or Cy2 region, more preferably a Cy1 region, or fragment thereof, and/or in a position of a C5 constant region, or fragment thereof, is an amino acid substitution by a cysteine. Such a modification can promote formation of cysteine bonds (cysteine bridges), improving the stability of the T-cell receptor chains. Preferred substitutions are Q58C in the case of a Cy constant region, preferably a Cy1 or Cy2 region, more preferably a Cy1 region, or fragment thereof, and V48C in the case of a C6 constant region or fragment thereof.

It is understood that amino acid modifications in amino acid positions corresponding to different domains described herein can be combined. As a non-limiting example, an amino acid modification may be introduced in the transmembrane domain of a Cy and/or C5 constant region and an amino acid modification may be introduced in the FG-loop of a Cy constant region and/or to an adjacent amino acid position. As another non-limiting example, an amino acid modification may be introduced in the transmembrane domain of a Cy and/or C5 constant region and an amino acid modification may be introduced in the a-loop of a Cy constant region. As another non-limiting example, an amino acid modification may be introduced in the transmembrane domain of a Cy and/or C5 constant region and an amino acid modification may be introduced in a CD3-interacting position of a Cy and/or C5 constant region. As another non-limiting example, an amino acid modification may be introduced in the FG-loop of a Cy constant region and/or to an adjacent amino acid position and an amino acid modification may be introduced in the a-loop of a Cy constant region. As another non-limiting example, an amino acid modification may be introduced in the FG-loop of a Cy constant region and/or to an adjacent amino acid position and an amino acid modification may be introduced in a CD3-interacting position of a Cy and/or Cb constant region. As another non-limiting example, an amino acid modification may be introduced in the a-loop of a Cy constant region and an amino acid modification may be introduced in a CD3-interacting position of a Cy and/or Cb constant region. As another non-limiting example, an amino acid modification may be introduced in the transmembrane domain of a Cy and/or Cb constant region and an amino acid modification may be introduced in a glycosylation position of a Cy and/or Cb constant region. As another non-limiting example, an amino acid modification may be introduced in the FG-loop of a Cy constant region and/or to an adjacent amino acid position and an amino acid modification may be introduced in a glycosylation position of a Cy and/or Cb constant region. As another non-limiting example, an amino acid modification may be introduced in the a-loop of a Cy constant region and an amino acid modification may be introduced in a glycosylation position of a Cy and/or Cb constant region. As another non-limiting example, an amino acid modification may be introduced in a CD3-interacting position of a Cy and/or Cb constant region and an amino acid modification may be introduced in a glycosylation position of a Cy and/or Cb constant region.

As another non-limiting example, an amino acid modification may be introduced in the transmembrane domain of a Cy and/or Cb constant region, an amino acid modification may be introduced in the FG-loop of a Cy constant region and/or to an adjacent amino acid position, and an amino acid modification may be introduced in a CD3-interacting position of a Cy and/or Cb constant region. As another non-limiting example, an amino acid modification may be introduced in the transmembrane domain of a Cy and/or Cb constant region, an amino acid modification may be Introduced In the a-loop of a Cy constant region, and an amino acid modification may be introduced in a CD3-interacting position of a Cy and/or Cb constant region. As another non-limiting example, an amino acid modification may be introduced in the transmembrane domain of a Cy and/or Cb constant region, an amino acid modification may be introduced in the FG-loop of a Cy constant region and/or to an adjacent amino acid position, and an amino acid modification may be introduced in the a-loop of a Cy constant region. As another non-limiting example, an amino acid modification may be introduced in the FG-loop of a Cy constant region and/or to an adjacent amino acid position, an amino acid modification may be introduced in a CD3-interacting position of a Cy and/or Cb constant region, and an amino acid modification may be introduced in the a-loop of a Cy constant region.

As another non-limiting example, an amino acid modification may be introduced in the transmembrane domain of a Cy and/or Cb constant region, an amino acid modification may be introduced in the FG-loop of a Cy constant region and/or to an adjacent amino acid position, and an amino acid modification may be introduced in a glycosylation position of a Cy and/or Cb constant region. As another non-limiting example, an amino acid modification may be introduced in the transmembrane domain of a Cy and/or Cb constant region, an amino acid modification may be introduced in the a-loop of a Cy constant region, and an amino acid modification may be introduced in a glycosylation position of a Cy and/or Cb constant region. As another non-limiting example, an amino acid modification may be introduced in the transmembrane domain of a Cy and/or Cb constant region, an amino acid modification may be introduced in a CD3-interacting position of a Cy and/or Cb constant region, and an amino acid modification may be introduced in a glycosylation position of a Cy and/or Cb constant region. As another non-limiting example, an amino acid modification may be introduced in the FG-loop of a Cy constant region and/or to an adjacent amino acid position, an amino acid modification may be introduced in the a-loop of a Cy constant region, and an amino acid modification may be introduced in a glycosylation position of a Cy and/or Cb constant region. As another non-limiting example, an amino acid modification may be introduced in the FG-loop of a Cy constant region and/or to an adjacent amino acid position, an amino acid modification may be introduced in a CD3- interacting position of a Cy and/or Cb constant region, and an amino acid modification may be introduced in a glycosylation position of a Cy and/or Cb constant region. As another non-limiting example, an amino acid modification may be introduced in the a-loop of a Cy constant region, an amino acid modification may be introduced in a CD3-interacting position of a Cy and/or C5 constant region, and an amino acid modification may be introduced in a glycosylation position of a Cy and/or C5 constant region.

As another non-limiting example, an amino acid modification may be introduced in the transmembrane domain of a Cy and/or C5 constant region, an amino acid modification may be introduced in the FG-loop of a Cy constant region and/or to an adjacent amino acid position, an amino acid modification may be introduced in a CD3-interacting position of a Cy and/or C5 constant region, and an amino acid modification may be introduced in the a-loop of a Cy constant region. As another non-limiting example, an amino acid modification may be introduced in the transmembrane domain of a Cy and/or C6 constant region, an amino acid modification may be introduced in the a-loop of a Cy constant region, an amino acid modification may be introduced in a CD3-interacting position of a Cy and/or C5 constant region, and an amino acid modification may be introduced in a glycosylation position of a Cy and/or C5 constant region. As another non-limiting example, an amino acid modification may be introduced in the transmembrane domain of a Cy and/or C5 constant region, an amino acid modification may be introduced in the FG-loop of a Cy constant region and/or to an adjacent amino acid position, an amino acid modification may be introduced in a CD3- interacting position of a Cy and/or C5 constant region, and an amino acid modification may be introduced in a glycosylation position of a Cy and/or C5 constant region. As another non-limiting example, an amino acid modification may be introduced in the transmembrane domain of a Cy and/or C5 constant region, an amino acid modification may be introduced in the FG-loop of a Cy constant region and/or to an adjacent amino acid position, an amino acid modification may be introduced in the a-loop of a Cy constant region, and an amino acid modification may be introduced in a glycosylation position of a Cy and/or C5 constant region. As another non-limiting example, an amino acid modification may be introduced in the FG-loop of a Cy constant region and/or to an adjacent amino acid position, an amino acid modification may be introduced in a CD3-interacting position of a Cy and/or C5 constant region, an amino acid modification may be introduced in the a-loop of a Cy constant region, and an amino acid modification may be introduced in a glycosylation position of a Cy and/or 05 constant region.

As another non-limiting example, an amino acid modification may be introduced in the transmembrane domain of a Cy and/or C5 constant region, an amino acid modification may be introduced in the FG-loop of a Cy constant region and/or to an adjacent amino acid position, an amino acid modification may be introduced in a CD3-interacting position of a Cy and/or C5 constant region, an amino acid modification may be introduced in the a-loop of a Cy constant region, and an amino acid modification may be introduced in a glycosylation position of a Cy and/or 05 constant region.

An amino acid substitution by a cysteine as discussed herein may also be introduced in addition, or as an alternative to the non-limiting examples discussed herein.

In some embodiments, the amino acid modification in step B) is selected from:

B1) an amino acid modification in a position corresponding to a position selected from D1 to V8 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1;

B2) an amino acid modification in a position corresponding to a position selected from K49 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 ;

B3) an amino acid modification in a position corresponding to a position selected from T136 to L161 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 ;

B6) a combination of any one of B1 ) to B5).

In some embodiments, the amino acid modification in B3), B5, or B6) is an amino acid substitution of a polar or an uncharged amino acid by a hydrophobic amino acid, preferably is a substitution of an S (serine), V (valine), F (phenylalanine), C (cysteine), or T (threonine) by a G (glycine), A (alanine), V (valine), L (leucine), I (isoleucine), P (proline), F (phenylalanine), M (methionine), or W (tryptophan). Preferably, in embodiments wherein a S, V, F, C, and/or T is substituted, a S is substituted by a G, A, V, L, I, P, F, M, or W, a V is substituted by a G, A, L, I, P, F, M, or W, a F is substituted by a G, A, V, L, I, P, M, or W, a C is substituted by a G, A, V, L, I, P, F, M, or W, and a T is substituted by a G, A, V, L, I, P, F, M, or W. In some embodiments, a T (threonine) is substituted by a L (leucine). In some embodiments, a S (serine) is substituted by an A (alanine). In some embodiments, a V (valine) is substituted by a L (leucine). In some embodiments, a F (phenylalanine) is substituted by an A (alanine). In some embodiments, a C (cysteine) is substituted by a F (phenylalanine). In some embodiments, a T (threonine) is substituted by a V (valine). In some embodiments, a F (phenylalanine) is substituted by a L (leucine).

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position K49 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1. Preferably, the substitution is a K49A substitution (substitution by an alanine).

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position K50 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1. Preferably, the substitution is a K50A substitution (substitution by an alanine).

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position Q58 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 . Preferably, the substitution is a Q58C substitution (substitution by a cysteine).

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position N66 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1. Preferably, the substitution is a N66S substitution (substitution by an serine).

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position E79 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1. Preferably, the substitution is an E79A substitution (substitution by an alanine) or an E79D substitution (substitution by an aspartic acid).

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position K80 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1. Preferably, the substitution is a K80A substitution (substitution by an alanine) or a K80R substitution (substitution by an arginine).

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position S81 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1. Preferably, the substitution is a S81 K substitution (substitution by a lysine).

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position K96 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1. Preferably, the substitution is a K96A substitution (substitution by an alanine) or a K96D substitution (substitution by an aspartic acid).

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position N97 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1. Preferably, the substitution is a N97A substitution (substitution by an alanine) or a N97K substitution (substitution by a lysine).

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position T136 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1. Preferably, the substitution is a T136L substitution (substitution by a leucine).

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position T152 of SEQ ID NO: 40 or SEQ ID NO: 41. Preferably, the substitution is a T152L substitution (substitution by a leucine).

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1. Preferably, the substitution is a S149A substitution (substitution by an alanine).

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position S165A of SEQ ID NO: 40 or SEQ ID NO: 41. Preferably, the substitution is a S165A substitution (substitution by an alanine).

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1. Preferably, the substitution is a V151 L substitution (substitution by a leucine).

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position V167 of SEQ ID NO: 40 or SEQ ID NO: 41. Preferably, the substitution is a V167L substitution (substitution by a leucine).

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position F153 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1. Preferably, the substitution is a F153A substitution (substitution by an alanine).

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position F169 of SEQ ID NO: 40 or SEQ ID NO: 41. Preferably, the substitution is a F169A substitution (substitution by an alanine).

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1. Preferably, the substitution is a C158F substitution (substitution by a phenylalanine).

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position C174 of SEQ ID NO: 40 or SEQ ID NO: 41. Preferably, the substitution is a C174F substitution (substitution by a phenylalanine).

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position C159 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1. Preferably, the substitution is a C159F substitution (substitution by a phenylalanine).

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position C175 of SEQ ID NO: 40 or SEQ ID NO: 41. Preferably, the substitution is a C175F substitution (substitution by a phenylalanine).

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position K49 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 , and a substitution in a position corresponding to position K50 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1. Preferably, the substitutions are respectively a K49A and a K50A substitution.

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position E79 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , and a substitution in a position corresponding to position K80 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1. Preferably, the substitutions are respectively a E79A and a K80A substitution.

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position K96 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 , and a substitution in a position corresponding to position N97 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1. Preferably, the substitutions are respectively a K96A or K96D, preferably K96D, and an N97A or N97K, preferably N97K, substitution.

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , and a substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1. Preferably, the substitutions are respectively a S149A and a V151L substitution.

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position S165A of SEQ ID NO: 40 or SEQ ID NO: 41 , and a substitution in a position corresponding to position V167 of SEQ ID NO: 40 or SEQ ID NO: 41. Preferably, the substitutions are respectively a S165A and a V167L substitution.

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , and a substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1. Preferably, the substitutions are respectively a S149A and a C158F substitution.

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position S165A of SEQ ID NO: 40 or SEQ ID NO: 41 , and a substitution in a position corresponding to position C174 of SEQ ID NO: 40 or SEQ ID NO: 41. Preferably, the substitutions are respectively a S165A and a C174F substitution.

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, and a substitution in a position corresponding to position F153 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1. Preferably, the substitutions are respectively a S149A and a F153A substitution.

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position S165A of SEQ ID NO: 40 or SEQ ID NO: 41 , and a substitution in a position corresponding to position F169 of SEQ ID NO: 40 or SEQ ID NO: 41. Preferably, the substitutions are respectively a S165A and a F169A substitution.

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position K49, K50, K96, and N97 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1. Preferably, the substitutions are respectively a K49A, a K50A, a K96D, and an N97K substitution. In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position S149, V151 , and C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1. Preferably, the substitutions are respectively a S149A, a V151 L, and a C158F substitution.

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution in a position corresponding to position S165, V167, and C174 of SEQ ID NO: 40 or SEQ ID NO: 41. Preferably, the substitutions are respectively a S165A, a V167L, and a C174F substitution.

In some embodiments, an amino acid modification in a C6 constant region or fragment thereof is a substitution in a position corresponding to position N14 of SEQ ID NO: 2. Preferably, the substitution is a N14S substitution (substitution by a serine).

In some embodiments, an amino acid modification in a C6 constant region or fragment thereof is a substitution in a position corresponding to position V48 of SEQ ID NO: 2. Preferably, the substitution is a V48C substitution (substitution by a cysteine).

In some embodiments, an amino acid modification in a C6 constant region or fragment thereof is a substitution in a position corresponding to position T128 of SEQ ID NO: 2. Preferably, the substitution is a T128L substitution (substitution by a leucine).

In some embodiments, an amino acid modification in a C6 constant region or fragment thereof is a substitution in a position corresponding to position T139 of SEQ ID NO: 2. Preferably, the substitution is a T139V substitution (substitution by a valine).

In some embodiments, an amino acid modification in a C6 constant region or fragment thereof is a substitution in a position corresponding to position F144 of SEQ ID NO: 2. Preferably, the substitution is an F144L substitution (substitution by a leucine).

In some embodiments, an amino acid modification in a C6 constant region or fragment thereof is a substitution in a position corresponding to position T128 of SEQ ID NO: 2 and a substitution in a position corresponding to position T139 of SEQ ID NO: 2. Preferably, the substitutions are respectively a T128L and a T139V substitution.

In some embodiments, an amino acid modification in a C5 constant region or fragment thereof is a substitution in a position corresponding to position T139 of SEQ ID NO: 2 and a substitution in a position corresponding to position F144 of SEQ ID NO: 2. Preferably, the substitutions are respectively a T139V and a F144L substitution.

In some embodiments, an amino acid modification in a 06 constant region or fragment thereof is a substitution in a position corresponding to position T128, T139, and F144 of SEQ ID NO: 2. Preferably, the substitutions are respectively a T128L, a T139V, and a F144L substitution.

In some embodiments, the amino acid modification in B2), B3), B4), B5), or B6) described herein is an amino acid substitution selected from: i. an amino acid substitution in a position corresponding to position K49 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine; ii. an amino acid substitution in a position corresponding to position K50 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine; iii. an amino acid substitution in a position corresponding to position Q58 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a cysteine; iv. an amino acid substitution in a position corresponding to position N66 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a serine; v. an amino acid substitution in a position corresponding to position K96 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine or an aspartic acid; vi. an amino acid substitution in a position corresponding to position N97 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine or a lysine; vii. an amino acid substitution in a position corresponding to position T136 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a leucine; viii. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine; ix. an amino acid substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a leucine; x. an amino acid substitution in a position corresponding to position F153 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine; xi. an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a phenylalanine; xii. an amino acid substitution in a position corresponding to position C159 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a phenylalanine; xiii. an amino acid substitution in a position corresponding to position K49 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position K50 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine; xiv. an amino acid substitution in a position corresponding to position E79 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position K80 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine; xv. an amino acid substitution in a position corresponding to position K96 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine or an aspartic acid, and an amino acid substitution in a position corresponding to position N97 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine or a lysine; xvi. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a leucine; xvii. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a phenylalanine; xviii. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position F153 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine; xix. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, an amino acid substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a leucine, and an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a phenylalanine; xx. an amino acid substitution in a position corresponding to position K49 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, an amino acid substitution in a position corresponding to position K50 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, an amino acid substitution in a position corresponding to position K96 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an aspartic acid, and an amino acid substitution in a position corresponding to position N97 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a lysine; xxi. an amino acid substitution in a position corresponding to position N14 of SEQ ID NO: 2, preferably a substitution by a serine; xxii. an amino acid substitution in a position corresponding to position V48 of SEQ ID NO: 2, preferably a substitution by a cysteine; xxiii. an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, preferably a substitution by a leucine; xxiv. an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, preferably a substitution by a valine; xxv. an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, preferably a substitution by a leucine; xxvi. an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, preferably a substitution by a leucine, and an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, preferably a substitution by a valine; xxvii. an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, preferably a substitution by a valine, and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, preferably a substitution by a leucine; xxviii. an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, preferably a substitution by a leucine, an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, preferably a substitution by a valine, and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, preferably a substitution by a leucine, or; xxix. a combination of any one of I) to xxviii).

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution of the positions corresponding to positions from D1 to V8 (DKQLDADV (SEQ ID NO: 42)) of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1 , by the amino acid sequence EDLKNVF (SEQ ID NO: 43), DKEDLKNVF (SEQ ID NO: 176), or a variant thereof, preferably by the amino acid sequence EDLKNVF (SEQ ID NO: 43) or a variant thereof.

In some embodiments, an amino acid modification in a Cy constant region or fragment thereof is a substitution of the FG-loop and/or its adjacent amino acids by the FG-loop of a C constant region and/or its adjacent amino acids, or a variant thereof. Preferably, the Cy constant region or fragment thereof is a Cy1 or Cy2 constant region or fragment thereof, more preferably a Cy1 constant region or fragment thereof. A definition of "adjacent” amino acids has been provided earlier herein. Modifications resulting in combinations of Cy FG-loop-adjacent amino acids with C0 FG-loop-adjacent amino acids are also encompassed by the disclosure. The FG-loop of a C0 constant region of a 0T-cell receptor chain is represented by amino acid sequence YGLSENDEWTQDRAKPVT (SEQ ID NO: 46). The FG-loop of a C0 constant region of a 0T-cell receptor chain including its adjacent amino acids is represented by amino acid sequence VQFYGLSENDEWTQDRAKPVTQIV (SEQ ID NO: 47).

In some embodiments, an amino acid modification in a Cy1 constant region or fragment thereof is a substitution of the positions corresponding to positions from E93 to D100 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, (ENNKNGVD (SEQ ID NO: 44)) by the amino acid sequence YGLSENDEWTQDRAKPVT (SEQ ID NO: 46) or a variant thereof.

In some embodiments, an amino acid modification in a Cy1 constant region or fragment thereof is a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , (VRHENNKNGVDQ (SEQ ID NO: 48)) by the amino acid sequence VQFYGLSENDEWTQDRAKPVTQIV (SEQ ID NO: 47) or a variant thereof.

In some embodiments, an amino acid modification in a Cy1 constant region or fragment thereof is a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , (VRHENNKNGVDQ (SEQ ID NO: 48)) by the amino acid sequence VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50) or a variant thereof.

In some embodiments, an amino acid modification in a Cy2 constant region or fragment thereof is a substitution of the positions corresponding to positions from E93 to D100 of SEQ ID NO: 40 or SEQ ID NO: 41 (ENNKNGID (SEQ ID NO: 45)) by the amino acid sequence YGLSENDEWTQDRAKPVT (SEQ ID NO: 46) or a variant thereof.

In some embodiments, an amino acid modification in a Cy2 constant region or fragment thereof is a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 40 or SEQ ID NO: 41 (VRHENNKNGIDQ (SEQ ID NO: 49)) by the amino acid sequence VQFYGLSENDEWTQDRAKPVTQIV (SEQ ID NO: 47) or a variant thereof.

In some embodiments, an amino acid modification in a Cy2 constant region or fragment thereof is a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 40 or SEQ ID NO: 41 (VRHENNKNGIDQ (SEQ ID NO: 49)) by the amino acid sequence VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50) or a variant thereof.

In some embodiments, the amino acid modification in B1), B2), or B6) described herein is an amino acid substitution selected from: a1 ) a substitution of the positions corresponding to positions from D1 to V8 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 , by EDLKNVF (SEQ ID NO: 43), DKEDLKNVF (SEQ ID NO: 176), or a variant thereof, preferably by EDLKNVF (SEQ ID NO: 43) or a variant thereof; b1 ) a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1, by VQFYGLSENDEWTQDRAKPVTQIV (SEQ ID NO: 47), VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50), or a variant thereof, or; c1) a combination of a1) and b1).

Step C)

In step C), the yT-cell receptor chain or fragment thereof and the 5T-cell receptor chain or fragment thereof obtained in step B) are expressed in an immunoresponsive cell. It is understood that, in some embodiments, in step B) only the yT-cell receptor chain or fragment thereof provided in step A) is modified. In these embodiments, the expressed 5T-cell receptor chain or fragment thereof is the one originally provided in step A). Likewise, in some embodiments, in step B) only the 5T-cell receptor chain or fragment thereof provided in step A) is modified. In these embodiments, the expressed yT-cell receptor chain or fragment thereof is the one originally provided in step A). The immunoresponsive cell may already express a yT-cell receptor chain or fragment thereof or a 5T-cell receptor or fragment thereof, as discussed earlier herein. Expression of the provided yT-cell receptor chains, 5T-cell receptor chains, or fragments thereof, may in some cases be introduced to cells that have not yet differentiated into an immunoresponsive cell, for example stem cells, precursor cells, or IPSC cells, which can later be differentiated into immunoresponsive cells.

An immunoresponsive cell expressing a yT-cell receptor chain or fragment thereof and a 6T-cell receptor chain or fragment thereof described herein may be called an engineered immunoresponsive cell. A cell as used herein is "engineered” when it has been transformed, modified or transduced to comprise a heterologous or exogenous nucleic acid molecule, and when it preferably expresses the polypeptide encoded by the nucleic acid molecule. As used herein, the term “engineered cell” may be replaced by “modified cell” or “transformed cell”. A further description is provided later herein.

In some embodiments, the immunoresponsive cell is a mammalian immunoresponsive cell, preferably a human immunoresponsive cell. Preferably, the immunoresponsive cell is selected from a T-cell, an a T- cell, a yST-cell, CD4+ T-cell, CD8+ T-cell, an induced pluripotent stem cell derived T-cell (IPSC-derived T- cell), a T-effector cell, a lymphocyte, a B-cell, an NK-cell, an NKT-cell, a myeloid cell, a monocyte, a macrophage, or a neutrophil. In preferred embodiments, the immunoresponsive cell is a T-cell, an induced pluripotent stem cell derived T-cell (IPSC-derived T-cell), an aPT-cell, a yST-cell, or an NK cell, more preferably a yST-cell or aPT-cell, most preferably an aPT-cell.

The immunoresponsive cells, such as T-cells, described herein may be primary cells, for example from a subject, such as described in the examples for a human subject. The T-cells may be a£ or y6T cells derived from a human subject. In some embodiments, a yST-cell is a primary yST-cell. In some embodiments, an aPT-cell is a primary aPT-cell. Alternatively, the T-cells may be a T-cell line, such as SupT-1 or Jurkat cells or any other widely available cell line. Alternatively, the T-cells may be derived from differentiated stem cells, progenitor cells, precursor cells, IPSC cells, and the like. Any cell type, being a primary cell, undifferentiated cell, differentiated cell, or any cell line can suffice, as long as the cell population, or a substantial part thereof, expresses or is able to express a T-cell receptor, i.e., such as being positive for the apTCR or the y5TCR in a FACS sorting or the like. The cell may also be a blood progenitor cell such as a thymocyte or a blood stem cell, which, after it has been provided with the right stimuli, can develop into a T-cell.

T-cells, alternatively called T-lymphocytes, belong to a group of white blood cells named lymphocytes, which play a role in cell-mediated immunity. T-cells originate from hematopoietic stem cells in the bone marrow, mature in the thymus, and gain their full function in peripheral lymphoid tissues. During T-cell development, CD4-CD8- T-cells (negative for both the CD4 and CD8 co-receptor) are committed either to an a or y6 fate as a result of an initial TCR or 6TCR gene rearrangement. Cells that undergo early -chain rearrangement express a pre-TCR structure composed of a complete p-chain and a pre-TCRa chain on the cell surface. Such cells switch to a CD4+CD8+ state, rearrange the TCRa-chain locus, and express a mature a TCR on the surface. CD4-CD8- T-cells that successfully complete the y gene rearrangement before the -gene rearrangement express a functional y5TCR and remain CD4-CD8- (Claudio Tripodo et al. Gamma delta T cell lymphomas Nature Reviews Clinical Oncology 6, 707-717, December 2009). The T-cell receptor associates with the CD3 protein complex. Mature T-cells, i.e., expressing an apTCR or a ySTCR, express the T-cell receptor complex on the cell surface. The y5T-cells, which constitute about 1-5% of the total population of T-cells, can be divided in further subpopulations which, in humans, is based on TCRS-chain expression. Within the extracellular domain of a T-cell receptor three complementarity determining regions (CDR1 , CDR2, CDR3) are located. CDR regions are composed during the development of a T-cell where so-called variable-(V), diverse-(D), and joining-(J)-gene segments are randomly combined to generate diverse TCRs. Of the three CDR regions CDR3, for both a[3T-cells and yST-cells, is the most variable one, and is therefore the key player in antigen/ligand recognition. CDR3 sequences can bind to their cognate antigens even in the absence of the remainder of the ySTCR sequence (Xu et al., Molecular Immunology 44 (2007) 302-310, incorporated herein by reference in its entirety). Further, transplanting of a y6CDR3 region to a different ySTCR can change the ySTCR’s binding characteristics to the ones of the transplanted y5CDR3 region (Adams et al., Nature Immunology (2008) (7): 777-784, incorporated herein by reference in its entirety). In contrast to the CDR3 regions which arise from somatic recombination of V(D)J loci, the CDR1 and CDR2 regions of y5T-cell receptors are generally conserved in each yT- and 6T-cell receptor chain type, and are not the determinant factors in specific antigen-recognition. The location of each of the CDR1 , CDR2, and CDR3 region in a respective yT-cell receptor chain or 5T-cell receptor chain can easily be identified using corresponding anchor amino acid positions according to IMGT numbering (Lefranc, M.- P., The Immunologist 7:132-136 (1999); Lefranc, M.-P., Dev Comp Immunol 29(3):185-203 (2005), both of which are incorporated herein by reference in their entireties).

APT-cells may be defined with respect to function as T lymphocytes that express an a0TCR, which recognize peptides bound to MHC molecules (major histocompatibility complex), which are expressed on the surface of various cells. MHC molecules present peptides derived from the proteins of a cell. When for example a cell is infected with a virus, the MHC will present viral peptides, and the interaction between the a TCR on the T-cell and the MHC-complex on the target cell (i.e., the virus infected cell) activates specific types of T-cells which initiate and immune responses to eliminate the infected cell. Hence, aPT-cells may be functionally defined as being cells capable of recognizing peptides bound to MHC molecules. ApT-cells may be selected from peripheral blood for example via the CD3 antigen. ApT-cells may also be selected with an antibody specific for the apTCR, many of which are commercially available such as the ones offered by ThermoFisher Scientific (Waltham, MA, USA). From such selected cells, the nucleic acid (or amino acid) sequence corresponding to the aT-cell receptor chain and/or the pT-cell receptor chain may be determined by sequencing using standard methods available in the art. Hence, apT cells may also be defined as being cells naturally comprising a nucleic acid (or amino acid) sequence corresponding to an aT-cell receptor chain and/or the PT-cell receptor chain. In some embodiments, apT-cells described herein express an endogenous apTCR. yST-cells may be functionally defined in that they are specifically and rapidly activated by e.g., (but not limited to) a set of non-peptidic phosphorylated isoprenoid precursors, collectively named phosphoantigens or stress signals medicated by non-classical HLA molecules like CD1 (this is for example the case for the Vy952 T-cell subset). Phosphoantigens are produced by virtually all living cells, though the levels are usually very low in healthy cells, and increased in transformed I malignant cells or cells infected by e.g., Mycobacterium tuberculosis, which deliver a derivate of phosphoantigens. Activation of yST-cells comprises clonal expansion, cytotoxic activity and expression and release of cytokines. yST-cells are also defined by expression of the y5T-cell receptor. For example, cells may be selected using an antibody specific for the yST-cell receptor, many of which are commercially available such as the ones offered by ThermoFisher Scientific (MA, USA). Hence, yST-cells may also be defined as being cells naturally comprising a nucleic acid (or amino acid) sequence corresponding to a yT-cell receptor chain and/or a 5T-cell receptor chain. In some embodiments, a y5T-cell expresses a y5TCR.

The person skilled in the art is well capable of selecting and/or identifying cell populations characterized by expression of an antigen or receptor on the surface of the cell such as described throughout herein. It is understood that with regard to expression on the surface of cells, such as expression of CD3, CD4, CD8, aPTCR, and ySTCR, and fragments thereof, this typically involves a population of cells of which a portion of cells have a much higher level of expression of the respective polypeptide when compared to cells having a lower level of expression. Hence, the terms positive or negative are to be understood as being relative, i.e., positive cells have a much higher expression level as compared to cells being negative. Cells being negative in this sense may thus still have an expression level which may be detectable.

Expression on the surface of cells may be analyzed using, for example, fluorescence activated cell sorting (FACS), and many specific antibodies are commercially available, e.g., targeting CD3, CD4, CD8, afTCR, ybTCR, that are suitable for such FACS analysis, such as the ones offered by ThermoFisher Scientific (MA, USA). As an example, a|3T cells can also be defined and selected as being positive for aPTCR expression in FACS. The same holds for y5T cells and ySTCR expression. Conditions that allow the selection of negative and/or positive cells may be according to the manufacturer’s protocols. Optionally, additional techniques such as magnetic bead separation may be utilized.

Further examples of antibodies that may be suitable for selection of T-cells described herein are available from BD Pharmingen (BD, Franklin Lakes, NJ USA) such as V52-FITC (clone B6, # 555738), or from ThermoFisher Scientific (Waltham, MA, USA) such as Vy1-PE-Cy7 (clone TS8.2, #25-5679-42), or from Biolegend (San Diego, CA, USA) such as aPTCR-BV785 (clone IP26, #306742), or from Beckman Coulter (Brea, CA, USA) such as pan-ySTCR-PE (clone IMMU510, # IM1418U), or from Miltenyi Biotec (Bergisch Gladbach, Germany) such as CD3-VioGreen (clone REA613, #130-113-142, or from Biolegend (San Diego, CA, USA) such as anti-biotin aPTCR (clone IP26, # 306704), with many others being commercially available.

The yT-cell receptor chain or fragment thereof and the 5T-cell receptor chain or fragment thereof may be expressed in an immunoresponsive cell (or undifferentiated cell as discussed above) using standard methods available in the art, including transformation, transfection, and transduction methods, discussed later herein. Expression of the yT-cell receptor chain or fragment thereof and the 5T-cell receptor chain or fragment thereof may be effected by transferring of the nucleic acid molecules encoding the polypeptides to the immunoresponsive cell. The nucleic acid molecules encoding the yT-cell receptor chain or fragment thereof and the 5T-cell receptor chain or fragment thereof may be comprised in a single or distinct nucleic acid molecules, nucleic acid constructs or vectors. Preferred nucleic acid constructs and vectors are described later herein. Preferred vectors are plasmids and viral vectors, with retroviral and lentiviral vectors being more preferred and lentiviral vectors being most preferred.

Nucleic acid molecules, constructs, and vectors described herein may comprise additional nucleotide sequences. Exemplary sequences are regulatory sequences, sequences encoding signal peptides, sequences encoding linker peptides, sequences facilitating the co-expression of the yT-cell receptor chain or fragment thereof and the 5T-cell receptor chain or fragment thereof (in embodiments wherein they are encoded by a single nucleic acid molecule, construct, or vector), and the like. A further description of additional nucleotide sequences is provided later herein.

It is understood that the nucleic acid molecules, nucleic acid constructs, or vectors encoding the yT-cell receptor chains or fragments thereof and the 5T-cell receptor chains or fragments thereof described herein do not have to be transferred simultaneously to an immunoresponsive cell (or to an undifferentiated cell that is later differentiated to an immunoresponsive cell). As a non-limiting example, an immunoresponsive cell which is transduced with a nucleic acid molecule encoding a yT-cell receptor chain or fragment thereof may have been priorly, simultaneously, or afterwards, transduced to express a 5T-cell receptor chain or a fragment thereof. As another non-limiting example, an immunoresponsive cell which is transduced with a nucleic acid molecule encoding a 5T-cell receptor chain or fragment thereof may have been priorly, simultaneously, or afterwards, transduced to express a yT-cell receptor chain or a fragment thereof. The yT-cell receptor chain, 5T-cell receptor chain, or fragment thereof may be modified as described in step B) or may correspond to the originally provided yT-cell receptor chain, 5T-cell receptor chain, or fragment thereof provided in step A) (in embodiments wherein only one of the chains or fragment thereof is modified).

The immunoresponsive cells may optionally be expanded before or after the transfer of the nucleic acids encoding the polypeptides described herein. Preferably, the expansion is after the transfer, allowing the amount of nucleic acids that needs to be transferred to be as low as possible. Expansion of T-cells may optionally be performed by stimulation with anti-CD3/CD28 polymeric nanomatrix beads, in the presence of IL-7 and IL-15. Expansion may be performed using commercially available kits, such as T-cell TransAct™ (M iltenyi Biotec, Bergisch Gladbach, Germany). A further example is provided in the experimental section herein.

Step D)

In step D) the anti-tumour or anti-infective response of the engineered immunoresponsive cell of step C), preferably T-cell, more preferably a0T-cell, is determined (measured). In this way, the yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, or fragment thereof, that mediates the anti-tumour or anti-infective response exhibited by the engineered immunoresponsive cell may be identified. Preferably, the determination of the anti-tumour or anti-infective response involves stimulation of the engineered immunoresponsive cells, more preferably yST-cell receptor dependent stimulation.

Stimulation as used herein refers to bringing the engineered immunoresponsive cells, preferably T-cells, more preferably a T-cells, into contact with a target cell, antigen, epitope, or another molecule which results in activation of TCR downstream signaling pathways and their mediated effect of proliferation, activation, and/or degranulation of the activated cell.

In some embodiments, the engineered immunoresponsive cells are T-cells stimulated by contacting with an antibody specific for a y5T-cell receptor or fragment thereof, for example the ones described earlier herein. In some embodiments, the engineered T-cells are stimulated by contacting with antibody variable regions (e.g., a variable chain heavy region (VH) and/or a variable chain light region (VL)), antibody short chain variable fragments (scFv), single domain antibodies, Fab, Fab', F(ab')2, dimers and trimers of Fab conjugates, Fv, minibodies, diabodies, triabodies, tetrabodies, affibodies, ankyrin proteins, ankyrin repeats, DARPins, monobodies, nanobodies, avimers, adnectins, anticalins, fynomers, kunitz domains, knottins, or P-hairpin mimetics. Manipulation of the orientation of the VH and VL domains of an antibody and the linker length can be used to create different forms of molecules that can be monomeric, dimeric (diabody), trimeric (triabody), or tetrameric (tetrabody). Nanobodies, also known as single-domain antibodies (sdAb), are antibody fragments consisting of a single monomeric variable antibody domain. Minibodies are scFv-CH3 fusion proteins that assemble into bivalent dimers. In some embodiments, the engineered T-cells are stimulated by contacting with an antigen or epitope specific for a yST-cell receptor or fragment thereof. In some embodiments, the engineered T-cells are stimulated by contacting with an antigen or epitope specific for a yST-cell receptor or fragment thereof which is a multimer, for example a dimer, trimer, tetramer, and the like. In some embodiments, the antigen is EPCR (Endothelial protein C receptor), preferably human EPCR. In some embodiments, the engineered T-cells are stimulated by contacting with a target cell, preferably a target cell expressing EPCR. A target cell may natively express an antigen, for example EPCR, and/or antigen expression may be introduced and/or enhanced in a target cell, for example via ectopic gene expression, gene overexpression, or any other genomic toolbox technique known to the skilled person. Examples of target cells are tumour cells, infected cells, or infectious agents as described later herein. In some embodiments, the contacting step has a duration of at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days. In some embodiments wherein the engineered T-cells are contacted with target cells, the contacting may involve any suitable effectontarget (E:T) ratio suitable for stimulation to occur. Non-limiting examples of suitable E:T ratios are 2:1 , 1:1 , 1:2, 1:4, 1:8, 1 :16, and others. The stimulation step may involve culturing of the engineered T-cells in order for proliferation, activation, and/or degranulation to occur. In some embodiments, culturing of the engineered T-cells involves co-culturing with target cells. Suitable growth media and culturing conditions will depend on the engineered cells used and will be known to the skilled person, with many media and protocols being commercially available, for example the TEXMACS™ medium (Miltenyi Biotec, Bergisch Gladbach, Germany), McCoy's 5a Medium, or the IMDM medium. Growth media may be supplemented with serum, for example fetal bovine serum (an exemplary value of which being 10% v/v) or human serum (an exemplary value of which being 2.5% or 5% v/v). A further example is provided in the experimental section herein.

The anti-tumour or anti-infective response of the engineered immunoresponsive cell, preferably T-cell, more preferably apT-cell, may be determined (measured) using any technique known to the skilled person. In some embodiments, determining an anti-tumour and/or anti-infective response or reactivity or activity comprises contacting the engineered cells with tumour cells, tumour cell lines, infected cells, or infectious agents such as e.g., fungal cells. Determining an anti-tumour or anti-infective activity may include any assay in which an anti-tumour or anti-infective effect may be determined, such as having an effect on tumour cell or infected cell or infectious agent division rate, i.e., the speed with which the tumour or infected cells or infectious agents divide, cell death, cytolysis/cytotoxicity of the tumour or infected cell or infectious agent, binding to the tumour or infected cells or infected agents, etc. T umour cells may be any kind of tumour cells. As a non-limiting example, they may be primary tumour cells from a patient. The tumour cells may be tumour cells from cell lines, such as (but not limited to) the cell lines listed hereafter: HT-29, RKO, MDA-MB-231, MZ1851 RC, and others, which are well known in the art. Tumour cell lines may be obtained from the American Type Culture Collection (ATCC, Manassas, Virginia) and the like. Infected cells may, for example, be cells that have been infected by a bacterium or a virus. The infection may result in the infected cell displaying an antigen or epitope that is a target of a yT-cell receptor chain, a 5T-cell receptor chain, a y5T- cell receptor, or a fragment thereof as described herein. Non-limiting examples are Plasmodium falciparum, Mycobacterium (M.) tuberculosis and M. leprae. Infectious agents may, for example be, bacteria or fungal cells. In some embodiments, a target cell expresses EPCR (Endothelial protein C receptor), preferably human EPCR.

The anti-tumour or anti-infective response that is mediated by a yT-cell receptor chain, 5T-cell receptor chain, y5T-cell receptor, or fragment thereof described herein may be improved (increased) relative to a control yT-cell receptor chain, 5T-cell receptor chain, y5T-cell receptor, or fragment thereof, which is used as a reference. In embodiments wherein the yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, or fragment thereof comprises an amino acid modification compared to a parent sequence (as provided in step A) of the method), the polypeptide comprising or consisting of the parent sequence may be used as a control (reference). Alternatively, a polypeptide comprising or consisting of a different sequence may be used as a control. A control may be a wild-type yT-cell receptor chain, 5T-cell receptor chain, y5T-cell receptor, or fragment thereof. A control may also be a comparable engineered cell not expressing a yT-cell receptor chain, 5T-cell receptor chain, y5T-cell receptor, or fragment thereof.

In some embodiments, a control yT-receptor chain comprises a Cy constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1. In some embodiments, a control ST-cell receptor chain comprises a C5 constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2. In some embodiments, a control yQT-cell receptor comprises a Cy constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1, and a C5 constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, a control yT-cell receptor chain comprises or consists of an amino acid sequence represented by SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 102, or SEQ ID NO: 120. Another control yT-cell receptor chain comprises or consists of an amino acid sequence represented by SEQ ID NO: 167. In some embodiments, a control 5T-cell receptor chain comprises or consists of an amino acid sequence represented by SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101 , SEQ ID NO: 103, or SEQ ID NO: 121. Another control 5T-cell receptor chain comprises or consists of an amino acid sequence represented by SEQ ID NO: 168. In some embodiments, a control yST-cell receptor comprises or consists of a yT-cell receptor chain comprising or consisting of an amino acid sequence represented by SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 102, or SEQ ID NO: 120, and a 5T-cell receptor chain comprising or consisting of an amino acid sequence represented by SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101 , SEQ ID NO: 103, or SEQ ID NO: 121. Another control yST-cell receptor comprises or consists of a yT-cell receptor chain comprising or consisting of an amino acid sequence represented by SEQ ID NO: 167 and a 3T-cell receptor chain comprising or consisting of an amino acid sequence represented by SEQ ID NO: 168. An anti-tumour or anti-infective response may be improved by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 100% (2-fold), at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8- fold, at least 9-fold, or at least 10-fold relative to a control yT-cell receptor chain, 5T-cell receptor chain, or yST-cell receptor.

In some embodiments, determining the anti-tumour or anti-infective response includes contacting an engineered T-cell as described herein with a tumour or infected cell or infectious agent and measuring its ability to lyse the tumour or infected cell or infectious agent (cytotoxicity). The contacting step may, for example, have a duration (incubation period) from 10 hours to 1, 2, 3, 4, 5, 6, 7 days, or longer. The measurement of the ability to lyse the tumour or infected cell or infectious agent may include initially providing a fixed number of tumour or infected cells or infectious agents with which the engineered T-cell is contacted and, after the incubation period, counting the number of the viable tumour or infected cells or infectious agents. An anti-tumour or anti-infective response may be considered to be present when the number of viable tumour or infected cells or infectious agents at the end of the incubation step is less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the initial number of tumour or infected cells or infectious agents at the onset of the incubation step.

Alternatively, an anti-tumour or anti-infective response may be considered to be present when the number of viable tumour or infected cells or infectious agents at the end of the incubation step with the engineered T-cell is lower than the number of tumour or infected cells or infectious agents at the end of a similar incubation/contacting step with a control (comparable) T-cell not expressing a yT-cell receptor chain, 5T- cell receptor chain, yST-cell receptor, or fragment thereof or expressing a control yT-cell receptor chain, 5T- cell receptor chain, y5T-cell receptor, or fragment thereof.

Lower in this context may mean at least 5% lower, at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower. Such cells may be considered to exhibit an improved anti-tumour or anti-infective response compared to the control cells.

In addition, or as alternative to the counting of the number of viable tumour or infected cells or infectious agents at the end of the incubation/contacting step, one may also perform a ⁵¹Chromium-release assay which is known to the skilled person. The amount of ⁵¹ Chromium release is a measure of the number of cells that have been lysed.

In some embodiments, one may determine the cytotoxicity of engineered immunoresponsive cells, preferably T-cells, more preferably aPT-cells using e.g., an xCELLigence assay (Agilent, CA, USA) and plotted as percentage of cytolysis relative to maximum cytolysis induced by treatment of the target cells with the detergent Triton-X-100. An example of this assay is provided in the experimental section herein. In some embodiments, the percentage of target cell cytolysis obtained by engineered T-cells is higher (preferably at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more) than the percentage of cytolysis obtained when the same target cells are contacted with control (comparable) T-cells not expressing a yT-cell receptor chain, 5T-cell receptor chain, y5T-cell receptor, or fragment thereof or expressing a control yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, or fragment thereof. Such cells may be considered to exhibit an improved anti-tumour or anti-infective response compared to the control cells.

Cytotoxicity may alternatively be measured using e.g., an LDH release assay from target cells, by calculating the LDH release fold-change when the target cells are incubated with engineered T-cells relative to when the same target cells are incubated with control T-cells. In some embodiments, the LDH release when the target cells are incubated with engineered T-cells is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 100% (2-fold), at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to when the same target cells are incubated with control T-cells.

Cytotoxicity may also be measured by a luciferase-based cytotoxicity assay, in which the target cells are pre-transduced with luciferase and cytotoxicity is measured by measuring decreased luciferase activity relative to target cells cultured alone or with control T-cells as described herein. These assays are known to the skilled person and examples are provided in the experimental section herein.

An anti-tumour response may also be determined by measuring the binding of an engineered immunoresponsive cell, preferably T-cell, more preferably apT-cell described herein to a tumour or infected cell after contacting both cells together. Such a contacting step may have a duration of from 10 hours to 1, 2, 3, 4, 5, 6, 7 days, or longer. When binding of the engineered immunoresponsive cell to the tumour cell or infected cell or infectious agent is detected at the end of the contacting step, said cell may be considered to exhibit an anti-tumour or anti-infective response.

Alternatively and preferably, the binding of the engineered immunoresponsive cell to the tumour or infected cell or infectious agent at the end of the contacting step is higher (preferably at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more) than the binding displayed by control (comparable) immunoresponsive cell not expressing the yT- cell receptor chain, 5T-cell receptor chain, yST-cell receptor, or fragment thereof, or expressing a control yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, or fragment thereof, to the same tumour or infected cell. Such cells may be considered to exhibit an improved anti-tumour or anti-infective response compared to the control cells.

As soon as an effect can be seen in any of the assays described above, or any other similar assay available to the skilled person, one can conclude that the yT-cell receptor chain, 5T-cell receptor chain, y5T-cell receptor, or fragment thereof mediates an anti-tumour or anti-infective response or activity. In these assays, the control cells may, for example, be T-cells that are untransduced or that are transduced with an empty viral vector or that are transduced with a control yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, or fragment thereof.

Optionally, any of the assays described above may be performed multiple times, for example by collecting the engineered immunoresponsive cells at the end of the contacting/incubation steps and re-exposing them to the same tumour or infected cells (serial stimulation). The engineered immunoresponsive cells may exhibit an improved anti-tumour or anti-infective response compared to control immunoresponsive cells not expressing the yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, or fragment thereof, or expressing a control yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, or fragment thereof after a subsequent stimulation. This improved response may be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% higher, or more compared to the control immunoresponsive cells.

In some embodiments wherein the engineered immunoresponsive cell is a T-cell, preferably an apT-cell, determining the anti-tumour or anti-infective response is performed by measuring the expression of a T-cell activation and/or degranulation marker. The expression of a T-cell activation and/or degranulation marker may be linked to the activation of a yQTCR; for example, expression of the marker may be linked to the transmittal of a signal via the ySTCR complex by a change in its conformation and/or position following its activation. Thus, assessment of the expression of a T-cell activation and/or degranulation marker can be used to assess the capacity of a yQTCR to transmit an activation signal in a T-cell.

Assessing the expression of a T-cell activation and/or degranulation marker after stimulation of the engineered T-cells as an alternative to the assays described above has an added benefit of further increasing the throughput with which mediation of an anti-tumour or anti-infective response by yT-cell receptor chains, 5T-cell receptor chains, yST-cell receptors, or fragments thereof, may be determined and thus the throughput with which the yT-cell receptor chains, 5T-cell receptor chains, yST-cell receptors, or fragments thereof mediating the activity are identified.

In some embodiments, a T-cell activation marker is a cytokine such as IFN-y, IL-2 or TNFa. Cytokine production may be determined, e.g. via antibody staining, ELISA and/or quantitative PCR for the expressed mRNA. Assays for determining the production of a cytokine such as IFN-y, IL-2 or TNFa are commercially widely available (for example from R&D Systems, Minneapolis, MN, US). When production of a cytokine such as IL-2, TNFa, or IFN-y is detected during or at the end of a contacting step with a tumour or infected cell or infectious agent such as described earlier herein, the engineered T-cell may be considered to exhibit an anti-tumour or anti-infective response. Alternatively and preferably, when the amount of IFN-y, IL-2 or TNFa produced during or at the end of the contacting step with the engineered T-cell is higher (preferably at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more) than the amount of IFN-y, IL-2 or TNFa produced when the tumour or infected cell or infectious agent is contacted with control (comparable) T-cells not expressing the yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, or expressing a control yT-cell receptor chain, 5T-cell receptor chain, y5T-cell receptor, or fragment thereof, the cells may be considered to exhibit an improved anti-tumour or anti-infective response compared to the control cells.

In some embodiments, the T-cell activation and/or degranulation marker is a surface expressed protein. In some embodiments, the T-cell activation and/or degranulation marker is selected from CD25 (for example Uniprot Ref: P01589), 41BB (for example Uniprot Ref: Q07011), CD62L (for example Uniprot Ref: P14151), Nur77 (for example Uniprot Ref: P22736), NOR1 (for example Uniprot Ref: Q92570), EGR2 (for example Uniprot Ref: P11161 ), LAG3 (for example Uniprot Ref: P18627), CD40L (for example Uniprot Ref:P29965), CD38 (for example Uniprot Ref: P28907), HLA-DR (for example Uniprot Ref: P01903), FASL (for example Uniprot Ref: P48023), CD63 (for example Uniprot Ref: P08962), CD69 (for example Uniprot Ref: Q07108), and/or CD107 (LAMP1, for example Uniprot Ref: P11279). Preferably, the T-cell activation and/or degranulation maker is CD69 (for example Uniprot Ref: Q07108) and/or CD107a (LAMP1, for example Uniprot Ref: P11279). Assessment of expression of a surface-expressed protein may be done using flow cytometry, for example using FACS as discussed elsewhere herein. As a non-limiting example, in the case of CD69 and/or CD107a, the engineered T-cells may be stained with anti-CD69 (e.g., CD69-APC (Clone REA824, Miltenyi Biotec, Gladback Germany)) and/or anti-CD107a (e.g., CD107a-BV421 (H4A3, Biolegend, CA, USA)) antibodies, allowing assessment (measurement) of their expression using flow cytometry.

When expression of a surface-expressed T-cell activation and/or degranulation marker is detected during or at the end of a contacting step with a tumour or infected cell or infectious agent such as described earlier herein, the engineered T-cell may be considered to exhibit an anti-tumour or anti-infective response. Alternatively and preferably, when the expression of a surface-expressed T-cell activation and/or degranulation marker in an engineered T-cell during or at the end of the contacting step with a tumour or infected cell or infectious agent is higher (preferably at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more) than its expression in a control (comparable) T-cell not expressing the yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, or expressing a control yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, or fragment thereof, the cells may be considered to exhibit an improved anti-tumour or anti-infective response compared to the control cells.

In some embodiments, determination of an anti-tumour or anti-infective response is performed by measuring the cell surface expression of CD69. In some embodiments, determination of an anti-tumour or anti-infective response is performed by measuring the cell surface expression of CD107a. In some embodiments, determination of an anti-tumour or anti-infective response is performed by measuring the cell surface expression of CD69 and CD107a. Simultaneous assessment of CD69 and CD107a may be associated with further increased throughput and identification accuracy.

In some embodiments, an engineered T-cell additionally expresses a reporter construct. The reporter construct may in some cases have been priorly introduced to a T-cell used in the method. A ’’reporter construct” as used herein refers to a nucleic acid construct comprising a promoter sequence which can drive transcription of a nucleic acid molecule following the activation of a y5TCR (e.g., a promoter which is activated following the transmittal of a signal via the y5TCR complex by a change in its conformation and/or position following its activation), and a nucleic acid molecule encoding a polypeptide the activity of which can be detected (reporter polypeptide), for example using flow cytometry. Such reporter constructs can be used to assess the activation of a yBTCR and convert it to a detectable signal via the expression of the reporter polypeptide.

Non-limiting examples of promoter sequences that can be comprised in a reporter construct are promoters from or derived from a response element protein selected from nuclear factor of activated T-cells (NFAT), Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-KB), Activator protein 1 (AP-I), Nur response element (NurRE), Interferon gamma (IFN-y), CD69, Early growth response protein 1 (EGR1), Early growth response protein 2 (EGR2), IL2, and any combination thereof.

In some embodiments, the promoter comprised in a reporter construct comprises, or is, an NFAT response element or a variant thereof. A NFAT response element may have a nucleotide sequence of WGGAAA, wherein "W” stands for A or T (see also Rao A, et al (1997), Annu. Rev. Immunol., 15: 707-747, incorporated herein by reference in its entirety). A promoter may comprise one or more of NFAT response elements or variants thereof, preferably one or more response elements having a nucleotide sequence of WGGAAA. A promoter comprising a variant of an NFAT response element may have at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% or 110%, 120%, 130%, 140%, 150% or more of the promoter activity of the original counterpart as measured under the same experimental conditions, for example using qPCR or any other suitable method known to the skilled person.

Non-limiting examples of a polypeptide the activity of which can be detected is a fluorescent or luminescent protein, for example green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), Blue fluorescent protein (BFP), cyan fluorescent protein (CFP), violet-excitable green fluorescent (Sapphire), or luciferase. In some embodiments, the polypeptide the activity of which can be detected is GFP. An exemplary GFP sequence comprises SEQ ID NO: 51. In some embodiments, the polypeptide the activity of which can be detected is luciferase. Exemplary luciferase sequences comprise SEQ ID NO: 52, 53, or 54. Detection of activity of a fluorescent or luminescent protein (such as luciferase) is discussed later herein.

Accordingly, in some embodiments, determination of an anti-tumour or anti-infective response is performed by measuring the expression of a reporter polypeptide. Stimulation of engineered T-cells may be performed as described earlier herein, for example by bringing the engineered T-cells into contact with a tumour or infected cell or infectious agent. In such embodiments, when the expression of the reporter polypeptide during or at the end of the contacting step with the engineered T-cell is higher (preferably at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more) than its expression when the tumour or infected cell or infectious agent is contacted with control (comparable) T-cells not expressing the yT-cell receptor chain, 5T-cell receptor chain, y5T-cell receptor, or expressing a control yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, or fragment thereof, the cells may be considered to exhibit an improved anti-tumour or anti-infective response compared to the control cells.

In some embodiments, determination of an anti-tumour or anti-infective response is performed measuring the expression of a T-cell activation and/or degranulation marker, preferably of CD69 and/or CD107a, in combination with assessing the expression of a reporter polypeptide. Preferably, the assessment of expression of the marker and the reporter polypeptide is performed using flow cytometry.

In some embodiments, the ability of a yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, or fragment thereof to mediate an anti-tumor or anti-infective response may be determined by measuring its surface expression in an immunoresponsive cell, preferably a T-cell, more preferably an apT-cell. Surface expression may be determined by flow cytometric methods known to the skilled person, for example in combination with using antibodies specific to a|3TCRs (e.g., clone IP26 as described above) and yQTCRs (e.g., clone IMMU510 as described above). An additional example of determination of ySTCR surface expression is given in the experimental section herein.

Step E)

In step E), the yT-cell receptor chain, ST-cell receptor chain, y5T-cell receptor, or fragment thereof that mediates the anti-tumour or anti-infective response determined in step D) is obtained.

Optionally, the obtained yT-cell receptor chain, ST-cell receptor chain, y5T-cell receptor, or fragment thereof is sequenced. Sequencing may be performed directly on amino acid sequences of yT-cell receptor chains, ST-cell receptor chains, y5T-cell receptors, or fragments thereof that are determined to mediate an antitumour or anti-infective response, using any protein sequencing method known to the skilled person, for example utilizing mass spectrometry or Edman degradation with a protein sequenator.

Alternatively, or in addition, sequencing may be performed on the nucleic acid molecule encoding the yT- cell receptor chain, ST-cell receptor chain, yST-cell receptor, or fragment thereof. Sequencing of a nucleic acid encoding a yT-cell receptor chain, ST-cell receptor chain, ybT-cell receptor, or fragment thereof may be performed using any nucleic acid sequencing method known to the skilled person. Non-limiting examples include Sanger sequencing, single-molecule real-time sequencing, NGS (next generation sequencing), ion torrent sequencing, pyrosequencing, lllumina-sequencing, combinatorial probe anchor synthesis, sequencing by ligation (SOLID sequencing), Nanopore sequencing, GenapSys sequencing, and the like. Sequencing sample preparation, instruments, and protocols are discussed in standard handbooks like Head, Ordoukhanian and Salomon (Eds), Next Generation Sequencing: Methods and Protocols, Humana Press, NJ, USA (2018), incorporated herein by reference in its entirety, with many being commercially available, e.g. from Illumina (CA, USA), Pacific Biosciences (CA, USA), and others.

In some embodiments, the obtained yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, or fragment thereof is expressed by a cell as described later herein. In some embodiments, the yT-cell receptor chain, 6T-cell receptor chain, yST-cell receptor, or fragment thereof is obtained in a soluble form, as described later herein. yT-cell receptor chains, 5T-cell receptor chains, v5T-cell receptors, or fragments thereof

Further provided herein are yT-cell receptor chains or fragments thereof, ST-cell receptor chains or fragments thereof, and ybT-cell receptors or fragments thereof comprising obtained by or obtainable by the method described herein. A yT-cell receptor chain or fragment thereof, a 6T-cell receptor chain or fragment thereof, and/or a y5T-cell receptor or fragment thereof is preferably able to mediate an anti-tumour or anti- infective response. Assessment of mediation of an anti-tumour or anti-infective response may be performed using any of the assays described earlier herein. A yT-cell receptor chain, yST-cell receptor, or fragment thereof comprises a Cy constant region or fragment thereof. In preferred embodiments, the Gy constant region or fragment thereof is a Cy1 constant region or fragment thereof. In some embodiments, the Cy constant region or fragment thereof is a Cy2 constant region or fragment thereof. A 6T-cell receptor chain, yST-cell receptor, or fragment thereof comprises a C5 constant region or fragment thereof.

A yT-cell receptor chain, 6T-cell receptor chain, y5T-cell receptor, or fragment thereof, may be an isolated polypeptide. AyT-cell receptor chain, 6T-cell receptor chain, y5T-cell receptor, or fragment thereof, may be synthetically made. A yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, or fragment thereof, may be a mutant (variant or derivative) of a parent yT-cell receptor chain, ST-cell receptor chain, yBT-cell receptor, or fragment thereof. A parent sequence may be a wild-type sequence. A parent sequence may be another mutant sequence. A parent sequence may be a sequence not available in nature. A yT-cell receptor chain, ST-cell receptor chain, ybT-cell receptor, or fragment thereof, is preferably comprised, more preferably expressed, by a cell, for example in a cellular membrane. In embodiments wherein the yT-cell receptor chain, ST-cell receptor chain, ybT-cell receptor, or fragment thereof is comprised, preferably expressed, by a cell, said yT-cell receptor chain, ST-cell receptor chain, ybT-cell receptor, or fragment thereof is preferably exogenous (heterologous) to said cell. Assessment of expression may be performed using any of the assays described herein. In some embodiments, a yT-cell receptor chain, 5T-cell receptor chain, ybT-cell receptor, or fragment thereof is or is comprised in a soluble polypeptide. A "soluble” polypeptide as used herein refers to the polypeptide not being embedded in the plasma membrane of an intact cell.

A yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, or fragment thereof may comprise an amino acid modification relative to a reference sequence. The reference sequence may in some embodiments be a parent sequence to which an amino acid modification is introduced. In some embodiments, the reference sequence is not a parent sequence. In some embodiments, the reference sequence is a wild-type sequence. In some embodiments, the reference sequence is a sequence not available in nature. In some embodiments, the reference sequence is a mutant of a wild-type sequence. A yT-cell receptor chain, yST-cell receptor, or fragment thereof may comprise an amino acid modification in the Cy constant region or fragment thereof relative to a reference sequence. A 6T-cell receptor chain, y5T- cell receptor, or fragment thereof may comprise an amino acid modification in the C6 constant region or fragment thereof relative to a reference sequence. The amino acid modification may be at a position corresponding to a position selected from one or more positions in the reference sequence.

Preferably, the amino acid modification is selected from an amino acid substitution, deletion, insertion, or a combination thereof, as described earlier herein. In some embodiments, the amino acid modification is an amino acid substitution. In some embodiments, the amino acid modification is an amino acid insertion. In some embodiments, the amino acid modification is an amino acid deletion.

In an aspect, there is provided a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or fragment thereof, wherein the Cy constant region or fragment thereof comprises an amino acid modification relative to a reference Cy constant region or fragment thereof. Preferably, the Cy constant region or fragment thereof is a Cy1 constant region or fragment thereof.

In some embodiments, there is provided a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or fragment thereof, wherein the Cy constant region or fragment thereof comprises an amino acid modification relative to SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1. In some embodiments, the Cy constant region or fragment thereof comprises at least two, at least three, at least four, or at least five amino acid modifications. In some embodiments, the Cy constant region or fragment thereof comprises at least two amino acid modifications. In some embodiments, the Cy constant region or fragment thereof comprises at least three amino acid modifications. In some embodiments, the Cy constant region or fragment thereof comprises at least four amino acid modifications. In some embodiments, the Cy constant region or fragment thereof comprises at least five amino acid modifications. A yT-cell receptor chain or fragment thereof may, for example, be a y2T-, y3T-, y4T-, y5T-, y8T-, y9T-, or y11 T-cell receptor chain or fragment thereof. In some embodiments, the yT-cell receptor chain or fragment thereof is a y4T-cell receptor chain or fragment thereof. In some embodiments, the yT-cell receptor chain or fragment thereof is a y9T-cell receptor chain or fragment thereof. In some embodiments, the yT-cell receptor chain or fragment thereof is a y5T-cell receptor chain or fragment thereof. In some embodiments, the yT-cell receptor chain or fragment thereof is a y2T-cell receptor chain or fragment thereof

An amino acid modification may be comprised in a specific position in a Cy (such as Cy1 or Cy2) constant region or fragment thereof relative to a reference sequence. Identification of the location of corresponding amino acid positions in different Cy constant region sequences is described earlier herein. In some embodiments, an amino acid modification is comprised in the transmembrane domain of a Cy constant region, preferably a Cy1 or Cy2 region, more preferably a Cy1 region, relative to a reference sequence. In some embodiments, an amino acid modification is comprised in the FG-loop of a Cy constant region, preferably a Cy1 or Cy2 region, more preferably a Cy1 region, and/or in an adjacent amino acid position, relative to a reference sequence. In some embodiments, an amino acid modification is comprised in the a- loop of a Cy constant region, preferably a Cy1 or Cy2 region, more preferably a Cy1 region, relative to a reference sequence. In some embodiments, an amino acid modification is comprised in a CD3-interacting position of a Cy constant region, preferably a Cy1 or Cy2 region, more preferably a Cy1 region, relative to a reference sequence. In some embodiments, an amino acid modification is comprised in a glycosylation position of a Cy constant region, preferably a Cy1 or Cy2 region, more preferably a Cy1 region, relative to a reference sequence. In some embodiments, an amino acid modification corresponding to a cysteine- bridge forming position is comprised in a Cy constant region, preferably a Cy1 or Cy2 region, more preferably a Cy1 region, relative to a reference sequence.

It is understood that a combination of amino acid modifications may be comprised in a Cy constant region, preferably a Cy1 or Cy2 region, more preferably a Cy1 region, or a fragment thereof relative to a reference sequence. As a non-limiting example, an amino acid modification in the transmembrane domain and an amino acid modification in the FG-loop and/or in an adjacent amino acid position may be comprised. As another non-limiting example, an amino acid modification in the transmembrane domain and an amino acid modification in the a-loop may be comprised. As another non-limiting example, an amino acid modification in the transmembrane domain and an amino acid modification in a CD3-interacting position may be comprised. As another non-limiting example, an amino acid modification in the FG-loop and/or in an adjacent amino acid position and an amino acid modification in the a-loop may be comprised. As another non-limiting example, an amino acid modification in the FG-loop and/or in an adjacent amino acid position and an amino acid modification in a CD3-interacting position may be comprised. As another non-limiting example, an amino acid modification in a-loop and an amino acid modification in a CD3-interacting position may be comprised. As another non-limiting example, an amino acid modification in the transmembrane domain and an amino acid modification in a glycosylation position may be comprised. As another nonlimiting example, an amino acid modification in the FG-loop and/or in an adjacent amino acid position and an amino acid modification in a glycosylation position may be comprised. As another non-limiting example, an amino acid modification in the a-loop and an amino acid modification in a glycosylation position may be comprised. As another non-limiting example, an amino acid modification in a CD3-interacting position and an amino acid modification in a glycosylation position may be comprised. As another non-limiting example, an amino acid modification in the transmembrane domain, an amino acid modification in the FG-loop and/or in an adjacent amino acid position, and an amino acid modification in a CD3-interacting position may be comprised. As another non-limiting example, an amino acid modification in the transmembrane domain, an amino acid modification in the a-loop, and an amino acid modification in a CD3-interacting position may be comprised. As another non-limiting example, an amino acid modification in the transmembrane domain, an amino acid modification in the FG-loop and/or in an adjacent amino acid position, and an amino acid modification in the a-loop may be comprised. As another non-limiting example, an amino acid modification in the FG-loop and/or in an adjacent amino acid position, an amino acid modification in a CD3-interacting position, and an amino acid modification in the a-loop may be comprised.

As another non-limiting example, an amino acid modification in the transmembrane domain, an amino acid modification in the FG-loop and/or in an adjacent amino acid position, and an amino acid modification in a glycosylation position may be comprised. As another non-limiting example, an amino acid in the transmembrane domain, an amino acid modification in the a-loop, and an amino acid modification in a glycosylation position may be comprised. As another non-limiting example, an amino acid modification in the transmembrane domain, an amino acid modification in a CD3-interacting position, and an amino acid modification in a glycosylation position may be comprised. As another non-limiting example, an amino acid modification in the FG-loop and/or in an adjacent amino acid position, an amino acid modification in the a- loop, and an amino acid modification in a glycosylation position may be comprised. As another non-limiting example, an amino acid modification in the FG-loop and/or in an adjacent amino acid position, an amino acid modification in a CD3-interacting position, and an amino acid modification in a glycosylation position may be comprised. As another non-limiting example, an amino acid modification in the a-loop, an amino acid modification in a CD3-interacting position, and an amino acid modification in a glycosylation position may be comprised.

As another non-limiting example, an amino acid modification in the transmembrane domain, an amino acid modification in the FG-loop and/or in an adjacent amino acid position, an amino acid modification in a CD3- interacting position, and an amino acid modification in the a-loop may be comprised. As another non-limiting example, an amino acid modification in the transmembrane domain, an amino acid modification in the a- loop of a Cy constant region, an amino acid modification in a CD3-interacting position, and an amino acid modification in a glycosylation position may be comprised. As another non-limiting example, an amino acid modification in the transmembrane domain, an amino acid modification in the FG-loop of a Cy constant region and/or in an adjacent amino acid position, an amino acid modification in a CD3-interacting position, and an amino acid modification in a glycosylation position may be comprised. As another non-limiting example, an amino acid modification in the transmembrane domain, an amino acid modification in the FG- loop and/or to in adjacent amino acid position, an amino acid modification in the a-loop, and an amino acid modification in a glycosylation position may be comprised. As another non-limiting example, an amino acid modification in the FG-loop of a Cy constant region and/or in an adjacent amino acid position, an amino acid modification in a CD3-interacting position, an amino acid modification in the a-loop, and an amino acid modification in a glycosylation position may be comprised.

As another non-limiting example, an amino acid modification in the transmembrane domain, an amino acid modification in the FG-loop and/or in an adjacent amino acid position, an amino acid modification in a CD3- interacting position, an amino acid modification in the a-loop, and an amino acid modification in a glycosylation position may be comprised.

In some embodiments, an amino acid modification introduced in a position of a Cy constant region, preferably a Cy1 or Cy2 region, more preferably a Cy1 region, or fragment thereof is an amino acid substitution by a cysteine, preferably a Q58C substitution (as corresponding to the Q58 position of SEQ ID NO: 1 or SEQ ID NO: 39). In preferred embodiments, a reference Cy constant region is a Cy1 constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 1 . In some embodiments, a reference Cy constant region is a Cy1 constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 39. In some embodiments, a reference Cy constant region is a Cy2 constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 40. In some embodiments, a reference Cy constant region is a Cy2 constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 41.

In some embodiments, a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof, comprises an amino acid modification in a position corresponding to a position selected from D1 to V8 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably of SEQ ID NO: 1. In some embodiments, a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof, comprises an amino acid modification in a position corresponding to a position selected from K49 to Q101 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably of SEQ ID NO: 1. Among positions corresponding to a position selected from K49 to Q101 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably of SEQ ID NO: 1 , positions corresponding to a position selected from K49 to E79 and from S81 to G98 are preferred.

In some embodiments, a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof, comprises an amino acid modification in a position corresponding to a position selected from K49 to S81 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably of SEQ ID NO: 1. In some embodiments, a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof, comprises an amino acid modification in a position corresponding to a position selected from V90 to Q101 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably of SEQ ID NO: 1. In some embodiments, a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof, comprises an amino acid modification in a position corresponding to a position selected from E93 to D100 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably of SEQ ID NO: 1 . In some embodiments, a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof, comprises an amino acid modification in a position corresponding to a position selected from E93 to D100 of SEQ ID NO: 40 or SEQ ID NO: 41.

In some embodiments, a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof, comprises an amino acid modification in a position corresponding to a position selected from T136 to L161 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably of SEQ ID NO: 1. Among positions corresponding to a position selected from T136 to L161 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably of SEQ ID NO: 1, positions corresponding to a position selected from T136 to Y140 and from Y142 to L161 are preferred.

In some embodiments, a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof, comprises an amino acid modification in a position corresponding to a position selected from T152 to L177 of SEQ ID NO: 40 or SEQ ID NO: 41. Among positions corresponding to a position selected from T152 to L177 of SEQ ID NO: 40 or SEQ ID NO: 41 , positions corresponding to a position selected from T152 to Y156 and from Y158 to L177 are preferred.

Preferably, the Cy region or fragment thereof comprises at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or similarity with SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably with SEQ ID NO: 1. In some embodiments, the Cy constant region or fragment thereof comprises: a) an amino acid modification in a position corresponding to a position selected from D1 to V8 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 ; b) an amino acid modification in a position corresponding to a position selected from K49 to Q101 of SEQ ID NO : 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 ; c) an amino acid modification in a position corresponding to a position selected from T136 to L161 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , or; d) a combination of any one of a) to c).

In some embodiments, the amino acid modification in c) or d) is an amino acid substitution of a polar or an uncharged amino acid by a hydrophobic amino acid, preferably is a substitution of an S (serine), V (valine), F (phenylalanine), C (cysteine), or T (threonine) by a G (glycine), A (alanine), V (valine), L (leucine), I (isoleucine), P (proline), F (phenylalanine), M (methionine), or W (tryptophan), more preferably a substitution of:

- a S by a G, A, V, L, I, P, F, M, or W;

- a V by a G, A, L, I, P, F, M, or W;

- a F by a G, A, V, L, I, P, M, or W;

- a C by a G, A, V, L, I, P, F, M, or W;

- a T by a G, A, V, L, I, P, F, M, or W, or;

- a combination thereof.

In some embodiments, it is a substitution of a T (threonine) by a L (leucine). In some embodiments, it is a substitution of a S (serine) by an A (alanine). In some embodiments, it is a substitution of a V (valine) by a L (leucine). In some embodiments, it is a substitution of an F (phenylalanine) by an A (alanine). In some embodiments, it is a substitution of a C (cysteine) by a F (phenylalanine). In some embodiments, it is a substitution of a T (threonine) by a V (valine). In some embodiments, it is a substitution of a F (phenylalanine) by a L (leucine).

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position K49 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 . Preferably, the substitution is a K49A substitution (substitution by an alanine).

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position K50 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1. Preferably, the substitution is a K50A substitution (substitution by an alanine).

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position Q58 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 . Preferably, the substitution is a Q58C substitution (substitution by a cysteine).

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position N66 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 . Preferably, the substitution is a N66S substitution (substitution by an serine).

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position E79 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 . Preferably, the substitution is an E79A substitution (substitution by an alanine) or an E79D substitution (substitution by an aspartic acid). In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position K80 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1. Preferably, the substitution is a K80A substitution (substitution by an alanine) or a K80R substitution (substitution by an arginine).

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position S81 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 . Preferably, the substitution is a S81K substitution (substitution by a lysine).

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position K96 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 . Preferably, the substitution is a K96A substitution (substitution by an alanine) or a K96D substitution (substitution by an aspartic acid).

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position N97 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 . Preferably, the substitution is a N97A substitution (substitution by an alanine) or a N97K substitution (substitution by a lysine).

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position T136 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1. Preferably, the substitution is a T136L substitution (substitution by a leucine).

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position T152 of SEQ ID NO: 40 or SEQ ID NO: 41. Preferably, the substitution is a T152L substitution (substitution by a leucine).

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1. Preferably, the substitution is a S149A substitution (substitution by an alanine).

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position S165A of SEQ ID NO: 40 or SEQ ID NO: 41. Preferably, the substitution is a S165A substitution (substitution by an alanine).

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1. Preferably, the substitution is a V151 L substitution (substitution by a leucine).

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position V167 of SEQ ID NO: 40 or SEQ ID NO: 41. Preferably, the substitution is a V167L substitution (substitution by a leucine).

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position F153 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1. Preferably, the substitution is a F153A substitution (substitution by an alanine).

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position F169 of SEQ ID NO: 40 or SEQ ID NO: 41. Preferably, the substitution is a F169A substitution (substitution by an alanine).

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1. Preferably, the substitution is a C158F substitution (substitution by a phenylalanine).

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position C174 of SEQ ID NO: 40 or SEQ ID NO: 41. Preferably, the substitution is a C174F substitution (substitution by a phenylalanine).

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position C159 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1. Preferably, the substitution is a C159F substitution (substitution by a phenylalanine).

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position C175 of SEQ ID NO: 40 or SEQ ID NO: 41. Preferably, the substitution is a C175F substitution (substitution by a phenylalanine).

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position K49 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1, and a substitution in a position corresponding to position K50 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1. Preferably, the substitutions are respectively a K49A and a K50A substitution.

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position E79 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 , and a substitution in a position corresponding to position K80 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1. Preferably, the substitutions are respectively a E79A and a K80A substitution.

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position K96 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1, and a substitution in a position corresponding to position N97 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 . Preferably, the substitutions are respectively a K96A or K96D, preferably K96D, and an N97A or N97K, preferably N97K, substitution.

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, and a substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1. Preferably, the substitutions are respectively a S149A and a V151 L substitution.

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position S165A of SEQ ID NO: 40 or SEQ ID NO: 41, and a substitution in a position corresponding to position V167 of SEQ ID NO: 40 or SEQ ID NO: 41. Preferably, the substitutions are respectively a S165A and a V167L substitution.

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, and a substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1. Preferably, the substitutions are respectively a S149A and a C158F substitution.

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position S165A of SEQ ID NO: 40 or SEQ ID NO: 41 , and a substitution in a position corresponding to position C174 of SEQ ID NO: 40 or SEQ ID NO: 41. Preferably, the substitutions are respectively a S165A and a C174F substitution.

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, and a substitution in a position corresponding to position F153 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1. Preferably, the substitutions are respectively a S149A and a F153A substitution.

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position S165A of SEQ ID NO: 40 or SEQ ID NO: 41 , and a substitution in a position corresponding to position F169 of SEQ ID NO: 40 or SEQ ID NO: 41. Preferably, the substitutions are respectively a S165A and a F169A substitution.

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position K49, K50, K96, and N97 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1. Preferably, the substitutions are respectively a K49A, a K50A, a K96D, and an N97K substitution.

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position S149, V151 , and C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1. Preferably, the substitutions are respectively a S149A, a V151 L, and a C158F substitution.

In some embodiments, the Cy constant region or fragment thereof comprises a substitution in a position corresponding to position S165, V167, and C174 of SEQ ID NO: 40 or SEQ ID NO: 41. Preferably, the substitutions are respectively a S165A, a V167L, and a C174F substitution.

In some embodiments, the Cy constant region or fragment comprises a substitution of the positions corresponding to positions from D1 to V8 (DKQLDADV (SEQ ID NO: 42)) of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1, by the amino acid sequence EDLKNVF (SEQ ID NO: 43), DKEDLKNVF (SEQ ID NO: 176), or a variant thereof, preferably by the amino acid sequence EDLKNVF (SEQ ID NO: 43) or a variant thereof.

In some embodiments, the Cy constant region or fragment thereof comprises a substitution of the positions corresponding to positions from E93 to D100 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , (ENNKNGVD (SEQ ID NO: 44)) by the amino acid sequence YGLSENDEWTQDRAKPVT (SEQ ID NO: 46) or a variant thereof.

In some embodiments, the Cy constant region or fragment thereof comprises a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , (VRHENNKNGVDQ (SEQ ID NO: 48)) by the amino acid sequence VQFYGLSENDEWTQDRAKPVTQIV (SEQ ID NO: 47) or a variant thereof.

In some embodiments, the Cy constant region or fragment thereof comprises a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , (VRHENNKNGVDQ (SEQ ID NO: 48)) by the amino acid sequence VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50) or a variant thereof.

In some embodiments, the Cy constant region or fragment thereof comprises a substitution of the positions corresponding to positions from E93 to D100 of SEQ ID NO: 40 or SEQ ID NO: 41 (ENNKNGID (SEQ ID NO: 45)) by the amino acid sequence YGLSENDEWTQDRAKPVT (SEQ ID NO: 46) or a variant thereof.

In some embodiments, the Cy constant region or fragment thereof comprises a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 40 or SEQ ID NO: 41 (VRHENNKNGIDQ (SEQ ID NO: 49)) by the amino acid sequence VQFYGLSENDEWTQDRAKPVTQIV (SEQ ID NO: 47) or a variant thereof.

In some embodiments, the Cy constant region or fragment thereof comprises a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 40 or SEQ ID NO: 41 (VRHENNKNGIDQ (SEQ ID NO: 49)) by the amino acid sequence VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50) or a variant thereof.

In some embodiments, the Cy constant region or a fragment thereof comprises a substitution of the positions corresponding to positions from D1 to V8 (DKQLDADV (SEQ ID NO: 42)) of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , by the amino acid sequence EDLKNVF (SEQ ID NO: 43), DKEDLKNVF (SEQ ID NO: 176), or a variant thereof, preferably by the amino acid sequence EDLKNVF (SEQ ID NO: 43) or a variant thereof, and a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, (VRHENNKNGVDQ (SEQ ID NO: 48)) by the amino acid sequence VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50) or a variant thereof.

In some embodiments, the Cy constant region or a fragment thereof comprises a substitution of the positions corresponding to positions from D1 to V8 (DKQLDADV (SEQ ID NO: 42)) of SEQ ID NO: 40 or SEQ ID NO: 41 by the amino acid sequence EDLKNVF (SEQ ID NO: 43), DKEDLKNVF (SEQ ID NO: 176), or a variant thereof, preferably by the amino acid sequence EDLKNVF (SEQ ID NO: 43) or a variant thereof, and a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 40 or SEQ ID NO: 41 (VRHENNKNGIDQ (SEQ ID NO: 49)) by the amino acid sequence VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50) or a variant thereof.

In some embodiments, the Cy constant region or fragment thereof comprises:

I. an amino acid substitution in a position corresponding to position K49 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by an alanine;

II. an amino acid substitution in a position corresponding to position K50 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by an alanine;

III. an amino acid substitution in a position corresponding to position Q58 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by a cysteine;

IV. an amino acid substitution in a position corresponding to position N66 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by a serine;

V. an amino acid substitution in a position corresponding to position K96 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by an alanine or an aspartic acid;

VI. an amino acid substitution in a position corresponding to position N97 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by an alanine or a lysine;

VII. an amino acid substitution in a position corresponding to position T136 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by a leucine;

VIII. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by an alanine;

IX. an amino acid substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by a leucine;

X. an amino acid substitution in a position corresponding to position F153 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by an alanine;

XI. an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by a phenylalanine;

XII. an amino acid substitution in a position corresponding to position C159 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by a phenylalanine;

XIII. an amino acid substitution in a position corresponding to position K49 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position K50 of SEQ ID NO: 1, preferably a substitution by an alanine;

XIV. an amino acid substitution in a position corresponding to position E79 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position K80 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by an alanine; XV. an amino acid substitution in a position corresponding to position K96 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by an alanine or an aspartic acid, and an amino acid substitution in a position corresponding to position N97 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by an alanine or a lysine;

XVI. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by a leucine;

XVII. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by a phenylalanine;

XVIII. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position F153 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, preferably a substitution by an alanine;

XIX. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by an alanine, an amino acid substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by a leucine, and an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by a phenylalanine;

XX. an amino acid substitution in a position corresponding to position K49 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by an alanine, an amino acid substitution in a position corresponding to position K50 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by an alanine, an amino acid substitution in a position corresponding to position K96 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by an aspartic acid, and an amino acid substitution in a position corresponding to position N97 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , preferably a substitution by a lysine;

XXI. a substitution of the positions corresponding to positions from D1 to V8 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , by EDLKNVF (SEQ ID NO: 43), DKEDLKNVF (SEQ ID NO: 176), or a variant thereof, preferably by EDLKNVF (SEQ ID NO: 43) or a variant thereof;

XXII. a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, by VQFYGLSENDEWTQDRAKPVTQIV (SEQ ID NO: 47), VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50), or a variant thereof, or;

XXIII. a combination of any one of I. to XXII.

Preferably, the Cy region or fragment thereof comprised by the yT-cell receptor chains or fragments thereof described herein comprises at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or similarity with SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably with SEQ ID NO: 1.

In some embodiments, the Cy constant region or a fragment thereof comprises or consists of an amino acid sequence represented by SEQ ID NO: 3. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 6. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 7. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 8. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 9. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 10. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 15. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 16. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 17. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 21. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 22. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 23. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 24. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 25. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 26. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 27. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 28. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 29. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 30. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 31. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 32. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 35. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 37. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 95. In some embodiments, it comprises or consists of an amino acid sequence represented by SEQ ID NO: 177. In some embodiments, the Cy constant region or a fragment thereof comprises or consists of an amino acid sequence represented by SEQ ID NO: 3, 6, 7, 8, 9, 10, 15, 16, 17, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 35, 37, 95, or 177. In preferred embodiments, the Cy constant region or a fragment thereof comprises or consists of an amino acid sequence represented by SEQ ID NO: 3, 6, 7, 8, 9, 10, 15, 16, 17, 21, 22, 23, 24, 25, 26, 27, 29, 32, 35, 37, 95, or 177.

In some embodiments, the yT-cell receptor chain or fragment thereof further comprises a variable region or fragment thereof comprising, consisting essentially of, or consisting of, preferably comprising, an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 108, 109, 110, 111 , 112, 113, or 122. In some embodiments, it further comprises a variable region or fragment thereof comprising, consisting essentially of, or consisting of, preferably comprising, an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 108, 109, 110, 111 , 112, 113, 122, or 150.

In some embodiments, the yT-cell receptor chain or fragment thereof further comprises a CDR3 region comprising, consisting essentially of, or consisting of, preferably comprising, an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 124, 125, 126, 127, 128, 129, or 130. In some embodiments, it further comprises a CDR3 region comprising, consisting essentially of, or consisting of, preferably comprising, an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 124, 125, 126, 127, 128, 129, 130, or 152.

A yT-cell receptor chain or fragment thereof described herein may comprise a modification, preferably a substitution, at a specific amino acid position corresponding to a position of the CDR3 region of a different yT-cell receptor chain or fragment thereof. The skilled person is aware of how to locate such positions utilizing standard numbering nomenclature, for example IMGT as discussed earlier herein. For example, according to IMGT, in yT-cell receptor chains the CDR3 region is delimited by the anchor positions C104 and F118 or (or W118). Using this information, the skilled person can locate the amino acid sequence encoding a yCDR3 region in any reference amino acid sequence (or specific positions within it) of a yT-cell receptor or fragment thereof as well as corresponding positions in other reference sequences, and subsequently introduce one or more amino acid modifications as described herein. The same approach can be applied to 5T-cell receptor chains or fragments thereof, the CDR3 regions of which are also delimited by the abovementioned anchor positions, described later herein.

In some embodiments, the yT-cell receptor chain or fragment thereof further comprises a CDR3 region comprising at least at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or similarity with SEQ ID NO: 124, wherein the CDR3 region comprises a modification relative to SEQ ID NO: 124 at an amino acid position corresponding to a position selected from one or more of positions 4-10, preferably one or more of positions 5-9, of SEQ ID NO: 124. Preferably, the one or more modifications are one or more amino acid substitutions.

In cases wherein the CDR3 region comprises a modification relative to SEQ ID NO: 124 at an amino acid position corresponding to one or more of positions 4-10 of SEQ ID NO: 124, the CDR3 region preferably comprises an amino acid sequence selected from WDAFYYK (SEQ ID NO: 138), WEAFYYK (SEQ ID NO: 139), WDGYFYK (SEQ ID NO: 140), WDGYYYK (SEQ ID NO: 141), WDGAYYK (SEQ ID NO: 142), or WDGSYYK (SEQ ID NO: 143). In cases wherein the CDR3 region comprises a modification relative to SEQ ID NO: 124 at an amino acid position corresponding to one or more of positions 5-9 of SEQ ID NO: 124, the CDR3 region preferably comprises an amino acid sequence selected from DAFYY (SEQ ID NO: 169), EAFYY (SEQ ID NO: 170), DGYFY (SEQ ID NO: 171), DGYYY (SEQ ID NO: 172), DGAYY (SEQ ID NO: 173), and DGSYY (SEQ ID NO: 174).

In some embodiments, the yT-cell receptor chain or fragment thereof further comprises a CDR3 region comprising a modification at an amino acid position corresponding to position 4 of SEQ ID NO: 124. Preferably, said modification is an amino acid substitution.

In some embodiments, the yT-cell receptor chain or fragment thereof further comprises a CDR3 region comprising a modification at an amino acid position corresponding to position 5 of SEQ ID NO: 124. Preferably, said modification is an amino acid substitution, more preferably an amino acid substitution by a glutamic acid, most preferably a substitution of an aspartic acid by a glutamic acid.

In some embodiments, the yT-cell receptor chain or fragment thereof further comprises a CDR3 region comprising a modification at an amino acid position corresponding to position 6 of SEQ ID NO: 124. Preferably, said modification is an amino acid substitution, more preferably an amino acid substitution by an alanine, most preferably a substitution of a glycine by an alanine.

In some embodiments, the yT-cell receptor chain or fragment thereof further comprises a CDR3 region comprising a modification at an amino acid position corresponding to position 7 of SEQ ID NO: 124. Preferably, said modification is an amino acid substitution, more preferably an amino acid substitution by an alanine, serine, or tyrosine, more preferably a substitution of a phenylalanine by an alanine, serine, or tyrosine. In some embodiments, the yT-cell receptor chain or fragment thereof further comprises a CDR3 region comprising a modification at an amino acid position corresponding to position 8 of SEQ ID NO: 124. Preferably, said modification is an amino acid substitution, more preferably an amino acid substitution by a phenylalanine, most preferably a substitution of a tyrosine by a phenylalanine.

In some embodiments, the yT-cell receptor chain or fragment thereof further comprises a CDR3 region comprising a modification at an amino acid position corresponding to position 9 of SEQ ID NO: 124. Preferably, said modification is an amino acid substitution.

In some embodiments, the yT-cell receptor chain or fragment thereof further comprises a CDR3 region comprising a modification at an amino acid position corresponding to position 10 of SEQ ID NO: 124. Preferably, said modification is an amino acid substitution.

In some embodiments, a preferred yT-cell receptor chain or fragment thereof further comprises a CDR1 region (yCDR1 region) comprising, consisting essentially of, or consisting of amino acid sequence SEQ ID NO: 154, 155, 156, or 157, or of an amino acid sequence comprising one, two, or three amino acid modifications relative to SEQ ID NO: 154, 155, 156, or 157, and a CDR2 region (yCDR2 region) comprising, consisting essentially of, or consisting of amino acid sequence SEQ ID NO: 158, 159, 160, or 161, or of an amino acid sequence comprising one, two, or three amino acid modifications relative to SEQ ID NO: 158, 159, 160, or 161. In embodiments wherein the yT-cell receptor chain or fragment thereof is a y4T-cell receptor chain or fragment thereof, the yCDR1 and yCDR2 regions preferably comprise (or consist essentially of or consist of), respectively, amino acid sequences SEQ ID NO: 155 and SEQ ID NO: 159, or an amino acid sequence comprising one, two, or three amino acid modifications relative to SEQ ID NO: 155 and SEQ ID NO: 159. In embodiments wherein the yT-cell receptor chain or fragment thereof is a y9T-cell receptor chain or fragment thereof, the yCDR1 and yCDR2 regions preferably comprise (or consist essentially of or consist of), respectively, amino acid sequences SEQ ID NO: 157 and SEQ ID NO: 161, or an amino acid sequence comprising one, two, or three amino acid modifications relative to SEQ ID NO: 157 and SEQ ID NO: 161. In embodiments wherein the yT-cell receptor chain or fragment thereof is a y5T-cell receptor chain or fragment thereof, the yCDR1 and yCDR2 regions preferably comprise (or consist essentially of or consist of), respectively, amino acid sequences SEQ ID NO: 156 and SEQ ID NO: 160, or an amino acid sequence comprising one, two, or three amino acid modifications relative to SEQ ID NO: 156 and SEQ ID NO: 160. In embodiments wherein the yT-cell receptor chain or fragment thereof is a y2T-cell receptor chain or fragment thereof, the yCDR1 and yCDR2 regions preferably comprise (or consist essentially of or consist of), respectively, amino acid sequences SEQ ID NO: 154 and SEQ ID NO: 158, or an amino acid sequence comprising one, two, or three amino acid modifications relative to SEQ ID NO: 154 and SEQ ID NO: 158.

In some embodiments, a yT-cell receptor chain or fragment thereof provided herein mediates an anti-tumour or anti-infective response that is improved relative to a control yT-cell receptor chain, preferably a yT-cell receptor chain comprising a Cy constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , more preferably SEQ ID NO: 1. In some embodiments, the improvement is of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 100% (2-fold), at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to a control yT-cell receptor chain, preferably a yT-cell receptor chain comprising a Cy constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, more preferably SEQ ID NO: 1 .

In some embodiments, a yT-cell receptor chain or fragment thereof provided herein demonstrates improved expression, preferably surface expression, in an immunoresponsive cell, preferably a T-cell, more preferably an apT-cell, relative to a control yT-cell receptor chain, preferably a yT-cell receptor chain comprising a Cy constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , more preferably SEQ ID NO: 1. In some embodiments, the improvement is of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 100% (2-fold), at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to a control yT-cell receptor chain, preferably a yT-cell receptor chain comprising a Cy constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , more preferably SEQ ID NO: 1.

In an aspect, there is provided a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or fragment thereof, wherein the C6 constant region or fragment thereof comprises an amino acid modification relative to a reference C6 constant region or fragment thereof. In some embodiments, there is provided a 6T-cell receptor chain or a fragment thereof, comprising a C6 constant region or fragment thereof, wherein the C6 constant region or fragment thereof comprises an amino acid modification relative to SEQ ID NO: 2. In some embodiments, the C6 constant region or fragment thereof comprises at least two, at least three, at least four, or at least five amino acid modifications. In some embodiments, the C6 constant region or fragment thereof comprises at least two amino acid modifications. In some embodiments, the C6 constant region or fragment thereof comprises at least three amino acid modifications. In some embodiments, the C6 constant region or fragment thereof comprises at least four amino acid modifications. In some embodiments, the C6 constant region or fragment thereof comprises at least five amino acid modifications.

A 6T-cell receptor chain or fragment thereof may, for example, be a 61T-, 62T-, 63T-, or 65T-cell receptor chain or fragment thereof. In some embodiments, the 6T-cell receptor chain or fragment thereof is a 65T- cell receptor chain or fragment thereof. In some embodiments, the 6T-cell receptor chain or fragment thereof is a 61T-cell receptor chain or fragment thereof. In some embodiments, the 6T-cell receptor chain or fragment thereof is a 62T-cell receptor chain or fragment thereof.

An amino acid modification may be comprised in a specific position in a C6 constant region or fragment thereof relative to a reference sequence. Identification of the location of corresponding amino acid positions in different C6 constant region sequences is described earlier herein. In some embodiments, an amino acid modification is comprised in the transmembrane domain of a C6 constant region relative to a reference sequence. In some embodiments, an amino acid modification is comprised in a CD3-interacting position of a C5 constant region relative to a reference sequence. In some embodiments, an amino acid modification is comprised in a glycosylation position relative to a reference sequence. In some embodiments, an amino acid modification corresponding to a cysteine-bridge forming position is comprised relative to a reference sequence. In some embodiments, a reference C5 constant region comprises or consists of an amino acid sequence represented by SEQ ID NO: 2.

It is understood that a combination of amino acid modifications may be comprised in a C6 constant region or a fragment thereof relative to a reference sequence. As a non-limiting example, an amino acid modification in the transmembrane domain and an amino acid modification in a CD3-interacting position may be comprised. As another non-limiting example, an amino acid modification in the transmembrane domain and an amino acid modification in a glycosylation position may be comprised. As another nonlimiting example, an amino acid modification in a CD3-interacting position and an amino acid modification in a glycosylation position may be comprised. As another non-limiting example, an amino acid modification in the transmembrane domain, an amino acid modification in a CD3-interacting position, and an amino acid modification in a glycosylation position may be comprised.

In some embodiments, a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof, comprises an amino acid modification in a position corresponding to a position selected from N14 to V48 of SEQ ID NO: 2. In some embodiments, a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof, comprises an amino acid modification in a position corresponding to a position selected from L127 to F152 of SEQ ID NO: 2.

In some embodiments, an amino acid modification introduced in a position of a C5 constant region or fragment thereof is an amino acid substitution by a cysteine, preferably a V48C substitution (as corresponding to the V48 position of SEQ ID NO: 2).

In some embodiments, the C5 constant region or fragment thereof comprises: e) an amino acid modification in a position corresponding to a position selected from N14 to V48 of SEQ ID NO: 2; f) an amino acid modification in a position corresponding to a position selected from L127 to L152 of SEQ ID NO: 2, or; g) a combination of e) and f).

In some embodiments, the amino acid modification in f) or g) is an amino acid substitution of a polar or an uncharged amino acid by a hydrophobic amino acid, preferably is a substitution of an S (serine), V (valine), F (phenylalanine), C (cysteine), or T (threonine) by a G (glycine), A (alanine), V (valine), L (leucine), I (isoleucine), P (proline), F (phenylalanine), M (methionine), or W (tryptophan), more preferably a substitution of:

- a S by a G, A, V, L, I, P, F, M, or W;

- a V by a G, A, L, I, P, F, M, or W;

- a F by a G, A, V, L, I, P, M, or W;

- a C by a G, A, V, L, I, P, F, M, or W;

- a T by a G, A, V, L, I, P, F, M, or W, or;

- a combination thereof.

In some embodiments, the C5 constant region or fragment thereof comprises a substitution in a position corresponding to position N14 of SEQ ID NO: 2. Preferably, the substitution is a N14S substitution (substitution by a serine).

In some embodiments, the C5 constant region or fragment thereof comprises a substitution in a position corresponding to position V48 of SEQ ID NO: 2. Preferably, the substitution is a V48C substitution (substitution by a cysteine).

In some embodiments, the C5 constant region or fragment thereof comprises a substitution in a position corresponding to position T128 of SEQ ID NO: 2. Preferably, the substitution is a T128L substitution (substitution by a leucine).

In some embodiments, the C5 constant region or fragment thereof comprises a substitution in a position corresponding to position T139 of SEQ ID NO: 2. Preferably, the substitution is a T139V substitution (substitution by a valine).

In some embodiments, the C5 constant region or fragment thereof comprises a substitution in a position corresponding to position F144 of SEQ ID NO: 2. Preferably, the substitution is an F144L substitution (substitution by a leucine).

In some embodiments, the C5 constant region or fragment thereof comprises a substitution in a position corresponding to position T128 of SEQ ID NO: 2 and a substitution in a position corresponding to position T139 of SEQ ID NO: 2. Preferably, the substitutions are respectively a T128L and a T139V substitution.

In some embodiments, the C6 constant region or fragment thereof comprises a substitution in a position corresponding to position T139 of SEQ ID NO: 2 and a substitution in a position corresponding to position F144 of SEQ ID NO: 2. Preferably, the substitutions are respectively a T139V and a F144L substitution.

In some embodiments, the C6 constant region or fragment thereof comprises a substitution in a position corresponding to position T128, T139, and F144 of SEQ ID NO: 2. Preferably, the substitutions are respectively a T128L, a T139V, and a F144L substitution.

In some embodiments, the C5 constant region or fragment thereof comprises:

XXIX. an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, preferably a substitution by a leucine, and an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, preferably a substitution by a valine; XXX. an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, preferably a substitution by a valine, and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, preferably a substitution by a leucine;

XXXII. a combination of any one of XXIV. to XXXII.

Preferably, the C5 region or fragment thereof comprised by the 5T-cell receptor chains or fragments thereof described herein comprises at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or similarity with SEQ ID NO: 2.

In some embodiments, the C5 constant region or a fragment thereof comprises or consists of an amino acid sequence represented by SEQ ID NO: 11. In some embodiments, the C5 constant region or a fragment thereof comprises or consists of an amino acid sequence represented by SEQ ID NO: 13. In some embodiments, the C5 constant region or a fragment thereof comprises or consists of an amino acid sequence represented by SEQ ID NO: 14. In some embodiments, the C5 constant region or a fragment thereof comprises or consists of an amino acid sequence represented by SEQ ID NO: 18. In some embodiments, the C5 constant region or a fragment thereof comprises or consists of an amino acid sequence represented by SEQ ID NO: 19. In some embodiments, the C5 constant region or a fragment thereof comprises or consists of an amino acid sequence represented by SEQ ID NO: 20. In some embodiments, the C5 constant region or a fragment thereof comprises or consists of an amino acid sequence represented by SEQ ID NO: 36. In some embodiments, the C5 constant region or a fragment thereof comprises or consists of an amino acid sequence represented by SEQ ID NO: 38. In some embodiments, the C5 constant region or a fragment thereof comprises or consists of an amino acid sequence represented by SEQ ID NO: 11, 13, 14, 18, 19, 20, 36, or 38.

In some embodiments, the 5T-cell receptor chain or a fragment thereof further comprises a variable region or fragment thereof comprising, consisting essentially of, or consisting of, preferably comprising, an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 114, 115, 116, 117, 118, 119, or 123. In some embodiments, it further comprises a variable region or fragment thereof comprising, consisting essentially of, or consisting of, preferably comprising, an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 114, 115, 116, 117, 118, 119, 123, or 151.

In some embodiments, the 5T-cell receptor chain or a fragment thereof further comprises a CDR3 region comprising, consisting essentially of, or consisting of, preferably comprising, an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 131, 132, 133, 134, 135, 136, or 137. In some embodiments, it further comprises a CDR3 region comprising, consisting essentially of, or consisting of, preferably comprising, an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence Identity or similarity with amino acid sequence SEQ ID NO: 131 , 132, 133, 134, 135, 136, 137, or 153.

A 5T-cell receptor chain or fragment thereof described herein may comprise a modification, preferably a substitution, at a specific amino acid position corresponding to a position of the CDR3 region of a different 5T-cell receptor chain or fragment thereof.

In some embodiments, the 5T-cell receptor chain or fragment thereof further comprises a CDR3 region comprising at least at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or similarity with SEQ ID NO: 131, wherein the CDR3 region comprises a modification relative to SEQ ID NO: 131 at an amino acid position corresponding to a position selected from one or more of positions 7-12 of SEQ ID NO: 131. Preferably, the one or more modifications are one or more amino acid substitutions.

In cases wherein the CDR3 region comprises a modification relative to SEQ ID NO: 131 at an amino acid position corresponding to one or more of positions 7-12 of SEQ ID NO: 131 , the CDR3 region preferably comprises an amino acid sequence selected from IRGFTG (SEQ ID NO: 144), IKGYTG (SEQ ID NO: 145), IKGFTG (SEQ ID NO: 146), LRGFTG (SEQ ID NO: 147), LKGFTG (SEQ ID NO: 148), or LKGYTG (SEQ ID NO: 149).

In some embodiments, the 5T-cell receptor chain or a fragment thereof further comprises a CDR3 region comprising a modification at an amino acid position corresponding to position 7 of SEQ ID NO: 131. Preferably, said modification is an amino acid substitution, more preferably an amino acid substitution by a leucine, most preferably a substitution of an isoleucine by a leucine.

In some embodiments, the 5T-cell receptor chain or a fragment thereof further comprises a CDR3 region comprising a modification at an amino acid position corresponding to position 8 of SEQ ID NO: 131. Preferably, said modification is an amino acid substitution, more preferably an amino acid substitution by a lysine, most preferably a substitution of an arginine by a lysine.

In some embodiments, the 5T-cell receptor chain or a fragment thereof further comprises a CDR3 region comprising a modification at an amino acid position corresponding to 9 of SEQ ID NO: 131. Preferably, said modification is an amino acid substitution.

In some embodiments, the 5T-cell receptor chain or a fragment thereof further comprises a CDR3 region comprising a modification at an amino acid position corresponding to position 10 of SEQ ID NO: 131. Preferably, said modification is an amino acid substitution, more preferably an amino acid substitution by a phenylalanine, most preferably a substitution of a tyrosine by a phenylalanine.

In some embodiments, the 5T-cell receptor chain or a fragment thereof further comprises a CDR3 region comprising a modification at an amino acid position corresponding to position 11 of SEQ ID NO: 131. Preferably, said modification is an amino acid substitution.

In some embodiments, the 5T-cell receptor chain or a fragment thereof further comprises a CDR3 region comprising a modification at an amino acid position corresponding to position 12 of SEQ ID NO: 131. Preferably, said modification is an amino acid substitution.

In some embodiments, a preferred 5T-cell receptor chain or fragment thereof further comprises a CDR1 region (5CDR1 region) comprising, consisting essentially of, or consisting of amino acid sequence SEQ ID NO: 162, 163, or 164, or of an amino acid sequence comprising one, two, or three amino acid modifications relative to SEQ ID NO: 162, 163, or 164, and a CDR2 region (5CDR2 region) comprising, consisting essentially of, or consisting of amino acid sequence EKD, QGS, or SEQ ID NO: 166, or of an amino acid sequence comprising one, two, or three amino acid modifications relative to amino acid sequence EKD, QGS, or SEQ ID NO: 166.

In embodiments wherein the 5T-cell receptor chain or fragment thereof is a 65T-cell receptor chain or fragment thereof, the 6CDR1 and 6CDR2 regions preferably comprise (or consist essentially of or consist of), respectively, amino acid sequences SEQ ID NO: 164 and SEQ ID NO: 166, or an amino acid sequence comprising one, two, or three amino acid modifications relative to SEQ ID NO: 164 and SEQ ID NO: 166. In embodiments wherein the 5T-cell receptor chain or fragment thereof is a 61 T-cell receptor chain or fragment thereof, the 6CDR1 and 6CDR2 regions preferably comprise (or consist essentially of or consist of), respectively, amino acid sequences SEQ ID NO: 162 and QGS, or an amino acid sequence comprising one, two, or three amino acid modifications relative to SEQ ID NO: 162 and QGS. In embodiments wherein the 6T-cell receptor chain or fragment thereof is a 62T-cell receptor chain or fragment thereof, the 6CDR1 and 6CDR2 regions preferably comprise (or consist essentially of or consist of), respectively, amino acid sequences SEQ ID NO: 163 and EKD, or an amino acid sequence comprising one, two, or three amino acid modifications relative to SEQ ID NO: 163 and EKD.

In some embodiments, the 6T-cell receptor chain or fragment thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281 , 282, 283, 284, 285, 286, 287, 288, or 289.

In some embodiments, a 6T-cell receptor chain or fragment thereof provided herein mediates an anti-tumour or anti-infective response that is improved relative to a control 6T-cell receptor chain, preferably a 6T-cell receptor chain comprising a C6 constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2. In some embodiments, the improvement is of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 100% (2-fold), at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to a control 6T-cell receptor chain, preferably a 61- cell receptor chain comprising a C6 constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, a 6T-cell receptor chain or fragment thereof provided herein demonstrates improved expression, preferably surface expression, in an immunoresponsive cell, preferably a T-cell, more preferably an apT-cell, relative to a control 6T-cell receptor chain, preferably a 6T-cell receptor chain comprising a C6 constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2. In some embodiments, the improvement is of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 100% (2-fold), at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to a control 6T-cell receptor chain, preferably a 6T-cell receptor chain comprising a C6 constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

It is understood that a yT-cell receptor chain or fragment thereof described herein may be paired with a 6T- cell receptor chain or fragment thereof. Pairing with any 6T-cell receptor chain or fragment thereof may be contemplated, as long as the chains or fragments thereof are able to form a ySTCR or fragment thereof that can mediate an anti-tumour or anti-infective response.

It is also understood that a ST-cell receptor chain or fragment thereof described herein may be paired with a yT -cell receptor chain or fragment thereof. Pairing with any yT -cell receptor chain or fragment thereof may be contemplated, as long as the chains or fragments thereof are able to form a ySTCR that can mediate an anti-tumour or anti-infective response.

Accordingly, in a further aspect, there is provided y5T-cell receptor or a fragment thereof comprising: a yT-cell receptor chain or fragment thereof as described herein; a 5T-cell receptor chain or fragment thereof as described herein, or; a yT-cell receptor chain or fragment thereof as described herein and a 5T-cell receptor chain or fragment thereof as described herein.

Thus, it is understood that a yST-cell receptor or a fragment thereof comprises a yT-cell receptor chain or fragment thereof as described herein and/or a 5T-cell receptor chain or fragment thereof as described herein. Preferably, the yST-cell receptor or a fragment thereof comprises a yT-cell receptor chain or fragment thereof as described herein and a 5T-cell receptor chain or fragment thereof as described herein.

In some embodiments, the yST-cell receptor or fragment thereof comprises: a yT-cell receptor chain or fragment thereof as described herein, and/or, preferably and; a 5T-cell receptor chain or fragment thereof comprising a C5 constant region or fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, the yST-cell receptor or fragment thereof comprises: a yT-cell receptor chain or fragment thereof comprising a Cy constant region or fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1, and/or, preferably and; a 6T-cell receptor chain or fragment thereof as described herein.

In some embodiments, the yST-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising:

X. an amino acid substitution in a position corresponding to position F153 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine;

XI. an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a phenylalanine;

XXIII. a combination of any one of I. to XXII, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising:

XXXII. a combination of any one of XXIV. To XXXI.

In some embodiments, the y5T-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, the substitution preferably being a C158F substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitution preferably being a F144L substitution.

In some embodiments, the y5T-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position C174 of SEQ ID NO: 40 or SEQ ID NO: 41 , the substitution preferably being a C174F substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitution preferably being a F144L substitution. In some embodiments, the y5T-cell receptor or fragment thereof comprises:

-a YT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 9, and/or, preferably and;

-a 5T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 14.

In some embodiments, the yQT-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, the substitution preferably being a S149A substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, the substitution preferably being a T139V substitution.

In some embodiments, the yBT-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S165 of SEQ ID NO: 40 or SEQ ID NO: 41 , the substitution preferably being a S165A substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, the substitution preferably being a T139V substitution.

In some embodiments, the yST-cell receptor or fragment thereof comprises:

-a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 6, and/or, preferably and;

-a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 13.

In some embodiments, the y5T-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , the substitution preferably being a S149A substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2 and an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, the substitutions preferably being respectively a T128L and a T139V substitution.

In some embodiments, the y5T-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S165 of SEQ ID NO: 40 or SEQ ID NO: 41 , the substitution preferably being a S165A substitution, and/or, preferably and; a 6T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2 and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T139V and a F144L substitution.

In some embodiments, the yST-cell receptor or fragment thereof comprises:

-a 5T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 18.

In some embodiments, the yBT-cell receptor or fragment thereof comprises:

-a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, and an amino acid substitution in a position corresponding to position F153 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , the substitutions preferably being respectively a S149A and a F153A substitution, and/or, preferably and;

-a 5T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, and an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, the substitutions preferably being respectively a T128L and a T139V substitution.

In some embodiments, the y5T-cell receptor or fragment thereof comprises:

-a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S165 of SEQ ID NO: 40 or SEQ ID NO: 41 , and an amino acid substitution in a position corresponding to position F169 of SEQ ID NO: 40 or SEQ ID NO: 41 , the substitutions preferably being respectively a S165A and a F169A substitution, and/or, preferably and;

-a 6T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, and an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, the substitutions preferably being respectively a T128L and a T139V substitution.

In some embodiments, the y5T-cell receptor or fragment thereof comprises:

-a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 175 and/or, preferably and;

-a 6T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 18.

In some embodiments, the y5T-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, the substitution preferably being a S149A substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2 and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T139V and a F144L substitution.

In some embodiments, the yST-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S165 of SEQ ID NO: 40 or SEQ ID NO: 41 , the substitution preferably being a S165A substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2 and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T139V and a F144L substitution.

In some embodiments, the yBT-cell receptor or fragment thereof comprises:

-a 5T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 19.

In some embodiments, the y5T-cell receptor or fragment thereof comprises:

-a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, the substitution preferably being a S149A substitution, and/or, preferably and;

-a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T128L, a T139V, and a F144L substitution.

In some embodiments, the y5T-cell receptor or fragment thereof comprises:

-a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S165 of SEQ ID NO: 40 or SEQ ID NO: 41, the substitution preferably being a S165A substitution, and/or, preferably and;

In some embodiments, the y5T-cell receptor or fragment thereof comprises: -a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 6, and/or, preferably and;

-a 5T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 20.

In some embodiments, the yST-cell receptor or fragment thereof comprises:

-a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , and an amino acid substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , the substitutions preferably being respectively a S149A and a V151 L substitution, and/or, preferably and;

-a 5T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T128L, a T139V, and a F144L substitution.

In some embodiments, the yBT-cell receptor or fragment thereof comprises:

-a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S165 of SEQ ID NO: 40 or SEQ ID NO: 41 , and an amino acid substitution in a position corresponding to position V167 of SEQ ID NO: 40 or SEQ ID NO: 41 , the substitutions preferably being respectively a S165A and a V167L substitution, and/or, preferably and;

In some embodiments, the y5T-cell receptor or fragment thereof comprises:

-a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 15, and/or, preferably and;

-a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 20.

In some embodiments, the y5T-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , an amino acid substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, and an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , the substitutions preferably being respectively a S149A, a V151 L, and a C158F substitution, and/or, preferably and; a 6T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2 and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T139V and a F144L substitution.

In some embodiments, the yST-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S165 of SEQ ID NO: 40 or SEQ ID NO: 41 , an amino acid substitution in a position corresponding to position V167 of SEQ ID NO: 40 or SEQ ID NO: 41, and an amino acid substitution in a position corresponding to position C174 of SEQ ID NO: 40 or SEQ ID NO: 41, the substitutions preferably being respectively a S165A, a V167L, and a C174F substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2 and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T139V and a F144L substitution.

In some embodiments, the yBT-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 17, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 19.

In some embodiments, the y5T-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, and an amino acid substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , the substitutions preferably being respectively a S149A and a V151 L substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2 and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T139V and a F144L substitution.

In some embodiments, the y5T-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S165 of SEQ ID NO: 40 or SEQ ID NO: 41 , and an amino acid substitution in a position corresponding to position V167 of SEQ ID NO: 40 or SEQ ID NO: 41, the substitutions preferably being respectively a S165A and a V167L substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2 and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T139V and a F144L substitution. In some embodiments, the yST-cell receptor or fragment thereof comprises: a YT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 15, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 19.

In some embodiments, the yST-cell receptor or fragment thereof comprises: a YT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, and an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, the substitutions preferably being respectively a S149A and a C158F substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2 and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T139V and a F144L substitution.

In some embodiments, the Y6T-cell receptor or fragment thereof comprises: a YT-cell receptor chain or a fragment thereof, comprising a CY constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S165 of SEQ ID NO: 40 or SEQ ID NO: 41, and an amino acid substitution in a position corresponding to position C174 of SEQ ID NO: 40 or SEQ ID NO: 41 , the substitutions preferably being respectively a S165A and a C174F substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2 and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T139V and a F144L substitution.

In some embodiments, the Y5T-cell receptor or fragment thereof comprises: a YT-cell receptor chain or a fragment thereof, comprising a CY constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 16, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 19.

In some embodiments, the Y6T-cell receptor or fragment thereof comprises: a YT-cell receptor chain or a fragment thereof, comprising a CY constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, and an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, the substitutions preferably being respectively a S149A and a C158F substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T128L, T139V, and a F144L substitution. In some embodiments, the y5T-cell receptor or fragment thereof comprises: a YT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S165 of SEQ ID NO: 40 or SEQ ID NO: 41, and an amino acid substitution in a position corresponding to position C174 of SEQ ID NO: 40 or SEQ ID NO: 41 , the substitutions preferably being respectively a S165A and a C174F substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T128L, T139V, and a F144L substitution.

In some embodiments, the yBT-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 16, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 20.

In some embodiments, the y5T-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position K49 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 , and an amino acid substitution in a position corresponding to position K50 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 , the substitutions preferably being respectively a K49A and a K50A substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, the y5T-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 26, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, the y5T-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position E79 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 , and an amino acid substitution in a position corresponding to position K80 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, the substitutions preferably being respectively a E79A and a K80A substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2. In some embodiments, the y5T-cell receptor or fragment thereof comprises: a YT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 29, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, the yST-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position K96 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1, and an amino acid substitution in a position corresponding to position N97 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1, the substitutions preferably being respectively a K96A and a N97A substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, the y5T-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 32, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, the y5T-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position K96 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1, and an amino acid substitution in a position corresponding to position N97 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1, the substitutions preferably being respectively a K96D and a N97K substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, the y5T-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 27, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, the y5T-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position K49 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1, an amino acid substitution in a position corresponding to position K50 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 , an amino acid substitution in a position corresponding to position K96 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1, and an amino acid substitution in a position corresponding to position N97 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1, the substitutions preferably being respectively a K49A, K50A, K96D, and a N97K substitution, and/or, preferably and; a ST-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, the ybT-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 95 and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, the yBT-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position N66 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 , the substitution preferably being a N66S substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position N14 of SEQ ID NO: 2, the substitution preferably being a N14S substitution.

In some embodiments, the y5T-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 35 and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 36.

In some embodiments, the yST-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position Q58 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 , the substitution preferably being a Q58C substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position V48 of SEQ ID NO: 2, the substitution preferably being a V48C substitution.

In some embodiments, the y5T-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 37 and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 38.

In some embodiments, the y6T-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from D1 to V8 (DKQLDADV (SEQ ID NO: 42)) of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 , by the amino acid sequence EDLKNVF (SEQ ID NO: 43), DKEDLKNVF (SEQ ID NO: 176), or a variant thereof, preferably by the amino acid sequence EDLKNVF (SEQ ID NO: 43) or a variant thereof, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, the yQT-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 21 , and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, the y5T-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from E93 to D100 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, (ENNKNGVD (SEQ ID NO: 44)) by the amino acid sequence YGLSENDEWTQDRAKPVT (SEQ ID NO: 46) or a variant thereof, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, the y6T-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from E93 to D100 of SEQ ID NO: 40 or SEQ ID NO: 41 (ENNKNGID (SEQ ID NO: 45)) by the amino acid sequence YGLSENDEWTQDRAKPVT (SEQ ID NO: 46) or a variant thereof, and/or, preferably and; a 6T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, the y6T-cell receptor or fragment thereof comprises : a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , (VRHENNKNGVDQ (SEQ ID NO: 48)) by the amino acid sequence VQFYGLSENDEWTQDRAKPVTQIV (SEQ ID NO: 47) or a variant thereof, and/or, preferably and; a 6T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, the y6T-cell receptor or fragment thereof comprises : a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 40 or SEQ ID NO: 41 (VRHENNKNGIDQ (SEQ ID NO: 49)) by the amino acid sequence VQFYGLSENDEWTQDRAKPVTQIV (SEQ ID NO: 47) or a variant thereof, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, the yST-cell receptor or fragment thereof comprises : a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , (VRHENNKNGVDQ (SEQ ID NO: 48)) by the amino acid sequence VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50) or a variant thereof, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, the yST-cell receptor or fragment thereof comprises : a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 40 or SEQ ID NO: 41 (VRHENNKNGIDQ (SEQ ID NO: 49)) by the amino acid sequence VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50) or a variant thereof, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, the y5T-cell receptor or fragment thereof comprises : a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from D1 to V8 (DKQLDADV (SEQ ID NO: 42)) of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, by the amino acid sequence EDLKNVF (SEQ ID NO: 43), DKEDLKNVF (SEQ ID NO: 176), or a variant thereof, preferably by the amino acid sequence EDLKNVF (SEQ ID NO: 43) or a variant thereof, and a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, (VRHENNKNGVDQ (SEQ ID NO: 48)) by the amino acid sequence VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50) or a variant thereof, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, the yST-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from D1 to V8 (DKQLDADV (SEQ ID NO: 42)) of SEQ ID NO: 40 or SEQ ID NO: 41 by the amino acid sequence EDLKNVF (SEQ ID NO: 43), DKEDLKNVF (SEQ ID NO: 176), or a variant thereof, preferably by the amino acid sequence EDLKNVF (SEQ ID NO: 43) or a variant thereof, and a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 40 or SEQ ID NO: 41 (VRHENNKNGIDQ (SEQ ID NO: 49)) by the amino acid sequence VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50) or a variant thereof, and/or, preferably and; a 6T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, the yST-cell receptor or fragment thereof comprises: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 177, preferably SEQ ID NO: 24 or SEQ ID NO: 177, more preferably SEQ ID NO: 24, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

Preferably, the Cy region or fragment thereof comprised by the y5T-cell receptors or fragments thereof described herein comprises at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or similarity with SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably with SEQ ID NO: 1 , and the C5 region or fragment thereof comprised by the yST-cell receptors or fragments thereof described herein comprises at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or similarity with SEQ ID NO: 2.

In some embodiments, the yST-cell receptor or fragment thereof further comprises:

- a y variable region or fragment thereof comprising, consisting essentially of, or consisting of, preferably comprising, an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 108, 109, 110, 111, 112, 113, or 122, and/or, preferably and;

- a 5 variable region or fragment thereof comprising, consisting essentially of, or consisting of, preferably comprising, an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 114, 115, 116, 117, 118, 119, or 123.

- a y variable region or fragment thereof comprising, consisting essentially of, or consisting of, preferably comprising, an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 108, 109, 110, 111, 112, 113, 122, or 150 and/or, preferably and;

- a 5 variable region or fragment thereof comprising, consisting essentially of, or consisting of, preferably comprising, an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 114, 115, 116, 117, 118, 119, 123, or 151.

In some embodiments, the y5T-cell receptor or fragment thereof further comprises:

- a yCDR3 region comprising, consisting essentially of, or consisting of, preferably comprising, an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 124, 125, 126, 127, 128, 129, or 130, and/or, preferably and;

- a 5CDR3 region comprising, consisting essentially of, or consisting of, preferably comprising, an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 131 , 132, 133, 134, 135, 136, or 137.

- a yCDR3 region comprising, consisting essentially of, or consisting of, preferably comprising, an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 124, 125, 126, 127, 128, 129, 130, or 152 and/or, preferably and;

- a 6CDR3 region comprising, consisting essentially of, or consisting of, preferably comprising, an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 131 , 132, 133, 134, 135, 136, 137, or 153.

- a yCDR3 region comprising at least at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or similarity with SEQ ID NO: 124, wherein the CDR3 region comprises a modification relative to SEQ ID NO: 124 at an amino acid position corresponding to a position selected from one or more of positions 4-10, preferably one or more of positions 5-9, of SEQ ID NO: 124, and/or, preferably and;

- a 6CDR3 region comprising at least at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or similarity with SEQ ID NO: 131, wherein the CDR3 region comprises a modification relative to SEQ ID NO: 131 at an amino acid position corresponding to a position selected from one or more of positions 7-12 of SEQ ID NO: 131.

Preferably, the one or more modifications are amino acid substitutions. In cases wherein the yCDR3 region comprises a modification relative to SEQ ID NO: 124 at an amino acid position corresponding to one or more of positions 4-10 of SEQ ID NO: 124, the yCDR3 region preferably comprises an amino acid sequence selected from WDAFYYK (SEQ ID NO: 138), WEAFYYK (SEQ ID NO: 139), WDGYFYK (SEQ ID NO: 140), WDGYYYK (SEQ ID NO: 141), WDGAYYK (SEQ ID NO: 142), or WDGSYYK (SEQ ID NO: 143). In cases wherein the yCDR3 region comprises a modification relative to SEQ ID NO: 124 at an amino acid position corresponding to one or more of positions 5-9 of SEQ ID NO: 124, the yCDR3 region preferably comprises an amino acid sequence selected from DAFYY (SEQ ID NO: 169), EAFYY (SEQ ID NO: 170), DGYFY (SEQ ID NO: 171), DGYYY (SEQ ID NO: 172), DGAYY (SEQ ID NO: 173), and DGSYY (SEQ ID NO: 174). In cases wherein the 5CDR3 region comprises a modification relative to SEQ ID NO: 131 at an amino acid position corresponding to one or more of positions 7-12 of SEQ ID NO: 131, the CDR3 region preferably comprises an amino acid sequence selected from IRGFTG (SEQ ID NO: 144), IKGYTG (SEQ ID NO: 145), IKGFTG (SEQ ID NO: 146), LRGFTG (SEQ ID NO: 147), LKGFTG (SEQ ID NO: 148), or LKGYTG (SEQ ID NO: 149).

-a yCDR3 region comprising a modification at an amino acid position selected from one or more of positions 4-10 of SEQ ID NO: 124, said modification selected from WDAFYYK (SEQ ID NO: 138), WEAFYYK (SEQ ID NO: 139), WDGYFYK (SEQ ID NO: 140), WDGYYYK (SEQ ID NO: 141), WDGAYYK (SEQ ID NO: 142), or WDGSYYK (SEQ ID NO: 143), and/or, preferably and; -a 5CDR3 region comprising a modification at an amino acid position corresponding to a position selected from one or more of positions 7-12 of SEQ ID NO: 131 , said modification selected from IRGFTG (SEQ ID NO: 144), IKGYTG (SEQ ID NO: 145), IKGFTG (SEQ ID NO: 146), LRGFTG (SEQ ID NO: 147), LKGFTG (SEQ ID NO: 148), or LKGYTG (SEQ ID NO: 149).

In some embodiments, a preferred yST-cell receptor or fragment thereof further comprises:

- a yCDR1 region comprising, consisting essentially of, or consisting of amino acid sequence SEQ ID NO: 154, 155, 156, or 157, or of an amino acid sequence comprising one, two, or three amino acid modifications relative to SEQ ID NO: 154, 155, 156, or 157, and a yCDR2 region comprising, consisting essentially of, or consisting of amino acid sequence SEQ ID NO: 158, 159, 160, or 161, or of an amino acid sequence comprising one, two, orthree amino acid modifications relative to SEQ ID NO: 158, 159, 160, or 161, and/or, preferably and;

-a 6CDR1 region comprising, consisting essentially of, or consisting of amino acid sequence SEQ ID NO: 162, 163, or 164, or of an amino acid sequence comprising one, two, or three amino acid modifications relative to SEQ ID NO: 162, 163, or 164, and a 5CDR2 region comprising, consisting essentially of, or consisting of amino acid sequence EKD, QGS, or SEQ ID NO: 166, or of an amino acid sequence comprising one, two, orthree amino acid modifications relative to amino acid sequence EKD, QGS, or SEQ ID NO: 166.

In some embodiments, the yST-cell receptor or fragment thereof comprises:

-a yT-cell receptor chain or fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251 , 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 , 262, 263, 264, or 265, and/or, preferably and;

-a 5T-cell receptor chain or fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, or 289.

In some embodiments, the yST-cell receptor or fragment thereof comprises:

-a yT-cell receptor chain or fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, or 223, and/or, preferably and;

-a 5T-cell receptor chain or fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 266, 267, 268, 269, 270, 271 , 272, or 273.

In some embodiments, the y5T-cell receptor or fragment thereof comprises: -a yT-cell receptor chain or fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, or 244, and/or, preferably and;

-a 5T-cell receptor chain or fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 274, 275, 276, 277, 278, 279, 280, or 281.

In some embodiments, the yST-cell receptor or fragment thereof comprises:

-a yT-cell receptor chain or fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 , 262, 263, 264, or 265, and/or, preferably and;

-a 5T-cell receptor chain or fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 282, 283, 284, 285, 286, 287, 288, or 289.

In some embodiments, a yST-cell receptor or fragment thereof provided herein mediates an anti-tumour or anti-infective response that is improved relative to a control yST-cell receptor, preferably a yST-cell receptor chain comprising a Cy constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , and a C6 constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2. In some embodiments, the improvement is of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 100% (2-fold), at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to a control yST-cell receptor, preferably a yST-cell receptor comprising a Cy constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , and C5 constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, a y5T-cell receptor or fragment thereof provided herein demonstrates improved expression, preferably surface expression, in an immunoresponsive cell, preferably a T-cell, more preferably an afJT-cell, relative to a control yST-cell receptor, preferably a y5T-cell receptor comprising a Cy constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, and a C6 constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2. In some embodiments, the improvement is of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 100% (2-fold), at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7- fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to a control y5T-cell receptor, preferably a y5T- cell receptor comprising a Cy constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, and a C5 constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

The disclosure further provides a chimeric antigen-recognition receptor (alternatively referred to herein as chimeric receptor or CAR) comprising a yT-cell receptor chain, ST-cell receptor chain, y6T-cell receptor chain, or fragment thereof, or comprising a Cy constant region, C5 constant region, or fragment thereof, as described herein. A chimeric antigen-recognition receptor as used herein refers to an artificial exogenous antigen-recognition receptor that can induce signaling in an engineered cell that expresses the CAR upon binding of the CAR to an antigen, for example, an antigen associated with a cancer or infectious disease. A CAR generally induces signaling in the engineered cell that expresses the CAR but not in a cell that expresses or presents the antigen bound by the CAR. A CAR comprises at least one extracellular targeting (antigen-binding) domain, at least one transmembrane domain, and at least one intracellular signaling domain. An intracellular signaling domain of a CAR can be or can comprise a fragment of a signaling domain of another molecule. In some embodiments, the intracellular domain of the CAR is involved in regulating primary activation of a TCR complex in either a stimulatory manner or an inhibitory manner. In some embodiments, the intracellular signaling domain of the CAR is involved in inducing T-cell activation and/or a cytotoxic response against cells that express the antigen that is bound by the CAR.

Accordingly, in a further aspect, there is provided a chimeric antigen-recognition receptor comprising: a Cy constant region of a yT-cell receptor chain or a fragment thereof as described herein; a C5 constant region of a 5T-cell receptor chain or a fragment thereof as described herein, or; a Cy constant region of a yT-cell receptor chain or a fragment thereof and a C6 constant region of a ST-cell receptor chain or a fragment thereof as described herein.

In some embodiments, the chimeric antigen-recognition receptor comprises: a yT-cell receptor chain or a fragment thereof as described herein; a 5T-cell receptor chain or fragment thereof as described herein, or; a yT-cell receptor chain or fragment thereof and a 5T-cell receptor chain or fragment thereof as described herein.

A Cy constant region, C6 constant region, yST-cell receptor chain, 5T-cell receptor chain, or a fragment thereof may be comprised in an extracellular targeting domain a CAR. A Cy constant region, C5 constant region, yST-cell receptor chain, 6T-cell receptor chain, or a fragment thereof may be comprised in a transmembrane domain of a CAR. A Cy constant region, C5 constant region, yST-cell receptor chain, 5T- cell receptor chain, or a fragment thereof may be comprised in an intracellular signaling domain of a CAR. The skilled person understands that the exact location in the CAR can vary depending on whether a complete Cy constant region, C6 constant region, yT-cell receptor chain, 5T-cell receptor chain, or a fragment thereof is comprised in the CAR. As a non-limiting example, when the transmembrane domain of a Cy constant region or a C5 constant region described herein is comprised in a CAR, it is preferably located in a transmembrane domain of the CAR. As another non-limiting example, when the variable domain of a yT-cell receptor chain or 5T-cell receptor chain is comprised in a CAR, it is preferably located in an extracellular targeting domain of the CAR.

A chimeric antigen-recognition receptor described herein may further comprise a Ca constant region of an aT-cell receptor chain or fragment thereof and/or a CP constant region of a T-cell receptor chain or a fragment thereof. Preferably, the Ca constant region of an aT-cell receptor chain or fragment thereof and/or a C constant region of a PT-cell receptor chain or a fragment thereof are of mammalian, preferably human origin.

In some embodiments, the chimeric antigen-recognition receptor comprises: a Ca constant region of an aT-cell receptor chain or a fragment thereof, and; a Cy constant region of a yT-cell receptor chain or a fragment thereof, preferably a transmembrane domain, as described herein.

In some embodiments, the chimeric antigen-recognition receptor comprises: a Ca constant region of an aT-cell receptor chain or a fragment thereof; a Cy constant region of a yT-cell receptor chain or a fragment thereof, preferably a transmembrane domain, as described herein, and a C5 constant region of a 5T-cell receptor chain or a fragment thereof, preferably a transmembrane domain, as described herein.

In some embodiments, the chimeric antigen-recognition receptor comprises: a Ca constant region of an aT-cell receptor chain or a fragment thereof, and; a yT-cell receptor chain or a fragment thereof as described herein.

In some embodiments, the chimeric antigen-recognition receptor comprises: a Ca constant region of an aT-cell receptor chain or a fragment thereof; a yT-cell receptor chain or a fragment thereof as described herein, and; a 5T-cell receptor chain or a fragment thereof as described herein.

In some embodiments, the chimeric antigen-recognition receptor comprises: a Ca constant region of an aT-cell receptor chain or a fragment thereof, and; a C5 constant region of a 5T-cell receptor chain or a fragment thereof, preferably a transmembrane domain, as described herein.

In some embodiments, the chimeric antigen-recognition receptor comprises: a Ca constant region of an aT-cell receptor chain or a fragment thereof, and; a 6T-cell receptor chain or a fragment thereof as described herein.

In some embodiments, the chimeric antigen-recognition receptor comprises: a CP constant region of an T-cell receptor chain or a fragment thereof, and; a Cy constant region of a yT-cell receptor chain or a fragment thereof, preferably a transmembrane domain, as described herein.

In some embodiments, the chimeric antigen-recognition receptor comprises: a C constant region of an pT-cell receptor chain or a fragment thereof; a Cy constant region of a yT-cell receptor chain or a fragment thereof, preferably a transmembrane domain, as described herein, and; a C6 constant region of a 5T-cell receptor chain or a fragment thereof, preferably a transmembrane domain, as described herein.

In some embodiments, the chimeric antigen-recognition receptor comprises: a CP constant region of an T-cell receptor chain or a fragment thereof, and; a yT-cell receptor chain or a fragment thereof as described herein.

In some embodiments, the chimeric antigen-recognition receptor comprises: a C constant region of an PT-cell receptor chain or a fragment thereof; a yT-cell receptor chain or a fragment thereof as described herein, and; a 5T-cell receptor chain or a fragment thereof as described herein.

In some embodiments, the chimeric antigen-recognition receptor comprises: a CP constant region of an PT-cell receptor chain or a fragment thereof, and; a C5 constant region of a 5T-cell receptor chain or a fragment thereof, preferably a transmembrane domain, as described herein.

In some embodiments, the chimeric antigen-recognition receptor comprises: a CP constant region of an PT-cell receptor chain or a fragment thereof, and; a 5T-cell receptor chain or a fragment thereof as described herein.

In some embodiments, the CP constant region is a Cpi constant region. In some embodiments, the Cp constant region is a CP2 constant region. A Ca constant region (or fragment thereof) of an aT-cell receptor chain (or fragment thereof) and a Cp constant region (or fragment thereof) of a PT-cell receptor chain (or fragment thereof) may easily be identified as the constant regions are generally conserved in apTCRs. For example, the human Ca constant region (TRAC) is represented by the amino acid sequence with Uniprot Ref: P01848. The human Cpi constant region (TRBC1) is represented by the amino acid sequence with Uniprot Ref: P01850. The human CP2 constant region (TRBC2) is represented by the amino acid sequence with Uniprot Ref: A0A5B9. Transmembrane domains of a Cy constant region and of a C5 constant region according to the disclosure have been described earlier herein.

In some embodiments, the extracellular targeting domain of a CAR comprises the variable domain of a yT- cell receptor chain and/or of a 5T-cell receptor chain described herein. In some embodiments, the extracellular targeting domain of a CAR comprises the CDR3 region of a yT-cell receptor chain and/or of a 5T-cell receptor chain described herein. In some embodiments, an extracellular targeting domain of a CAR further comprises an antigen-binding domain from or derived from an antibody, an antibody variable region, an scFv, a single domain antibody, a Fab, a Fab', a F(ab’)2, a dimer or a trimer of Fab conjugates, an Fv, a minibody, a diabody, a triabody, a tetrabody, an affibody, an ankyrin protein, an ankyrin repeat, a DARPin, a monobody, a nanobody, an avimer, an adnectin, an anticalin, a fynomer, a Kunitz domain, a knottin, a p- hairpin mimetic, or a combination thereof.

In some embodiments, a CAR further comprises a hinge or spacer. A hinge or spacer can be located at any region of a CAR. In some embodiments, a hinge or spacer is located between an extracellular targeting domain and a transmembrane domain. In some embodiments, a hinge can be used to provide flexibility to an extracellular targeting domain. In some embodiments, a hinge can be used to detect the expression of a CAR on the surface of a cell, for example via binding of labelled antibodies to it. In some embodiments, the hinge is derived from an immunoglobulin molecule and may require optimization depending on the location of the first epitope or second epitope on the target. In some embodiments, a hinge may not belong to an immunoglobulin molecule but instead to another molecule such the native hinge of a CD8 alpha molecule. A CD8 alpha hinge can contain cysteine and proline residues which many play a role in the interaction of a CD8 co-receptor and MHC molecule.

The transmembrane domain anchors the CAR to the plasma membrane of a cell expressing the CAR. Preferably, a transmembrane domain comprises a Cy constant region, C5 constant region, or fragment thereof, preferably a transmembrane domain of a Cy constant region or C6 constant region, described herein. In some embodiments, a transmembrane domain further comprises a polypeptide sequence of a membrane spanning portion of an endogenous or wild-type membrane spanning protein, or a variant thereof. In some embodiments, a transmembrane domain comprises a non-natural polypeptide sequence, such as the sequence of a linker peptide.

In some embodiments, a transmembrane domain comprises a sequence from an alpha chain of a T-cell receptor (TCR), beta chain of a TCR, CD8, CD4, CD28, CD45, ICOS, PD-1 and/or CD152. A native transmembrane portion of CD28 can be used in a CAR. In other cases, a native transmembrane portion of CD8 alpha can also be used in a subject CAR. In some embodiments, the transmembrane domain of a CAR comprises a sequence from an alpha chain of a TCR. In some embodiments, the transmembrane domain of a CAR comprises a sequence from CD8, preferably from CD8a.

In some embodiments, the CAR comprises a linker peptide. A linker peptide can allow for separation and flexibility of the domains it separates, for example, a transmembrane domain and extracellular targeting domain. The length of a peptide linker can be adjusted to alter the ability of a domain to bind to, for example, an antigen, or a factor that participates in a signaling pathway (e.g., for the intracellular signaling domain). A linker peptide sequence can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, or more, amino acid residues in length. In some embodiments, a peptide linker is at least 1, at least 3, at least 5, at least 7, at least 9, at least 11, or at least 15 amino acids in length. In some embodiments, a linker is at most 5, at most 7, at most 9, at most 11 , at most 15, at most 20, at most 25, or at most 50 amino acids in length. A flexible linker can have a sequence containing stretches of glycine and serine residues. The small size of the glycine and serine residues provides flexibility and allows for mobility of the connected functional domains. The incorporation of serine or threonine can maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, thereby reducing unfavourable interactions between the linker and protein moieties. Flexible linkers can also contain additional amino acids such as threonine and alanine to maintain flexibility, as well as polar amino acids such as lysine and glutamine. A rigid linker can have, for example, an alpha helix-structure. An alphahelical rigid linker can act as a spacer between protein domains. Non-limiting examples of linker peptides are represented by SEQ ID NO: 80-94 and SEQ ID NO: 182-193. Other examples of flexible linker peptides are Gly-Gly and Gly-Gly-Ser. A CAR may comprise multiple copies of a linker peptide. In some embodiments, a CAR comprises a linker peptide with at least 1 , at least 2, at least 3, at least 4, or at least 5 amino acid insertions, deletions, or substitutions relative to any of SEQ ID NOs: 80-94 or SEQ ID NO: 182-193. The insertions, deletions, or substitutions can be at the N-temninus, the C-terminus, within the sequence, or a combination thereof. The insertions, deletions, or substitutions can be contiguous or noncontiguous. In some cases, the substitutions are conservative. In some cases, the substitutions are nonconservative. In some embodiments, a CAR does not comprise any linker peptides, for example, the domains of the CAR are directly fused with no intervening amino acid sequence.

An intracellular signaling domain of a CAR can comprise a signaling domain, or any derivative, variant, or fragment thereof, involved in cell signaling. The intracellular signaling domain of a CAR can induce activity of an engineered cell comprising the CAR. While usually the signaling domain of another molecule can be employed in a CAR, in many cases it is not necessary to use the entire chain. In some cases, a truncated portion of the signaling domain is used in a CAR of an engineered cell provided herein.

In some embodiments, an intracellular signaling domain comprises multiple signaling domains involved in cell signaling, or any derivatives, variants, or fragments thereof. A CAR intracellular signaling domain may be that of a TCR complex. A CAR intracellular signaling domain can comprise a signaling domain of an Fey receptor (FcyR), an Fee receptor (FCER), an Fea receptor (FcaR), neonatal Fc receptor (FcRn), CD3, CD3 <, CD3 y, CD35, CD3 E, CD4, CD5, CD8, CD21 , CD22, CD26, CD28, CD32, CD40L (CD154), CD45, CD46, 41 BB, 0X40, GITR, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also known as ICOS), CD247 , CD247 , DAP10, DAP12, FYN, LAT, Lek, MAPK, MHC complex, NFAT, NF-KB, PLC-y, iC3b, C3dg, C3d, and Zap70. In some embodiments, a CAR signaling domain includes an immunoreceptor tyrosine-based activation motif or ITAM. A CAR signaling domain comprising an ITAM can comprise two repeats of the amino acid sequence YxxL/l separated by 6-8 amino acids, wherein each x is independently any amino acid. A CAR signaling domain comprising an ITAM can be modified, for example, by phosphorylation when the targeting moiety is bound to an epitope. A phosphorylated ITAM can function as a docking site for other proteins, for example proteins involved in various signaling pathways. In some embodiments, the primary CAR signaling domain comprises a modified ITAM domain, e.g., a mutated, truncated, and/or optimized ITAM domain, which has altered (e.g., increased or decreased) activity compared to the native ITAM domain.

In some embodiments, the intracellular signaling domain of a CAR comprises an FcyR signaling domain (e.g., ITAM). The FcyR signaling domain can be selected from FcyRI (CD64), FcyRIIA (CD32), FcyRIIB (CD32), FcyRIIIA (CD16a), and FcyRIIIB (CD16b). In some embodiments, the CAR intracellular signaling domain comprises an FCER signaling domain (e.g., ITAM). The FCER signaling domain can be selected from FCERI and FCERII (CD23). In some embodiments, the CAR intracellular signaling domain comprises an FcaR signaling domain (e.g., ITAM). The FcaR signaling domain can be selected from FcaRI (CD89) and Fca/pR. In some embodiments, the CAR intracellular signaling domain comprises a CD3 signaling domain. In some embodiments, the primary CAR signaling domain comprises an ITAM of CD3

In some embodiments, an intracellular signaling domain of a subject CAR comprises an immunoreceptor tyrosine-based inhibition motif or ITIM. A signaling domain comprising an ITIM can comprise a conserved sequence of amino acids that is found in the cytoplasmic tails of some inhibitory receptors of the immune system. A primary CAR signaling domain comprising an ITIM can be modified, for example phosphorylated, by enzymes such as a Src kinase family member (e.g., Lek). Following phosphorylation, other proteins, including enzymes, can be recruited to the ITIM. These other proteins include, but are not limited to, enzymes such as the phosphotyrosine phosphatases SHP-1 and SHP-2, the inositol-phosphatase called SHIP, and proteins having one or more SH2 domains (e.g., ZAP70). A CAR intracellular signaling domain can comprise a signaling domain (e.g., ITIM) of BTLA, CD5, CD31 , CD66a, CD72, CMRF35H, DCIR, EPO- R, FcyRIIB (CD32), Fc receptor-like protein 2 (FCRL2), Fc receptor-like protein 3 (FCRL3), Fc receptor-like protein 4 (FCRL4), Fc receptor-like protein 5 (FCRL5), Fc receptor-like protein 6 (FCRL6), protein G6b (G6B), interleukin 4 receptor (IL4R), immunoglobulin superfamily receptor translocation-associated 1(IRTA1), immunoglobulin superfamily receptor translocation-associated 2 (IRTA2), killer cell immunoglobulin-like receptor 2DL1 (KIR2DL1), killer cell immunoglobulin-like receptor 2DL2 (KIR2DL2), killer cell immunoglobulin-like receptor 2DL3 (KIR2DL3), killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), killer cell immunoglobulin-like receptor 2DL5 (KI R2DL5), killer cell immunoglobulin-like receptor 3DL1 (KIR3DL1), killer cell immunoglobulin-like receptor 3DL2 (KIR3DL2), leukocyte immunoglobulin-like receptor subfamily B member 1 (LIR1), leukocyte immunoglobulin-like receptor subfamily B member 2 (LIR2), leukocyte immunoglobulin-like receptor subfamily B member 3 (LIR3), leukocyte immunoglobulin- like receptor subfamily B member 5 (LIR5), leukocyte immunoglobulin-like receptor subfamily B member 8 (LIR8), leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1), mast cell function-associated antigen (MAFA), NKG2A, natural cytotoxicity triggering receptor 2 (NKp44), NTB-A, programmed cell death protein 1 (PD-1), PILR, SIGLECL1, sialic acid binding Ig like lectin 2 (SIGLEC2 or CD22), sialic acid binding Ig like lectin 3 (SIGLEC3 or CD33), sialic acid binding Ig like lectin 5 (SIGLEC5 or CD170), sialic acid binding Ig like lectin 6 (SIGLEC6), sialic acid binding Ig like lectin 7 (SIGLEC7), sialic acid binding Ig like lectin 10 (SIGLEC10), sialic acid binding Ig like lectin 11 (SIGLEC11), sialic acid binding Ig like lectin 4 (SIGLEC4), sialic acid binding Ig like lectin 8 (SIGLEC8), sialic acid binding Ig like lectin 9 (SIGLEC9), platelet and endothelial cell adhesion molecule 1 (PECAM-1), signal regulatory protein (SIRP 2), and signaling threshold regulating transmembrane adaptor 1 (SIT). In some embodiments, a CAR intracellular signaling domain comprises a modified ITIM domain, e.g., a mutated, truncated, and/or optimized ITIM domain, which has altered (e.g., increased or decreased) activity compared to the native ITIM domain.

In some embodiments, a CAR intracellular signaling domain comprises at least 2 ITAM domains (e.g., at least 3, 4, 5, 6, 7, 8, 9, or 10 ITAM domains). In some embodiments, a CAR intracellular signaling domain comprises at least 2 ITIM domains (e.g., at least 3, 4, 5, 6, 7, 8, 9, or 10 ITIM domains) (e.g., at least 2 primary signaling domains). In some embodiments, the CAR intracellular signaling domain comprises both ITAM and ITIM domains. In some embodiments, an intracellular signaling domain of a CAR is from an Fey receptor (FcyR), an Fee receptor (FceR), an Fea receptor (FcaR), neonatal Fc receptor (FcRn), CD3, CD3 , CD3y, CD35, CD3e, CD4, CD5, CD8, CD21 , CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also known as ICOS), CD247 CD247 q, DAP10, DAP12, FYN, LAT, Lek, MAPK, MHC complex, NFAT, NF-KB, PLC-y, iC3b, C3dg, C3d, and Zap70. In some embodiments, the intracellular signaling domain of a CAR is from CD3, CD3 , CD3y, CD35, and/or CD3e. In some embodiments, the intracellular signaling domain of a CAR is from CD3 .

In some embodiments, a CAR intracellular signaling domain comprises a co-stimulatory domain. In some embodiments, a CAR co-stimulatory domain, for example from a cellular co-stimulatory molecule, can provide co-stimulatory signals for engineered cell signaling, such as signaling from ITAM and/or ITIM domains, e.g., for the activation and/or deactivation of engineered cell activity. In some embodiments, a CAR costimulatory domain is operable to regulate a proliferative and/or survival signal in the engineered cell. In some embodiments, a CAR co-stimulatory signaling domain comprises a signaling domain of a MHC class I protein, MHC class II protein, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphocytic activation molecule (SLAM protein), activating NK cell receptor, BTLA, or a Toll ligand receptor. In some embodiments, a CAR costimulatory domain comprises a signaling domain of a molecule selected from the group consisting of: 2B4/CD244/SLAMF4, 4-1 BB/TNFSF9/CD137, CD137L, B7-1/CD80, B7-2/CD86, B7-H1/PD-L1 , B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BAFF R/TNFRSF13C, BAFF/BLyS/TNFSF13B, BLAME/SLAMF8, BTLA/CD272, CD100 (SEMA4D), CD103, CD11a, CD11b, CD11c, CD11d, CD150, CD160 (BY55), CD18, CD19, CD2, CD200, CD229/SLAMF3, CD27 Ligand/TNFSF7, CD27/TNFRSF7, CD28, CD29, CD2F-10/SLAMF9, CD30 Ligand/TNFSF8, CD30/TNFRSF8, CD300a/LMIR1 , CD4, CD40 Ligand/TNFSF5, CD40/TNFRSF5, CD46, CD48/SLAMF2, CD49a, CD49D, CD49f, CD5, CD53, CD58/LFA-3, CD69, CD7, CD8 a, CD8 P, CD82/Kai-1 , CD84/SLAMF5, CD90/Thy1, CD96, CDS, CEACAM1 , CRACC/SLAMF7, CRTAM, CTLA-4, DAP12, Dectin- 1/CLEC7A, DNAM1 (CD226), DPPIV/CD26, DR3/TNFRSF25, EphB6, GADS, Gi24/VISTA/B7-H5, GITR Ligand/TNFSF18, GITR/TNFRSF18, HLA Class I, HLA-DR, HVEM/TNFRSF14, IA4, ICAM-1, ICOS/CD278, Ikaros, IL2R , IL2R y, IL7R a, Integrin a4/CD49d, Integrin a4pi, Integrin a4P7/LPAM-1 , IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT/TNFSF14, LTBR, Ly108, Ly9 (CD229), lymphocyte function associated antigen-1 (LFA-1), Lymphotoxin-a/TNF-P, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), NTB-A/SLAMF6, 0X40 Ligand/TNFSF4, OX40/TNFRSF4, PAG/Cbp, PD-1 , PDCD6, PD-L2/B7-DC, PSGL1, RELT/TNFRSF19L, SELPLG (CD162), SLAM (SLAMF1), SLAM/CD150, SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP- 76, TACI/TNFRSF13B, TCL1A, TCL1B, TIM-1/KIM-1/HAVCR, TIM-4, TL1A/TNFSF15, TNF RII/TNFRSF1 B, TNF-a, TRANCE/RANKL, TSLP, TSLP R, VLA1 , and VLA-6. In some embodiments, a CAR costimulatory domain comprises a signaling domain of a molecule selected from the group consisting of: CD3 and CD28.

In some embodiments, a CAR intracellular signaling domain comprises multiple costimulatory domains, for example at least two, e.g., at least 3, 4, or 5 costimulatory domains. In an aspect, a CAR comprises at least 2 or 3 co-stimulatory domains. In an aspect, a CAR comprises at least 2 costimulatory domains, and the at least 2 costimulatory domains are CD28 and CD137. In an aspect, a CAR comprises at least 3 costimulatory domains, wherein the at least 3 costimulatory domains are CD28, CD137, and 0X40. Co-stimulatory signaling regions may provide a signal synergistic with the primary effector activation signal and can complete the requirements for activation of an immunoresponsive cell, for example a T-cell. In some embodiments, the addition of co-stimulatory domains to the CAR can enhance the efficacy and persistence of the engineered cells provided herein.

The CAR can be a CAR that binds to an antigen that is associated with a cancer, for example, an antigen that is over-expressed in a cancer, or a neoantigen. In some embodiments, the exogenous antigenrecognition receptor binds to an infected cell or an infectious agent.

In some embodiments, a yT-cell receptor chain, 5T-cell receptor chain, y5T-cell receptor, or fragment thereof is a soluble polypeptide. A soluble yT-cell receptor chain or fragment thereof, soluble 5T-cell receptor chain or fragment thereof, and/or soluble yST-cell receptor or fragment thereof is preferably able to mediate an anti-tumour or anti-infective response. Assessment of mediation of an anti-tumour or anti- infective response may be performed using any of the assays described herein, for example as described in step D) of the methods of the invention and the Examples, with the difference that the assessment comprises contacting the soluble yT-cell receptor chains, 5T-cell receptor chains, yST-cell receptors, or fragments thereof with an immunoresponsive cell, for example selected from a T-cell, an IPSC-derived T- cell, an apT-cell, a yST-cell, or an NKcell, and with tumour cells, tumour cell lines, infected cells, or infectious agents such as e.g., fungal cells. Control cells, such as T-cells that have not been contacted with soluble polypeptides of the disclosure, may similarly be used for the comparisons. Optionally, different amounts of soluble polypeptides and/or ratios of soluble polypeptides to T-cells may be tested.

Alternatively, the assessment of mediation of an anti-tumour or anti-infective response may be performed by assessing the binding of the soluble yT-cell receptor chains, soluble 5T-cell receptor chains, soluble y5T- cell receptors, or fragments thereof to a target. Binding may be assessed by any suitable method known to the skilled person, such as the methods described herein. Additional examples of such methods are described in WO2018/234319A1 and US20210355188A1 , incorporated herein by reference in their entireties. Briefly, target binding analysis of soluble yT-cell receptor chains, soluble 6T-cell receptor chains, soluble y5T-cell receptors, or fragments thereof may be carried out by surface plasmon resonance, e.g., using a BIAcore 3000 or BIAcore T200 instrument (Cytiva Lifesciences, MA, USA), or by biolayer interferometry, e.g., using a ForteBio Octet instrument (Sartorius, DE).

In some embodiments, the contacting step has a duration of at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, or at least 5 days. Optionally, varying amounts of soluble polypeptide may be utilized, for example from 0.01 pg to 100 pg, from 0.01 pg to 10 pg, from 0.1 pg to 10 pg, from 1 pg to 10 mg, or any other suitable amount.

In some embodiments, a soluble yT-cell receptor chain or fragment thereof mediates an anti-tumour or anti- infective response that is improved relative to a control soluble yT-cell receptor chain comprising a Cy constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , or a fragment thereof. In some embodiments, a soluble ST-cell receptor chain or fragment thereof mediates an anti-tumour or anti-infective response that is improved relative to a control soluble 5T-cell receptor chain comprising a C5 constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2 or a fragment thereof. In some embodiments, a soluble ybT-cell receptor or fragment thereof mediates an anti-tumour or anti-infective response that is improved relative to a control soluble ybT-cell receptor comprising a Cy constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, or a fragment thereof, and a C5 constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2 or a fragment thereof. In some embodiments, the improvement is of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 100% (2-fold), at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relative to the control soluble yT-cell receptor chain, soluble 5T-cell receptor chain, or soluble yST-cell receptor.

In some embodiments, a soluble yT-cell receptor chain, soluble 5T-cell receptor chain, soluble y5T-cell receptor, or fragment thereof comprises or consists of the extracellular domain of a yT-cell receptor chain, a 5T-cell receptor chain, a y5T-cell receptor, or a fragment thereof.

In some embodiments, the Cy constant region or C5 constant region comprised (as applicable) in the soluble yT-cell receptor chains, soluble 5T-cell receptor chains, soluble y5T-cell receptors, or fragments thereof, lacks the transmembrane and/or cytoplasmic region or a fragment thereof. In some embodiments, a Cy constant region or fragment thereof does not comprise SEQ ID NO: 178, SEQ ID NO: 179, or a fragment thereof. In some embodiments, a C5 constant region or a fragment thereof region does not comprise SEQ ID NO: 180 or a fragment thereof.

In some embodiments, a soluble yT-cell receptor chain, soluble 5T-cell receptor chain, soluble y5T-cell receptor, or fragment thereof is in single chain format. Suitable single chain formats include, but are not limited to, soluble y5TCR polypeptides of the Vy - L - V5, V5 - L - Vy, Vy - Cy (Cy1 or Cy2) - L - V5, Vy - L - V5 - C5, or Vy - Cy (Cy1 or Cy2) - L - V5 - C5 types, wherein Vy and V5 correspond to, respectively, the yT- and 5T-cell receptor chain variable regions (or fragments thereof), Cy (Cy1 or Cy2) and C5 correspond to, respectively, the yT- and 5T-cell receptor chain constant regions (or fragments thereof), and L is a linker peptide sequence, such as the linker peptide sequences described herein. Examples of suitable Vy (gamma variable) and V5 (delta variable) regions are provided elsewhere herein. In some embodiments, a preferred soluble yT-cell receptor chain or fragment thereof comprises a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position Q58 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 , the substitution preferably being a Q58C substitution. In some embodiments, a preferred soluble yT-cell receptor chain or fragment thereof comprises a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 37 or a fragment thereof.

In some embodiments, a preferred soluble 5T-chain or fragment thereof comprises a C6 constant region or fragment thereof comprising an amino acid substitution in a position corresponding to position V48 of SEQ ID NO: 2, the substitution preferably being a V48C substitution. In some embodiments, a preferred soluble ST-chain or fragment thereof comprises a C5 constant region or fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 38 or a fragment thereof.

In some embodiments, a preferred soluble yST-cell receptor or fragment thereof comprises: a soluble yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position Q58 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 , the substitution preferably being a Q58C substitution, and/or, preferably and; a soluble 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position V48 of SEQ ID NO: 2, the substitution preferably being a V48C substitution.

In some embodiments, a preferred soluble yST-cell receptor or fragment thereof comprises: a soluble yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 37 or a fragment thereof and/or, preferably and; a soluble 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 38 or a fragment thereof.

In some embodiments, a soluble yT-cell receptor chain or fragment thereof comprises a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from D1 to V8 (DKQLDADV (SEQ ID NO: 42)) of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1, by the amino acid sequence EDLKNVF (SEQ ID NO: 43), DKEDLKNVF (SEQ ID NO: 176), or a variant thereof, preferably by the amino acid sequence EDLKNVF (SEQ ID NO: 43) or a variant thereof.

In some embodiments, a soluble yT-cell receptor chain or fragment thereof comprises a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from E93 to D100 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, (ENNKNGVD (SEQ ID NO: 44)) by the amino acid sequence YGLSENDEWTQDRAKPVT (SEQ ID NO: 46) or a variant thereof.

In some embodiments, a soluble yT-cell receptor chain or fragment thereof comprises a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , (VRHENNKNGVDQ (SEQ ID NO: 48)) by the amino acid sequence VQFYGLSENDEWTQDRAKPVTQIV (SEQ ID NO: 47) or a variant thereof. In some embodiments, a soluble yT-cell receptor chain or fragment thereof comprises a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , (VRHENNKNGVDQ (SEQ ID NO: 48)) by the amino acid sequence VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50) or a variant thereof.

In some embodiments, a soluble yT-cell receptor chain or fragment thereof comprises a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from E93 to D100 of SEQ ID NO: 40 or SEQ ID NO: 41 (ENNKNGID (SEQ ID NO: 45)) by the amino acid sequence YGLSENDEWTQDRAKPVT (SEQ ID NO: 46) or a variant thereof.

In some embodiments, a soluble yT-cell receptor chain or fragment thereof comprises a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 40 or SEQ ID NO: 41 (VRHENNKNGIDQ (SEQ ID NO: 49)) by the amino acid sequence VQFYGLSENDEWTQDRAKPVTQIV (SEQ ID NO: 47) or a variant thereof.

In some embodiments, a soluble yT-cell receptor chain or fragment thereof comprises a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 40 or SEQ ID NO: 41 (VRHENNKNGIDQ (SEQ ID NO: 49)) by the amino acid sequence VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50) or a variant thereof.

In some embodiments, a soluble yT-cell receptor chain or fragment thereof comprises a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from D1 to V8 (DKQLDADV (SEQ ID NO: 42)) of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, by the amino acid sequence EDLKNVF (SEQ ID NO: 43), DKEDLKNVF (SEQ ID NO: 176), or a variant thereof, preferably by the amino acid sequence EDLKNVF (SEQ ID NO: 43) or a variant thereof, and a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, (VRHENNKNGVDQ (SEQ ID NO: 48)) by the amino acid sequence VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50) or a variant thereof.

In some embodiments, a soluble yT-cell receptor chain or fragment thereof comprises a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from D1 to V8 (DKQLDADV (SEQ ID NO: 42)) of SEQ ID NO: 40 or SEQ ID NO: 41 by the amino acid sequence EDLKNVF (SEQ ID NO: 43), DKEDLKNVF (SEQ ID NO: 176), or a variant thereof, preferably by the amino acid sequence EDLKNVF (SEQ ID NO: 43) ora variant thereof, and a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 40 or SEQ ID NO: 41 (VRHENNKNGIDQ (SEQ ID NO: 49)) by the amino acid sequence VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50) or a variant thereof. Preferably, the Cy constant region or fragment thereof comprises at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or similarity with SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1.

In some embodiments, a soluble yST-cell receptor or fragment thereof comprises: a soluble yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from D1 to V8 (DKQLDADV (SEQ ID NO: 42)) of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 , by the amino acid sequence EDLKNVF (SEQ ID NO: 43), DKEDLKNVF (SEQ ID NO: 176), or a variant thereof, preferably by the amino acid sequence EDLKNVF (SEQ ID NO: 43) or a variant thereof, and/or, preferably and; a soluble 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2 or a fragment thereof.

In some embodiments, a soluble y6T-cell receptor or fragment thereof comprises: a soluble yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 21 or a fragment thereof, and/or, preferably and; a soluble 5T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2 or a fragment thereof.

In some embodiments, a soluble y5T-cell receptor or fragment thereof comprises: a soluble yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from E93 to D100 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, (ENNKNGVD (SEQ ID NO: 44)) by the amino acid sequence YGLSENDEWTQDRAKPVT (SEQ ID NO: 46) or a variant thereof, and/or, preferably and; a soluble 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2 or a fragment thereof.

In some embodiments, a soluble yST-cell receptor or fragment thereof comprises: a soluble yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from E93 to D100 of SEQ ID NO: 40 or SEQ ID NO: 41 (ENNKNGID (SEQ ID NO: 45)) by the amino acid sequence YGLSENDEWTQDRAKPVT (SEQ ID NO: 46) or a variant thereof, and/or, preferably and; a soluble 5T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2 or a fragment thereof.

In some embodiments, a soluble y6T-cell receptor or fragment thereof comprises : a soluble yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, (VRHENNKNGVDQ (SEQ ID NO: 48)) by the amino acid sequence VQFYGLSENDEWTQDRAKPVTQIV (SEQ ID NO: 47) or a variant thereof, and/or, preferably and; a soluble 6T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2 or a fragment thereof.

In some embodiments, a soluble y6T-cell receptor or fragment thereof comprises : a soluble yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 40 or SEQ ID NO: 41 (VRHENNKNGIDQ (SEQ ID NO: 49)) by the amino acid sequence VQFYGLSENDEWTQDRAKPVTQIV (SEQ ID NO: 47) or a variant thereof, and/or, preferably and; a soluble 6T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2 or a fragment thereof.

In some embodiments, a soluble yST-cell receptor or fragment thereof comprises : a soluble yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, (VRHENNKNGVDQ (SEQ ID NO: 48)) by the amino acid sequence VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50) or a variant thereof, and/or, preferably and; a soluble 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2 or a fragment thereof.

In some embodiments, a soluble y5T-cell receptor or fragment thereof comprises : a soluble yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 40 or SEQ ID NO: 41 (VRHENNKNGIDQ (SEQ ID NO: 49)) by the amino acid sequence VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50) or a variant thereof, and/or, preferably and; a soluble 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2 or a fragment thereof.

In some embodiments, a soluble yST-cell receptor or fragment thereof comprises : a soluble yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from D1 to V8 (DKQLDADV (SEQ ID NO: 42)) of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, bythe amino acid sequence EDLKNVF (SEQ ID NO: 43), DKEDLKNVF (SEQ ID NO: 176), or a variant thereof, preferably bythe amino acid sequence EDLKNVF (SEQ ID NO: 43) or a variant thereof, and a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , (VRHENNKNGVDQ (SEQ ID NO: 48)) by the amino acid sequence VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50) or a variant thereof, and/or, preferably and; a soluble 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2 or a fragment thereof.

In some embodiments, a soluble yST-cell receptor or fragment thereof comprises: a soluble yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from D1 to V8 (DKQLDADV (SEQ ID NO: 42)) of SEQ ID NO: 40 or SEQ ID NO: 41 by the amino acid sequence EDLKNVF (SEQ ID NO: 43), DKEDLKNVF (SEQ ID NO: 176), or a variant thereof, preferably by the amino acid sequence EDLKNVF (SEQ ID NO: 43) or a variant thereof, and a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 40 or SEQ ID NO: 41 (VRHENNKNGIDQ (SEQ ID NO: 49)) by the amino acid sequence VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50) or a variant thereof, and/or, preferably and; a soluble 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2 or a fragment thereof.

In some embodiments, a soluble yST-cell receptor or fragment thereof comprises: a soluble yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 177, preferably SEQ ID NO: 24 or SEQ ID NO: 177, more preferably SEQ ID NO: 24, and/or, preferably and; a soluble 5T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

Preferably, the Cy constant region or fragment thereof comprised in the soluble yT-cell receptor chains, y5T-cell receptors, or fragments thereof described herein comprises at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or similarity with SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 . Preferably, the C5 constant region or fragment thereof comprised in the soluble 5T-cell receptor chains, yST-cell receptors, or fragments thereof described herein comprises at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or similarity with SEQ ID NO: 2.

In some embodiments, the soluble yST-cell receptor or fragment thereof further comprises:

- a 6 variable region or fragment thereof comprising, consisting essentially of, or consisting of, preferably comprising, an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 114, 115, 116, 117, 118, 119, 123, or 151.

- a 5CDR3 region comprising, consisting essentially of, or consisting of, preferably comprising, an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 131 , 132, 133, 134, 135, 136, 137, or 153.

In some embodiments, the soluble y5T-cell receptor or fragment thereof further comprises:

-a yCDR3 region comprising a modification at an amino acid position selected from one or more of positions 4-10 of SEQ ID NO: 124, said modification selected from WDAFYYK (SEQ ID NO: 138), WEAFYYK (SEQ ID NO: 139), WDGYFYK (SEQ ID NO: 140), WDGYYYK (SEQ ID NO: 141), WDGAYYK (SEQ ID NO: 142), or WDGSYYK (SEQ ID NO: 143), and/or, preferably and;

-a 5CDR3 region comprising a modification at an amino acid position corresponding to a position selected from one or more of positions 7-12 of SEQ ID NO: 131 , said modification selected from IRGFTG (SEQ ID NO: 144), IKGYTG (SEQ ID NO: 145), IKGFTG (SEQ ID NO: 146), LRGFTG (SEQ ID NO: 147), LKGFTG (SEQ ID NO: 148), or LKGYTG (SEQ ID NO: 149).

In some embodiments, a preferred soluble yST-cell receptor or fragment thereof further comprises:

- a yCDR1 region comprising, consisting essentially of, or consisting of amino acid sequence SEQ ID NO: 154, 155, 156, or 157, or of an amino acid sequence comprising one, two, or three amino acid modifications relative to SEQ ID NO: 154, 155, 156, or 157, and a yCDR2 region comprising, consisting essentially of, or consisting of amino acid sequence SEQ ID NO: 158, 159, 160, or 161, or of an amino acid sequence comprising one, two, or three amino acid modifications relative to SEQ ID NO: 158, 159, 160, or 161, and/or, preferably and;

-a 5CDR1 region comprising, consisting essentially of, or consisting of amino acid sequence SEQ ID NO: 162, 163, or 164, or of an amino acid sequence comprising one, two, or three amino acid modifications relative to SEQ ID NO: 162, 163, or 164, and a 5CDR2 region comprising, consisting essentially of, or consisting of amino acid sequence EKD, QGS, or SEQ ID NO: 166, or of an amino acid sequence comprising one, two, or three amino acid modifications relative to amino acid sequence EKD, QGS, or SEQ ID NO: 166. In some embodiments, a soluble yT-cell receptor chain, soluble 5T-cell receptor chain, soluble y5T-cell receptor, or fragment thereof is comprised in a soluble polypeptide, preferably comprising an additional binding domain. Preferably, the additional binding domain is a T-cell- and/or NK-cell binding domain. The additional binding domain may be fused (i.e. , physically linked) to the soluble yT-cell receptor chain, soluble ST-cell receptor chain, soluble yST-cell receptor, or fragment thereof. Such a soluble polypeptide may be called a bispecific polypeptide. The binding domain may be fused to the soluble yT-cell receptor chain, soluble ST-cell receptor chain, soluble yST-cell receptor, or fragment thereof directly, or via a linker peptide (for example via the linker peptides described herein) or an additional domain as described later herein. In embodiments wherein the soluble polypeptide comprises a y5T-cell receptor or a fragment thereof fused with a T-cell- and/or NK-cell-binding domain, said polypeptide may be alternatively called a ybTCR bispecific engager. Such a bispecific polypeptide may be advantageous, as it may first bind to a T- and/or NK-cell and then recruit the cell to a tumour cell, or to an infection site, thus mediating an anti-tumour or an anti-infective response without the requirement for its expression in a cellular membrane of an engineered T- and/or NK- cell.

A T-cell- and/or NK-cell-binding domain is to be understood as a domain that specifically binds to a T-cell and/or NK-cell, for example via binding to a receptor, an antigen, or epitope that is present on or displayed by the T-cell and/or NK-cell. In some embodiments, the T-cell and/or NK-cell is a mammalian cell, preferably a human cell. Preferably, binding of a T-cell- or NK-cell-binding domain to the respective T-cell or NK-cell results in the stimulation and/or activation of the T-cell or NK-cell.

In some embodiments, the T-cell- and/or NK-cell-binding domain is derived from, or is, an antibody, a single heavy chain variable domain antibody (such as for example a camelid VHH), a shark immunoglobulinderived variable new antigen receptor, an scFv, a tandem scFv, a Fab, an Fc domain of an antibody, an scFab, an antibody mimetic (such as for example a designed ankyrin repeat protein), a binding protein based on a Z domain of protein A, a binding protein based on a fibronectin type III domain, a lipocalin, or a combination thereof.

A T-cell- and/or NK-cell-binding domain may also be derived from, or is, a minibody, a F(ab’)2 fragment, a dsFv, a nanobody (these constructs, marketed by Ablynx (Belgium), comprise synthetic single immunoglobulin variable heavy domains derived from a camelid (e.g. camel or llama) antibody), a Domain Antibody (Domantis (Belgium), comprising an affinity matured single immunoglobulin variable heavy domain or immunoglobulin variable light domain), or an alternative protein scaffold that exhibits antibody like binding characteristics such as affibodies (Affibody (Sweden), comprising engineered protein A scaffold) or anticalins (Pieris (Germany)), comprising engineered anticalins).

In some embodiments, the T-cell- and/or NK-cell-binding domain is of mammalian origin, preferably of human origin. In some embodiments, the T-cell- and/or NK-cell-binding domain is selected from the group of CD3-, CD4-, CD8-, CD16-, CD56-, CD103-, CD154-, CD314-binding domains, and combinations thereof. In some embodiments, a preferred T-cell- and/or NK-cell binding domain is a CD3-binding domain, also referred to herein as an "anti-CD3” binding domain.

In preferred embodiments, a soluble polypeptide comprises a yT-cell receptor chain, a 5T-cell receptor chain, a yST-cell receptor, or a fragment thereof (such as e.g., an extracelllular domain or fragment thereof) as described herein, and an anti-CD3 binding domain.

Such anti-CD3 binding domains are known to the skilled person, and are further described in e.g., W02007/062245, Liao et al., 2000 (Gene Ther 7: 339-47), WO2001/051644, Arakawa et al., 1996 (J Biochem 120: 657-62), Adair et al., 1994 (Human Antibodies 5: 41-47), Kipriyanov et al., 1997 (Protein Engin Design Selection 10:445), van Diest et al., 2021 (J Immunother Cancer 2021;9:e003850), and WO2019/156566, all of which are incorporated herein by reference in their entireties. For example, such binding domains include commercially available binding domains such as the ones offered by Creative Biolabs (Shirley, NY, USA). The anti-CD3 binding domain may, for example, be an OKT3, UCHT-1 , BMA- 031 , or 12F6 binding domain.

In some embodiments, a soluble polypeptide comprises a yT-cell receptor chain, a 5T-cell receptor chain, a y5T-cell receptor, or a fragment thereof (such as e.g., an extracelllular domain or fragment thereof) as described herein, and an scFv domain, preferably an anti-CD3 scFv domain. A non-limiting example of a suitable anti-CD3 scFv domain is SEQ ID NO: 181.

Linkage of the soluble yT-cell receptor chain, soluble 5T-cell receptor chain, soluble yST-cell receptor, or fragment thereof may, for example, be via covalent or non-covalent attachment. Binding of soluble polypeptides comprising T-cell- and/or NK-cell-binding domains to the T-cells and/or NK-cells may be assessed utilizing any assay suitable for measuring an anti-tumour or anti-infective response known to the skilled person, such as the ones described herein, by bringing the soluble polypeptides into contact with the T-cells and/or NK-cells and measuring their anti-tumour or anti-infective response. As an example, T-cell activation mediated by soluble TCR-anti-CD3 chimeric polypeptides may be determined by measuring IFNy secretion using an ELISpot assay. Assays can be performed e.g., using a human IFN-yELISPOT kit (BD Biosciences, NJ, USA) according to the manufacturer's instructions. Peripheral blood mononuclear cells (PBMC), isolated from fresh donor blood, isolated T-cells, or other suitable T-cell or NK-cells may be used as effector cells. To further determine suitability for therapeutic use, the TCR-anti-CD3 chimeric polypeptides may be tested for non-specific activation in the presence of normal cells derived from healthy human tissues using the same ELISPOT methodology as described above.

In some embodiments, a soluble polypeptide comprises yT-cell receptor chain, a 5T-cell receptor chain, a y5T-cell receptor, or a fragment thereof (such as e.g., an extracellular domain or fragment thereof) as described herein, and an Fc domain of an antibody. In some embodiments, the yT-cell receptor chain, 5T- cell receptor chain, y5T-cell receptor, or fragment thereof (such as e.g., extracellular domain or fragment thereof) comprised in the soluble polypeptides described herein is fused to an extracellular domain of an immune checkpoint-related molecule (or fragment thereof), such as for example an immune checkpoint inhibitor. The term "immune checkpoint inhibitor” as used herein refers to polypeptides, such as, but not limited to, inhibitory receptors, expressed by T- and/or NK-cells.

Accordingly, in some embodiments, a soluble polypeptide comprises a yT-cell receptor chain, a 5T-cell receptor chain, a yST-cell receptor, or a fragment thereof (such as e.g., an extracellular domain or fragment thereof) as described herein, an extracellular immune checkpoint inhibitor domain, and a T-cell- and/or NK- cell-binding domain, preferably an anti-CD3 scFv or Fc domain. Such a soluble polypeptide may be called a trispecific polypeptide. Suitable extracellular immune checkpoint inhibitor domains may be derived from, but are not limited to, the group consisting of the adenosine A2A receptor, programmed death 1 (PD1) receptor, T-cell immunoglobulin domain, mucin domain 3, and V-domain Ig suppressor of T cell activation (TIGIT). Among the suitable immune checkpoint inhibitor domains, the extracellular domain of PD1 (or fragment thereof) is preferred. Such trispecific polypeptides may be advantageous in mediating an enhanced anti-tumour or anti-infective response. As a non-limiting example, the presence of the extracellular PD1 domain (or fragment thereof) in a trispecific polypeptide may interact with the PD-L1 ligand of a tumour cell, thereby enhancing the anti-tumour response of the T- and/or NK-cell that is recruited to the tumour cell via the binding domain of the polypeptide. Accordingly, in some embodiments, a soluble polypeptide is comprises a yT-cell receptor chain, a 5T-cell receptor chain, a y5T-cell receptor, or a fragment thereof (such as e.g., an extracellular domain or fragment thereof), an extracellular domain of PD1 , and a T-cell- and/or NK-cell-binding domain, preferably an anti-CD3 scFv or Fc domain. In some embodiments, the anti-CD3 scFv domain is represented by SEQ ID NO: 181 or a variant thereof.

In embodiments wherein an additional binding domain is fused to a soluble yT-cell receptor chain, soluble 5T-cell receptor chain, soluble y5T-cell receptor, or fragment thereof via a linker peptide, the linker peptide may for example be as described earlier herein for the case of chimeric antigen receptors, non-limiting examples of which being the linker peptides represented by SEQ ID NO: 80-94 and SEQ ID NO: 182-193. Additional examples of suitable linker peptides comprise or consist of Gly-Gly or Gly-Gly-Ser or Gly-Ser- Gly or Tyr-Gly-Ser, which may in some cases be repeated.

A soluble polypeptide may be synthetically made or made in vitro. A soluble polypeptide may alternatively be produced by an engineered cell, and optionally subsequently isolated and/or purified.

A soluble polypeptide as described herein may optionally comprise further additional domains, for example a domain facilitating polypeptide excretion (in embodiments wherein the soluble polypeptide is produced by a cell, i.e., a signal peptide), and/or polypeptide isolation and/or purification and/or stability. Such domains and their applications are known in the art. Additional examples of signal peptides are represented by SEQ ID NO: 194 (y chain) and SEQ ID NO: 195 (5 chain).

Examples of suitable domains facilitating polypeptide isolation and/or purification, and/or stability, may be derived from an Avi-tag, His-tag, c-myc domain, hemagglutinin tag, glutathione-S-transferase, maltose- binding protein, FLAG tag peptide, biotin acceptor peptide, streptravidin-binding peptide, calmodulin-binding peptide, bovine serum albumin, and others. Additional examples of suitable domains facilitating polypeptide isolation and/or purification, and/or stability, are encoded by SEQ ID NO: 196 (Avi-tag, suitable for biotinylation purposes) and SEQ ID NO: 197 (His-tag, suitable for purification purposes) or are represented by SEQ ID NO: 198-200.

In soluble polypeptides described herein, a T-cell- and/or NK-cell-binding domain, an immune checkpoint inhibitor domain, and/or an additional domain may be fused to a yT-cell receptor chain, a 6T-cell receptor chain, a y5T-cell receptor, or a fragment thereof (such as e.g., an extracellular domain or fragment thereof) at the N-terminus or C-terminus ofthe receptor chain, receptor, or fragment thereof. In cases where multiple domains are comprised in a soluble polypeptide, the skilled person understands that each additional domain may also be fused at the N-terminus or C-terminus of the domain it is fused to. Optionally, linker peptides may be comprised between the domains as described earlier herein.

In some embodiments, a soluble polypeptide is a dimer, or a higher multimer such as a trimer. In some embodiments, dimerization or multimerization is facilitated by the inclusion of a dimerization or multimerization domain in the polypeptide, for example a leucine zipper, a Jun-Fos interaction motif (such as for example described in Pack and Pluckthun, 1992, Biochemistry 31, 1579-1584; de Kruif and Logtenberg, 1996. JBC 271: 7630-7634, incorporated herein by reference in their entireties), or any other suitable such domain known to the skilled person. Additional examples of dimerization domains are represented by SEQ ID NO: 201 (c-Jun dimerization motif) and SEQ ID NO: 202 (c-Fos dimerization motif). Optionally, a soluble polypeptide comprising a yT-cell receptor chain or fragment thereof comprises a c-Jun dimerization motif. Optionally, a soluble polypeptide comprising a 5T-cell receptor chain or fragment thereof comprises a c-Fos dimerization motif.

Alternatively, a dimer or higher multimer may be generated via chemical cross-linking using standard methods. The skilled person understands that a dimer or a higher multimer as described herein may be a dimer or multimer of soluble polypeptides comprising the same or different yT-cell receptor chains, 5T-cell receptor chains, y5T-cell receptors, or fragment s thereof (such as e.g., extracellular domains or fragments thereof), and/or T-cell and/or NK-cell-binding domains, said polypeptides and/or domains optionally having different targets.

In embodiments wherein the soluble polypeptide comprises a yQT-cell receptor or a fragment thereof, formation of the receptor heterodimer (or fragment thereof) from the yT-cell receptor and 6T-cell receptor chain (or fragments thereof) may be promoted by inclusion of any of the dimerization domains, or any other dimerization method discussed above. Alternatively, or in addition, it can be promoted by inclusion of cysteines in the soluble polypeptide, for example in the constant Cy and/or C6 regions described herein or in any other region, to promote formation of cysteine bonds (cysteine bridges).

A soluble polypeptide described herein may be subject to post translational modifications. Glycosylation is an example of such a modification, which comprises the covalent attachment of oligosaccharide moieties to defined amino acids in the ySTCR chain. For example, asparagine residues, or serine/threonine residues are well-known locations for oligosaccharide attachment. The glycosylation status of a particular protein depends on a number of factors, including protein sequence, protein conformation and the availability of certain enzymes. Furthermore, glycosylation status (i.e. oligosaccharide type, covalent linkage and total number of attachments) can influence protein function. Therefore, when producing recombinant proteins, controlling glycosylation is often desirable. Controlled glycosylation has been used to improve antibody based therapeutics. (Jefferis etal., (2009) Nat Rev Drug Discov Mar;8(3):226-34, incorporated herein by reference in its entirety). For soluble ybTCRs of the invention glycosylation may be controlled, as an example, by using particular cell lines for their production (including but not limited to mammalian cell lines such as Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells as discussed elsewhere herein), or by chemical modification. Such modifications may be desirable, since glycosylation can improve pharmacokinetics, reduce immunogenicity and more closely mimic a native human protein (Sinclair and Elliott, (2005) Pharm Sci.Aug; 94(8): 1626-35, incorporated herein by reference in its entirety).

The skilled person may arrive at the soluble polypeptides described herein utilizing standard molecular toolbox techniques. A soluble polypeptide described herein may be synthetic, may be produced in vitro, or may be produced by an engineered cell. Optionally, the polypeptide may be isolated and/or purified after its production. Suitable downstream processing methods for isolation and/or purification of polypeptides from cell cultures are well-known in the art. Examples of suitable isolation and/or purification techniques are chromatographic methods such as high performance liquid chromatography, size exclusion chromatography, ion exchange chromatography, affinity chromatography (such as for example utilizing His- tags or protein A), immunoaffinity chromatography, immunoprecipitation, and the like. As a non-limiting example, a soluble polypeptide comprising a yT-cell receptor chain, a 5T-cell receptor chain, a yST-cell receptor, or a fragment thereof (such as e.g., an extracellular domain or fragment thereof), and a T- and/or NK-cell-binding domain (for example an anti-CD3 scFv or Fc domain) may be produced by a cell, for example a cell line such as HEK293 or HEK293F cells, and subsequently purified using affinity chromatography (for example via a His-tag peptide). A proper folding of the chimeric polypeptide can be probed using conformational-specific antibodies that can target y and 5 variable domains. Purity can be assessed by standard methods, for example SDS-PAGE and Coomassie staining. The soluble polypeptides may then be used in antibody dependent cell mediated cytotoxicity (ADCC), complement dependent cytotoxicity (GDC) and antibody drug conjugate (ADC) assays to determine functional efficacy. After performing in vitro assays, functional efficacy of soluble polypeptides can be tested in vitro and/or in vivo. Accordingly, in some embodiments, a soluble, polypeptide described herein is expressed by a cell. In some embodiments, the cell is selected from a bacterium (e.g., E. coli), a yeast (e.g., P. pastoris, S. cerevisiae), an insect cell (e.g. an Sf9 cell), a mammalian cell, or a human cell, for example a human cells line (e.g., HEK293 or HEK293F or derivatives thereof). In some embodiments, the cell is an immunoresponsive cell, preferably selected from T-cells, iPSC-derived T-cells, a T-cells, ybT-cells, or NK cells, more preferably selected from y5T-cells or a T-cells, most preferably aPT-cells. In some embodiments, the cell is an undifferentiated cell, for example a stem cell, a progenitor cell, a precursor cell, or an IPSC cell.

In a further aspect, there is provided a conjugate comprising a soluble yT-cell receptor chain, a ST-cell receptor chain, a yST-cell receptor, or a fragment thereof (such as e.g., an extracellular domain or fragment thereof), as described herein, which is linked to an agent. The skillled person understands that the type of agent used depends on the type of applications envisaged. Such a conjugate may, for example, comprise a substrate (e.g. chemicals, nanoparticles) and may be used e.g., to administer chemotherapy to a target of interest. As another example, in diagnostics expression of defined ligands may be tested by taking advantage of soluble ySTCRs linked to fluorochromes which are used as staining tool or for the biochemical isolation of the ligand.

In some embodiments, the agent is selected from the group consisting of a diagnostic agent, a therapeutic agent, an anti-cancer agent, a chemical, a nanoparticle, a chemotherapeutic agent, a radioligand, a fluorescent protein, or an enzyme whose catalytic activity could be detected.

Examples of suitable agents include, but are not limited to:

-small molecule cytotoxic agents, i.e. compounds with the ability to kill mammalian cells having a molecular weight of less than 700 Daltons. Such compounds could also contain toxic metals capable of having a cytotoxic effect. Furthermore, it is to be understood that these small molecule cytotoxic agents also include pro-drugs, i.e. compounds that decay or are converted under physiological conditions to release cytotoxic agents. Examples of such agents include cis-platin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodiumphotofrin II, temozolomide, topotecan, trimetreate 21arbour21ate, auristatin E vincristine and doxorubicin;

-peptide cytotoxins, i.e. proteins or fragments thereof with the ability to kill mammalian cells. Examples of such agents include ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, Dnase and Rnase;

- radio-nuclides, i.e. unstable isotopes of elements which decay with the concurrent emission of one or more of a or p particles, or y rays. Examples of such agents include iodine 131 , rhenium 186, indium 111, yttrium 90, bismuth 210 and 213, actinium 225 and astatine 213; chelating agents may be used to facilitate the association of these radio-nuclides to the high affinity y5TCRs, or multimers thereof;

-immuno-stimulants, i.e., immune effector molecules which stimulate immune response. Examples of such agents include cytokines such as IL-2 and IFN-y;

-superantigens and mutants thereof;

-chemokines such as IL-8, platelet factor 4, melanoma growth stimulatory protein, etc;

-alternative protein scaffolds with antibody like binding characteristics;

-complement activators;

-xenogeneic protein domains, allogeneic protein domains, viral/bacterial protein domains, viral/bacterial peptides.

In some embodiments, the conjugate is linked to a liposome containing the agent. This can prevent damaging effects during the transport in the body and can ensure that the agent exerts its activity after binding of the soluble yT-cell receptor chain, 6T-cell receptor chain, y5T-cell receptor, or fragment thereof to its target.

In some embodiments, the fluorescent protein is selected from the group consisting of: green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), cyan fluorescent variant known (CFP), yellow fluorescent protein (YFP), violet-excitable green fluorescent protein known as Sapphire, and cyan-excitable green fluorescent protein known as enhanced green fluorescent protein (eGFP). The presence of a fluorescent protein can be assessed by live cell imaging, flow cytometry, and/or fluorescent spectrophotometry. Fluorescent reporters can be detected using various means including but not limited to microscopy, visual observation, flow cytometry, Luminex, and the like. In some embodiments, a fluorescent reporter is detected using flow cytometry.

In some embodiments, the enzyme whose activity could be detected is selected from the group consisting of luciferase, beta galactosidase, beta-lactamase, catalase, alkaline phosphatase, and the like. As a nonlimiting example, luciferase activity can be detected by commercially available assays, e.g., by the Luciferase 1000 Assay System, Nano-Gio or the Bio-Gio (Promega, Wl, US). The Luciferase 1000 Assay System contains coenzyme A (CoA) besides luciferin as a substrate, resulting in a strong light intensity lasting for at least one minute when the manufacturer’s protocol is followed. Alternatively, D-luciferin can also be utilized. In some cases, prior to an intracellular luciferase assay, it may be helpful to lyse the cells prior to detection. In some embodiments a luciferase assay is used wherein the luciferase is secreted from the cells. Hence, in some cases, the assay can be performed without lysis of the cells.

The disclosure further provides nucleic acid molecules encoding the polypeptides, such as yT-cell receptor chains, ST-cell receptor chains, yST-cell receptors, fragments thereof, soluble polypeptides, or chimeric antigen-recognition receptors, described herein. A nucleic acid molecule described herein may in some cases be a synthetic nucleic acid molecule or be part of a synthetic construct. A nucleic acid molecule described herein may in some cases be a codon optimized molecule, preferably for expression in a mammalian cell, more preferably in a human cell. A definition of codon optimization is provided later herein. Accordingly, in a further aspect, there is provided a nucleic acid molecule encoding a yT-cell receptor chain or fragment thereof, comprising a Cy constant region or fragment thereof, as described herein. In a further aspect, there is provided a nucleic acid molecule encoding a 6T-cell receptor chain or fragment thereof, comprising a C5 constant region or fragment thereof, as described herein. In a further aspect, there is provided a nucleic acid molecule encoding a yST-cell receptor or fragment thereof, comprising a Cy constant region or fragment thereof and a C6 constant region or fragment thereof, as described herein.

A nucleic acid molecule described herein may be comprised in a nucleic acid construct. A nucleic acid construct may be alternatively referred to herein as an "expression construct”. Accordingly, in a further aspect, there is provided a nucleic acid construct comprising a nucleic acid molecule as described herein. The skilled person understands that the nucleic acid molecule comprised in the nucleic acid constructs described herein may be operably linked to a regulatory sequence. A definition of "operably linked” is provided later herein. As used herein, a regulatory sequence refers to any genetic element that is known to the skilled person to drive or otherwise regulate expression of nucleic acids in a cell. Such sequences include without limitation promoters, transcription terminators, enhancers, repressors, silencers, kozak sequences, polyA sequences, and the like. A regulatory sequence can, for example, be inducible, noninducible, constitutive, cell-cycle regulated, metabolically regulated, and the like. A regulatory sequence may be a promoter. More information on promoters is provided later herein. Accordingly, in some embodiments, the nucleic acid construct comprises a nucleic acid molecule operably linked to a promoter. Non-limiting examples of suitable promoters include EF1a, MSCV, EIF alpha-HTLV-1 hybrid promoter, Moloney murine leukemia virus (MoMuLV or MMLV), Gibbon Ape Leukemia virus (GALV), murine mammary tumor virus (MuMTV or MMTV), Rous sarcoma virus (RSV), MHO class II, clotting Factor IX, insulin promoter, PDX1 promoter, CD11 , CD4, CD2, gp47 promoter, PGK, Beta-globin, UbC, MND, and derivatives (i.e. variants) thereof, of which the MSCV promoter is preferred. An example of an MSCV promoter comprises SEQ ID NO: 57.

A nucleic acid construct may comprise additional nucleic acid molecules, for example encoding a 2A-self cleaving peptide as described later herein.

A nucleic acid molecule or nucleic acid construct described herein may be comprised in a vector. Accordingly, in a further aspect, there is provided a vector comprising a nucleic acid molecule or nucleic acid construct as described herein. A preferred vector is a viral vector, preferably a retroviral or lentiviral vector. Suitable vectors are known to the skilled person and further information is provided later herein.

In some embodiments, the vector is a good manufacturing practices (GMP) compatible vector. For example, a GMP vector can be purer than a non-GMP vector. In some cases, purity can be measured by bioburden. For example, bioburden can be the presence or absence of aerobes, anaerobes, sporeformers, fungi, or combinations thereof in a vector composition. In some cases, a pure vector can be endotoxin low or endotoxin free. Purity can also be measured by double-stranded primer-walking sequencing. Plasmid identity can be a source of determining purity of a vector. A GMP vector of the disclosure can be from 10% to 99% more pure than a non-GMP vector. A GMP vector can be from 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% more pure than a non-GMP vector as measured by the presence of bioburden, endotoxin, sequencing, or combinations thereof.

Each of the yT-cell receptor chains, 5T-cell receptor chains, y5T-cell receptors, fragments thereof and polypeptides comprising them (such as soluble polypeptides or chimeric antigen-recognition receptors), described herein, or encoded by the nucleic acid molecules, constructs, and vectors described herein, are preferably able to mediate an anti-tumour activity/response and/or an anti-infective activity/response. Accordingly, they are preferably suitable for designing a medicament, such as for example for preventing, treating, regressing, curing and/or delaying a cancer or an infection in a subject, preferably in a human being.

Table 1 provides non-limiting examples of sequences according to the disclosure.

Cells

In a further aspect, there Is provided a cell, for example an Immunoresponslve cell, expressing a polypeptide and/or comprising a nucleic acid molecule, a nucleic acid construct, and/or a vector as described herein. Preferably, a cell, for example an immunoresponslve cell, is selected from a T-cell, an IPSC-derived T-cell, an aPT-cell, a yST-cell, or an NK cell, more preferably is selected from a y5T-cell or aPT-cell, most preferably is an aPT-cell. Such a cell may alternatively be called an engineered cell. In some embodiments, a cell, for example an immunoresponslve cell, is a primary cell Isolated from a donor. A cell expressing a polypeptide and/or comprising a nucleic acid molecule, a nucleic acid construct, and/or a vector as described herein may also be from a cell line, for example a Jurkat cell line, or the like. Alternatively, the cell may be, or may be derived from an undifferentiated cell, for example a stem cell, a progenitor cell, a precursor cell, or an IPSC cell, preferably a blood progenitor cell such as a thymocyte or a blood stem cell.

In some embodiments, the cell, for example the immunoresponslve cell, expresses a yT-cell receptor chain, a 5T-cell receptor chain, a yBT-cell receptor, fragment thereof, or chimeric-antigen recognition receptor as described herein. In some embodiments, the cell, for example the Immunoresponslve cell, Is a mammalian cell, preferably a human cell, more preferably a human y5T-cell or apT-cell, most preferably is a human apT-cell.

It is understood that in embodiments wherein a cell, for example an immunoresponslve cell, expresses a yT-cell receptor chain or fragment thereof as described herein, the cell may also express a 5T-cell receptor chain or fragment thereof. Expression of any 5T-cell receptor chain or fragment thereof may be contemplated, as long as a ySTCR complex which is able to mediate an anti-tumour or anti-infective response is assembled on the cell surface. It is also understood that in embodiments wherein a cell, for example an immunoresponslve cell, expresses a 5T-cell receptor chain or fragment thereof as described herein, the cell may also express a yT-cell receptor chain or fragment thereof. Expression of any yT-cell receptor chain or fragment thereof may be contemplated, as long as a y5TCR complex which is able to mediate an anti-tumour or anti-infective response is assembled on the cell surface. In some embodiments, a cell, for example an immunoresponsive cell, expresses a y5T-cell receptor or a fragment thereof comprising: a yT-cell receptor chain or fragment thereof as described herein; a 5T-cell receptor chain or fragment thereof as described herein, or; a yT-cell receptor chain or fragment thereof as described herein and a ST-cell receptor chain or fragment thereof as described herein.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a ybT-cell receptor or fragment thereof comprising: a yT-cell receptor chain or fragment thereof as described herein, and/or, preferably and; a 5T-cell receptor chain or fragment thereof comprising a C5 constant region or fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a y5T-cell receptor or fragment thereof comprising: a yT-cell receptor chain or fragment thereof comprising a Cy constant region or fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1, and/or, preferably and; a 5T-cell receptor chain or fragment thereof as described herein.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a ybT-cell receptor or fragment thereof comprising: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising:

XXIV. an amino acid substitution in a position corresponding to position N14 of SEQ ID NO: 2, preferably a substitution by a serine; XXV. an amino acid substitution in a position corresponding to position V48 of SEQ ID NO: 2, preferably a substitution by a cysteine;

XXXII. a combination of any one of XXIV. To XXXI.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yBT-cell receptor or fragment thereof comprising: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, the substitution preferably being a C158F substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitution preferably being a F144L substitution.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a y5T-cell receptor or fragment thereof comprising: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position C174 of SEQ ID NO: 40 or SEQ ID NO: 41 , the substitution preferably being a C174F substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitution preferably being a F144L substitution.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or fragment thereof comprising:

-a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 14.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a ybT-cell receptor or fragment thereof comprising: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, the substitution preferably being a S149A substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, the substitution preferably being a T139V substitution.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or fragment thereof comprising: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S165 of SEQ ID NO: 40 or SEQ ID NO: 41 , the substitution preferably being a S165A substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, the substitution preferably being a T139V substitution.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a ydT-cell receptor or fragment thereof comprising:

-a 5T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 13.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a y5T-cell receptor or fragment thereof comprising: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, the substitution preferably being a S149A substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2 and an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, the substitutions preferably being respectively a T128L and a T139V substitution.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a y5T-cell receptor or fragment thereof comprising: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S165 of SEQ ID NO: 40 or SEQ ID NO: 41 , the substitution preferably being a S165A substitution, and/or, preferably and; a 6T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2 and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T139V and a F144L substitution.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a y5T-cell receptor or fragment thereof comprising:

In some embodiments, a cell, for example an immunoresponsive cell, expresses a ybT-cell receptor or fragment thereof comprising:

-a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, and an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, the substitutions preferably being respectively a T128L and a T139V substitution.

-a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 18. In some embodiments, a cell, for example an immunoresponsive cell, expresses a y6T-cell receptor or a fragment thereof comprising: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, the substitution preferably being a S149A substitution, and/or, preferably and; a ST-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2 and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T139V and a F144L substitution.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a y6T-cell receptor or a fragment thereof comprising: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S165 of SEQ ID NO: 40 or SEQ ID NO: 41 , the substitution preferably being a S165A substitution, and/or, preferably and; a ST-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2 and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T139V and a F144L substitution.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yBT-cell receptor or a fragment thereof comprising: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 6, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 19.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a y6T-cell receptor or a fragment thereof comprising: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, the substitution preferably being a S149A substitution, and/or, preferably and; a 6T-cell receptor chain or a fragment thereof, comprising a 06 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T128L, a T139V, and a F144L substitution.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a ybT-cell receptor or a fragment thereof comprising: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S165 of SEQ ID NO: 40 or SEQ ID NO: 41 , the substitution preferably being a S165A substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T128L, a T139V, and a F144L substitution.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a ybT-cell receptor or a fragment thereof comprising: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 6, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 20.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or a fragment thereof comprising: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , and an amino acid substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , the substitutions preferably being respectively a S149A and a V151 L substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T128L, a T139V, and a F144L substitution.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a y6T-cell receptor or a fragment thereof comprising: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S165 of SEQ ID NO: 40 or SEQ ID NO: 41, and an amino acid substitution in a position corresponding to position V167 of SEQ ID NO: 40 or SEQ ID NO: 41 , the substitutions preferably being respectively a S165A and a V167L substitution, and/or, preferably and; a 6T-cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T128L, a T139V, and a F144L substitution.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a y6T-cell receptor or a fragment thereof comprising: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 15, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 20.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or a fragment thereof comprising: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , an amino acid substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, and an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, the substitutions preferably being respectively a S149A, a V151 L, and a C158F substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2 and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T139V and a F144L substitution.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yBT-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S165 of SEQ ID NO: 40 or SEQ ID NO: 41, an amino acid substitution in a position corresponding to position V167 of SEQ ID NO: 40 or SEQ ID NO: 41, and an amino acid substitution in a position corresponding to position C174 of SEQ ID NO: 40 or SEQ ID NO: 41, the substitutions preferably being respectively a S165A, a V167L, and a C174F substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2 and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T139V and a F144L substitution.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 17, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 19.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, and an amino acid substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , the substitutions preferably being respectively a S149A and a V151 L substitution, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2 and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T139V and a F144L substitution.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S165 of SEQ ID NO: 40 or SEQ ID NO: 41 , and an amino acid substitution in a position corresponding to position V167 of SEQ ID NO: 40 or SEQ ID NO: 41, the substitutions preferably being respectively a S165A and a V167L substitution, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2 and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T139V and a F144L substitution.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 15, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 19.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, and an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, the substitutions preferably being respectively a S149A and a C158F substitution, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2 and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T139V and a F144L substitution.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S165 of SEQ ID NO: 40 or SEQ ID NO: 41, and an amino acid substitution in a position corresponding to position C174 of SEQ ID NO: 40 or SEQ ID NO: 41 , the substitutions preferably being respectively a S165A and a C174F substitution, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2 and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T139V and a F144L substitution.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 16, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 19.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a ybT-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, and an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, the substitutions preferably being respectively a S149A and a C158F substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T128L, T139V, and a F144L substitution.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position S165 of SEQ ID NO: 40 or SEQ ID NO: 41, and an amino acid substitution in a position corresponding to position C174 of SEQ ID NO: 40 or SEQ ID NO: 41 , the substitutions preferably being respectively a S165A and a C174F substitution, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, the substitutions preferably being respectively a T128L, T139V, and a F144L substitution.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 16, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 20. In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position K49 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 , and an amino acid substitution in a position corresponding to position K50 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 , the substitutions preferably being respectively a K49A and a K50A substitution, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a ybT-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 26, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a Y^T-cell receptor or a fragment thereof comprising: a YT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position E79 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 , and an amino acid substitution in a position corresponding to position K80 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, the substitutions preferably being respectively a E79A and a K80A substitution, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or a fragment thereof comprising: a YT -cell receptor chain or a fragment thereof, comprising a CY constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 29, and/or, preferably and; a 6T -cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or a fragment thereof comprising: a YT -cell receptor chain or a fragment thereof, comprising a CY constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position K96 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1, and an amino acid substitution in a position corresponding to position N97 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1, the substitutions preferably being respectively a K96A and a N97A substitution, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a ybT-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 32, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a ybT-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position K96 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1, and an amino acid substitution in a position corresponding to position N97 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1, the substitutions preferably being respectively a K96D and a N97K substitution, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a ybT-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 27, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position K49 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1, an amino acid substitution in a position corresponding to position K50 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 , an amino acid substitution in a position corresponding to position K96 of SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1, and an amino acid substitution in a position corresponding to position N97 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 , the substitutions preferably being respectively a K49A, K50A, K96D, and a N97K substitution, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2. In some embodiments, a cell, for example an immunoresponsive cell, expresses a y5T-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 95, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or fragment thereof comprising: a yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position N66 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 , the substitution preferably being a N66S substitution, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position N14 of SEQ ID NO: 2, the substitution preferably being a N14S substitution.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yBT-cell receptor or fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 35 and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 36.

In some embodiments, a cell, for example an immunoresponsive cell, expresses y5T-cell receptor or fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position Q58 of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 , the substitution preferably being a Q58C substitution, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising an amino acid substitution in a position corresponding to position V48 of SEQ ID NO: 2, the substitution preferably being a V48C substitution.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a y5T-cell receptor or fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 37 and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 38.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from D1 to V8 (DKQLDADV (SEQ ID NO: 42)) of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41 , preferably SEQ ID NO: 1 or SEQ ID NO: 39, more preferably SEQ ID NO: 1 , by the amino acid sequence EDLKNVF (SEQ ID NO: 43), DKEDLKNVF (SEQ ID NO: 176), or a variant thereof, preferably by the amino acid sequence EDLKNVF (SEQ ID NO: 43) or a variant thereof, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a ybT-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 21 , and/or, preferably and; a ST-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from E93 to D100 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1, (ENNKNGVD (SEQ ID NO: 44)) by the amino acid sequence YGLSENDEWTQDRAKPVT (SEQ ID NO: 46) or a variant thereof, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from E93 to D100 of SEQ ID NO: 40 or SEQ ID NO: 41 (ENNKNGID (SEQ ID NO: 45)) by the amino acid sequence YGLSENDEWTQDRAKPVT (SEQ ID NO: 46) or a variant thereof, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , (VRHENNKNGVDQ (SEQ ID NO: 48)) by the amino acid sequence VQFYGLSENDEWTQDRAKPVTQIV (SEQ ID NO: 47) or a variant thereof, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C6 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 40 or SEQ ID NO: 41 (VRHENNKNGIDQ (SEQ ID NO: 49)) by the amino acid sequence VQFYGLSENDEWTQDRAKPVTQIV (SEQ ID NO: 47) or a variant thereof, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a yST-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , (VRHENNKNGVDQ (SEQ ID NO: 48)) by the amino acid sequence VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50) or a variant thereof, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a Y6T-cell receptor or a fragment thereof comprising: a YT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 40 or SEQ ID NO: 41 (VRHENNKNGIDQ (SEQ ID NO: 49)) by the amino acid sequence VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50) or a variant thereof, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a Y^T-cell receptor or fragment thereof comprising: a YT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from D1 to V8 (DKQLDADV (SEQ ID NO: 42)) of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , by the amino acid sequence EDLKNVF (SEQ ID NO: 43), DKEDLKNVF (SEQ ID NO: 176), or a variant thereof, preferably by the amino acid sequence EDLKNVF (SEQ ID NO: 43) or a variant thereof, and a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably SEQ ID NO: 1 , (VRHENNKNGVDQ (SEQ ID NO: 48)) by the amino acid sequence VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50) or a variant thereof, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a Y^T-cell receptor or fragment thereof comprising: a YT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising a substitution of the positions corresponding to positions from D1 to V8 (DKQLDADV (SEQ ID NO: 42)) of SEQ ID NO: 40 or SEQ ID NO: 41 by the amino acid sequence EDLKNVF (SEQ ID NO: 43), DKEDLKNVF (SEQ ID NO: 176), or a variant thereof, preferably by the amino acid sequence EDLKNVF (SEQ ID NO: 43) or a variant thereof, and a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 40 or SEQ ID NO: 41 (VRHENNKNGIDQ (SEQ ID NO: 49)) by the amino acid sequence VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50) or a variant thereof, and/or, preferably and; a 5T -cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a ybT-cell receptor or a fragment thereof comprising: a yT -cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 177, preferably SEQ ID NO: 24 or SEQ ID NO: 177, more preferably SEQ ID NO: 24, and/or, preferably and; a 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2.

Preferably, the Cy region or fragment thereof comprised by the yST-cell receptors or fragments thereof expressed by the cells described herein comprises at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or similarity with SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably with SEQ ID NO: 1, and the C6 region or fragment thereof comprised by the yST-cell receptors or fragments thereof expressed by the cells described herein comprises at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or similarity with SEQ ID NO: 2.

In some embodiments, the y5T-cell receptor or fragment thereof expressed by the cell, for example immunoresponsive cell, further comprises:

- a y variable region or fragment thereof comprising, consisting essentially of, or consisting of, preferably comprising, an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 108, 109, 110, 111 , 112, 113, or 122, and/or, preferably and;

- a y variable region or fragment thereof comprising, consisting essentially of, or consisting of, preferably comprising, an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 108, 109, 110, 111, 112, 113, 122, or 150 and/or, preferably and; - a 5 variable region or fragment thereof comprising, consisting essentially of, or consisting of, preferably comprising, an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 114, 115, 116, 117, 118, 119, 123, or 151.

- a 6CDR3 region comprising, consisting essentially of, or consisting of, preferably comprising, an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 131 , 132, 133, 134, 135, 136, or 137.

In some embodiments, the yST-cell receptor or fragment thereof expressed by the cell, for example immunoresponsive cell, further comprises:

- a 5CDR3 region comprising at least at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity or similarity with SEQ ID NO: 131, wherein the CDR3 region comprises a modification relative to SEQ ID NO: 131 at an amino acid position corresponding to a position selected from one or more of positions 7-12 of SEQ ID NO: 131.

-a 6CDR3 region comprising a modification at an amino acid position corresponding to a position selected from one or more of positions 7-12 of SEQ ID NO: 131 , said modification selected from IRGFTG (SEQ ID NO: 144), IKGYTG (SEQ ID NO: 145), IKGFTG (SEQ ID NO: 146), LRGFTG (SEQ ID NO: 147), LKGFTG (SEQ ID NO: 148), or LKGYTG (SEQ ID NO: 149).

In some embodiments, a preferred yST-cell receptor or fragment thereof expressed by the cell, for example immunoresponsive cell, further comprises:

-a 5CDR1 region comprising, consisting essentially of, or consisting of amino acid sequence SEQ ID NO: 162, 163, or 164, or of an amino acid sequence comprising one, two, or three amino acid modifications relative to SEQ ID NO: 162, 163, or 164, and a 5CDR2 region comprising, consisting essentially of, or consisting of amino acid sequence EKD, QGS, or SEQ ID NO: 166, or of an amino acid sequence comprising one, two, orthree amino acid modifications relative to amino acid sequence EKD, QGS, or SEQ ID NO: 166.

In some embodiments, the yST-cell receptor or fragment thereof expressed by the cell, for example immunoresponsive cell, comprises:

In some embodiments, the y5T-cell receptor or fragment thereof expressed by the cell, for example immunoresponsive cell, comprises:

-a yT-cell receptor chain or fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, or 244, and/or, preferably and;

In some embodiments, a cell, for example an immunoresponsive cell, expresses a chimeric antigenrecognition receptor comprising: a Cy constant region of a yT-cell receptor chain or a fragment thereof as described herein; a C5 constant region of a 5T-cell receptor chain or a fragment thereof as described herein, or; a Cy constant region of a yT-cell receptor chain or a fragment thereof and a C5 constant region of a 5T-cell receptor chain or a fragment thereof as described herein. in some embodiments, a cell, for example an immunoresponsive cell, expresses a chimeric antigenrecognition receptor comprising: a yT-cell receptor chain or a fragment thereof as described herein; a 5T-cell receptor chain or fragment thereof as described herein, or; a yT-cell receptor chain or fragment thereof and a 5T-cell receptor chain or fragment thereof as described herein.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a chimeric antigenrecognition receptor comprising: a Ca constant region of an aT-cell receptor chain or a fragment thereof, and; a Cy constant region of a yT-cell receptor chain or a fragment thereof, preferably a transmembrane domain, as described herein.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a chimeric antigenrecognition receptor comprising: a Ca constant region of an aT-cell receptor chain or a fragment thereof; a Cy constant region of a yT-cell receptor chain or a fragment thereof, preferably a transmembrane domain, as described herein, and a C5 constant region of a 5T-cell receptor chain or a fragment thereof, preferably a transmembrane domain, as described herein.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a chimeric antigenrecognition receptor comprising: a Ca constant region of an aT-cell receptor chain or a fragment thereof, and; a yT-cell receptor chain or a fragment thereof as described herein.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a chimeric antigenrecognition receptor comprising: a Ca constant region of an aT-cell receptor chain or a fragment thereof; a yT-cell receptor chain or a fragment thereof as described herein, and; a 5T-cell receptor chain or a fragment thereof as described herein.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a chimeric antigenrecognition receptor comprising: a Ca constant region of an aT-cell receptor chain or a fragment thereof, and; a C5 constant region of a 5T-cell receptor chain or a fragment thereof, preferably a transmembrane domain, as described herein.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a chimeric antigenrecognition receptor comprising: a Ca constant region of an aT-cell receptor chain or a fragment thereof, and; a 5T-cell receptor chain or a fragment thereof as described herein.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a chimeric antigen recognition receptor comprising: a Cp constant region of an PT-cell receptor chain or a fragment thereof, and; a Cy constant region of a yT-cell receptor chain or a fragment thereof, preferably a transmembrane domain, as described herein.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a chimeric antigenrecognition receptor comprising: a CP constant region of an T-cell receptor chain or a fragment thereof; a Cy constant region of a yT-cell receptor chain or a fragment thereof, preferably a transmembrane domain, as described herein, and; a C5 constant region of a 5T-cell receptor chain or a fragment thereof, preferably a transmembrane domain, as described herein.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a chimeric antigenrecognition receptor comprising: a CP constant region of an PT-cell receptor chain or a fragment thereof, and; a yT-cell receptor chain or a fragment thereof as described herein.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a chimeric antigenrecognition receptor comprising: a CP constant region of an PT-cell receptor chain or a fragment thereof; a yT-cell receptor chain or a fragment thereof as described herein, and; a 5T-cell receptor chain or a fragment thereof as described herein.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a chimeric antigenrecognition receptor comprising: a Cp constant region of an PT-cell receptor chain or a fragment thereof, and; a C5 constant region of a 5T-cell receptor chain or a fragment thereof, preferably a transmembrane domain, as described herein.

In some embodiments, a cell, for example an immunoresponsive cell, expresses a chimeric antigenrecognition receptor comprising: a c constant region of an pT-cell receptor chain or a fragment thereof, and; a 6T-cell receptor chain or a fragment thereof as described herein.

In some embodiments, the cell, for example immunoresponsive cell, is an apT-cell having decreased or no expression of an endogenous apTCR. Decreased expression may correspond to at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, decreased expression of an apTCR relative to an otherwise comparable apT-cell not comprising a genomic modification or not having been subjected to selective expansion as described later herein. In some embodiments, an apT-cell comprises a higher ratio of an exogenous ySTCR or fragment thereof as compared to an endogenous (naturally expressed) apTCR. In certain cases, decreased expression of an endogenous apTCR and/or a higher ratio of an exogenous y5TCR or fragment thereof to an endogenous apTCR may be achieved by way of preferential expansion of said T-cells, benefiting the growth and survival of said T-cells. Decreased expression of an endogenous apTCR and/or a higher ratio of an exogenous y5TCR or fragment thereof to an endogenous a TCR may be promoted by improved expression of the exogenous ybTCR or fragment thereof as described herein and as demonstrated in the experimental section.

As non-limiting examples, aPT-cells comprising an exogenous ySTCR or fragment thereof may be stimulated by anti-CD3/CD28 antibodies, by contact with antigens that are specific for the exogenous ySTCR or fragment thereof, or with cells expressing such antigens, optionally the stimulation being serial stimulation. The preferential expansion may result in a population of said aPT-cells with limited or absent cell surface expression of the endogenous aPTCR, while expressing sufficient amounts of the exogenous ySTCR or fragment thereof (referred to as a population enriched for a single positive phenotype). Such cells may have reduced alloreactivity (e.g., graft versus host disease) as compared to cells having surface expression of the endogenous apTCR. Reduced alloreactivity may result in improved therapeutic applications with decreased side-effects.

In other cases, decreased expression of an endogenous apTCR and/or a higher ratio of an exogenous ybTCR or fragment thereof to an endogenous apTCR, can be achieved by positively or negatively selecting for cells that express the exogenous ySTCR or fragment thereof and have reduced surface expression of the endogenous receptor or lack the endogenous receptor.

In other cases, decreased expression of an endogenous apTCR and/or a higher ratio of an exogenous ySTCR or fragment thereof to an endogenous apTCR can be achieved via genomic modification, for example a genomic modification which results in the reduction or elimination of surface expression of the endogenous apTCR. A genomic modification may be combined with preferential expansion and/or selection as discussed above. Genomic modification techniques are discussed later herein.

In some embodiments, an aPT-cell of the disclosure comprises a ratio of an exogenous ySTCR or fragment thereof to an endogenous apTCR that is at least 0.5:1 , at least 1:1, at least 2:1, at least 3:1 , at least 4:1 , at least 5: 1 , at least 6: 1 , at least 7: 1 , at least 8: 1 , at least 9: 1 , at least 10: 1 , at least 11 : 1 , at least 12: 1 , at least 13:1, at least 14, :1 at least 15:1 , at least 20:1 , at least 30:1 , at least 40:1, at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least 90:1, at least 100:1 , at least 150:1, at least 200:1, at least 250:1, or at least 300:1.

In some embodiments, expression of a yT-cell receptor chain, a 5T-cell receptor chain, a y5T-cell receptor, a fragment thereof, or a chimeric antigen-recognition receptor, by a cell (for example an immunoresponsive cell), preferably selected from a T-cell, an iPSC-derived T-cell, an apT-cell, a y5T-cell, or an NK cell, more preferably selected from a y5T-cell or apT-cell, most preferably apT-cell, as described herein may result in increased anti-tumour or anti-infective response, increased expansion, fitness and/or survival, and/or decreased exhaustion of the cell relative to an otherwise comparable cell not expressing the yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, fragment thereof, or chimeric antigen-recognition receptor of the disclosure. Assays for measuring the anti-tumour or anti-infective response of cells, for example immunoresponsive cells, have been described earlier herein, and further examples are given in the experimental section.

In some embodiments, the anti-tumour or anti-infective response of a cell, for example an immunoresponsive cell, expressing a yT-cell receptor chain, a 5T-cell receptor chain, a y5T-cell receptor, a fragment thereof, or a chimeric antigen-recognition receptor as described herein, is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7- fold, at least 8-fold, at least 9-fold, at least 10-fold, or more, relative to an otherwise comparable cell not expressing the yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, fragment thereof, or chimeric antigen-recognition receptor.

The expansion (proliferative ability), fitness and/or survival (lifespan), and/or exhaustion of the provided cell, for example immunoresponsive cell, may be assessed using any technique known to the skilled person. As a non-limiting example, in the case of T-cells and for assessment of expansion, T-cells may be optionally stimulated with anti-CD3/CD28 polymeric nanomatrix beads, in the presence of IL-7 and IL-15. This may be performed using commercially available kits as discussed herein. The T-cells may be stimulated by bringing them into contact with an antigen, epitope, or target cells as described earlier herein. The skilled person may measure the T-cell number prior to and post-stimulation and thus determine the proliferative ability of the cells. Alternatively, expansion may be monitored via e.g., cell trace violet (or any other suitable dye) dilution when T-cells are stained at the start of a proliferative assay.

Fitness of T-cells may, for example, be monitored based on staining for various markers including, but not limited to CD4, CD8a, CD3, apTCR, ySTCR, 4-1 BB, 0X40, PD-1 , TIM-3, LAG-3, 4-1 BBL, OX40L, CD86, Fab2, CD107a and CD69, for example staining with fluorescent-labelled antibodies targeting these markers in combination with flow cytometry.

In some embodiments, expansion of a cell, for example an immunoresponsive cell, expressing a yT-cell receptor chain, a 5T-cell receptor chain, a yST-cell receptor, a fragment thereof, or chimeric-antigen recognition receptor as described herein, is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or more, relative to an otherwise comparable cell not expressing the yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, fragment thereof, or chimeric antigen-recognition receptor.

In some embodiments, fitness of a cell, for example an immunoresponsive cell, expressing a yT-cell receptor chain, a 5T-cell receptor chain, a yST-cell receptor, a fragment thereof, or a chimeric-antigen recognition receptor, as described herein, is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or more, relative to an otherwise comparable cell not expressing the yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, fragment thereof, or chimeric antigen-recognition receptor.

Survival of cells, for example immunoresponsive cells, may, for example, be monitored based on any cell viability assay known to the skilled person, many of which are commercially available (see for example the assays offered by ThermoFisher Scientific, WA, MA, USA). Non-limiting examples of cell viability assays involve the use of dyes such as calcein AM, ethidium-homodimer-1 , SYTOX Deep Red, DiOC 19(3), propidium iodide, SYBR 14, SYTO 10, green ethidium homodimer-2, SYTOX Green, C-12 resazurin, BOBO-3 iodide, DAPI, and others. By knowing a starting number of cells, for example immunoresponsive cells, the skilled person may monitor their survival by measuring the number of viable cells over time.

In some embodiments, survival of a cell, for example an immunoresponsive cell, expressing a yT-cell receptor chain, a 5T-cell receptor chain, a y5T-cell receptor, a fragment thereof, or a chimeric antigenrecognition receptor, as described herein, is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or more, relative to an otherwise comparable cell not expressing the yT-cell receptor chain, 5T-cell receptor chain, y5T-cell receptor, fragment thereof, or chimeric antigen-recognition receptor. Survival may be measured over a defined period, for example over about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, or about 10 weeks.

In some embodiments, upon exposure of a cell, for example an immunoresponsive cell, expressing a yT- cell receptor chain, a ST-cell receptor chain, a y5T-cell receptor, a fragment thereof, or a chimeric antigenrecognition receptor, as described herein, to target cells for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 14 days, at least 21 days, or at least 28 days, expression of an exhaustion marker by the cell is at least 5%, least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, or at least 1000 fold lower compared to upon exposure of a corresponding control (comparable) cell not expressing the yT-cell receptor chain, 5T-cell receptor chain, y5T-cell receptor, fragment thereof, or chimeric antigen-recognition receptor.

In some embodiments, the increased anti-tumour or anti-infective response, increased expansion, fitness and/or survival, and/or decreased exhaustion may persist over multiple stimulations of the cells, for example immunoresponsive cells, such as at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine stimulations or more, for example over multiple exposures to target antigens, epitopes, or target cells (serial stimulation). The persisting improvement may be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more, relative to control (comparable) cells not expressing the yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, fragment thereof, or chimeric antigen-recognition receptor. Stimulation of cells, for example immunoresponsive cells, has been described earlier herein.

Population of cells

In a further aspect, there is provided a population of cells comprising a cell, for example an immunoresponsive cell, as defined earlier herein. In some embodiments, such a cell expresses a yT-cell receptor chain, a 5T-cell receptor chain, yST-cell receptor, a fragment thereof, or chimeric-antigen recognition receptor as described herein. In some embodiments, such a cell comprises a nucleic acid molecule, construct, or vector encoding a yT-cell receptor chain, a 5T-cell receptor chain, y5T-cell receptor, a fragment thereof, or a chimeric antigen-recognition receptor as described herein.

In some embodiments, within the cell population, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the cells are expressing a yT-cell receptor chain, a 5T-cell receptor chain, a y5T-cell receptor, a fragment thereof, a chimeric antigen-recognition receptor, or are comprising a nucleic acid molecule, construct, or vector encoding a yT-cell receptor chain, a 5T-cell receptor chain, a yST-cell receptor, a fragment thereof, or chimeric antigen-recognition receptor as described herein. The person skilled in the art is well capable of selecting and/or identifying cell populations, or cells within cell populations, characterized by expression of the yT-cell receptor chain, 5T-cell receptor chain, y5T-cell receptor, fragment thereof, or chimeric antigen-recognition receptor using, e.g., flow cytometric methods such as FACS as explained earlier herein.

In a further aspect, there is provided a composition, preferably a pharmaceutical composition, comprising a yT-cell receptor chain, a 5T-cell receptor chain, a yST-cell receptor, a fragment thereof (for example a soluble yT-cell receptor chain, 6T-cell receptor chain, y5T-cell receptor, or fragment thereof), a chimeric antigen-recognition receptor, a nucleic acid molecule, a nucleic acid construct, a vector, or a cell (for example an immunoresponsive cell), as described herein.

Pharmaceutical compositions of the present disclosure comprise an effective amount of one or more molecules (a yT-cell receptor chain, a 5T-cell receptor chain, a yST-cell receptor, a fragment thereof, a chimeric antigen-recognition receptor, a nucleic acid molecule, a nucleic acid construct, a vector, or a cell (for example an immunoresponsive cell) as described herein), optionally dissolved or dispersed in a pharmaceutically acceptable carrier.

The term "effective amount" as used herein is defined as the amount of the molecules of the present disclosure that are necessary to result in the desired physiological change in the cell or tissue to which it is administered. The term "therapeutically effective amount" as used herein is defined as the amount of the molecules of the present disclosure that achieves a desired effect with respect to cancer. In this context, a “desired effect" is synonymous with “an anti-tumour or anti-infective” activity/response as earlier defined herein. A skilled artisan readily recognizes that in many cases the molecules may not provide a cure but may provide a partial benefit, such as alleviation or improvement of at least one symptom or parameter. In some embodiments, a physiological change having some benefit is also considered therapeutically beneficial. Thus, in some embodiments, an amount of molecules that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount."

The phrases "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce or produce acceptable adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. Whether certain adverse effects are acceptable is determined based on the severity of the disease. The preparation of a pharmaceutical composition that contains at least one active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference in its entirety. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, supra). Except insofar as any conventional carrier is incompatible with the molecules described herein, its use in the therapeutic or pharmaceutical compositions is contemplated.

In certain embodiments, a pharmaceutical composition described herein further comprises a suitable amount of an antifungal agent. In some cases, a pharmaceutical composition described herein comprises an antifungal agent in an amount sufficient for the pharmaceutical composition to retain at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of its desired activity for a period of at least 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years.

The actual dosage amount of a composition of the present disclosure administered to an animal or a patient (e.g., a human) can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

In some embodiments, a preferred pharmaceutical composition comprises a cell (for example an immunoresponsive cell), preferably selected from a T-cell, an iPSC-derived T-cell, an apT-cell, a y5T-cell, or an NK cell, more preferably selected from a yQT-cell or a T-cell, most preferably apT-cell, expressing a yT-cell receptor chain, a ST-cell receptor chain, a yST-cell receptor, a fragment thereof, a chimeric antigenrecognition receptor, or comprising a nucleic acid molecule, a nucleic acid construct, or a vector as described herein. The composition may comprise a population of said cells as described herein. In some embodiments, a pharmaceutical composition comprises a soluble yT-cell receptor chain, 6T-cell receptor chain, ybT-cell receptor, or a fragment thereof, as described herein. These compositions may easily be obtained using the information of the disclosure.

As a non-limiting example, in the case of T-cells (for example aPT-cells) prior to expansion and genetic modification of the T-cells, a source of T-cells may be obtained from a subject. T-cells can be obtained from a number of sources, including PBMCs, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumours. In some embodiments, T-cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. In some embodiments, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T- cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, a T-cell may be an effector (TEFF), effector-memory (TEM), central-memory (TCM), T memory stem (TSCM), naive (TN), or CD4+ or CD8+ T-cell. The T-cells can also be selected from a bulk population, for example, selecting T-cells from whole blood. The T-cells can also be expanded from a bulk population. The T-cells, preferably aPT-cells, may be transduced with a yT-cell receptor chain, a 6T-cell receptor chain, a yST-cell receptor, a fragment thereof, or a chimeric antigen-recognition receptor as described herein, and can be formulated into a pharmaceutical composition. A T-cell, preferably apT-cell, may alternatively be obtained by differentiation of a stem cell, progenitor cell, precursor cell, or IPSC cell, performed according to standard procedures in the art. In such cases, transduction with the yT-cell receptor chain, 6T-cell receptor chain, yST-cell receptor, fragment thereof, or chimeric antigen-recognition receptor may be performed before or after the differentiation of the stem cell, progenitor cell, precursor cell, or iPSC cell into a T-cell.

A cell (for example an immunoresponsive cell), preferably selected from a T-cell, an iPSC-derived T-cell, an apT-cell, a yST-cell, or an NK cell, more preferably selected from a yST-cell or apT-cell, most preferably aPT-cell, used in compositions, pharmaceutical compositions, and therapeutic uses/methods described herein can be autologous to a subject in need of therapy. The cell (for example immunoresponsive cell) can be allogeneic to a subject in need of therapy. In some embodiments, the cell (for example immunoresponsive cell) does not exhibit alloreactivity. The cell (for example immunoresponsive cell) can be part of a combination therapy to treat a subject in need thereof. The cell (for example immunoresponsive cell) can be a human cell. The subject that is being treated can be a human. A method of attaining suitable cells (for example immunoresponsive cells) can comprise sorting of cells. In some cases, a cell can comprise a marker that can be selected for the cell. For example, such marker can comprise GFP (such as e.g., represented by SEQ ID NO: 51), a resistance gene, a cell surface marker, or an endogenous tag. Cells can be selected using any endogenous marker. Suitable cells can be selected or sorted using any technology. Such technology can comprise flow cytometry and/or magnetic columns. The selected cells can then be infused into a subject or differentiated into immunoresponsive cells as applicable. The selected cells can also be expanded to large numbers. The selected cells can be expanded prior to infusion. For genetic modification, vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, T cells, bone marrow aspirates, tissue biopsy), followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector. Prior to or after selection, the cells can be expanded.

Ex vivo cell transfection can also be used for diagnostics, research, or for gene therapy (e.g. via re-infusion of the transfected cells into the host organism). In some cases, cells (for example immunoresponsive cells) are isolated from the subject organism, transfected with a nucleic acid (e.g., gene or DNA), and re-infused back into the subject organism (e.g. patient). Further, also in vivo cell transfection can be used for gene therapy, in order to reduced immune reactions of the patient.

In some cases, populations of cells (for example immunoresponsive cells), preferably selected from T-cells, IPSC-derived T-cells, aPT-cells, yST-cells, or NK cells, more preferably selected from yST-cells or apT-cells, most preferably apT-cells, expressing a yT-cell receptor chain, a 5T-cell receptor chain, a yST-cell receptor, a fragment thereof, or a chimeric antigen-recognition receptor described herein, may be formulated for administration to a subject using techniques known to the skilled artisan. Formulations comprising populations of cells (for example immunoresponsive cells) may include pharmaceutically acceptable excipient(s). Excipients included in the formulations will have different purposes depending, for example, on the subpopulation of cell used and the mode of administration. Examples of generally used excipients included, without limitation: saline, buffered saline, dextrose, water-for-injection, glycerol, ethanol, and combinations thereof, stabilizing agents, solubilizing agents and surfactants, buffers and preservatives, tonicity agents, bulking agents, and lubricating agents. The formulations comprising populations of cells (for example immunoresponsive cells) will typically have been prepared and cultured in the absence of any nonhuman components, such as animal serum.

A formulation may include one population of cells (for example immunoresponsive cells), or more than one, such as two, three, four, five, six or more populations of cells. The formulations comprising population(s) of cells (for example immunoresponsive cells) may be administered to a subject using modes and techniques known to the skilled artisan. Exemplary modes include, but are not limited to, intravenous injection. Other modes include, without limitation, intratumoural, intradermal, subcutaneous (s.c., s.q., sub-Q, Hypo), intramuscular (i.m.), intraperitoneal (i.p.), intra-arterial, intramedullary, intracardiac, intra-articular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluids). Any known device useful for parenteral injection of infusion of the formulations can be used to effect such administration. The formulations comprising population(s) of cells (for example immunoresponsive cells) that are administered to a subject comprise a number of cells that is effective for the treatment and/or prophylaxis of the specific indication or disease. Similar formulations and modes of administration may be contemplated for the case of soluble yT-cell receptor chains, 5T-cell receptor chains, y5T-cell receptors, or fragments thereof described herein.

Thus, therapeutically-effective populations of cells (for example immunoresponsive cells) are administered to subjects when the methods of the present disclosure are practiced. In general, formulations are administered that comprise between about 1 x 10⁴ and about 1 x 10¹⁰ cells. In most cases, the formulation may comprise between about 1 x 10⁵ and about 1 x 10⁹ cells, from about 5 x 10⁵to about 5 x 10® cells, or from about 1 x 10® to about 1 x 10⁷ cells. However, the number of cells administered to a subject will vary between wide limits, depending upon the location, source, identity, extent and severity of the cancer or infection, the age and condition of the individual to be treated etc. A physician will ultimately determine appropriate dosages to be used.

Therapy

The yT-cell receptor chains, ST-cell receptor chains, ybTCRs, fragments thereof (for example soluble yT- cell receptor chains, ST-cell receptor chains, ySTCRs, or fragments thereof), chimeric antigen-recognition receptors, cells (for example immunoresponsive cells), populations of cells, and compositions (such as pharmaceutical compositions), all as described herein, are preferably able to mediate (or exhibit) an antitumour activity/response and/or an anti-infective activity/response. The yT-cell receptor chains, 6T-cell receptor chains, ySTCRs, fragments thereof (for example soluble yT-cell receptor chains, ST-cell receptor chains, ySTCRs, or fragments thereof), chimeric antigen-recognition receptors, cells, populations of cells, and compositions (such as pharmaceutical compositions), all as described herein, are preferably suitable for use in therapy.

Accordingly, in a further aspect, there is provided a yT-cell receptor chain or fragment thereof, comprising a Cy constant region, a ST-cell receptor chain or fragment thereof, comprising a C6 constant region, a y5T- cell receptor or fragment thereof, comprising a Cy and a C5 constant region (for example a soluble yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, or fragment thereof), a chimeric antigenrecognition receptor, a nucleic acid molecule, a nucleic acid construct, a vector, a cell (for example an immunoresponsive cell), or a composition, all as described herein, for use as a medicament. The medicament is preferably for the treatment, regression, curing, and/or delaying of cancer or an infection, more preferably of cancer, in a subject, preferably a human being.

In a further aspect, there is provided the use of a yT-cell receptor chain or fragment thereof, comprising a Cy constant region, a 5T-cell receptor chain or fragment thereof, comprising a C5 constant region, a y5T- cell receptor or fragment thereof, comprising a Cy and a C5 constant region (for example a soluble yT-cell receptor chain, 6T-cell receptor chain, yST-cell receptor, or fragment thereof), a chimeric antigenrecognition receptor, a nucleic acid molecule, a nucleic acid construct, a vector, a cell (for example an immunoresponsive cell), or a composition, all as described herein, for the manufacture of a medicament. The medicament is preferably for the treatment, regression, curing, and/or delaying of cancer or an infection, more preferably of cancer, in a subject, preferably a human being.

In a further aspect, there is provided a method of treatment, regression, curing, and/or delaying of a disease comprising providing a yT-cell receptor chain or fragment thereof, comprising a Cy constant region, a 5T- cell receptor chain or fragment thereof, comprising a C5 constant region, a yST-cell receptor or fragment thereof, comprising a Cy and a C6 constant region (for example a soluble yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, or fragment thereof), a chimeric antigen-recognition receptor, a nucleic acid molecule, a nucleic acid construct, a vector, a cell (for example an immunoresponsive cell), or a composition, all as described herein, to a subject in need thereof, preferably to a human. The method is preferably a method of treatment, regression, curing, and/or delaying of a cancer or infection.

In therapeutic uses, uses, and methods of treatment described herein, the yT-cell receptor chain or fragment thereof, comprising a Cy constant region, the 5T-cell receptor chain or fragment thereof, comprising a C6 constant region, the y5T-cell receptor or fragment thereof, comprising a Cy and a C5 constant region (for example the soluble yT-cell receptor chain, 5T-cell receptor chain, y5T-cell receptor, or fragment thereof), the chimeric antigen-recognition receptor, the nucleic acid molecule, the nucleic acid construct, the vector, the cell (for example immunoresponsive cell), or the composition, are administered to a subject in need thereof, i.e., a subject that is afflicted by or is at risk of developing the disease or infection. A preferred subject is a human being. The amount administered is preferably a therapeutically effective amount, as described earlier herein.

In some embodiments, a cancer is a liquid cancer. In some embodiments, a cancer is a solid cancer. In some embodiments, a cancer is a colon cancer. In some embodiments, a cancer is a breast cancer. In some embodiments, a cancer is a renal cancer.

In some embodiments, a cancer is a metastatic cancer. In some embodiments, a cancer is a relapsed or refractory cancer. In some embodiments, a cancer is a solid tumour or a hematologic malignancy. In some embodiments, a cancer is a solid tumour. In some embodiments, a cancer is a hematologic malignancy.

The following general part dedicated to the definitions provides more information as to the aspects and embodiments described herein.

General part dedicated to definitions

Polypeptide/nuclelc acid

A “wild type” protein/polypeptide amino acid sequence can refer to a sequence that is naturally occurring and encoded by a germline genome. A species can have one wild type sequence, or two or more wild type sequences (for example, with one canonical wild type sequence and one or more non-canonical wild type sequences). A wild type protein amino acid sequence can be a mature form of a protein that has been processed to remove N-terminal and/or C-terminal residues, for example, to remove a signal peptide. In some embodiments, a reference sequence used herein is a wild type sequence. In some embodiments, a reference sequence used herein is isolated from a healthy individual. In some embodiments, a reference sequence used herein is isolated from a patient, such as a cancer patient. An amino acid sequence that is “derived from” a wild type sequence or reference sequence or another amino acid sequence disclosed herein can refer to an amino acid sequence that comprises an amino acid modification, for example an amino acid sequence that differs by one or more amino acids compared to the wild type or reference amino acid sequence, for example, containing one or more amino acid insertions, deletions, or substitutions as disclosed herein.

In the context of the disclosure, a polypeptide comprises an amino acid sequence. In the context of the disclosure, a nucleic acid molecule such as a nucleic acid molecule encoding a yT-cell receptor chain, a 6T-cell receptor chain, a ySTCR, a fragment thereof (for example a soluble yT-cell receptor chain, 5T-cell receptor chain, yST-cell receptor, or fragment thereof), or chimeric antigen-recognition receptor comprises a nucleic acid or nucleotide sequence which encodes such a polypeptide. A nucleic acid molecule may comprise a regulatory region.

It is to be understood that each nucleic acid molecule or polypeptide or construct as identified herein by a given Sequence Identity Number (SEQ ID NO) is not limited to this specific sequence as disclosed. Throughout this application, each time one refers to a specific nucleotide sequence SEQ ID NO (take SEQ ID NO: X as example) encoding a given polypeptide, one may replace it by: i. a nucleotide sequence comprising a nucleotide sequence that has at least 60% or at least 80% sequence identity with SEQ ID NO: X; ii. a nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence of (I); iii. a nucleotide sequence the sequence of which differs from the sequence of a nucleic acid molecule of (i) or (II) due to the degeneracy of the genetic code; or, iv. a nucleotide sequence that encodes an amino acid sequence that has at least 60% or at least 80% amino acid identity or similarity with an amino acid sequence encoded by a nucleotide sequence SEQ ID NO: X.

Throughout this application, each time one refers to a specific amino acid sequence SEQ ID NO (take SEQ ID NO: Y as example), one may replace it by: a polypeptide comprising an amino acid sequence that has at least 60% sequence identity or similarity with amino acid sequence SEQ ID NO: Y. In the context of the application, the minimum identity or similarity in relation to a yT-cell receptor chain or fragment thereof may mean an identity or a similarity of at least 60%. In the context of the application, the minimum identity or similarity in relation to a 5T-cell receptor chain or fragment thereof may mean an identity or a similarity of at least 60%.

Each nucleotide sequence or amino acid sequence described herein by virtue of its identity or similarity percentage (e.g. at least 60%) with a given nucleotide sequence or amino acid sequence respectively has in a further preferred embodiment an identity (or a similarity where applicable) of at least 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with the given nucleotide or amino acid sequence respectively. In a preferred embodiment, sequence identity or similarity is determined by comparing the whole length of the sequences as identified herein. In other words, sequence identity is preferably calculated based on the full length of two given sequences being compared (for example of a sequence represented by a SEQ ID NO herein and of another sequence it is being compared to). Unless otherwise indicated herein, identity or similarity with a given SEQ ID NO means identity or similarity based on the full length of said sequence (i.e. over its whole length or as a whole).

Sequence identity

"Sequence identity" is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. The identity between two amino acid or two nucleic acid sequences is typically defined by assessing their identity within a whole length SEQ ID NO as identified herein or part thereof. Part thereof in terms of comparing the identity or similarity of two or more sequences may mean at least 50% of the length of the SEQ ID NO, or at least 60%, or at least 70%, or at least 80%, or at least 90%.

In the art, "identity" also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in Bioinformatics and the Cell: Modern Computational Approaches in Genomics, Proteomics and transcriptomics, Xia X., Springer International Publishing, New York, 2018; and Bioinformatics: Sequence and Genome Analysis, Mount D., Cold Spring Harbor Laboratory Press, New York, 2004, each incorporated herein by reference in its entirety.

“Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g. Needleman-Wunsch) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith- Waterman). Sequences may then be referred to as "substantially identical” or “essentially similar'’ when they (when optimally aligned by for example the program EMBOSS needle or EMBOSS water (EMBLI-EBI) using default parameters share at least a certain minimal percentage of sequence identity (as described herein).

A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths. When sequences have a substantially different overall length, local alignments, such as those using the Smith-Waterman algorithm, are preferred. EMBOSS needle uses the Needleman-Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. EMBOSS water uses the Smith-Waterman local alignment algorithm. Generally, the EMBOSS needle and EMBOSS water default parameters are used, with a gap open penalty = 10 (nucleotide sequences) / 10 (proteins) and gap extension penalty = 0.5 (nucleotide sequences) / 0.5 (proteins). For nucleotide sequences the default scoring matrix used is DNAfull and for amino acid sequences the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915- 919, incorporated herein by reference).

Alternatively percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc. Thus, the nucleotide and amino acid sequences of some embodiments of the present disclosure can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10, incorporated herein by reference in its entirety. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences having a certain identity with nucleic acid molecules of the disclosure. BLAST protein searches can be performed with the BLASTx program, score = 50, wordlength = 3 to obtain amino acid sequences having a certain identity or similarity with polypeptides of the disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402, incorporated herein by reference. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information accessible on the world wide web at www.ncbi.nlm.nih.gov/.

Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. As used herein, “conservative” amino acid substitutions refer to the interchangeability of residues having similar side chains. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. Examples of classes of amino acid residues for conservative substitutions are given in the Tables below.

Alternative conservative amino acid residue substitution classes :

Alternative physical and functional classifications of amino acid residues:

For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine- leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gin or His; Asp to Glu; Cys to Ser or Ala; Gin to Asn; Glu to Asp; Gly to Pro; His to Asn or Gin; lie to Leu or Vai; Leu to lie or Vai; Lys to Arg; Gin or Glu; Met to Leu or lie; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and, Vai to lie or Leu.

Antigen

A yT-cell receptor chain, a 5T-cell receptor chain, a ySTCR, a fragment thereof (for example a soluble yT- cell receptor chain, 5T-cell receptor chain, y5T-cell receptor, or fragment thereof), or a chimeric antigenrecognition receptor, as disclosed herein may, for example, bind to an antigen comprised in or displayed by a target cell. An “antigen” is a molecule or molecular structure that an antigen receptor or an antigen-binding protein can recognize (for example, bind to). An antigen can be or can comprise, for example, a peptide, a polypeptide, a carbohydrate, a chemical, a moiety, a non-peptide antigen, a phosphoantigen, a tumour- associated antigen, a neoantigen, a tumour microenvironment antigen, a microbial antigen, a viral antigen, a bacterial antigen, an autoantigen, a glycan-based antigen, a peptide-based antigen, a lipid-based antigen, or any combination thereof. In some embodiments, an antigen is capable of inducing an immune response. In some examples, an antigen binds to an antigen receptor or antigen-binding protein, or induces an immune response, when present in a complex e.g., presented by MHC. In some cases, an antigen adopts a certain conformation in order to bind to an antigen receptor or antigen-binding protein, and/or to induce an immune response, e.g., adopts a conformation in response to the presence or absence of one or more metabolites. Antigen can refer to a whole target molecule, a whole complex, a or a fragment of a target molecule or complex that binds to an antigen receptor or an antigen-binding protein. Antigen receptors that recognize antigens include ybTCRs and chimeric antigen-recognition receptors disclosed herein and other receptors, such as endogenous T cell receptors. In some embodiments, the antigen is EPCR (Endothelial protein C receptor), preferably human EPCR. In some embodiments, the antigen is a phosphoantigen. In some embodiments, the antigen is CD277, preferably CD277 in a J-configuration. In some embodiments, the antigen is an HLA molecule, preferably an HLA-A molecule, more preferably an HLA-A*24 molecule, most preferably an HLA-A*24:02 molecule.

Gene or coding sequence

“Gene” or “coding sequence” or “nucleic acid” or “nucleic acid molecule” refers to a DNA or RNA region (the transcribed region) which “encodes” a particular polypeptide such as a 5T-cell receptor or a yT-cell receptor or a ySTCR or parts thereof. A coding sequence is transcribed (DNA) and translated (RNA) into a polypeptide when placed under the control of an appropriate regulatory region, such as a promoter. A gene may optionally comprise several operably linked fragments, such as a promoter, a 5’ leader sequence, an intron, a coding sequence and a 3' nontranslated sequence, comprising a polyadenylation site or a signal sequence. A chimeric or recombinant gene (such as the ones described herein) is a gene not normally found in nature, such as a gene in which for example the promoter is not associated in nature with part or all of the transcribed DNA region, or genes comprising nucleotide sequences encoding domains from multiple polypeptides. “Expression of a gene” refers to the process wherein a gene is transcribed into an RNA and/or translated into an active protein.

Codon optimization

“Codon optimization”, as used herein, refers to the processes employed to modify an existing coding sequence, or to design a coding sequence, for example, to improve translation in an expression host cell or organism of a transcript RNA molecule transcribed from the coding sequence, or to improve transcription of a coding sequence. Codon optimization includes, but is not limited to, processes including selecting codons for the coding sequence to suit the codon preference of the expression host cell. For example, to suit the codon preference of mammalian, insect, plant, or microbial cells, preferably human cells. Codon optimization also eliminates elements that potentially impact negatively RNA stability and/or translation (e. g. termination sequences, TATA boxes, splice sites, ribosomal entry sites, repetitive and/or GC rich sequences and RNA secondary structures or instability motifs). Codon optimization may be done according to standard methods available to skilled person.

Promoter

As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes (or coding sequence), located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive” promoter is a promoter that is active under most physiological and developmental conditions. An "inducible" promoter is a promoter that is regulated depending on physiological or developmental conditions. A "tissue specific" promoter is preferentially active in specific types of differentiated cells/tissues, such as preferably in a T cell. A preferred promoter is the MSCV promoter. An example of an MSCV promoter comprises or consists of SEQ ID NO: 57.

Operably linked

“Operably linked” is defined herein as a configuration in which a control sequence such as a promoter sequence or regulating sequence is appropriately placed at a position relative to the nucleotide sequence of interest. For instance, a promoter is operably linked to a coding sequence if the promoter is able to initiate or regulate the transcription or expression of a coding sequence, in which case the coding sequence should be understood as being “under the control of’ the promoter.

Nucleic acid construct

An "expression construct” or "nucleic acid construct” comprises a nucleic acid molecule, such as the ones described herein, which may be expressed in a host cell. In some cases, such a construct is a viral expression construct. A viral expression construct comprises parts of a virus' genome, as further described later herein.

Within the context of the disclosure, a host cell encompasses a cell used to make the construct or a cell to which the construct will be administered. Alternatively, a nucleic acid construct is capable of integrating into a cell's genome, e.g., through homologous recombination or otherwise. A particularly preferred expression construct is one wherein a nucleotide sequence encoding a yT-cell receptor chain, a 6T-cell receptor chain, a ySTCR, a fragment thereof (for example a soluble yT-cell receptor chain, 5T-cell receptor chain, y5T-cell receptor, or fragment thereof), or a chimeric antigen-recognition receptor as described herein, is operably linked to a promoter as defined herein wherein said promoter is capable of directing expression of said nucleotide sequence (i.e. , coding sequence) in a cell (for example an immunoresponsive cell), preferably a T-cell, an iPSC-derived T-cell, an apT-cell, a y5T-cell, or an NK cell, more preferably a y5T-cell or apT-cell, most preferably an apT-cell. Such a preferred expression construct is said to comprise an expression cassette.

An expression construct may comprise two expression cassettes to allow the expression of two polypeptides such as a yTCR and a 5TCR chain, or fragments thereof. An expression construct may further comprise a sequence encoding a 2A-self cleaving peptide. These selfcleaving peptides are known to the skilled person and are further described, for example, in Xu Y., et al (2019), Cancer Immunology, Immunotherapy, 68: 1979-1993 and Pincha M., et al, (2011), Gene Therapy, 18: 750-764, both of which incorporated herein by reference in their entireties. Non-limiting examples of suitable 2A peptides are F2A (2A peptide derived from the foot-and-mouth disease virus), E2A (2A peptide derived from the equine rhinitis virus), P2A (2A peptide derived from the porcine teschovirus-1), or T2A (2A peptide derived from the Thosea asigna virus). In some embodiments, the 2A self-cleaving peptide is a F2A peptide. In some embodiments, the 2A self-cleaving peptide is an E2A peptide. In some embodiments, the 2A self-cleaving peptide is a P2A peptide. In some embodiments, the 2A self-cleaving peptide is a T2A peptide. The skilled person understands that an expression construct described herein may also comprise nucleotide sequences encoding different 2A self-cleaving peptides. In expression constructs encoding a y5TCR or a fragment thereof, a sequence encoding a 2A self-cleaving peptide may in some cases be inserted between the sequence encoding the yT-cell receptor chain or fragment thereof and the 5T-cell receptor chain or fragment thereof. An exemplary T2A self-cleaving peptide is represented by SEQ ID NO: 55. An exemplary P2A self-cleaving peptide is represented by SEQ ID NO: 56.

Expression constructs disclosed herein could be prepared using recombinant techniques which result in nucleotide sequences being expressed in a suitable cell, e.g., cultured cells or cells of a multicellular organism, such as described in Ausubel et al., and in Sambrook and Green (supra). Also see, Kunkel (1985) Proc. Natl. Acad. Sci. 82:488 (describing site directed mutagenesis) and Roberts et al. (1987) Nature 328:731 734 or Wells, J.A., et al. (1985) Gene 34: 315 (describing cassette mutagenesis).

Typically, a nucleic acid molecule or construct is used in a vector. A vector may alternatively be called an expression vector. The phrase "expression vector" generally refers to a nucleotide sequence that is capable of effecting expression of a gene in a host compatible with such sequences. These expression vectors typically include at least suitable promoter sequences and optionally, transcription termination signals. An additional factor necessary or helpful in effecting expression can also be used as described herein. An expression vector may optionally be suitable for replication in a prokaryotic host, such as bacteria, e.g., E. coli, or may be introduced into a cultured mammalian, plant, insect, (e.g., Sf9), yeast, fungi or other eukaryotic cell lines.

A nucleic acid molecule, construct, or vector, prepared for introduction into a particular host may include a replication system recognized by the host, an intended DNA segment encoding a desired polypeptide, and transcriptional and translational initiation and termination regulatory sequences operably linked to the polypeptide-encoding segment. The term “operably linked” has already been defined herein. DNA signal sequences may be included. DNA for a signal sequence is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of a polypeptide. Generally, DNA sequences that are operably linked are contiguous, and, in the case of a signal sequence, both contiguous and in reading frame. However, enhancers need not be contiguous with a coding sequence whose transcription they control. Linking is accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof, or by gene synthesis.

The selection of an appropriate promoter sequence generally depends upon the host cell selected for the expression of a DNA segment. Examples of suitable promoter sequences include prokaryotic, and eukaryotic promoters well known in the art (see, e.g., Sambrook and Green, supra). Additional examples have been provided earlier herein. A transcriptional regulatory sequence typically includes a heterologous enhancer or promoter that is recognised by the host. The selection of an appropriate promoter depends upon the host, but promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters are known and available (see, e.g. Sambrook and Green, supra). For example, an expression vector which includes the replication system and transcriptional and translational regulatory sequences together with the insertion site for the polypeptide encoding segment can be employed. In most cases, the replication system is only functional in the cell that is used to make the vector (e.g., bacterial cell as E. coli). Most plasmids and vectors do not replicate in the cells infected with the vector. Examples of workable combinations of cell lines and expression vectors are for example described in Sambrook and Green (supra). For example, suitable expression vectors can be expressed in, yeast, e.g. S. cerevisiae, e.g., insect cells, e.g., Sf9 cells, mammalian cells, e.g., CHO cells and bacterial cells, e.g., E. coli. A cell may thus be a prokaryotic or eukaryotic host cell. A cell may be a cell that is suitable for culture in liquid or on solid media. A cell may be a cell line, e.g. a HEK293 or HEK293F cell line or a derivative thereof. In some cases, a host cell is a cell that is part of a multicellular organism such as a transgenic plant or animal. Commercial kits comprising cells for vector expression are available, for example the LV-Max system from Thermo Fisher Scientific (Waltham, MA, USA).

A vector as described herein may be selected from any genetic element known in the art which can facilitate transfer of nucleic acids between cells, such as, but not limited to, plasmids, transposons, cosmids, chromosomes, artificial chromosomes, viruses, virions, and the like. A vector may also be a chemical vector, such as a lipid complex or naked DNA. "Naked DNA” or "naked nucleic acid” refers to a nucleic acid molecule that is not contained in encapsulating means that facilitates delivery of a nucleic acid into the cytoplasm of a target host cell. Naked DNA may be circular or linear (linearized DNA sequence). Optionally, a naked nucleic acid can be associated with standard means used in the art for facilitating its delivery of the nucleic acid to the target host cell, for example to facilitate the transport of the nucleic acid through the cell membrane.

A preferred vector is a viral vector. Non-limiting examples of viral vectors are adenoviral vectors, adeno- associated viral vectors, retroviral vectors, and lentiviral vectors. Among viral vectors, retroviral and lentiviral vectors are preferred, with lentiviral vectors being further preferred. In an embodiment, a single bicistronic viral vector is used. In an embodiment, a single bicistronic lentiviral vector further comprising a 2A selfcleaving peptide sequence is used, as described earlier herein.

Adenoviral and adeno associated viral (AAV) vectors infect a wide number of dividing and non-dividing cell types including synovial cells and liver cells. The episomal nature of the adenoviral and AAV vectors after cell entry makes these vectors suited for therapeutic applications (Russell, 2000, J. Gen. Virol. 81 : 2573- 2604; Goncalves, 2005, Virol J. 2(1):43). AAV vectors are known to result in very stable long term expression of transgene expression (up to 9 years in dog (Niemeyer et al, Blood. 2009 Jan 22; 113(4):797- 806) and ~ 2 years in human cells (Nathwani et al, N Engl J Med. 2011 Dec 22;365(25):2357-65, Simonelli et al, Mol Ther. 2010 Mar;18(3):643-50. Epub 2009 Dec 1). Preferred adenoviral vectors are modified to reduce the host response as reviewed by Russell (2000, supra).

Lentiviral vectors have the ability to infect and to stably integrate into the genome of dividing and nondividing cells (Amado and Chen, 1999 Science 285: 674-6). Methods for the construction and use of lentiviral based expression constructs are described in U.S. Patent No.'s 6,165,782, 6,207,455, 6,218,181 , 6,277,633 and 6,323,031 and in Federico (1999, Curr Opin Biotechnol 10: 448-53) and Vigna et al. (2000, J Gene Med 2000; 2: 308-16). Other suitable viral vectors include a herpes virus vector, a polyoma virus vector or a vaccinia virus vector. Transposon or other non-viral delivery systems may also be used in this context. All systems can be used in vitro or in vivo.

A viral vector may optionally comprise a further nucleotide sequence coding for a further polypeptide. A further polypeptide may be a (selectable) marker polypeptide that allows for the identification, selection and/or screening for cells containing the expression construct. Suitable marker proteins for this purpose are e.g. the fluorescent protein GFP, and the selectable marker genes HSV thymidine kinase (for selection on HAT medium), bacterial hygromycin B phosphotransferase (for selection on hygromycin B), Tn5 aminoglycoside phosphotransferase (for selection on G418), and dihydrofolate reductase (DHFR) (for selection on methotrexate), CD20, the low affinity nerve growth factor gene. Sources for obtaining these marker genes and methods for their use are provided in standard handbooks, for example in Sambrook and Green (supra). An exemplary GFP sequence is represented by SEQ ID NO: 51

In some embodiments, a viral vector may be formulated in a pharmaceutical composition as defined herein. In this context, a pharmaceutical composition may comprise a suitable pharmaceutical carrier as earlier defined herein.

Transgene

A "transgene" is herein defined as a gene or a nucleic acid molecule (/.e. a molecule encoding a 5TCR or a yTCR chain or a ySTCR or a fragment thereof or a chimeric antigen-recognition receptor) that has been newly introduced into a cell. The transgene may comprise sequences that are native to the cell, sequences that naturally do not occur in the cell and it may comprise combinations of both. A transgene may contain sequences coding for a yTCR or a 5TCR chain or a ySTCR or a fragment thereof or chimeric antigenrecognition receptor as earlier defined herein that may be operably linked to appropriate regulatory sequences for expression of the sequences coding for the yTCR or 5TCR chain or y5TCR or fragment thereof or chimeric antigen-recognition receptor.

Transduction

“Transduction” refers to the delivery of a yTCR or a 5TCR chain or a y6TCR or a fragment thereof (for example of a soluble yT-cell receptor chain, 5T-cell receptor chain, y5T-cell receptor, or fragment thereof) or chimeric antigen-recognition receptor as earlier defined herein into a recipient host cell by a viral vector. For example, transduction of a cell by e.g., a retroviral or lentiviral vector as described herein leads to transfer of the genome contained in that vector into the transduced cell. In an embodiment, the vector is a retroviral or lentiviral vector. An example of transduction is provided in Pirona et al., 2020 (Biology Methods and Protocols, Volume 5, Issue 1 , 2020, bpaa005). Additional examples are provided in the experimental section herein.

Engineered cells

The term "engineered cells" refers herein to cells having been engineered, e.g., by the introduction of an exogenous nucleic acid sequence as defined herein. Such a cell has been genetically modified for example by the introduction of for example one or more mutations, insertions and/or deletions in an endogenous gene and/or insertion of a nucleic acid construct in the genome. The modification may have been introduced using recombinant DNA technology. An engineered cell may refer to a cell in isolation or in culture. Engineered cells may be "transduced cells" wherein the cells have been infected with e.g. a modified virus, for example, a retrovirus may be used but other suitable viruses may also be contemplated such as lentiviruses. Non-viral methods may also be used, such as transfections. Engineered cells may thus also be "stably transfected cells" or "transiently transfected cells". Transfection refers to non-viral methods to transfer DNA (or RNA) to cells such that a gene is expressed. Transfection methods are widely known in the art, such as calcium phosphate transfection, PEG transfection, and liposomal or lipoplex transfection of nucleic acids. Such a transfection may be transient, but may also be a stable transfection wherein cells can be selected that have the gene construct integrated in their genome. In some cases genetic engineering systems such as CRISPR or Argonaute may be utilized to design engineered cells that express a polypeptide described herein.

A variety of enzymes can catalyze insertion of foreign DNA into a host genome. Non-limiting examples of gene editing tools and techniques include CRISPR, TALEN, zinc finger nuclease (ZFN), meganuclease, Mega-TAL, and transposon-based systems.

A CRISPR system can be utilized to facilitate insertion of a polynucleotide sequence encoding a membrane protein or a component thereof into a cell genome. For example, a CRISPR system can introduce a double stranded break at a target site in a genome. There are at least five types of CRISPR systems which all incorporate RNAs and CRISPR-associated proteins (Cas). Types I, III, and IV assemble a multi-Cas protein complex that is capable of cleaving nucleic acids that are complementary to the crRNA. Types I and III both require pre-crRNA processing prior to assembling the processed crRNA into the multi-Cas protein complex. Types II and V CRISPR systems comprise a single Cas protein complexed with at least one guiding RNA. Genome editing tools as described above may also be used to introduce a genomic modification which results in the reduction or elimination of surface expression of an endogenous a0TCR in an aPT-cell as discussed earlier herein.

In some embodiments, an “engineered cell” has been transformed, modified or transduced to comprise a heterologous or exogenous nucleic acid molecule. In the application, the wording “engineered cell” may be replaced by “modified cell” or “transformed cell” or “transduced cell”. In an embodiment, said cell expresses a protein encoded by said nucleic acid molecule. In an embodiment, said cell is a TEG.

TEG

A “TEG” as used herein refers to a T-cell engineered to express a defined yTCR chain, 5TCR chain, yBTCR, a fragment thereof, or chimeric antigen-recognition receptor, as disclosed herein, preferably embedded in its cellular membrane. In preferred embodiments, a TEG is an apT-cell that is engineered to express a defined yTCR chain, 6TCR chain, ySTCR, fragment thereof, or chimeric antigen-recognition receptor, as described herein, preferably embedded in its cellular membrane.

In some embodiments, the cells can be cultured for extended periods without stimulation or with stimulation. Stimulation may comprise contact with an anti-CD3 antibody or antigen binding fragment thereof immobilized on a surface. For co-stimulation of an accessory molecule on the surface of the T-cells, a ligand that binds the accessory molecule can be used. In some cases a population of T-cells can be CD3- CD28 co-stimulated, for example, contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions that can stimulate proliferation of the T-cells.

Conditions appropriate for T-cell culture can include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640, TexMACS (Miltenyi Biotec, Bergisch Gladbach, Germany), X-vivo 5 (Lonza), or McCoy’s 5a Medium), that may contain factors necessary for proliferation and viability, including serum (such as e.g., human serum or fetal bovine serum as earlier described herein). In some embodiments, cells can be maintained under conditions necessary to support growth; for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% CO2). Further examples of suitable media and conditions are provided in the experimental section herein.

Cells can be obtained from any suitable source for the generation of engineered cells. Cells can be primary cells. Cells can be recombinant cells. Cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumours. Cells can be derived from a healthy donor or from a patient diagnosed with cancer. Cells can also be obtained from a cell therapy bank. Cells can also be obtained from differentiation of stem cells, progenitor cells, precursor cells, iPSC cells, and the like. Cells can also be obtained from whole blood, apheresis, or a tumour sample of a subject. A cell can be a tumour infiltrating lymphocytes (TIL). In some cases an apheresis can be a leukapheresis.

A desirable cell population can also be selected prior to modification. A selection can include at least one of: magnetic separation, flow cytometric selection, antibiotic selection. The one or more cells can be any blood cells, such as peripheral blood mononuclear cell (PBMC), lymphocytes, monocytes or macrophages. The one or more cells can be any immune cells such as a lymphocyte, an alpha-beta T cell, a gamma-delta T cell, CD4+ T cell, CD8+ T cell, a T effector cell, a lymphocyte, a B cell, an NK cell, an NKT cell, a myeloid cell, a monocyte, a macrophage, or a neutrophil.

General

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb “to consist” may be replaced by “to consist essentially of’ meaning that a method, respectively component as defined herein may comprise additional step(s), respectively component(s) than the ones specifically identified, said additional step(s), respectively component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

The word “about” when used in association with an integer (about 10) preferably means that the value may be the given value of 10 more or less 1 of the value: about 10 preferably means from 9 to 11. The word “about” when used in association with a numerical value (about 10.6) preferably means that the value may be the given value of 10.6 more or less 0.1 of the value 10.6: about 10.6 preferably means from 10.5 to 10.7.

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. The following examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

Examples

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Unless specified, reagents employed in the examples are commercially available or can be prepared using commercially available instrumentation, methods, or reagents known in the art. The examples illustrate various aspects of the invention and practice of the methods of the invention. The examples are not intended to provide an exhaustive description of the many different embodiments of the invention. Thus, although the invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, those of ordinary skill in the art will realize readily that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Materials and methods pertaining to the Examples

Plasmid and lentivirus production

The cloning plasmid used to produce lentivirus, contained the tricistronic yTCR-eGFP-6TCR transgene where the ribosomal skipping T2A (SEQ ID NO: 55) and P2A self-cleaving peptide (SEQ ID NO: 56) motifs separated the translation of the y-TCR, eGFP (SEQ ID NO: 51) and 5-TCR proteins. Plasmid transcription was driven by an MSCV promoter (SEQ ID NO: 57) and viral genome packaging and enhanced transgene expression were regulated with the LTR/M^J and WPRE regulatory elements, respectively. Lentiviral particles were produced using the LV-Max system from Thermo Fisher Scientific (Waltham, MA, USA). LV-MAX producer cells (A35827) were transfected with pLenti 6.3 y5TCR transfer construct and packaging mix (pLP1, pLP2, pLP-VSVG). Lentiviral titers were assessed in a|3TCR-deficient Jurkat-76 cells by flow cytometry analysis, measuring the percentage of CD3/y6TCR among live cells. ySTCR DNA mutagenesis and subcloninq

Mutated amino acids in the transmembrane regions were numbered based on their corresponding position in the yTCR constant 1 region (TRGC1; SEQ ID NO:1) or constant 5TCR (TRDC; SEQ ID NO: 2) region. The same holds for mutated amino acids in the a-loop and FG-loop regions of TRGC1 , and mutated amino acids in CD3-interacting, glycosylation, and cysteine-bridge forming positions in TRGC1 and TRDC. Single amino acid mutations were introduced in the TRGC1 and TRDC of various ySTCRs (E57 (SEQ ID NO: 96, 99); clone 4 (SEQ ID NO: 97, 100); clone 5 (SEQ ID NO: 98, 101); cl5 (SEQ ID NO: 102, 103); G115 (SEQ ID NO: 167, 168)) via Gibson assembly HiFi (ThermoFisher Scientific, USA), cloning or side-directed Q5 QuickChange side-directed mutagenesis (New England Biolabs, Ipswich, MA, UK) using specific primers according to manufacturer protocol. Primers used to introduce mutations in the case of transmembrane region mutations are represented by SEQ ID NO: 58-79. In short, for site-directed mutagenesis, forward (FW) and reverse (RV) primers were added to plasmids containing the wild-type y5TCR sequence and linear PCR fragments were produced by PCR followed by an enzymatic ligation step KLD where the parental plasmid was degraded simultaneously For Gibson cloning, DNA fragments containing the mutations were ordered at IDT (Leuven, BE) or PCR fragments containing the mutations were produced. Single plasmids were assembled from multiple overlapping DNA fragments, including the mutated DNA fragments according to manufacturer protocol (Thermofisher scientific, Waltham, MA, USA). Newly synthesized plasmids were transduced into XL1 blue bacteria and bacteria were grown on agar plates supplemented with Luria Broth (LB) and carbenicillin. Colonies were further grown in LB and DNA from bacteria was extracted with NucleoBond xtra Mini kit (Macherey-Nagel, PA, USA) according to manufacturer instructions. The full open reading frames from plasmids were Sanger sequenced and plasmids containing the correct sequences were further subjected to a second round of expansion in DH5a E. coli bacteria, isolation with NucleoBond xtra Midi kit, followed by a lentivirus production. TEG production

TEGs were manufactured starting from apT-cells enriched by MACS separation (Miltenyi Biotec, Bergisch Gladbach, Germany) from healthy donor leukapheresis material, according to manufacturer instructions. Purified apT-cells were cultured in TEXMACS medium supplemented with 2.5% human serum (Sanquin, Amsterdam, NL), rhlL-7 (20-2000 lU/mL) and rh IL15 (20-200 lU/mL) (both from Miltenyi Biotec), and 1% Penicillin/Streptomycin, and activated using TransAct (Miltenyi Biotec) per manufacturer’s recommendations. Cells were transduced with lentivirus containing the ySTCR transgene with an multiplicity of infection ranging from 3-5 as determined on Jurkat-76 cells. TEGs were then expanded for 12 days in TEXMACS medium, 2.5% human serum, rhlL-7 (20-2000 lU/mL) and rh IL15 (20-200 lU/mL), 1% Penicillin/Streptomycin. At the end of the production, transduction efficiency (% ySTCR and or eGFP, >40% in all cases), T-cell purity (>90% in all cases), and relative expression of T-cell markers CD4 and CD8 were measured by flow cytometry. Cells were then cryopreserved in 1 volume of NaCI 0.9%/5% human serum albumin and 1 volume of Cryostor CSIO (Sigma-Aldrich).

Luciferase-based (serial) cytotoxicity assay

Luc-Tom HT-29 tumour cells were harvested, counted and seeded to the appropriate number of cells per well, in triplicate in 96-well E-plates, and cultured in McCoy's 5a Medium, 10% fetal bovine serum and 1% Penicillin/Streptomycin (ThermoFisher Scientific) for 24 hours at 37°C. TEGs expressing a yST-cells receptor comprising the E57, E57 G5D6, clone 4 and 5, cl5, and G115 reference and mutant sequences were then harvested, counted, resuspended in IMDM medium, 5% human serum, and 1% Penicillin/Streptomycin, and added to the tumour target cells at E:T ratio of 2:1, 1 :1 , 1:2, 1 :4, 1:8, 1 :16. The resulting co-culture was maintained at 37°C for 72 hour or for one week (or for 48 hours in the case of cocultures with TEGs expressing G115 reference and mutant sequences). In experiments involving cl5- or G115-based yST-cell receptors (reference and mutant sequences), 10 pM pamidronate was added to the co-cultures. At the end of the co-culture, supernatant depleted from TEGs by centrifugation force, was collected and used for IFN-y ELISA. Luciferase activity of Luc-Tom HT-29 tumour cells from the co-culture plate was determined by the addition of D-luciferine substrate (ThermoFisher Scientific) and reading the luminescence in endpoint mode was performed using Glomax luminometer according to the manufacturer’s instructions (Promega, Madison, Wl, USA). Cytolysis/cytotoxicity was calculated using the following formula: 100x [1-(Luminescence from target cells in co-culture with effector T-cells/Luminescence from target cells cultured alone)]. Samples were corrected (normalized) for transduction efficiency and total cell numbers. xCELLiqence cytotoxicity assay

TEG anti-tumour activity towards several tumour cell lines was evaluated in vitro by measuring the killing of tumour target cells in a xCELLigence co-culture assay (Agilent, Santa Clara, CA, USA). First, cell lines were harvested, counted and seeded to the appropriate number of cells per well in triplicate in 96-well E-plates, and then placed in the xCELLigence cradles. Target cell adhesion and proliferation was measured for 24 hours. TEG or negative control untransduced apT-cells were then harvested, counted, resuspended in IMDM medium, 5% human serum, and 1% Penicillin/Streptomycin, and added to the tumour target cells at Effector/Target ratio of 1:1, 1:2, 1:6. In experiments involving cl5-based y5T-cell receptors (reference and mutant sequences), 10 pM pamidronate was added to the co-cultures. Loss of target cell adherence, as a readout for cytotoxicity, was measured for 72 hours. Cytotoxicity was calculated as percentage of cytolysis relative to maximum cytolysis induced by treatment of the target cells with the detergent T riton-X-100. IFN-v ELISA assay

Cell culture supernatants from Luciferase-based cytotoxicity assays were harvested at the end of the coculture to measure IFN-y secretion using a commercial Human IFN-gamma DuoSet ELISA assay (cat nr. DY285B-05, R&D Systems, Minneapolis, MN, US), according to manufacturer’s instructions. This is a standard sandwich ELISA using a plate-bound capture antibody and a detection antibody both specific for IFN-y. The detection antibody is linked to an enzyme which can convert a substrate into an absorbance signal which is measured with a plate reader. The internal standard curve was to calculate the IFN-y concentration (pg/mL) from supernatants.

Example 1. Exchanging single non-hydrophobic amino acids for hydrophobic amino acids in the v5TCR transmembrane on TEGs positively correlates with augmented cvtolvsis and IFN release.

Based on folding of alpha helixes that are embedded in the lipid bilayer, and the interaction of the yb-TCR transmembrane regions interacting within the eight helix bundle of the TCR-CD3 complex, we mutated a number of amino acids to stabilize or destabilize the y5-TCR complex in the transmembrane region or in close proximity of this region. Selected number of amino acids were explored for the constant 1 region of the gamma TOR (TRGC1, SEQ ID NO: 1) or constant delta region (TRDC, SEQ ID NO: 2). For the y-TCR: T136L (SEQ ID NO: 3), L143E (SEQ ID NO: 4), L143D (SEQ ID NO: 5) S149A (SEQ ID NO: 6), V151 L (SEQ ID NO: 7), F153A (SEQ ID NO: 8), C158F (SEQ ID NO: 9) and C159F (SEQ ID NO: 10) substitutions based on the amino acid positioning in the TRGC1 (SEQ ID NO: 1) region were examined. Specifically amino acids with polar uncharged side chains, positive or negative charged side chains, or with no side charge were exchanged for hydrophobic side chain amino acids. Moreover, amino acids with hydrophobic side chains were exchanged for other amino acids with hydrophobic side chains that established additional hydrogen bond interactions with neighboring alpha helixes. In addition, the leucin at position 143 was exchanged for a negatively charged amino acid to establish an interaction with CD3e as observed in the aP-TCR-CD3 complex. For the 5-TCR: T128L (SEQ ID NO: 11), G131V (SEQ ID NO: 12), T139V (SEQ ID NO: 13) and F144L (SEQ ID NO: 14) substitutions based on the positioning in the TRDC (SEQ ID NO: 2) region, were tested to stabilize the eight helix TCR-CD3 complex according to the same strategy as listed for the y-TCR. To determine whether the variety of transmembrane mutants had an impact on y5TCR expression, we introduced the mutations in the E57 y6TCR (Vy4V55, SEQ ID NO: 96, 99; wild-type), as template. TEGs expressing E57 wild-type and TCRs with single transmembrane mutations were produced and TCR expression was monitored (Fig. 2A-2C). The L143D, L143E (TRGC1), and G131V (TRDC) substitutions reduced ySTCR expression, whereas F144L improved expression in terms of a higher frequency of single positive ySTCR-expressing TEGs (Fig. 4A). From the E57 wild-type and single transmembrane mutants, cytolysis was determined in a co-culture assay against two colorectal tumour cell lines, HT-29 and RKO (Fig. 5A and 5B) as described in the materials and methods. The percentage of TEGs for each condition was equalized with untransduced donor matched T-cells. All tested transmembrane mutants increased the average cytolysis when targeting the HT-29 tumour cell line, with G153V as exception which showed a reduction in cytolysis (Fig. 5A). For the RKO tumour cell line, the F153A, C158F, T128L, T139Vand, F144L substitutions increased cytolysis and G131V decreased cytolysis (Fig. 5B).

Example 2. Combining modifications augments v5TCR expression in TEGs and increases target specific cvtolvsis and IFN release.

To study whether individual substitutions could synergize mutant ySTCRs with double, triple and multiple amino acid substitutions in the transmembrane region were generated using the E57 y5TCR (SEQ ID NO: 96, 99) as template. Expression profiles of these mutant y5TCRs were analyzed with FACS flow cytometry (Fig. 3A-3C). Increasing the number of substitutions increased the number of single positive y5TCR TEGs over yb-aPTCR expressing TEGs (Fig. 4B, 4C, and 4D). By changing the transmembrane region, up to 300- 400% more single positive ySTCRs were observed for respectively S149A-V151 L/T128L-T139V-F144L (SEQ ID NO: 15/SEQ ID NO: 20) and S149A-V151 L-C158F/T139V-F144L (SEQ ID NO: 17/SEQ ID NO: 19). HT-29 (Fig. 6A) and RKO (Fig. 6B) cytolysis was determined for a number of double, triple and multiple transmembrane TEG mutants. Increasing the number of substitutions increased the ability of TEGs to cytolyze both HT-29 and RKO cells. From the tested combinations, S149A-V151 L/T128L-T139V-F144L (SEQ ID NO: 15 / SEQ ID NO: SEQ ID NO: 20), S149A-C158F/T139V-F144L (SEQ ID NO: 16/SEQ ID NO:

19), and S149A-V151 L-C158F/T139V-F144L (SEQ ID NO: 17/SEQ ID NO: 19) showed the strongest potential to cytolyze HT-29 cells (Fig. 6A). Cytolysis of RKO tumour cells was highest with the S149A- V151 L-C158F/T139V-F144L (SEQ ID NO: 17/SEQ ID NO: 19) mutant (Fig. 6B). The IFNy release by TEGs in response to contacting with tumour target cells HT-29 was the highest with the S149A/T128L-T139V- F144L (SEQ ID NO: 6/SEQ ID NO: 20), S149A-V151L/T128L-T139V-F144L (SEQ ID NO: 15/SEQ ID NO:

20) and S149A-C158F/T139V-F144L (SEQ ID NO: 16/SEQ ID NO: 19) mutants, with a 3-fold increase (Fig. 6C). For RKO cells multiple combinations gave a 2- to 2,8-fold augmented IFNy response in comparison to the wild-type E57 ySTCR (Fig. 6D).

Example 3. Combined modifications augments v5TCR expression comprising other variable regions.

Two combinations of amino acid substitutions in the transmembrane constant regions, S149A- C158F/T139V-F144L (SEQ ID NO: 16/SEQ ID NO: 19) and S149A-C158F/T128L-T139V-F144L (SEQ ID NO: 16/SEQ ID NO: 20) were explored using other ySTCRs as templates. Clone 4 (Vy9V61; SEQ ID NO: 97, 100) and clone 5 (Vy5V51; SEQ ID NO: 98, 101) ySTCR transmembrane region mutants were generated and produced as TEGs, according to the materials and methods, and the TCR expression profile was analyzed by FACS flow cytometry (Fig. 7A). For clone 5 mutants the total percentage of apT-cells expressing a y5-TCR was increased from 23% to 40% (Fig. 7A). For both clone 4 and 5 ySTCRs having the mutated transmembrane regions, the percentage of single positive expressing y5TCR TEGs was strongly upregulated (Fig. 7B). Up to 2-fold increase was observed for clone 5 mutants and 2,5-fold for clone 4 mutants. No effect in cytolysis and IFNy production was observed for clone 4 when targeting the triple negative breast cell line MDA-MB-231 in co-culture (Fig. 8A, 8C). For done 5, when normalized on ySTCR expression a 2-fold enhanced production of IFNy was observed (Fig. 8D). In a more accurate comparison normalizing the TEG input based on the percentage of eGFP expression (refleding the number of integrations) cytolysis of MDA-MB-231 cells was augmented up to -130% and a 3-fold increase of IFNy release was measured (Fig. 8C-8D).

Example 4. Introduction of a prolonged FG-loop in the v5TCR increases TCR fundion.

The FG-loop in the PTCR constant region of apT cells regulates T cell development and mediates downstream T cell signalling processes (Das et al., 2015; Hwang et al., 2020; Sasada et al., 2002; Touma et al., 2006). Comparatively, in pTCRs a larger FG-loop is present than in the yTCR constant region of ySTCRs (Mallis et al., 2021). The FG-loop in the yTCR constant region lacks proper coverage of the CD3ey subunits and might be less efficient in inducing strong T cell signaling compared to the one that is induced by the FG-loop from PT cell receptor chains. To strengthen TCR-signaling of y5T-cell receptors we exchanged the smaller native y-FG loop (SEQ ID NO: 44) for the longer p-FG loop (SEQ ID NO: 46) in the Cy1 chain (SEQ ID NO: 1 ) of the E57 ySTCR (SEQ ID NO: 96, 99), resulting in SEQ ID NO: 22 and 23. To graft the introduced P-FG loop onto the yTCR FG-beta sheet we replaced the adjacent 5’-end and 3’-start of the yTCR FG-beta (SEQ ID NO: 47, 50). In addition, positions D1 to V8 (a-loop) from the Cy1 region (SEQ ID NO: 42) were swapped with the a-loop of the -TCR (SEQ ID NO: 43) to keep the interaction between the newly introduced FG-0 loop with the a-loop obtained from the 0-TCR, resulting in SEQ ID NO: 24, 25. Four E57 TOR FG-loop mutants were compared to wild-type E57 TCR (yFG, SEQ ID NO : 22, £FG, SEQ ID NO: 23), yFG con0 (SEQ ID NO: 24), 0FG con0 (SEQ ID NO: 25)) and expression of ySTCR in a|3T cells was monitored (Fig. 9A). All E57 TCR FG-mutants showed a reduced percentage of single positive y5-TCR expressing cells compared to wild-type E57 TCR (Fig. 9B). Similar results were obtained when the FG-loop mutations were introduced in other ySTCRs with different variable TCRy and TCR5 regions compared to E57, respectively clone 4 (SEQ NO ID: 97, 100), clone 5 (SEQ NO ID: 98, 101) and E57 G5D6 (SEQ NO ID: 120, 121 ) (Fig. 10A-10C). To examine whether the FG-loop mutations improved yQTCR function co-culture experiments were conducted with TEGs expressing the E57 TCR mutants and two tumour target cells, respectively HT-29 and RKO. Tumour cytolysis was increased with —115% (Fig. 11A, 11 B) and release of IFNy in culture supernatant was increased with 140-160% (Fig. 11 A, 11 B). Additionally, increased TCR function of clone 5 ySTCR with introduced FG-loop mutations in the yTCR (yFG con0; SEQ ID NO: 24) showed higher levels of IFN release (up to -175%) when targeting two other tumour cell lines, respectively MDA-MB-231 and MZ1851 RC (Fig. 12A and 12B).

Example 5. Introduction of mutations in the constant TCRy chain that can impact the TCR-CD3 interaction increases TCR function and uprequlates IFN release.

Some flexible loops in the constant yTCR region are positioned in close proximity across flexible loops of CD3 subunits. To examine whether the interaction between the ySTCR with CD3 subunits can be improved we mutated amino acids in the constant yTCR that are close to CD3 subunits. The impact of the mutations on decreasing or increasing the stability of the CD3-TCR complex was explored in the E57 yBTCR (SEQ ID NO: 96, 99) and TCR expression and TCR function was determined. From the tested mutations (SEQ ID NO: 26, 27, 28, 29, 30, 31 , and 32) no impact on y6TCR expression was observed as E57 TEGs were generated from a T cells (Fig. 13). A co-culture experiment with wild-type y6TCR E57 or different E57 CD3- TCR mutants was conducted with two targeted tumour cell lines, respectively HT-29 and RKO. A number of single CD3-TCR mutants showed altered release of IFN in culture supernatant after the co-culture of TEGs with the indicated cell line (Fig. 14A and 14B). The combination of K49A and K50A together with K96D and N97K substitutions (SEQ ID NO: 95) increased IFN release with -135-185%.

Example 6. Introduction of mutations in the constant TCRy and TCR5 chain that disable attachment of potential N-linked glycans increase the percentage of TEGs single positive for ySTCR expression.

In total, six potential N-linked glycans can be attached to constant regions of the ySTCR. The N-linked glycans are connected to the asparagine amino acid when a NxT/S motif is present in the amino acid sequence. Attachment of the N-linked glycan to such a motif can be disrupted by substituting the asparagine by a serine. To determine whether negating N-linked glycan attachment to the ySTCR can impact TCR- CD3 expression, two mutations were introduced; N66S (TRGC1 SEQ ID NO: 35) and/or N14S (TRDC SEQ ID NO: 36) in the E57 y5TCR (SEQ ID NO: 96 ,99) and cl5 y5TCR (SEQ ID NO: 102, 103), based on the amino acid positioning in the TRGC and TRDC regions, and expression was monitored in TEGs (Fig. 15A). Especially N66S (TRGC1) combined with N14S (TRDC) showed an increase in the number of TEGs expressing solely the y5TCR. For cl5-based mutants an -145% increase in the number of single positive y5TCR expressing TEGs was observed and for E57-based mutants this was -250% (Fig. 15A-15B). Example 7. Introduction of mutations in the constant TCRv and TCR5 chain that introduce an extra cysteine bond between the two subunits.

The TCR constant gamma domain 1 (TRGC1 , SEQ ID NO: 1) is able to form a cysteine bridge with the TCR constant delta subunit (TRDC, SEQ ID NO: 2), whilst the TCR constant gamma 2 (TRGC2, SEQ ID NO: 40) that has a duplication in the connecting peptide lacks this cysteine and is unable to form a cysteine bridge between the subunits. Stabilization of the ySTCR complex could be important to transfer changes in energy that occur during docking of its ligand to other parts of the TCR leading to downstream TCR signalling processes. To further stabilize the y5TCR we mutated Q58C (TRGC1 , SEQ ID NO: 37) and V48C (TRDC, SEQ ID NO: 38), based on the amino acid positioning in the TRGC and TRDC regions, to form an additional disulfide bridge between both subunits in the cl5 ybTCR (SEQ ID NO: 102, 103). Expression of the y5TCR of wild-type cl5 and cysteine mutated cl5 in TEGs was determined and no altered y5TCR expression profile was observed (Fig. 16). Changes in functionality was determined in a experiment, where cysteine mutated cl5-expressing TEGs and control wild-type cl5-expressing TEGs were co-cultured with HT-29 and MZ1851 Re target cells. The Q58C/V48C mutation increased cytolysis up to -120% (Fig. 17).

Example 8. Combined modifications increases cvtolvsis mediated by vSTCRs comprising other variable regions.

Amino acid substitutions described in Examples 1 -3 were additionally explored using another ybTCR (G115; SEQ ID NO: 167, 168) as template. Transmembrane region mutants were generated and produced as TEGs according to the materials and methods and tested for Luc-Tom HT-29 cell cytolysis together with TEGs expressing reference G115 sequences via luciferase assay as described above. Combinations of amino acid substitutions tested included C158F/F144L (SEQ ID NO: 9/ SEQ ID NO: 14), S149A/T139V (SEQ ID NO: 6/ SEQ ID NO: 13), S149A-C158F/T139V (SEQ ID NO: 16/ SEQ ID NO: 13), S149A/T128L- T139V (SEQ ID NO: 6/ SEQ ID NO: 18), S149A-F153A/T128L-T139V (SEQ ID NO: 175/SEQ ID NO: 18), S149A/T139V-F144L (SEQ ID NO: 6/SEQ ID NO: 19), S149A-V151 L/T139V-F144L (SEQ ID NO: 15/ SEQ ID NO: 19), S149A/T128L-T139V-F144L (SEQ ID NO: 6/SEQ ID NO: 20), S149A-V151 L/T128L-T139V- F144L (SEQ ID NO: 15/SEQ ID NO: 20), S149A-C158F/T139V-F144L (SEQ ID NO: 16/SEQ ID NO: 19), and S149A-V151 L-C158F/T139V-F144L (SEQ ID NO: 17/SEQ ID NO: 19). All mutants demonstrated increased cytolysis compared to TEGs expressing the reference G115 sequences (Fig. 18).

References

Das, D. K., Feng, Y., Mallis, R. J., Li, X., Keskin, D. B., Hussey, R. E., Brady, S. K., Wang, J. H., Wagner, G., Reinherz, E. L., & Lang, M. J. (2015). Force-dependent transition in the T-cell receptor -subunit allosterically regulates peptide discrimination and pMHC bond lifetime. Proceedings of the National Academy of Sciences of the United States of America, 112(5), 1517-1522. https://doi.Org/10.1073/pnas.1424829112.

Hwang, W., Mallis, R. J., Lang, M. J., & Reinherz, E. L. (2020). The a TCR mechanosensor exploits dynamic ectodomain allostery to optimize its ligand recognition site. Proceedings of the National Academy of Sciences of the United States of America, 117(35), 21336-21345. https://doi.Org/10.1073/pnas.2005899117.

Mallis, R. J., Duke-cohan, J. S., Kumar, D., Akitsu, A., & Luoma, A. M. (2021). Molecular design of the y5 T cell receptor ectodomain encodes biologically fit ligand recognition in the absence of mechanosensing. Proceedings of the National Academy of Sciences of the United States of America, 118(26), 1-12. https://doi.Org/10.1073/pnas.2023050118. Sasada, T., Touma, M., Chang, H. C., Clayton, L. K., Wang, J. H., & Reinherz, E. L. (2002). Involvement of the TCR CP FG loop in thymic selection and T cell function. Journal of Experimental Medicine, 195(11), 1419-1431. https://doi.Org/10.1084/jem .20020119.

Touma, M., Chang, H.-C., Sasada, T., Handley, M., Clayton, L. K., & Reinherz, E. L. (2006). The TCR C FG Loop Regulates ap T Cell Development. The Journal of Immunology, 176(11), 6812-6823. https://doi.org/10.4049/jimmunol.176.11.6812.

Claims

1. A method for obtaining a yT-cell receptor chain, a 5T-cell receptor chain, a yBT-cell receptor, or a fragment thereof that mediates an anti-tumour or anti-infective response, said method comprising the steps of:

A) providing a yT-cell receptor chain or fragment thereof, comprising a Cy constant region or a fragment thereof, and a 5T-cell receptor chain or fragment thereof, comprising a C6 constant region or a fragment thereof;

C) expressing the yT-cell receptor chain or fragment thereof and the 6T-cell receptor chain or fragment thereof obtained in step B) in an immunoresponsive cell;

2. A method according to claim 1, wherein said immunoresponsive cell is selected from a T-cell, an aPT-cell, a y5T-cell, CD4+ T-cell, CD8+ T-cell, an induced pluripotent stem cell derived T-cell, a T-effector cell, a lymphocyte, a B-cell, an NK-cell, an NKT-cell, a myeloid cell, a monocyte, a macrophage, or a neutrophil.

3. A method according to claim 1 or 2, wherein in step B) at least two, at least three, at least four, or at least five amino acid modifications are introduced in said Cy constant region or fragment thereof and/or in said C5 constant region or fragment thereof.

4. A method according to any one of claims 1 to 3, wherein in step A) said Cy constant region or fragment thereof comprises or consists of an amino acid sequence represented by SEQ ID NO: 1 , SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably SEQ ID NO: 1, and/or said C5 constant region or fragment thereof comprises or consists of an amino acid sequence represented by SEQ ID NO: 2.

5. A method according to claim 4, wherein the amino acid modification in step B) is selected from: B1 ) an amino acid modification in a position corresponding to a position selected from D1 to V8 of SEQ ID NO: 1 or SEQ ID NO: 39;

B2) an amino acid modification in a position corresponding to a position selected from K49 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39;

B3) an amino acid modification in a position corresponding to a position selected from T136 to L161 of SEQ ID NO: 1 or SEQ ID NO: 39;

B4) an amino acid modification in a position corresponding to a position selected from N14 to V48 of SEQ ID NO: 2; B5) an amino acid modification in a position corresponding to a position selected from L127 to F152 of SEQ ID NO: 2, or;

B6) a combination of any one of B1) to B5).

6. A method according to claim 5, wherein the amino acid modification in B3), B5), or B6) is an amino acid substitution of a polar or an uncharged amino acid by a hydrophobic amino acid, preferably is a substitution of:

- a S by a G, A, V, L, I, P, F, M, or W;

- a V by a G, A, L, I, P, F, M, orW;

- a F by a G, A, V, L, I, P, M, orW;

- a C by a G, A, V, L, I, P, F, M, or W;

- a T by a G, A, V, L, I, P, F, M, or W, or;

- a combination thereof.

7. A method according to claim 5 or 6, wherein the amino acid modification in B2), B3), B4), B5), or B6) is an amino acid substitution selected from: i. an amino acid substitution in a position corresponding to position K49 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine; ii. an amino acid substitution in a position corresponding to position K50 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine; ill. an amino acid substitution in a position corresponding to position Q58 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a cysteine; iv. an amino acid substitution in a position corresponding to position N66 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a serine; v. an amino acid substitution in a position corresponding to position K96 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine or an aspartic acid; vi. an amino acid substitution in a position corresponding to position N97 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine or a lysine; vii. an amino acid substitution in a position corresponding to position T136 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a leucine; viii. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine; lx. an amino acid substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a leucine; x. an amino acid substitution in a position corresponding to position F153 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine; xi. an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a phenylalanine; xii. an amino acid substitution in a position corresponding to position C159 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a phenylalanine; xiii. an amino acid substitution in a position corresponding to position K49 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position K50 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine; xiv. an amino acid substitution in a position corresponding to position E79 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position K80 of SEQ ID NO: 1 , preferably a substitution by an alanine; xv. an amino acid substitution in a position corresponding to position K96 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine or an aspartic acid, and an amino acid substitution in a position corresponding to position N97 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine or a lysine; xvi. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a leucine; xvii. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a phenylalanine; xviii. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position F153 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine; xix. an amino acid substitution in a position corresponding to position S149 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, an amino acid substitution in a position corresponding to position V151 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a leucine, and an amino acid substitution in a position corresponding to position C158 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a phenylalanine; xx. an amino acid substitution in a position corresponding to position K49 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, an amino acid substitution in a position corresponding to position K50 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, an amino acid substitution in a position corresponding to position K96 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an aspartic acid, and an amino acid substitution in a position corresponding to position N97 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a lysine; xxi. an amino acid substitution in a position corresponding to position N14 of SEQ ID NO: 2, preferably a substitution by a serine; xxii. an amino acid substitution in a position corresponding to position V48 of SEQ ID NO: 2, preferably a substitution by a cysteine; xxiii. an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, preferably a substitution by a leucine; xxiv. an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, preferably a substitution by a valine; xxv. an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, preferably a substitution by a leucine; xxvi. an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, preferably a substitution by a leucine, and an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, preferably a substitution by a valine; xxvii. an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, preferably a substitution by a valine, and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, preferably a substitution by a leucine; xxviii. an amino acid substitution in a position corresponding to position T128 of SEQ ID NO: 2, preferably a substitution by a leucine, an amino acid substitution in a position corresponding to position T139 of SEQ ID NO: 2, preferably a substitution by a valine, and an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, preferably a substitution by a leucine, or; xxix. a combination of any one of I) to xxviii).

8. A method according to any one of claims 5 to 7, wherein the amino acid modification in B1), B2), or B6) is an amino acid substitution selected from: a1) a substitution of the positions corresponding to positions from D1 to V8 of SEQ ID NO: 1 or SEQ ID NO: 39 by EDLKNVF (SEQ ID NO: 43), DKEDLKNVF (SEQ ID NO: 176), or a variant thereof; b1) a substitution of the positions corresponding to positions from V90 to Q101 of SEQ ID NO: 1 or SEQ ID NO: 39 by VQFYGLSENDEWTQDRAKPVTQIV (SEQ ID NO: 47), VRFYGLSENDEWTQDRAKPVTQDQ (SEQ ID NO: 50), or a variant thereof, or; c1) a combination of a1) and b1).

9. A yT-cell receptor chain or a fragment thereof, comprising a Cy constant region or a fragment thereof, wherein said Cy constant region or fragment thereof comprises: a) an amino acid modification in a position corresponding to a position selected from D1 to V8 of SEQ ID NO: 1 or SEQ ID NO: 39; b) an amino acid modification in a position corresponding to a position selected from K49 to Q101 of SEQ ID NO : 1 or SEQ ID NO: 39; c) an amino acid modification in a position corresponding to a position selected from T136 to L161 of SEQ ID NO: 1 or SEQ ID NO: 39, or; d) a combination of any one of a) to c).

10. A yT-cell receptor chain or a fragment thereof according to claim 9, wherein said amino acid modification is an amino acid substitution, and wherein said Cy constant region or fragment thereof comprises:

11. an amino acid substitution in a position corresponding to position K50 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine;

VI. an amino acid substitution in a position corresponding to position N97 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine or a lysine; VII. an amino acid substitution in a position corresponding to position T136 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by a leucine;

XIII. an amino acid substitution in a position corresponding to position K49 of SEQ ID NO: 1 or SEQ ID NO: 39, preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position K50 of SEQ ID NO: 1 , preferably a substitution by an alanine;

XIV. an amino acid substitution in a position corresponding to position E79 of SEQ ID NO: 1 , preferably a substitution by an alanine, and an amino acid substitution in a position corresponding to position K80 of SEQ ID NO: 1, preferably a substitution by an alanine;

XXI. a substitution of the positions corresponding to positions from D1 to V8 of SEQ ID NO: 1 or SEQ ID NO: 39 by EDLKNVF (SEQ ID NO: 43), DKEDLKNVF (SEQ ID NO: 176), or a variant thereof;

XXIII. a combination of any one of I. to XXII.

11. A yT-cell receptor chain or fragment thereof according to claim 9 or 10, wherein said Cy constant region or fragment thereof comprises at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or similarity with SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41, preferably with SEQ ID NO: 1.

12. A yT-cell receptor chain or fragment thereof according to any one of claims 9 to 11 , wherein said Cy constant region or a fragment thereof comprises or consists of an amino acid sequence represented by SEQ ID NO: 3, 6, 7, 8, 9, 10, 15, 16, 17, 21 , 22, 23, 24, 25, 26, 27, 29, 32, 35, 37, 95, or 177.

13. A yT-cell receptor chain or fragment thereof according to any one of claims 9 to 12, wherein said yT-cell receptor chain or fragment thereof further comprises a variable region or fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 108, 109, 110, 111, 112, 113, 122, or 150.

14. A yT-cell receptor chain or fragment thereof according to any one of claims 9 to 13, wherein said yT-cell receptor chain or fragment thereof further comprises a CDR3 region comprising, consisting essentially of, or consisting of an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 124, 125, 126, 127, 128, 129, 130, or 152.

15. A yT-cell receptor chain or fragment thereof according to any one of claims 9 to 14, wherein said yT-cell receptor chain or fragment thereof further comprises a CDR3 region comprising: an amino acid modification, preferably an amino acid substitution, at an amino acid position corresponding to position 4 of SEQ ID NO: 124, an amino acid modification, preferably an amino acid substitution, more preferably a substitution by a glutamic acid, most preferably a substitution of an aspartic acid by a glutamic acid, at an amino acid position corresponding to position 5 of SEQ ID NO: 124; an amino acid modification, preferably an amino acid substitution, more preferably a substitution by an alanine, most preferably a substitution of a glycine by an alanine, at an amino acid position corresponding to position 6 of SEQ ID NO: 124; an amino acid modification, preferably an amino acid substitution, more preferably a substitution by an alanine, serine, or tyrosine, most preferably a substitution of a phenylalanine by an alanine, serine, or tyrosine, at an amino acid position corresponding to position 7 of SEQ ID NO: 124; an amino acid modification, preferably an amino acid substitution, more preferably a substitution by a phenylalanine, most preferably a substitution of a tyrosine by a phenylalanine, at an amino acid position corresponding to position 8 of SEQ ID NO: 124; an amino acid modification, preferably an amino acid substitution, at an amino acid position corresponding to position 9 of SEQ ID NO: 124; an amino acid modification, preferably an amino acid substitution, at an amino acid position corresponding to position 10 of SEQ ID NO: 124, or; any combination thereof.

16. A yT-cell receptor chain or fragment thereof according to any one of claims 9 to 15, wherein said yT-cell receptor chain or fragment thereof further comprises a CDR3 region comprising a modification at an amino acid position corresponding to a position selected from one or more of positions 4-10 of SEQ ID NO: 124, said modification selected from WDAFYYK, WEAFYYK, WDGYFYK, WDGYYYK, WDGAYYK, or WDGSYYK.

17. A yT-cell receptor chain or fragment thereof according to any one of claims 9 to 16, wherein said yT-cell receptor chain or fragment thereof further comprises a CDR1 region comprising, consisting essentially of, or consisting of amino acid sequence SEQ ID NO: 154, 155, 156, or 157, or of an amino acid sequence comprising one, two, or three amino acid modifications relative to SEQ ID NO: 154, 155, 156, or 157, and a CDR2 region comprising, consisting essentially of, or consisting of amino acid sequence SEQ ID NO: 158, 159, 160, or 161 , or of an amino acid sequence comprising one, two, or three amino acid modifications relative to SEQ ID NO: 158, 159, 160, or 161.

18. A yT-cell receptor chain or fragment thereof according to any one of claims 9 to 17, wherein said yT-cell receptor chain or fragment thereof is soluble, preferably wherein said yT-cell receptor chain or fragment thereof comprises a T-cell- and/or NK-cell binding domain, more preferably a CD3- binding domain.

19. A yT cell receptor chain or fragment thereof according to any one of claims 9 to 18, wherein said yT-cell receptor chain or fragment thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251 , 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 , 262, 263, 264, or 265.

20. A 5T-cell receptor chain or a fragment thereof, comprising a C5 constant region or a fragment thereof, wherein said C5 constant region or fragment thereof comprises: e) an amino acid modification in a position corresponding to a position selected from N14 to V48 of SEQ ID NO: 2; f) an amino acid modification in a position corresponding to a position selected from L127 to L152 of SEQ ID NO: 2, or; g) a combination of e) and f).

21. A 5T-cell receptor chain or a fragment thereof according to claim 20, wherein said amino acid modification is an amino acid substitution, and wherein said C5 constant region or fragment thereof comprises:

XXVIII. an amino acid substitution in a position corresponding to position F144 of SEQ ID NO: 2, preferably a substitution by a leucine;

XXXII. a combination of any one of XXIV. To XXXI.

22. A 5T-cell receptor chain or a fragment thereof according to claim 20 or 21 , wherein said C5 constant region or fragment thereof comprises at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or similarity with SEQ ID NO: 2.

23. A 5T-cell receptor chain or a fragment thereof according to any one of claims 20 to 22, wherein said C5 constant region or a fragment thereof comprises or consists of an amino acid sequence represented by SEQ ID NO: 11, 13, 14, 18, 19, 20, 36, or 38.

24. A 5T-cell receptor chain or fragment thereof according to any one of claims 20 to 23, wherein said 5T-cell receptor chain or fragment thereof further comprises a variable region or fragment thereof comprising, consisting essentially of, or consisting of an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence Identity or similarity with amino acid sequence SEQ ID NO: 114, 115, 116, 117, 118, 119, 123, or 151.

25. A 5T-cell receptor chain or fragment thereof according to any one of claims 20 to 24, wherein said 5T-cell receptor chain orfragmentthereof further comprises a CDR3 region comprising, consisting essentially of, or consisting of an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 131, 132, 133, 134, 135, 136, 137, or 153.

26. A 5T-cell receptor chain or fragment thereof according to any one of claims 20 to 25, wherein said 5T-cell receptor chain or fragment thereof further comprises a CDR3 region comprising: an amino acid modification, preferably an amino acid substitution, more preferably a substitution by a leucine, most preferably a substitution of an isoleucine by a leucine, at an amino acid position corresponding to position 7 of SEQ ID NO: 131; an amino acid modification, preferably an amino acid substitution, more preferably a substitution by a lysine, most preferably a substitution of an arginine by a lysine, at an amino acid position corresponding to position 8 of SEQ ID NO: 131 ; an amino acid modification, preferably an amino acid substitution, at an amino acid position corresponding to position 9 of SEQ ID NO: 131 ; an amino acid modification, preferably an amino acid substitution, more preferably a substitution by a phenylalanine, most preferably a substitution of a tyrosine by a phenylalanine, at an amino acid position corresponding to position 10 of SEQ ID NO: 131; an amino acid modification, preferably an amino acid substitution, at an amino acid position corresponding to position 11 of SEQ ID NO: 131 ; an amino acid modification, preferably an amino acid substitution, at an amino acid position corresponding to position 12 of SEQ ID NO: 131 , or; any combination thereof.

27. A 5T-cell receptor chain or fragment thereof according to any one of claims 20 to 26, wherein said 5T-cell receptor chain or fragment thereof further comprises a CDR3 region comprising a modification at an amino acid position corresponding to a position selected from one or more of positions 7-12 of SEQ ID NO: 131 , said modification selected from IRGFTG, IKGYTG , IKGFTG, LRGFTG, LKGFTG, or LKGYTG.

28. A 5T-cell receptor chain or fragment thereof according to any one of claims 20 to 27, wherein said 5T-cell receptor chain orfragmentthereof further comprises a CDR1 region comprising, consisting essentially of, or consisting of amino acid sequence SEQ ID NO: 162, 163, or 164, or of an amino acid sequence comprising one, two, or three amino acid modifications relative to SEQ ID NO: 162, 163, or 164, and a CDR2 region comprising, consisting essentially of, or consisting of amino acid sequence EKD, QGS, or SEQ ID NO: 166, or of an amino acid sequence comprising one, two, or three amino acid modifications relative to amino acid sequence EKD, QGS, or SEQ ID NO: 166.

29. A 6T-cell receptor chain or fragment thereof according to any one of claims 20 to 28, wherein said 6T-cell receptor chain or fragment thereof is soluble, preferably wherein said 5T-cell receptor chain or fragment thereof comprises a T-cell- and/or NK-cell binding domain, more preferably a CD3- binding domain.

30. A 5T cell receptor chain or fragment thereof according to any one of claims 20 to 29, wherein said 5T-cell receptor chain or fragment thereof comprises, consists essentially of, or consists of an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity or similarity with amino acid sequence SEQ ID NO: 266, 267, 268, 269, 270, 271 , 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, or 289.

31. A y5T-cell receptor or a fragment thereof, wherein said receptor or fragment thereof comprises: a yT-cell receptor chain or fragment thereof as defined in any one of claims 9 to 19; a 5T-cell receptor chain or fragment thereof as defined in any one of claims 20 to 30, or; a yT-cell receptor chain or fragment thereof as defined in any one of claims 9 to 19 and a 5T-cell receptor chain or fragment thereof as defined in any one of claims 20 to 30.

32. A chimeric antigen-recognition receptor comprising: a Cy constant region of a yT-cell receptor chain or a fragment thereof as defined in any one of claims 9 to 19; a C5 constant region of a 5T-cell receptor chain or a fragment thereof as defined in any one of claims 20 to 30, or; a Cy constant region of a yT-cell receptor chain or a fragment thereof as defined in any one of claims 9 to 19 and a C5 constant region of a 5T -cell receptor chain or fragment thereof as defined in any one of claims 20 to 30, preferably wherein said chimeric antigen-recognition receptor further comprises an antigenbinding domain from or derived from an antibody, an antibody variable region, an scFv, a single domain antibody, a Fab, a Fab', a F(ab')2, a dimer or a trimer of Fab conjugates, an Fv, a minibody, a diabody, a triabody, a tetrabody, an affibody, an ankyrin protein, an ankyrin repeat, a DARPin, a monobody, a nanobody, an avimer, an adnectin, an anticalin, a tynomer, a Kunitz domain, a knottin, a 0-hairpin mimetic, or a combination thereof.

33. A nucleic acid molecule encoding a yT-cell receptor chain or a fragment thereof as defined in any one of claims 9 to 19, a 5T-cell receptor chain or a fragment thereof as defined in any one of claims 20 to 30, a y5T-cell receptor or fragment thereof as defined in claim 31, or a chimeric antigenrecognition receptor as defined in claim 32.

34. A cell, preferably an engineered cell, expressing the yT-cell receptor chain or fragment thereof as defined in any one of claims 9 to 19, the 5T-cell receptor chain or fragment thereof as defined in any one of claims 20 to 30, the y5T-cell receptor or fragment thereof as defined in claim 31 , or the chimeric antigen-recognition receptor as defined in claim 32, or comprising a nucleic acid molecule as defined in claim 33, preferably wherein said cell is selected from a T-cell, an IPSC-derived T- cell, an apT-cell, a y5T-cell, or an NK cell, more preferably from a yST-cell or apT-cell, most preferably Is an apT-cell.

35. A composition, preferably a pharmaceutical composition, comprising a yT-cell receptor chain or a fragment thereof as defined in any one of claims 9 to 19, a 5T-cell receptor chain or a fragment thereof as defined in any one of claims 20 to 30, a y5T-cell receptor or fragment thereof as defined in claim 31 , a chimeric antigen-recognition receptor as defined in claim 32, a nucleic acid molecule as defined in claim 33, or a cell as defined in claim 34.

36. A yT-cell receptor chain or a fragment thereof as defined in any one of claims 9 to 19, a 5T-cell receptor chain or a fragment thereof as defined in any one of claims 20 to 30, a y5T-cell receptor or fragment thereof as defined in claim 31, a chimeric antigen-recognition receptor as defined in claim 32, a nucleic acid molecule as defined in claim 33, a cell as defined in claim 34, or a composition as defined in claim 35, for use as a medicament, preferably for use in the treatment, regression, curing, and/or delaying of cancer or an infection.