Proteins comprising t cell receptor constant domains

Abstract

The present invention relates to a protein comprising: a first polypeptide comprising a T cell receptor (TCR) alpha constant domain (Cα) comprising at least one of the following residues: asparagine at position 134, arginine, glutamate or serine at position 156, serine or tyrosine at position 169, leucine at position 175, leucine or tryptophane at position 178, serine at position 190, or threonine at position 199 (residues numbered according to Kabat numbering); and / or a second polypeptide comprising a TCR beta constant domain (Cβ) comprising at least one of the following residues: threonine at position 128, tryptophan at position 150, or histidine at position 184 (residues numbered according to Kabat numbering). The present invention further relates to therapeutic uses of said protein.

Description

[0001] PROTEINS COMPRISING T CELL RECEPTOR CONSTANT DOMAINS

[0002] The present disclosure relates to proteins comprising T cell receptor (TCR) constant domains with one or more stabilization mutations, nucleic acids encoding such proteins, and methods of making and using such proteins.

[0003] BACKGROUND OF THE INVENTION

[0004] Protein-based drugs require sufficient levels of stability to ensure that their native folded conformation is maintained for the duration of manufacturing, storage and patient dosing, and at the temperatures associated with these activities. This presents a significant challenge for TCRs, which are poorly stable in solution and begin to unfold (or 'melt') at temperatures as low as 40°C. In addition to a loss of activity, protein unfolding can initiate a process of irreversible protein aggregation, leading to the formation of protein multimers (or aggregates) that may further compromise the homogeneity and shelf-life of the drug product, both of which are subject to strict regulatory quality requirements. An additional aspect correlated to both thermal stability and aggregation propensity is recombinant expression yield, with poorly stable proteins typically displaying titers that are not sufficiently high for upscaling of manufacturing. In line with this observation, it has been reported that only around one third of TCRs are expressible from E. coli inclusion bodies, a process that is further complicated by the requirement of chemical denaturation and refolding steps.

[0005] Several molecular engineering strategies have been applied to universally stabilise TCRs for enhanced soluble expression. For example, initial approaches relying on the introduction of leucine zipper domains at the C-termini of TCRa and TCR chains have been shown to increase TCR expressibility in E. coli (Willcox, B. E. etal. Production of soluble alpha beta T-cell receptor heterodimers suitable for biophysical analysis of ligand binding. Protein Sci.Ji, 2418-2423 (1999)). A subsequent approach by Boulter eta / . 2003 engineered an artificial disulphide bond between the TCRa and TCR chain constant domains enabled by the introduction of a pair of cysteine residues at positions 166 and 173 of each chain (Boulter, J. M. et al. Stable, soluble T- cell receptor molecules for crystallization and therapeutics. Protein Eng. 16, 707-711 (2003)). In this study, cysteine introduction was targeted to the inter-chain residues in closest proximity to each other, as determined from the crystal structure of the model human TCRB? (PDB: 1BD2) (Ding, Y. H. et al. Two human T cell receptors bind in a similar diagonal mode to the HLA-A2 / Tax peptide complex using different TCR amino acids. Immunity 8, 403-411 (1998)). Testing of thirteen pairs of candidate cysteine substitutions by expression from E. coli inclusion bodies led to the selection of T166C (TCRa) and S173C (TCR ) as the combination with highest expression yield. The successful formation of an artificial disulphide bond by the introduced cysteines was confirmed subsequently via X-ray crystallography.

[0006] Despite providing a viable approach for soluble TCR manufacturing, expression from bacterial inclusion bodies followed by denaturation and refolding is a labour intensive process associated with a substantial loss of material. In this context, expression of soluble TCRs from mammalian cells offers streamlined manufacturing processes, expression of fully glycosylated proteins and cost-of-goods that are not dissimilar to bacterial production, as the increased direct production costs (e.g., cell line development, media) are counterbalanced by direct secretion of fully folded proteins into the culture supernatant, thus circumventing the need for protein refolding. Furthermore, different to bacterial expression, mammalian expression enables facile expression of multiple polypeptide chains to assemble the final protein drug, with four different polypeptide chains being routinely expressed for bispecific antibody manufacturing. Despite its advantages, a major limitation of mammalian expression of soluble TCRs is a low expression yield (typically <30 mg / L for transient expression) even when including the Boulter disulphide.

[0007] A recent study applied a structure-guided computational approach to identify seven stabilising mutations in the constant TCR domains (three in TCRa, and four in TCR ) (Froning, K. et al. Computational stabilization of T cell receptors allows pairing with antibodies to form bispecifics. Nat. Commun. 11, 2330 (2020)). This approach consisted in subjecting the crystal structure of the model human TCRIG (PDB: 2F53) to in silica simulations using the Rossetta energy function in order to identify substitutions providing the highest theoretical reduction in free energy. The authors identified 52 candidate substitutions, from which seven substitutions were identified to provide both increased mammalian expression yield and Tm over wild-type controls in the context of constant domain TCR fragments. Combination of these 7 mutations into full-length soluble TCRs resulted in a ~ 3-fold increase in mammalian expression yield and 2-5°C increases in Tm(first melting event) depending on the TCR tested. This work further resulted in patent application WO 2021 / 046072.

[0008] In a different study, Sadio and colleagues tested the introduction of artificial disulphide bonds in the TCR constant domains for TCR stabilization (Sadio, F. et al. Stabilization of soluble high- affinity T-cell receptor with de novo disulfide bonds. FEBS Lett. 594, 477-490 (2020)). Pairs of candidate residues for targeted cysteine introduction were selected in silica based on their intermolecular distances. Notably, only eight candidates out of eighteen tested were expressible in mammalian cells, and a single artificial disulphide enhanced TCR thermal stability, albeit to a moderate level (2°C increase in Tm).

[0009] WO 2024 / 036166 discloses engineered TCR molecules in which amino acid residues forming the interface between the alpha chain constant domain and the beta chain constant domain have been substituted to enhance the functions of the TCR. WO 2022 / 133592 discloses TCRs comprising engineered interchain disulfide bonds and additional stabilizing mutations. US 2022 / 0306720 is a further document disclosing engineered TCRs comprising stabilizing mutations.

[0010] While the studies cited above were able to identify TCR stabilising mutations, they also highlight important challenges of structural modelling approaches, namely low testing throughput, inability to select for variant expressibility and difficulty to identify synergistic effects between mutations. This is particularly relevant when seeking to identify minimal sets of stabilising mutations that limit the immunogenicity potential of the resulting stabilised TCR drug.

[0011] There is thus still a need in the art for highly stable TCR drugs that can be efficiently expressed in mammalian cells and possess a low risk of immunogenicity.

[0012] The objective technical problem underlying the present invention can thus be formulated as the provision of improved TCR drugs.

[0013] SUMMARY OF THE INVENTION

[0014] The present invention is characterized in the herein provided embodiments and claims. In particular, the present invention relates, inter alia, to the following embodiments:

[0015] 1. A protein comprising: a first polypeptide comprising a T cell receptor (TCR) alpha constant domain (Ca) comprising at least one of the following residues: asparagine at position 134, arginine, glutamate or serine at position 156, serine or tyrosine at position 169, leucine at position 175, leucine or tryptophane at position 178, serine at position 190, or threonine at position 199 (residues numbered according to Kabat numbering); and / or a second polypeptide comprising a TCR beta constant domain (CP) comprising at least one of the following residues: threonine at position 128, tryptophan at position 150, or histidine at position 184 (residues numbered according to Kabat numbering).

[0016] 2. The protein of embodiment 1, wherein the first polypeptide comprises a Ca domain comprising at least one of the following residues: glutamate at position 156, tyrosine at position 169, leucine or tryptophane at position 178, or serine at position 190 (residues numbered according to Kabat numbering); and / or the second polypeptide comprises a CP domain comprising at least one of the following residues: threonine at position 128, or histidine at position 184 (residues numbered according to Kabat numbering).

[0017] 3. The protein of embodiment 1 or 2, wherein the CP domain further comprises a glutamate at position 170 (residues numbered according to Kabat numbering).

[0018] 4. The protein of any one of embodiments 1-3, wherein: the first polypeptide comprises a Ca domain comprising the following residues: tryptophane at position 178 and serine at position 190 and, optionally, threonine at position 199 (residues numbered according to Kabat numbering); and / or the second polypeptide comprises a CP domain comprising the following residues: tryptophan at position 150 and glutamate at position 170 (residues numbered according to Kabat numbering).

[0019] 5. The protein of any one of embodiments 1-3, wherein the first polypeptide comprises a Ca domain comprising at least one of the following residues: leucine or tryptophane at position 178, and / or serine at position 190 (residues numbered according to Kabat numbering); and / or the second polypeptide comprises a CP domain comprising at least one of the following residues: glutamate at position 170, and / or histidine at position 184 (residues numbered according to Kabat numbering).

[0020] 6. The protein of embodiment 5, wherein the first polypeptide comprises a Ca domain comprising a leucine at position 178, and a CP domain comprising a glutamate at position 170 (residues numbered according to Kabat numbering); or wherein the first polypeptide comprises a Ca domain comprising a tryptophan at position 178 and a serine at position 190, a CP domain comprising a glutamate at position 170 (residues numbered according to Kabat numbering); or wherein the first polypeptide comprises a Ca domain comprising a tryptophan at position 178, and a CP domain comprising a glutamate at position 170 (residues numbered according to Kabat numbering); or wherein the first polypeptide comprises a Ca domain comprising a tryptophan at position 178, and a CP domain comprising a histidine at position 184 (residues numbered according to Kabat numbering); or wherein the first polypeptide comprises a Ca domain comprising a tryptophan at position 178 and a serine at position 190, and a CP domain (residues numbered according to Kabat numbering); or wherein the first polypeptide comprises a Ca domain comprising a serine at position 190, and a CP domain comprising a glutamate at position 170 (residues numbered according to Kabat numbering).

[0021] 7. The protein of any one of embodiments 1-6, wherein: the Cot domain comprises or consists of SEQ ID NO: 5 and the CP domain comprises or consists of SEQ ID NO: 6.

[0022] 8. The protein of any one of embodiments 1-6, wherein: the Cot domain comprises or consists of SEQ ID NO: 7 and the CP domain comprises or consists of SEQ ID NO: 8.

[0023] 9. The protein of any one of embodiments 1-6, wherein: the Cot domain comprises or consists of SEQ ID NO: 9 and the CP domain comprises or consists of SEQ ID NO: 10.

[0024] 10. The protein of any one of embodiments 1-6, wherein: the Cot domain comprises or consists of SEQ ID NO: 11 and the CP domain comprises or consists of SEQ ID NO: 12.

[0025] 11. The protein of any one of embodiments 1-6, wherein: the Cot domain comprises or consists of SEQ ID NO: 5 and the CP domain comprises or consists of SEQ ID NO: 38.

[0026] 12. The protein of any one of embodiments 1-6, wherein: the Cot domain comprises or consists of SEQ ID NO: 39 and the CP domain comprises or consists of SEQ ID NO: 6.

[0027] 13. The protein of any one of embodiments 1-12, wherein the protein has increased thermal stability compared to a protein having the same amino acid sequence except that: the TCR Ca comprises lysine at position 134, lysine at position 156, aspartate at position 169, phenylalanine at position 175, asparagine at position 178, alanine at position 190, and isoleucine at position 199, and the TCR CP comprises alanine at position 128, threonine at position 150, serine at position 170, and alanine at position 184 (residues numbered according to Kabat numbering).

[0028] 14. The protein of any one of embodiments 1-13, wherein the first polypeptide comprising the TCR Ca and the second polypeptide comprising the TCR CP form a quaternary structure having a melting temperature (Tm) of at least 55°C, at least 56°C, at least 57°C, at least 58°C, at least 59°C, at least 60°C, at least 61°C, at least 62°C, preferably wherein the Tm is determined by differential scanning fluorimetry (DSF).

[0029] 15. The protein of any one of embodiments 1-14, wherein the first polypeptide comprising the TCR Ca and the second polypeptide comprising the TCR CP form a quaternary structure having a melting temperature onset (Tonset) of at least 49°C, at least 50°C, at least 51°C, at least 52°C, at least 53°C, at least 54°C, or at least 55°C, preferably wherein the Tonset is determined by differential scanning fluorimetry (DSF). 16. The protein of any one of embodiments 1-15, wherein the first polypeptide comprising the TCR Cot and the second polypeptide comprising the TCR cp form a quaternary structure having an aggregation temperature (Tagg) of at least 51°C, at least 52°C, at least 53°C, at least 54°C, at least 55°C, at least 56°C, or at least 57°C, preferably wherein the Tagg determined by differential scanning fluorimetry (DSF) and / or static light scattering (SLS).

[0030] 17. The protein of any one of embodiments 1-16, wherein the protein has an increased expression level when expressed under the same conditions compared to a protein having the same amino acid sequence except that: the TCR Cot comprises lysine at position 134, lysine at position 156, aspartate at position 169, phenylalanine at position 175, asparagine at position 178, alanine at position 190, and isoleucine at position 199, and the TCR CP comprises alanine at position 128, threonine at position 150, serine at position 170, and alanine at position 184 (residues numbered according to Kabat numbering).

[0031] 18. The protein of any one of embodiments 1-17, wherein the first polypeptide is linked to the second polypeptide by an inter-chain disulfide bond.

[0032] 19. The protein of any one of embodiments 1-18, wherein the first and second polypeptide are linked by an inter-chain disulfide bond between a cysteine residue at position 213 of the TCR Cot domain and a cysteine residue at position 247 of the TCR CP domain (residues numbered according to Kabat numbering).

[0033] 20. The protein of any one of embodiments 1-19, wherein: the Ca domain further comprises a cysteine residue at position 166 (residue numbered according to Kabat numbering), and the CP domain further comprises a cysteine residue at position 173 (residue numbered according to Kabat numbering), wherein the first polypeptide and the second polypeptide are linked by an inter-chain disulfide bond between the cysteine residue at position 166 of Ca and the cysteine residue at position 173 of cp.

[0034] 21. The protein of any one of embodiments 1-20, wherein: the first polypeptide further comprises a TCR alpha variable domain (Va); and the second polypeptide further comprises a TCR beta variable domain (VP), wherein the Va and VP form an antigen binding domain that binds an antigen.

[0035] 22. The protein of embodiment 21, wherein: the Va is fused to the N-terminus of Ca; and the VP is fused to the N-terminus of cp.

[0036] 23. The protein of embodiments 21 or 22, wherein the antigen is a tumor antigen, a tissue-specific antigen or a viral antigen.

[0037] 24. The protein of any one of embodiments 1-23, wherein the protein further comprises additional antigen-binding domain(s).

[0038] 25. The protein of embodiment 24, wherein the additional antigen binding domain(s) bind(s) one or more antigen(s) on a T cell surface or an NK cell surface.

[0039] 26. The protein of embodiment 24 or 25, wherein the additional antigen binding domain binds CD3 or NKp46.

[0040] 27. The protein of any one of embodiments 24-26, wherein the additional antigen binding domain is a scFv, Fab, Fab', (Fab')2, single domain antibody, or camelid VHH domain.

[0041] 28. The protein of any one of embodiments 24-27, wherein the additional antigen binding domain is a Fab.

[0042] 29. The protein of embodiment 28, wherein the Fab comprises a Fab heavy chain comprising a heavy chain variable domain (VH) and a human IgG CHI domain, and a Fab light chain comprising a light chain variable domain (VL) and a human light chain constant domain (CL), wherein the VH and VL form the second antigen binding domain.

[0043] 30. The protein of any one of embodiments 1-29, wherein the protein further comprises a human IgG Fc region (Fc).

[0044] 31. The protein of embodiment 30, wherein the human IgG Fc region is a modified human IgG Fc region with reduced effector function compared to the corresponding wild type human IgG Fc region.

[0045] 32. The protein of any one of embodiments 1-31, wherein the protein is a soluble protein.

[0046] 33. The protein of any one of embodiments 1-32, wherein the protein is linked to a detectable label or to a therapeutic agent.

[0047] 34. The protein of embodiment 33, wherein the therapeutic agent is a cytotoxic agent, an anti-inflammatory agent, an immunostimulatory agent or an immunosuppressive agent.

[0048] 35. The protein of embodiment 34, wherein the immunostimulatory agent is IL-2, or an engineered variant of IL-2.

[0049] 36. The protein of embodiment 34, wherein the immunosuppressive agent is a PD-1 agonist or a Fas ligand.

[0050] 37. A nucleic acid encoding one or more polypeptides of the protein of any one of embodiments 1-36.

[0051] 38. A vector comprising the nucleic acid of embodiment 37.

[0052] 39. A cell comprising the nucleic acid of embodiment 37 or the vector of embodiment 38.

[0053] 40. A pharmaceutical composition comprising the protein of any one of embodiments 1-36, the nucleic acid of embodiment 37, the vector of embodiment 38, or the cell of embodiment 39.

[0054] 41. A method of treating cancer or infection or autoimmune disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the protein of any one of embodiments 1-36, the nucleic acid of embodiment 37, the vector of embodiment 38, the cell of embodiment 39, or the pharmaceutical composition of embodiment 40.

[0055] 42. The protein of any one of embodiments 1-36, the nucleic acid of embodiment 37, the vector of embodiment 38, the cell of embodiment 39, or the pharmaceutical composition of embodiment 40, for use in the treatment of cancer or infection or autoimmune disease.

[0056] The present invention is directed to proteins comprising at least a TCR alpha and a TCR beta chain constant domain (Ca / cp). Preferably, the protein according to the invention is or comprises a fragment of a TCR and thus further comprises the TCR alpha and beta variable regions (Va / vp) to for an antigen-binding domain.

[0057] In certain embodiments, the protein according to the invention is a soluble TCR fragment comprising two polypeptide chains, wherein the first polypeptide chain comprises the variable region and the constant domain of the TCR alpha chain (Va-Ca) and the second polypeptide chain comprises the variable region and the constant domain of the TCR beta chain (VP-CP).

[0058] In certain embodiments, the protein according to the invention comprises a soluble TCR fragment comprising a first polypeptide chain comprising a variable region and a constant domain of a TCR alpha chain and a second polypeptide chain comprising a variable region and a constant domain of a TCR beta chain. In such embodiments, the protein according to the invention may comprise one or more further protein-based component(s), as defined in more detail herein below.

[0059] Herein, the inventors developed and applied an unbiased approach combining saturation mutagenesis, yeast surface display, thermal cycling selections and deep sequencing to successfully identify a minimal set of stabilising mutations in the constant TCR domains. This minimal set of mutations confers increased stability to soluble TCR fragments or constructs comprising soluble TCR fragments while reducing the risk of immunogenicity. Moreover, the mutations identified herein were shown to improve expression of the soluble TCR fragments.

[0060] Accordingly, provided herein are proteins comprising one or more stabilising mutations in the TCR constant domains (Cct / C|3 domains) that increase the thermal stability of TCRs and / or improve the expression and / or assembly of TCRs.

[0061] In a particular embodiment, the invention relates to a protein comprising a first polypeptide comprising a T cell receptor (TCR) alpha constant domain (Cot) and a second polypeptide comprising a TCR beta constant domain (CP), wherein: the TCR Ca comprises at least one of the following residues: asparagine at position 134, arginine, glutamate or serine at position 156, serine or tyrosine at position 169, leucine at position 175, leucine or tryptophane at position 178, serine at position 190, or threonine at position 199 (residues numbered according to Kabat numbering); and / or the TCR C|3 comprises at least one of the following residues: threonine at position 128, tryptophan at position 150, or histidine at position 184 (residues numbered according to Kabat numbering).

[0062] The numbering of the amino acid residues in the TCR Ca and CP domains used herein follows the Kabat numbering system (Kabat, et al. Sequences of Immunological Interest Vol. 1 Fifth Edition 1991 US Department of Health and Human Services, Public Health Service, NIH).

[0063] The TCR Ca residues recited above correspond to the following residues in the TCR Ca domain (based on Kabat numbering):

[0064] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSD

[0065] FACANAFNNSIIPEDTFFPSPESSC (SEQ ID NO:1); Position 134 of Kabat numbering corresponds to position 15 of SEQ ID NO: 1; position 156 of Kabat numbering corresponds to position 37 of SEQ ID NO: 1; position 169 of Kabat numbering corresponds to position 50 of SEQ ID NO: 1; position 175 of Kabat numbering corresponds to position 56 of SEQ ID NO: 1; position 178 of Kabat numbering corresponds to position 59 of SEQ ID NO: 1; position 190 of Kabat numbering corresponds to position 71 of SEQ ID NO: 1; and position 199 of Kabat numbering corresponds to position 80 of SEQ ID NO: 1.

[0066] The TCR CP residues recited above correspond to the following residues in the TCR CP domain encoded by the TRBC1 or TRBC2 gene (based on Kabat numbering):

[0067] TRBC1:

[0068] DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALN DSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC (SEQ ID NO:2);

[0069] TRBC2:

[0070] DLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALN DSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC (SEQ ID NO:3);

[0071] Position 128 of Kabat numbering corresponds to position 11 of SEQ ID NO: 2 or 3; position 150 of Kabat numbering corresponds to position 33 of SEQ ID NO: 2 or 3; and position 184 of Kabat numbering corresponds to position 67 of SEQ ID NO: 2 or 3.

[0072] In one aspect, provided herein are proteins comprising: a first polypeptide comprising a TCR Ca comprising at least one of the following residues: asparagine at position 134, arginine, glutamate or serine at position 156, serine or tyrosine at position 169, leucine at position 175, leucine or tryptophane at position 178, serine at position 190, or threonine at position 199 (residues numbered according to Kabat numbering); and / or a second polypeptide comprising a TCR C|3 comprising at least one of the following residues: threonine at position 128, tryptophan at position 150, or histidine at position 184 (residues numbered according to Kabat numbering). These mutations were identified by the inventors to result at least in improved expression of TCRs; see e.g. Fig. 3b.

[0073] In another aspect, provided herein are proteins comprising: a first polypeptide comprising a TCR Ca comprising at least one of the following residues: glutamate at position 156, tyrosine at position 169, leucine or tryptophane at position 178, or serine at position 190 (residues numbered according to Kabat numbering); and / or a second polypeptide comprising a TCR C comprising at least one of the following residues: threonine at position 128, or histidine at position 184 (residues numbered according to Kabat numbering). It has been shown that these mutations improve the expression of TCRs independently of other mutations; see e.g. Fig. 3b and Table 5.

[0074] In a particular embodiment, the invention relates to the protein according to the invention, wherein the CP domain further comprises a glutamate at position 170 (residues numbered according to Kabat numbering). Position 170 of Kabat numbering corresponds to position 53 of SEQ ID NO:2 or 3.

[0075] The TCR|3 S170E mutation was previously reported by Froning et al. (Nat. Commun. 11, 2330 (2020)) and the beneficial effect of this mutation on TCR stability was confirmed in the experimental examples. Therefore, this mutation may be combined with one or more of the additional mutations disclosed herein above.

[0076] In a particular embodiment, the invention relates to the protein according to the invention, wherein the first polypeptide comprises a Cot domain comprising the following residues: tryptophane at position 178 and serine at position 190 and, optionally, threonine at position 199 (residues numbered according to Kabat numbering); and / or the second polypeptide comprises a C domain comprising the following residues: tryptophan at position 150 and glutamate at position 170 (residues numbered according to Kabat numbering). These combinations of mutations were shown to improve the expression and / or the thermal stability of TCRs; see e.g., Fig.3b and Tables 5 and 6.

[0077] In a particular embodiment, the invention relates to the protein according to the invention, wherein the first polypeptide comprises a Ca domain comprising at least one of the following residues: leucine or tryptophane at position 178, and / or serine at position 190 (residues numbered according to Kabat numbering); and / or the second polypeptide comprises a CP domain comprising at least one of the following residues: glutamate at position 170, and / or histidine at position 184 (residues numbered according to Kabat numbering). These mutations were shown to have a particularly high impact on expression and / or stability of TCRs; see e.g., Fig.3b and Tables 5 and 6.

[0078] In a particular embodiment, the invention relates to the protein according to the invention, wherein the first polypeptide comprises a Ca domain comprising a leucine at position 178, and a CP domain comprising a glutamate at position 170 (also referred to as Var4 herein, residues numbered according to Kabat numbering); or wherein the first polypeptide comprises a Ca domain comprising a tryptophan at position 178 and a serine at position 190, and a CP domain comprising a glutamate at position 170 (also referred to as Var3 herein, residues numbered according to Kabat numbering); or wherein the first polypeptide comprises a Ca domain comprising a tryptophan at position 178, and a CP domain comprising a glutamate at position 170 (also referred to as Varl2 herein, residues numbered according to Kabat numbering); or wherein the first polypeptide comprises a Ca domain comprising a tryptophan at position 178, and a CP domain comprising a histidine at position 184 (also referred to as Varl4 herein, residues numbered according to Kabat numbering); or wherein the first polypeptide comprises a Ca domain comprising a tryptophan at position 178 and a serine at position 190, and a CP domain (also referred to as Vari herein, residues numbered according to Kabat numbering); or wherein the first polypeptide comprises a Ca domain comprising a serine at position 190, and a CP domain comprising a glutamate at position 170 (also referred to as #46 herein, residues numbered according to Kabat numbering).

[0079] These combinations of mutations were shown to result in particularly high expressibility and / or stability of TCRs; see e.g., Fig.3b; Fig.7 and Tables 5, 6 and 8.

[0080] In certain embodiments, the protein of the invention comprises a TCR Ca domain based on SEQ ID NO: 1 and a TCR CP domain based on SEQ ID NO: 2 or 3, wherein the TCR Ca domain and / or the TCR CP domain comprise one or more of the mutations disclosed herein.

[0081] In certain embodiments, the protein of the invention comprises a TCR Ca domain based on SEQ ID NO: 1 and a TCR CP domain based on SEQ ID NO: 4, wherein the TCR Ca domain and / or the TCR CP domain comprise one or more of the mutations disclosed herein.

[0082] TRBCx:

[0083] DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALN DSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC (SEQ ID NO:4)

[0084] In certain embodiments, the protein according to the invention comprises a first polypeptide comprising a Ca domain comprising or consisting of the amino acid sequence:

[0085] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSWSAVAWSNKS DFSCANAFNNSIIPEDTFFPSPESSC (SEQ ID NO:5); and a second polypeptide comprising a CP domain comprising or consisting of the amino acid sequence: DLKNVFPPEVAVFEPSEAEISHTQKATLVCLAEGFYPDHVELSWWVNGKEVHEGVCTDPQPLKEQPALN DSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC (SEQ ID NO:6).

[0086] In certain embodiments, the protein according to the invention comprises a first polypeptide comprising a Ca domain comprising or consisting of the amino acid sequence:

[0087] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSLSAVAWSNKSD FACANAFNNSIIPEDTFFPSPESSC (SEQ ID NO:7); and a second polypeptide comprising a CP domain comprising or consisting of the amino acid sequence:

[0088] DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHEGVCTDPQPLKEQPALN DSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC (SEQ ID NO:8).

[0089] In certain embodiments, the protein according to the invention comprises a first polypeptide comprising a Ca domain comprising or consisting of the amino acid sequence:

[0090] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSWSAVAWSNKS DFACANAFNNSIIPEDTFFPSPESSC (SEQ ID NO:9); and a second polypeptide comprising a CP domain comprising or consisting of the amino acid sequence:

[0091] DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHEGVCTDPQPLKEQPALN DSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC (SEQ ID NQ:10).

[0092] In certain embodiments, the protein according to the invention comprises a first polypeptide comprising a Ca domain comprising or consisting of the amino acid sequence:

[0093] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSWSAVAWSNKS DFACANAFNNSIIPEDTFFPSPESSC (SEQ ID NO:11); and a second polypeptide comprising a CP domain comprising or consisting of the amino acid sequence: DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPHLN DSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC (SEQ ID N0:12).

[0094] In certain embodiments, the protein according to the invention comprises a first polypeptide comprising a Ca domain comprising or consisting of the amino acid sequence:

[0095] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSWSAVAWSNKS DFSCANAFNNSIIPEDTFFPSPESSC (SEQ ID NO:5); and a second polypeptide comprising a CP domain comprising or consisting of the amino acid sequence:

[0096] DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALN DSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC (SEQ ID NO:38).

[0097] In certain embodiments, the protein according to the invention comprises a first polypeptide comprising a Ca domain comprising or consisting of the amino acid sequence:

[0098] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSD FSCANAFNNSIIPEDTFFPSPESSC (SEQ ID NO:39); and a second polypeptide comprising a CP domain comprising or consisting of the amino acid sequence:

[0099] DLKNVFPPEVAVFEPSEAEISHTQKATLVCLAEGFYPDHVELSWWVNGKEVHEGVCTDPQPLKEQPALN DSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC (SEQ ID NO:6).

[0100] In certain embodiments, the protein according to the invention comprises any of the mutations or combinations of mutations in the TCR Ca and / or CP domain provided in Table 1 or 2.

[0101] Table 1:

[0102] Table 2: The mutations disclosed herein have in common that they contribute to the thermal stability of TCRs. Thermal stability of a protein, or more specifically a TCR, may be quantified in different ways. Most commonly, the melting temperature (Tm) may be used to quantify the thermal stability of a protein. The term melting temperature (Tm) refers to the temperature at which 50% of a protein is unfolded.

[0103] Alternatively or in addition, the melting temperature onset (Tonset) may be used to quantify the thermal stability of a protein. The melting temperature onset (Tonset) refers to the temperature at which unfolding of the protein begins. Moreover, the aggregation temperature (Tagg) may be used to quantify the thermal stability of a protein. The aggregation temperature (Tagg) refers to the temperature at which aggregation of the protein begins. The skilled person is aware of methods that may be used to determine the thermal stability (including Tm, Tonset and Tagg) of a protein. For example, melting and aggregation temperatures may be determined by differential scanning fluorometry (DSF) or nanoDSF, for example and without limitation, on an UNcle instrument (Unchained Labs). Therefore, TCR variants may be diluted to a concentration of 0.5 mg / mL in PBS at pH 7.5 and loaded in triplicates in a Uni. Sealed Unis may be loaded into the instrument and analysed using the Tm & Tagg with optional DLS method. Full spectra may be collected from 250—720 nm, and a thermal ramp from 25 - 95 °C with a ramp rate of 0.6 °C / minute may be used. Melting temperature (Tm), unfolding onset (Tonset) and aggregation onset (Tagg) may be calculated using the UNcle analysis software. For Tm and Tonset, the barycentric mean (BCM) from 300 to 430 nm may be plotted against the temperature and the values may be calculated from the first derivative of the curve. Tagg values may be calculated from the static light scattering (SLS) signal at 266 nm, as the temperature at which a 5% signal increase is observed.

[0104] When determining the thermal stability of the protein of the invention, it is to be understood that the thermal stability of the entire protein, potentially comprising further non-TCR domains, may be determined, or that only the thermal stability of the TCR domain may be determined. In certain embodiments, thermal stability measurements are performed with a purified TCR fragment, preferably a TCR fragment comprising an alpha chain that is truncated at cysteine residue 213 of Ca (position 94 in SEQ ID NO:1) and a beta chain that is truncated at cysteine residue 247 of CP (position 130 in SEQ ID NO:2-4) (residues numbered according to Kabat numbering).

[0105] In certain embodiments, the invention relates to proteins comprising mutated TCR Ca / cp domains having a higher thermal stability than their unmutated counterparts.

[0106] Accordingly, in a particular embodiment, the invention relates to the protein according to the invention, wherein the protein has increased thermal stability compared to a protein having the same amino acid sequence except that: the TCR Ca comprises lysine at position 134, lysine at position 156, aspartate at position 169, phenylalanine at position 175, asparagine at position 178, alanine at position 190, and isoleucine at position 199, and the TCR CP comprises alanine at position 128, threonine at position 150, and alanine at position 184 (residues numbered according to Kabat numbering).

[0107] In certain embodiments, the protein according to the invention comprises a first polypeptide comprising a TCR Ca and second polypeptide comprising a TCR CP, wherein the TCR Ca comprises at least one of the following residues: asparagine at position 134, arginine, glutamate or serine at position 156, serine or tyrosine at position 169, leucine at position 175, leucine or tryptophane at position 178, serine at position 190, or threonine at position 199 (residues numbered according to Kabat numbering); and / or the TCR CP comprises at least one of the following residues: threonine at position 128, tryptophan at position 150, or histidine at position 184 (residues numbered according to Kabat numbering), wherein the protein has increased thermal stability compared to a protein having the same amino acid sequence except that: the TCR Ca comprises lysine at position 134, lysine at position 156, aspartate at position 169, phenylalanine at position 175, asparagine at position 178, alanine at position 190, and isoleucine at position 199, and the TCR CP comprises alanine at position 128, threonine at position 150, and alanine at position 184 (residues numbered according to Kabat numbering).

[0108] In certain embodiments, the protein according to the invention comprises a first polypeptide comprising a TCR Ca and second polypeptide comprising a TCR CP, wherein a) the TCR CP comprises a glutamate at position 170 (residues numbered according to Kabat numbering); and b) the TCR Ca comprises at least one of the following residues: asparagine at position 134, arginine, glutamate or serine at position 156, serine or tyrosine at position 169, leucine at position 175, leucine or tryptophane at position 178, serine at position 190, or threonine at position 199 (residues numbered according to Kabat numbering); and / or the TCR CP comprises at least one of the following residues: threonine at position 128, tryptophan at position 150, or histidine at position 184 (residues numbered according to Kabat numbering); wherein the protein has increased thermal stability compared to a protein having the same amino acid sequence except that: the TCR Ca comprises lysine at position 134, lysine at position 156, aspartate at position 169, phenylalanine at position 175, asparagine at position 178, alanine at position 190, and isoleucine at position 199, and the TCR CP comprises alanine at position 128, threonine at position 150, serine at position 170 and alanine at position 184 (residues numbered according to Kabat numbering).

[0109] In certain embodiments, the TCR portion of the protein according to the invention, i.e., the portion comprising a first polypeptide comprising Ca and Va and a second polypeptide comprising CP and VP, has a melting temperature (Tm) of at least 55°C, at least 56°C, at least 57°C, at least 58°C, at least 59°C, at least 60°C, at least 61°C, at least 62°C.

[0110] Thus, in a particular embodiment, the invention relates to the protein of the invention, wherein the first polypeptide comprising the TCR Ca and the second polypeptide comprising the TCR CP form a quaternary structure having a melting temperature (Tm) of at least 55°C, at least 56°C, at least 57°C, at least 58°C, at least 59°C, at least 60°C, at least 61°C, at least 62°C, preferably wherein the Tm is determined by differential scanning fluorometry (DSF) as described herein.

[0111] In certain embodiments, the protein according to the invention comprises a first polypeptide comprising a TCR Ca and second polypeptide comprising a TCR CP, wherein the TCR Ca comprises at least one of the following residues: asparagine at position 134, arginine, glutamate or serine at position 156, serine or tyrosine at position 169, leucine at position 175, leucine or tryptophane at position 178, serine at position 190, or threonine at position 199 (residues numbered according to Kabat numbering); and / or the TCR CP comprises at least one of the following residues: threonine at position 128, tryptophan at position 150, or histidine at position 184 (residues numbered according to Kabat numbering), wherein the first polypeptide comprising the TCR Ca and the second polypeptide comprising the TCR C|3 form a quaternary structure having a melting temperature (Tm) of at least 55°C, at least 56°C, at least 57°C, at least 58°C, at least 59°C, at least 60°C, at least 61°C, at least 62°C, preferably wherein the Tm is determined by differential scanning fluorometry (DSF) as described herein.

[0112] In certain embodiments, the protein according to the invention comprises a first polypeptide comprising a TCR Ca and second polypeptide comprising a TCR CP, wherein a) the TCR CP comprises a glutamate at position 170 (residues numbered according to Kabat numbering); and b) the TCR Ca comprises at least one of the following residues: asparagine at position 134, arginine, glutamate or serine at position 156, serine or tyrosine at position 169, leucine at position 175, leucine or tryptophane at position 178, serine at position 190, or threonine at position 199 (residues numbered according to Kabat numbering); and / or the TCR CP comprises at least one of the following residues: threonine at position 128, tryptophan at position 150, or histidine at position 184 (residues numbered according to Kabat numbering); wherein the first polypeptide comprising the TCR Ca and the second polypeptide comprising the TCR CP form a quaternary structure having a melting temperature (Tm) of at least 55°C, at least 56°C, at least 57°C, at least 58°C, at least 59°C, at least 60°C, at least 61°C, at least 62°C, preferably wherein the Tm is determined by differential scanning fluorometry (DSF) as described herein.

[0113] In certain embodiments, the TCR portion of the protein according to the invention, i.e., the portion comprising a first polypeptide comprising Ca and Va and a second polypeptide comprising CP and VP, has a melting temperature onset (Tonset) of at least 49°C, at least 50°C, at least 51°C, at least 52°C, at least 53°C, at least 54°C, or at least 55°C.

[0114] That is, in certain embodiments, the invention relates to the protein according to the invention, wherein the first polypeptide comprising the TCR Ca and the second polypeptide comprising the TCR CP form a quaternary structure having a melting temperature onset (Tonset) of at least 49°C, at least 50°C, at least 51°C, at least 52°C, at least 53°C, at least 54°C, or at least 55°C, preferably wherein the Tonset is determined by differential scanning fluorometry (DSF) as described herein.

[0115] In certain embodiments, the protein according to the invention comprises a first polypeptide comprising a TCR Ca and second polypeptide comprising a TCR CP, wherein the TCR Ca comprises at least one of the following residues: asparagine at position 134, arginine, glutamate or serine at position 156, serine or tyrosine at position 169, leucine at position 175, leucine or tryptophane at position 178, serine at position 190, or threonine at position 199 (residues numbered according to Kabat numbering); and / or the TCR CP comprises at least one of the following residues: threonine at position 128, tryptophan at position 150, or histidine at position 184 (residues numbered according to Kabat numbering), wherein the first polypeptide comprising the TCR Ca and the second polypeptide comprising the TCR CP form a quaternary structure having a melting temperature onset (Tonset) of at least 49°C, at least 50°C, at least 51°C, at least 52°C, at least 53°C, at least 54°C, or at least 55°C, preferably wherein the Tonset is determined by differential scanning fluorometry (DSF) as described herein.

[0116] In certain embodiments, the protein according to the invention comprises a first polypeptide comprising a TCR Ca and second polypeptide comprising a TCR CP, wherein a) the TCR CP comprises a glutamate at position 170 (residues numbered according to Kabat numbering); and b) the TCR Ca comprises at least one of the following residues: asparagine at position 134, arginine, glutamate or serine at position 156, serine or tyrosine at position 169, leucine at position 175, leucine or tryptophane at position 178, serine at position 190, or threonine at position 199 (residues numbered according to Kabat numbering); and / or the TCR CP comprises at least one of the following residues: threonine at position 128, tryptophan at position 150, or histidine at position 184 (residues numbered according to Kabat numbering); wherein the first polypeptide comprising the TCR Ca and the second polypeptide comprising the TCR CP form a quaternary structure having a melting temperature onset (Tonset) of at least 49°C, at least 50°C, at least 51°C, at least 52°C, at least 53°C, at least 54°C, or at least 55°C, preferably wherein the Tonset is determined by differential scanning fluorometry (DSF) as described herein.

[0117] In certain embodiments, the invention relates to the protein of the invention, wherein the first polypeptide comprising the TCR Ca and the second polypeptide comprising the TCR CP form a quaternary structure having a melting temperature (Tm) of at least 60°C and / or a melting temperature onset (Tonset) of at least 52°C, preferably wherein the Tm and Tonset are determined by differential scanning fluorometry (DSF) as described herein.

[0118] In certain embodiments, the protein according to the invention comprises a first polypeptide comprising a TCR Ca and second polypeptide comprising a TCR CP, wherein the TCR Ca comprises at least one of the following residues: asparagine at position 134, arginine, glutamate or serine at position 156, serine or tyrosine at position 169, leucine at position 175, leucine or tryptophane at position 178, serine at position 190, or threonine at position 199 (residues numbered according to Kabat numbering); and / or the TCR CP comprises at least one of the following residues: threonine at position 128, tryptophan at position 150, or histidine at position 184 (residues numbered according to Kabat numbering), wherein the first polypeptide comprising the TCR Ca and the second polypeptide comprising the TCR CP form a quaternary structure having a melting temperature (Tm) of at least 60°C and / or a melting temperature onset (Tonset) of at least 52°C, preferably wherein the Tm and Tonset are determined by differential scanning fluorometry (DSF) as described herein.

[0119] In certain embodiments, the protein according to the invention comprises a first polypeptide comprising a TCR Ca and second polypeptide comprising a TCR CP, wherein a) the TCR C p comprises a glutamate at position 170 (residues numbered according to Kabat numbering); and b) the TCR Ca comprises at least one of the following residues: asparagine at position 134, arginine, glutamate or serine at position 156, serine or tyrosine at position 169, leucine at position 175, leucine or tryptophane at position 178, serine at position 190, or threonine at position 199 (residues numbered according to Kabat numbering); and / or the TCR CP comprises at least one of the following residues: threonine at position 128, tryptophan at position 150, or histidine at position 184 (residues numbered according to Kabat numbering); wherein the first polypeptide comprising the TCR Ca and the second polypeptide comprising the TCR C|3 form a quaternary structure having a melting temperature (Tm) of at least 60°C and / or a melting temperature onset (Tonset) of at least 52°C, preferably wherein the Tm and Tonset are determined by differential scanning fluorometry (DSF) as described herein. In certain embodiments, the TCR portion of the protein according to the invention, i.e., the portion comprising a first polypeptide comprising Cot and Va and a second polypeptide comprising CP and VP, has an aggregation temperature (Tagg) of at least 51°C, at least 52°C, at least 53°C, at least 54°C, at least 55°C, at least 56°C, or at least 57°C.

[0120] That is, in certain embodiments, the invention relates to the protein of the invention, wherein the first polypeptide comprising the TCR Cot and the second polypeptide comprising the TCR CP form a quaternary structure having an aggregation temperature (Tagg) of at least 51°C, at least 52°C, at least 53°C, at least 54°C, at least 55°C, at least 56°C, or at least 57°C, preferably wherein the Tagg determined by differential scanning fluorometry (DSF) and / or static light scattering (SLS) as described herein.

[0121] In certain embodiments, the protein according to the invention comprises a first polypeptide comprising a TCR Cot and second polypeptide comprising a TCR CP, wherein the TCR Ca comprises at least one of the following residues: asparagine at position 134, arginine, glutamate or serine at position 156, serine or tyrosine at position 169, leucine at position 175, leucine or tryptophane at position 178, serine at position 190, or threonine at position 199 (residues numbered according to Kabat numbering); and / or the TCR CP comprises at least one of the following residues: threonine at position 128, tryptophan at position 150, or histidine at position 184 (residues numbered according to Kabat numbering), wherein the first polypeptide comprising the TCR Ca and the second polypeptide comprising the TCR CP form a quaternary structure having an aggregation temperature (Tagg) of at least 51°C, at least 52°C, at least 53°C, at least 54°C, at least 55°C, at least 56°C, or at least 57°C, preferably wherein the Tagg determined by differential scanning fluorometry (DSF) and / or static light scattering (SLS)as described herein.

[0122] In certain embodiments, the protein according to the invention comprises a first polypeptide comprising a TCR Ca and second polypeptide comprising a TCR CP, wherein a) the TCR CP comprises a glutamate at position 170 (residues numbered according to Kabat numbering); and b) the TCR Ca comprises at least one of the following residues: asparagine at position 134, arginine, glutamate or serine at position 156, serine or tyrosine at position 169, leucine at position 175, leucine or tryptophane at position 178, serine at position 190, or threonine at position 199 (residues numbered according to Kabat numbering); and / or the TCR CP comprises at least one of the following residues: threonine at position 128, tryptophan at position 150, or histidine at position 184 (residues numbered according to Kabat numbering); wherein the first polypeptide comprising the TCR Cot and the second polypeptide comprising the TCR C|3 form a quaternary structure having an aggregation temperature (Tagg) of at least 51°C, at least 52°C, at least 53°C, at least 54°C, at least 55°C, at least 56°C, or at least 57°C, preferably wherein the Tagg determined by differential scanning fluorometry (DSF) and / or static light scattering (SLS)as described herein.

[0123] Alternatively or in addition, the mutations disclosed herein contribute to the expressibility of TCRs. Expression of TCRs may be quantified directly in the culture medium (pre-purification) or after a purification step (post-purification). For that, TCRa and TCR chains may be expressed from a single plasmid separated by a P2A peptide, containing a 6x His tag at the N- terminus of the TCRP chain for purification. Suspension-adapted HEK293 cells (Expi293, Thermo Fisher) may be transfected with the TCR plasmids and secreted soluble TCRs may be purified from culture supernatants using a HisTrap excel column (5 mL, Cytiva, #17371206 on an Akta pure system (Cytiva) with a 20 mM sodium phosphate, 0.5 M NaCI and 25 mM imidazole wash buffer and a single step elution with 20 mM sodium phosphate, 0.5 M NaCI, 500 mM imidazole elution buffer. Purified TCRs may be buffer exchanged into PBS pH 7.5. Protein concentration in the culture supernatant (pre-purification) or in the eluted fraction with methods known in the art.

[0124] In certain embodiments, the invention relates to the protein of the invention, wherein the protein has an increased expression level when expressed under the same conditions compared to a protein having the same amino acid sequence except that: the TCR Ca comprises lysine at position 134, lysine at position 156, aspartate at position 169, phenylalanine at position 175, asparagine at position 178, alanine at position 190, and isoleucine at position 199, and the TCR Cp comprises alanine at position 128, threonine at position 150, and alanine at position 184 (residues numbered according to Kabat numbering).

[0125] In certain embodiments, the protein according to the invention comprises a first polypeptide comprising a TCR Ca and second polypeptide comprising a TCR CP, wherein the TCR Ca comprises at least one of the following residues: asparagine at position 134, arginine, glutamate or serine at position 156, serine or tyrosine at position 169, leucine at position 175, leucine or tryptophane at position 178, serine at position 190, or threonine at position 199 (residues numbered according to Kabat numbering); and / or the TCR C comprises at least one of the following residues: threonine at position 128, tryptophan at position 150, or histidine at position 184 (residues numbered according to Kabat numbering), wherein the protein has increased expression level when expressed under the same conditions compared to a protein having the same amino acid sequence except that: the TCR Ca comprises lysine at position 134, lysine at position 156, aspartate at position 169, phenylalanine at position 175, asparagine at position 178, alanine at position 190, and isoleucine at position 199, and the TCR CP comprises alanine at position 128, threonine at position 150, and alanine at position 184 (residues numbered according to Kabat numbering).

[0126] In certain embodiments, the protein according to the invention comprises a first polypeptide comprising a TCR Ca and second polypeptide comprising a TCR CP, wherein a) the TCR CP comprises a glutamate at position 170 (residues numbered according to Kabat numbering); and b) the TCR Ca comprises at least one of the following residues: asparagine at position 134, arginine, glutamate or serine at position 156, serine or tyrosine at position 169, leucine at position 175, leucine or tryptophane at position 178, serine at position 190, or threonine at position 199 (residues numbered according to Kabat numbering); and / or the TCR CP comprises at least one of the following residues: threonine at position 128, tryptophan at position 150, or histidine at position 184 (residues numbered according to Kabat numbering); wherein the protein has increased expression level when expressed under the same conditions compared to a protein comprising the same amino acid sequence except that: the TCR Ca comprises lysine at position 134, lysine at position 156, aspartate at position 169, phenylalanine at position 175, asparagine at position 178, alanine at position 190, and isoleucine at position 199, and the TCR CP comprises alanine at position 128, threonine at position 150, serine at position 170 and alanine at position 184 (residues numbered according to Kabat numbering).

[0127] In certain embodiments, the protein according to the invention comprises not more than 5, not more than 4, not more than 3 or not more than 2 of the mutations in the TCR Ca and / or CP disclosed herein.

[0128] That is, in certain embodiments, the protein according to the invention comprises not more than 4, not more than 3 or not more than 2 of the mutations aK134N, aK156R, aK156E, aK156S, aD169S, aD169Y, aF175L, aN178L, aN178W, aA190S, all99T, PA128T, PT150W, and PA184H (residues numbered according to Kabat numbering), preferably wherein the protein has increased thermal stability or increased expression levels when expressed under the same conditions compared to a reference protein not comprising the respective mutations, i.e., compared to a protein comprising the same amino acid sequence except that: the TCR Ca comprises lysine at position 134, lysine at position 156, aspartate at position 169, phenylalanine at position 175, asparagine at position 178, alanine at position 190, and isoleucine at position 199, and the TCR CP comprises alanine at position 128, threonine at position 150, serine at position 170 and alanine at position 184 (residues numbered according to Kabat numbering). In certain embodiments, the protein according to the invention comprises the mutation PS170E and not more than 3, not more than 2 or not more than 1 of the mutations aK134N, aK156R, aK156E, aK156S, aD169S, aD169Y, aF175L, aN178L, aN178W, aA190S, all99T, PA128T, PT150W, and PA184H (residues numbered according to Kabat numbering), preferably wherein the protein has increased thermal stability or increased expression levels when expressed under the same conditions compared to a reference protein not comprising the respective mutations.

[0129] The protein of the invention may comprise one or more TCR fragments through which the protein of the invention can engage with cognate peptide-MHC (pMHC) ligands. As used herein, the term " T cell receptor fragment" preferably refers to heterodimeric truncated variants of native TCRs comprising extracellular portions of the TCR a-chain and p-chain linked by one or more disulfide bonds, wherein the TCR a-chain and p-chain comprise one or more of the Ca and / or CP mutations disclosed herein. Preferably, the TCR fragment lacks the transmembrane and cytosolic domains of the native TCR.

[0130] The terms " T cell receptor a-chain sequence and T-cell receptor p-chain sequence" refer to TCR a-chain and p-chain sequences that preferably lack the transmembrane and cytosolic domains. The sequence (amino acid or nucleic acid) of the TCR a-chain and P-chains may be identical to the corresponding sequences in a native TCR or may comprise variant TCR a-chain and P-chain sequences, as compared to the corresponding native TCR sequences.

[0131] The term " T cell receptor" as used herein encompasses soluble TCRs with variant or nonvariant soluble TCR a-chain and P-chain sequences. The variations may be in the variable or constant regions of the TCR a-chain and P-chain sequences and can include, but are not limited to, amino acid deletion, insertion, substitution mutations as well as changes to the nucleic acid sequence, which do not alter the amino acid sequence. TCRs of the invention preferably retain the binding functionality of their parent molecules.

[0132] Within the present invention, reference is made to the first and second polypeptide chain comprised in the TCR fragment. In certain embodiments, the first polypeptide chain comprised in the TCR fragment is derived from the TCR a-chain and the second polypeptide chain comprised in the TCR fragment is derived from the TCR P-chain, or vice versa.

[0133] The TCR fragment may have been derived from any TCR. However, it is preferred herein that the TCR fragment has been derived from a genetically engineered TCR. Thus, in a particular embodiment, the invention relates to the protein according to the invention, wherein the TCR fragment has been engineered for increased affinity, avidity, specificity and / or stability. T1

[0134] That is, in certain embodiments, the TCR fragment has been engineered for increased affinity, (functional / structural) avidity, specificity and / or stability.

[0135] The term "affinity" as used herein means the strength of a single interaction between, for example, a peptide (presented in the context of an MHC) and a TCR. Affinity is measured in a cell-free context, for example, where the MHC-presented peptide is immobilized on a solid interface and the TCR of interest is in solution.

[0136] The term "avidity" as used herein refers to a measure of multiple affinities and reflects the overall binding strength or the observed functional response between a target peptide-MHC of interest and a TCR. In this context, the peptide-MHC and the TCR can be presented on the surface of a cell or may be provided as soluble binding reagents (e.g., monomers or multimers). Preferably, the TCR is presented on the surface of the cell according to the invention, and the peptide-MHC target is in the form of a soluble reagent (i.e., peptide-MHC multimers) or presented by an APC.

[0137] The term "functional avidity" as used herein is the concentration of an antigen (e.g. peptide) required to achieve 50% of maximal response in a functional assay. For each functional assay, the maximal response is determined. Functions measured include for example antigen- induced signaling as described in the invention, cytokine secretion and lysis of target cells (i.e., cytotoxicity). The maximal response is obtained when T cells are maximally stimulated. Therefore, maximal responses depend on the functional capabilities of given T cell(s) and can for example be expressed as EC50 in pM or other molar units.

[0138] The term "structural avidity" or "binding avidity" as used herein, which can be expressed as EC50 in pg / mL or other concentration units, is the concentration of an antigen (e.g. a peptide bound by an MHC multimer) required to achieve half-maximal antigen staining (e.g. multimer staining). Functional and structural avidity can for example be calculated with software known in the art such as GraphPad prism 6.

[0139] Functional avidity can be measured for example using an enzyme-linked immunospot (ELISpot) assay and measuring, for example, interferon-gamma (I FNy) and / or IL-2 production and / or a combination thereof. Structural avidity can be measured by staining TCR expressing cells, optionally T cells or the cells according to the invention expressing a TCR, using graded concentrations of peptide-MHC multimers. In some embodiments, avidity may be assessed by flow cytometric analysis for specific TCR binding to fluorochrome-labeled multimeric synthetic peptide-MHC complexes, and / or functional assessment with peptide-pulsed APCs and screening for IFN-gamma production using standard enzyme-linked immunosorbent assay (ELISA) or ELISpot assays. In some embodiments, T cell avidity may be detected via a dual parameter cell sorting protocol that detects cells bound to the fluorescently labeled multimer in conjunction with, indicating that the cell was activated by the recognition of the TCR- multimer complex.

[0140] In certain embodiments, the TCR fragment has been engineered to exhibit increased specificity for a desired pMHC ligand. Increased specificity for a desired pMHC ligand may be achieved by increasing the affinity / avidity for said pMHC ligand and / or by reducing the affinity / avidity for an off-target ligand.

[0141] Methods for increasing the affinity, avidity and / or specificity of a TCR have been described in detail in WO 2021 / 074249, which is incorporated by reference herein.

[0142] In a particular embodiment, the invention relates to the protein according to the invention, wherein the first polypeptide is linked to the second polypeptide by an inter-chain disulfide bond.

[0143] That is, in some embodiments, the first polypeptide is linked to the second polypeptide by one or more inter-chain disulfide bonds. Disulfide bonds can be formed by pairs of engineered cysteine residues in the TCR a and chains, and such disulfide bonds link the TCR a and 0 chains together (see WO 2003 / 020763, WO 2004 / 033685, WO 2004 / 074322, Li, et ah, Nat. Biotechnol. 2005, 23(3): 349-354; Boulter, et al., Protein Eng. 2003, 16(9): 707-11).

[0144] In certain embodiments, the TCR Ca domain and the TCR C0 domain comprised in the protein of the invention are linked by an inter-chain disulfide bond formed between two native cysteine residues. Thus, in a particular embodiment, the invention relates to the protein of the invention, wherein the first and second polypeptide are linked by an inter-chain disulfide bond between a cysteine residue at position 213 of the TCR Ca domain and a cysteine residue at position 247 of the TCR C0 domain (residues numbered according to Kabat numbering).

[0145] In some embodiments, the Ca domain further comprises a cysteine residue at position 166 (residue numbered according to Kabat numbering; corresponding to position 47 of SEQ ID NO:1), and the C0 domain further comprises a cysteine residue at position 173 (residue numbered according to Kabat numbering; corresponding to position 56 of SEQ ID NO:2-4), wherein the first polypeptide and the second polypeptide are linked by an inter-chain disulfide bond between the cysteine residue at position 166 of Ca and the cysteine residue at position 173 of C0. In a preferred embodiment, the first and second polypeptide chains of the protein according to the invention are linked together by an artificial inter-chain disulfide bond between the cysteine residue at position 166 of Ca and the cysteine residue at position 173 of CP and by a naturally occurring inter-chain disulfide bond between the cysteine residue at position 213 of Ca and the cysteine residue at position 247 of C .

[0146] In certain embodiments, the protein of the invention only comprises a single inter-chain disulfide bond formed between the cysteine residue at position 166 of Ca and the cysteine residue at position 173 of Cp. In such embodiments, it is preferred that the C-terminal ends of the TCR alpha and beta constant domains are truncated such that the native inter-chain disulfide bond between the cysteine residue at position 213 of Ca and the cysteine residue at position 247 of CP can no longer be formed.

[0147] Accordingly, in certain embodiments, the protein according to the invention may comprise a TCR Ca domain based on SEQ ID NO:13:

[0148] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSD FACANAFNNSIIPEDT (SEQ ID NO:13); and a TCR CP domain based on any one of SEQ ID NOs: 14-16:

[0149] DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALN DSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD (SEQ ID NO:14);

[0150] DLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALN DSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD (SEQ ID NO:15);

[0151] DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALN DSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD (SEQ ID NO:16); wherein the TCR Ca domain and / or the TCR cp domain comprise one or more of the mutations disclosed herein.

[0152] In some embodiments, the first polypeptide further comprises a TCR a chain variable domain (Va); and the second polypeptide further comprises a TCR p chain variable domain (VP), wherein the Va and VP form an antigen binding domain that binds an antigen, e.g., a tumor antigen, a tissue-specific antigen or a viral antigen. In some embodiments, the Va is fused to the N-terminus of Ca, forming a Va-Ca domain; and the VP is fused to the N-terminus of CP, forming a V -C domain.

[0153] The TCR variable regions (Va / vp) may bind any tumor or viral antigen, including but not limited to, a viral antigen, a neoantigen (the antigens expressed only in cancer cells but not in normal cells), a tumor-associated antigen (the processed fragments of proteins that are expressed at low levels in normal cells, but are overexpressed in cancer cells), or a cancer / testis (CT) antigen (derived from proteins usually only expressed by reproductive tissues, e.g. testes, fetal ovaries, and placenta, and have limited / no expression in all other adult tissues) (see Pritchard, et al., BioDrugs, 2018, 32:99-109).

[0154] In some embodiments, the TCR variable region (Va / vp) binds an antigen selected from any one of the following: ERBB2, CD19, NY-ESO-1, MAGE (e.g., MAGE-A1, A2, A3, A4, A6, A10, A12), gplOO, MART-l / Melan-A, gp75 / TRP-l, TRP-2, Tyrosinase, BAGE, CAMEL, SSX-2, p- Catenin, Caspase-8, CDK4, MUM-2 (TRAPPCI), MUM-3, MART-2, OS-9, pl4ARF (CDKN2A), GAS7, GAPDH, SIRT2, GPNMB, SNRP116, RBAF600, SNRPD1, PRDX5, CLPP, PPP1R3B, EF2 (see Pritchard, et al., BioDrugs, 2018, 32:99-109; and Wang, et al., Cell Research, 2017, 27:11-37).

[0155] Preferably, the TCR variable regions may bind to an antigenic peptide derived from any one of the antigens MAGE-A3, EBNA-1, GPC3, KRAS proto-oncogene neoantigens, TCF-1, AFP or PSA. That is, in a particular embodiment, the invention relates to the protein according to the invention, wherein the TCR fragment specifically binds to MAGE-A3, EBNA-1, GPC3, KRAS proto-oncogene neoantigens, TCF-1, AFP or PSA.

[0156] In a preferred embodiment, the TCR variable region (Va / vp) binds to MAGE-A3, in particular to the peptide EVDPIGHLY (SEQ ID NO:17; MAGE-A3I68-I76) derived from MAGE-A3. In a particularly preferred embodiment, the TCR variable region (Va / vp) binds to the peptide EVDPIGHLY (SEQ ID NO:17; MAGE-A3I68-I76) in the context of HLA-A*01:01.

[0157] In certain embodiments, the TCR fragment comprised in the protein of the invention has been engineered to exhibit increased affinity, avidity and / or specificity for an antigenic peptide derived from MAGE-A3, preferably the peptide EVDPIGHLY (SEQ ID NO:17). In certain embodiments, the TCR fragment comprised in the protein of the invention is derived from any one of the engineered MAGE-A3 specific TCRs disclosed in WO 2021 / 074249.

[0158] In certain embodiments, the TCR fragment comprised in the protein of the invention may specifically bind to a tissue-specific antigen. "Tissue-specific antigen" as used herein refers to an antigen that is characteristic of a tissue type, including specific tumor tissues. That is, the TCR fragment may specifically bind to a marker that is predominantly expressed by cells that are part of a specific tissue. With that, the TCR fragment may be used to direct the protein of the invention to said tissue. The tissue may be a tissue belonging to a specific organ. For example, the TCR fragment may be used to direct the protein of the invention to a specific organ, such as an organ that is affected by a disease. Accordingly, the tissue-specific antigen may also be an organ-specific antigen. Suitable markers for targeting specific tissues / organs are known in the art and the skilled person is capable of identifying such markers as targets of the TCR fragment.

[0159] In certain embodiments, the protein of the invention comprises two different TCR fragments or more. In certain embodiments, a first TCR fragment specifically binds to a first pMHC ligand comprising an antigenic peptide derived from an antigen and a second TCR fragment specifically binds to a second pMHC ligand comprising another antigenic peptide derived from the same antigen. In certain embodiments, a first TCR fragment specifically binds to a first pMHC ligand comprising an antigenic peptide derived from a first antigen and a second TCR fragment specifically binds to a second pMHC ligand comprising an antigenic peptide derived from a second antigen.

[0160] The protein according to the invention may be a heterodimeric a / p-TCR, preferably a soluble heterodimeric a / p-TCR, comprising one or more of the Ca / Cp mutations disclosed herein.

[0161] In certain embodiments, the protein according to the invention may be a multi-domain protein comprising a soluble TCR fragment comprising one or more of the Ca / cp mutations disclosed herein and at least one further protein-based component.

[0162] In certain embodiments, the protein according to the invention comprises at least a soluble TCR fragment comprising one or more of the Ca / cp mutations disclosed herein and an additional antigen-binding domain.

[0163] In certain embodiments, the protein of the invention comprises a second antigen-binding domain. That is, in certain embodiments, the protein of the invention comprises at least a TCR fragment and a second antigen-binding domain.

[0164] However, the protein of the invention may comprise two or more additional antigen-binding domains in addition to the TCR fragment. That is, in certain embodiments, the protein of the invention comprises, at least, a soluble TCR fragment and two additional antigen-binding domains. In certain embodiments, the protein of the invention comprises, at least, a soluble TCR fragment and three or more additional antigen-binding domains. In embodiments where the protein comprises two or more additional antigen-binding domains, the two or more additional antigen-binding domains may bind to the same or different antigens.

[0165] In certain embodiments, the protein of the invention is a bispecific molecule comprising a TCR fragment binding to a first antigen and a second antigen-binding domain binding to a second antigen.

[0166] In certain embodiments, the protein of the invention is a trispecific molecule comprising a TCR fragment binding to a first antigen, a second antigen-binding domain binding to a second antigen and an additional antigen-binding domain binding to a third antigen.

[0167] In certain embodiments, the protein of the invention is a trispecific molecule comprising a TCR fragment binding to a first antigen and a second antigen-binding domain having crossspecificity for a second antigen and a third antigen.

[0168] In some embodiments, the proteins described herein further comprise a second or additional antigen-binding domain. The term "antigen-binding domain" is to be understood in the broadest sense and refers to a protein or fragment thereof capable of binding an antigen or an epitope. In certain embodiments, the antigen-binding domain is an antibody or a fragment thereof, as defined in more detail herein below. However, the antigen-biding domain may also be a naturally occurring ligand that specifically binds to a receptor, a fragment thereof or an engineered variant thereof.

[0169] In certain embodiments, the second or additional antigen-binding domain binds to an antigen on the T cell surface, e.g., CD3, CD4, orCD8; or an antigen on the NK cell surface, e.g., NKp46. In some embodiments, the second or additional antigen binding domain binds CD3 or NKp46.

[0170] In some embodiments, the second and / or additional antigen binding domain may be an antibody or antibody fragment, e.g., an scFv, Fab, Fab', (Fab')2, single domain antibody, or camelid VHH domain.

[0171] In some embodiments, the second and / or additional antigen binding domain is a Fab. In some embodiments, the Fab comprises a Fab heavy chain comprising a heavy chain variable domain (VH) and a human IgG CHI domain, and a Fab light chain comprising a light chain variable domain (VL) and a human light chain constant domain (CL), wherein the VH and VL domains form the second antigen binding domain that binds an antigen on the T cell or NK cell surface, e.g., CD3, CD4, CD8 or NKp46. In certain embodiments, the protein according to the invention may comprise a soluble TCR fragment comprising a Va-Ca domain in a first polypeptide and a VP-CP domain in a second polypeptide comprising any of the mutations in the Cot and / or CP domain disclosed herein, and a second or additional antigen-binding domain. The soluble TCR fragment and the second or additional antigen-binding domain may be fused or conjugated in any way known in the art.

[0172] Preferably, the TCR fragment is fused to the second or additional antigen-binding domain. That is, one polypeptide chain comprised in the TCR fragment may be fused to the second or additional antigen-binding domain, preferably via a peptide linker, such as one of the linkers disclosed herein. The second or additional antigen-binding domain may be fused to the N- or C-terminal end of a polypeptide chain, i.e., the alpha or beta chain, comprised in the TCR fragment.

[0173] In a preferred embodiment, the second or additional antigen-binding domain may be a CD3 agonist.

[0174] A CD3 agonist is a molecule that interacts with CD3 on the surface of T cells and induces T cell activation. Preferably, the CD3 agonist is a protein. Even more preferably, the CD3 agonist is an agonist anti-CD3 antibody or is derived from an agonist anti-CD3 antibody. Thus, in a particular embodiment, the invention relates to the protein according to the invention, wherein the second or additional antigen-binding domain is an anti-CD3 antibody, or an antigen-binding fragment thereof.

[0175] That is, in certain embodiments, the CD3 agonist may be a full length anti-CD3 antibody. In certain embodiments, the CD3 agonist may be an antigen-binding fragment derived from an anti-CD3 antibody.

[0176] To reduce the risk of immunogenicity towards the protein of the invention, it is preferred herein that the CD3 agonist is a human or humanized anti-CD3 antibody or an antigen-binding fragment thereof. In certain embodiments, the CD3 agonist may be a humanized variant of the anti-CD3 antibody OKT3 or a humanized antigen-binding fragment thereof, as described by Adair et al. (Hum Antibodies Hybridomas, 1994, 5(l-2):41-7), which is fully incorporated herein by reference.

[0177] In certain embodiments, the CD3 agonist may be a humanized variant of the anti-CD3 antibody SP34 or a humanized antigen-binding fragment thereof, as disclosed in W02016020444, which is fully incorporated herein by reference. In certain embodiments, the CD3 agonist may be a humanized variant of the anti-CD3 antibody UCHT1 or a humanized antigen-binding fragment thereof. UCHT1 was initially described by Beverley and Callard (Eur J Immunol, 1981, ll(4):329-34), which is fully incorporated herein by reference.

[0178] Preferably, the humanized variant of UCHT1 comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:18 and / or a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:19.

[0179] In certain embodiments, the CD3 agonist may be a humanized variant of the anti-CD3 antibody BMA031 a humanized antigen-binding fragment thereof. BMA031 was initially described by Borst et al. (Hum Immunol, 1990, 29(3):175-88), which is fully incorporated herein by reference.

[0180] In certain embodiments, the CD3 agonist may be a humanized variant of the anti-CD3 antibody 12F6 or a humanized antigen-binding fragment thereof, as described by Li et al. (Immunology, 2005, 116(4): 487-498), which is fully incorporated herein by reference.

[0181] Preferably, the humanized variant of 12F6 comprises a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NQ:20 and / or a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:21.

[0182] In a particularly preferred embodiment, the CD3 agonist may be a humanized variant of the anti-CD3 antibody UCHT1 or a humanized antigen-binding fragment thereof.

[0183] It is further preferred herein that the CD3 agonist comprised in the protein of the invention is a human or humanized Fab fragment, nanobody or scFv fragment.

[0184] In certain embodiments, the second or additional antigen-binding domain may be an NKp46 agonist.

[0185] An NKp46 agonist is a molecule that interacts with NKp46 on the surface of NK cells and induces NK cell activation. Preferably, the NKp46 agonist is a protein. Even more preferably, the NKp46 agonist is an agonist anti-NKp46 antibody or is derived from an agonist anti-NKp46 antibody. Thus, in a particular embodiment, the invention relates to the protein according to the invention, wherein the second or additional antigen-binding domain is an anti-NKp46 antibody, or an antigen-binding fragment thereof. That is, in certain embodiments, the NKp46 agonist may be a full length anti-NKp46 antibody. In certain embodiments, the NKp46 agonist may be an antigen-binding fragment derived from an anti-NKp46 antibody.

[0186] In certain embodiments, the NKp46 agonist may be a humanized variant or a humanized antigen-binding fragment of the anti-NKp46 antibody disclosed in WO2015 / 197593, which is fully incorporated herein by reference.

[0187] It is further preferred herein that the NKp46 agonist comprised in the protein of the invention is a human or humanized Fab fragment, nanobody or scFv fragment.

[0188] As used herein, "antibody" refers to immunoglobulin, a structure of four-peptide chains connected together by disulfide bonds between two identical heavy chains and two identical light chains. Different immunoglobulin heavy chain constant regions exhibit different amino acid compositions and rank orders, hence present different kinds of antigenicity. Accordingly, immunoglobulin can be divided into five categories, or immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, with heavy chain p, 6, y, a and E, respectively. According to its amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds, the same type of Ig can be divided into different sub-categories. For example, IgG can be divided into IgGl, lgG2, lgG3, and lgG4. Light chains can be divided into K or A chain, due to different constant regions. Each IgG among the five types has K or A chain.

[0189] The sequence of about 110 amino acids closest to the N-terminus of the antibody heavy and light chains is commonly referred to as the variable region (Fv region). The sequence of amino acids closest to the C-terminus is commonly referred to as the constant region. The variable region comprises three hypervariable regions (HVR) and four framework regions (FR) having relatively conserved sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDRs). Each light chain variable region (LCVR) and each heavy chain variable region (HCVR) comprises three CDR regions and four FR regions. Sequentially ordered from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three light chain CDRs are referred to as LCDR1, LCDR2, and LCDR3. The three heavy chain CDRs are referred to as HCDR1, HCDR2 and HCDR3. The number and location of the CDR amino acid residues in the LCVR and HCVR regions of the antibody or antigen binding fragment herein comply with known Kabat numbering criteria (e.g., LCDR1-3, HCDR2-3), or comply with Kabat and Chothia numbering criteria (e.g., HCDR1).

[0190] In certain embodiment, the antibody may be a human or human derived antibody. The terms "human antibody" and "human derived antibody" are used interchangeably and refer to an antibody comprising one or more variable and constant regions derived from a human immunoglobulin sequence. In a preferred embodiment of the invention, all of the variable and constant regions are derived from human immunoglobulin sequences, i.e., "fully human derived antibody" or "fully human antibody". These antibodies can be obtained in a variety of ways, including antibodies obtained by using phage display technology, including isolating B cells from human PBMC, spleen, lymph node tissue and constructing natural single-stranded phage human antibody library, or by immunizing transgenic mice expressing human antibody light and heavy chain and screening.

[0191] In certain embodiments, the antibody may be a murine antibody. The term "murine antibody" used in the present invention refers to a monoclonal antibody against human CD3 prepared according to the knowledge and skill in the art. During preparation, the test subject is injected with a CD3 antigen, and then the hybridoma expressing antibodies having desired sequences or functional properties are isolated. In a preferred embodiment of the invention, the murine anti-CD3 antibody or antigen-binding fragment thereof further comprises a light chain constant region of a murine kappa, lambda chain or a variant thereof, or further comprises a heavy chain constant region of murine IgGl, lgG2, lgG3 or variants thereof.

[0192] In certain embodiments, the antibody may be a chimeric antibody. The term "chimeric antibody" is an antibody which is formed by fusing the variable region of a murine antibody with the constant region of a human antibody, so as to alleviate the murine antibody-induced immune response. To establish a chimeric antibody, a hybridoma secreting a specific murine monoclonal antibody is established and a variable region gene is cloned from the murine hybridoma cells. Then a desired constant region gene of a human antibody is cloned and connected with the murine variable region genes to form a chimeric gene which can be subsequently inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in eukaryotic or prokaryotic system. In a preferred embodiment of the present invention, the light chain of the anti-CD3 chimeric antibody further comprises a light chain constant region derived from the human kappa, lambda chain or a variant thereof. The heavy chain of the anti-CD3 chimeric antibody further comprises a heavy chain constant region derived from human IgGl, lgG2, lgG3 or lgG4 or a variant thereof.

[0193] In certain embodiments, the antibody may be a humanized antibody. The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody generated by grafting murine CDR sequences into a variable region framework of a human antibody (i.e., antibodies produced within different types of human germline antibody framework sequences). A humanized antibody overcomes the heterologous response induced by a chimeric antibody that carries a large amount of murine protein components. Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or 31 published references. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in e.g., "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk / vbase), as well as found in Kabat, E A, et al, 1991 Sequences of Proteins of Immunological Interest, 5th edition. The CDR graft can reduce the affinity of the anti-CD3 antibody or antigen-binding fragment thereof to the antigen, due to the framework residues that are in contact with the antigen. Such interaction can be the result of hyper-mutation in somatic cells. Therefore, it may still be necessary to graft such donor framework amino acids onto the framework of humanized antibodies. Amino acid residues from a non-human anti-CD3 antibody or antigen-binding fragment thereof which are involved in antigen binding can be identified by examining the murine monoclonal antibody variable region sequences and structures. Each residue in the CDR donor framework that differs from the germline can be considered to be relevant. If the closest germline cannot be determined, the sequence can be compared with the common sequence of a subtype or the sequence of the murine with a high similarity percentage. Rare framework residues are thought to be the result of somatic hyper-mutation and thus play an important role in binding.

[0194] The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen {e.g., CD3).

[0195] Thus, in a particular embodiment, the invention relates to protein according to the invention, wherein the second antigen-binding domain is a nanobody, a Fab fragment, a F(ab')2 fragment, a Fab' fragment, a single-chain antibody (scFv), a dimerized V region (diabody), a disulfide- stabilized V region (dsFv), or a CDR-containing peptide.

[0196] The term "nanobody", as used herein, refers to a single-domain antibody. The single-domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. With a molecular weight of only 12-15 kDa, single-domain antibodies are much smaller than common antibodies (150-160 kDa) which are composed of two heavy protein chains and two light chains, and even smaller than Fab fragments (~50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (~25 kDa, two variable domains, one from a light and one from a heavy chain).

[0197] It should be noted that the term "nanobody," as used herein in its broadest sense, is not limited to a specific biological source or to a specific method of preparation. For example, the nanobodies hereof can generally be obtained: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by "humanization" of a naturally occurring VHH domain or by expression of a nucleic acid encoding such a humanized VHH domain; (4) by "camelization" of a naturally occurring VH domain from any animal species, and, in particular, from a mammalian species, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by "camelization" of a "domain antibody" or "Dab" as described in the art, or by expression of a nucleic acid encoding such a camelized VH domain; (6) by using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences known per se; (7) by preparing a nucleic acid encoding a nanobody using techniques for nucleic acid synthesis known per se, followed by expression of the nucleic acid thus obtained; and / or (8) by any combination of one or more of the foregoing.

[0198] Fab is an antibody fragment having a molecular weight of about 50,000 and having antigenbinding activity, such fragments are obtained by treating an IgG antibody molecule with protease papain (cleaving amino acid residue at position 224 of H chain), wherein about half of the N-terminal side of the H chain and the entire L chain are bound by disulfide bond. The Fab of the present invention may be produced by treating a monoclonal antibody with papain. Furthermore, the Fab may be produced by inserting a DNA encoding the Fab of the antibody into a prokaryotic expression vector or eukaryotic expression vector and introducing the vector into prokaryote or eukaryote to express the Fab. The Fab fragment may be linked to another component of the protein, i.e., a TCR fragment, an antibody Fc region or an IL-2 variant, via the polypeptide chain that comprises the heavy or light chain variable region. Preferably, the Fab fragment may be linked to another component of the protein, i.e., a TCR fragment, an antibody Fc region or an IL-2 variant, via the polypeptide chain that comprises the heavy chain variable region.

[0199] F(ab')2 is an antibody fragment obtained by digesting the lower part of two disulfide bonds in IgG hinge region with pepsin. It has a molecular weight of about 100,000 and antigen-binding activity, and comprises two Fab regions linked at the hinge position.

[0200] The F(ab')2 of the present invention can be produced by treating the monoclonal antibody of the present invention, which specifically recognizes human CD3 and binds to the extracellular region amino acid sequence or three-dimensional structure thereof, with pepsin. Furthermore, the F(ab')2 can be produced by linking the Fab' described below with a thioether bond or a disulfide bond.

[0201] Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigenbinding activity. It is obtained by cleaving the disulfide bond in the hinge region of the F(ab')2 mentioned above. The Fab' of the present invention may be produced by treating the F(ab')2 of the present invention, which specifically recognizes human CD3 and binds to the extracellular region amino acid sequence or three-dimensional structure thereof, with a reducing agent (such as dithiothreitol).

[0202] Furthermore, the Fab' can be produced by inserting a DNA encoding a Fab' fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into prokaryote or eukaryote to express the Fab'.

[0203] The term "single-chain antibody", "single-chain Fv" or "scFv" refers to a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable linkers in prior art consist of repeated GGGGS amino acid sequence or variants thereof, for example a variant having 1-4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used in the present invention are described in Alfthan et al. (1995), Protein Eng. 8: 725- 731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055- 3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.

[0204] The scFv of the present invention can be produced by the following steps: obtaining the cDNA encoding VH and VL of the monoclonal antibody of the present invention which specifically recognizes human CD3 and binds to the extracellular region amino acid sequence or three- dimensional structure thereof; constructing a DNA encoding the scFv; inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector; and then introducing the expression vector into prokaryote or eukaryote to express said scFv.

[0205] A diabody is an antibody fragment in which the scFv is dimerized. Diabodies may have bivalent antigen-binding activity.

[0206] The diabody of the present invention can be produced by the following steps: obtaining the cDNA encoding VH and VL of the monoclonal antibody of the present invention which specifically recognizes human CD3 and binds to the extracellular region amino acid sequence or three-dimensional structure thereof; constructing a DNA encoding scFv such that the length of the linker peptide is 8 or less amino acid residues; inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector; and then introducing the expression vector into prokaryote or eukaryote to express the diabody.

[0207] The dsFv is obtained by substituting one amino acid residue in each of the VH and the VL with cysteine residue, and then linking the polypeptides via disulfide bond between the two cysteine residues. The amino acid residue to be substituted with a cysteine residue can be selected based on a three-dimensional structure prediction of the antibody in accordance with known methods (Protein Engineering, 1, 697 (1994)).

[0208] The dsFv of the present invention can be produced by the following steps: obtaining the cDNA encoding the VH and the VL of the monoclonal antibody of the present invention which specifically recognizes human CD3 and binds to the extracellular region amino acid sequence or three-dimensional structure thereof; constructing a dsFv-encoding DNA; inserting the DNA into prokaryotic expression vector or eukaryotic expression vector; and then introducing the expression vector into prokaryote or eukaryote to express said dsFv.

[0209] The CDR-containing peptide is constructed by one or more regions of CDRs of VH or VL. Peptides comprising several CDRs can be linked directly or via a suitable peptide linker.

[0210] The CDR-containing peptide of the present invention can be produced by the following steps: constructing a DNA encoding CDRs of the VH and the VL of the monoclonal antibody of the present invention which specifically recognizes human CD3 and binds to the extracellular region amino acid sequence or three-dimensional structure thereof; inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector; and then introducing the expression vector into prokaryote or eukaryote to express said peptide. The CDR-containing peptide can also be produced by chemical synthesis methods such as Fmoc method or tBoc method.

[0211] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding. One of the most commonly used definitions for the six CDRs is provided by Kabat E. A. et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242. As used herein, the Kabat definition of CDR only applies to CDR1, CDR2 and CDR3 of the light chain variable domain (CDR LI, CDR L2, CDR L3 or LI, L2, L3), as well as CDR2 and CDR3 of heavy chain variable domain (CDR H2, CDR H3 or H2, H3).

[0212] The term "antibody framework" as used herein, refers to a portion of the variable domain VL or VH, which serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain without CDRs.

[0213] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on CD3 molecule). Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996). The terms "specific binding", "selective binding", "selectively bind" and "specifically bind" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody binds with an affinity (KD) of less than about 10“7M, such as approximately less than about 10“8M, 10“9M or 1010M or less.

[0214] Exemplary formats comprising at least a soluble TCR fragment comprising one or more of the Ca / Cp mutations disclosed herein and a second antigen-binding domain are disclosed herein below:

[0215] In a particular embodiment, the invention relates to the protein according to the invention, wherein the second antigen-binding domain, e.g., the CD3 or NKp46 agonist, is linked to the C-terminal end of a polypeptide chain comprised in the TCR fragment. More specifically, the TCRfragment is linked to a polypeptide chain comprised in the second antigen-binding domain via a C-terminal end of a polypeptide chain comprised in the TCR fragment, wherein the TCR fragment is linked to an N-terminal end of a polypeptide chain comprised in the second antigen-binding domain via a C-terminal end of a polypeptide chain comprised in the TCR fragment.

[0216] In certain embodiments, the TCR fragment is a heterodimer consisting of a first and second polypeptide chain and the second antigen-binding domain is an scFv fragment. In such embodiments, the TCR fragment may be linked to the scFv fragment via a C-terminal end of the first or second polypeptide chain comprised in the TCR fragment. Preferably, the C- terminal end of the first or second polypeptide chain comprised in the TCR fragment is linked to the N-terminal end of the scFv fragment.

[0217] In certain embodiments, the TCR fragment may be linked to the scFv fragment via the C- terminal end of the alpha or beta chain of the TCR fragment. Preferably, the C-terminal end of the alpha or beta chain of the TCR fragment is linked to the N-terminal end of the scFv fragment. Even more preferably, the C-terminal end of the alpha chain of the TCR fragment is linked to the N-terminal end of the scFv fragment.

[0218] In certain embodiments, the TCR fragment is a heterodimer consisting of a first and second polypeptide chain and the second antigen-binding domain is a Fab fragment derived from an antibody. In such embodiments, the TCR fragment may be linked to a polypeptide chain comprised in the Fab fragment via a C-terminal end of the first or second polypeptide chain comprised in the TCR fragment. Preferably, the C-terminal end of the first or second polypeptide chain comprised in the TCR fragment is linked to an N-terminal end of a polypeptide chain comprised in the Fab fragment, in particular to an N-terminal end of a polypeptide comprising the heavy chain variable region or the light chain variable region.

[0219] In certain embodiments, the TCR fragment may be linked to a polypeptide chain comprised in the Fab fragment via the C-terminal end of the alpha or beta chain of the TCR fragment. Preferably, the C-terminal end of the alpha or beta chain of the TCR fragment is linked to an N-terminal end of a polypeptide chain comprised in the Fab fragment, in particular to an N- terminal end of a polypeptide comprising the heavy chain variable region or the light chain variable region. Even more preferably, the C-terminal end of the alpha chain of the TCR fragment is linked to an N-terminal end of a polypeptide chain comprised in the Fab fragment, in particular to an N-terminal end of a polypeptide comprising the heavy chain variable region or the light chain variable region.

[0220] In a particular embodiment, the invention relates to the protein according to the invention, wherein the second antigen-binding domain, e.g., the CD3 or NKp46 agonist, is linked to the N-terminal end of a polypeptide chain comprised in the TCR fragment. More specifically, the TCRfragment is linked to a polypeptide chain comprised in the second antigen-binding domain via an N-terminal end of a polypeptide chain comprised in the TCR fragment, wherein the TCR fragment is linked to a C-terminal end of a polypeptide chain comprised in the second antigenbinding domain via an N-terminal end of a polypeptide chain comprised in the TCR fragment.

[0221] In certain embodiments, the TCR fragment is a heterodimer consisting of a first and second polypeptide chain and the second antigen-binding domain is a scFv fragment. In such embodiments, the TCR fragment may be linked to the scFv fragment via an N-terminal end of the first or second polypeptide chain comprised in the TCR fragment. Preferably, the N- terminal end of the first or second polypeptide chain comprised in the TCR fragment is linked to the C-terminal end of the scFv fragment.

[0222] In certain embodiments, the TCR fragment may be linked to the scFv fragment via the N- terminal end of the alpha or beta chain of the TCR fragment. Preferably, the N-terminal end of the alpha or beta chain of the TCR fragment is linked to the C-terminal end of the scFv fragment. Even more preferably, the N-terminal end of the beta chain of the TCR fragment is linked to the C-terminal end of the scFv fragment.

[0223] In certain embodiments, the TCR fragment is a heterodimer consisting of a first and second polypeptide chain and the second antigen-binding domain is a Fab fragment derived from an antibody. In such embodiments, the TCR fragment may be linked to a polypeptide chain comprised in the Fab fragment via an N-terminal end of the first or second polypeptide chain comprised in the TCR fragment. Preferably, the N-terminal end of the first or second polypeptide chain comprised in the TCR fragment is linked to a C-terminal end of a polypeptide chain comprised in the Fab fragment, in particular to a C-terminal end of a polypeptide comprising the heavy chain variable region or the light chain variable region.

[0224] In certain embodiments, the TCR fragment may be linked to a polypeptide chain comprised in the Fab fragment via the N-terminal end of the alpha or beta chain of the TCR fragment. Preferably, the N-terminal end of the alpha or beta chain of the TCR fragment is linked to a C- terminal end of a polypeptide chain comprised in the Fab fragment, in particular to a C- terminal end of a polypeptide comprising the heavy chain variable region or the light chain variable region. Even more preferably, the N-terminal end of the alpha chain of the TCR fragment is linked to a C-terminal end of a polypeptide chain comprised in the Fab fragment, in particular to a C-terminal end of a polypeptide comprising the heavy chain variable region or the light chain variable region.

[0225] Preferable formats comprising a TCR fragment and a second antigen-binding domain are shown in Fig.6.

[0226] In certain embodiments, the protein according to the invention comprises more than one TCR fragment. In certain embodiments, the protein according to the invention comprises 2, 3, 4, 5 or more TCR fragments. Accordingly, in a particular embodiment, the invention relates to the protein according to the invention, wherein the protein comprises at least one additional TCR fragment.

[0227] In certain embodiments, the at least one additional TCR fragment is linked to the first TCR fragment and / or to the second antigen-binding domain. Accordingly, in a particular embodiment, the invention relates to a protein according to the invention, wherein the at least one additional TCR fragment is linked to a polypeptide chain comprised in the first TCR fragment or to a polypeptide chain comprised in the second antigen-binding domain.

[0228] In certain embodiments, the first TCR fragment is an a -heterodimeric TCR fragment comprising an alpha and beta chain as defined herein and the second antigen-binding domain is a Fab fragment or an scFv fragment derived from an antibody. In such embodiments, (i) a polypeptide chain comprised in the second antigen-binding domain (the polypeptide chain comprising the heavy chain variable region, the polypeptide chain comprising the light chain variable region, or the polypeptide chain of the scFv) may be linked via its N-terminal end to the C-terminal end of the alpha or beta chain of the first TCR fragment and (ii) a polypeptide chain comprised in a further TCR fragment may be linked via its C-terminal end to the N- terminal end of the alpha or beta chain of the first TCR fragment. In certain embodiments, the first TCR fragment is an ap-heterodimeric TCR fragment comprising an alpha and beta chain and the second antigen-binding domain is a Fab fragment or an scFv fragment derived from an antibody. In such embodiments, (i) a polypeptide chain comprised in the second antigen-binding domain (the polypeptide chain comprising the heavy chain variable region, the polypeptide chain comprising the light chain variable region, or the polypeptide chain of the scFv) may be linked via its N-terminal end to the C-terminal end of the alpha chain of the first TCR fragment and (ii) a polypeptide chain comprised in a further TCR fragment may be linked via its C-terminal end to the N-terminal end of the beta chain of the first TCR fragment.

[0229] In these embodiments, the further TCR fragment may be an a -heterodimeric TCR fragment as defined herein, and the further TCR fragment may be linked to a polypeptide chain comprised in the first TCR fragment via the C-terminal end of the first polypeptide chain comprised in the further TCR fragment, i.e., the alpha chain, or the second polypeptide chain comprised in the further TCR fragment, i.e., the beta chain.

[0230] In a particular embodiment, the invention relates to a protein according to the invention, wherein the at least one additional TCR fragment is linked to a polypeptide chain comprised in the second antigen-binding domain, wherein the at least one additional TCR fragment is linked to a C-terminal end of a polypeptide chain comprised in the second antigen-binding domain via an N-terminal end of a polypeptide chain comprised in the at least one additional TCR fragment.

[0231] In certain embodiments, the first TCR fragment is an ap-heterodimeric TCR fragment comprising an alpha and beta chain as defined herein and the second antigen-binding domain is a Fab fragment or an scFv fragment derived from an antibody. In such embodiments, (i) a polypeptide chain comprised in the second antigen-binding domain (the polypeptide chain comprising the heavy chain variable region, the polypeptide chain comprising the light chain variable region, or the polypeptide chain of the scFv) may be linked via its N-terminal end to the C-terminal end of the alpha or beta chain of the first TCR fragment and (ii) a polypeptide chain comprised in a further TCR fragment may be linked via its N-terminal end to the C- terminal end of the alpha or beta chain of a polypeptide chain comprised in the second antigen-binding domain.

[0232] In certain embodiments, the first TCR fragment is a ap-heterodimeric TCR fragment comprising an alpha and beta chain as defined herein and the second antigen-binding domain is an scFv fragment derived from an antibody. In such embodiments, (i) the polypeptide chain of the scFv may be linked via its N-terminal end to the C-terminal end of the alpha or beta chain of the first TCR fragment and (ii) a polypeptide chain comprised in a further TCR fragment may be linked via its N-terminal end to the C-terminal end of the scFv fragment.

[0233] In these embodiments, the further TCR fragment may be an ap-heterodimeric TCR fragment, and the further TCR fragment may be linked to a polypeptide chain comprised in the second antigen-binding domain via the N-terminal end of the alpha chain or the beta chain.

[0234] In any embodiment where the protein comprises two or more TCR fragments, the two or more TCR fragments may be identical or may be different. In a preferred embodiment, the protein comprises two or more heterodimeric TCR fragments, in particular two or more a - heterodimeric TCR fragments comprising one or more of the mutations defined herein. The two or more TCR fragments may bind to different target antigens or to the same target antigens. In a preferred embodiment, the two or more TCR fragments bind to the same target antigen. In certain embodiments, the target antigen is derived from MAGE-A3.

[0235] In some embodiments, the proteins described herein may be linked to a detectable label. In some embodiments, such detectable label may be a fluorescent label, a radioactive label, a chemiluminescent label, a bioluminescent label, a paramagnetic label, an MRI contrast agent, an organic dye, or a quantum dot.

[0236] In some embodiments, the proteins described herein may be linked to a therapeutic agent, e.g., a cytotoxic agent, an anti-inflammatory agent, an immunostimulatory agent or an immunosuppressive agent.

[0237] That is, in certain embodiments, the protein according to the invention may be linked to an anti-inflammatory agent. In certain embodiments, the protein of the invention may be used to deliver an anti-inflammatory agent to a site of inflammation. For example, an inflamed tissue may be targeted with a TCR fragment that specifically bind to a tissue-specific antigen.

[0238] The term "anti-inflammatory agent" as used herein refers to a compound for treating an inflammatory disease or a symptom related thereto. Non-limiting examples of the antiinflammatory agents include: but not limited thereto, a non-steroidal anti-inflammatory drug (NSAID; e.g., aspirin, ibuprofnaproxen, methyl salicylate, diflunisal, indometacin, sulindac, diclofenac, ketoprofen, ketorolac, carprofen, fenoprofen, mefenamic acid, piroxicam, meloxicam, methotrexate, celecoxib, valdecoxib, parecoxib, etoricoxib, and nimesulide), corticosteroid (e.g., prednisone, betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methyl prednisolone, prednisolone, triamcinolone, and fluticasone), rapamycin (e.g., see document [Migita et al., Clin. Exp. Immunol. (1997) 108:199-203]; [Migita et al., Clin. Exp. Immunol. (1996) 104:86-91]; [Foroncewicz et al., Transpl. Int. (2005) 18:366- 368]), a compound increasing levels of high-density lipoprotein (HDL) and HDL-cholesterol (e.g., see documents [Birjmohun et al. (2007) Arterioscler. Thromb. Vase. Biol., 27:1153-1158]; [Nieland et al. (2007) J. Lipid Res., 48:1832-1845]; [Bloedon et al. (2008) J. Lipid Res., Samaha et al. (2006) Arterioscler. Thromb. Vase. Biol., 26:1413-1414] disclosing use of rosiglitazone as an anti-inflammatory agent; and [Duffy et al. (2005) Curr. Opin. Cardiol., 20:301-306]), a rho- kinase inhibitor (e.g., see document [Hu, E. (2006) Rec. Patents Cardiovasc. Drug Discov., 1:249-263]), an antimalarial drug (e.g., hydroxychloroquine and chloroquine), acetaminophen, glucocorticoid, steroid, beta-agonist, anticholinergic, methyl xanthine, gold injection (e.g., sodium aurothiomalate), sulfasalazine, penicillamine, antiangiogenic drug, dapsone, psoralen, antiviral drug, statin (e.g., see document [Paraskevas et al. (2007) Curr. Pharm. Des., 13:3622-36]; [Paraskevas, K.l. (2008) Clin. Rheumatol. 27:281-287]), and an antibiotic (e.g., tetracycline).

[0239] In certain embodiments, the protein according to the invention may be linked to an immunosuppressive agent. The immunosuppressive agent may be any agent that inhibits or kills immune cells, in particular autoreactive immune cells.

[0240] In certain embodiments, the immunosuppressive agent, i.e., the agent that inhibits or kills immune cells, may be an antibody or a fragment thereof. In certain embodiments, the immunosuppressive agent, i.e., the agent that inhibits or kills immune cells, may be an ectodomain of an immunoligand.

[0241] In certain embodiments, the immunosuppressive agent is a PD-1 agonist. The PD- 1 agonist may be the soluble extracellular form (ectodomain) of PD-L1 or a functional fragment thereof. The PD-L1 may comprise or consist of the sequence:

[0242] FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLK DQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPY (SEQ ID NO:22).

[0243] Alternatively, the PD-1 agonist may be a full-length antibody or fragment thereof, such as a scFv antibody or a Fab fragment, or a nanobody. Examples of such antibodies are provided in W02011 / 110621, W02010 / 029434 and WO2018 / 024237.

[0244] In certain embodiments, the immunosuppressive agent is a Fas ligand or a functional fragment thereof.

[0245] The immunosuppressive agent, in particular the PD-1 agonist or the Fas ligand, may be fused to the C- or N- terminus of the TCR alpha or beta chain, preferably with a linker, such as any of the peptide linkers disclosed herein. Immunosuppressive agents are particularly relevant for the treatment of autoimmune diseases. That is, the protein of the invention may be used to deliver immunosuppressive agents to a tissue or organ that is affected by an autoimmune disease, such that the immunosuppressive agent can inhibit or kill autoreactive T cells at the target site. Accordingly, in a preferred embodiment, the immunosuppressive agent is linked to a tissue- or organspecific TCR fragment. In certain embodiments, a PD-1 agonist is linked to a tissue- or organspecific TCR fragment.

[0246] In certain embodiments, the protein of the invention comprises an immunostimulatory agent. The term "immunostimulatory agent" as used herein may refer to any substance or molecule that can trigger an immune response, in particular any substance or molecule that can active T cells or NK cells. That is, in certain embodiments, the immunostimulatory agent may be a T cell agonist, such as a CD3 agonist or a CD2 agonist. In certain embodiments, the immunostimulatory agent may be an NK cell agonist, such as an NKp46 agonist, an Nkp44 agonist, a CD16a agonist, a 2B4 agonist, or an IL-2R agonist.

[0247] It is to be understood that the immunosuppressive and immunostimulatory agents disclosed herein may be protein-based components, such as antibodies or antibody fragments, or ectodomains of naturally occurring ligands, or engineered variants thereof. Such immunosuppressive or immunostimulatory agents may also be referred to as (second or additional) antigen-binding domains as defined herein and may thus be incorporated into the protein of the invention as disclosed herein.

[0248] In a particular embodiment, the immunostimulatory agent is IL-2, or an engineered variant of IL-2.

[0249] That is, the protein of the invention may further comprise an IL-2 molecule. Preferably, the IL- 2 molecule is human IL-2 or an engineered variant thereof.

[0250] In certain embodiments, the IL-2 molecule is human IL-2 comprising the amino acid sequence set forth in SEQ ID NO:23.

[0251] Human IL-2 (SEQ ID NO:23)

[0252] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT Preferably, the IL-2 molecule is an engineered variant of human IL-2. Thus, in a particular embodiment, the invention relates to the protein according to the invention, wherein the IL- 2 molecule is an engineered IL-2 molecule.

[0253] The IL-2 molecule may be engineered for increased stability and / or for improved / altered biological activity. For example, the IL-2 molecule may be engineered to disrupt the interaction with CD25. Thus, in a particular embodiment, the invention relates to the protein according to the invention, wherein the IL-2 molecule has been engineered for disrupted interaction with CD25.

[0254] Vazquez-Lombardi and colleagues showed that IL-2-Fc variants engineered for disrupted interaction with the high-affinity IL-2 receptor alpha subunit (CD25) mediate robust and specific expansion of CD8+ T cells and NK cells, with no activation of immunosuppressive regulatory T cells (Tregs) (Nature Communications, 2017, 8:15373). This particular no-alpha IL-2 molecule incorporates three charge-reversal mutations, namely R38D, K43E and E61R and offers not only enhanced selectivity towards cytotoxic subsets but also increased expression yield relative to wild-type IL-2. Furthermore, this variant, also known as IL-23X, provides advantages in terms of biodistribution as it displays increased serum plasma concentrations and half-life, and a reduced association with multiple splenic immune cell subsets, indicating reduced retention in secondary lymphoid organs, and a better potential for targeted tumor delivery (e.g., through genetic fusion with an affinity enhanced TCR) than wild-type, unmodified IL-2.

[0255] Thus, in a particular embodiment, the invention relates to the protein according to the invention, wherein the engineered IL-2 molecule comprises the mutations R38D, K43E and E61R with respect to SEQ ID NO:23.

[0256] Of relevance, a different no-alpha IL-2 variant, namely IL-2v, containing mutations F42A, Y45A and L72G, has been identified and is the subject of clinical trials as a fusion to antibodies targeting PD-1 (for reactivation of exhausted T cells), and antibodies targeting the tumor antigens CEA / FAP (for delivery of cytokine to the tumor site). Furthermore, a recent study using a no-alpha IL-2 peptide fused to an anti-NKp46 antibody aims to NK cells has shown promising activity in preclinical models.

[0257] Thus, in a particular embodiment, the invention relates to the protein according to the invention, wherein the engineered IL-2 molecule comprises the mutations F42A, Y45A and L72G with respect to SEQ ID NO:23. In a particular embodiment, the invention relates to the protein according to the invention, wherein the engineered IL-2 molecule further comprises one or more mutations in positions N88, V91, T123, Q126 and / or S127 of SEQ ID NO:23.

[0258] In certain embodiments, the engineered IL-2 variant comprised in the protein of the invention comprises the mutations R38D, K43E and E61R and at least one additional mutation in positions N88, V91, T123, Q126 and / or S127 with respect to SEQ ID NO:23. In certain embodiments, the engineered IL-2 variant comprised in the protein of the invention comprises the mutations R38D, K43E and E61R and at least two additional mutations in positions N88, V91, T123, Q126 and / or S127 with respect to SEQ ID NO:23. In certain embodiments, the engineered IL-2 variant comprised in the protein of the invention comprises the mutations R38D, K43E and E61R and up to two additional mutations in positions N88, V91, T123, Q126 and / or S127 with respect to SEQ ID NO:23.

[0259] In certain embodiments, the engineered IL-2 variant comprised in the protein of the invention comprises the mutations R38D, K43E, E61R and N88A with respect to SEQ ID NO:23.

[0260] In certain embodiments, the engineered IL-2 variant comprised in the protein of the invention comprises the mutations F42A, Y45A and L72G and at least one additional mutations in positions N88, V91, T123, Q126 and / or S127 with respect to SEQ ID NO:23. In certain embodiments, the engineered IL-2 variant comprised in the protein of the invention comprises the mutations F42A, Y45A and L72G and at least two additional mutations in positions N88, V91, T123, Q126 and / or S127 with respect to SEQ ID NO:23. In certain embodiments, the engineered IL-2 variant comprised in the protein of the invention comprises the mutations F42A, Y45A and L72G and up to two additional mutations in positions N88, V91, T123, Q126 and / or S127 with respect to SEQ ID 23.

[0261] In certain embodiments, the protein according to the invention comprises at least a TCR fragment comprising any one of the mutations in TCR Ca and / or CP disclosed herein, and an IL-2 molecule (including engineered variants thereof). Exemplary formats of proteins comprising a TCR fragment comprising any one of the mutations in TCR Ca and / or CP disclosed herein, and an IL-2 molecule are disclosed herein below:

[0262] In certain embodiments, the protein of the invention consists of one or more TCR fragments and one or more IL-2 molecules.

[0263] In a particular embodiment, the invention relates to the protein according to the invention, wherein the IL-2 molecule is linked to an N-terminal or C-terminal end of a polypeptide chain comprised in the TCR fragment. In certain embodiments, the protein of the invention consists of a TCR fragment and an IL-2 molecule, wherein the IL-2 molecule is linked to a C-terminal end of a polypeptide chain comprised in the TCR fragment. In certain embodiments, the protein of the invention consists of a TCR fragment and an IL-2 molecule, wherein the IL-2 molecule is linked to an N-terminal end of a polypeptide chain comprised in the TCR fragment.

[0264] In certain embodiments, the protein of the invention consists of two TCR fragments and one IL-2 molecule, wherein a first TCR fragment is linked to the N-terminal end of the IL-2 molecule and wherein a second TCR fragment is linked to the C-terminal end of the IL-2 molecule.

[0265] Preferably, the protein of the invention comprises an ap-heterodimeric TCR fragment and an IL-2 molecule, wherein the IL-2 molecule is linked to the N- or C-terminal end of the alpha or beta chain comprised in the a -heterodimeric TCR fragment. Such protein may further comprise a second antigen-binding domain and / or an antibody Fc region, as described herein.

[0266] In certain embodiments, the protein according to the invention comprises a TCR fragment comprising any one of the mutations in TCR Ca and / or CP disclosed herein, a second antigenbinding domain and an IL-2 molecule (including engineered variants thereof). Exemplary formats of proteins comprising a TCR fragment comprising any one of the mutations in TCR Ca and / or CP disclosed herein, a second antigen-binding domain and an IL-2 molecule are disclosed herein below:

[0267] In certain embodiments, the second antigen-binding domain is linked to an N-terminal end of a polypeptide chain comprised in the TCR fragment and the IL-2 molecule is linked to a C- terminal end of a polypeptide chain comprised in the TCR fragment. For example, the second antigen-binding domain may be linked to the N-terminal end of a first polypeptide chain comprised in the TCR fragment and the IL-2 molecule may be linked to the C-terminal end of a second polypeptide chain comprised in the TCR fragment. Alternatively, the second antigenbinding domain may be linked to the N-terminal end and the IL-2 molecule may be linked to the C-terminal end of the same polypeptide chain comprised in the TCR fragment.

[0268] In certain embodiments, the second antigen-binding domain is linked to a C-terminal end of a polypeptide chain comprised in the TCR fragment and the IL-2 molecule is linked to an N- terminal end of a polypeptide chain comprised in the TCR fragment. For example, the IL-2 molecule may be linked to the N-terminal end of a first polypeptide chain comprised in the TCR fragment and the second antigen-binding domain may be linked to the C-terminal end of a second polypeptide chain comprised in the TCR fragment. Alternatively, the IL-2 molecule may be linked to the N-terminal end and the second antigen-binding domain may be linked to the C-terminal end of the same polypeptide chain comprised in the TCR fragment. In certain embodiments, the TCR fragment is an ap-heterodimeric TCR fragment and the second antigen-binding domain is an scFv fragment. In such embodiments, the IL-2 molecule may be linked via its C-terminal end to the N-terminal end of the alpha or beta chain of the TCR fragment and the scFv fragment may be linked via its N-terminal end to the C-terminal end of the alpha or beta chain of the TCR fragment.

[0269] In certain embodiment, the TCR fragment is an ap-heterodimeric TCR fragment and the second antigen-binding domain is a Fab fragment. In such embodiments, the IL-2 molecule may be linked via its C-terminal end to the N-terminal end of the alpha or beta chain of the TCR fragment and one of the polypeptide chains comprised in the Fab fragment may be linked via its N-terminal end to the C-terminal end of the alpha or beta chain of the TCR fragment.

[0270] In certain embodiments, the IL-2 molecule may be linked via its C-terminal end to the N- terminal end of the alpha chain of the TCR fragment and the second antigen-binding domain may be linked via an N-terminal end to the C-terminal end of the beta chain of the TCR fragment. In certain embodiments, the IL-2 molecule may be linked via its C-terminal end to the N-terminal end of the beta chain of the TCR fragment and the second antigen-binding domain may be linked via an N-terminal end to the C-terminal end of the alpha chain of the TCR fragment. In certain embodiments, the IL-2 molecule may be linked via its C-terminal end to the N-terminal end of the alpha chain of the TCR fragment and the second antigen-binding domain may be linked via an N-terminal end to the C-terminal end of the alpha chain of the TCR fragment. In certain embodiments, the IL-2 molecule may be linked via its C-terminal end to the N-terminal end of the beta chain of the TCR fragment and the second antigen-binding domain may be linked via an N-terminal end to the C-terminal end of the beta chain of the TCR fragment.

[0271] In a particularly preferred embodiment, the invention relates to a protein comprising an ap- heterodimeric TCR fragment, a Fab fragment and an IL-2 molecule, wherein the IL-2 molecule is linked via its C-terminal end to the N-terminal end of the alpha chain of the ap- heterodimeric TCR fragment and wherein the Fab fragment is linked via the N-terminal end of the heavy or light chain to the C-terminal end of the alpha chain of the ap-heterodimeric TCR fragment.

[0272] In another particularly preferred embodiment, the invention relates to a protein comprising an ap-heterodimeric TCR fragment, a Fab fragment and an IL-2 molecule, wherein the IL-2 molecule is linked via its C-terminal end to the N-terminal end of the beta chain of the ap- heterodimeric TCR fragment and wherein the Fab fragment is linked via the N-terminal end of the heavy or light chain to the C-terminal end of the alpha chain of the ap-heterodimeric TCR fragment. In certain embodiments, the second antigen-binding domain and the IL-2 molecule are linked to a C-terminal end of a polypeptide chain comprised in the TCR fragment, preferably wherein the second antigen-binding domain is linked to a C-terminal end of a polypeptide chain comprised in the TCR fragment and wherein the IL-2 molecule is linked to the second antigenbinding domain.

[0273] In certain embodiments, the second antigen-binding domain may be linked to the C-terminal end of a first polypeptide chain comprised in the TCR fragment and the IL-2 molecule may be linked to the C-terminal end of a second polypeptide chain comprised in the TCR fragment.

[0274] For example, the second antigen-binding domain, in particular a Fab fragment, may be linked to the C-terminal end of the alpha chain of an ap-heterodimeric TCR fragment via the N- terminal end of the heavy or light chain of the Fab fragment and the IL-2 molecule may be linked to the C-terminal end of the beta chain of the ap-heterodimeric TCR fragment via its N- terminal end.

[0275] Alternatively, a second antigen-binding domain, in particular a Fab fragment, may be linked to the C-terminal end of the beta chain of an ap-heterodimeric TCR fragment via the N- terminal end of the heavy or light chain of the Fab fragment and the IL-2 molecule may be linked to the C-terminal end of the alpha chain of the ap-heterodimeric TCR fragment via its N-terminal end.

[0276] Alternatively, an scFv fragment may be linked to the C-terminal end of the alpha or beta chain of an ap-heterodimeric TCR fragment via its N-terminal end and the IL-2 molecule may be linked to the C-terminal end of the other one of the alpha or beta chain of the ap- heterodimeric TCR fragment via its N-terminal end.

[0277] In certain embodiments, the second antigen-binding domain and the IL-2 molecule may be sequentially linked to the C-terminal end of one of the polypeptide chains comprised in the TCR fragment. That is, in certain embodiments, the second antigen-binding domain may be linked to the C-terminal end of a polypeptide chain comprised in the TCR fragment and the IL- 2 molecule may be linked to the second antigen-binding domain.

[0278] Accordingly, in a particular embodiment, the invention relates to the protein according to the invention, wherein the second antigen-binding domain is linked to a C-terminal end of a polypeptide chain comprised in the TCR fragment and the IL-2 molecule is linked to an N- or C-terminal end of a polypeptide chain comprised in the second antigen-binding domain. In certain embodiments, the second antigen-binding domain is an scFv fragment. In such embodiments, the scFv fragment may be linked to the C-terminal end of the TCR fragment via its N-terminal end and the IL-2 molecule may be linked to the C-terminal end of the scFv fragment.

[0279] In certain embodiments, the second antigen-binding domain is a Fab fragment. In such embodiments, the Fab fragment may be linked to a C-terminal end of the TCR fragment via an N-terminal end of a first polypeptide chain comprised in the Fab fragment and the IL-2 molecule may be linked to the N-terminal end of the second polypeptide chain comprised in the Fab fragment.

[0280] For example, in some embodiments, the N-terminal end of the heavy chain of the Fab fragment may be linked to the C-terminal end of the TCR fragment and the N-terminal end of the light chain of said Fab fragment may be linked to the IL-2 molecule. Alternatively, the N- terminal end of the light chain of the Fab fragment may be linked to the C-terminal end of the TCR fragment and the N-terminal end of the heavy chain of said Fab fragment may be linked to the IL-2 molecule.

[0281] In a particular embodiment, the N-terminal end of the heavy chain of a Fab fragment may be linked to the C-terminal end of the alpha chain of an a|3-heterodimeric TCR fragment and the N-terminal end of the light chain of said Fab fragment may be linked to the IL-2 molecule.

[0282] In certain embodiments, the second antigen-binding domain may be linked to a C-terminal end of the TCR fragment via an N-terminal end of a first polypeptide chain comprised in the second antigen-binding domain and the IL-2 molecule may be linked to a C-terminal end of the second antigen-binding domain.

[0283] Accordingly, in a particular embodiment, the invention relates to the protein according to the invention, wherein the second antigen-binding domain is linked to a C-terminal end of a polypeptide chain comprised in the TCR fragment via the N-terminal end of a first polypeptide chain comprised in the second antigen-binding domain and the IL-2 molecule is linked to a C- terminal end of a first or second polypeptide chain comprised in the second antigen-binding domain.

[0284] For example, in some embodiments, the N-terminal end of the heavy chain of a Fab fragment may be linked to the C-terminal end of the TCR fragment and the IL-2 molecule may be linked to the N- or C-terminal end of the heavy or light chain of said Fab fragment. Alternatively, the N-terminal end of the light chain of a Fab fragment may be linked to the C-terminal end of the TCR fragment and the IL-2 molecule may be linked to the N- or C-terminal end of the heavy or light chain of said Fab fragment.

[0285] In a preferred embodiment, the N-terminal end of the heavy or light chain of a Fab fragment may be linked to the C-terminal end of the alpha or beta chain of an ap-heterodimeric TCR fragment and the C-terminal end of the light or heavy chain of said Fab fragment may be linked to the IL-2 molecule.

[0286] In a particularly preferred embodiment, the N-terminal end of the heavy chain of a Fab fragment may be linked to the C-terminal end of the alpha chain of an ap-heterodimeric TCR fragment and the C-terminal end of the light or heavy chain of said Fab fragment may be linked to the IL-2 molecule.

[0287] Alternatively, the IL-2 molecule may be linked to the C-terminal end of a polypeptide chain comprised in the TCR fragment and the second antigen-binding domain may be linked to the IL-2 molecule.

[0288] Accordingly, the TCR fragment and the second antigen-binding domain may be connected via the IL-2 molecule. In certain embodiments, the N-terminal end of the IL-2 molecule is linked to a C-terminal end of a polypeptide chain comprised in the TCR fragment and the C-terminal end of the IL-2 molecule is linked to an N-terminal end of a polypeptide chain comprised in the second antigen-binding domain.

[0289] In certain embodiments, the TCR fragment is an ap-heterodimeric TCR fragment and the second antigen-binding domain is an scFv fragment. In such embodiments, the IL-2 molecule may be linked via its N-terminal end to the C-terminal end of the alpha or beta chain of the ap-heterodimeric TCR fragment and via its C-terminal end to the N-terminal end of the scFv fragment.

[0290] In certain embodiments, the TCR fragment is an ap-heterodimeric TCR fragment and the second antigen-binding domain is a Fab fragment. In such embodiments, the IL-2 molecule may be linked via its N-terminal end to the C-terminal end of the alpha or beta chain of the ap-heterodimeric TCR fragment and via its C-terminal end to the N-terminal end of the first or second polypeptide chain comprised in the Fab fragment, e.g., to the N-terminal end of the light or heavy chain comprised in the Fab fragment. For example, in some embodiments, the IL-2 molecule may be linked via its N-terminal end to the C-terminal end of the alpha chain of the ap-heterodimeric TCR fragment and via its C-terminal end to the N-terminal end of the heavy chain comprised in the Fab fragment. In certain embodiments, the protein of the invention may comprise an antibody Fc region. The term "Fc region" or "Fc domain" refers to the region(s) of an antibody constant region (e.g., IgGl, lgG2, lgG3, or lgG4) that is involved in the binding interaction of the Fc region to one or more Fey receptors (e.g., FcyRI (CD64), FcyRllb (CD32b) or FcyRllla (CD16). The locations of the regions or domains of IgG isotype constant regions are known in the art and are described, for example, in Shields et al., 2001, J. Biol. Chem. 276:6591-6604, and Canfield and Morrison, 1991, J. Exp. Med. 173:1483-1491 and Sondermann et al., 2000, Nature 406(6793): 267-73. The Fc regions or domains include, for example and not for limitation, the hinge region and the CH2 domain.

[0291] The Fc-region is a dimeric molecule comprising two disulfide-linked antibody heavy chain fragments (heavy chain Fc-region polypeptide chains). An Fc-region can be generated by papain digestion, or IdeS digestion, or trypsin digestion of an intact (full length) antibody or can be produced recombinantly.

[0292] The Fc-region obtainable from a full length antibody or immunoglobulin comprises at least residues 226 (Cys) to the C-terminus of the full length heavy chain and, thus, comprises a part of the hinge region and two or three constant domains, i.e. a CH2 domain, a CH3 domain, and an additional / extra CH4 domain on IgE and IgM class antibodies.

[0293] In a particular embodiment, the invention relates to the protein according to the invention, wherein the antibody Fc region is derived from a human IgG antibody heavy chain, in particular a human IgGl antibody.

[0294] The antibody Fc region may be a naturally occurring antibody Fc region or may be an engineered variant of a naturally occurring antibody Fc region. For example, the antibody Fc region may comprise mutations that reduce immune effector functions. Exemplary mutations that may be comprised in the antibody region are summarized in Liu et al. (Antibodies, 2020, 9(4):64).

[0295] Thus, in a particular embodiment, the invention relates to the protein according to the invention, wherein the antibody Fc region comprises mutations that reduce immune effector functions.

[0296] In some embodiments, the antibody Fc region is a modified human IgG Fc region with reduced effector function compared to the corresponding wild type human IgG Fc region. In some embodiments, the Fc region is a modified human IgGl Fc region. IgGl is well known to bind to the proteins of the Fc-gamma receptor family (FcyR) as well as Clq. Interaction with these receptors can induce antibody-dependent cell cytotoxicity (ADCC) and complement- dependent cytotoxicity (CDC). Therefore, certain amino acid substitutions are introduced into human IgGl Fc region to ablate immune effector function. In some embodiments, the Fc region is a modified human IgGl Fc region comprising one or more of the following mutations: N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L, M252Y, S254T, T256E (residues numbered according to the EU Index Numbering). In some embodiments, the Fc region is a modified human IgGl Fc region comprising the following mutations: L234A, L235A and N297Q (residues numbered according to the EU Index Numbering). In some embodiments, the Fc region is a modified human IgGl Fc region and further comprises a human IgGl hinge region at the N-terminus of the modified human IgGl Fc region.

[0297] In some embodiments, the Fc region is a modified human lgG4 Fc region comprising one or more of the following mutations: E233P, F234V, F234A, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q (residues numbered according to the EU Index Numbering). In some embodiments, the Fc region is a modified human lgG4 Fc region comprising the following mutations: F234A and L235A (residues numbered according to the EU Index Numbering). In some embodiments, the Fc region is a modified human lgG4 Fc region and further comprises a modified human lgG4 hinge region comprising the S228P mutation (according to the EU Index Numbering) at the N-terminus of the modified human lgG4 Fc region. Such modified human lgG4 hinge region reduces the lgG4 Fab-arm exchange in vivo (see Labrijn, et al., Nat Biotechnol 2009, 27(8):767).

[0298] In some embodiments, the proteins described herein comprise a hinge region comprising SEQ ID NO: 24 or 25. In some embodiments, the proteins described herein comprise an Fc region comprising SEQ ID NO: 26 or 27. In some embodiments, the proteins described herein comprise a hinge region comprising SEQ ID NO: 24 and a first and second Fc region comprising SEQ ID NO: 26. In some embodiments, the proteins described herein comprise a hinge region comprises SEQ ID NO: 25 and a first and second Fc region comprising SEQ ID NO: 27.

[0299] Furthermore, the antibody Fc region may comprise mutations that allow heterodimerization of the antibody Fc region. Normally, the two polypeptide chains of the antibody Fc region have an identical sequence and are expressed from the same gene. Thus, in certain embodiments, both polypeptide chains comprised in the antibody Fc region are linked to the same components. In such embodiments, no modification of the antibody Fc region is required. However, in some embodiments, the first polypeptide chain comprised in the antibody Fc region is linked to different components than the second polypeptide chain comprised in the antibody Fc region. To allow heterodimerization of heavy chain fragments that are linked to different components, certain modifications have to be introduced into the heavy chain fragments. The most commonly applied strategy to achieve heterodimerization of heavy chain fragments is by introducing knobs-into-holes mutations into the antibody Fc region.

[0300] The term "knobs-into holes mutations" refers to mutations, including those in the CHS domain of an Fc region, that facilitate heterodimerization of the first and second polypeptide chains in an antibody Fc region. Exemplary mutations useful for this heterodimerization are described in Ridgway et al. (1996) Protein Engin. 9(7): 617- 21, Atwell et al. (1997) J, Mol. Biol 270:26-35, and PCT Publication No. W02014 / 106015, which are each incorporated by reference herein in their entirety. For instance, electrostatic or hydrophobic interactions can be altered to create knobs and corresponding holes in the two polypeptide chains. For instance, a "protuberance" comprising one or more amino acid modifications may be added to one chain to increase the bulk (e.g., the total volume) taken up by the amino acids. For instance, smaller amino acids can be modified or replaced by those having larger side chains which projects from the interface of the first polypeptide chain and can therefore be positioned in a related cavity in the adjacent second polypeptide chain so as to stabilize the heterodimer, and thereby favor heterodimer formation over homodimer formation. The protuberance may exist in the original interface or may be introduced synthetically (e.g., by altering one or more nucleic acid encoding the amino acid(s) at the interface). In some embodiments, a protuberance is introduced by modifying the nucleic acid encoding at least one '"original" amino acid residue in the interface of the first polypeptide with a nucleic acid encoding at least one "engineered" amino acid residue which has a larger side chain volume than die original amino acid residue, it will be appreciated that there can be more than one original and corresponding engineered residue. The upper limit for the number of original residues which are replaced is the total number of residues in the interface of the first polypeptide.

[0301] In addition to a knob (protuberance) added to one chain, a "cavity"' (hole) may be added to the second chain, comprising to at least one amino acid side chain which is recessed from the interface of the first or second polypeptide chain and therefore accommodates a corresponding protuberance on the adjacent second polypeptide chain. The cavity may exist in the original interface or may be introduced synthetically (e.g., by altering one or more nucleic acid encoding the amino acid(s) at the interface). In some embodiments, a protuberance is introduced by modifying the nucleic acid encoding at least one "original" amino acid residue in the interface of the first polypeptide with a nucleic acid encoding at least one "engineered" amino acid residue which has a smaller side chain volume than the original amino acid residue. It will be appreciated that there can be more than one original and corresponding engineered residue. The upper limit for the number of original residues which are replaced is the total number of residues in the interface of the first polypeptide. In some embodiments, the first and second Fc regions comprise a set of heterodimerization mutations, e.g., a set of CH2 and / or CH3 heterodimerization mutations.

[0302] In some embodiments, the first and second Fc regions comprise a set of CH3 heterodimerization mutations, e.g., knobs-in-holes (Ridgway, et al., Protein Eng. 1996, 9:617- 621), electrostatic mutations, and other CH3 dimerization mutations described in Verdino, et al., Current Opinion in Chemical Engineering 2018, 19:107-123; WO2016118742; US Patent Nos. 9605084, 9701759, 10106624; US Patent Application Publication No. 20180362668.

[0303] In some embodiments, one of the first or second Fc region comprises a CH3 domain comprising an alanine at residue 407; and the other of the first or second Fc region comprises a CH3 domain comprising a valine or methionine at residue 366 and a valine at residue 409 (residues numbered according to the EU Index Numbering). In some embodiments, one of the first or second Fc region comprises a CH3 domain comprising an alanine at residue 407, a methionine at residue 399, and an aspartic acid at residue 360; and the other of said first or second Fc region comprises a CH3 domain comprising a valine at residue 366, a valine at residue 409, and an arginine at residues 345 and 347 (residues numbered according to the EU Index Numbering).

[0304] Exemplary formats of proteins comprising a TCR fragment comprising any one of the mutations in TCR Ca and / or CP disclosed herein and an antibody Fc region and, optionally, a second antigen-binding domain and / or an IL-1 molecule are disclosed herein below:

[0305] In certain embodiments, the invention relates to the protein according to the invention comprising a T cell receptor (TCR) fragment and a second antigen-binding domain, wherein the TCR fragment and the second antigen-binding domain are linked to each other via an antibody Fc region. That is in certain embodiments, the invention relates to a protein comprising at least a TCR fragment, a second antigen-binding domain and an antibody Fc region.

[0306] The TCR fragment, the second antigen-binding domain and the antibody Fc region may be linked together in any way that ensures functionality of the individual components. However, it is preferred that both the first TCR fragment and the second antigen-binding domain are linked to the N-terminal end and / or the C-terminal end of the antibody Fc region.

[0307] In certain embodiments, the antibody Fc region is a heterodimeric antibody Fc region. That is, the amino acid sequence of one of the polypeptide chains of the antibody Fc region has been modified such that the two polypeptide chains of the antibody Fc region are no longer identical. Heterodimeric antibody Fc regions have the advantage that asymmetrical proteins can be generated in which different components are linked to the first and second polypeptide chain of the heterodimeric antibody Fc region. Methods for generating heterodimeric antibody Fc regions are disclosed elsewhere herein.

[0308] In a particular embodiment, the invention relates to the protein according to the invention, wherein the antibody Fc region is a heterodimeric Fc region comprising a first and second polypeptide chain, wherein the TCR fragment is linked to the first polypeptide chain comprised in the antibody Fc region and the second antigen-binding domain is linked to the second polypeptide chain comprised in the antibody Fc region.

[0309] In certain embodiments, the protein according to the invention comprises a heterodimeric antibody Fc region, wherein the first polypeptide chain comprised in the heterodimeric antibody Fc region is linked to the TCR fragment, preferably via the N-terminal end of the first polypeptide chain of the heterodimeric antibody Fc region, and / or wherein the second polypeptide chain comprised in the heterodimeric antibody Fc region is linked to the second antigen-binding domain, preferably via the N-terminal end of the second polypeptide chain of the heterodimeric antibody Fc region

[0310] That is, in a particular embodiment, the invention relates to the protein according to the invention, wherein the TCR fragment is linked to the N-terminal end of the first polypeptide chain comprised in the antibody Fc region via a C-terminal end of a polypeptide chain comprised in the first TCR fragment and / or wherein the second antigen-binding domain is linked to the N-terminal end of the second polypeptide chain comprised in the antibody Fc region via a C-terminal end of a polypeptide chain comprised in the second antigen-binding domain.

[0311] In certain embodiments, the TCR fragment is an ap-heterodimeric TCR fragment comprising an alpha and beta chain, and the second antigen-binding domain is an scFv fragment. In such embodiments, the TCR fragment may be linked to the N-terminal end of the first polypeptide chain of the heterodimeric antibody Fc region via the C-terminal end of the alpha or beta chain of the ap-heterodimeric TCR fragment and the second antigen-binding domain may be linked to the N-terminal end of the second polypeptide chain of the heterodimeric antibody Fc region via the C-terminal end of the scFv fragment.

[0312] In certain embodiments, the TCR fragment is an ap-heterodimeric TCR fragment comprising an alpha and beta chain, and the second antigen-binding domain is a Fab fragment. In such embodiments, the TCR fragment may be linked to the N-terminal end of the first polypeptide chain of the heterodimeric antibody Fc region via the C-terminal end of the alpha or beta chain of the ap-heterodimeric TCR fragment and the second antigen-binding domain may be linked to the N-terminal end of the second polypeptide chain of the heterodimeric antibody Fc region via a C-terminal end of a polypeptide chain comprised in the Fab fragment.

[0313] The protein comprising the heterodimeric antibody Fc region, the TCR fragment and the second antigen-binding domain may comprise further TCR fragments. That is, in a particular embodiment, the invention relates to the protein according to the invention, wherein the protein comprises at least one additional TCR fragment.

[0314] In a particular embodiment, the invention relates to the protein according to the invention, wherein the at least one additional TCR fragment is linked to an N-terminal end of a polypeptide chain comprised in the first TCR fragment via a C-terminal end of a polypeptide chain comprised in the at least one additional TCR fragment; and / or linked to a C-terminal end of the first or second polypeptide chain comprised in the antibody Fc region via an N-terminal end of a polypeptide chain comprised in the at least one additional TCR fragment.

[0315] Accordingly, in certain embodiments, the invention relates to the protein according to the invention, wherein the at least one additional TCR fragment is linked to an N-terminal end of a polypeptide chain comprised in the first TCR fragment via a C-terminal end of a polypeptide chain comprised in the at least one additional TCR fragment.

[0316] That is, the first polypeptide chain of the heterodimeric antibody Fc region may comprise two or more TCR fragments linked to its N-terminal end.

[0317] In certain embodiments, two afJ-heterodimeric TCR fragments may be linked to the N- terminal end of the first polypeptide chain of the heterodimeric antibody Fc region. That is, a first a|3-heterodimeric TCR fragment may be linked via the C-terminal end of its alpha or beta chain to the N-terminal end of the first polypeptide chain of the heterodimeric antibody Fc region and a further afJ-heterodimeric TCR fragment may be linked via the C-terminal end of its alpha or beta chain to the N-terminal end of the alpha or beta chain of the first heterodimeric TCR fragment.

[0318] In such embodiments where two or more TCR fragments are linked to the N-terminal end of the first polypeptide chain of the heterodimeric antibody Fc region, the second antigenbinding domain may be linked to the N-terminal end of the second polypeptide chain of the heterodimeric antibody Fc region as described elsewhere herein.

[0319] In a particular embodiment, the invention relates to the protein according to the invention, wherein the at least one additional TCR fragment is linked to a C-terminal end of the first and / or second polypeptide chain comprised in the antibody Fc region via an N-terminal end of a polypeptide chain comprised in the at least one additional TCR fragment.

[0320] That is, the further TCR fragment may also be linked to the C-terminal end of the first and / or second polypeptide chain of the heterodimeric antibody Fc region. In such embodiments, the first TCR fragment and the second antigen-binding domain, and optionally further TCR fragments, may be simultaneously attached to the N- and C-terminal ends of the first and second polypeptide chain of the heterodimeric antibody Fc region as described herein.

[0321] The further TCR fragment that is attached to the C-terminal end of the first and / or second polypeptide chain of the heterodimeric antibody Fc region is preferably an ap-heterodimeric TCR fragment. In such embodiments, the further TCR fragment may be linked to the C-terminal end of the first and / or second polypeptide chain of the heterodimeric antibody Fc region via the N-terminal end of the alpha or beta chain of the ap-heterodimeric TCR fragment.

[0322] In certain embodiments, the further TCR fragment that is attached to the C-terminal end of the first and / or second polypeptide chain of the heterodimeric antibody Fc region is an ap- heterodimeric TCR fragment. In such embodiments, the further TCR fragment may be linked to the C-terminal end of the first and / or second polypeptide chain of the heterodimeric antibody Fc region via the N-terminal end of the alpha chain of the ap-heterodimeric TCR fragment.

[0323] In certain embodiments, the further TCR fragment that is attached to the C-terminal end of the first and / or second polypeptide chain of the heterodimeric antibody Fc region is an ap- heterodimeric TCR fragment. In such embodiments, the further TCR fragment may be linked to the C-terminal end of the first and / or second polypeptide chain of the heterodimeric antibody Fc region via the N-terminal end of the beta chain of the ap-heterodimeric TCR fragment.

[0324] In certain embodiments, further TCR fragments may be linked to the N-terminal end of the first TCR fragment and to the C-terminal end of the antibody Fc region. Such a protein may comprise three or more TCR fragments. In certain embodiments, the first TCR fragment and the second antigen-binding domain may be linked to the N-terminal ends of the heterodimeric antibody Fc region as described herein above. In such embodiments, a second TCR fragment may be linked via a C-terminal end of a polypeptide chain comprised in the second TCR fragment to an N-terminal end of a polypeptide chain comprised in the first TCR fragment and a third TCR fragment may be linked via an N-terminal end of a polypeptide chain comprised in the third TCR fragment to a C-terminal end of a polypeptide chain comprised in the antibody Fc region. In certain embodiments, the third TCR fragment is linked to the C-terminal end of the polypeptide chain of the heterodimeric antibody Fc region that is linked to the second antigen-binding domain via its N-terminal end. In certain embodiments, the third TCR fragment is linked to the C-terminal end of the polypeptide chain of the heterodimeric antibody Fc region that is linked to the first and second TCR fragment via its N-terminal end.

[0325] In certain embodiments, the antibody Fc region is a homodimeric antibody Fc region. That is, the resulting protein is symmetric and both polypeptide chains of the antibody Fc region are linked to the same components, in the same order.

[0326] Accordingly, in a particular embodiment, the invention relates to the prtoein according to the invention, wherein the antibody Fc region is a homodimeric Fc region comprising a first and second polypeptide chain, wherein the first TCR fragment is linked to an N-terminal end of a polypeptide chain comprised in the antibody Fc region via a C-terminal end of a polypeptide chain comprised in the first TCR fragment; and the second antigen-binding domain is linked to a C-terminal end of a polypeptide chain comprised in the antibody Fc region via an N-terminal end of a polypeptide chain comprised in the second antigen-binding domain.

[0327] That is, both polypeptide chains of the antibody Fc region may be linked to the first TCR fragment and the second antigen-binding domain.

[0328] Preferably, the TCR fragment is linked to the N-terminal end of the antibody Fc region and the second antigen-binding domain is linked to the C-terminal end of the antibody Fc region.

[0329] In certain embodiments, the first TCR fragment is an ap-heterodimeric TCR fragment that is linked to the N-terminal end of a polypeptide chain comprised in the antibody Fc region via the C-terminal end of the alpha or beta chain of the heterodimeric TCR fragment and the second antigen-binding domain is an scFv fragment that is linked to the C-terminal end of a polypeptide chain comprised in the antibody Fc region via the N-terminal end of the scFv fragment.

[0330] In certain embodiments, the first TCR fragment is an ap-heterodimeric TCR fragment that is linked to the N-terminal end of a polypeptide chain comprised in the antibody Fc region via the C-terminal end of the alpha chain of the heterodimeric TCR fragment and the second antigen-binding domain is an scFv fragment that is linked to the C-terminal end of a polypeptide chain comprised in the antibody Fc region via the N-terminal end of the scFv fragment.

[0331] In certain embodiments, the first TCR fragment is an ap-heterodimeric TCR fragment that is linked to the N-terminal end of a polypeptide chain comprised in the antibody Fc region via the C-terminal end of the alpha or beta chain of the heterodimeric TCR fragment and the second antigen-binding domain is a Fab fragment that is linked to the C-terminal end of a polypeptide chain comprised in the antibody Fc region via the N-terminal end of a polypeptide chain comprised in the Fab fragment.

[0332] In certain embodiments, the first TCR fragment is an ap-heterodimeric TCR fragment that is linked to the N-terminal end of a polypeptide chain comprised in the antibody Fc region via the C-terminal end of the alpha chain of the heterodimeric TCR fragment and the second antigen-binding domain is a Fab fragment that is linked to the C-terminal end of a polypeptide chain comprised in the antibody Fc region via the N-terminal end of a polypeptide chain comprised in the Fab fragment.

[0333] In certain embodiments, the TCR fragment may be linked to the C-terminal end of the homodimeric antibody Fc region and the second antigen-binding domain may be linked to the N-terminal end of the homodimeric antibody Fc region.

[0334] The protein comprising a TCR fragment, a second antigen-binding domain and an antibody Fc region disclosed herein above may further comprise an IL-2 variant at any suitable position.

[0335] In certain embodiments, the protein of the invention comprises a TCR fragment comprising one or more mutations disclosed herein, a second antigen-binding domain and an antibody Fc region.

[0336] In certain embodiments, protein of the invention may have the format of a bispecific antibody, wherein a TCR fragment is linked to the first heavy chain comprised in the antibody Fc region and a second antigen-binding domain is linked to the second heavy chain comprised in the antibody Fc region. Preferably, the TCR fragment and the second antigen-binding domain are linked to the N-terminal hinge region of the antibody Fc region.

[0337] In certain embodiments, a first TCR fragment is linked to a first polypeptide chain comprised in the antibody Fc region and a second TCR fragment is linked to a second polypeptide chain comprised in the antibody Fc region. Preferably, the two TCR fragments are linked to the N- terminal ends of the two heavy chains comprised in the antibody Fc region, more preferably via the hinge region.

[0338] In certain embodiments, the two TCR fragments are identical TCR fragments. In certain embodiments, the two TCR fragments are different TCR fragments, i.e., TCR fragments that recognize different antigens or different epitopes of the same antigen. In this case, the protein of the invention may function as a bispecific TCR. An IL-2 molecule and / or a second antigen-binding domain may be linked to such a protein at any suitable position. In certain embodiments, the IL-2 molecule and / or the second antigenbinding domain are linked to an accessible end of a TCR fragment. Accessible ends may be the N-terminal ends of the polypeptide chains comprised in the TCR fragment or the C-terminal end of the polypeptide chain that is not linked to the antibody Fc region.

[0339] In certain embodiments, the IL-2 molecule is linked to an N-terminal end of the TCR fragment. In certain embodiments, the IL-2 molecule is linked to the same polypeptide chain of the TCR fragment that is linked to the antibody Fc region. In certain embodiments, the IL-2 molecule is linked to the polypeptide chain of the TCR fragment that is not linked to the antibody Fc region.

[0340] In certain embodiments, the IL-2 molecule is linked to a C-terminal end of the TCR fragment, in particular the C-terminal end of the polypeptide chain that is not linked to the antibody Fc region.

[0341] In certain embodiments, the second antigen-binding domain is linked to an N-terminal end of the TCR fragment. In certain embodiments, the second antigen-binding domain is linked to the same polypeptide chain of the TCR fragment that is linked to the antibody Fc region. In certain embodiments, the second antigen-binding domain is linked to the polypeptide chain of the TCR fragment that is not linked to the antibody Fc region.

[0342] In certain embodiments, the second antigen-binding domain is linked to a C-terminal end of the TCR fragment, in particular the C-terminal end of the polypeptide chain that is not linked to the antibody Fc region.

[0343] Alternatively, a TCR fragment and an IL-2 molecule may be linked to the N-terminal ends of the antibody Fc region. Thus, in certain embodiments, the TCR fragment is linked to a first polypeptide chain comprised in the antibody Fc region and the IL-2 molecule in linked to a second polypeptide chain comprised in the antibody Fc region. Preferably, the TCR fragment is linked to the N-terminal end of the first polypeptide chain of the antibody Fc region and the IL-2 molecule is linked to the N-terminal end of second polypeptide chain of the antibody Fc region via the hinge region.

[0344] In certain embodiments, an additional second antigen-binding domain may be linked to an accessible N- or C-terminal end of the TCR fragment. In certain embodiments, the second antigen-binding domain may be linked to the N-terminal end of the same polypeptide chain of the TCR fragment that is linked to the antibody Fc region. In certain embodiments, the second antigen-binding domain may be linked to the N- or C-terminal end of the other polypeptide chain comprised in the TCR fragment.

[0345] In certain embodiments, a further TCR fragment may be linked to the antibody Fc region via the IL-2 molecule. That is, the first polypeptide chain comprised in the antibody Fc region may be directly linked to a first TCR fragment via the N-terminal end of said first polypeptide chain and the second polypeptide chain comprised in the antibody Fc region may be indirectly linked to a second TCR fragment via the IL-2 molecule that is linked to the N-terminal end of said second polypeptide chain.

[0346] In certain embodiments, the first polypeptide chain comprised in the antibody Fc region may be directly linked to a TCR fragment via the N-terminal end of said first polypeptide chain and the second polypeptide chain comprised in the antibody Fc region may be indirectly linked to a second antigen-binding domain via the IL-2 molecule that is linked to the N-terminal end of said second polypeptide chain.

[0347] In certain embodiments, both the TCR fragment and the second antigen-binding domain may be linked to the N-terminal end of the same polypeptide chain of the antibody Fc region.

[0348] This may be achieved by linking the antibody Fc region to the C-terminal end of a first polypeptide chain comprised in the TCR fragment and by linking the second antigen-binding domain to the C-terminal end of a second polypeptide chain comprised in the TCR fragment.

[0349] In certain embodiments, the antibody Fc region may be linked to a C-terminal end of the TCR fragment and the second antigen-binding domain may be linked to an N-terminal end of the TCR fragment. In certain embodiments, the second antigen-binding domain and the antibody Fc region may be linked to the same polypeptide chain of the TCR fragment. Alternatively, the second antigen-binding domain may be linked to a first polypeptide chain comprised in the TCR fragment and the antibody Fc region may be linked to a second polypeptide chain comprised in the TCR fragment. Such molecules may further comprise and IL-2 molecule at any suitable position.

[0350] In certain embodiments, two TCR fragments may be sequentially linked to the same polypeptide chain of the antibody Fc region.

[0351] In certain embodiments, the TCR fragment may be linked to the antibody Fc region via the second antigen-binding domain. That is, in certain embodiments, the antibody Fc region may be linked to the C-terminal end of a first polypeptide chain comprised in the second antigenbinding domain and the TCR fragment may be linked to the N-terminal end of the same polypeptide chain. Alternatively, the antibody Fc region may be linked to the C-terminal end of a first polypeptide chain comprised in the second antigen-binding domain and the TCR fragment may be linked to the N-terminal end of a second polypeptide chain comprised in the second antigen-binding domain.

[0352] In certain embodiments, the second antigen-binding domain consists of a single polypeptide chain, such as an scFv. In such embodiments, the antibody Fc region may be linked to the C- terminal end of the scFv and the TCR fragment may be linked to the N-terminal end of the scFv, or vice versa.

[0353] It is to be understood that the first and second polypeptide chain comprised in the antibody Fc region are preferably derived from the heavy chain of an IgG antibody and comprise the hinge region.

[0354] In a particular embodiment, the invention relates to the protein according to the invention, wherein the TCR fragment is directly linked to the antibody Fc region or indirectly linked to the antibody Fc region via the second antigen-binding domain and / or the IL-2 molecule.

[0355] Within the present invention, a TCR fragment is said to be directly linked to an antibody Fc region if there is no biologically active molecule located between the TCR fragment and the antibody Fc region. However, a TCR fragment may still be said to be directly linked to an antibody Fc region if it is linked to the antibody Fc region via a short peptide linker, such as a flexible glycine-serine-rich linker.

[0356] Alternatively, a TCR fragment may be indirectly linked to the antibody Fc region via one or more additional components, i.e., a second antigen-binding domain and / or an IL-2 molecule. For example, the TCR fragment may be linked to the antibody Fc region via an IL-2 molecule and / or a second antigen-binding domain.

[0357] In a particular embodiment, the invention relates to the protein according to the invention, wherein the second antigen-binding domain and / or the IL-2 molecule are linked to a C- terminal end of a first polypeptide chain comprised in the TCR fragment, preferably wherein the TCR fragment is linked to the antibody Fc region via a C-terminal end of a second polypeptide chain comprised in the TCR fragment.

[0358] In certain embodiments, the second antigen-binding domain and / or the IL-2 molecule may be linked to a C-terminal end of a first polypeptide chain comprised in the TCR fragment. More preferably, the second polypeptide chain comprised in the TCR fragment is simultaneously linked to the antibody Fc region. &7

[0359] That is, in certain embodiments, the first polypeptide chain comprised in the TCR fragment is linked to a second antigen-binding domain and the second polypeptide chain comprised in the TCR fragment is linked to an antibody Fc region. In certain embodiments, such a molecule may further comprise an IL-2 molecule in a suitable position.

[0360] In certain embodiments, the first polypeptide chain comprised in the TCR fragment is linked to an IL-2 molecule and the second polypeptide chain comprised in the TCR fragment is linked to an antibody Fc region.

[0361] It is preferred herein that the TCR fragment, the second antigen-binding domain and / or the IL-2 molecule are linked to the N-terminal end of the antibody Fc region, preferably via the hinge region, either directly or indirectly. However, one or more of these components may be alternatively or additionally linked to the C-terminal end of the antibody Fc region. For example, in certain embodiments, an IL-2 molecule may be linked to the C-terminal end of the antibody Fc region. In other embodiments, a second antigen-binding domain may be linked to the C-terminal end of the antibody Fc region. In certain embodiments, the IL-2 molecule may be linked to a first polypeptide chain comprised in the antibody Fc region, preferably via the C-terminal end of the polypeptide chain comprised in the antibody Fc region, and the second antigen-binding domain may be linked to a second polypeptide chain comprised in the antibody Fc region, preferably via the C-terminal end of the polypeptide chain comprised in the antibody Fc region.

[0362] In other embodiments, a TCR fragment may be linked to the C-terminal end of the antibody Fc region. In certain embodiments, a first TCR fragment may be linked to the N-terminal end of the antibody Fc region and a second TCR fragment may be linked to the C-terminal end of the antibody Fc region. In certain embodiments, the first TCR fragment may be linked to the N-terminal end of a first heavy chain comprised in the antibody Fc region and the second TCR fragment may be linked to the C-terminal end of the same heavy chain. In certain embodiments, the first TCR fragment may be linked to the N-terminal end of a first heavy chain comprised in the antibody Fc region and the second TCR fragment may be linked to the C-terminal end of a second heavy chain comprised in the antibody Fc region. Such molecules may further comprise an IL-2 molecule at suitable positions.

[0363] It is to be understood that in all embodiments in which the protein of the invention comprises two TCR fragments or more, these TCR fragment may recognize the same target or different targets. Within the present invention, the individual components of the protein of the invention (the TCR fragment and one or more of the IL-2 molecule, the second antigen-binding domain, and / or the antibody Fc region) are linked to each other. The individual components may be linked in any way known in the art. It is, however, preferred that the individual components are covalently linked.

[0364] The protein according to the invention may be a fusion protein. That is, two or more polypeptide chains derived from individual components are genetically fused together. Here, the term "genetic fusion" refers to a co-linear, covalent linkage of two or more proteins or fragments thereof via their individual peptide backbones, through genetic expression of a polynucleotide molecule encoding those proteins. Genetic engineering methods that may be used for obtaining genetic fusions are well known in the art.

[0365] Any of the protein-based components may be linked with a peptide linker. The term "peptide linker", "linker" or "spacer" as used herein refers to a spacer acting as a hinge region between polypeptide domains, allowing them to move independently from one another while maintaining the three-dimensional form of the individual domains. In a preferred embodiment, all protein-based components, i.e., the TCR fragment and, optionally, the second antigen-binding domain, the IL-2 molecule and / or the antibody Fc region, are linked with a peptide linker.

[0366] The length of the spacer can vary; typically, the number of amino acids in the spacer is 100 or less amino acids, preferably 50 or less amino acids, more preferably 40 or less amino acids, still more preferably, 30 or less amino acids, or even more preferably 20 or less amino acids. Preferred ranges are from 5 to 50 amino acids.

[0367] In preferred embodiments, said spacer is a peptide having structural flexibility (i.e., a flexible linking peptide or "flexible linker") and comprises 2 or more amino acids selected from the group consisting of glycine, serine, alanine and threonine. Preferably, wherein at least 65%, preferably 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the amino acids in said flexible peptide linker are selected from the group consisting of glycine, serine, alanine and threonine.

[0368] In certain embodiments, the flexible linker comprises the sequence (T / S)-(NxGGGGS)-G-(S / T) where N = 1,2, 3, 4, 5. That is, in certain embodiments, the flexible linker comprises the sequence (T / S)-(lxGGGGS)-G-(S / T) (SEQ ID NO:28), (T / S)-(2xGGGGS)-G-(S / T) (SEQ ID NO:29), (T / S)-(3xGGGGS)-G-(S / T) (SEQ ID NO:30), (T / S)-(4xGGGGS)-G-(S / T) (SEQ ID NO:31), or (T / S)- (5xGGGGS)-G-(S / T) (SEQ ID NO:32). In certain embodiments, all protein-based components, i.e., the TCR fragment and one or more of the second antigen-binding domain, the IL-2 molecule and / or the antibody Fc region, are linked with a flexible linker.

[0369] In certain embodiments, the linker is an alpha-helical linker. Due to their rigid structure, alphahelical linkers can be used to establish a defined spatial distance between the two components of the protein that are linked with the alpha helical linker. Alpha-helical linkers are known in the art and the skilled person is capable of identifying alpha-helical linkers. Non-limiting examples of alpha-helical linkers include (T / S)-A(3xEAAAK)A-G-(S / T) (SEQ ID NO:33), (T / S)- A(6xEAAAK)A-G-(S / T) (SEQ ID NO:34) and (T / S)-(A-4xEAAAK-A)-LE-(A-4xEAAAK-A)-G-(S / T) (SEQ ID NO:35).

[0370] Alternatively, linkers from naturally occurring proteins may be used to connect two components of the protein of the invention. Non limiting examples are the naturally occurring linker derived from the EPO receptor (T / S-(G-INEVVLLDAP)-G-(S / T) (SEQ ID NO:36)) and the natural occurring linker derived from T LYMPHOCYTE ACTIVATION ANTIGEN (CD80) (T / S-(G- LSVKADF)-G-(S / T) / SEQ ID NO:37)).

[0371] Proteins comprising three or more components may comprise two or more identical or different peptide linkers to connect the individual components. That is, the protein of the invention may comprise different types of linkers, including flexible, alpha-helical and / or naturally occurring linkers. Preferably, all components of the protein are linked with a peptide linker.

[0372] In some embodiments, the protein described herein may be a transmembrane protein. In some embodiments, the first polypeptide of the protein may further comprise the transmembrane and intracellular domains of the TCR a chain; and the second polypeptide may further comprise the transmembrane and intracellular domains of the TCR chain. Also provided herein are T cells comprising a TCR that comprises a Ca domain and C0 domain comprising any of the mutations disclosed herein. Such TCR may also include an antigen binding domain, e.g., an antibody fragment or TCR Va / V0 domain.

[0373] However, it is preferred herein that the protein according to the invention is a soluble protein, such as a soluble TCR or a fusion protein comprising a soluble TCR. Particularly preferred is that the soluble TCR comprises a fragment of a TCR alpha chain and a fragment of a TCR beta chain, wherein the constant regions are truncated at positions Cys213 (TRAC) and Cys247 (TRBC) (Kabat numbering).

[0374] In another aspect, provided herein are nucleic acids encoding one or more polypeptides of the proteins described herein. Such nucleic acid may encode a polypeptide comprising a TCR Ca domain comprising one or more of the mutations disclosed herein and / or a polypeptide comprising a TCR CP domain comprising one or more of the mutations disclosed herein.

[0375] That is, the present invention further relates to a nucleic acid molecule encoding the protein provided and described herein. As used herein, unless specifically defined otherwise, the term "nucleic acid" or "nucleic acid molecule" is used synonymously with "oligonucleotide", "nucleic acid strand", "polynucleotide", or the like, and means a polymer comprising one, two, or more nucleotides. The term "nucleic acid molecule" relates to the sequence of bases comprising purine- and pyrimidine bases which are comprised by polynucleotides, whereby said bases represent the primary structure of a nucleic acid molecule. Herein, the term "nucleic acid molecule" includes all kinds of nucleic acid, including DNA, cDNA, genomic DNA, RNA, synthetic forms of DNA and mixed polymers comprising two or more of these molecules, and preferably relates to DNA and cDNA. As readily understood by those of skill in the art, the nucleic acid sequences provided herein represent sequences of DNA and also comprise corresponding RNA sequences where T is replaced by U. The term "nucleic acid molecule" generally comprises sense and antisense strands. "Nucleic acid molecule" may further comprise non-natural or derivatized nucleotide bases as well as natural or artificial nucleotide analogues, e.g., in order to protect the nucleic acid molecule against endo- and / or exonucleases as will be readily appreciated by those skilled in the art.

[0376] In another aspect, provided herein are vectors comprising nucleic acids encoding a polypeptide of the proteins described herein. Such vectors can further include an expression control sequence operably linked to the nucleic acid encoding a polypeptide of the proteins described herein. Expression vectors capable of direct expression of coding sequences to which they are operably linked are well known in the art. Expression vectors may encode a signal peptide that facilitates secretion of the polypeptides from a host cell. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide. The expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Expression vectors may contain selection markers, e.g., tetracycline, neomycin, and dihydrofolate reductase, to permit detection of those cells transformed with the desired DNA sequences. The vectors containing the polynucleotide sequences of interest may be transferred into the host cell by well-known methods (e.g., stable or transient transfection, transformation, transduction or infection), which vary depending on the type of cellular host.

[0377] The term "vector" as used herein generally comprises all kinds of linear or circular nucleic acid molecules which can replicate autonomously is a suitable host cell. Such vectors comprise, but are not limited to, plasmids, cosmids, phages, virus (e.g., adeno-, adeno-associated-, lenti-, or preferably retroviral vectors), and other vectors or shuttles known in the art which are suitable to carry and transfer genes into host cells in order to allow stable or transient translation and constitutive or conditional expression of the inventive fusion protein in the host cell. The vector is usually not integrated into the cell genome, but may also be integrated. Vectors according to the present invention which comprise nucleic acid molecules as described and provided herein preferably allow stable expression of the fusion protein of the present invention in the host cell (expression vector). Vectors of the present invention may further comprise marker genes, promoter and / or enhancer sequences (operably linked to the nucleic acid molecule of the present invention), replication origin suitable for the respective host cell, restriction sited, multiple cloning sites, labels and further functional units as known in the art. The vectors may inter alia be transferred into host cells via a shuttle such as a virus (which may itself be considered a vector), or be nakedly transformed or transduced into host cells. The vector is preferably adapted to suit to the respective host cell where it is to be transformed or transduced into. The skilled person will readily understand that different host cells will require different kinds of vectors. For example, plasmids are suitable vectors for transformation of bacterial cells, while retroviral vectors are suitable for transduction into eukaryotic cells (e.g., T cells). In one embodiment of the present invention, the vector of the present invention is a viral vector, e.g., a retroviral or lentiviral vector.

[0378] Also provided herein are host cells, e.g., mammalian cells, comprising a nucleic acid or vector described herein; such host cells may express the proteins described herein. Mammalian host cells known to be capable of expressing functional proteins include CHO cells, HEK293 cells, COS cells, and NSO cells. The present disclosure further provides a process for producing a protein described herein by cultivating the host cell described above under conditions such that the protein is expressed, and recovering the expressed protein.

[0379] In another aspect, provided herein are pharmaceutical compositions comprising a protein, nucleic acid, vector, or cell described herein. Such pharmaceutical compositions can also comprise one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0380] That is, the present invention further relates to a pharmaceutical composition comprising a protein, a nucleic acid molecule, a vector, and / or a host cell as described and provided by the present invention. Such pharmaceutical composition is suitable to be administered to a patient (preferably, a human patient).

[0381] Medicaments in accordance with the invention will usually be supplied as part of a sterile, pharmaceutical composition which will normally include a pharmaceutically acceptable carrier. This pharmaceutical composition may be in any suitable form, (depending upon the desired method of administering it to a patient). It may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms.

[0382] The pharmaceutical composition may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such compositions may be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

[0383] Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solution which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Excipients which may be used for injectable solutions include water, alcohols, polyols, glycerine and vegetable oils, for example. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

[0384] The pharmaceutical compositions may contain preserving agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, colourants, odourants, salts (substances of the present invention may themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents or antioxidants. They may also contain therapeutically active agents in addition to the substance of the present invention.

[0385] Dosages of the substances of the present invention can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used. The dosage may be repeated as often as appropriate. If side effects develop the amount and / or frequency of the dosage can be reduced, in accordance with normal clinical practice.

[0386] As is common in anti-cancer and autoimmune therapy the protein of this invention may be used in combination with other agents for the treatment of cancer and autoimmune disease, and other related conditions found in similar patient groups. 13

[0387] Accordingly, the present invention also relates to methods for treating a disease or disorder by administering comprising a pharmaceutical composition comprising a protein, a nucleic acid molecule, a vector, and / or a host cell as described and provided by the present invention.

[0388] Thus, in a particular embodiment, the invention relates to the protein according to the invention, the nucleic acid according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention for use in medicine, in particular for use in the treatment of cancer.

[0389] The protein of the invention may be used in the treatment of various diseases, including cancer, autoimmune diseases and viral infections.

[0390] Thus, in certain embodiments, the invention relates to the protein according to the invention, the nucleic acid according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention for use in treating cancer.

[0391] Various cancer antigens, including neoantigens, have been described in the art. The skilled person would thus have no difficulties generating a protein of the invention comprising a TCR fragment that binds to a specific cancer antigen.

[0392] The term "cancer" as used herein refers to any malignant neoplasm. The malignant neoplasm refers to diseases resulting from the undesired growth, the invasion, and under certain conditions metastasis of impaired cells in an organism. The cells giving rise to cancer are genetically impaired and have usually lost their ability to control cell division, cell migration behavior, differentiation status and / or cell death machinery. Most cancers form a tumor but some hematopoietic cancers, such as leukemia, do not. Symptoms and staging systems forthe different cancers are well known in the art and described in standard textbooks of pathology. Cancer as used herein encompasses any stage, grade, morphological feature, invasiveness, aggressiveness or malignancy of the cancer or the tissue or organ affected thereby.

[0393] In certain embodiments, the invention relates to the protein according to the invention, the nucleic acid according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention for use in treating autoimmune diseases. As explained herein above, a protein for the treatment of autoimmune diseases preferably comprise a tissue- or organ-specific TCR fragment and / or an immunosuppressive agent. In certain embodiments, the invention relates to the protein according to the invention, the nucleic acid according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention for use in treating viral infections.

[0394] The skilled person is aware of soluble TCR fragments that may be used in the context of the present invention for treating autoimmune diseases and viral infections.

[0395] In certain embodiments, the invention relates to a method of treating cancer in a subject in need, wherein the method comprises the administration of a protein according to the invention to said subject.

[0396] In certain embodiments, the invention relates to a method of treating a viral infection in a subject in need, wherein the method comprises the administration of a protein according to the invention to said subject.

[0397] In certain embodiments, the invention relates to a method of treating an autoimmune disease in a subject in need, wherein the method comprises the administration of a protein according to the invention to said subject.

[0398] The term "subject," as used herein, generally refers to any animal, e.g., a mammal or marsupial. A subject may be a patient. A subject may be symptomatic or asymptomatic with respect to a disease or ailment. A subject may be primate (e.g., a human), non-human primate (e.g., rhesus or other types of macaques), dog, cat, mouse, pig, horse, donkey, cow, sheep, rat, and fowl. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Preferably, the protein of the invention is used for the treatment of humans. To reduce the risk of immunogenic reactions, it is thus preferred that all components of the protein of the invention, i.e., the soluble TCR fragment, the IL-2 molecule and, optionally, the antibody Fc region and / or the second antigen-binding domain are of human origin.

[0399] The terms "treatment" and "treating," as used herein, refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and / or a prophylactic benefit. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, a composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested. IS

[0400] In another aspect, provided herein are methods of treating cancer or infection in a subject in need thereof by administering to the subject a therapeutically effective amount of a protein, nucleic acid, vector, cell, or pharmaceutical composition described herein.

[0401] The term "therapeutically effective amount," as used herein, refers to an amount of a protein or nucleic acid or vector or cell or composition of the invention that will elicit the biological or medical response of a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc. In one non-limiting embodiment, the term "a therapeutically effective amount" refers to the amount of a protein or nucleic acid or vector or cell or composition of the invention that, when administered to a subject, is effective to at least partially alleviate, inhibit, prevent and / or ameliorate a condition, or a disorder or a disease.

[0402] Also provided are proteins, nucleic acids, vectors, cells, and pharmaceutical compositions described herein for use in a therapy. Furthermore, the present disclosure also provides proteins, nucleic acids, vectors, cells, or pharmaceutical compositions described herein for use in the treatment of cancer or infection. Additionally, the present disclosure provides the use of a protein, nucleic acid, vector, cell, or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer or infection.

[0403] As used herein, the term "a," "an," "the" and similar terms used in the context of the present invention (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.

[0404] All numeric ranges provided herein are inclusive of narrower ranges; delineated upper and lower range limits are interchangeable to create further ranges not explicitly delineated. The number of significant digits conveys neither limitation on the indicated amounts nor on the accuracy of the measurements.

[0405] The term "about," as used herein, means approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 10%. Therefore, about 50% means in the range of 45%-55%. Numerical ranges recited herein by endpoints include all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about." BRIEF DESCRIPTION OF THE DRAWINGS

[0406] Fig. 1. Characterisation of unbiased TRAC and TRBC saturation mutagenesis libraries by deep sequencing. A-B. Distribution of observed TCR variants according to their number of amino acid substitutions relative to wild-type TRAC (A) or TRBC (B) in equimolar pools of rounds 1, 2, 3, 4 and 5 saturation mutagenesis amplicons. C-D. Observed cumulative frequencies of TRAC (C) or TRBC (D) variants according to the distance between pairs of mutations. A random distribution is represented by a dotted line. E-F. Distribution and frequency of mutated positions across the TRAC (E) or TRBC (F) domains within an equimolar pool of rounds 1, 2, 3, 4 and 5 saturation mutagenesis amplicons.

[0407] Fig. 2. Enrichment and sequencing of yeast-displayed TCR variants retaining antigen binding following thermal cycling. A. Schematic representation of the selection strategy of TRAC+TRBC pooled libraries by FACS enrichment then thermal cycling followed by MACS enrichment. B. Flow cytometry plots showing TCR clones with retained antigen binding following thermal cycling to 60°C.

[0408] Fig. 3. Candidate TCR stabilising substitutions enhance TCR pre-purification expression yields. Heat maps showing pre-purification expression levels from filtered HEK293 supernatants as determined by His-tag capture using biolayer interferometry. (A) Prepurification expression yields (1 mL scale) of a panel of soluble TCRMOH3 variants containing Boulter and native disulphides (BN) plus additional 90 candidate sets of stabilising mutations. Controls included TCRMOF containing native disulfide only (N, well H12), Boulter and native disulphides (BN, well G12), seven Froning mutations plus Boulter and native disulphides (FBN, well E6), Froning alpha mutations (FaBN, well F6) and Froning beta mutations (F BN, well F12). Sample in well A2 (marked with an "X") was lost due to filtration membrane clogging. (B) Pre-purification expression yields (3 mL scale) of selected expression-enhancing substitutions from panel A.

[0409] Fig. 4. Sample melting curves and aggregation curves showing increased heat resistance and aggregation resistance for selected variants.

[0410] Fig. 5. Biophysical characteristics of soluble TCRsioth-6 containing identified sets of stabilising substitutions. TCRsioth-6 expressed in TCR-Fab format in the context of Boulter-Native disulphide (BN), or with additional sets of stabilising mutations (Var3 [n=3], Var4 [n=2], FBN control [n=7]) were analysed by SDS-PAGE (A), size-exclusion chromatography following 4°C incubation (B) and biolayer interferometry (C). Fig. 6. Exemplary embodiments of the present invention comprising a soluble TCR fragment (dark grey) and a CD3 agonist (light grey). The left chain of each TCR fragment represents the TCR alpha chain and the right chain of each TCR fragment represents the TCR beta chain. IL-2 molecule(s) and, optionally, an antibody Fc region may be attached at suitable positions. Flexible linkers are shown as curved lines.

[0411] Fig. 7. Production of TCRsioth-6 variants containing different combinations of the V3 mutations. Comparison of TCRsioth-6 expressed in TCR-Fab format in the context of Boulter- Native disulphide (BN), or with additional single or double mutations from the V3 mutation set. Yields were determined after purification.

[0412] Fig. 8. Production yields of 4 TCRs originating from diverse germlines containing only the boulter-disulfide (BN) or the Var3 mutations in addition. TCR4 corresponds to TCRsioth-6. Yields were determined after purification.

[0413] EXAMPLES

[0414] The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. 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.

[0415] EXAMPLE 1: IDENTIFICATION OF STABILIZING MUTATIONS

[0416] 1.1 Generation of unbiased TCR constant domain mutagenesis libraries

[0417] A monocistronic TCR-Fab expression vector suitable for yeast surface display was generated comprising TRA and TRB sequences encoding TCR alpha and beta chains separated by a P2A self-processing peptide, with both chains fused to the yeast surface protein Aga2p for tethering to the cell membrane.

[0418] To obtain unbiased mutagenesis libraries of the constant TCR alpha (TRAC) and constant TCR beta (TRBC) chains, the inventors adapted the method reported by Bloom and colleagues (Bloom, J. D. An experimentally determined evolutionary model dramatically improves phylogenetic fit. Mol. Biol. Evol. 31, 1956-1978 (2014)) with the aim of generating variants that collectively have mutations in all codons across both domains. In brief, two sets of mutagenic primers (i.e., a forward set and a reverse set) were designed to span each entire domain, with each primer being on average 37 nucleotides in length and containing a single NNK degenerate codon roughly in the middle of its sequence. Next, each set was used separately for PCR amplification using TRAC(T166C) or TRBC(S173C) dsDNA sequence as a template and a corresponding reverse or forward non-mutagenic primer. This process resulted in the generation of two truncated pools of mutagenised dsDNA, which were then combined to perform a second PCR amplification step in which the generated pools prime each other. The end result of this process (i.e., a single round of saturation mutagenesis) was the generation of dsDNA in which the wild-type TRAC orTRBC sequence is mutagenised across the entire domain. Of note, two rounds of saturation mutagenesis have been previously reported by Bloom et al. to introduce an average of 1.4 mutations per gene.

[0419] In order to explore the effect of multiple mutations per variant on thermal stability, the inventors performed 5 sequential rounds of saturation mutagenesis for each TRAC and TRBC domains. Material from rounds 1-5 for each chain was then pooled in an equimolar fashion in order to obtain a more homogenous distribution of number of mutations per variant. The inventors assembled the resulting DNA fragments for each chain into a pool of expression vectors encoding the variable TCR domains of five affinity-enhanced TCRs recognising MAGE- A3168-176 with target affinities ranging from 10 pM to 350 nM. The resulting TRAC plasmid library and TRBC plasmid library were subjected to amplicon sequencing in order to assess the mutagenesis efficiency and distribution. Analysis of deep sequencing data revealed that the TRAC library contained a median number of mutations between 3 and 4, (Fig. 1A). The TRBC library contained a median number of mutations between 5 and 6 (Fig. IB). This median corresponds to the minimal number of mutations required to cover 50% of the pooled deep sequencing data from rounds 1-5.

[0420] Assessment of the distance between pairs of mutations followed a random cumulative distribution for both libraries, indicating a lack of bias for proximal or distant pairs of mutations (Figs. 1C and ID). The frequency of variants per position was largely homogeneous across domains, although some codons showed a lower than average mutation rate (e.g., codons 22- 23 for TRAC and 29 for TRBC), suggesting a lower performance of mutagenic primers in that region (Figs. IE and IF). In summary, the inventors successfully generated saturation mutagenesis libraries containing substitutions across every possible codon of TRAC and TRBC in a variety of mutational contexts (i.e., variants with a median of 3-6 substitutions per variant).

[0421] 1.2 Yeast surface display and thermal cycling selection of constant TCR domain mutagenesis libraries identifies heat-resistant clones In preparation for screening, the generated TRAC and TRBC plasmid libraries were pooled and used to generate an HDR amplicon for transformation of yeast cells, leading to the generation of >107transformants. After transformation, the inventors amplified the TRAC and TRBC transgenes by PCR in order to obtain deep sequencing reference datasets capturing the TCR variant frequencies in the unselected library. Due to the nature of the constructed saturation mutagenesis libraries (Table 3), the inventors expected full sampling of all variants with one substitution but inevitable undersampling of variants with two substitutions and above.

[0422] Table 3. Theoretical library diversities according to the number of substitutions per variant.

[0423] Following induction, cells were first enriched for retained antigen binding by staining with MAGE-A3 tetramer followed by fluorescence activated cell sorting (FACS). These populations were then grown to saturation in non-inducing medium, then induced again for 16-20 hours at 30°C. The inventors subjected these yeast cells to thermal cycling (10 cycles of: 1 min at 20°C followed by 1 min at 58°C). Magnetic-activated cell sorting (MACS) was performed immediately following thermal cycling to enrich for MAGE-A3 binders. In this workflow, retention of antigen binding served as a proxy for correct TCR folding at elevated temperatures (i.e., heat resistance). Since yeast cells lose their ability to multiply following incubation at elevated temperatures (>46°C), the inventors recovered the genetic material encoding TRAC and TRBC transgenes by means of PCR amplification, pooled the resulting amplicons, and used them to transform and induce fresh yeast cells, after which the genetic material of enriched yeast cells was recovered by PCR amplification of TRAC and TRBC transgenes in preparation for final validation and deep sequencing (Fig. 2A). Given that TRAC and TRBC libraries were constructed separately and pooled prior to transformation, amplification of transgenes from yeast genetic material was always performed separately for each chain, with pooling performed prior to re-transformation. In the case of deep sequencing libraries, the TRAC and TRBC amplicons were sequenced separately.

[0424] To assess the efficiency of the workflow, the inventors used the amplicons obtained from MACS-enriched fractions to transform and induce fresh yeast cells. Thermal cycling of these cells at 56°C, 58°C or 60°C, revealed a substantial enrichment of heat resistant TCR variants relative to the parental TRAC+TRBC library across all tested temperatures and, as expected, an inverse correlation between thermal cycling temperature and retained antigen binding (Fig. 2B).

[0425] 1.3 Identification of candidate stabilising mutations from heat resistant fractions

[0426] Following thermal cycling selections the inventors proceeded to analyse the resulting deep sequencing data from separate TRAC and TRBC amplicons. Enrichment of individual substitutions, regardless of their mutational context, was determined by comparing the frequency of deep sequencing reads containing them in the FACS-sorted fraction (sorted library) over that observed prior to MACS selection (unselected library). Read frequency enrichment heatmaps of individual substitutions across the entire TRAC and TRBC domains revealed a number of TRAC regions with a high prevalence of enriched mutations (e.g., positions 129-139 and 149-159, Kabat numbering), while the TRBC dataset showed no obvious regions with a preferential accumulation of enriched substitutions. Cumulative enrichment analysis per position, highlighted a number of positions in both TRAC and TRBC that appeared to be preferentially mutated following selection, most notably positions 139, 156, 169, 178 and 187 in TRAC and 134, 141, 156 in TRBC. In certain positions, multiple substitutions drove cumulative enrichment (e.g., TRAC position 178), while in others a single dominant substitution was responsible for this effect (e.g., TRBC substitution H156V).

[0427] While initial analysis of deep sequencing data provided a general overview of positions and substitutions of potential influence on heat resistance, these were examined in the absence of a mutational context. In order to address this limitation, the inventors analysed the enrichment of variants in the FACS-selected fraction containing pairs of substitutions. The inventors identified clusters of pairwise substitutions in TRAC and TRBC that were highly enriched relative to most other pairwise combinations and used this clustering visualisation to select sets of mutations for experimental testing.

[0428] The selection was performed in an iterative manner by:

[0429] (i) selecting highly enriched positions and substitutions,

[0430] (ii) including other substitutions / positions which are enriched as a pair, (iii) plotting clustered heatmaps and choosing sets of mutations belonging to the same cluster.

[0431] The resulting selected sets of substitutions for TRAC and TRBC are listed in Table 4.

[0432] Table 4. Selected sets of substitutions following single and pairwise enrichment analysis of deep sequencing data of heat-resistant TRAC and TRBC libraries.

[0433] Following this selection strategy, the inventors proceeded to design individual TCR variant constructs encoding every possible combination of substitutions from within a selected substitution set, which amounted to 89 individual TCR variants containing between 1 and 4 substitutions. 1.4 Recombinant expression in mammalian cells identifies stabilising mutations leading to increased expression yield

[0434] In order to identify stabilising mutations that also increase expression yield, the inventors proceeded to clone the selected 89 TCR variants in a His-tagged TCR-Fab format containing the Boulter disulphide (B) and native C-terminal disulphide (N) into a mammalian expression vector. For these studies, the inventors cloned TRAC and TRBC candidate variants into constructs encoding the variable domains of an affinity-enhanced MAGE-A3-specific TCR with two-digit nanomolar affinity for its target (TCRMOHS). A negative control containing the Boulter and native disulphides (BN) and a positive control containing in addition seven stabilising mutations previously reported by Froning et al. (FBN) were included throughout the study.

[0435] The inventors first performed a small-scale transient transfection experiment in suspension- adapted HEK293 cells (Expi293) using deep well 96-well plates (i.e., 1 mL transient transfections) (Fig. 3A). Following an expression time of 5 days, the inventors collected and filtered cell culture supernatants for quantification of His-tagged TCR-Fab fragments by means of biolayer interferometry (BLI). This analysis revealed a number of variants in which expression was substantially enhanced relative to the BN negative control (well G12, 5 pg / mL yield). The inventors proceeded to select the variants with highest expression yields (i.e., eight TRAC variants and 4 TRBC variants) and re-expressed them at 3 mL scale in 24-well deep well plates / microplates to validate the results (Fig. 3B). Indeed, all re-expressed variants showed substantially increased expression yields over both Froning alpha (FaBN 3 mutations, 18.6 pg / mL) and Froning beta (F BN 4 mutations, 25.7 pg / mL) controls, with the highest expressing variants showing expression levels similarto those observed for the FBN (7 mutations) positive control (58.8 pg / mL).

[0436] Following initial candidate selection based on estimated pre-purification yields in small scale experiments, the inventors next investigated expressibility at a medium scale and in a postpurification setting. Accordingly, the inventors selected the substitutions present in the top three expressing unrelated TRAC variants (i.e., N178W / A190S, K134N / K156R / F175L and K156S / D169S / N178L) and the top two unrelated TRBC variants (i.e., S170E and A184H) to design a panel of variants for testing. This panel consisted of thirteen variants containing between one and three substitutions, five of which contained substitutions in both chains (Table 5). Designed variants were cloned into constructs encoding the variable domains of a different affinity-enhanced MAGE-A3-specific TCR with one-digit nanomolar affinity for its target (TCRsioth-e). Expression constructs and relevant controls in the same format (i.e., TCRsioth- 6 BN negative control and TCRsioth-6 FBN positive control) were transfected into Expi293 cells at a 30 mL scale using 125 mL Erlenmeyer flasks and a 5-day expression protocol. Following expression, recombinant TCRs were purified from cell culture supernatants by immobilised metal affinity chromatography and expression yields determined from 280 nm absorbance values. The inventors found that the vast majority of variants had increased post-purification expression yields over BN negative control, with Var3, Var4, VarlO, Varl2 and Varl4 showing an increase in yield of at least 2-fold. In this experiment, the FBN positive control increased yield by 3-fold relative to the BN negative control. Interestingly, Var5 (aK134N, aK156R, aF175L) and Var6 (aK156S, aD169S, aN178L) showed no increase in post-purification yield over control despite having shown an increase in pre-purification yield in previous experiments, highlighting the potential impact of TCR variable domain use, expression scale and purification on quantified expression yield.

[0437] Table 5. List of candidate stabilising mutation sets and their corresponding post-purification expression yields. All TCRs incorporated TCRsioth-6 variable domains and were expressed in a

[0438] 1.5 Stabilising mutations result in enhanced melting and aggregation temperatures

[0439] Having expressed and purified candidate stabilised TCRs, the inventors proceeded to determine the melting temperature (Tm), melting temperature onset (Tonset) and aggregation temperature (Tagg) in a subset of them. The inventors selected five variants in which expression yield over the negative BN control was either increased (Vari, Var3 and Var 4) or unaffected (Var5 and Var6). For Tm and Tonset determination, the mean intrinsic fluorescence signal in the 250-720 nm range during a 25-95’C temperature ramp was assessed (i.e., differential scanning fluorimetry, DSF), while for Tagg determination, static light scattering (SLS) at 260 nm during the same temperature ramp was applied (Fig. 4). The inventors found that all tested variants had increased Tm, Tonset and Tagg relative to the BN negative control, with the largest increases observed for Var3 (7°C, 6.2°C and 6°C) and Var4 (6.5°C, 5.1°C and 5.4°C) across the three parameters (Table 6). Notably, despite having a higher expression yield (see Table 5), the FBN positive control showed markedly lower increases in Tm and Tonset (2.6°C, 2°C) compared to Var3 and Var4, but a similar increase in Tagg (6°C).

[0440] The results thus highlight that expression yield is not necessarily correlated with thermal stability of individual TCR molecules, and importantly, identify novel combinations of 2-3 mutations in the TCR constant domains that enhance the thermal stability of soluble TCRs to a higher extent than a previously reported set of 7 rationally-designed mutations. Different to Tm and Tonset, the inventors found that expression yield was correlated with Tagg, as evidenced by Var5, Var6 and the BN negative control having similarly low yields (see Table 5) and low Tagg values of around 50°C. This finding may help rationalise the observed increase in pre-purification yields for Var5 and Var6 (see Fig. 3B) but a lack of enhancement in postpurification yields (Table 5), as they may suffer from a higher propensity to aggregate during the purification process than other tested variants. Table 6. Comparison of yield, melting temperature and aggregation temperature of the different stability mutations.

[0441] Further characterisation of TCRsioth-6 recombinant protein containing Var3 or Var4 substitutions revealed migration patterns similar to those observed for corresponding BN and FBN control molecules on SDS-PAGE, with all molecules displaying a molecular weight above the expected 50 kDa due to glycosylation (Fig. 5A). Notably, TCRsioth-6 proteins containing Var3 and Var4 substitutions showed an SDS-PAGE profile that was similar to that of the BN control, indicating a similar glycosylation pattern, while the FBN molecule displayed a noticeably lower molecular weight suggesting that larger differences in glycosylation pattern are introduced by this set of mutations. Size-exclusion chromatography (SEC) following incubation at 4°C for 10 days revealed negligible aggregation for TCRsioth-6 proteins containing Var3 or Var4 substitutions (Fig. 5B). Furthermore, the major elution peak of TCRsioth-6 proteins containing Var3 and Var4 substitutions occurred closer in time to the BN control peak than TCRsioth-6 expressed with the FBN set of stabilising mutations, which is consistent with SDS-PAGE results. Finally, affinity measurements to the MAGE-A3i68-i76 peptide-MHC target by BLI revealed the expected singledigit nanomolar affinity of TCRsioth-6 for its target regardless of whether it was expressed to incorporate Var3 or Var4 substitutions or if expressed in the context of corresponding BN and FBN controls (Fig. 5C), thus showing that the identified stabilising mutations have no impact on antigen binding kinetics.

[0442] 1.6 Identified stabilising mutations show a low potential for immunogenicity

[0443] In order to assess the immunogenicity potential of Var3 and Var4 substitutions, the inventors determined the number of peptide fragments in their sequence predicted as strong binders to the most common 27 MHC class II alleles in humans using the NetMHCHpan - 4.1 tool (Reynisson, B. et al. Improved Prediction of MHC II Antigen Presentation through Integration and Motif Deconvolution of Mass Spectrometry MHC Eluted Ligand Data. J. Proteome Res. 19, 2304-2315 (2020) and Greenbaum, J. et al. Functional classification of class II human leukocyte antigen (HLA) molecules reveals seven different supertypes and a surprising degree of repertoire sharing across supertypes. Immunogenetics 63, 325-335 (2011); Table 7). Here, the number of predicted high affinity peptides to MHC class II serves as an indication for the potential of a protein drug to elicit affinity matured anti-drug antibodies (through CD4+ T cell help to antibody-producing B cells). In this analysis, the inventors also evaluated the immunogenicity potential of TCR constant domains containing a previously reported set of seven stabilising mutations (i.e., the FBN control), and benchmarked all evaluated sequences over the negative control BN sequence, which lacks stabilising mutations other than the Boulter disulphide. The inventors found that FBN substitutions resulted in the emergence of 3 strong and 10 weak predicted binders relative to the BN control. By contrast, analysis of Var3 and Var4 sequences revealed no emergence of strong binders and the emergence of 4-6 weak binders relative to the BN control indicating a lower potential for immunogenicity.

[0444] Table 7. Emergence of predicted MHC class II restricted peptides in TCRs containing stabilising mutations. List of strong binding (top 1%) and weak binding (top 5%) peptide sequences to the most common 27 MHC class II alleles in humans (covering 98% of the human population) as predicted by the NetMHCilpan tool. Strong and weak binding peptides that also occur in the parental BN control sequence are excluded from this enumeration.

[0445] 1.7 Stabilising mutations increase expression yield and thermal stability individually and in combination

[0446] The inventors further investigated the relative contributions of each mutation comprising the Var3 substitutions towards the observed enhancement of protein expression yield and stability. To this end, the inventors generated constructs encoding the TCRsioth-6 variable domains in the context of different constant TCR domains, including the Boulter-Native (BN) control and BN plus each individual substitution of Var3 (aN178W, aA190S, S17OE), as well as every pair of substitutions (aN178W+aA190S, aN178W+pS170E, aA190S+pS170E), and the triple mutant (aN178W+aA190S+pS170E).

[0447] Recombinant expression and purification from Expi293 cells revealed a synergistic effect in which individual substitutions caused a moderate increase in expression yield (10% to 29% increase over BN), double substitutions elicited larger increase in expression yield (30% to 110% increase over BN) and the triple substitution displayed the highest improvement in yield (262% increase over BN) (Fig.7).

[0448] The inventors further assessed the melting temperatures of the purified recombinant TCRs by means of DSF (Table 8). In this experiment, the inventors found that individual substitutions increased Tm by 1.5°C to 2.3°C, double substitutions increased Tm by 1.7°C to 4.6°C and the triple substitution increased Tm by 4.4°C. These results both highlighted a trend towards higher Tm with an increasing number of substitutions, and that the double substitution aA190S+|3S170E was sufficient to drive the highest observed increase in Tm (but not in expression yield).

[0449] Together, our results reveal a synergistic effect of Var3 substitutions on enhanced TCR expression yield and an additive effect on the enhancement of TCR thermal stability.

[0450] Table 8. Comparison of melting temperatures of TCRsioth-6 variants containing different combinations of the Var3 substitutions.

[0451] 1.8 Identified stabilising mutations are generally applicable across TCR germlines

[0452] In order to assess the transferability of identified Var3 mutations to alternative TCR germlines, the inventors generated constructs encoding the variable domains of three model TCRs (TCR- 1, TCR-2 and TCR-3) in the context of the BN control constant domain as well as in the context of BN plus all three Var3 substitutions (Fig.8). In this recombinant expression test, the inventors included TCRsioth-6 with BN or BN + Var3 constant domains as a control (labelled TCR- 4). Following transfection and purification from Expi293 supernatants, the inventors observed a substantial increase in expression yield across all TCRs containing Var3 substitutions. This increase ranged from 3.1- to 4.6-fold increase in expression yield depending on the TCR (fold increase in expression yield: TCR-1 < TCR-4 < TCR-3 < TCR-2). Finally, the inventors assessed the impact of Var3 substitutions on the melting and aggregation temperatures of recombinant TCR-1, TCR-2, TCR-3 and TCR-4 by means of DSF and SLS. In these experiments, the inventors confirmed substantial increases in both Tm (4.4°C) and Tagg (4.9°C) for TCR-4 (i.e., TCRsioth-e) containing Var3 mutations, as well as similar increases in both parameters across all model TCRs when Var3 substitutions were present (with the exception of Tm increase for TCR2_Var3, which was only 0.7°C, see Table 9).

[0453] Taken together, our results demonstrate the general applicability of Var3 mutations to enhance the expression yield, thermal stability and aggregation resistance of soluble TCR molecules.

[0454] Table 9.. Melting and aggregation temperatures of the 4 TCRs containing the boulter disulfide (BN) with or without additional Var3 mutations.

[0455] 1.9 Materials and methods

[0456] Generation of Libraries in Yeast

[0457] Mutations were introduced into the TCR alpha and beta constant chains stochastically by polymerase chain reaction (PCR) using oligonucleotides with degenerate codons encoding all 20 amino acids ("NNK" codon), as described previously (Bloom, J. D. An experimentally determined evolutionary model dramatically improves phylogenetic fit. Mol. Biol. Evol. 31, 1956-1978 (2014)). Two rounds of successive mutagenesis were performed, after which the DNA from each round was pooled to produce a library of mutant genes. Mutant genes were cloned into a yeast display vector, either in combination ("OE12" library) or paired with wildtype ("OE10" and "OE11" libraries), yielding variant libraries exceeding 107variants.

[0458] Yeast Thermal Cycling and Selection

[0459] Libraries and wild-type controls were induced for expression by growth in galactose- containing medium (SG-UT) for 16-20 hours at 30°C. Induced cells were washed twice with FACS buffer (Dubelleco's phosphate-buffered saline with 0.05% Tween20 (v / v), 2 mM EDTA, and 0.5% bovine serum albumin (w / v)). Washed cells were stained with 2.63 nM phycoerythrin (PE)-labeled HLA-A*0101 / MAGEA3i68-i76 tetramer and 2 pg / ml anti-FLAG APC (Biolegend 637307) for 30 minutes at room temperature. Double positive cells were enriched by flow cytometry, after which sorted cells were grown to saturation in non-inducing medium. After re-induction, washed cells were then cycled at 56°, 58°, or 60°C 10 times (20C 1 min, 56758760°C min). After thermal cycling, cells were incubated with 2.63 nM phycoerythrin (PE)-labeled HLA-A*0101 / MAGEA3i68-i76 tetramer for 30 minutes at room temperature. Cells were then washed twice to remove excess tetramer. PE-labeled cells were then isolated with anti-PE magnetic beads according to manufacturer's instructions (Biolegend 480091).

[0460] Deep sequencing ofTCR mutagenesis libraries

[0461] TCR genes from pre- and post-selection cell populations were isolated from yeast using a commercial kit according to manufacturer's instructions (Zymo D2004). Adaptors for Illumina MiSeq were then added by PCR amplification of the TRAC or TRBC with custom oligonucletides, followed by a second PCR round with Illumina indexing primers and sequencing on the Illumina MiSeq platform.

[0462] Read frequency enrichment analysis

[0463] Fastq files were preprocessed for each chain separately as the alpha and the beta chains were not paired in the sequenced data. The data were filtered based on read quality and the constant region was extracted. Read frequency enrichment analysis was performed using deep sequencing datasets originating from the sorted library over the unselected library. The enrichment of a sequence is defined as the frequency of the sequence in the sorted dataset divided by its frequency in the unselected dataset. If a sequence was absent from the unselected dataset, a pseudo-frequency (defined as the minimum frequency in this set) was used. Additionally, an enrichment matrix was computed to capture general enrichment patterns between positions and amino acids. This matrix is defined as the frequency matrix of the sorted dataset divided by the frequency matrix of the unselected dataset.

[0464] Recombinant expression and purification

[0465] TCRs were re-formatted for soluble expression. All variants contained an additional interchain disulphide bond (Boulter disulphide) by adding two further mutations (i.e., T166C in TRAC and S173C in TRBC). Constant regions were truncated at positions Cys 231 (TRAC) and Cys 247 (TRBC1), thus retaining the native disulphide bond. Designed TCR cassettes were generated by gene synthesis (IDT DNA eBlocks) and cloned into the mammalian expression vector pTwist_CMV_WPRE (Twist Biosciences).

[0466] TCRa and TCR chains were expressed from a single plasmid separated by a P2A peptide, containing a 6x His tag at the N-terminus of the TCR chain for purification.

[0467] Suspension-adapted HEK293 cells (Expi293, Thermo Fisher) were transfected with the TCR plasmids and secreted soluble TCRs were purified from culture supernatants using a HisTrap excel column (5 mL, Cytiva, #17371206 on an Akta pure system (Cytiva) with a 20 mM sodium phosphate, 0.5 M NaCI and 25 mM imidazole wash buffer and a single step elution with 20 mM sodium phosphate, 0.5 M NaCI, 500 mM imidazole elution buffer. Purified TCRs were buffer exchanged into PBS pH 7.5.

[0468] Determination of melting and aggregation temperatures

[0469] Melting and aggregation temperatures were determined by nanoDSF on an UNcle instrument (Unchained Labs). All TCR variants were diluted to a concentration of 0.5 mg / mL in PBS at pH 7.5 and loaded in triplicates in a Uni. Sealed Unis were loaded into the instrument and analysed using the Tm & Tagg with optional DLS method. Full spectra were collected from 250—720 nm, and a thermal ramp from 25 - 95 °C with a ramp rate of 0.6 °C / minute was used. Melting temperature (Tm), unfolding onset (Tonset) and aggregation onset (Tagg) were calculated using the UNcle analysis software. For Tmand Tonset, the barycentric mean (BCM) from 300 to 430 nm was plotted against the temperature and the values were calculated from the first derivative of the curve.

[0470] Taggvalues were calculated from the static light scattering (SLS) signal at 266 nm, as the temperature at which a 5% signal increase is observed.

[0471] UPLC-SEC measurements To compare size and aggregation levels TCR variants were analysed via UPLC-SEC (Vanquish Flex, Thermo) using a 300 mm MAbPac SEC-1 column (Thermo). Samples were diluted to 0.5 mg / mL in PBS and 10 pL were injected onto the column with a constant flow of 0.2 mL / min of PBS for 20 min. Absorbance was measured at 280 nm.

[0472] Affinity measurements

[0473] Affinity measurements with a single concentration were performed for variant 3 and 4, as well as, Froning and Boulter only constructs as controls. Biolayer interferometry (BLI) measurements were performed using the Octet instrument (ForteBio). Biotinylated MAGE-A3 peptide-MHC class I monomers (Biolegend, #280013) were loaded onto streptavidin biosensors at 1 pg mL1for 300 s. Binding curves were obtained using soluble TCRs at a concentration of 500 nM with 120 s association and 300 s dissociation times. Kinetic curves were fitted with a 1:1 binding model using BLI Discovery 12.0 software and re-plotted using the Prism GraphPad software.

[0474] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.

Claims

CLAIMS1. A protein comprising: a first polypeptide comprising a T cell receptor (TCR) alpha constant domain (Ca) comprising at least one of the following residues: leucine or tryptophane at position 178, asparagine at position 134, arginine, glutamate or serine at position 156, serine or tyrosine at position 169, leucine at position 175, serine at position 190, or threonine at position 199 (residues numbered according to Kabat numbering); and / or a second polypeptide comprising a TCR beta constant domain (C|3) comprising at least one of the following residues: threonine at position 128, tryptophan at position 150, or histidine at position 184 (residues numbered according to Kabat numbering).

2. The protein of claim 1, wherein the first polypeptide comprises a Ca domain comprising at least one of the following residues: leucine or tryptophane at position 178, glutamate at position 156, tyrosine at position 169, or serine at position 190 (residues numbered according to Kabat numbering); and / or the second polypeptide comprises a c domain comprising at least one of the following residues: threonine at position 128, or histidine at position 184 (residues numbered according to Kabat numbering).

3. The protein of claim 1 or 2, wherein the Cp domain further comprises a glutamate at position 170 (residues numbered according to Kabat numbering).

4. The protein of any one of claims 1-3, wherein: the first polypeptide comprises a Ca domain comprising the following residues: tryptophane at position 178 and serine at position 190 and, optionally, threonine at position 199 (residues numbered according to Kabat numbering); and / or the second polypeptide comprises a Cp domain comprising the following residues: tryptophan at position 150 and glutamate at position 170 (residues numbered according to Kabat numbering).

5. The protein of any one of claims 1-3, wherein the first polypeptide comprises a Ca domain comprising at least one of the following residues: leucine or tryptophane at position 178, and / or serine at position 190 (residues numbered according to Kabat numbering); and / or the second polypeptide comprises a CP domain comprising at least one of the following residues: glutamate at position 170, and / or histidine at position 184 (residues numberedaccording to Kabat numbering).

6. The protein of claim 5, wherein the first polypeptide comprises a Ca domain comprising a leucine at position 178, and a CP domain comprising a glutamate at position 170 (residues numbered according to Kabat numbering); or wherein the first polypeptide comprises a Ca domain comprising a tryptophan at position 178 and a serine at position 190, a CP domain comprising a glutamate at position 170 (residues numbered according to Kabat numbering); or wherein the first polypeptide comprises a Ca domain comprising a tryptophan at position 178, and a CP domain comprising a glutamate at position 170 (residues numbered according to Kabat numbering); or wherein the first polypeptide comprises a Ca domain comprising a tryptophan at position 178, and a CP domain comprising a histidine at position 184 (residues numbered according to Kabat numbering); or wherein the first polypeptide comprises a Ca domain comprising a tryptophan at position 178 and a serine at position 190, and a CP domain (residues numbered according to Kabat numbering); or wherein the first polypeptide comprises a Ca domain comprising a serine at position 190, and a CP domain comprising a glutamate at position 170 (residues numbered according to Kabat numbering).

7. The protein of any one of claims 1-6, wherein: the Ca domain comprises or consists of SEQ ID NO: 5 and the CP domain comprises or consists of SEQ ID NO: 6.

8. The protein of any one of claims 1-6, wherein: the Ca domain comprises or consists of SEQ ID NO: 7 and the CP domain comprises or consists of SEQ ID NO: 8.

9. The protein of any one of claims 1-6, wherein: the Ca domain comprises or consists of SEQ ID NO: 9 and the CP domain comprises or consists of SEQ ID NO: 10.

10. The protein of any one of claims 1-6, wherein: the Ca domain comprises or consists of SEQ ID NO: 11 and the CP domain comprises or consists of SEQ ID NO: 12.

11. The protein of any one of claims 1-6, wherein: the Ca domain comprises or consists of SEQ ID NO: 5 and the CP domain comprises or consists of SEQ ID NO: 38.

12. The protein of any one of claims 1-6, wherein: the Ca domain comprises or consists of SEQ ID NO: 39 and the CP domain comprises or consists of SEQ ID NO: 6.

13. The protein of any one of claims 1-12, wherein the protein has increased thermal stability compared to a protein having the same amino acid sequence except that: the TCR Cot comprises lysine at position 134, lysine at position 156, aspartate at position 169, phenylalanine at position 175, asparagine at position 178, alanine at position 190, and isoleucine at position 199, and the TCR CP comprises alanine at position 128, threonine at position 150, serine at position 170, and alanine at position 184 (residues numbered according to Kabat numbering).

14. The protein of any one of claims 1-13, wherein the first polypeptide comprising the TCR Ca and the second polypeptide comprising the TCR CP form a quaternary structure having a melting temperature (Tm) of at least 55°C, at least 56°C, at least 57°C, at least 58°C, at least 59°C, at least 60°C, at least 61°C, at least 62°C, preferably wherein the Tm is determined by differential scanning fluorimetry (DSF).

15. The protein of any one of claims 1-14, wherein the first polypeptide comprising the TCR Ca and the second polypeptide comprising the TCR CP form a quaternary structure having a melting temperature onset (Tonset) of at least 49°C, at least 50°C, at least 51°C, at least 52°C, at least 53°C, at least 54°C, or at least 55°C, preferably wherein the Tonset is determined by differential scanning fluorimetry (DSF).

16. The protein of any one of claims 1-15, wherein the first polypeptide comprising the TCR Ca and the second polypeptide comprising the TCR CP form a quaternary structure having an aggregation temperature (Tagg) of at least 51°C, at least 52°C, at least 53°C, at least 54°C, at least 55°C, at least 56°C, or at least 57°C, preferably wherein the Tagg determined by differential scanning fluorimetry (DSF) and / or static light scattering (SLS).

17. The protein of any one of claims 1-16, wherein the protein has an increased expression level when expressed under the same conditions compared to a protein having the same amino acid sequence except that: the TCR Ca comprises lysine at position 134, lysine at position 156, aspartate at position 169, phenylalanine at position 175, asparagine at position 178, alanine at position 190, and isoleucine at position 199, and the TCR CP comprises alanine at position 128, threonine at position 150, serine at position 170, and alanine at position 184 (residues numbered according to Kabat numbering).

18. The protein of any one of claims 1-17, wherein the first polypeptide is linked to the second polypeptide by an inter-chain disulfide bond.

19. The protein of any one of claims 1-18, wherein the first and second polypeptide are linked by an inter-chain disulfide bond between a cysteine residue at position 213 of the TCRCa domain and a cysteine residue at position 247 of the TCR cp domain (residues numbered according to Kabat numbering).

20. The protein of any one of claims 1-19, wherein: the Ca domain further comprises a cysteine residue at position 166 (residue numbered according to Kabat numbering), and the CP domain further comprises a cysteine residue at position 173 (residue numbered according to Kabat numbering), wherein the first polypeptide and the second polypeptide are linked by an inter-chain disulfide bond between the cysteine residue at position 166 of Ca and the cysteine residue at position 173 of cp.

21. The protein of any one of claims 1-20, wherein: the first polypeptide further comprises a TCR alpha variable domain (Va); and the second polypeptide further comprises a TCR beta variable domain (VP), wherein the Va and VP form an antigen binding domain that binds an antigen.

22. The protein of claim 21, wherein: the Va is fused to the N-terminus of Ca; and the VP is fused to the N-terminus of cp.

23. The protein of claims 21 or 22, wherein the antigen is a tumor antigen, a tissuespecific antigen or a viral antigen.

24. The protein of any one of claims 1-23, wherein the protein further comprises additional antigen-binding domain(s).

25. The protein of claim 24, wherein the additional antigen binding domain(s) bind(s) one or more antigen(s) on a T cell surface or an NK cell surface.

26. The protein of claim 24 or 25, wherein the additional antigen binding domain binds CD3 or NKp46.

27. The protein of any one of claims 23-26, wherein the additional antigen binding domain is a scFv, Fab, Fab', (Fab')2, single domain antibody, or camelid VHH domain.

28. The protein of any one of claims 24-27, wherein the additional antigen binding domain is a Fab.

29. The protein of claim 28, wherein the Fab comprises a Fab heavy chain comprising a heavy chain variable domain (VH) and a human IgG CHI domain, and a Fab light chain comprising a light chain variable domain (VL) and a human light chain constant domain (CL),wherein the VH and VL form the second antigen binding domain.

30. The protein of any one of claims 1-29, wherein the protein further comprises a human IgG Fc region (Fc).

31. The protein of claim 30, wherein the human IgG Fc region is a modified human IgG Fc region with reduced effector function compared to the corresponding wild type human IgG Fc region.

32. The protein of any one of claims 1-31, wherein the protein is a soluble protein.

33. The protein of any one of claims 1-32, wherein the protein is linked to a detectable label or to a therapeutic agent.

34. The protein of claim 33, wherein the therapeutic agent is a cytotoxic agent, an antiinflammatory agent, an immunostimulatory agent or an immunosuppressive agent.

35. The protein of claim 34, wherein the immunostimulatory agent is IL-2, or an engineered variant of IL-2.

36. The protein of claim 34, wherein the immunosuppressive agent is a PD-1 agonist or a Fas ligand.

37. A nucleic acid encoding one or more polypeptides of the protein of any one of claims 1-36.

38. A vector comprising the nucleic acid of claim 37.

39. A cell comprising the nucleic acid of claim 37 or the vector of embodiment 38.

40. A pharmaceutical composition comprising the protein of any one of claims 1-36, the nucleic acid of claim 37, the vector of embodiment 38, or the cell of claim 39.

41. A method of treating cancer or infection or autoimmune disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the protein of any one of claims 1-36, the nucleic acid of claim 37, the vector of claim 38, the cell of claim 39, or the pharmaceutical composition of claim 40.

42. The protein of any one of claims 1-36, the nucleic acid of claim 37, the vector of claim38, the cell of claim 39, or the pharmaceutical composition of claim 40, for use in the treatment of cancer or infection or autoimmune disease.

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