Stabilized TCR constructs and methods of use

Abstract

Stabilized TCR constructs are described, comprising a TCR α chain polypeptide having a variable α (Vα) domain and a constant α (Cα) domain, and a TCR β chain polypeptide having a variable β (Vβ) domain and a constant β (Cβ) domain. The TCR constructs are stabilized by introducing stabilizing mutations into the Cα and / or Cβ domains, such as non-native disulfide bonds between the Cα and Cβ domains (interchain disulfide bonds), non-native intrachain disulfide bonds, point mutations, loop truncation mutations, and combinations thereof. TCR fusion proteins are also described, comprising one or more of the TCR constructs and a scaffold and / or other biologically active moiety.

Description

[Technical Field]

[0001] Field The present disclosure relates to the field of soluble T cell receptors for use as pharmaceuticals, and in particular to stabilized TCR constructs and TCR fusion proteins. [Background technology]

[0002] background T cell receptors (TCRs) are proteins found on the surface of T cells. TCRs regulate immune responses by binding to class I and class II major histocompatibility complexes (MHC) present on the cell surface. MHCs that present peptide sequences and activate T cells via TCRs induce immune responses. In cancer, mutated or overexpressed peptide sequences may be presented on the surface of cancer cells. TCRs can recognize peptides with single amino acid mutations, providing an opportunity to specifically target these mutant peptide-MHC complexes.

[0003] TCRs belong to the immunoglobulin superfamily (IgSF) of proteins and share certain structural similarities with antibodies. Similar to the Fab portion of an antibody, TCRs contain two unique chains, each containing one variable and one constant domain, with highly variable loops (CDRs) in the variable domains that confer the binding selectivity of the TCR.

[0004] TCRs are membrane-bound proteins containing transmembrane domains. While there has been interest in developing soluble forms of TCRs as pharmaceuticals, soluble TCRs are inherently unstable proteins, resulting in poor expression and stability. Modifications to improve the stability of soluble TCRs have been described. International Publication No. 2004 / 074322 (Patent Document 1) describes a stabilized soluble TCR containing a disulfide bond between constant domain residues that is not present in native TCRs. International Publication No. 2016 / 070814 (Patent Document 2) describes a highly stable soluble TCR containing an artificial interchain disulfide bond connecting the constant domains of the TCR α chain and the TCR β chain. International Publication No. 2016 / 184258 (Patent Document 3) describes a stabilized soluble heterodimeric TCR containing an artificial interchain disulfide bond between the variable region of the α chain and the constant region of the β chain. Point mutations that improve the stability of soluble TCRs have also been described (see Shusta, et al., 2000, Nature Biotechnol., 18:754-759 (Non-Patent Document 1) and Gunnarsen, et al., 2013, Scientific Reports, 3:1162 (Non-Patent Document 2)).

[0005] Fusing the extracellular portion of the TCR to various domains of human immunoglobulin (Ig) has also been used as a strategy to increase TCR expression and / or improve TCR stability (see Lunde, et al., 2010, BMC Biotechnol., 10:61 (Non-Patent Document 3); Ozawa, et al., 2012, Biochem. Biophys. Res. Commun., 422:245-249 (Non-Patent Document 4); and Wu, et al., 2015, MAbs, 7:364-376 (Non-Patent Document 5)).

[0006] WO 1999 / 018129 (Patent Document 4) describes a single-chain TCR (sc-TCR) in which the α and β chains of the TCR are linked by a flexible linker. This sc-TCR exhibits improved stability. Also described are sc-TCR fusion proteins containing covalently linked TCR Vα and TCR Vβ chains fused to an immunoglobulin light chain constant region. U.S. Patent No. 6,534,633 (Patent Document 5) and U.S. Patent No. 8,105,830 (Patent Document 6) describe sc-TCRs covalently linked to at least one single-chain antibody (sc-Ab) via a peptide linker sequence.

[0007] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present disclosure. No admission is necessarily intended, nor should it be construed, that any of the preceding information constitutes prior art against the claimed invention. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2004 / 074322 [Patent Document 2] International Publication No. 2016 / 070814 [Patent Document 3] International Publication No. 2016 / 184258 [Patent Document 4] International Publication No. 1999 / 018129 [Patent Document 5] U.S. Patent No. 6,534,633 [Patent Document 6] U.S. Patent No. 8,105,830 [Non-patent literature]

[0009] [Non-Patent Document 1] Shusta,et al.,2000,Nature Biotechnol.,18:754-759 [Non-Patent Document 2] Gunnarsen,et al.,2013,Scientific Reports,3:1162 [Non-Patent Document 3] Lunde,et al.,2010,BMC Biotechnol.,10:61 [Non-Patent Document 4] Ozawa,et al.,2012,Biochem.Biophys.Res.Commun.,422:245-249 [Non-Patent Document 5] Wu,et al.,2015,MAbs,7:364-376 [Summary of the Invention]

[0010] Summary Described herein are stabilized TCR constructs and methods of use thereof. In one aspect, the disclosure provides a TCR construct comprising a TCR α chain polypeptide and a TCR β chain polypeptide, wherein the TCR α chain polypeptide comprises a variable α (Vα) domain and a constant α (Cα) domain, and the TCR β chain polypeptide comprises a variable β (Vβ) domain and a constant β (Cβ) domain, wherein the Cα domain and the Cβ domain comprise stabilizing mutations, the stabilizing mutations comprising a first interchain disulfide bond between the Cα domain and the Cβ domain, and one or more additional stabilizing mutations, the one or more additional stabilizing mutations being: a) an interchain disulfide bond formed between i) a cysteine ​​residue comprised in an amino acid extension at the C-terminus of the Cβ domain of the TCR β chain polypeptide, the amino acid extension being from 1 to about 10 amino acids in length, and ii) a cysteine ​​residue at position TRAC128 of the Cα domain of the TCR α chain polypeptide; b) an interchain disulfide bond between a cysteine ​​residue substituent at position TRAC84.2 and a cysteine ​​residue substituent at position TRBC79. c) an interchain disulfide bond between a cysteine ​​residue substituent at position 122 of TRAC and a cysteine ​​residue substituent at position 12 of TRBC; d) an interchain disulfide bond between a cysteine ​​residue substituent at position 124 of TRAC and a cysteine ​​residue substituent at position 11 of TRBC; e) an interchain disulfide bond between a cysteine ​​residue substituent at position 125 of TRAC and a cysteine ​​residue substituent at position 11 of TRBC; f) a cysteine ​​residue substituent at position 126 of TRAC and a cysteine ​​residue substitution at position 11 of TRBC. g) an interchain disulfide bond between the cysteine ​​residue substituent at position 127 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; h) an interchain disulfide bond between the cysteine ​​residue substituent at position 128 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; i) an intrachain disulfide bond between the cysteine ​​residue substituent at position 39 of TRAC and the cysteine ​​residue substituent at position 85 of TRAC; j) a cysteine ​​residue substituent at position 26 of TRAC and the cysteine ​​residue substituent at position 85 of TRAC.k) an intrachain disulfide bond between the cysteine ​​residue substituent at position 1 in TRAC; k) an amino acid substitution of Val to Ala, Thr, Ile, Leu, or Met at position 4 in TRAC; l) an amino acid substitution of Thr to Ala, Val, Ile, Leu, or Met at position 26 in TRAC; m) an amino acid substitution of Val to Ala, Thr, Ile, Leu, or Met at position 39 in TRAC; n) an amino acid substitution of Ala to Ser, Thr, Val, Ile, or Met at position 85 in TRAC; o) an amino acid substitution of Ala to Ser, Thr, Glu, Gln, Asp, Asn, His, Lys, or Arg at position 105 in TRAC; p) an amino acid substitution of Phe to Tyr or His at position 120 in TRAC; q) an amino acid substitution of Val to Ala, Thr, Ile, Leu, or Met at position 6 in TRBC; r) an amino acid substitution of His to Phe at position 36 in TRBC the amino acid numbering is that of IMGT, and the TCR construct has an increased TCR melting temperature (Tm) compared to a corresponding TCR construct comprising only the first non-natural disulfide bond.

[0011] In another aspect, the disclosure provides a TCR construct comprising a TCR α chain polypeptide and a TCR β chain polypeptide, wherein the TCR α chain polypeptide comprises a variable α (Vα) domain and a constant α (Cα) domain, and the TCR β chain polypeptide comprises a variable β (Vβ) domain and a constant β (Cβ) domain, wherein the Cα domain and / or the Cβ domain comprises a) an interchain disulfide bond formed between i) a cysteine ​​residue contained in an amino acid extension at the C-terminus of the Cβ domain of the TCR β chain polypeptide, wherein the amino acid extension is from 1 to about 10 amino acids in length, and ii) a cysteine ​​residue at position TRAC128 of the Cα domain of the TCR α chain polypeptide; b) an interchain disulfide bond between a cysteine ​​residue substituent at position TRAC84.2 and a cysteine ​​residue substituent at position TRBC79; c) an interchain disulfide bond formed between a cysteine ​​residue substituent at position TRAC84.2 and a cysteine ​​residue substituent at position TRBC79 of the TCR α chain polypeptide; a) an interchain disulfide bond between the cysteine ​​residue substituent at position 124 and the cysteine ​​residue substituent at position 11 of TRBC; b) an interchain disulfide bond between the cysteine ​​residue substituent at position 125 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; c) an interchain disulfide bond between the cysteine ​​residue substituent at position 125 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; e) an interchain disulfide bond between the cysteine ​​residue substituent at position 126 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; f) an interchain disulfide bond between the cysteine ​​residue substituent at position 127 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; g) an interchain disulfide bond between the cysteine ​​residue substituent at position 128 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; h) an intrachain disulfide bond between the cysteine ​​residue substituent at position 39 of TRAC and the cysteine ​​residue substituent at position 85 of TRAC; i) a cysteine ​​residue substituent at position 26 of TRAC and TRAC85.j) an intrachain disulfide bond between the cysteine ​​residue substituent at position 1 in TRAC; k) an amino acid substitution of Thr at position 26 in TRAC with Ala, Val, Ile, Leu, or Met; l) an amino acid substitution of Val at position 39 in TRAC with Ala, Thr, Ile, Leu, or Met; m) an amino acid substitution of Ala at position 85 in TRAC with Ser, Thr, Val, Ile, or Met; n) an amino acid substitution of Ala at position 105 in TRAC with Ser, Thr, Glu, Gln, Asp, Asn, His, Lys, or Arg; o) an amino acid substitution of Phe at position 120 in TRAC with Tyr or His; p) an amino acid substitution of Val at position 6 in TRBC with Ala, Thr, Ile, Leu, or Met; q) an amino acid substitution of His at position 36 in TRBC with Phe , Tyr, or Trp; r) an amino acid substitution of Ser at position 86 of TRBC with Ala or Thr; s) an amino acid substitution of Val at position 45.3 of TRBC with Ser, Thr, Glu, Gln, Asp, Asn, His, Lys, or Arg; t) a deletion of 1 to 4 consecutive amino acids in the DE loop of the Cβ domain of the TCR construct; and u) a replacement of amino acids at positions 84.4 to 85.4 of TRBC with an amino acid sequence consisting of 2 to 4 amino acids that enables β-turn formation, wherein the amino acid numbering is that of IMGT, and the TCR construct has an increased TCR melting temperature (Tm) compared to a corresponding TCR construct that does not contain the one or more stabilizing mutations.

[0012] In another aspect, the disclosure provides a TCR construct comprising a TCR α chain polypeptide and a TCR β chain polypeptide, wherein the TCR α chain polypeptide comprises a variable α (Vα) domain and a constant α (Cα) domain, and the TCR β chain polypeptide comprises a variable β (Vβ) domain and a constant β (Cβ) domain, wherein the Cα domain and the Cβ domain together comprise: a) an interchain disulfide bond formed between i) a cysteine ​​residue contained in an amino acid extension at the C-terminus of the Cβ domain of the TCR β chain polypeptide, wherein the amino acid extension is from 1 to about 10 amino acids in length, and ii) a cysteine ​​residue at position TRAC128 of the Cα domain of the TCR α chain polypeptide; b) an interchain disulfide bond between the cysteine ​​residue substituent at position TRAC84 and the cysteine ​​residue substituent at position TRBC79; c) an interchain disulfide bond between the cysteine ​​residue substituent at position TRAC84.2 and the cysteine ​​residue substituent at position TRBC79; d) an interchain disulfide bond between the cysteine ​​residue substituent at position TRAC128 and the cysteine ​​residue substituent at position TRBC79; a) an interchain disulfide bond between the cysteine ​​residue substituent at position 24 and the cysteine ​​residue substituent at position 11 of TRBC; b) an interchain disulfide bond between the cysteine ​​residue substituent at position 22 of TRAC and the cysteine ​​residue substituent at position 12 of TRBC; c) an interchain disulfide bond between the cysteine ​​residue substituent at position 125 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; d) an interchain disulfide bond between the cysteine ​​residue substituent at position 126 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; interchain disulfide bond, h) interchain disulfide bond between the cysteine ​​residue substituent at position 127 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC, i) interchain disulfide bond between the cysteine ​​residue substituent at position 128 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC, j) intrachain disulfide bond between the cysteine ​​residue substituent at position 39 of TRAC and the cysteine ​​residue substituent at position 85 of TRAC, k) cysteine ​​residue substituent at position 26 of TRAC and TRAC85.l) an intrachain disulfide bond between the cysteine ​​residue substituent at position 1 in TRAC; m) an amino acid substitution of Thr at position 26 in TRAC with Ala, Val, Ile, Leu, or Met; n) an amino acid substitution of Val at position 39 in TRAC with Ala, Thr, Ile, Leu, or Met; o) an amino acid substitution of Ala at position 85 in TRAC with Ser, Thr, Val, Ile, or Met; p) an amino acid substitution of Ala at position 105 in TRAC with Ser, Thr, Glu, Gln, Asp, Asn, His, Lys, or Arg; q) an amino acid substitution of Phe at position 120 in TRAC with Tyr or His; r) an amino acid substitution of Val at position 6 in TRBC with Ala, Thr, Ile, Leu, or Met; s) an amino acid substitution of His at position 36 in TRBC with Phe , Tyr, or Trp; t) an amino acid substitution of Ser at position 86 of TRBC with Ala or Thr; u) an amino acid substitution of Val at position 45.3 of TRBC with Ser, Thr, Glu, Gln, Asp, Asn, His, Lys, or Arg; v) a deletion of 1 to 4 consecutive amino acids in the DE loop of the Cβ domain of the TCR construct; and w) a replacement of amino acids at positions 84.4 to 85.4 of TRBC with an amino acid sequence consisting of 2 to 4 amino acids that enables β-turn formation, wherein the amino acid numbering is that of IMGT, and the TCR construct has an increased TCR melting temperature (Tm) compared to a corresponding TCR construct that does not contain the two or more stabilizing mutations.

[0013] In another aspect, the disclosure relates to a TCR construct comprising a TCR α chain polypeptide and a TCR β chain polypeptide, wherein the TCR α chain polypeptide comprises a variable α (Vα) domain and a constant α (Cα) domain, and the TCR β chain polypeptide comprises a variable β (Vβ) domain and a constant β (Cβ) domain, and wherein the TCR construct comprises a combination of amino acid mutations as described for any one of the variants shown in Table 2, and wherein the amino acid numbering is that of IMGT.

[0014] In another aspect, the disclosure relates to a TCR construct comprising a TCR α chain polypeptide and a TCR β chain polypeptide, wherein the TCR α chain polypeptide comprises a variable α (Vα) domain and a constant α (Cα) domain, and the TCR β chain polypeptide comprises a variable β (Vβ) domain and a constant β (Cβ) domain, and wherein the TCR construct comprises a combination of amino acid mutations as described for any one of the variants shown in Table 3, and wherein the amino acid numbering is that of IMGT.

[0015] In another aspect, the present disclosure relates to a TCR fusion protein comprising one or more of the TCR constructs described herein and a scaffold, wherein at least one of the TCR constructs is fused to the scaffold.

[0016] In another aspect, the present disclosure relates to a pharmaceutical composition comprising a TCR construct or TCR fusion protein as described herein and a pharmaceutically acceptable carrier or diluent.

[0017] In another aspect, the disclosure relates to a polynucleotide or set of polynucleotides that encodes a TCR construct or a TCR fusion protein as described herein.

[0018] In another aspect, the disclosure relates to a method of preparing a TCR construct or a TCR fusion protein as described herein, comprising transfecting a cell with a polynucleotide or set of polynucleotides encoding said TCR construct or TCR fusion protein, and culturing said cell under conditions suitable for expression of said TCR construct or TCR fusion protein.

[0019] In another aspect, the present disclosure relates to a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of a TCR construct or TCR fusion protein as described herein.

[0020] In another aspect, the disclosure relates to the use of a TCR construct or TCR fusion protein as described herein in therapy. [Brief explanation of the drawings]

[0021] [Figure 1-1] Figure 1 shows a schematic representation of exemplary TCR fusion proteins and controls as described herein and in the Examples. (A) Control one-arm antibody (OA-scFv). (B) Control antibody containing two scFv arms (dual-scFv). (C) TCR-scFv. (D) One-arm alpha fusion. [Figure 1-2] Figure 1 shows a schematic representation of exemplary TCR fusion proteins and controls as described herein and in the Examples. (E) Single-arm alpha fusion-DS. (F) Dual alpha fusion-DS. (G) Dual fusion. (H) Single-arm beta fusion. [Figure 1-3] Figure 1 shows a schematic representation of exemplary TCR fusion proteins and controls as described herein and in the Examples. (I) Single-arm beta fusion-DS. (J) Dual beta fusion-DS. (K) Bispecific alpha fusion. (L) Bispecific beta fusion. [Figure 1-4] Figure 1 shows a schematic representation of exemplary TCR fusion proteins and controls as described herein and in the Examples. (M) Bispecific chimeras. (N) Bispecific tandem beta fusions. (O) 2x1 bispecific beta fusions. [Figure 1-5] Figure 1 shows a schematic representation of exemplary TCR fusion proteins and controls as described herein and in the Examples. (P) 3x1 bispecific β-fusion. (Q) 4x1 bispecific C-terminal β-fusion. [Figure 1-6] Figure 1 shows a schematic representation of exemplary TCR fusion proteins and controls as described herein and in the Examples. (R) 4x1 bispecific light chain beta fusion. (S) 1-arm alpha fusion. (T) 1-arm beta fusion. The black bar between the TRAC and TRBC domains represents the interchain disulfide bond ("DS"). [Figure 2] 1 shows differential scanning calorimetry (DSC) thermograms of TCR fusion proteins containing exemplary stabilizing mutations as described herein (v21230 is the IC disulfide, v22712 is the TRBC / 6.VAL→LEU+IC disulfide, and v28881 is the TRBC / 6.VAL→ILE+IC disulfide). Each peak in this DSC thermogram corresponds to a thermal transition. There are three predicted thermal transitions: TCR, CH2 (approximately 71°C), and CH3 (approximately 80°C). The TCR transition includes the TRAV-TRBV interface and the TRAC-TRBC interface. [Figure 3] Figure 1 shows a comparison of UPLC-SEC traces of an exemplary TCR fusion protein described herein (variant v21230 containing an IC disulfide) after expression at (A) 37°C and (B) 32°C. Monodisperse species elutes at 2.6 minutes. High molecular weight species (aggregates) elute before the monodisperse species. The peak at 2.8 minutes corresponds to mispaired anti-CD3 scFv homodimers, and the peak at 3.25 minutes corresponds to unpaired anti-CD3 scFv. [Figure 4]1 shows differential scanning calorimetry (DSC) thermograms illustrating the change in Tm of an exemplary TCR fusion protein containing the TRAC-hinge disulfide described herein (v22752: IC disulfide + TRAC-hinge disulfide) compared to a TCR fusion protein containing only the IC disulfide (v21230). Each peak in the DSC thermogram corresponds to a thermal transition as illustrated in FIG. [Figure 5-1] Figure 5 shows flow cytometry binding of exemplary TCR fusion proteins to target cell surface peptide-MHC complexes. (A) Variant v21230 (control containing IC disulfides) and variants v22705, v22707, and v22709. (B) Variant v21230 (control containing IC disulfides) and variants v22712, v22716, v22720, and v22722. (See Tables 4.1 and 8.1 for variant details.) [Figure 5-2] Figure 5 shows flow cytometry binding of exemplary TCR fusion proteins to target cell surface peptide-MHC complexes. (C) Variant v21230 (control containing IC disulfides) and variants v22729, v22730, v22748, and v22752. (D) Variant v21230 (control containing IC disulfides) and variants v22772, v22837, v22840, and v22842. (See Tables 4.1 and 8.1 for variant details.) [Figure 6] Figure 1 depicts the EC50 values ​​measured for binding of exemplary TCR fusion proteins to target cell surface peptide-MHC complexes (assessed by flow cytometry). The dashed line corresponds to the EC50 of the control variant v21230, which contains the IC disulfide. [Figure 7]The results of evaluating avidity effects are presented using low-affinity TCR components of exemplary TCR fusion proteins with various formats. Variants v30972 (bispecific tandem β-fusion), v30964 (bispecific β-fusion), v30975 (single-arm β-fusion), and v30968 (2x1 bispecific β-fusion) contain low-affinity anti-gp100 TCR components. Variants v29011 (single-arm β-fusion) and v31327 (bispecific β-fusion) contain high-affinity anti-gp100 TCR components. Variant v30968 contains two copies of the TCR components, while all other variants contain one copy of the TCR components. [Figure 8] Figure 1 shows the UPLC-SEC traces of two exemplary TCR fusion proteins: (A) variant v32548, which is a "4x1 tandem" format containing anti-NY-ESO1 TCR, and (B) variant v32549, which is a "4x1LC" format containing anti-NY-ESO1 TCR. [Figure 9-1] Figure 9 shows the amino acid sequences of the TCR α chain constant region (TRAC (SEQ ID NO: 1)) and TCR β chain constant regions (TRBC1 (SEQ ID NO: 2) and TRBC2 (SEQ ID NO: 3)) with standard IMGT numbering. Sequence differences between TRBC1 and TRBC2 are shown in bold italics. [Figure 9-2] See description of Figure 9-1. [Figure 10] Figure 1 shows the cytotoxicity of TCR fusion proteins with various formats, as measured by a T2 T cell-dependent cytotoxicity assay. These TCR fusion proteins included the stabilized, affinity-matured 1G4-33A anti-NY-ESO1 TCR and an anti-CD3 Fab or anti-CD3 scFv. Variant v31185 is a control one-arm TCR construct that does not contain the anti-CD3 component. [Figure 11-1] FIG. 11 depicts a table of TCR fusion protein variants (Table 2) containing exemplary combinations of stabilizing mutations. [Figure 11-2] See description of Figure 11-1. [Figure 11-3] See description of Figure 11-1. [Figure 12] 1 depicts a table (Table 3) of TCR fusion protein variants containing exemplary combinations of stabilizing mutations. DETAILED DESCRIPTION OF THE INVENTION

[0022] Detailed Description The present disclosure relates to stabilized TCR constructs. The TCR constructs comprise a TCR α chain polypeptide having a variable α (Vα) domain and a constant α (Cα) domain, and a TCR β chain polypeptide having a variable β (Vβ) domain and a constant β (Cβ) domain, and are stabilized by introducing stabilizing mutations into the Cα and / or Cβ domains. The stabilizing mutations may include one or more non-native interchain disulfide bonds, one or more non-native intrachain disulfide bonds, one or more point mutations, one or more loop truncation mutations, or a combination thereof.

[0023] In certain embodiments, the stabilizing mutations include a non-native disulfide bond (interchain disulfide bond) between the Cα and Cβ domains, along with one or more additional stabilizing mutations, which may include an additional non-native interchain disulfide bond, a non-native intrachain disulfide bond, a point mutation, a loop truncation mutation, or a combination thereof.

[0024] Also disclosed herein are TCR fusion proteins that include one or more of the TCR constructs described herein fused to a scaffold, such as an immunoglobulin (Ig) Fc region, which can be, for example, the Fc region of an IgG or IgA.

[0025] The TCR constructs and TCR fusion proteins of the present invention find use, for example, as therapeutic or diagnostic agents.

[0026] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0027] As used herein, the term "about" refers to approximately a ±10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not the variation is specifically referred to.

[0028] The use of the words "a" or "an" when used in conjunction with the word "comprising" herein may mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more," unless the context clearly dictates otherwise.

[0029] As used herein, the terms "comprising," "having," "including," and "containing," as well as grammatical variations thereof, are inclusive, i.e., open-ended, and do not exclude additional, unrecited elements and / or method steps. The term "consisting essentially of," when used in connection with a composition, use, or method, indicates that additional elements and / or method steps may be present, but that these additions do not materially affect the manner in which the recited composition, method, or use functions. The term "consisting of," when used in connection with a composition, use, or method, excludes the presence of additional elements and / or method steps. When a composition, use, or method described herein is said to include particular elements and / or steps, it may, in certain embodiments, consist essentially of those elements and / or steps, and in other embodiments, consist of those elements and / or steps, whether or not those embodiments are specifically referred to.

[0030] When two components of a TCR construct or TCR fusion protein described herein are "fused," it means that the components are linked either directly by a peptide bond or via a peptide linker.

[0031] The terms "derived from" and "based on," when used in reference to a recombinant amino acid sequence, mean that the recombinant amino acid sequence is substantially identical to the sequence of the corresponding wild-type amino acid sequence. For example, an amino acid sequence of an Ig Fc derived from (or based on) the sequence of a wild-type Ig Fc is substantially identical to the sequence of the wild-type Ig Fc (e.g., has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity).

[0032] Where a range of values ​​is given herein, for example, where a value is defined as from an upper limit to a lower limit, it is understood that the range includes both the upper and lower limits, as well as each value therebetween.

[0033] It is contemplated that any embodiment discussed herein with respect to a TCR construct may be implemented in the context of any fusion protein, method, use or composition disclosed herein.

[0034] The amino acid residues of the extracellular TCR domain are numbered according to the IMGT numbering system (Lefranc, et al., 2005, Developmental and Comparative Immunology, 29:185-203; see also Figure 9).

[0035] TCR constructs The TCR constructs of the present disclosure are based on the αβTCR heterodimer, which comprises a TCR α chain polypeptide having a variable α (Vα) domain and a constant α (Cα) domain, and a TCR β chain polypeptide having a variable β (Vβ) domain and a constant β (Cβ) domain, and are stabilized by introducing stabilizing mutations into the Cα and / or Cβ domains.

[0036] Human wild-type αβ TCR contains a Cα domain (T cell receptor alpha constant region (TRAC)) and a Cβ domain (either T cell receptor beta constant region 1 (TRBCl) or T cell receptor beta constant region 2 (TRBC2)). The sequences of TRBCl and TRBC2 differ by only three residues: position TRBC / 1.4 is Asn in TRBCl and Lys in TRBC2; position TRBC / 1.3 is Lys in TRBCl and Asn in TRBC2; and position TRBC / 29 is Phe in TRBCl and Tyr in TRBC2. The amino acid sequences of human TRAC (SEQ ID NO: 1), TRBCl (SEQ ID NO: 2), and TRBC2 (SEQ ID NO: 3) are shown in Figure 9.

[0037] As used herein, the term "Cα domain" refers to the amino acid sequence of the TRAC Cα domain, excluding any transmembrane sequence. The Cα domain included in the TCR constructs described herein may optionally include a naturally occurring cysteine ​​residue at TRAC / position 128. In some embodiments, the Cα domain included in the TCR construct has the amino acid sequence of the human TRAC Cα domain as set forth in SEQ ID NO: 1 (i.e., the sequence ending at TRAC / position 127). In some embodiments, for example, when the TCR construct includes a disulfide bond with the naturally occurring cysteine ​​residue at TRAC / position 128, the Cα domain included in the TCR construct has the amino acid sequence of the human TRAC Cα domain as set forth in SEQ ID NO: 4 (i.e., including a cysteine ​​residue at TRAC / position 128).

[0038] As used herein, the term "Cβ domain" refers to the amino acid sequence of the TRBC Cβ domain, excluding any transmembrane sequence. In some embodiments, the Cβ domain included in a TCR construct of the present invention has the amino acid sequence of the human TRBC1 Cβ domain or the human TRBC2 Cβ domain ending at position TRBC126. In some embodiments, the Cβ domain included in a TCR construct has the amino acid sequence of the human TRBC1 Cβ domain as set forth in SEQ ID NO:2. In some embodiments, the Cβ domain included in a TCR construct has the amino acid sequence of the human TRBC2 Cβ domain as set forth in SEQ ID NO:3. In some embodiments, the Cβ domain included in a TCR construct has the amino acid sequence of the human TRBC1 Cβ domain, with a cysteine ​​to alanine mutation at position 85.1, as set forth in SEQ ID NO:43.

[0039] In certain embodiments, the TCR β chain polypeptide further comprises a cysteine ​​residue at the C-terminus of the Cβ domain that forms a non-native disulfide bond with a cysteine ​​residue in the Cα domain of the TCR α chain polypeptide as described herein. The cysteine ​​residue at the C-terminus of the Cβ domain of the TCR β chain polypeptide may be an additional amino acid, or the cysteine ​​residue may be part of a short amino acid stretch at the C-terminus of the Cβ domain of the TCR β chain polypeptide, as described herein. In some embodiments, the cysteine ​​residue may be part of a short amino acid stretch, e.g., 1 to about 10 amino acids in length, at the C-terminus of the Cβ domain of the TCR β chain polypeptide. In some embodiments, the cysteine ​​residue may be part of a short amino acid stretch, e.g., 1 to about 10 amino acids in length, at the C-terminus of the Cβ domain of the TCR β chain polypeptide, which amino acid stretch includes all or part of the sequence of an IgG1 hinge region, e.g., three or more consecutive amino acids (e.g., EPKSCDKTHT [SEQ ID NO: 16] or EPKSCDKTHTCPPCP [SEQ ID NO: 21]).

[0040] Stabilizing mutations introduced into the Cα and / or Cβ domains of the TCR constructs of the present disclosure may include non-native interchain disulfide bonds, non-native intrachain disulfide bonds, point mutations, loop truncation mutations, and combinations thereof, as described in more detail below. The stabilizing mutations included in the TCR constructs of the present invention improve the stability of the TCR construct compared to a TCR construct that does not include the stabilizing mutation(s). In this regard, improved stability of the TCR construct may include improved thermal stability of the TCR construct, improved colloidal stability of the TCR construct, or both.

[0041] In certain embodiments, the TCR constructs of the present disclosure exhibit improved thermal stability compared to corresponding TCR constructs that do not include the stabilizing mutation(s). The thermal stability of a TCR construct may be assessed, for example, by measuring the melting temperature (Tm) of the TCR construct. Thus, in some embodiments, the TCR constructs of the present invention have an increased Tm compared to corresponding TCR constructs that do not include the stabilizing mutation(s).

[0042] In certain embodiments, the TCR constructs of the present disclosure have a Tm that is increased by 0.5° C. or more compared to the Tm of the corresponding TCR construct that does not comprise the stabilizing mutation(s). In some embodiments, the TCR constructs of the present disclosure have a Tm that is increased by 1° C. or more compared to the Tm of the corresponding TCR construct that does not comprise the stabilizing mutation(s), for example, an increase of 2° C. or more, 3° C. or more, 4° C. or more, or 5° C. or more compared to the Tm of the corresponding TCR construct that does not comprise the stabilizing mutation(s).

[0043] In certain embodiments, the TCR constructs of the present disclosure have a Tm that is increased by 0.5°C to about 10°C relative to the Tm of the corresponding TCR construct that does not comprise the stabilizing mutation(s). In some embodiments, the TCR constructs of the present disclosure have a Tm that is increased by 1°C to about 10°C relative to the Tm of the corresponding TCR construct that does not comprise the stabilizing mutation(s), e.g., increased by 1°C to about 9°C, 1°C to about 8°C, or 1°C to about 7°C relative to the Tm of the corresponding TCR construct that does not comprise the stabilizing mutation(s). In some embodiments, the TCR constructs of the present disclosure have a Tm that is increased by 2°C to about 10°C, 2°C to about 9°C, 2°C to about 8°C, or 2°C to about 7°C relative to the Tm of the corresponding TCR construct that does not comprise the stabilizing mutation(s).

[0044] In certain embodiments, the TCR constructs of the present disclosure comprise a non-native disulfide bond between the Cα and Cβ domains (the "first non-native interchain disulfide bond") along with one or more additional stabilizing mutations as described herein, and have an increased Tm compared to a corresponding TCR construct comprising only the first non-native interchain disulfide bond.

[0045] In certain embodiments, the TCR construct comprises a first non-native interchain disulfide bond and one or more additional stabilizing mutations and has a Tm that is increased by 0.5°C or more compared to the Tm of a corresponding TCR construct comprising only the first non-native interchain disulfide bond, e.g., an increase of 1°C or more, 2°C or more, 3°C or more, 4°C or more, or 5°C or more compared to the Tm of a corresponding TCR construct comprising only the first non-native interchain disulfide bond.

[0046] In certain embodiments, a TCR construct of the invention comprises a first non-native interchain disulfide bond and one or more additional stabilizing mutations and has a Tm that is increased by 0.5°C to about 10°C relative to the Tm of a corresponding TCR construct comprising only the first non-native interchain disulfide bond, e.g., an increase of 1°C to about 10°C, 1°C to about 9°C, 1°C to about 8°C, or 1°C to about 7°C relative to the Tm of a corresponding TCR construct comprising only the first non-native interchain disulfide bond. In some embodiments, a TCR construct comprising a first non-native interchain disulfide bond and one or more additional stabilizing mutations has a Tm that is increased by 2°C to about 10°C, 2°C to about 9°C, 2°C to about 8°C, or 2°C to about 7°C relative to the Tm of a corresponding TCR construct comprising only the first non-native interchain disulfide bond.

[0047] The Tm of a TCR construct may be measured using standard techniques, for example, by circular dichroism (CD), differential scanning calorimetry (DSC), or differential scanning fluorimetry (DSF). In certain embodiments, a TCR construct of the invention has an elevated Tm relative to a given corresponding control TCR construct (e.g., a corresponding TCR construct that does not include the stabilizing mutation(s), or a corresponding TCR construct that includes only the first non-native interchain disulfide bond), where the Tm is measured by DSC.

[0048] In certain embodiments, the TCR constructs of the present disclosure exhibit improved colloidal stability compared to a corresponding TCR construct that does not contain the stabilizing mutation(s). Colloidal stability may be assessed, for example, by measuring the amount of high molecular weight (HMW) species (or aggregation) of the TCR construct that occurs during expression of the TCR construct. Thus, in some embodiments, the TCR constructs of the present disclosure exhibit a reduced amount of HMW species (aggregation) when expressed under the same conditions compared to a corresponding TCR construct that does not contain the stabilizing mutation(s). In certain embodiments, the TCR constructs of the present disclosure comprise a first non-native interchain disulfide bond and one or more additional stabilizing mutations, and exhibit a reduced amount of HMW species (aggregation) when expressed under the same conditions compared to a corresponding TCR construct that contains only the first non-native interchain disulfide bond.

[0049] The amount of HMW species present in a preparation of a TCR construct may be assessed by a variety of standard techniques known in the art. For example, the amount of HMW species in a preparation of a TCR construct may be assessed by size exclusion chromatography (SEC), e.g., using UPLC-SEC, or dynamic light scattering (DLS). In certain embodiments, the TCR constructs of the present invention exhibit a reduced amount of HMW species compared to a corresponding control TCR construct, as assessed by UPLC-SEC.

[0050] A non-native interchain disulfide bond between cysteine ​​substituents at positions TRAC / 84.THR and TRBC / 79.SER that stabilizes soluble αβ TCRs has been previously described (Boulter, et al., 2003, PEDS, 16:707-711). This disulfide bond between TRAC / 84.THR and TRBC / 79.SER is referred to herein as the "IC disulfide." In certain embodiments, the TCR constructs of the present disclosure have an increased Tm compared to a corresponding TCR construct comprising only the IC disulfide. In certain embodiments, the TCR constructs of the present disclosure comprise a first non-native disulfide bond and one or more additional stabilizing mutations as described herein, wherein the first non-native disulfide bond is an IC disulfide and has an increased Tm compared to a corresponding TCR construct comprising only the IC disulfide.

[0051] In certain embodiments, the TCR constructs of the present disclosure have a Tm that is increased by 0.5° C. or more compared to the Tm of the corresponding TCR construct comprising only the IC disulfide. In some embodiments, the TCR constructs of the present disclosure have a Tm that is increased by 1° C. or more compared to the Tm of the corresponding TCR construct comprising only the IC disulfide, e.g., by 2° C. or more, 3° C. or more, 4° C. or more, or 5° C. or more compared to the Tm of the corresponding TCR construct comprising only the IC disulfide.

[0052] In certain embodiments, the TCR constructs of the present disclosure have a Tm that is increased by 0.5°C to about 10°C relative to the Tm of the corresponding TCR construct comprising only the IC disulfide, e.g., increased by 1°C to about 10°C, 1°C to about 9°C, 1°C to about 8°C, or 1°C to about 7°C relative to the Tm of the corresponding TCR construct comprising only the IC disulfide. In some embodiments, the TCR constructs of the present disclosure have a Tm that is increased by 2°C to about 10°C, 2°C to about 9°C, 2°C to about 8°C, or 2°C to about 7°C relative to the Tm of the corresponding TCR construct comprising only the IC disulfide.

[0053] In certain embodiments, the TCR constructs of the present disclosure have substantially the same or reduced amount of HMW species when expressed under the same conditions as a corresponding TCR construct comprising only the IC disulfide. "Substantially the same," with respect to the amount of HMW species, means that the amount of HMW species measured in the test TCR construct is ±10% of the amount of HMW species measured in the corresponding TCR construct comprising only the IC disulfide when expressed under the same conditions. In certain embodiments, the amount of HMW species is measured by UPLC-SEC.

[0054] In certain embodiments, the TCR constructs of the present disclosure comprise an IC disulfide and one or more additional stabilizing mutations and have one or both of the following properties: (i) an increased Tm compared to a corresponding TCR construct comprising only the IC disulfide, and / or (ii) a reduced amount of HMW species compared to a corresponding TCR construct comprising only the IC disulfide when expressed under the same conditions.

[0055] In certain embodiments, a TCR construct of the present disclosure comprises a first non-native interchain disulfide bond and one or more additional stabilizing mutations and has one or both of the following properties: (i) an increased Tm compared to a corresponding TCR construct comprising only the first non-native interchain disulfide bond, and / or (ii) a reduced amount of HMW species when expressed under the same conditions compared to a corresponding TCR construct comprising only the first non-native interchain disulfide bond.

[0056] Stabilizing mutations Stabilizing mutations included in the TCR constructs of the present disclosure were identified through iterative structural and computationally guided design, as well as experimental screening, as described herein. Based on structures from the Protein Database (PDB), in silico modeling with TCR models was used to identify mutation designs in the TRAC and TRBC domains that may potentially improve the thermal and / or colloidal stability of the TCR, which were then tested experimentally.

[0057] Mutation designs identified by this approach that successfully improved the thermal and / or colloidal stability of TCR constructs included disulfide bonds (non-native inter-chain disulfide bonds and / or non-native intra-chain disulfide bonds), point mutations, loop truncation mutations, and combinations thereof.

[0058] I. Disulfide bonds The stabilizing disulfide bonds identified by the above approaches include non-natural interchain disulfide bonds and non-natural intrachain disulfide bonds. In this context, a non-natural interchain disulfide bond is a disulfide bond between a cysteine ​​residue in the Cα domain of a TCR construct and a cysteine ​​residue in the Cβ domain of a TCR construct, which is a disulfide bond not found in a wild-type TCR. One or both of the cysteine ​​residues in this non-natural interchain disulfide bond are introduced by substituting a cysteine ​​residue for the wild-type residue at a predetermined position ("cysteine ​​residue substitution" or "cysteine ​​substitution"), or by adding a cysteine ​​residue to the C-terminus of the Cα domain or Cβ domain.

[0059] A non-native intrachain disulfide bond is a disulfide bond between two cysteine ​​residues in the Cα domain of a TCR construct or between two cysteine ​​residues in the Cβ domain of a TCR construct that is not found in a wild-type TCR. One or both of the cysteine ​​residues in this non-native intrachain disulfide bond are introduced by substituting a cysteine ​​residue for the wild-type residue at a given position (a "cysteine ​​residue substitution" or "cysteine ​​substitution"), or by adding a cysteine ​​residue to the C-terminus of the Cα or Cβ domain.

[0060] a) Interchain disulfide bonds In certain embodiments, the TCR constructs of the present disclosure comprise at least one non-native interchain disulfide bond.

[0061] In certain embodiments, a non-native interchain disulfide bond that may be included in a TCR construct of the present invention comprises a "hinge disulfide bond," which, as used herein, refers to a disulfide bond formed between a cysteine ​​residue added to the C-terminus of the Cβ domain of a TCR β chain polypeptide and a cysteine ​​residue in the Cα domain of a TCR α chain polypeptide.

[0062] The cysteine ​​residue added to the C-terminus of the Cβ domain of the TCR β chain polypeptide may be a single amino acid addition, or the cysteine ​​residue may be part of a short amino acid extension at the C-terminus of the Cβ domain of the TCR β chain polypeptide. In those embodiments in which the cysteine ​​residue is added to the C-terminus of the Cβ domain of the TCR β chain polypeptide as part of an amino acid extension, the amino acid extension is typically 10 amino acids or less in length. In some embodiments, the cysteine ​​residue added to the C-terminus of the Cβ domain of the TCR β chain polypeptide is part of an amino acid extension that is 10 amino acids or less in length, e.g., 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, or 5 amino acids or less in length.

[0063] In some embodiments, the cysteine ​​residue added to the C-terminus of the Cβ domain of a TCR β chain polypeptide is part of an amino acid extension that is 1 to about 10 amino acids in length. In some embodiments, the cysteine ​​residue added to the C-terminus of the Cβ domain of a TCR β chain polypeptide is part of an amino acid extension that is 1 to about 9 amino acids in length, for example, 1 to about 8 amino acids in length, 1 to about 7 amino acids in length, 1 to about 6 amino acids in length, or 1 to about 5 amino acids in length. In some embodiments, the cysteine ​​residue added to the C-terminus of the Cβ domain of a TCR β chain polypeptide is part of an amino acid extension that is 2 to about 10 amino acids in length, for example, 2 to about 9 amino acids in length, for example, 2 to about 8 amino acids in length, 2 to about 7 amino acids in length, 2 to about 6 amino acids in length, or 2 to about 5 amino acids in length.

[0064] In certain embodiments, the amino acid extension comprises a cysteine ​​residue and one or two "linkers," where the linker(s) are composed of amino acids other than cysteine. That is, in some embodiments, the Cβ domain of the TCR β chain polypeptide comprises at its C-terminus the following structure (N-terminal to C-terminal): Linker-Cys, Cys-linker, or Linker-Cys-Linker The amino acid stretch comprises one of:

[0065] When present, the linker allows the cysteine ​​residue of the amino acid extension to assume the correct conformation relative to its cognate cysteine ​​residue in the Cα domain of the TCR α chain polypeptide to form the desired disulfide bond. Linker peptides may have one of many amino acid sequences known in the art to function well as linkers or spacers in polypeptide and protein sequences.

[0066] In some embodiments, the linker peptide may comprise the amino acid residues Gly, Ser, Ala, or Thr, or a combination thereof. Examples of useful linkers include (GS) n , (GSGGS) n , (GGGGS) n and (GGGS) n (wherein n is an integer of 1 to 4), as well as glycine-alanine linkers and alanine-serine linkers having similar configurations.

[0067] In some embodiments, the amino acid extension may comprise all or part of an immunoglobulin or TCR hinge region sequence, in which case the cysteine ​​residues contained in the amino acid extension may be naturally present in the hinge region sequence or may have been introduced by amino acid substitution. Non-limiting examples of hinge region sequences, all or a portion of which (e.g., at least 2, 3, 4, 5, 6 or more contiguous amino acids) may be comprised in the amino acid extension include ESSCDVKLVEKSFET (SEQ ID NO: 5) (TCR alpha), DCGFTS (SEQ ID NO: 6) (TCR beta), DVITMDPKDNCSKDAN (SEQ ID NO: 7) (TCR gamma), DHVKPKETENTKQPSKSCHKPK (SEQ ID NO: 8) (TCR delta), EPKSCDKTHTCPPCP (SEQ ID NO: 9) (IgG1), ERKCCVECPPCP (SEQ ID NO: 10) (IgG2), ELKTPLGDTTHTCPRCP (SEQ ID NO: 11) (IgG3-H1), EPKSCDTPPPCPRCP (SEQ ID NO: 12) (IgG3-H2, IgG3-H3 and IgG3-H4), ESKYGPPCPSCP (SEQ ID NO: 13) (IgG4), VPPPPP (SEQ ID NO: 14) (IgA2).

[0068] Immunoglobulin hinge region sequences may be divided into an "upper," a "core," and a "lower" hinge region. For example, in IgG1, the complete hinge region sequence is EPKSCDKTHTCPPCPAPELLGG (SEQ ID NO: 15). The upper hinge region of an IgG hinge is generally defined as the region extending from Glu216 to Thr225 (EU numbering) (i.e., EPKSCDKTHT (SEQ ID NO: 16)), the core hinge region is generally defined as the region extending from Cys226 to Pro230 (EU numbering) (i.e., CPPCP (SEQ ID NO: 17)), and the lower hinge region is generally defined as the region extending from Ala231 to Pro238 (EU numbering) (i.e., APELLGG (SEQ ID NO: 18)) (see Burton, 1985, Molec. Immunol., 22:161-206).

[0069] In certain embodiments, the amino acid extension comprises all or a portion of an immunoglobulin hinge region sequence. In some embodiments, the amino acid extension comprises all or a portion of an immunoglobulin upper hinge region sequence and / or core hinge region sequence. In some embodiments, the amino acid extension comprises all or a portion of an immunoglobulin upper hinge region sequence.

[0070] In some embodiments, the amino acid extension comprises all or a portion of the hinge region sequence of IgG1. In some embodiments, the amino acid extension comprises all or a portion of the upper hinge region sequence and / or core hinge region sequence of IgG1. In some embodiments, the amino acid extension comprises all or a portion of the upper hinge region sequence of IgG1. In some embodiments, the amino acid extension comprises the sequence: EPKSC [SEQ ID NO: 19]. In some embodiments, the amino acid extension comprises the sequence: EPKSCDKTHT [SEQ ID NO: 16].

[0071] The cysteine ​​residue in the Cα domain of the TCR α chain polypeptide that forms a hinge disulfide with the cysteine ​​residue added to the C-terminus of the Cβ domain of the TCR β chain polypeptide may be a naturally occurring cysteine ​​residue (e.g., a naturally occurring cysteine ​​residue at TRAC position 128) or may be a cysteine ​​substituent located adjacent to the C-terminus of the TCR α chain polypeptide. "Adjacent to" in this context means within 10 amino acids of the C-terminus of the TCR α chain polypeptide.

[0072] In a specific embodiment, the cysteine ​​residue in the Cα domain of the TCR α chain polypeptide that forms a hinge disulfide with the cysteine ​​residue added to the C-terminus of the Cβ domain of the TCR β chain polypeptide is the naturally occurring cysteine ​​residue at TRAC position 128.

[0073] In a specific embodiment, the TCR construct of the invention comprises: i) a cysteine ​​residue contained in an amino acid extension at the C-terminus of the Cβ domain of the TCR β chain polypeptide (the amino acid extension is 1 to about 10 amino acids in length); ii) a natural cysteine ​​residue at position 128 of TRAC in the Cα domain of the TCR α chain polypeptide; The non-native interchain disulfide bond formed between the

[0074] In a specific embodiment, the TCR construct of the invention comprises: i) a cysteine ​​residue in an amino acid extension at the C-terminus of the Cβ domain of a TCR β chain polypeptide (the amino acid extension has the sequence: EPKSC [SEQ ID NO: 19] or EPKSCDKTHT [SEQ ID NO: 16]); ii) a natural cysteine ​​residue at position 128 of TRAC in the Cα domain of the TCR α chain polypeptide; The non-native interchain disulfide bond formed between the

[0075] Other non-native interchain disulfide bonds that may be included in the TCR constructs of the invention include: i) a disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; ii) a disulfide bond between the cysteine ​​residue substituent at position 122 of TRAC and the cysteine ​​residue substituent at position 12 of TRBC; iii) a disulfide bond between the cysteine ​​residue substituent at position 124 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; iv) a disulfide bond between the cysteine ​​residue substituent at position 125 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; v) a disulfide bond between the cysteine ​​residue substituent at position 126 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; vi) a disulfide bond between the cysteine ​​residue substituent at position 127 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; and vii) Disulfide bond between the cysteine ​​residue substituent at position 28 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC Examples include:

[0076] In certain embodiments, the non-natural interchain disulfide bond contained in the TCR constructs of the present invention may include an IC disulfide (i.e., a disulfide bond between a cysteine ​​residue substituent at position TRAC84 of a TCR α chain polypeptide and a cysteine ​​residue substituent at position TRBC79 of a TCR β chain polypeptide).

[0077] In a specific embodiment, the TCR construct of the invention comprises: (a) i) a disulfide bond formed between a cysteine ​​residue contained in an amino acid extension at the C-terminus of the Cβ domain of a TCR β chain polypeptide (the amino acid extension is 1 to about 10 amino acids in length (e.g., an amino acid extension comprising all or part of an immunoglobulin or TCR hinge region sequence, such as an amino acid extension having the sequence: EPKSC [SEQ ID NO: 19] or EPKSCDKTHT [SEQ ID NO: 16])) and ii) a naturally occurring cysteine ​​residue at position TRAC128 of the Cα domain of a TCR α chain polypeptide; b) a disulfide bond between the cysteine ​​residue substituent at position 84 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; c) a disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; d) a disulfide bond between the cysteine ​​residue substituent at position 122 of TRAC and the cysteine ​​residue substituent at position 12 of TRBC; e) a disulfide bond between the cysteine ​​residue substituent at position 124 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; f) a disulfide bond between the cysteine ​​residue substituent at position 125 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; g) a disulfide bond between the cysteine ​​residue substituent at position 126 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; h) a disulfide bond between the cysteine ​​residue substituent at position 127 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; and i) A disulfide bond between the cysteine ​​residue substituent at position 28 of TRAC1 and the cysteine ​​residue substituent at position 11 of TRBC1 The present invention further comprises a non-native interchain disulfide bond selected from the group consisting of:

[0078] In a specific embodiment, the TCR construct of the invention comprises: (a) i) a disulfide bond formed between a cysteine ​​residue contained in an amino acid extension at the C-terminus of the Cβ domain of a TCR β chain polypeptide (the amino acid extension is 1 to about 10 amino acids in length (e.g., an amino acid extension comprising all or part of an immunoglobulin or TCR hinge region sequence, such as an amino acid extension having the sequence: EPKSC [SEQ ID NO: 19] or EPKSCDKTHT [SEQ ID NO: 16])) and ii) a naturally occurring cysteine ​​residue at position TRAC128 of the Cα domain of a TCR α chain polypeptide; b) a disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; c) a disulfide bond between the cysteine ​​residue substituent at position 124 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; d) a disulfide bond between the cysteine ​​residue substituent at position 125 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; e) a disulfide bond between the cysteine ​​residue substituent at position 126 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; f) a disulfide bond between the cysteine ​​residue substituent at position 127 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; and g) Disulfide bond between the cysteine ​​residue substituent at position 128 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC The present invention further comprises a non-native interchain disulfide bond selected from the group consisting of:

[0079] b) intrachain disulfide bonds Intrachain disulfide bonds that may be included as stabilizing mutations in the TCR constructs of the invention include a disulfide bond between two cysteine ​​residues in the Cα domain of the TCR construct and a disulfide bond between two cysteine ​​residues in the Cβ domain of the TCR construct. Typically, at least one of the cysteine ​​residues that make up the intrachain disulfide bond is a cysteine ​​substitute for a naturally occurring residue in the Cα or Cβ domain.

[0080] In certain embodiments, the TCR constructs of the present disclosure comprise one or more intrachain disulfide bonds. In some embodiments, the TCR constructs of the present disclosure comprise an intrachain disulfide bond between two cysteine ​​residues in the Cα domain of the TCR construct. In some embodiments, the TCR constructs of the present disclosure comprise an intrachain disulfide bond between two cysteine ​​residues in the Cα domain of the TCR construct, and both cysteine ​​residues involved in the disulfide bond are cysteine ​​substituents.

[0081] Non-native intrachain disulfide bonds that may be included in the TCR constructs of the invention include, in particular embodiments, i) a disulfide bond between the cysteine ​​residue substituent at TRAC position 39 and the cysteine ​​residue substituent at TRAC position 85, and ii) a disulfide bond between the cysteine ​​residue substituent at TRAC position 26 and the cysteine ​​residue substituent at TRAC position 85.1.

[0082] In certain embodiments in which the TCR constructs of the present disclosure comprise interchain disulfide bonds, the TCR constructs may also comprise one or more intrachain disulfide bonds as additional stabilizing mutations.

[0083] II. Point Mutations and Loop Truncation Mutations Many of the stabilizing point mutations and stabilizing loop truncation mutations were identified by the approaches outlined above and described in the Examples, and the TCR constructs of the present disclosure may include one or more of these stabilizing point mutations and / or stabilizing loop truncation mutations.

[0084] "Point mutation," as used herein, refers to the substitution of one amino acid found in the wild-type sequence with a different amino acid. In certain embodiments, the TCR constructs of the invention comprise one or more stabilizing point mutations that are amino acid substitutions at one or more of the following positions: TRAC4, TRAC26, TRAC39, TRAC85, TRAC105, TRAC120, TRBC6, TRBC36, TRBC86, and TRBC45.3.

[0085] In a specific embodiment, the TCR construct of the invention comprises: i) an amino acid substitution at position 4 of TRAC from Val to Ala, Thr, Ile, Leu, or Met; ii) an amino acid substitution at TRAC position 26 from Thr to Ala, Val, Ile, Leu, or Met; iii) an amino acid substitution at TRAC position 39 from Val to Ala, Thr, Ile, Leu, or Met; iv) an amino acid substitution at TRAC position 85 from Ala to Ser, Thr, Val, Ile, or Met; v) an amino acid substitution at TRAC position 105 from Ala to Ser, Thr, Glu, Gln, Asp, Asn, His, Lys, or Arg; vi) an amino acid substitution of Phe at position 120 of TRAC with Tyr or His; vii) an amino acid substitution at TRBC position 6 from Val to Ala, Thr, Ile, Leu, or Met; viii) an amino acid substitution of His at position 36 of TRBC with Phe, Tyr, or Trp; ix) an amino acid substitution at position 86 of TRBC from Ser to Ala or Thr, and x) an amino acid substitution of Val at position TRBC45.3 with Ser, Thr, Glu, Gln, Asp, Asn, His, Lys, or Arg The present invention further comprises one or more stabilizing point mutations selected from the group consisting of:

[0086] In certain embodiments, the amino acid substitution at position TRAC4 is a Val to Ile substitution. In certain embodiments, the amino acid substitution at position TRAC26 is a Thr to Ile substitution. In certain embodiments, the amino acid substitution at position TRAC39 is a Val to Ile substitution. In certain embodiments, the amino acid substitution at position TRAC85 is an Ala to Val substitution. In certain embodiments, the amino acid substitution at position TRAC105 is an Ala to Ser substitution. In certain embodiments, the amino acid substitution at position TRAC120 is a Phe to Tyr substitution. In certain embodiments, the amino acid substitution at position TRBC6 is a Val to Ile or Leu substitution. In certain embodiments, the amino acid substitution at position TRBC36 is a His to Phe substitution. In certain embodiments, the amino acid substitution at position TRBC86 is a Ser to Thr substitution. In certain embodiments, the amino acid substitution at position TRBC45.3 is a Val to Thr substitution.

[0087] In a specific embodiment, the TCR construct of the invention comprises: i) an amino acid substitution from Val to Ile at position 4 of TRAC; ii) an amino acid substitution at TRAC position 26 from Thr to Ile; iii) an amino acid substitution from Val to Ile at TRAC position 39; iv) an amino acid substitution of Ala to Val at position 85 of TRAC; v) an amino acid substitution of Ala to Ser at position 105 of TRAC; vi) an amino acid substitution of Phe to Tyr at position 120 of TRAC; vii) an amino acid substitution of Val to Ile or Leu at position 6 of TRBC; viii) an amino acid substitution of His to Phe at position 36 of TRBC; ix) an amino acid substitution from Ser to Thr at position 86 of TRBC, and x) Amino acid substitution from Val to Thr at position TRBC45.3 The present invention further comprises one or more stabilizing point mutations selected from the group consisting of:

[0088] As used herein, the term "loop truncation mutation" refers to a mutation that shortens a native loop of a wild-type TCR sequence by deleting one or more amino acids within the loop or by replacing all or part of the loop with a shorter amino acid sequence. In certain embodiments, a TCR construct of the invention may comprise a loop truncation mutation that shortens the DE loop of the Cβ domain of the TCR construct. The DE loop of the Cβ domain is 13 amino acids in length and spans positions TRBC84.1 to TRBC85.1. In some embodiments, the loop truncation mutation shortens the DE loop of the Cβ domain of the TCR construct by 1-10 amino acids. In some embodiments, the loop truncation mutation shortens the DE loop of the Cβ domain of the TCR construct by 1-9 amino acids, 1-8 amino acids, 1-7 amino acids, 1-6 amino acids, 1-5 amino acids, or 1-4 amino acids.

[0089] In certain non-human species, the DE loop of the TCR Cβ domain differs in amino acid sequence from the DE loop of the human TCR Cβ domain, and notably is three or four residues shorter than the human TCR DE loop. Thus, in certain embodiments, the TCR constructs of the present invention comprise a loop truncation mutation, which is a deletion of one or more amino acids, e.g., one to four consecutive amino acids or one to three consecutive amino acids, in the DE loop of the Cβ domain of the TCR construct. In some embodiments, the TCR constructs of the present invention comprise a loop truncation mutation, which is a deletion of amino acids at positions TRBC84.5-85.6.

[0090] In certain embodiments, the TCR constructs of the present invention comprise a loop truncation mutation in which all or part of the DE loop of the Cβ domain of the TCR construct is replaced with a shorter amino acid sequence, shortening the DE loop by 1 to 8 amino acids, e.g., 1 to 7 amino acids or 1 to 6 amino acids.

[0091] In some embodiments, the TCR constructs of the invention comprise a loop truncation mutation in which 5 to 13 consecutive amino acids of the DE loop are replaced with a shorter amino acid sequence, hi some embodiments, the TCR constructs of the invention comprise a loop truncation mutation in which 5 to 10 consecutive amino acids, e.g., 5 to 9, 5 to 8, or 5 to 7 consecutive amino acids of the DE loop are replaced with a shorter amino acid sequence.

[0092] In some embodiments, the shorter amino acid sequence is selected so that, alone or in combination with the amino acids flanking the insertion site, it provides a β-turn motif, thereby enabling β-turn formation. Amino acid sequences that enable β-turn formation can be readily identified, for example, using sequence analysis software and programs known in the art. In such embodiments, the shorter amino acid sequence is typically 2-4 amino acids in length. In some embodiments, the TCR constructs of the present invention comprise a loop truncation mutation in which 5-13 contiguous amino acids, e.g., 5-10 contiguous amino acids, 5-9 contiguous amino acids, 5-8 contiguous amino acids, or 5-7 contiguous amino acids, of the DE loop are replaced with a shorter amino acid sequence that enables β-turn formation. In some embodiments, the TCR constructs of the present invention comprise a loop truncation mutation in which 5-13 contiguous amino acids, e.g., 5-10 contiguous amino acids, 5-9 contiguous amino acids, 5-8 contiguous amino acids, or 5-7 contiguous amino acids, of the DE loop are replaced with a shorter sequence of 2-4 amino acids that enables β-turn formation. In some embodiments, the TCR constructs of the present invention comprise a loop truncation mutation in which the amino acids at TRBC positions 84.4 to 85.4 are replaced with a shorter sequence of 2 to 4 amino acids that allows for β-turn formation. In some embodiments, the TCR constructs of the present invention comprise a loop truncation mutation in which the amino acids at TRBC positions 84.4 to 85.4 are replaced with the amino acids Gly-Asn.

[0093] In a specific embodiment, the TCR construct of the invention comprises: Deletion of 1 to 4 consecutive amino acids in the DE loop of the Cβ domain of the TCR construct, and Replacement of 5-7 consecutive amino acids in the DE loop of the Cβ domain of the TCR construct with an amino acid sequence of 2-4 amino acids that allows for the formation of a β-turn. The present invention further comprises one or more loop truncation mutations selected from the group consisting of:

[0094] In a specific embodiment, the TCR construct of the invention comprises: Deletion of 1 to 4 consecutive amino acids in the DE loop of the Cβ domain of the TCR construct, and Replacement of the amino acids at positions 84.4-85.4 of TRBC with an amino acid sequence consisting of 2-4 amino acids that allows for β-turn formation The present invention further comprises one or more loop truncation mutations selected from the group consisting of:

[0095] In a specific embodiment, the TCR construct of the invention comprises: Deletion of amino acids at positions 84.5 to 85.6 in TRBC, and Replacement of the amino acids at positions 84.4-85.4 of TRBC with Gly-Asn The present invention further comprises one or more loop truncation mutations selected from the group consisting of:

[0096] III. Combinations of stabilizing mutations The TCR constructs of the present disclosure may include one or a combination of the stabilizing mutations described herein.

[0097] In certain embodiments, the TCR construct comprises one or more of the following stabilizing mutations: a) a disulfide bond formed between i) a cysteine ​​residue contained in an amino acid extension at the C-terminus of the Cβ domain of the TCR β chain polypeptide (the amino acid extension is 1 to about 10 amino acids in length) and ii) a naturally occurring cysteine ​​residue at position TRAC128 of the Cα domain of the TCR α chain polypeptide; b) a disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; c) a disulfide bond between the cysteine ​​residue substituent at position 124 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; d) a disulfide bond between the cysteine ​​residue substituent at position 125 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; e) a disulfide bond between the cysteine ​​residue substituent at position 126 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; f) a disulfide bond between the cysteine ​​residue substituent at position 127 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; g) a disulfide bond between the cysteine ​​residue substituent at position 128 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; h) a disulfide bond between the cysteine ​​residue substituent at position 39 of TRAC and the cysteine ​​residue substituent at position 85 of TRAC; i) a disulfide bond between the cysteine ​​residue substituent at position 26 of TRAC and the cysteine ​​residue substituent at position 85.1 of TRAC; j) an amino acid substitution at TRAC position 4 from Val to Ala, Thr, Ile, Leu or Met; k) an amino acid substitution at TRAC position 26 from Thr to Ala, Val, Ile, Leu, or Met; l) an amino acid substitution at TRAC position 39 from Val to Ala, Thr, Ile, Leu, or Met; m) an amino acid substitution at TRAC position 85 from Ala to Ser, Thr, Val, Ile, or Met; n) an amino acid substitution at TRAC position 105 from Ala to Ser, Thr, Glu, Gln, Asp, Asn, His, Lys, or Arg; o) an amino acid substitution at TRAC position 120 from Phe to Tyr or His; p) an amino acid substitution at position 6 of TRBC from Val to Ala, Thr, Ile, Leu or Met; q) an amino acid substitution at TRBC position 36 from His to Phe, Tyr or Trp; r) an amino acid substitution at position 86 of TRBC from Ser to Ala or Thr; s) an amino acid substitution at position TRBC45.3 from Val to Ser, Thr, Glu, Gln, Asp, Asn, His, Lys, or Arg; t) deletion of 1 to 4 consecutive amino acids in the DE loop of the Cβ domain of the TCR construct; and u) Replacement of the amino acids at positions 84.4 to 85.4 of TRBC with an amino acid sequence consisting of 2 to 4 amino acids that allows the formation of a β-turn.

[0098] In some embodiments, the amino acid extension at the C-terminus of the Cβ domain of the TCR β chain polypeptide comprises all or part of an immunoglobulin or TCR hinge region sequence, hi some embodiments, the amino acid extension at the C-terminus of the Cβ domain of the TCR β chain polypeptide has the sequence: EPKSC [SEQ ID NO: 19] or EPKSCDKTHT [SEQ ID NO: 16].

[0099] In some embodiments, the amino acid substitution at position TRAC4 is a Val to Ile substitution. In some embodiments, the amino acid substitution at position TRAC26 is a Thr to Ile substitution. In some embodiments, the amino acid substitution at position TRAC39 is a Val to Ile substitution. In some embodiments, the amino acid substitution at position TRAC85 is an Ala to Val substitution. In some embodiments, the amino acid substitution at position TRAC105 is an Ala to Ser substitution. In some embodiments, the amino acid substitution at position TRAC120 is a Phe to Tyr substitution. In some embodiments, the amino acid substitution at position TRBC6 is a Val to Leu or Ile substitution. In some embodiments, the amino acid substitution at position TRBC36 is a His to Phe substitution. In some embodiments, the amino acid substitution at position TRBC86 is a Ser to Thr substitution. In some embodiments, the amino acid substitution at position TRBC45.3 is a Val to Thr substitution.

[0100] In some embodiments, the deletion of 1 to 4 consecutive amino acids in the DE loop of the Cβ domain of the TCR construct is a deletion of amino acids at TRBC positions 84.5 to 85.6.

[0101] In some embodiments, the substitution of the amino acids at positions 84.4 to 85.4 of TRBC is a substitution of the amino acids Gly-Asn.

[0102] Combinations of any of the above embodiments for the TCR constructs are also envisaged, with each combination forming a separate embodiment for the purposes of this disclosure.

[0103] In some embodiments, the TCR constructs of the invention comprise one or more of the following stabilizing mutations: a) a disulfide bond formed between i) a cysteine ​​residue contained in an amino acid extension at the C-terminus of the Cβ domain of a TCR β chain polypeptide (the amino acid extension has the sequence: EPKSC [SEQ ID NO: 19] or EPKSCDKTHT [SEQ ID NO: 16]) and ii) a naturally occurring cysteine ​​residue at position TRAC128 of the Cα domain of a TCR α chain polypeptide; b) a disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; c) a disulfide bond between the cysteine ​​residue substituent at position 124 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; d) a disulfide bond between the cysteine ​​residue substituent at position 125 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; e) a disulfide bond between the cysteine ​​residue substituent at position 126 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; f) a disulfide bond between the cysteine ​​residue substituent at position 127 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; g) a disulfide bond between the cysteine ​​residue substituent at position 128 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; h) a disulfide bond between the cysteine ​​residue substituent at position 39 of TRAC and the cysteine ​​residue substituent at position 85 of TRAC; i) a disulfide bond between the cysteine ​​residue substituent at position 26 of TRAC and the cysteine ​​residue substituent at position 85.1 of TRAC; j) an amino acid substitution from Val to Ile at position 4 of TRAC; k) an amino acid substitution of Thr to Ile at position 26 of TRAC; l) an amino acid substitution of Val to Ile at position 39 of TRAC; m) an amino acid substitution of Ala to Val at position 85 of TRAC; n) an amino acid substitution of Ala to Ser at position 105 of TRAC; o) an amino acid substitution of Phe to Tyr at position 120 of TRAC; p) an amino acid substitution of Val to Leu or Ile at position 6 of TRBC; q) an amino acid substitution of His to Phe at position 36 of TRBC; r) an amino acid substitution from Ser to Thr at position 86 of TRBC; s) an amino acid substitution from Val to Thr at position 45.3 of TRBC; t) TRBC deletion of amino acids at positions 84.5 to 85.6, and u) Replacement of the amino acids at positions 84.4 to 85.4 of TRBC with the amino acids Gly-Asn.

[0104] In certain embodiments, the TCR constructs of the present disclosure comprise a combination of two or more of the stabilizing mutations described herein. Combinations of stabilizing mutations that may be included in the TCR constructs of the present invention include combinations of non-native interchain disulfide bonds, combinations of non-native intrachain disulfide bonds, combinations of point mutations and loop truncation mutations, and combinations comprising different categories of stabilizing mutations.

[0105] In certain embodiments, the TCR constructs of the invention comprise two or more of the following stabilizing mutations: a) a disulfide bond formed between i) a cysteine ​​residue contained in an amino acid extension at the C-terminus of the Cβ domain of the TCR β chain polypeptide (the amino acid extension is 1 to about 10 amino acids in length) and ii) a naturally occurring cysteine ​​residue at position TRAC128 of the Cα domain of the TCR α chain polypeptide; b) a disulfide bond between the cysteine ​​residue substituent at position 84 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; c) a disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; d) a disulfide bond between the cysteine ​​residue substituent at position 122 of TRAC and the cysteine ​​residue substituent at position 12 of TRBC; e) a disulfide bond between the cysteine ​​residue substituent at position 124 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; f) a disulfide bond between the cysteine ​​residue substituent at position 125 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; g) a disulfide bond between the cysteine ​​residue substituent at position 126 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; h) a disulfide bond between the cysteine ​​residue substituent at position 127 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; i) a disulfide bond between the cysteine ​​residue substituent at position 128 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; j) a disulfide bond between the cysteine ​​residue substituent at position 39 of TRAC and the cysteine ​​residue substituent at position 85 of TRAC; k) a disulfide bond between the cysteine ​​residue substituent at position 26 of TRAC and the cysteine ​​residue substituent at position 85.1 of TRAC; l) an amino acid substitution at position 4 of TRAC from Val to Ala, Thr, Ile, Leu, or Met; m) an amino acid substitution at TRAC position 26 from Thr to Ala, Val, Ile, Leu, or Met; n) an amino acid substitution at TRAC position 39 from Val to Ala, Thr, Ile, Leu or Met; o) an amino acid substitution at TRAC position 85 from Ala to Ser, Thr, Val, Ile, or Met; p) an amino acid substitution at TRAC position 105 from Ala to Ser, Thr, Glu, Gln, Asp, Asn, His, Lys, or Arg; q) an amino acid substitution at position 120 of TRAC from Phe to Tyr or His; r) an amino acid substitution at position 6 of TRBC from Val to Ala, Thr, Ile, Leu, or Met; s) an amino acid substitution at TRBC position 36 from His to Phe, Tyr or Trp; t) an amino acid substitution at position 86 of TRBC from Ser to Ala or Thr; u) an amino acid substitution at position TRBC45.3 from Val to Ser, Thr, Glu, Gln, Asp, Asn, His, Lys, or Arg; v) deletion of 1 to 4 consecutive amino acids in the DE loop of the Cβ domain of the TCR construct; and w) Replacement of the amino acids at positions 84.4 to 85.4 of TRBC with an amino acid sequence consisting of 2 to 4 amino acids that allows the formation of a β-turn.

[0106] In some embodiments, the amino acid extension at the C-terminus of the Cβ domain of the TCR β chain polypeptide comprises all or part of an immunoglobulin or TCR hinge region sequence, hi some embodiments, the amino acid extension at the C-terminus of the Cβ domain of the TCR β chain polypeptide has the sequence: EPKSC [SEQ ID NO: 19] or EPKSCDKTHT [SEQ ID NO: 16].

[0107] In some embodiments, the amino acid substitution at position TRAC4 is a Val to Ile substitution. In some embodiments, the amino acid substitution at position TRAC26 is a Thr to Ile substitution. In some embodiments, the amino acid substitution at position TRAC39 is a Val to Ile substitution. In some embodiments, the amino acid substitution at position TRAC85 is an Ala to Val substitution. In some embodiments, the amino acid substitution at position TRAC105 is an Ala to Ser substitution. In some embodiments, the amino acid substitution at position TRAC120 is a Phe to Tyr substitution. In some embodiments, the amino acid substitution at position TRBC6 is a Val to Leu or Ile substitution. In some embodiments, the amino acid substitution at position TRBC36 is a His to Phe substitution. In some embodiments, the amino acid substitution at position TRBC86 is a Ser to Thr substitution. In some embodiments, the amino acid substitution at position TRBC45.3 is a Val to Thr substitution.

[0108] In some embodiments, the deletion of 1 to 4 consecutive amino acids in the DE loop of the Cβ domain of the TCR construct is a deletion of amino acids at TRBC positions 84.5 to 85.6.

[0109] In some embodiments, the substitution of the amino acids at positions 84.4 to 85.4 of TRBC is a substitution of the amino acids Gly-Asn.

[0110] Combinations of any of the above embodiments for the TCR constructs are also envisaged, with each combination forming a separate embodiment for the purposes of this disclosure.

[0111] In some embodiments, the TCR constructs of the invention comprise: a) a disulfide bond formed between i) a cysteine ​​residue contained in an amino acid extension at the C-terminus of the Cβ domain of a TCR β chain polypeptide (the amino acid extension has the sequence: EPKSC [SEQ ID NO: 19] or EPKSCDKTHT [SEQ ID NO: 16]) and ii) a naturally occurring cysteine ​​residue at position TRAC128 of the Cα domain of a TCR α chain polypeptide; b) a disulfide bond between the cysteine ​​residue substituent at position 84 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; c) a disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; d) a disulfide bond between the cysteine ​​residue substituent at position 122 of TRAC and the cysteine ​​residue substituent at position 12 of TRBC; e) a disulfide bond between the cysteine ​​residue substituent at position 124 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; f) a disulfide bond between the cysteine ​​residue substituent at position 125 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; g) a disulfide bond between the cysteine ​​residue substituent at position 126 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; h) a disulfide bond between the cysteine ​​residue substituent at position 127 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; i) a disulfide bond between the cysteine ​​residue substituent at position 128 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; j) a disulfide bond between the cysteine ​​residue substituent at position 39 of TRAC and the cysteine ​​residue substituent at position 85 of TRAC; k) a disulfide bond between the cysteine ​​residue substituent at position 26 of TRAC and the cysteine ​​residue substituent at position 85.1 of TRAC; l) an amino acid substitution of Val to Ile at position 4 of TRAC; m) an amino acid substitution of Thr to Ile at position 26 of TRAC; n) an amino acid substitution of Val to Ile at position 39 of TRAC; o) an amino acid substitution from Ala to Val at position 85 of TRAC; p) an amino acid substitution from Ala to Ser at position 105 of TRAC; q) an amino acid substitution of Phe to Tyr at position 120 of TRAC; r) an amino acid substitution at position 6 of TRBC from Val to Ile or Leu; s) an amino acid substitution from His to Phe at position 36 of TRBC; t) an amino acid substitution from Ser to Thr at position 86 of TRBC; u) an amino acid substitution from Val to Thr at position TRBC45.3; v) deletion of amino acids at positions 84.5 to 85.6 of TRBC; and w) Replacement of the amino acids at positions 84.4 to 85.4 of TRBC with the amino acids Gly-Asn It contains two or more of the following stabilizing mutations:

[0112] In certain embodiments, the TCR constructs of the invention comprise a first non-native interchain disulfide bond in combination with one or more additional stabilizing mutations, which may be additional non-native interchain disulfide bonds, non-native intrachain disulfide bonds, point mutations, loop truncation mutations, or combinations thereof, as described herein.

[0113] In some embodiments in which the TCR construct comprises a first non-native interchain disulfide bond and one or more additional stabilizing mutations, the first non-native interchain disulfide bond may be a known stabilizing interchain disulfide bond or may be one of the stabilizing interchain disulfide bonds described herein. Examples of known stabilizing interchain disulfide bonds include, but are not limited to, the interchain disulfide bonds listed in Table 1.

[0114] [Table 1]

[0115] In certain embodiments, the first non-native interchain disulfide bond comprised in the TCR construct of the present invention is an IC disulfide (i.e., a disulfide bond between a cysteine ​​residue substituent at position TRAC84 of a TCR α chain polypeptide and a cysteine ​​residue substituent at position TRBC79 of a TCR β chain polypeptide).

[0116] In certain embodiments, the first non-native interchain disulfide bond comprised in the TCR construct of the invention is a) a disulfide bond formed between i) a cysteine ​​residue contained in an amino acid extension at the C-terminus of the Cβ domain of the TCR β chain polypeptide (the amino acid extension is 1 to about 10 amino acids in length) and ii) a naturally occurring cysteine ​​residue at position TRAC128 of the Cα domain of the TCR α chain polypeptide; b) a disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; c) a disulfide bond between the cysteine ​​residue substituent at position 122 of TRAC and the cysteine ​​residue substituent at position 12 of TRBC; d) a disulfide bond between the cysteine ​​residue substituent at position 124 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; e) a disulfide bond between the cysteine ​​residue substituent at position 125 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; f) a disulfide bond between the cysteine ​​residue substituent at position 126 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; g) a disulfide bond between the cysteine ​​residue substituent at position 127 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; and h) A disulfide bond between the cysteine ​​residue substituent at position 128 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC is selected from.

[0117] In some embodiments, the C-terminal amino acid extension of the Cβ domain of the TCR β chain polypeptide comprises all or part of an immunoglobulin or TCR hinge region sequence.

[0118] In some embodiments, the first non-native interchain disulfide bond comprised in the TCR construct of the invention is a) a disulfide bond formed between i) a cysteine ​​residue in an amino acid extension at the C-terminus of the Cβ domain of a TCR β chain polypeptide (the amino acid extension has the sequence: EPKSC [SEQ ID NO: 19] or EPKSCDKTHT [SEQ ID NO: 16]) and ii) a naturally occurring cysteine ​​residue at position TRAC128 in the Cα domain of a TCR α chain polypeptide; b) a disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; c) a disulfide bond between the cysteine ​​residue substituent at position 122 of TRAC and the cysteine ​​residue substituent at position 12 of TRBC; d) a disulfide bond between the cysteine ​​residue substituent at position 124 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; e) a disulfide bond between the cysteine ​​residue substituent at position 125 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; f) a disulfide bond between the cysteine ​​residue substituent at position 126 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; g) a disulfide bond between the cysteine ​​residue substituent at position 127 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; and h) A disulfide bond between the cysteine ​​residue substituent at position 128 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC is selected from.

[0119] In certain embodiments, the first non-native interchain disulfide bond comprised in the TCR construct of the invention is a) a disulfide bond formed between i) a cysteine ​​residue contained in an amino acid extension at the C-terminus of the Cβ domain of a TCR β chain polypeptide (the amino acid extension is 1 to about 10 amino acids in length) and ii) a naturally occurring cysteine ​​residue at position TRAC128 of the Cα domain of a TCR α chain polypeptide; and b) A disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC is selected from.

[0120] In some embodiments, the C-terminal amino acid extension of the Cβ domain of the TCR β chain polypeptide comprises all or part of an immunoglobulin or TCR hinge region sequence.

[0121] In some embodiments, the first non-native interchain disulfide bond comprised in the TCR construct of the invention is a) a disulfide bond formed between i) a cysteine ​​residue in an amino acid extension at the C-terminus of the Cβ domain of a TCR β chain polypeptide (the amino acid extension has the sequence: EPKSC [SEQ ID NO: 19] or EPKSCDKTHT [SEQ ID NO: 16]) and ii) a naturally occurring cysteine ​​residue at position TRAC128 in the Cα domain of a TCR α chain polypeptide; and b) A disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC is selected from.

[0122] In certain embodiments, the first non-native interchain disulfide bond comprised in the TCR construct of the invention is a) a disulfide bond formed between i) a cysteine ​​residue contained in an amino acid extension at the C-terminus of the Cβ domain of the TCR β chain polypeptide (the amino acid extension is 1 to about 10 amino acids in length) and ii) a naturally occurring cysteine ​​residue at position TRAC128 of the Cα domain of the TCR α chain polypeptide; b) a disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; and c) Disulfide bond between the cysteine ​​residue substituent at position 84 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC (IC disulfide) is selected from.

[0123] In some embodiments, the C-terminal amino acid extension of the Cβ domain of the TCR β chain polypeptide comprises all or part of an immunoglobulin or TCR hinge region sequence.

[0124] In certain embodiments, the first non-native interchain disulfide bond comprised in the TCR construct of the invention is a) a disulfide bond formed between i) a cysteine ​​residue in an amino acid extension at the C-terminus of the Cβ domain of a TCR β chain polypeptide (the amino acid extension has the sequence: EPKSC [SEQ ID NO: 19] or EPKSCDKTHT [SEQ ID NO: 16]) and ii) a naturally occurring cysteine ​​residue at position TRAC128 in the Cα domain of a TCR α chain polypeptide; b) a disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; and c) Disulfide bond between the cysteine ​​residue substituent at position 84 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC (IC disulfide) is selected from.

[0125] In certain embodiments, the one or more additional stabilizing mutations in combination with the first non-native interchain disulfide bond are: a) a disulfide bond formed between i) a cysteine ​​residue contained in an amino acid extension at the C-terminus of the Cβ domain of the TCR β chain polypeptide (the amino acid extension is 1 to about 10 amino acids in length) and ii) a naturally occurring cysteine ​​residue at position TRAC128 of the Cα domain of the TCR α chain polypeptide; b) a disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; c) a disulfide bond between the cysteine ​​residue substituent at position 122 of TRAC and the cysteine ​​residue substituent at position 12 of TRBC; d) a disulfide bond between the cysteine ​​residue substituent at position 124 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; e) a disulfide bond between the cysteine ​​residue substituent at position 125 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; f) a disulfide bond between the cysteine ​​residue substituent at position 126 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; g) a disulfide bond between the cysteine ​​residue substituent at position 127 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; h) a disulfide bond between the cysteine ​​residue substituent at position 128 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; i) a disulfide bond between the cysteine ​​residue substituent at position 39 of TRAC and the cysteine ​​residue substituent at position 85 of TRAC; j) a disulfide bond between the cysteine ​​residue substituent at position 26 of TRAC and the cysteine ​​residue substituent at position 85.1 of TRAC; k) an amino acid substitution at position 4 of TRAC from Val to Ala, Thr, Ile, Leu or Met; l) an amino acid substitution at TRAC position 26 from Thr to Ala, Val, Ile, Leu, or Met; m) an amino acid substitution at TRAC position 39 from Val to Ala, Thr, Ile, Leu or Met; n) an amino acid substitution at TRAC position 85 from Ala to Ser, Thr, Val, Ile, or Met; o) an amino acid substitution at TRAC position 105 from Ala to Ser, Thr, Glu, Gln, Asp, Asn, His, Lys, or Arg; p) an amino acid substitution of Phe at position 120 of TRAC with Tyr or His; q) an amino acid substitution at position 6 of TRBC from Val to Ala, Thr, Ile, Leu, or Met; r) an amino acid substitution of His at position 36 of TRBC with Phe, Tyr, or Trp; s) an amino acid substitution at position 86 of TRBC from Ser to Ala or Thr; t) an amino acid substitution at position TRBC45.3 from Val to Ser, Thr, Glu, Gln, Asp, Asn, His, Lys, or Arg; u) deletion of 1 to 4 consecutive amino acids in the DE loop of the Cβ domain of the TCR construct of the present invention; and v) Replacement of the amino acids at positions 84.4 to 85.4 of TRBC with an amino acid sequence consisting of 2 to 4 amino acids that allows the formation of a β-turn. wherein the first non-native interchain disulfide bond is different from any additional interchain disulfide bonds.

[0126] In some embodiments, the amino acid extension at the C-terminus of the Cβ domain of the TCR β chain polypeptide comprises all or part of an immunoglobulin or TCR hinge region sequence, hi some embodiments, the amino acid extension at the C-terminus of the Cβ domain of the TCR β chain polypeptide has the sequence: EPKSC [SEQ ID NO: 19] or EPKSCDKTHT [SEQ ID NO: 16].

[0127] In some embodiments, the amino acid substitution at position TRAC4 is a Val to Ile substitution. In some embodiments, the amino acid substitution at position TRAC26 is a Thr to Ile substitution. In some embodiments, the amino acid substitution at position TRAC39 is a Val to Ile substitution. In some embodiments, the amino acid substitution at position TRAC85 is an Ala to Val substitution. In some embodiments, the amino acid substitution at position TRAC105 is an Ala to Ser substitution. In some embodiments, the amino acid substitution at position TRAC120 is a Phe to Tyr substitution. In some embodiments, the amino acid substitution at position TRBC6 is a Val to Leu or Ile substitution. In some embodiments, the amino acid substitution at position TRBC36 is a His to Phe substitution. In some embodiments, the amino acid substitution at position TRBC86 is a Ser to Thr substitution. In some embodiments, the amino acid substitution at position TRBC45.3 is a Val to Thr substitution.

[0128] In some embodiments, the deletion of 1 to 4 consecutive amino acids in the DE loop of the Cβ domain of the TCR construct is a deletion of amino acids at TRBC positions 84.5 to 85.6.

[0129] In some embodiments, the substitution of the amino acids at positions 84.4 to 85.4 of TRBC is a substitution of the amino acids Gly-Asn.

[0130] Combinations of any of the above embodiments for the TCR constructs are also envisaged, with each combination forming a separate embodiment for the purposes of this disclosure.

[0131] In some embodiments, the one or more additional stabilizing mutations in combination with the first non-native interchain disulfide bond are: a) a disulfide bond formed between i) a cysteine ​​residue in an amino acid extension at the C-terminus of the Cβ domain of a TCR β chain polypeptide (the amino acid extension has the sequence: EPKSC [SEQ ID NO: 19] or EPKSCDKTHT [SEQ ID NO: 16]) and ii) a naturally occurring cysteine ​​residue at position TRAC128 in the Cα domain of a TCR α chain polypeptide; b) a disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; c) a disulfide bond between the cysteine ​​residue substituent at position 122 of TRAC and the cysteine ​​residue substituent at position 12 of TRBC; d) a disulfide bond between the cysteine ​​residue substituent at position 124 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; e) a disulfide bond between the cysteine ​​residue substituent at position 125 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; f) a disulfide bond between the cysteine ​​residue substituent at position 126 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; g) a disulfide bond between the cysteine ​​residue substituent at position 127 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; h) a disulfide bond between the cysteine ​​residue substituent at position 128 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; i) a disulfide bond between the cysteine ​​residue substituent at position 39 of TRAC and the cysteine ​​residue substituent at position 85 of TRAC; j) a disulfide bond between the cysteine ​​residue substituent at position 26 of TRAC and the cysteine ​​residue substituent at position 85.1 of TRAC; k) an amino acid substitution from Val to Ile at position 4 of TRAC; l) an amino acid substitution of Thr to Ile at position 26 of TRAC; m) an amino acid substitution of Val to Ile at position 39 of TRAC; n) an amino acid substitution of Ala to Val at position 85 of TRAC; o) an amino acid substitution from Ala to Ser at position 105 of TRAC; p) an amino acid substitution of Phe to Tyr at position 120 of TRAC; q) an amino acid substitution from Val to Ile or Leu at position 6 of TRBC; r) an amino acid substitution of His to Phe at position 36 of TRBC; s) an amino acid substitution from Ser to Thr at position 86 of TRBC; t) an amino acid substitution of Val to Thr at position 45.3 of TRBC; u) TRBC deletion of amino acids at positions 84.5 to 85.6, and v) Replacement of the amino acids at positions 84.4 to 85.4 of TRBC with the amino acids Gly-Asn wherein the first non-native interchain disulfide bond is different from any additional interchain disulfide bonds.

[0132] In certain embodiments, the one or more additional stabilizing mutations in combination with the non-native interchain disulfide bond are: a) an amino acid substitution at TRAC position 39 from Val to Ala, Thr, Ile, Leu or Met; b) an amino acid substitution at TRAC position 85 from Ala to Ser, Thr, Val, Ile, or Met; c) an amino acid substitution of Val at position 6 of TRBC with Ala, Thr, Ile, Leu, or Met; and d) Amino acid substitution of His at position 36 of TRBC with Phe, Tyr, or Trp is selected from.

[0133] In certain embodiments, the one or more additional stabilizing mutations in combination with the non-native interchain disulfide bond are: a) an amino acid substitution from Val to Ile at TRAC position 39; b) an amino acid substitution of Ala to Val at position 85 of TRAC; c) an amino acid substitution from Val to Ile at position 6 of TRBC; and d) Amino acid substitution from His to Phe at position 36 of TRBC is selected from.

[0134] In certain embodiments, the additional stabilizing mutations in combination with the non-native interchain disulfide bond are: a) an amino acid substitution from Val to Ile at TRAC position 39; b) an amino acid substitution of Ala to Val at position 85 of TRAC; c) an amino acid substitution from Val to Ile at position 6 of TRBC; and d) Amino acid substitution from His to Phe at position 36 of TRBC is.

[0135] In a specific embodiment, a TCR construct of the invention comprises a first non-naturally occurring interchain disulfide bond and one or more additional stabilizing mutations, wherein the first non-naturally occurring disulfide bond is a disulfide bond formed between i) a cysteine ​​residue contained in an amino acid extension at the C-terminus of the Cβ domain of a TCR β chain polypeptide, the amino acid extension being 1 to about 10 amino acids in length, and ii) a naturally occurring cysteine ​​residue at position TRAC128 of the Cα domain of a TCR α chain polypeptide, and the one or more additional stabilizing mutations are a) a disulfide bond between the cysteine ​​residue substituent at position 84 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; b) a disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; c) a disulfide bond between the cysteine ​​residue substituent at position 122 of TRAC and the cysteine ​​residue substituent at position 12 of TRBC; d) a disulfide bond between the cysteine ​​residue substituent at position 124 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; e) a disulfide bond between the cysteine ​​residue substituent at position 125 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; f) a disulfide bond between the cysteine ​​residue substituent at position 126 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; g) a disulfide bond between the cysteine ​​residue substituent at position 127 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; and h) A disulfide bond between the cysteine ​​residue substituent at position 128 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC The present invention further comprises an additional non-native interchain disulfide bond selected from:

[0136] In some embodiments, the C-terminal amino acid extension of the Cβ domain of the TCR β chain polypeptide comprises all or part of an immunoglobulin or TCR hinge region sequence.

[0137] In some embodiments, the first non-natural interchain disulfide bond comprised in the TCR construct of the present invention is a disulfide bond formed between i) a cysteine ​​residue in an amino acid extension at the C-terminus of the Cβ domain of a TCR β chain polypeptide (the amino acid extension has the sequence: EPKSC [SEQ ID NO: 19] or EPKSCDKTHT [SEQ ID NO: 16]) and ii) a natural cysteine ​​residue at position TRAC128 in the Cα domain of a TCR α chain polypeptide.

[0138] In a specific embodiment, a TCR construct of the invention comprises a first non-native interchain disulfide bond and one or more additional stabilizing mutations, wherein the first non-native disulfide bond is a disulfide bond between a cysteine ​​residue substituent at position TRAC84.2 and a cysteine ​​residue substituent at position TRBC79, and the one or more additional stabilizing mutations are a) a disulfide bond formed between i) a cysteine ​​residue contained in an amino acid extension at the C-terminus of the Cβ domain of the TCR β chain polypeptide (the amino acid extension is 1 to about 10 amino acids in length) and ii) a naturally occurring cysteine ​​residue at position TRAC128 of the Cα domain of the TCR α chain polypeptide; b) a disulfide bond between the cysteine ​​residue substituent at position 122 of TRAC and the cysteine ​​residue substituent at position 12 of TRBC; c) a disulfide bond between the cysteine ​​residue substituent at position 124 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; d) a disulfide bond between the cysteine ​​residue substituent at position 125 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; e) a disulfide bond between the cysteine ​​residue substituent at position 126 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; f) a disulfide bond between the cysteine ​​residue substituent at position 127 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; and g) Disulfide bond between the cysteine ​​residue substituent at position 128 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC The present invention further comprises an additional non-native interchain disulfide bond selected from:

[0139] In certain embodiments, the TCR constructs of the present disclosure comprise a combination of a first non-native interchain disulfide bond and one or more additional stabilizing mutations, where the combination is one of the combinations described for any one of the variants listed in Table 2 (Figure 11). In some embodiments, the TCR constructs of the present disclosure comprise a combination of a first non-native interchain disulfide bond and one or more additional stabilizing mutations, where the combination is one of the combinations described for any one of the variants listed in Table 3 (Figure 12).

[0140] In certain embodiments, the TCR constructs of the present disclosure comprise a combination of two or more stabilizing mutations, wherein the stabilizing mutations are point mutations and / or loop truncation mutations. In some embodiments, the TCR constructs of the present invention comprise: i) an amino acid substitution at position 4 of TRAC from Val to Ala, Thr, Ile, Leu, or Met; ii) an amino acid substitution at TRAC position 26 from Thr to Ala, Val, Ile, Leu, or Met; iii) an amino acid substitution at TRAC position 39 from Val to Ala, Thr, Ile, Leu, or Met; iv) an amino acid substitution at TRAC position 85 from Ala to Ser, Thr, Val, Ile, or Met; v) an amino acid substitution at TRAC position 105 from Ala to Ser, Thr, Glu, Gln, Asp, Asn, His, Lys, or Arg; vi) an amino acid substitution of Phe at position 120 of TRAC with Tyr or His; vii) an amino acid substitution at TRBC position 6 from Val to Ala, Thr, Ile, Leu, or Met; viii) an amino acid substitution of His at position 36 of TRBC with Phe, Tyr, or Trp; ix) an amino acid substitution at position 86 of TRBC from Ser to Ala or Thr; x) an amino acid substitution of Val at position TRBC45.3 with Ser, Thr, Glu, Gln, Asp, Asn, His, Lys, or Arg; xi) deletion of 1 to 4 consecutive amino acids in the DE loop of the Cβ domain of the TCR construct; and xii) Replacement of the amino acids at positions 84.4 to 85.4 of TRBC with an amino acid sequence consisting of 2 to 4 amino acids that allows for the formation of a β-turn. The compound comprises two or more stabilizing mutations selected from:

[0141] In some embodiments, the TCR constructs of the invention comprise: i) an amino acid substitution from Val to Ile at position 4 of TRAC; ii) an amino acid substitution at TRAC position 26 from Thr to Ile; iii) an amino acid substitution from Val to Ile at TRAC position 39; iv) an amino acid substitution of Ala to Val at position 85 of TRAC; v) an amino acid substitution of Ala to Ser at position 105 of TRAC; vi) an amino acid substitution of Phe to Tyr at position 120 of TRAC; vii) an amino acid substitution of Val to Ile or Leu at position 6 of TRBC; viii) an amino acid substitution of His to Phe at position 36 of TRBC; ix) an amino acid substitution from Ser to Thr at position 86 of TRBC; x) an amino acid substitution of Val to Thr at position TRBC45.3; xi) deletion of amino acids at positions 84.5 to 85.6 of TRBC; and xii) Replacement of the amino acids at positions 84.4 to 85.4 of TRBC with the amino acids Gly-Asn The compound comprises two or more stabilizing mutations selected from:

[0142] TCR fusion proteins Certain embodiments of the present disclosure relate to TCR fusion proteins comprising one or more TCR constructs as described herein operably linked to a scaffold and / or one or more additional biologically active moieties. The term "operably linked," as used herein, means that the described components are in a relationship that allows each to function in its intended manner. The TCR constructs may be directly or indirectly linked to the scaffold or additional biologically active moiety in the TCR fusion protein. Indirectly linked means that a given TCR construct is linked to the scaffold or biologically active moiety via another component, such as a linker or a second TCR construct. Various configurations of TCR fusion proteins are contemplated, as described in more detail below.

[0143] The TCR fusion proteins of the present disclosure may comprise one TCR construct or multiple TCR constructs. The number of TCR constructs that may be included in the TCR fusion protein will depend on the nature of the scaffold and / or additional biologically active moieties included in the fusion protein. Typically, the TCR fusion proteins of the present disclosure comprise between 1 and 24 TCR constructs. In specific embodiments, the TCR fusion proteins may comprise between 1 and 12 TCR constructs, between 1 and 8 TCR constructs, between 1 and 6 TCR constructs, between 1 and 4 TCR constructs, or between 1 and 3 TCR constructs.

[0144] scaffold In certain embodiments, the TCR fusion proteins of the present disclosure include a scaffold. The scaffold may be, for example, a protein (including a peptide or polypeptide), a polymer, a nanoparticle, or another chemical substance. When the scaffold is a protein, each TCR construct included in the TCR fusion protein is typically linked to either the N-terminus or C-terminus of the protein scaffold; however, in certain embodiments, it is also envisioned that the TCR constructs may be linked to a region other than the N-terminus or C-terminus via an amino acid side chain, with or without a linker.

[0145] In embodiments where the scaffold is a protein, the TCR construct may be linked to the scaffold by genetic fusion or chemical conjugation. In embodiments where the scaffold is a polymer or nanoparticle, the TCR construct is typically linked to the scaffold by chemical conjugation.

[0146] In certain embodiments, the TCR fusion proteins of the invention comprise a protein scaffold. Examples of protein scaffolds include immunoglobulin (Ig) Fc regions, albumin, albumin analogs and derivatives, toxins, cytokines, chemokines, growth factors, and protein pairs such as leucine zipper domains (e.g., Fos and Jun) (Kostelny, et al., 1992, J. Immunol., 148:1547-53; Wranik, et al., 2012, J. Biol. Chem., 287:43331-43339), barnase and barstar (Deyev, et al., 2003, Nat. Biotechnol., 21:1486-1492), and split fluorescent protein pairs (WO 2011 / 13504). Examples of albumin derivatives include those described in WO2012 / 116453 and WO2014 / 012082, which may be fused to up to four different TCR constructs, optionally via linkers.

[0147] In certain embodiments, other protein scaffolds described in combination with various binding domains are also contemplated (see, e.g., Muller et al., 2007, J Biol Chem, 282:12650-12660; McDonaugh et al., 2012, Mol Cancer Ther, 11:582-593; Vallera et al., 2005, Clin Cancer Res, 11:3879-3888; Song et al., 2006, Biotech Appl Biochem, 45:147-154; U.S. Patent Application Publication No. US2009 / 0285816; and International Publication Nos. 2012 / 116453 and WO2014 / 012082).

[0148] Fc area In certain embodiments, the TCR fusion proteins described herein comprise a scaffold based on an immunoglobulin (Ig) Fc region. The terms "Fc region," "Fc," and "Fc domain," when used interchangeably herein, refer to the C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of its constant region. The term includes native-sequence Fc regions and variant Fc regions. The "Fc polypeptide" of a dimeric Fc refers to one of the two polypeptides that form the dimeric Fc region, which comprises the C-terminal constant region of an immunoglobulin heavy chain that is capable of stably self-associating.

[0149] Unless otherwise specified herein, numbering of amino acid residues in the Fc or constant region of an Ig is according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0150] The Fc region may comprise only a CH3 domain or both a CH3 domain and a CH2 domain. The CH3 domain comprises two CH3 sequences, each of which is contained in one of the two Fc polypeptides of the dimeric Fc. Similarly, the CH2 domain comprises two CH2 sequences, each of which is contained in one of the two Fc polypeptides of the dimeric Fc.

[0151] The Ig Fc region included in the TCR fusion protein may be based on the Fc region of IgG, IgA, or IgM. In certain embodiments, the TCR fusion protein comprises an IgG Fc-based scaffold. In some embodiments, the TCR fusion protein comprises an IgG1 Fc-based scaffold. In some embodiments, the TCR fusion protein comprises a human IgG Fc-based scaffold. In some embodiments, the TCR fusion protein comprises a human IgG1 Fc-based scaffold.

[0152] In certain embodiments, the Ig Fc region included in the TCR fusion protein may be a variant Fc region comprising one or more amino acid modifications in the CH3 domain, the CH2 domain, or both. In some embodiments, the Ig Fc region included in the TCR fusion protein may be a variant Fc region that is a heterodimeric Fc comprising two different Fc polypeptides.

[0153] In certain embodiments, the TCR fusion protein comprises a variant IgG Fc-based scaffold in which the CH3 domain comprises one or more amino acid modifications ("modified CH3 domain"). In some embodiments, the TCR fusion protein comprises a variant IgG Fc-based scaffold in which the CH2 domain comprises one or more amino acid modifications ("modified CH2 domain"). In some embodiments, the TCR fusion protein comprises a variant IgG Fc-based scaffold in which the CH3 domain comprises one or more amino acid modifications and the CH2 domain comprises one or more amino acid modifications.

[0154] Engineered CH3 domain In certain embodiments, the TCR fusion proteins described herein comprise a heterodimeric Ig Fc comprising a modified CH3 domain. In some such embodiments, the modified CH3 domain comprises one or more asymmetric amino acid modifications. As used herein, "asymmetric amino acid modifications" refer to modifications in which an amino acid at a given position in a first Fc polypeptide differs from the amino acid at the corresponding position in a second Fc polypeptide. These asymmetric amino acid modifications may involve modification of only one of the two amino acids at the corresponding position in each Fc polypeptide, or may involve modification of both amino acids at the corresponding positions in each of the first and second Fc polypeptides.

[0155] In certain embodiments, the TCR fusion protein comprises a heterodimeric Fc comprising a modified CH3 domain, wherein the modified CH3 domain comprises one or more asymmetric amino acid modifications that favor the formation of a heterodimeric Fc over the formation of a homodimeric Fc. Amino acid modifications that may be made to the CH3 domain of an Fc to promote heterodimeric Fc formation are known in the art and include, for example, those described in WO 96 / 027011 ("knobs into holes"), Gunasekaran et al., 2010, J Biol Chem, 285, 19637-46 ("electrostatic steering"), Davis et al., 2010, Prot Eng Des Sel, 23(4):195-202 (strand exchange engineered domain (SEED) technology), and Labrijn et al., 2013, Proc Natl Acad Sci USA, 110(13):5145-50 (Fab arm exchange). Other examples include approaches that combine positive and negative design strategies to create asymmetrically engineered stable Fc regions, as described in International Publication Nos. WO2012 / 058768 and WO2013 / 063702.

[0156] In certain embodiments, the TCR fusion proteins of the invention comprise a scaffold that is a heterodimeric Fc with a modified CH3 domain, as described in WO2012 / 058768 or WO2013 / 063702.

[0157] In some embodiments, the TCR fusion proteins of the invention comprise a scaffold that is a heterodimeric human IgG1 Fc with a modified CH3 domain. Table 4 below shows the amino acid sequence of the human IgG1 Fc sequence corresponding to amino acids 231-447 of the full-length human IgG1 heavy chain. The CH2 domain is typically defined as the domain comprising amino acids 231-340 of the full-length human IgG1 heavy chain, and the CH3 domain is typically defined as the domain comprising amino acids 341-447 of the full-length human IgG1 heavy chain.

[0158] In certain embodiments, the TCR fusion proteins of the invention comprise a heterodimeric Fc having a modified CH3 domain comprising one or more asymmetric amino acid modifications that favor heterodimeric Fc formation over homodimeric Fc formation, wherein the modified CH3 domain comprises a first Fc polypeptide comprising amino acid substitutions at positions F405 and Y407 and a second Fc polypeptide comprising amino acid substitutions at positions T366 and T394. In some embodiments, the amino acid substitution at position F405 in the first Fc polypeptide of the modified CH3 domain is F405A, F405I, F405M, F405S, F405T, or F405V. In some embodiments, the amino acid substitution at position Y407 in the first Fc polypeptide of the modified CH3 domain is Y407I or Y407V. In some embodiments, the amino acid substitution at position T366 in the second Fc polypeptide of the modified CH3 domain is T366I, T366L, or T366M. In some embodiments, the amino acid substitution at position T394 of the second Fc polypeptide of the modified CH3 domain is T394W. In some embodiments, the first Fc polypeptide of the modified CH3 domain further comprises an amino acid substitution at position L351, which amino acid substitution is L351Y. In some embodiments, the second Fc polypeptide of the modified CH3 domain further comprises an amino acid substitution at position K392 selected from K392F, K392L, and K392M. In some embodiments, one or both of the first Fc polypeptide and the second Fc polypeptide of the modified CH3 domain further comprise the amino acid substitution T350V.

[0159] In certain embodiments, the TCR fusion proteins of the invention comprise a heterodimeric Fc having a modified CH3 domain comprising one or more asymmetric amino acid modifications that favor heterodimeric Fc formation over homodimeric Fc formation, wherein the modified CH3 domain comprises a first Fc polypeptide comprising an amino acid substitution of F405A, F405I, F405M, F405S, F405T, or F405V, along with an amino acid substitution of Y407I or Y407V, and a second Fc polypeptide comprising an amino acid substitution of T366I, T366L, or T366M, along with an amino acid substitution of T394W. In some embodiments, the first Fc polypeptide of the modified CH3 domain further comprises an amino acid substitution of L351Y, and / or the second Fc polypeptide of the modified CH3 domain further comprises an amino acid substitution of K392F, K392L, or K392M. In some embodiments, one or both of the first and second Fc polypeptides of the modified CH3 domain further comprises the amino acid substitution T350V.

[0160] In a specific embodiment, the TCR fusion proteins of the invention comprise a heterodimeric Fc comprising a modified CH3 domain having a first Fc polypeptide comprising amino acid substitutions at positions F405 and Y407, and optionally further comprising an amino acid substitution at position L351, and a second Fc polypeptide comprising amino acid substitutions at positions T366 and T394, and optionally further comprising an amino acid substitution at position K392, as described above, wherein the first Fc polypeptide has an amino acid substitution at one or both of positions S400 or Q347. and / or the second Fc polypeptide further comprises an amino acid substitution at one or both of positions K360 or N390, wherein the amino acid substitution at position S400 is S400E, S400D, S400R or S400K, the amino acid substitution at position Q347 is Q347R, Q347E or Q347K, the amino acid substitution at position K360 is K360D or K360E, and the amino acid substitution at position N390 is N390R, N390K or N390D.

[0161] In certain embodiments, the TCR fusion proteins of the invention comprise a heterodimeric Fc comprising a modified CH3 domain comprising any one of variant 1, variant 2, variant 3, variant 4, or variant 5 amino acid substitutions as shown in Table 4.

[0162] [Table 4]

[0163] Modified CH2 domain In certain embodiments, the TCR fusion proteins of the invention comprise an IgG Fc-based scaffold having a modified CH2 domain, e.g., a CH2 domain comprising amino acid modifications that alter binding to one or more Fc receptors (FcRs). In some embodiments, the amino acid modifications in the CH2 domain alter binding to one or more of the Fc receptor subclasses, FcγRI, FcγRII, and FcγRIII.

[0164] Many amino acid modifications to the CH2 domain that selectively alter the affinity of Fc for various Fcγ receptors are known in the art. Amino acid modifications that increase binding and amino acid modifications that decrease binding can each be useful in certain indications. For example, increasing the binding affinity of Fc to FcγRIIIa (an activating receptor) improves antibody-dependent cell-mediated cytotoxicity (ADCC) and, in turn, increases target cell lysis. Similarly, decreasing binding to FcγRIIb (an inhibitory receptor) can be beneficial in some situations. Reducing or eliminating ADCC and complement-mediated cytotoxicity (CDC) may be desirable in certain indications. In such cases, modified CH2 domains containing amino acid modifications that increase binding to FcγRIIb or that reduce or eliminate binding of the Fc region to all Fcγ receptors ("knockout" variants) may be useful.

[0165] Among the amino acid modifications in the CH2 domain, examples of amino acid modifications that alter Fc binding via the Fcγ receptor include S298A / E333A / K334A and S298A / E333A / K334A / K326A (improved affinity for FcγRIIIa) (Lu, et al., 2011, J Immunol Methods, 365(1-2):132-41), F243L / R292P / Y300L / V305I / P396L (improved affinity for FcγRIIIa) (Stavenhagen, et al., 2007, Cancer Res, 67(18):8882-90), and F243L / R292P / Y300L / L235V / P396L (improved affinity for FcγRIIIa) (Nordstrom JL, et al. al., 2011, Breast Cancer Res, 13(6):R123), F243L (improved affinity for FcγRIIIa) (Stewart, et al., 2011, Protein Eng Des Sel., 24(9):671-8), S298A / E333A / K334A (improved affinity for FcγRIIIa) (Shields, et al., 2001, J Biol Chem, 276(9):6591-604), S239D / I332E / A330L and S239D / I332E (improved affinity for FcγRIIIa) (Lazar, et al., 2006, Proc Natl Acad Sci USA, 103(11):4005-10), and S239D / S267E and S267E / L328F (improved affinity for FcγRIIb) (Chu, et al., 2008, Mol Immunol, 45(15):3926-33).

[0166] Additional modifications that affect Fc binding to Fcγ receptors are described in Therapeutic Antibody Engineering (Strohl & Strohl, Woodhead Publishing series in Biomedicine No 11, ISBN 1 907568 37 9, October 2012, page 283).

[0167] In certain embodiments, the TCR fusion proteins of the invention comprise an IgG Fc-based scaffold with a modified CH2 domain that comprises one or more amino acid modifications that reduce or abolish binding of the Fc region to all Fcγ receptors (i.e., a "knockout" variant).

[0168] Various publications describe strategies that have been used to engineer Fc regions to create "knockout" variants (see, for example, Strohl, 2009, Curr Opin Biotech 20:685-691 and Strohl & Strohl, "Antibody Fc engineering for optimal antibody performance" In Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing, 2012, pp 225-249). These strategies include reducing effector function through modified glycosylation (described in more detail below), using an IgG2 / IgG4 scaffold, or introducing mutations in the hinge or CH2 domain of the Fc (see, e.g., U.S. Patent Application Publication No. 2011 / 0212087, WO 2006 / 105338, U.S. Patent Application Publication No. 2012 / 0225058, U.S. Patent Application Publication No. 2012 / 0251531, and Strop et al., 2012, J. Mol. Biol., 420:204-219).

[0169] Non-limiting examples of known amino acid modifications that decrease FcγR and / or complement binding to Fc include those defined in Table 5.

[0170] [Table 5]

[0171] Additional examples include Fc regions engineered to contain the amino acid substitutions L235A / L236A / D265S and asymmetric amino acid modifications in the CH2 domain, as described in WO 2014 / 190441.

[0172] Glycosylation variants In certain embodiments, the TCR fusion proteins described herein may comprise a scaffold based on a native glycosylation-altered IgG Fc. As is known in the art, glycosylation of the Fc may be altered to increase or decrease effector function.

[0173] For example, mutating the conserved asparagine residue at position 297 to alanine, glutamine, lysine, or histidine (i.e., N297A, N297Q, N297K, or N297H) results in an aglycosylated Fc that lacks all effector function (Bolt et al., 1993, Eur. J. Immunol., 23:403-411; Tao & Morrison, 1989, J. Immunol., 143:2595-2601).

[0174] Conversely, removal of fucose from the glycan linked to N297 of the heavy chain has been shown to enhance ADCC due to increased binding to FcγRIIIa (see, e.g., Shields et al., 2002, J. Biol. Chem., 277:26733-26740 and Niwa et al., 2005, J. Immunol. Methods, 306:151-160). Such low-fucose antibodies may be produced, for example, in knockout Chinese hamster ovary (CHO) cells deficient in fucosyltransferase (FUT8) (Yamane-Ohnuki et al., 2004, Biotechnol. Bioeng., 87:614-622), in the variant CHO cell line Lec13, which has a reduced ability to add fucose to N297-linked glycans (WO 2003 / 035835), or in other cells that produce afucosylated antibodies (see, e.g., Li et al., 2006, Nat Biotechnol, 24:210-215; Shields et al., 2002, ibid.; and Shinkawa et al., 2003, J. Biol. Chem., 278:3466-3473). Additionally, WO 2009 / 135181 describes the addition of a fucose analog to the culture medium during antibody production to inhibit the introduction of fucose into the sugar chains of the antibody.

[0175] Other methods for generating antibodies with little or no fucose at the Fc glycosylation site (N297) are known in the art, such as GlymaX® technology (ProBioGen AG) (see von Horsten et al., 2010, Glycobiology, 20(12):1607-1618 and U.S. Pat. No. 8,409,572).

[0176] Other glycosylation variants include those with bisected sugar chains, such as variants in which N-acetylglucosamine (GlcNAc) is attached to a branch of a biantennary sugar chain attached to the Fc region of an antibody. Such glycosylation variants may have reduced fucosylation and / or improved ADCC function (see, e.g., WO 2003 / 011878, U.S. Pat. No. 6,602,684, and U.S. Patent Application Publication No. 2005 / 0123546). Useful glycosylation variants also include variants with at least one galactose residue in the sugar chain attached to the Fc region, which may have improved CDC function (see, e.g., WO 1997 / 030087, WO 1998 / 58964, and WO 1999 / 22764).

[0177] Additional biologically active moieties In certain embodiments, the TCR fusion proteins of the present disclosure may comprise one or more additional biologically active moieties fused or covalently attached to the TCR construct and / or, when the TCR fusion protein comprises a scaffold, to the scaffold. Examples of additional biologically active moieties that may be included in a TCR fusion protein include, but are not limited to, antigen-binding domains, ligands, receptors, receptor fragments (such as extracellular portions), cytokines, and antigens. When the TCR fusion proteins of the present invention comprise two or more additional biologically active moieties, the moieties may be the same or different.

[0178] In certain embodiments, the TCR fusion proteins of the present disclosure comprise one or more additional biologically active moieties. In some embodiments, the TCR fusion proteins comprise 1 to 6 additional biologically active moieties. In some embodiments, the TCR fusion proteins comprise 1 to 4 additional biologically active moieties.

[0179] In certain embodiments, the TCR fusion protein may comprise one or more additional biologically active moieties that are antigen-binding domains. In some embodiments, the TCR fusion protein may comprise two or more antigen-binding domains, for example, two, three, four, five, or six antigen-binding domains. When the TCR fusion protein comprises two or more antigen-binding domains, the antigen-binding domains may bind to the same antigen or different antigens.

[0180] Non-limiting examples of antigen binding domains that may be included in the TCR fusion proteins include, in some embodiments, Fab fragments, Fv fragments, single chain Fv fragments (scFv), and single domain antibodies (sdAbs).

[0181] In certain embodiments, the TCR fusion proteins of the present invention may comprise one or more additional biologically active moieties that are cytokines or biologically active fragments thereof.

[0182] format The TCR fusion proteins of the present disclosure may have a variety of formats. Within the TCR fusion protein, the TCR construct may be fused or covalently linked to the second element of the fusion protein via a TCR α chain polypeptide, a TCR β chain polypeptide, or both. For example, the TCR construct may be fused or covalently linked to a scaffold, an additional biologically active moiety, or another TCR construct via a TCR α chain polypeptide, a TCR β chain polypeptide, or both. The TCR construct may be fused or covalently linked to the second element of the fusion protein through the N-terminus or C-terminus of the relevant TCR polypeptide(s), and may be linked to the second element directly or indirectly (e.g., by a linker).

[0183] In certain embodiments, the TCR fusion proteins of the present invention comprise a TCR construct fused or covalently linked to a scaffold or additional biologically active moiety via a TCR β chain polypeptide. In certain embodiments, the TCR fusion protein comprises a TCR construct fused or covalently linked to a scaffold or additional biologically active moiety via the C-terminus of one of the polypeptides of the TCR construct. In certain embodiments, the TCR fusion protein comprises a TCR construct fused or covalently linked to a scaffold or additional biologically active moiety via the C-terminus of a TCR β chain polypeptide of the TCR construct.

[0184] In those embodiments in which the TCR fusion protein comprises a scaffold and multiple TCR constructs, the TCR constructs may be fused or covalently linked in tandem to the scaffold, or may be fused or covalently linked to different portions of the scaffold. In those embodiments in which the TCR fusion protein comprises a scaffold, a TCR construct, and an additional biologically active moiety, the TCR construct and the additional biologically active moiety may be fused or covalently linked in tandem to the scaffold, or may be fused or covalently linked to different portions of the scaffold. In those embodiments in which the TCR fusion protein comprises three or more TCR constructs, some of the TCR constructs may be fused or covalently linked in tandem to the scaffold, while other TCR constructs may be fused or covalently linked to different portions of the scaffold, or all of the TCR constructs may be fused or covalently linked to different portions of the scaffold.

[0185] In certain embodiments, the TCR fusion proteins of the present disclosure comprise a TCR construct and an additional biologically active moiety, hi some embodiments, the TCR fusion protein comprises a TCR construct fused directly or indirectly to an antigen-binding domain or cytokine.

[0186] In certain embodiments, the TCR fusion proteins of the present disclosure comprise one or more TCR constructs and a scaffold. In some embodiments, the TCR fusion proteins comprise one or more TCR constructs and an Ig Fc scaffold. In some embodiments, the TCR fusion proteins comprise one or more TCR constructs, an Ig Fc scaffold, and one or more additional biologically active moieties. In some embodiments, the TCR fusion proteins comprise one or more TCR constructs, an Ig Fc scaffold, and one or more antigen-binding domains. In some embodiments, the TCR fusion proteins comprise 1-12 TCR constructs, an Ig Fc scaffold, and 1-6 antigen-binding domains. In some embodiments, the TCR fusion proteins comprise 1-8 TCR constructs, an Ig Fc scaffold, and 1-6 antigen-binding domains. In some embodiments, the TCR fusion proteins comprise 1-6 TCR constructs, an Ig Fc scaffold, and 1-4 antigen-binding domains.

[0187] Figure 1 shows various non-limiting examples of TCR fusion protein formats comprising an IgG Fc scaffold and one or more TCR constructs, as well as non-limiting examples of TCR fusion protein formats comprising an IgG Fc scaffold, one or more TCR constructs, and one or more additional biologically active moieties (exemplified by antigen-binding domains). Figure 1(C) shows a TCR fusion protein ("TCR-scFv") comprising a TCR fused to an antigen-binding domain (scFv). Figures 1(D) and 1(E) show one-arm TCR fusion proteins ("one-arm α-fusion" (D) and "one-arm α-fusion-DS" (E)) comprising a TCR α polypeptide fused to an Fc scaffold. Figure 1(F) shows a TCR fusion protein ("Dual α Fusion-DS") comprising two TCR constructs, each independently fused to an Fc scaffold polypeptide via a TCR α polypeptide and each comprising an interchain disulfide bond between the TCR α and TCR β polypeptides. Figure 1(G) shows a TCR fusion protein ("Dual Fusion") having a TCR α polypeptide and a TCR β polypeptide independently fused to an Fc scaffold. Figures 1(H) and (I) show one-arm TCR fusion proteins ("One-Arm β Fusion" (H) and "One-Arm β Fusion-DS" (I)) comprising an interchain disulfide bond between the TCR α and β polypeptides. Figure 1(J) shows a TCR fusion protein comprising two TCR constructs independently fused to an Fc scaffold via a TCR β polypeptide, each comprising an interchain disulfide bond between the TCR α and TCR β polypeptides ("dual β fusion-DS"). Figure 1(K) shows a TCR fusion protein comprising a TCR construct fused to one polypeptide of an Fc scaffold via the TCR α chain and an scFv fused to the other Fc polypeptide, wherein the TCR comprises an interchain disulfide bond between the TCR α and TCR β polypeptides ("bispecific α fusion").Figure 1(L) shows a TCR fusion protein comprising a TCR construct fused via a TCR β polypeptide to one polypeptide of an Fc scaffold and an scFv fused to another Fc polypeptide, where the TCR comprises an interchain disulfide bond between the TCR α and TCR β polypeptides (a "bispecific β fusion"). Figure 1(M) shows a TCR fusion protein where one arm comprises a TRAV domain fused to a CH1 domain and a TRBC domain fused to a CL domain, and the second arm comprises an scFv, where each arm is independently fused to a polypeptide of an Fc scaffold (a "bispecific chimera"). Figure 1(N) shows a one-arm TCR fusion protein comprising a Fab fused to one polypeptide of an Fc scaffold and a TCR construct fused to the Fab via a TCR β polypeptide, where the TCR construct comprises an interchain disulfide bond between the TCR α and TCR β polypeptides (a "bispecific tandem β fusion"). Figure 1(O) shows a TCR fusion protein comprising a Fab fused to one polypeptide of an Fc scaffold and a first TCR construct fused to the Fab via a TCR β polypeptide, and a second TCR construct fused to the other Fc polypeptide via a TCR β polypeptide, where each TCR comprises an interchain disulfide bond between the TCR α and TCR β polypeptides (a "2x1 bispecific β fusion"). FIG. 1(P) shows a TCR fusion protein comprising a Fab fused to one polypeptide of an Fc scaffold, a first TCR construct fused to the Fab via a TCR β polypeptide, a second TCR construct fused to a second Fc polypeptide via a TCR β polypeptide, and a third TCR construct fused to the C-terminus of the second Fc polypeptide via a TCR β polypeptide, wherein each TCR comprises an interchain disulfide bond between the TCR α and TCR β polypeptides (a "3x1 bispecific β fusion").Figure 1(Q) shows a TCR fusion protein comprising a Fab fused to one polypeptide of an Fc scaffold, a first TCR construct fused to the Fab via a TCR β polypeptide, a second TCR construct fused to a second Fc polypeptide via a TCR β polypeptide, a third TCR construct fused to the second TCR via a TCR β polypeptide, and a fourth TCR construct fused to the C-terminus of the second Fc polypeptide via a TCR β polypeptide, wherein each TCR comprises an interchain disulfide bond between the TCR α and TCR β polypeptides (a "4x1 bispecific C-terminal β fusion"). Figure 1(R) shows a TCR fusion protein comprising a Fab fused to one polypeptide of an Fc scaffold, a first TCR construct fused to the Fab via a TCR β polypeptide, a second TCR construct fused to a second Fc polypeptide via a TCR β polypeptide, a third TCR construct fused to the second TCR via a TCR β polypeptide, and a fourth TCR construct fused to the C-terminus of the light chain of the Fab, where each TCR comprises an interchain disulfide bond between the TCR α and TCR β polypeptides (a "4x1 bispecific light chain β fusion"). Figure 1(S) shows a TCR fusion protein comprising a TCR fused to the C-terminus of one polypeptide of an Fc scaffold via a TCR α polypeptide (a "one-arm α fusion"). FIG. 1(T) shows a TCR fusion protein comprising a TCR fused via a TCRβ polypeptide to the C-terminus of one polypeptide of an Fc scaffold (a "one-arm β fusion").

[0188] The TCR fusion protein embodiments shown in Figure 1 are illustrative only and not limiting. Other conformations are contemplated and included in the present disclosure, including TCR fusion proteins as illustrated in Figure 1 with other antigen-binding domains or other biologically active moieties replacing the illustrated antigen-binding domains or biologically active moieties, and TCR constructs that include stabilizing mutations as described herein other than or in addition to the illustrated interchain disulfide bonds.

[0189] Preparation of TCR constructs and TCR fusion proteins The TCR constructs and TCR fusion proteins described herein may be produced using standard recombinant methods known in the art. Typically, recombinant production of a TCR construct or TCR fusion protein involves generating a polynucleotide or set of polynucleotides encoding the TCR construct or TCR fusion protein and inserting them into one or more vectors for further cloning and / or expression in a host cell. The polynucleotide(s) encoding the TCR construct or TCR fusion protein may be produced by standard methods known in the art (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1994 & updates and "Antibodies: A Laboratory Manual," 2002). nd(See, e.g., The Journal of Experimental Biology, Vol. 1, No. 1, pp. 111-114, 2014.) The number of polynucleotides required for expression of a TCR construct or TCR fusion protein will depend on the format of the construct or protein, including whether the construct includes a scaffold. When multiple polynucleotides are required, the polynucleotides may be introduced into a single vector (e.g., a multicistronic vector) or two or more vectors.

[0190] Generally, for expression, a polynucleotide or set of polynucleotides encoding a TCR construct or TCR fusion protein is introduced into an expression vector along with one or more regulatory elements, such as transcription elements (required for efficient transcription of the polynucleotide). Examples of such regulatory elements include, but are not limited to, promoters, enhancers, terminators, and polyadenylation signals. The choice of regulatory element will depend on the host cell selected for expression of the TCR construct or TCR fusion protein, and it will be apparent to those skilled in the art that such regulatory elements can be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. Optionally, the expression vector may further comprise a heterologous nucleic acid sequence that facilitates expression or purification of the expressed protein. Examples include, but are not limited to, signal peptides and affinity tags such as metal affinity tags, histidine tags, avidin / streptavidin coding sequences, glutathione-S-transferase (GST) coding sequences, and biotin coding sequences. Expression vectors can be extrachromosomal or integrating vectors.

[0191] Suitable host cells for cloning or expressing TCR constructs or TCR fusion proteins include a variety of prokaryotic or eukaryotic cells as known in the art. Prokaryotic host cells include, for example, E. coli, A. salmonicida, or B. subtilis cells. Eukaryotic host cells include, for example, mammalian cells, plant cells, insect cells, and yeast cells (such as Saccharomyces or Pichia cells). In certain embodiments, eukaryotic microorganisms such as filamentous fungi or yeast may be suitable expression host cells. Fungal and yeast strains have been developed that have "humanized" glycosylation pathways to produce antibody constructs with partially or fully human glycosylation patterns (see, e.g., Gerngross, 2004, Nat. Biotech. 22:1409-1414 and Li et al., 2006, Nat. Biotech. 24:210-215) and may be useful in certain embodiments.

[0192] In certain embodiments, the TCR constructs or TCR fusion proteins of the present invention are expressed in eukaryotic host cells. In some embodiments, the TCR constructs or TCR fusion proteins are expressed in mammalian cell lines. In this regard, mammalian cell lines adapted to growth in suspension are particularly useful. Examples include SV40-transformed monkey kidney line CV1 (COS-7), human embryonic kidney (HEK) line 293 ("293 cells") (see, e.g., Graham et al., 1977, J. Gen Virol., 36:59), baby hamster kidney cells (BHK), mouse Sertoli TM4 cells (see, e.g., Mather, 1980, Biol Reprod., 23:243-251), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma (HeLa) cells, canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary carcinoma cells (MMT060562), TRI cells (see, e.g., Mather et al., 1982, Annals of NY Acad. Sci. 199:101-104). Sci, 383:44-68), MRC5 cells, FS4 cells, Chinese hamster ovary (CHO) cells (including DHFR-CHO cells; see Urlaub et al., 1980, Proc Natl Acad Sci USA, 77:4216), and myeloma cell lines (such as Y0, NS0, and Sp2 / 0). Exemplary mammalian host cell lines are reviewed in Yazaki & Wu, Methods in Molecular Biology, Vol. 248, pp. 255-268 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003).

[0193] In certain embodiments, the TCR constructs or TCR fusion proteins of the present invention are expressed in transient or stable mammalian cell lines. In some embodiments, the TCR constructs or TCR fusion proteins are expressed in HEK293 cells, CHO cells, HeLa cells, NS0 cells, or COS cells. In some embodiments, the TCR constructs or TCR fusion proteins are expressed in HEK293 cells.

[0194] Host cells containing expression vector(s) encoding the TCR constructs or TCR fusion proteins of the invention may be cultured using conventional methods for producing TCR constructs or TCR fusion proteins. In certain embodiments, host cells containing expression vector(s) encoding the TCR constructs or TCR fusion proteins may be cultured at lower temperatures to improve expression of the TCR constructs or TCR fusion proteins and / or reduce the amount of HMW species (aggregates) of the TCR constructs or TCR fusion proteins. In certain embodiments, host cells containing expression vector(s) encoding the TCR constructs or TCR fusion proteins may be cultured at temperatures below 37°C. In some embodiments, host cells containing expression vector(s) encoding the TCR constructs or TCR fusion proteins may be cultured at temperatures between about 30°C and about 36°C, e.g., between about 30°C and about 35°C or between about 30°C and about 34°C. In some embodiments, host cells containing expression vector(s) encoding the TCR constructs or TCR fusion proteins may be cultured at a temperature of about 32°C.

[0195] Typically, the TCR constructs and TCR fusion proteins of the present invention are purified after expression. Proteins may be isolated or purified by a variety of methods known to those skilled in the art (see, for example, Protein Purification: Principles and Practice, 3). rd(See, Ed., Scopes, Springer-Verlag, NY, 1994). Standard purification methods include chromatographic techniques (including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reversed-phase chromatography) performed at atmospheric or elevated pressure using systems such as FPLC and HPLC. Additional purification methods include electrophoretic, immunological, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques in conjunction with protein concentration may also be useful.

[0196] Purification may be facilitated by the specific fusion partner. For example, TCR constructs and TCR fusion proteins may be purified using glutathione resin when a GST fusion is used, or Ni when a His-tag is used. +2 It may be purified by affinity chromatography or, if a FLAG tag is used, by immobilized anti-FLAG antibodies.

[0197] In certain embodiments where the TCR fusion proteins of the invention comprise an Ig Fc scaffold and / or an antibody Fab region, purification may involve using one of a variety of naturally occurring proteins that bind to the Fc or Fab. For example, bacterial protein A and protein G bind to the Fc region. Similarly, bacterial protein L binds to the Fab region of some antibodies.

[0198] The degree of purification required will vary depending on the intended use of the TCR construct or TCR fusion protein. In some cases, purification may not be necessary. In certain embodiments, the TCR constructs and TCR fusion proteins of the present invention are substantially pure. The term "substantially pure" (or "substantially purified"), when used with respect to the TCR constructs or TCR fusion proteins described herein, means that the TCR construct or TCR fusion protein is substantially or essentially free from components that typically accompany or interact with that protein in the host cell in which the TCR construct or TCR fusion protein is expressed. In certain embodiments, a substantially pure preparation of a TCR construct or TCR fusion protein is a protein preparation that contains less than about 10% contaminating proteins. In this context, contaminating proteins refer to any protein that is not a TCR construct or TCR fusion protein but that does not contain aggregated forms of the TCR construct or TCR fusion protein (i.e., HMW species). In some embodiments, a substantially pure preparation of a TCR construct or TCR fusion protein is a protein preparation that has less than about 8% contaminating proteins, e.g., less than about 7%, less than about 6%, or less than about 5% contaminating proteins.

[0199] In certain embodiments, preparations of TCR constructs or TCR fusion proteins of the invention contain minimal amounts of HMW species (aggregates). In some embodiments, preparations of TCR constructs or TCR fusion proteins contain about 40% or less HMW species. In some embodiments, preparations of TCR constructs or TCR fusion proteins contain about 35% or less HMW species, e.g., about 30% or less, about 25% or less, about 20% or less, or about 15% or less HMW species. In certain embodiments, the amount of HMW species is determined by size exclusion chromatography (SEC), e.g., UPLC-SEC.

[0200] Certain embodiments of the present disclosure relate to methods for preparing a TCR construct or TCR fusion protein as described herein, comprising culturing host cells into which one or more polynucleotides or one or more expression vectors encoding the TCR construct or TCR fusion protein have been introduced under conditions suitable for expression of the TCR construct or TCR fusion protein, and optionally recovering the TCR construct or TCR fusion protein from the host cells (or from the host cell culture medium). In certain embodiments, the method comprises culturing the host cells containing the polynucleotide(s) or expression vector(s) encoding the TCR construct or TCR fusion protein at a temperature below 37°C. In some embodiments, the method comprises culturing the host cells containing the polynucleotide(s) or expression vector(s) encoding the TCR construct or TCR fusion protein at a temperature between about 30°C and about 36°C, e.g., between about 30°C and about 35°C or between about 30°C and about 34°C. In some embodiments, the method comprises culturing a host cell comprising a polynucleotide(s) or expression vector(s) encoding a TCR construct or a TCR fusion protein at a temperature of about 32° C. In certain embodiments, the host cell is a human embryonic kidney (HEK) cell, such as a HEK273 cell.

[0201] Polynucleotides, Vectors, and Host Cells Certain embodiments of the present disclosure relate to isolated polynucleotides or sets of isolated polynucleotides that encode the TCR constructs or TCR fusion proteins described herein. A polynucleotide in this context may encode all or part of a TCR construct or TCR fusion protein.

[0202] The terms "nucleic acid," "nucleic acid molecule," and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, isolated DNA, isolated RNA, nucleic acid probes, and primers.

[0203] A polynucleotide "encoding" a given polypeptide is one that is transcribed (in the case of DNA) or translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A transcription termination sequence may be located 3' to the coding sequence.

[0204] Certain embodiments of the present disclosure relate to vectors (such as expression vectors) that contain one or more polynucleotides encoding a TCR construct or TCR fusion protein as described herein. The polynucleotide(s) may be contained in a single vector or in two or more vectors. In some embodiments, the polynucleotides are contained in a multicistronic vector.

[0205] Certain embodiments of the present disclosure relate to host cells comprising polynucleotide(s) encoding a TCR construct or a TCR fusion protein, or one or more vectors comprising the polynucleotide(s). In some embodiments, the host cell is a eukaryotic cell, e.g., a mammalian cell. In some embodiments, the host cell is a human cell. In some embodiments, the host cell is a human embryonic kidney (HEK) cell.

[0206] Pharmaceutical Composition For therapeutic applications, the TCR constructs and TCR fusion proteins of the invention may be supplied in the form of compositions comprising the TCR construct or TCR fusion protein and a pharmaceutically acceptable carrier or diluent. The compositions may be prepared by known procedures using well-known and readily available ingredients.

[0207] Pharmaceutical compositions may be formulated for administration to a subject, for example, by oral (e.g., buccal or sublingual), topical, parenteral, rectal, or vaginal routes, or by inhalation or spray. The term "parenteral," as used herein, includes subcutaneous injection, and intradermal, intraarticular, intravenous, intramuscular, intravascular, intrasternal, or intrathecal injection or infusion. Pharmaceutical compositions will typically be formulated in a manner suitable for administration to a subject, for example, as a syrup, elixir, tablet, troche, lozenge, hard capsule, soft capsule, pill, suppository, oily suspension, aqueous suspension, dispersible powder, dispersible granule, emulsion, injection, or solution. Pharmaceutical compositions may be supplied as a unit-dosage formulation.

[0208] In certain embodiments, pharmaceutical compositions comprising the TCR constructs or TCR fusion proteins of the invention are formulated for parenteral administration, hi some embodiments, pharmaceutical compositions comprising the TCR constructs or TCR fusion proteins of the invention are formulated for parenteral administration in a unit dosage injectable form, for example, as a lyophilized formulation or aqueous solution.

[0209] Pharmaceutically acceptable carriers and diluents are generally non-toxic to recipients at the dosages and concentrations employed. Examples of components that may be contained in such carriers and diluents include buffers such as phosphate, citrate, and other organic acids, antioxidants such as ascorbic acid and methionine, preservatives such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkylparabens (such as methylparaben or propylparaben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol, low molecular weight (less than about 10 residues) polypeptides, These include, but are not limited to, proteins such as serum albumin or gelatin, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides such as glucose, mannose, or dextrin, disaccharides, and other sugar chains, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes such as Zn-protein complexes, and non-ionic surfactants such as polyethylene glycol (PEG).

[0210] In certain embodiments, compositions containing the TCR constructs or TCR fusion proteins of the present invention may be in the form of a sterile injectable aqueous solution, a sterile injectable aqueous suspension, a sterile injectable oleaginous solution, or a sterile injectable oleaginous suspension. Such suspensions may be formulated using suitable dispersing or wetting agents and / or suspending agents known in the art. The sterile injectable solutions or suspensions may contain the TCR constructs or TCR fusion proteins of the present invention in a non-toxic parenterally acceptable carrier or diluent. Acceptable carriers and diluents that may be used include, for example, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution. In addition, sterile fixed oils may be used. For this purpose, various bland fixed oils may be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectable solutions. Adjuvants such as local anesthetics, preservatives, and / or buffers may also be included in the injectable solutions or suspensions.

[0211] In certain embodiments, compositions comprising the TCR constructs or TCR fusion proteins of the present invention may be formulated for intravenous administration to a subject. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the compositions may also include a solubilizing agent and / or a local anesthetic such as lignocaine to ease pain at the injection site. Generally, the ingredients may be supplied separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container (such as an ampule or sachet) indicating the quantity of active agent. Where the composition is to be administered by infusion, it may be dispensed using an infusion bottle containing pharmaceutical-grade sterile water or sterile saline. Where the composition is to be administered by injection, an ampule of sterile water for injection or sterile saline may be provided so that the ingredients can be mixed prior to administration.

[0212] Other pharmaceutical compositions and methods for preparing pharmaceutical compositions are known in the art and are described, for example, in "Remington: The Science and Practice of Pharmacy" (formerly "Remington's Pharmaceutical Sciences"), Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).

[0213] How to use Certain embodiments of the present disclosure relate to therapeutic uses of the TCR constructs and TCR fusion proteins described herein. For example, TCR constructs and TCR fusion proteins as described herein may be used to target various types of diseased or infected cells, or specific types of tissues or organs. Accordingly, certain embodiments of the present disclosure relate to methods of treating a disease or condition, comprising administering a TCR construct or TCR fusion protein to a subject in need thereof. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0214] Depending on the particular diseased cell, infected cell, tissue, or organ targeted by the TCR constructs or TCR fusion proteins of the present invention, a variety of diseases, disorders, and conditions may be treated. Examples include, but are not limited to, cancer, bacterial infections, viral infections, infectious diseases, and immune disorders (including immunodeficiency disorders, immunodeficiency diseases, autoimmune diseases, and autoimmune conditions). In some embodiments, the TCR constructs and TCR fusion proteins may be used in methods for treating cancer. In some embodiments, the TCR constructs and TCR fusion proteins may be used in methods for treating bacterial, viral, or infectious diseases. In some embodiments, the TCR constructs and TCR fusion proteins may be used in methods for treating immune disorders. In some embodiments, the TCR constructs and TCR fusion proteins may be used in methods for treating immunodeficiency disorders or immunodeficiency diseases. In certain embodiments, the TCR constructs and TCR fusion proteins may be used in methods for treating autoimmune diseases or autoimmune conditions.

[0215] The methods of treatment described herein involve administering to a subject in need thereof a TCR construct or TCR fusion protein of the invention. The TCR construct or TCR fusion protein will be administered to the subject by an appropriate route of administration. The route and / or method of administration will vary depending on the disease or condition being treated and the desired outcome, and can be readily determined by one of ordinary skill in the medical arts.

[0216] Alternatively, in some embodiments, host cells comprising an expression vector(s) encoding a TCR construct or TCR fusion protein of the invention may be used therapeutically or prophylactically to deliver the TCR construct or TCR fusion protein to a subject, or a polynucleotide or expression vector encoding a TCR construct or TCR fusion protein may be administered ex vivo to cells obtained from a subject and then the cells are returned to the subject's body.

[0217] Treatment is carried out by administering a "therapeutically effective amount" of the TCR construct or TCR fusion protein of the present invention. A "therapeutically effective amount" refers to an amount that is effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount may vary according to factors such as the condition, age, sex, and weight of the subject. A therapeutically effective amount may be one in which the therapeutically beneficial effects of the TCR construct or TCR fusion protein outweigh any toxic or adverse effects.

[0218] A skilled medical practitioner will be able to determine appropriate dosages for the TCR constructs or TCR fusion proteins of the invention. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular TCR construct or TCR fusion protein used, the route of administration, the time of administration, the rate of excretion of the construct or protein, the duration of treatment, other drugs, compounds and / or substances used in combination with the TCR construct or TCR fusion protein, the age, sex, weight, condition, general health and past medical history of the subject being treated, and similar factors well known in the medical arts.

[0219] Certain embodiments of the present disclosure relate to diagnostic uses of the TCR constructs and TCR fusion proteins of the present invention. For example, the TCR constructs or TCR fusion proteins may be used in vitro or in vivo to detect antigen-presenting cells bearing peptides to which the TCR constructs or TCR fusion proteins specifically bind. For diagnostic uses, the TCR constructs or TCR fusion proteins are typically labeled with a suitable detectable label.

[0220] The following examples are offered for illustrative purposes and are not intended to limit the scope of the present disclosure in any way. [Example]

[0221] Example 1: Design and cloning of TCR fusion proteins The Examples below describe various formats of TCR fusion proteins and controls. The following outlines the design and construction of TCR fusion proteins and controls.

[0222] All TCR fusion proteins contained extracellular Vα and Vβ TCR domains, along with either extracellular Cα and Cβ TCR domains, or IgG1 CL domain, IgG1 CH domain, and IgG1 Fc. Unless otherwise indicated, controls also contained extracellular TCR domains and IgG1 Fc. Amino acid residues in the Fc region are numbered according to the EU index. Amino acid residues in the extracellular TCR domains are numbered according to the IMGT numbering system (Lefranc, et al., 2005, Developmental and Comparative Immunology, 29:185-203; see Figure 9).

[0223] To optimize the preparation of these TCR fusion proteins, the IgG1 Fc contained amino acid substitutions in the CH3 domain that promote the formation of a heterodimeric Fc. Specifically, the IgG1 Fc contained in these TCR fusion proteins is a human IgG1 heterodimeric Fc containing the following amino acid substitutions in the CH3 domain (referred to throughout the examples as "Het Fc" modifications): Chain A (“Het FcA”): T350V / L351Y / F405A / Y407V Chain B (“Het FcB”): T350V / T366L / K392L / T394W

[0224] Some TCR fusion proteins also contained amino acid substitutions in CH2 (referred to throughout the examples as "FcKO"), ie, L234A / L235A / D265S, in both chains of the IgG1 Fc, which prevent FcγR binding.

[0225] Monovalent, bivalent, trivalent, and tetravalent TCR fusion proteins were prepared. Schematics of each TCR fusion protein format are shown in Figure 1. To generate each of the final TCR fusion proteins in the format shown in Figure 1, the following heavy chain constructs were prepared (N- to C-terminally): a) Vα-Cα-hinge-CH2-CH3 b) Vβ-Cβ-hinge-CH2-CH3 c) Vα-Cα-hinge-CH2-CH3 d) Vβ-Cβ-hinge-CH2-CH3-Vβ-Cβ-hinge e) Vβ-Cβ-hinge-Vβ-Cβ-hinge-CH2-CH3-Vβ-Cβ-hinge f) Vβ-Cβ-hinge-VH-CH1-CH2-CH3 g) Vβ-Cβ-hinge-VH-CH1-CH2-CH3-Vβ-Cβ-hinge h) VL-VH-hinge-CH2-CH3 i) Hinge - CH2-CH3

[0226] All heavy chain constructs contained the HetFc modification. Most TCR fusion proteins also contained complementary α or β TCR chains containing the corresponding variable and constant domains in addition to the two heavy chains. Those TCR fusion proteins containing antibody Fabs also contained a single antibody light chain.

[0227] The "hinge" sequence corresponds to the upper region of the human IgG1 hinge sequence (EPKSCDKTHT [SEQ ID NO: 16]), except when "hinge" is followed by "CH2," in which case the lower region of the human IgG1 hinge is also included (i.e., the sequence is EPKSCDKTHT CPPCPThe TCR α chain consists of an extracellular Vα domain and a Cα domain. The sequence ends with TRAC / 127. The TCR β chain consists of an extracellular Vβ domain and a Cβ domain. The sequence ends with TRCB / 126. The constant region of the wild-type TCR β chain contains a cysteine ​​residue (residue 85.1 in exon 1 of TRBC1*01 and TRBC2*01) that is not involved in the formation of either inter- or intra-chain disulfide bonds. This position is generally mutated to Ala to eliminate the possibility of mispairing. All TCR fusion proteins described in the Examples below have this TRBC / 85.1.CYS→ALA mutation introduced.

[0228] As described in the Examples below, some TCR fusion proteins contain the known disulfide bond between TRAC / 84.THR and TRBC / 79.SER, which has been shown to improve expression and stability of TCR proteins (Boulter, et al., 2003, PEDS, 16:707-711). The disulfide bond between TRAC / 84.THR and TRBC / 79.SER is referred to throughout the Examples as the "IC disulfide."

[0229] As described in the Examples below, some of the TCR fusion proteins contain the TRAC / 1.5.GLN→LYS mutation and the TRBC / 97.GLN→ASP mutation, which do not alter either the activity or stability of the TCR.

[0230] Table 1.1 provides an overview of the TCR fusion proteins that were prepared. The number of TCR "arms" (peptide-MHC targeting domains) and the number of anti-CD3 "arms" (CD3 targeting domains) contained in each TCR fusion protein is indicated in the "Format" column. For example, "1x0" indicates that the TCR fusion protein contains one TCR arm and no anti-CD3 arm, "1x1" indicates that the TCR fusion protein contains one TCR arm and one anti-CD3 arm, etc. The anti-CD3 arm is present in either scFv or Fab format, as indicated.

[0231] [Table 1.1] TIFF2023554119000006.tif103159

[0232] Vectors expressing the TCR fusion proteins of the present invention were constructed as follows: All constructs used the pTT5 vector (Durocher et al., 2002, Nucl. Acids Res., 30(2):e9) and the signal sequence MRPTWAWWLFLVLLLALWAPARG [SEQ ID NO: 22] (Barash et al., 2002, Biochem and Biophys Res. Comm., 294:835-842).

[0233] The vector encoding the TCR α chain contained the following insert: 5'-EcoRI restriction site-signal peptide-α chain-stop codon TGA-BamH1 restriction site-3'.

[0234] The vector encoding the TCR β chain contained the following insert: 5'-EcoRI restriction site-signal peptide-β chain-stop codon TGA-BamH1 restriction site-3'.

[0235] The vector encoding the IgG1 heavy chain contained the following insert: 5'-EcoR1 restriction site-signal peptide-IgG1 CH2 domain terminating at G446 (EU numbering) and IgG1 CH3 domain-stop codon TGA-BamH1 restriction site-3'.

[0236] The vector encoding the IgG1 light chain contained the following insert: 5'-EcoRI restriction site-signal peptide-IgG1 light chain-stop codon TGA-BamH1 restriction site-3'.

[0237] The TCR chain fused to the N-terminus of IgG1 Fc was linked to the upper hinge of the IgG1 heavy chain at position E216 (EU numbering) and contained a stop codon TGA and a BamH1 restriction site after the CH2 and CH3 domains terminating at position G446 (EU numbering).

[0238] In the case of the TCR chain fused to the C-terminus of IgG1 Fc, the TCR chain was followed by a short sequence of the upper hinge starting at E216 and ending at T225 (EU numbering).

[0239] All expression vectors were sequenced to confirm the correct reading frame and sequence of the coding DNA.

[0240] Example 2: Comparison of expression of TCR fusion proteins in various formats A mammalian transient transfection protocol was used to generate TCR fusion proteins in the formats shown in Table 2.1. To determine whether orientation affected fusion protein production, the formats tested included TCR domains fused in various orientations. The fusion proteins used a modified anti-gp100 TCR domain and an anti-CD3 scFv. All TCR fusion proteins containing both the TRAC and TRBC domains also included an IC disulfide (see Example 1).

[0241] The relevant TCRα, TCRβ, IgG1 heavy chain, and scFv chains of these TCR fusion proteins were co-expressed in 2.5 mL cultures of Expi293F™ cells (Thermo Fisher, Waltham, Mass.) as described below.

[0242] Expi293™ cells were cultured at 37°C in Expi293™ Expression Medium (Thermo Fisher, Waltham, MA) on an orbital shaker rotating at 125 rpm in a humidified atmosphere of 8% CO2. A total cell number of 7.5 x 10 7 A total of 2.5 μg of DNA was transfected into a 2.5 mL volume of cells. Prior to transfection, the DNA was diluted with 0.15 mL of Opti-MEM™ I Reduced Serum Medium (Thermo Fisher, Waltham, MA) to create a DNA transfection mix. 8 μL of ExpiFectamine™ 293 Reagent (Thermo Fisher, Waltham, MA) was diluted with 0.15 mL of Opti-MEM™ I Reduced Serum Medium. After 5 minutes of incubation, the solution was combined with the DNA transfection mix to a total volume of 0.30 mL. After 10–20 minutes, the DNA-ExpiFectamine™ 293 Reagent mixture was added to the cell culture. After incubation at 37°C for 18 to 22 hours, 15 μL of ExpiFectamine™ 293 Enhancer 1 and 0.15 mL of ExpiFectamine™ 293 Enhancer 2 (Thermo Fisher, Waltham, MA) were added to each culture. The cells were incubated for 5 days, and the supernatants were collected.

[0243] Protein levels in the supernatants were quantified using an Octet™ RED96 (ForteBio, Fremont, CA) with a Protein A chip. 200 μL of each culture supernatant was transferred to a 96-well plate. Samples were measured in triplicate for 120 seconds. After each reading, the chip was regenerated in 100 mM glycine (pH 1.5) for 5 seconds and then neutralized in PBS for 5 seconds. Measurements were compared to a standard curve to obtain the protein concentration of each sample. Results are shown in Table 2.1.

[0244] [Table 2.1]

[0245] As can be seen from Table 2.1, particularly high expression titers were observed for the TCR fusion proteins linked to the Fc through the β-strand (v21230 and v21232). Overall, the expression titers of these TCR fusion proteins were lower than those of the scFv constructs. Of these variants listed in Table 2.1, those containing the suffix "-B" did not contain an Fc domain, i.e., consisted of only two TCRs or two scFvs. In both cases, these homodimeric variants had lower expression titers.

[0246] Example 3: Identification of potential stabilizing mutations in TCR constant domains Using structural and computational guided approaches, we generated a library of mutation designs in the TRAC and TRBC domains that could potentially improve the thermal and / or colloidal stability of the TCR. These mutations were selected based on improved surface properties, interfacial interactions, and internal packing, as described in more detail below.

[0247] The design of these mutations focused on stabilizing the TRAC and TRBC domains, as these domains contain little variation among TCRs. The TRAC domain is identical in all human TCRs, and the TRBC domain has only two distinct allotypes. The TRBC sequences differ between TRBC1 and TRBC2 by only three residues: at position TRBC / 1.4, Lys in TRBC1 and Gln in TRBC2; at position TRBC / 1.3, Gln in TRBC1 and Lys in TRBC2; and at position TRBC / 29, Tyr in TRBC1 and Phe in TRBC2.

[0248] An in silico TCR model was prepared using the H27-14 TCR (Protein Data Bank reference number: pdb:3VXS) as a framework.

[0249] Identification of hydrophobic patch mutations Hydrophobic patches were identified using the Protein Patches tool in the Molecular Operating Environment (version 2019.01, Chemical Computing Group, Montreal, QC). All nonpolar residues in the TRAC or TRBC domains with surface-exposed side chains and an area greater than 50 Å were flagged.

[0250] Identification of stabilizing point mutations The crystal structure residues of H27-14 were analyzed by in silico mutagenesis and packing / modeling. Mutations were made in silico at every position in the TCR TRAC and TRBC domains. Each residue was substituted with every possible amino acid except proline and cysteine. These analyses identified a list of hotspot positions for manipulating preferential α-β pairing. The mutations used to improve stability were divided into different categories: 1) Cavities - Located inside the core of the domain, they enhance hydrophobic packing 2) Interface - Located at the interface between two domains, it enhances interfacial interactions 3) Patches - located on the surface, reducing exposed hydrophobic patches

[0251] The cavity, interface, and patch mutations were ranked based on the improvement in force field index. The patch mutations were further ranked based on the reduction in hydrophobic patches.

[0252] Identification of loop truncation mutants Truncation of the TRBC FG loop in Fab / TCR chimeras has previously been reported to improve expression of the chimeras (Wu, et al., 2015, MABS, 7:364-376). We investigated various truncations of the FG loop to determine whether these truncations improve expression in intact TCRs. Because the TRBC DE loop is short in many non-human TCRs, we also investigated truncations of the human TRBC DE loop and truncations with the addition of a Gly-Asn β-turn motif.

[0253] Ranking of stabilizing mutants Potential stabilizing mutations and designs at identified hotspot locations and near the hotspots in the 3D crystal structure were simulated and identified through in silico mutagenesis and packing / modeling, and scored based on a number of factors, including steric and electrostatic improvements. Steric improvements were modeled and calculated based on energy factors such as van der Waals packing, cavitation, and close hydrophobic group spacing. Similarly, electrostatic interaction energies were modeled and evaluated based on charge-charge Coulombic interactions, hydrogen bonding, and desolvation. Potential stabilizing mutations identified through this analysis are listed in Table 3.1.

[0254] [Table 3.1]

[0255] Example 4: Expression and characterization of TCR fusion proteins containing potential stabilizing mutations To determine the effect of the potential stabilizing mutations identified in Example 3 on TCR stability, TCR fusion proteins containing those mutations were constructed in a one-arm (OA) format with a β-strand fused to Fc, as described in Example 1. The TCR fusion proteins were expressed and tested for stability in vitro to identify mutations that provided particularly large improvements in expression, thermal stability, and / or colloidal stability. The fusion proteins used a modified anti-gp100 TCR domain. All TCR fusion proteins contained an IC disulfide (see Example 1).

[0256] The cells were grown at a density of 1.5–2.2 × 10 in FreeStyle™ F17 medium (GIBCO catalog number A13835-01) supplemented with G418 sulfate (Wisent Bioproducts catalog number 400-130-IG), 4 mM glutamine, and 0.1% Pluronic™ F-68 (GIBCO catalog number 24040-032). 6HEK293-6E cells were cultured at 37°C at 1000 cells / ml. A total of 1 μg of DNA (50% variant DNA, 5% GFP, 15% AKT, 30% ssDNA) was transfected per ml of HEK293-6E cells using PEI-max (Polysciences catalog no. 24765-2) at a DNA:PEI ratio of 1:2.5, with a 40:30:30 ratio for α-chain, β-chain-Fc(A), and Fc(B). The cells were then incubated at 37°C for 24 hours. After incubation, 0.5 mM valproic acid (final concentration) and 0.5% (w / v) tryptone N1 (final concentration) were added to the cells. The cells were then incubated at 37°C for 7 days before harvesting. Culture medium was harvested by centrifugation and vacuum filtered using a 0.22 μM Stericup® filter (Millipore catalog number SCGPU05RE). Samples were initially tested for expression in a volume of 0.8 mL. Samples that showed protein bands on SDS-PAGE gels were scaled up to 250-500 mL cultures.

[0257] To purify the TCR fusion protein, cells were first removed from the supernatant by centrifugation at 1000 rcf for 15 minutes. A Protein A Gravitrap™ column was prepared by equilibrating with 10 ml of PBS, and then the protein supernatant was applied in 10 ml batches. Once all the supernatant had loaded onto the column, the column was washed with 2 x 10 ml of PBS. The TCR fusion protein was eluted by adding 3 ml of 0.1 M glycine-HCl (pH 2.7). The eluted TCR fusion protein was then neutralized with 1 M Tris-HCl (pH 9). Protein yield was quantified based on absorbance at 280 nm (A280 nm). (If a precipitate was present upon neutralization of the sample, the sample was briefly centrifuged before measuring A280 nm.)

[0258] The homogeneity of the TCR fusion protein was assessed by UPLC-SEC. UPLC-SEC was performed using a Waters ACQUITY BEH200 SEC column (2.5 mL, 4.6 x 150 mm, stainless steel, 1.7 μm particles) (Waters Ltd, Mississauga, ON) attached to a Waters ACQUITY UPLC H-Class Bio system set at 30 °C and equipped with a photodiode array (PDA) detector. The run time was 7 min and the total volume per injection was 2.8 mL, using a running buffer of Dulbecco's phosphate-buffered saline (DPBS) with 0.02% Tween 20 (pH 7.4) or DPBS at 0.4 mL / min. Elution was monitored by UV absorbance in the range of 210–500 nm, and chromatograms were extracted at 280 nm. Peak integration was performed using Empower3 software (Waters Ltd, Mississauga, ON).

[0259] Samples with acceptable homogeneity (>95%) were buffer-exchanged into DPBS and purified with Protein A followed by sterile filtration. Samples with less homogeneity underwent SEC purification as follows: Samples were loaded onto a Superdex® 200 10 / 30 Increase column (GE Healthcare Life Sciences, Marlborough, MA) on an Akta™ Avant 25 Chromatography System (GE Healthcare Life Sciences, Marlborough, MA) in DBPS at a flow rate of 0.5 mL / min. Eluted protein fractions were collected based on absorbance at 280 nm and evaluated by the High Throughput Protein Express assay in both non-reduced and reduced forms using a Caliper LabChip® GXII (Perkin Elmer, Waltham, MA). The procedure was performed according to the HT Protein Express LabChip® User Guide version 2 and the LabChip GXII User Manual, with the following modifications: Either 2 μl or 5 μl of TCR fusion protein sample (concentration range 5-2000 ng / μl) was added to separate wells of a 96-well plate (BioRad, Hercules, CA) along with 7 μl of HT Protein Express Sample Buffer (Perkin Elmer catalog number 760328). The TCR fusion protein samples were then denatured at 70°C for 15 minutes. The LabChip® instrument was operated using the HT Protein Express Chip (Perkin Elmer, Waltham, MA) and Ab-200 assay settings.

[0260] The thermal stability of the TCR fusion proteins was determined by differential scanning calorimetry (DSC). Each purified TCR fusion protein was diluted to 1 mg / mL with PBS. A total of 950 μL was used for DSC analysis using a NanoDSC (TA Instruments, New Castle, DE). A buffer blank injection was performed at the start of each DSC run to stabilize the baseline, and buffer injections were performed before each TCR fusion protein injection for reference. Each sample was scanned from 25°C to 95°C at a rate of 60°C / h and 60 psi nitrogen pressure. The resulting thermograms were referenced and analyzed using a NanoAnalyze (TA Instruments, New Castle, DE). Each peak on the DSC thermogram corresponds to a thermal transition. There are three predicted thermal transitions: TCR, CH2 (approximately 71°C), and CH3 (approximately 80°C). The TCR transitions include the TRAV-TRBV interface and the TRAC-TRBC interface.

[0261] The yield, homogeneity and Tm determined for each of the TCR fusion proteins are shown in Table 4.1. Note that the DNA ratios of α chain, β chain-Fc(A) and Fc(B) used in the transfection step were not optimized (which would have affected the exact percentage of species observed).

[0262] [Table 4.1] TIFF2023554119000010.tif186165

[0263] Variants that exhibited a reduction in high molecular weight (HMW) species (aggregation) or an increase in Tm compared to the control (v21230) were considered stabilized. As can be seen from Table 4.1, TCR fusion proteins v22705, v22707, v22709, v22712, v22716, v22722, v22837, v22840, and v22772 exhibited both a reduction in HMW species and an increase in Tm compared to the control v21230. TCR fusion protein v28881 also exhibited a reduction in HMW species and an increase in Tm compared to the control v21230; however, since this protein was expressed at 32°C, a reduction in HMW species was expected. A decrease in HMW species was observed in several TCR fusion proteins, with particularly large decreases in HMW species observed in v22709 and v22837 (from 37.4% to 20.2% for v22709 and 20.1% for v22837). Among the TCR fusion proteins that showed an increase in Tm, particularly large increases were observed in v22706, v22709, and v28881. Both v22706 and v22709 showed a 3°C increase in Tm (from 53.7°C to 56.7°C), whereas v22706 showed no decrease in HMW species, and v28881 showed a 2.7°C increase in Tm (from 53.7°C to 56.4°C).

[0264] Both TCR fusion proteins contain identical sequences except for the point mutations listed in Table 4.1, and the observed increase in homogeneity and / or Tm may be due to these point mutations.

[0265] Example 5: Effect of temperature and cell line on expression of TCR fusion proteins To identify conditions that result in the highest yields and the fewest HMW species (aggregates), the TCR fusion protein v21232 was produced under a variety of expression conditions as described herein.

[0266] A TCR bispecific beta-fusion TCR fusion protein (v21232; see Example 2) comprising an anti-gp100 TCR and an anti-CD3 scFv was expressed in 2.5 mL of Expi293F™ cells (Thermo Fisher, Waltham, MA) or ExpiCHO™ cells (Thermo Fisher, Waltham, MA) at a 30:30:40 H1 DNA:H2 DNA:L1 DNA ratio.

[0267] For expression in Expi293F™ cells, cultures were prepared as described in Example 2, except that a 40:40:20 α-chain, β-chain-Fc(A), and Fc(B) DNA ratio was used during the transfection step. After 18–22 h of incubation at 37°C, 15 μL of ExpiFectamine™ 293 Enhancer 1 and 0.25 mL of ExpiFectamine™ 293 Enhancer 2 (Thermo Fisher, Waltham, MA) were added to each culture. The cultures were then placed at 37°C or 32°C for the remainder of the incubation period, after which the supernatants were harvested.

[0268] For expression in ExpiCHO™ cells, cells were cultured at 37°C in ExpiCHO™ Expression Medium (Thermo Fisher, Waltham, MA) on an orbital shaker rotating at 125 rpm in a humidified atmosphere of 8% CO2. A total cell number of 1.5 x 10 8A total of 2 μg of DNA was transfected into a 2.5 ml culture of cells at a DNA ratio of 40:40:20 (α-chain, β-chain-Fc(A), and Fc(B)). Prior to transfection, the DNA was diluted with 0.1 mL of OptiPRO™ SFM (Thermo Fisher, Waltham, MA) to obtain the DNA transfection mix. Eight μL of ExpiFectamine™ CHO Reagent (Thermo Fisher, Waltham, MA) was diluted with 92 μL of OptiPRO™ SFM and, after 1–5 min of incubation, combined with the DNA transfection mix to a total volume of 0.2 mL. After 1–5 min, the DNA and ExpiFectamine™ CHO Reagent mixture was added to the cell culture. After incubation at 37°C for 18–22 hours, 15 μL of ExpiCHO™ Enhancer and 0.6 mL of ExpiCHO™ Feed (Thermo Fisher, Waltham, MA) were added to each culture. The cultures were then warmed to 37°C or 32°C and incubated for 7 days. The supernatants were then harvested.

[0269] Protein titers in the collected supernatants were measured by Biolayer Interferometry as described in Example 2. Because titer measurements in Expi293F™ cells were significantly higher than in ExpiCHO™ cells (see Table 5.1), samples expressed in Expi293F™ cells were purified by Protein A as described in Example 4. Homogeneity was determined by UPLC-SEC as described in Example 4. Peak areas corresponding to the exact molecular weights were used to estimate the percentage of the desired monodisperse species. The results are shown in Table 5.1 and Figure 3.

[0270] [Table 5.1]

[0271] As can be seen from Table 5.1, expression of variant v21232 in Expi293F™ cells at a low temperature of 32°C produced the highest amount of desired protein. At higher temperatures (37°C), while total protein production increased, a significant portion of the protein formed high molecular weight species. As can be seen from Table 5.1 and Figure 3, the amount of high molecular weight species contained in the TCR fusion protein produced at 32°C was greatly reduced, resulting in an increased amount of desired protein. Expi293F™ cells consistently produced more protein than ExpiCHO™ cells. Therefore, only samples produced from Expi293F™ were purified and characterized by UPLC-SEC.

[0272] Example 6: Addition of a hinge-like disulfide bond to stabilize TCR fusion proteins To improve the stability of the TCR fusion proteins of the present invention, fusion proteins containing a disulfide bond mimicking the disulfide bond between the light chain and the upper hinge found in IgG1 antibodies were prepared as follows.

[0273] The native TCR sequence contains a C-terminal disulfide bond located in the linker region between the constant and transmembrane domains. A potential stability-enhancing modification was designed to introduce a disulfide bond between the native C-terminal cysteine ​​on the TCR α chain (referred to as position TRAC / 128.CYS, which is typically deleted from soluble TCR constructs) and the native cysteine ​​located in the upper hinge region of Fc in the β-Fc fusion chain (position 220.CYS (EU numbering)). The disulfide bond formed between these two cysteines (i.e., TRAC / 128.CYS and hinge 220.CYS) is referred to herein as the "TRAC-hinge disulfide."

[0274] This TRAC-hinge disulfide was first tested in two TCR fusion proteins: an anti-gp100 TCR fused to Fc and an anti-NY-ESO1 1G4-HA (high affinity) TCR fused to Fc. Both TCR fusion proteins also contained the IC disulfide. These TCR fusion proteins were constructed in a one-arm format with a β-strand fused to Fc, as described in Example 1. Both sequences were preceded by the signal peptide MRPTWAWWLFLVLLLALWAPARG [SEQ ID NO: 22]. Vector inserts were prepared as described in Example 1 and cloned into the pTT5 vector for expression.

[0275] The TCR fusion proteins were first expressed in 0.8 mL of HEK293-6E cells as described in Example 4, and expression was confirmed by SDS-PAGE. The fusion proteins were then expressed in 250 mL of HEK293-6E cells as described in Example 4, except that the DNA ratio of α chain, β chain-Fc(A), and Fc(B) was 40:40:20. After an initial 18-22 hour incubation at 37°C, the cells were incubated at 32°C and then purified and characterized by UPLC-SEC as described in Example 4.

[0276] The thermal stability of these TCR fusion proteins was determined by differential scanning calorimetry (DSC). Each purified TCR fusion protein was diluted to 0.4 mg / mL with PBS. For DSC analysis, a total of 400 μL was used with a MicroCal™ VP-Capillary DSC (GE Healthcare Life Sciences, Chicago, IL). Five buffer blank injections were performed at the start of each DSC run to stabilize the baseline, and buffer injections were performed before each TCR fusion protein injection for reference. Each sample was scanned from 20°C to 100°C at a rate of 60°C / h with low feedback, an 8-second filter, a 5-minute preTstat, and 70 psi nitrogen pressure. The resulting thermograms were referenced and analyzed using Origin7 software (OriginLab Corporation, Northampton, MA). Each peak in the DSC thermogram corresponds to a thermal transition. There are three predicted thermal transitions: TCR, CH2 (approximately 71° C.), and CH3 (approximately 80° C.). The TCR transition involves the TRAV-TRBV interface and the TRAC-TRBC interface.

[0277] The results are shown in Table 6.1 and Figure 4.

[0278] [Table 6.1]

[0279] As can be seen in Table 6.1 and Figure 4, the anti-gp100 TCR fusion protein containing the TRAC-hinge disulfide exhibited a greater than 3°C increase in Tm compared to the same fusion protein without this additional disulfide bond (comparison of variants v21230 and v22752). Similarly, the anti-NY-ESO1 1G4-HA TCR fusion protein containing the TRAC-hinge disulfide exhibited an approximately 2°C increase in Tm compared to the same fusion protein without this additional disulfide (comparison of variants v30930 and v31185). The anti-NY-ESO1 1G4-HA TCR fusion protein containing the TRAC-hinge disulfide exhibited a 29.4% increase in correct species compared to the TCR fusion protein without the TRAC-hinge disulfide. These results also indicate that the TRAC-hinge disulfide can stabilize TCR fusion proteins and is transferable to a variety of TCR fusion proteins.

[0280] Example 7: Identification of additional disulfide bonds to stabilize TCR fusion proteins Mutations were identified by in silico modeling for inclusion in the TRAC and / or TRBC domains to introduce new disulfide bonds to improve the stability of the TCR fusion proteins.

[0281] The TCR model described in Example 3 was used to identify locations for introducing novel disulfide bonds. Pairwise Cβ-Cβ and Cα-Cα distances were calculated for all non-cysteine ​​residues located in the TRAC and TRBC domains, as well as all other residues in both the α and β chains. Residue pairs with a Cβ-Cβ distance of less than 5 Å or a Cα-Cα distance of less than 7 Å, and separated by more than five residues in sequence space, were considered positive hits for potential disulfide bonds. All flagged residue pairs were visually inspected to confirm that the two residues were in the correct orientation to form a disulfide bond. Potential disulfide bonds that met the above criteria were modeled in silico.

[0282] The lowest energy conformation of each disulfide bond was used to rank the models using the following criteria: Disulfide dihedral angle energy functions as described by Katz & Kossiakoff, 1986, J Biol Chem, 261(33):15480-15485 Structural perturbation as measured by the root mean square deviation (RMSD) of the main chain and side chains The number of steric clashes in the structure Changes in either folding energy or interaction energy upon introduction of intrachain disulfide bonds and interchain disulfide bonds, respectively

[0283] The top hits that were further investigated experimentally are listed in Table 7.1.

[0284] [Table 7.1]

[0285] Example 8: Generation and characterization of TCR fusion proteins containing novel interchain disulfide bonds The effect of the interchain disulfide bond identified in Example 7 on stability was investigated by analyzing the expression, thermal stability, and colloidal stability of the TCR fusion proteins in a one-arm format.

[0286] Each of the top-ranked interchain disulfide bonds identified in Example 7 (see Table 7.1) was introduced into an anti-gp100 TCR fused to Fc. These TCR fusion proteins were constructed in a one-arm format with the β-chain fused to Fc as described in Example 1. Both sequences were preceded by the signal peptide MRPTWAWWLFLVLLLALWAPARG [SEQ ID NO: 22]. Vector inserts were prepared as described in Example 1 and cloned into the pTT5 vector for expression.

[0287] TCR fusion proteins were first tested for expression in 0.8 mL of HEK293-6E cells as described in Example 4. Expression was confirmed by SDS-PAGE, and variants for which protein expression bands were observable were then expressed in 250 mL of HEK293-6E cells as described in Example 4, except that the DNA ratio of α chain, β chain-Fc(A), and Fc(B) was 40:40:20. After the initial 18-22 hour incubation, cells were incubated at either 32°C or 37°C, then purified and characterized by UPLC-SEC as described in Example 4 and DSC as described in Example 6.

[0288] The results are shown in Table 8.1.

[0289] [Table 8.1]

[0290] Two of the identified interchain disulfide bonds (TRAC / 84.2_TRBC / 79 (v22729) and TRAC / 122_TRBC / 12 (v31093)) were observed to express and stabilize TCR fusion proteins. Both variants had lower Tm and more HMW species than v21230 (IC disulfide).

[0291] Example 9: Generation and characterization of TCR fusion proteins containing novel intrachain disulfide bonds The effect of the intrachain disulfide bond identified in Example 7 on stability was investigated by analyzing the expression, thermal stability, and colloidal stability of TCR fusion proteins in one-arm format.

[0292] Each of the top-ranked intrachain disulfide bonds identified in Example 7 (see Table 7.1) was introduced into an anti-gp100 TCR fused to Fc. The TCR fusion proteins were constructed in a one-arm format with a β-chain fused to Fc as described in Example 1. All variants included the TRAC / 84.2_TRBC / 79 disulfide (see Example 8) and the TRAC-hinge disulfide. All sequences were preceded by the signal peptide MRPTWAWWLFLVLLLALWAPARG [SEQ ID NO: 22]. Vector inserts were prepared as described in Example 1 and cloned into the pTT5 vector for expression.

[0293] The TCR fusion proteins were first tested for expression in 0.8 mL of HEK293-6E cells as described in Example 4. Expression was confirmed by SDS-PAGE, and variants for which protein expression bands were observable were then expressed in 250 mL of HEK293-6E cells as described in Example 4, except that the DNA ratio of α chain, β chain-Fc(A), and Fc(B) was 40:40:20. After the initial 18-22 hour incubation, the cells were incubated at 32°C and then purified and characterized by UPLC-SEC as described in Example 4 and DSC as described in Example 6.

[0294] The results are shown in Table 9.1.

[0295] [Table 9.1]

[0296] As can be seen from Table 9.1, variant v31085, which contains the disulfide at TRAC / 39.VAL_TRAC / 85.ALA in combination with the interchain disulfide at TRAC / 84.2.THR_TRBC / 79.LEU and the TRAC-hinge disulfide, showed an increase in Tm of approximately 5°C.

[0297] Variant v31086, which contains a combination of the disulfide at the TRAC / 26.THR_TRAC / 85.1.SER position with the disulfide of TRAC / 84.2_TRBC / 79 (Example 8) and the TRAC-hinge disulfide, showed an increase in Tm of approximately 2°C.

[0298] Example 10: Binding of stabilized TCR fusion proteins to target peptide-MHC complexes The TCR fusion proteins of Example 4, and variants v22729 and v22730 (containing a new disulfide bond; see Example 8), in which the TCR Tm is equal to or greater than that of the parent TCR fusion protein, were tested for binding to their target peptide-MHC complexes by flow cytometry as described below.

[0299] T2 cells (ATCC CRL-1992) were cultured in RPMI-1640 medium with 10% FCS and 1% penicillin-streptomycin. The cells were then centrifuged, resuspended, and mixed with 10 μM of gp100 peptide (YLEPGPVTA [SEQ ID NO: 24]). After 2 hours of incubation at 37°C to allow peptide binding, the cells were washed, resuspended in PBS with 1% FCS, and added to wells of a 96-well plate at 25 μL per well. Using parallel plates, TCR fusion proteins were prepared by serially diluting the stock starting from a 1:20 dilution (stock concentration range: 0.54–1.09 mg / ml) in 1:3 steps (resulting in a minimum concentration of 3–8 pM). The cell plates were then spun down, the supernatant removed, and the TCR fusion protein solution added. Plates were kept on ice for 30 minutes, washed once with PBS 1% FCS, and resuspended in 1:200 anti-human IgG Alexa647 conjugate (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Plates were kept on ice for another 30 minutes, then washed twice with PBS 1% FCS. Subsequently, 100 μl of PBS 1% FCS was added to the wells, and plates were analyzed by flow cytometry using a BD Fortessa™ X-20 (BD Biosciences, San Jose, CA).

[0300] The results are shown in Figures 5 and 6. As can be seen in Figure 5, all TCR fusion proteins except variant v22730 were found to bind to the target peptide-MHC complex. However, the UPLC-SEC trace of variant v22730 suggested that the majority of the sample was not the desired species (see Example 8). The EC 50 were comparable (see Figure 6). These results indicate that the stabilizing mutations introduced into the TCR fusion proteins do not affect the binding of the proteins to the target peptide-MHC complex.

[0301] Example 11: TCR fusion proteins containing combinations of stabilizing mutations Examples 4 and 6-9 describe the identification of various mutations that improve the thermal and / or colloidal stability of TCR fusion proteins. TCR fusion proteins containing various combinations of these mutations were constructed to determine whether the combined mutations could further improve the thermal and / or colloidal stability of the TCR fusion proteins.

[0302] As outlined in Table 11.1, individual mutations identified in Examples 4 and 6-9 were combined and introduced into anti-gp100 TCRs fused to Fc. These TCR fusion proteins were constructed in a one-arm format with the β-strand fused to Fc as described in Example 1. Both sequences were preceded by the signal peptide MRPTWAWWLFLVLLLALWAPARG [SEQ ID NO: 22]. Vector inserts were prepared as described in Example 1 and cloned into the pTT5 vector for expression.

[0303] The TCR fusion proteins were first tested for expression in 0.8 mL of HEK293-6E cells as described in Example 4. Expression was confirmed by SDS-PAGE, and variants with observable protein expression bands were then expressed in 250 mL of HEK293-6E cells as described in Example 4, except that the DNA ratio of α chain, β chain-Fc(A), and Fc(B) was 40:40:20. After the initial 18-22 hour incubation, the cells were incubated at 32°C for 5 days, then purified and characterized by UPLC-SEC as described in Example 4 and DSC as described in Example 6. Binding of the TCR fusion proteins to target peptide-MHC complexes was confirmed using flow cytometry as described in Example 10.

[0304] The results are shown in Table 11.1. Table 12 also includes variants v33047 and v33048, which are further discussed in Example 12.

[0305] As can be seen in Table 11.1, TCR fusion proteins containing various combinations of stabilizing mutations were successfully generated. The only combinations that were unsuccessful were those contained in variants v23953, v28893, v28903, and v23398.

[0306] When combined with the IC disulfides described above, many stabilizing mutations were observed to improve thermal stability, with several variants, including combinations containing IC disulfides, showing an increase in Tm of approximately 5°C compared to the IC disulfide alone. Specifically: v28897 (TRAC / 85 Ala→Val, TRAC-hinge disulfide and IC disulfide) v29002 (TRAC / 85 Ala→Val, TRBC / 45.3 Val→Thr, TRAC-hinge disulfide and IC disulfide) v29003 (TRAC / 85 Ala→Val, TRBC / 6 Val→Leu, TRAC-hinge disulfide and IC disulfide) v28914 (TRAC / 4 Val→Ile, TRAC / 85 Ala→Val, TRBC / 6 Val→Leu, TRAC-hinge disulfide and IC disulfide) v28915 (TRAC / 4 Val→Ile, TRAC / 85 Ala→Val, TRBC / 6 Val→Leu, ΔTRBC / 84.4-85.4→Gly-Asn, TRAC-hinge disulfide and IC disulfide) v29012 (TRAC / 4 Val→Ile, TRAC / 85 Ala→Val, TRAC / 105 Ala→Ser, TRBC / 6 Val→Leu, TRBC / 36 His→Phe, TRBC / 86 Ser→Thr, TRBC / 45.3 Val→Thr, TRAC-hinge disulfide and IC disulfide)

[0307] The combination of the TRAC / 84.2_TRBC / 79 interchain disulfide bond, but not the IC disulfide, with stabilizing mutations also improved the thermal stability of TCR fusion proteins. As can be seen from Table 11.1, all TCR fusion proteins containing the TRAC / 84.2_TRBC / 79 interchain disulfide bond and the TRAC-hinge disulfide in combination with three or more other stabilizing mutations exhibited higher thermal stability than that of the IC disulfide alone (see variants v29011, v31095, v31096, v31097, v31098, v31099, v31100, v31101, v31102, v31103, v31104, and v33048).

[0308] Among the TCR fusion proteins that do not have IC disulfides, the following proteins were particularly thermostable. v31099 (TRAC / 39 Val→Ile, TRAC / 85 Ala→Val, TRBC / 36 His→Phe, TRAC / 84.2 Leu→Cys, TRBC / 79 Ser→Cys (disulfide) and TRAC-hinge disulfide) v33048 (TRBC / 6 Val → Ile, TRBC / 36 His → Phe, TRAC / 84.2 Leu → Cys, TRBC / 79 Ser → Cys (disulfide), TRAC-hinge disulfide and TRAC / 39 Val → Cys, TRAC / 85 Ala → Cys (disulfide))

[0309] [Table 11.1] TIFF2023554119000017.tif210155TIFF2023554119000018.tif209162

[0310] Example 12: TCR fusion proteins containing alternative disulfide bonds in place of the TRAC-hinge disulfide Example 11 describes various combinations of mutations that can improve the thermal and / or colloidal stability of TCR fusion proteins in the absence of IC disulfides. All of the identified stabilizing combinations included the TRAC-hinge disulfide. Because certain formats of TCR fusion proteins cannot contain the TRAC-hinge disulfide, combinations of mutations using alternative disulfide bonds were examined to identify combinations that can improve the thermal and / or colloidal stability of TCR fusion proteins.

[0311] TCR fusion proteins were constructed containing a combination of stabilizing point mutations that replaced the TRAC-hinge disulfide and mimicked the IgG4 disulfide. The replacement disulfides consisted of TRBC / 11.CYS, which served as the equivalent of the IgG4 CH1 cysteine, and one of TRAC / 124, TRAC / 125, TRAC / 126, TRAC / 127, or TRAC / 128, which served as the equivalent of the light chain (LC) cysteine.

[0312] The disulfide introduced at the TRAC / 122.PRO_TRBC / 12.ALA position described in Example 8 was also placed near the C-terminus of the TCR and tested as another alternative to the TRAC-hinge disulfide, along with a combination of stabilizing point mutations.

[0313] A variant containing the intrachain disulfide TRAC / 39.VAL_TRAC / 85.ALA (see Example 9) along with a combination of stabilizing point mutations was also tested with (variant v33048) and without (variant v33047) the TRAC-hinge disulfide. These two variants served as controls, as described in detail below.

[0314] Both TCR fusion proteins contained the TRAC / 84.2_TRBC / 79 disulfide. The mutation combinations tested are shown in Table 12.1.

[0315] Each combination of mutations was introduced into an anti-gp100 TCR fused to Fc. These TCR fusion proteins were constructed in a one-arm format with the β-strand fused to Fc as described in Example 1. Both sequences were preceded by the signal peptide MRPTWAWWLFLVLLLALWAPARG [SEQ ID NO: 22]. Vector inserts were prepared as described in Example 1 and cloned into the pTT5 vector for expression.

[0316] The TCR fusion proteins were first tested for expression in 0.8 mL of HEK293-6E cells as described in Example 4. Expression was confirmed by SDS-PAGE, and variants with observable protein expression bands were then expressed in 250 mL of HEK293-6E cells as described in Example 4, except that the DNA ratio of α chain, β chain-Fc(A), and Fc(B) was 40:40:20. After the initial 18-22 hour incubation, the cells were incubated at 32°C for 5 days, then purified and characterized by UPLC-SEC as described in Example 4 and DSC as described in Example 6. Binding of the TCR fusion proteins to target peptide-MHC complexes was confirmed using flow cytometry as described in Example 10.

[0317] The results are shown in Table 12.1. As can be seen from Table 12.1, all of the combinations tested were successfully expressed as TCR fusion proteins. TCR fusion proteins containing the tested combinations were compared to variant v33048, which contains a TRAC-hinge disulfide. Expression yields of TCR fusion proteins containing alternative disulfide bonds were generally lower than those of variant v33048. The amount of HMW species observed in TCR fusion proteins containing alternative disulfide bonds was similar to that of variant v33048, and the amounts observed in variants v33049 and v33055 were very similar. All other variants contained less than 15% HMW species.

[0318] All TCR fusion proteins containing alternative C-terminal disulfide bonds (i.e., variants v33048, v33049, v33050, v33051, v33052, v33053, v33054, v33056, and v33057) exhibited increased Tm compared to variant v33047, which does not have an alternative C-terminal disulfide bond. In addition, several variants containing alternative C-terminal disulfide bonds exhibited increased Tm compared to variant v33048, which contains a TRAC-hinge disulfide (see variants v33050, v33053, v33054, v33056, and v33057 in Table 12.1).

[0319] In summary, all alternative C-terminal disulfide bonds tested were successful in improving the stability of TCR fusion proteins and can be used in place of the TRAC-hinge disulfide.

[0320] [Table 12.1]

[0321] Example 13: Bispecific TCR fusion proteins containing stabilizing mutations This example describes the construction, expression and characterization of CD3-binding bispecific TCR fusion proteins comprising an anti-CD3 scFv or anti-CD3 Fab and one or more TCRs.

[0322] The TCR fusion proteins were expressed as heterodimers with one or two TCR components and one anti-CD3 component, which was either humanized OKT3 or humanized UCHT-1 as the paratope in scFv or canonical Fab format (see WO 2017 / 008169 and WO 2010 / 133828).

[0323] The TCR components were either anti-gp100 TCR or anti-NY-ESO1 1G4-HA with substitutions in the CDRs to confer different affinities, as shown in Table 13.1 (see Li et al., 2005, Nature Biotechnology, 23(3):349-354 and WO 2011 / 001152).

[0324] Both TCR components contained the following stabilizing mutations in the TCR constant domain: TRAC / 4.VAL→ILE, TRAC / 85.ALA→VAL, TRAC / 105.ALA→SER, TRBC / 6.VAL→LEU, TRBC / 36.HIS→PHE, TRBC / 86.SER→THR, TRBC / 45.3→THR, Δ_TRBC / 84.4-85.4→GLY-ASN, TRAC / 84.2→CYS_TRBC / 79.SER→CYS (disulfide), and TRAC-hinge disulfide.

[0325] The TCR fusion proteins included a human IgG1 heterodimeric Fc containing CH3 domain amino acid substitutions that promote heterodimeric Fc formation, as described in Example 1. Both bispecific variants included CH2 domain amino acid substitutions (L234A, L235A, and D265S) that knock out FcγR binding.

[0326] The following TCR fusion protein formats were used: one-arm beta fusion ("OA"; see Figure 1H), bispecific beta fusion ("hybrid"; see Figure 1L), 2x1 bispecific beta fusion ("2x1"; see Figure 1O), and bispecific tandem beta fusion ("1x1 tandem"; see Figure 1N).

[0327] [Table 13.1]

[0328] Vector inserts encoding each bispecific TCR fusion protein were prepared and cloned into the pTT5 vector for expression as described in Example 1. Each sequence was preceded by the signal peptide MRPTWAWWLFLVLLLALWAPARG [SEQ ID NO: 22].

[0329] TCR fusion proteins were first tested for expression in 0.8 mL of HEK293-6E cells as described in Example 4. Expression was confirmed by SDS-PAGE, and variants with observable protein expression bands were then expressed in 250 mL of HEK293-6E cells as described in Example 4. After an initial 18-22 hour incubation, cells were incubated at 32°C and purified as described in Example 4. The amount of HMW species was measured by UPLC-SEC as described in Example 4. Binding of anti-gp100 TCR fusion proteins to target peptide-MHC complexes was measured by flow cytometry as described in Example 10.

[0330] The results are shown in Table 13.2 and Figure 7.

[0331] [Table 13.2]

[0332] As can be seen from Table 13.2, all of the formats tested that were successfully expressed had yields purified by Protein A ranging from 24 mg / L to 54 mg / L. The amount of high molecular weight species present ranged from 3.7% to 23.9%. Formats containing Fab or a second TCR maintained similar expression yields and HMW species.

[0333] All variants containing the anti-gp100 TCR maintained binding to target MHC-peptide complexes. The OA variant v29011 and the bispecific variant v31327 had nearly identical binding curves (see Figure 7), suggesting that the addition of a second binding moiety (such as an anti-CD3 component) to the TCR fusion protein does not affect binding of the TCR component to the target MHC-peptide complex. Figure 7 also shows that variants containing lower affinity anti-gp100 TCR components (v30972, v30964, v30975, and v30968) appeared to exhibit reduced binding responsiveness compared to the higher affinity anti-gp100 TCR controls (v29011 and v31327).

[0334] Increased binding responses were observed with increasing numbers of TCR moieties in the fusion proteins. As can be seen in Table 13.2, all 2x1 variants (v31307, v30966, v30967, and v30968) exhibited improved affinity over the corresponding variants containing a single TCR component. The 2x1 variant v30968, which contains two copies of the lowest affinity anti-gp100 TCR component, exhibited a significantly higher EC than variants v30975, 30964, and 30972, which contain a single copy of this TCR component. 50 A decrease in the β-glucan concentration was observed (see Figure 7).

[0335] Example 14: Effect of different germline TCR sequences on the expression of TCR fusion proteins To determine whether germline sequences affect the expression and stability of TCR fusion proteins, the CDRs of the anti-gp100 TCR were grafted into various germline TCR framework sequences as follows:

[0336] To determine the effect of different germline sequences on TCR stability, only the framework regions of the variable domains of the TCR components of the TCR fusion proteins were varied. CDR boundaries were selected based on the IMGT definition, and the CDR sequences of the α and β chains of the anti-gp100 TCR were used to replace the corresponding CDR regions in the alternative germline sequences. The top five α and top five β germline sequences were identified based on frequency using the IMGT / GeneFrequency tool accessed through the International ImMunoGeneTics Information System (IMGT®) website. The top five α sequences were identified as TRAV19, TRAV14 / DV4, TRAV17, TRAV9-2, and TRAV8-4, and the top five β sequences were identified as TRBV28, TRBV30, TRBV19, TRBV27, and TRBV5-1. The natural germline sequences in the anti-gp100 TCRs are TRAV17 and TRBV19.

[0337] The α and β sequences containing the grafted CDR sequences were then combined, with each α sequence paired with each β sequence, for a total of 25 combinations. TCR fusion proteins containing each combination were constructed in a single-arm format with a β chain fused to Fc, as described in Example 1. All TCR fusion proteins contained an IC disulfide and a TRAC-hinge disulfide. Each sequence was preceded by the signal peptide MRPTWAWWLFLVLLLALWAPARG [SEQ ID NO: 22]. Vector inserts were prepared as described in Example 1 and cloned into the pTT5 vector for expression.

[0338] The TCR fusion proteins were first tested for expression in 0.8 mL of HEK293-6E cells as described in Example 4. Expression was confirmed by SDS-PAGE, and variants for which protein expression bands were observable were then expressed in 250 mL of HEK293-6E cells as described in Example 4, except that the DNA ratio of α chain, β chain-Fc(A), and Fc(B) was 40:40:20. After the initial 18-22 hour incubation, the cells were incubated at 32°C and then purified and characterized by UPLC-SEC as described in Example 4 and DSC as described in Example 6.

[0339] The results are shown in Table 14.1.

[0340] [Table 14.1]

[0341] As can be seen from Table 14.1, only two variants produced monodisperse, soluble proteins: v31129 and v31131. Variant v31131 contains the germline sequences TRAV17 and TRBV19, which are native to the anti-gp100 TCR and therefore were expected to be expressed. Variant v31129 contains the germline sequences TRAV17 and TRBV28, and therefore contains the same TRAV sequences as the native anti-gp100 TCR. Although the amount of HMW species observed with variant v31129 was somewhat higher than with v31131, the thermal stability was similar, at 53.5°C and 53.3°C. Therefore, TRBV28 appears to be a match for this set of TCR CDRs and TRAV17. Overall, the results in Table 14.1 suggest that the CDR set derived from this anti-gp100 TCR is not compatible with most other germlines, and that the native germline sequence is likely to provide optimal stability for a given TCR sequence.

[0342] Example 15: Long-term stability of stabilized TCR fusion proteins To assess the long-term stability of the TCR fusion proteins, selected variants described in Examples 4, 6, 9, 11 and 14 were incubated at 40°C and the amount of HMW species assessed by UPLC-SEC, as described below.

[0343] The TCR fusion proteins tested are listed in Table 15.1. The protein concentration of these selected stabilized TCR fusion proteins was adjusted to 1 mg / mL with PBS buffer. 200 μl of each variant solution was sealed in an Eppendorf tube and incubated in an incubator set at 40°C for 14 or 30 days. 40 μl samples of each variant were taken at days 0, 3, 7, 10, and 14 (for the 14-day incubation) or days 0, 5, 20, and 30 (for the 30-day incubation). Samples were immediately frozen at -80°C. After the completion of the incubation period, all samples were thawed, and the amount of HMW species was assessed by UPLC-SEC as described in Example 4.

[0344] The results are shown in Table 15.1.

[0345] [Table 15.1] TIFF2023554119000024.tif117165

[0346] The percent change for each species was determined by the difference between the first measurement (day 0) and the final measurement (day 14 or 30) and averaged over the length of incubation. For example, a recorded increase of 0.70% for HMW species equates to a 9.8% increase after 14 days of incubation.

[0347] As can be seen from Table 15.1, of all the variants tested, the TCR fusion protein containing only the IC disulfide (v21230) had the highest rate of increase in HMW species (aggregation). All TCR fusion proteins containing combinations of stabilizing mutations reduced the amount of HMW species compared to variant v21230, with variants v28897, v31097, and v31099 showing a greater than 10-fold reduction in the rate of HMW species formation compared to v21230.

[0348] The results shown in Table 15.1 demonstrate that including a combination of the stabilizing mutations described in the previous examples in a TCR fusion protein improves the long-term colloidal stability of the fusion protein compared to TCR fusion proteins containing only the IC disulfide.

[0349] Example 16: Generation and characterization of multivalent TCR fusion proteins Increasing the number of TCR moieties in a TCR fusion protein may enhance binding to target peptide-MHC complexes (as described in Example 13) or provide additional biological functions by allowing simultaneous binding to multiple peptide-MHC complexes. Multivalent TCR fusion proteins with valencies of up to four TCRs were generated and characterized as described below.

[0350] The TCR fusion proteins were constructed in the following format: (1) Bispecific β-fusion (“1x1”; see Figure 1L) (2) 2x1 bispecific β-fusion (“2x1”, see Figure 1O) (3) 3x1 bispecific beta fusion (“3x1”; see Figure 1P) (4) 4x1 bispecific C-terminal β-fusion (“4x1 tandem,” see Figure 1Q) (5) 4x1 bispecific light chain β fusion (“4x1 LC”; see Figure 1R)

[0351] The antibody component in each case was an anti-CD3 Fab or anti-CD3 scFv. The TCR components were the wild-type 1G4-WT anti-NY-ESO1 TCR ("NY-ESO1-WT"), the affinity-matured 1G4-33A anti-NY-ESO1 TCR ("NY-ESO1-33A") (Li, et al., 2005, Nat. Biotechnol., 23:349-354), or the anti-PRAME TCR, as outlined in Table 16.1. All TCR sequences contained the following stabilizing mutations: TRAC / 84.2_TRBC / 79 disulfide, TRAC / 39.VAL→CYS_TRAC / 85.ALA→CYS (disulfide), and TRAC-hinge disulfide.

[0352] Vector inserts encoding each TCR fusion protein were prepared and cloned into the pTT5 vector for expression as described in Example 1. Each sequence was preceded by the signal peptide MRPTWAWWLFLVLLLALWAPARG [SEQ ID NO: 22].

[0353] TCR fusion proteins were first tested for expression in 0.8 mL of HEK293-6E cells as described in Example 4. Expression was confirmed by SDS-PAGE, and variants for which protein expression bands were observable were then expressed in 250 mL of HEK293-6E cells as described in Example 4. After an initial 18-22 hour incubation, cells were incubated at 32°C and purified as described in Example 4. Monodispersity was measured by UPLC-SEC as described in Example 4.

[0354] The results are shown in Table 16.1.

[0355] [Table 16.1]

[0356] As can be seen from Table 16.1, TCR fusion proteins containing WT anti-NY-ESO1 TCR, high-affinity anti-NY-ESO1 TCR, or anti-PRAME TCR were successfully expressed in a 1x1 format. TCR fusion proteins containing up to four anti-NY-ESO1 TCRs were expressed in sufficient quantities for analysis and then purified as monodisperse samples. Exemplary UPLC-SEC traces of two 4x1 TCR fusion proteins, v32548 and v32549, are shown in Figure 8. Fusion proteins containing anti-PRAME TCRs generally appeared to be less stable than fusion proteins containing anti-NY-ESO1 TCRs. While the stabilizing mutations described above sufficiently stabilized the anti-PRAME TCR for successful expression in a 1x1 format, expression levels were lower than those of either anti-NY-ESO1 TCR in the same 1x1 format, indicating that the anti-PRAME TCR is likely an inherently poorly expressed TCR.

[0357] Example 17: T2 T cell dependent cytotoxicity assay of stabilized TCR fusion proteins The TCR fusion proteins of Example 16 specific for the NY-ESO1 peptide were tested for cell killing against cells bearing target peptide-MHC complexes in a T2 T cell-dependent cytotoxicity assay (TDCC). The TCR fusion proteins tested are listed in Table 17.1.

[0358] T2 cells (ATCC CRL-1992) were cultured in a T75 flask in RPMI 1640 + 10% FBS at 37°C + 5% CO2 for at least two passages before use. Cells were pulsed by resuspending in culture medium containing 10 μM NY-ESO-1 peptide (SLLMWITQC [SEQ ID NO: 23]) and 100 ng / mL β-2-microglobulin (Sino Biological, Beijing, China) and incubated for 24 hours at 37°C + 5% CO2. The TDCC assay solution was prepared in 96-well U-bottom plates in RPMI 1640 + 10% FBS + 1% penicillin / streptomycin assay medium. TCR fusion proteins were serially diluted 1:5 to a concentration range of 1 μM to 0.05 pM, at 60 μL per well. T2 cells were stained with 2 μM carboxyfluorescein succinimidyl ester (CFSE) (ThermoFisher, Waltham, MA) using the manufacturer's protocol. After staining, T2 cells were resuspended in RPMI 1640 + 10% FBS and mixed with freshly thawed T cells (BioIVT, Westbury, NY) at a 5:1 ratio. 60 μL of the cell mixture was added per well, and the plate was incubated at 37°C + 5% CO2 for 48 hours. After incubation, the samples were transferred to a 96-well V-bottom plate and washed twice with FACS buffer (PBS + 2% FBS). The samples were resuspended in 2 μg / mL 7-aminoactinomycin D (7-AAD) (BioLegend, San Diego, CA) at 50 μL per well. The plate was incubated at room temperature for 15 minutes and then washed twice with FACS buffer. Samples were resuspended in 50 μL / well of FACS buffer, and plates were analyzed by flow cytometry using a BD Fortessa™ X-20 (BD Biosciences, San Jose, Calif.).

[0359] EC 50The measurements are summarized in Table 17.1. See also Figure 10. The TCR fusion proteins contain either the NY-ESO1-WT paratope (affinity: 32000 nM) or the NY-ESO1-33A paratope (affinity: 254 nM), as shown in Table 17.1.

[0360] [Table 17.1]

[0361] As can be seen from Table 17.1 and Figure 10, the TCR-anti-CD3 bispecific proteins were able to selectively kill target cells presenting the desired MHC-peptide complex. Target cell killing was observed only with the bispecific molecule; a one-arm TCR molecule (v31185) lacking the anti-CD3 paratope did not produce killing at any of the concentrations observed. This indicates that a multivalent format is required for cell killing. Additionally, the 3x variant increased cell killing by approximately 10-fold compared to the 1x variant, suggesting that the avidity of the 3x variant is responsible for the greatest enhancement of cell killing. The 4x variant increased cell killing by approximately 10-fold compared to the 1x variant, suggesting that the avidity of the 3x variant is responsible for the greatest enhancement of cell killing. 50 No further increase in response was observed, likely because maximum potency had already been reached or the 4x format is not a suitable format for the NY-ESO1 TCR. All variants containing the lower affinity TCR (NY-ESO1-WT) elicited minimal responses, regardless of TCR valency.

[0362] Taken together, these results demonstrate that TCR-anti-CD3 bispecific proteins are capable of selectively killing target cells and that this activity can be enhanced through increased valency of the TCR components.

[0363] The disclosures of all patents, patent applications, publications, and database entries mentioned in this specification are specifically incorporated herein by reference in their entirety to the same extent as if each individual patent, patent document, publication, and database entry was specifically and individually indicated to be incorporated by reference.

[0364] Modifications of the specific embodiments described herein that are obvious to those skilled in the art are intended to be included within the scope of the appended claims.

[0365] TIFF2023554119000027.tif192163TIFF2023554119000028.tif196163

Claims

1. 1. A TCR construct comprising a TCR alpha chain polypeptide and a TCR beta chain polypeptide, the TCR α chain polypeptide comprises a variable α (Vα) domain and a constant α (Cα) domain, and the TCR β chain polypeptide comprises a variable β (Vβ) domain and a constant β (Cβ) domain; the Cα domain and the Cβ domain comprise stabilizing mutations, the stabilizing mutations comprising a first interchain disulfide bond between the Cα domain and the Cβ domain and one or more additional stabilizing mutations, the one or more additional stabilizing mutations being selected from the following: a) an interchain disulfide bond formed between i) a cysteine ​​residue contained in an amino acid stretch of 1 to about 10 amino acids in length at the C-terminus of the Cβ domain of the TCR β chain polypeptide, and ii) a cysteine ​​residue at TRAC position 128 of the Cα domain of the TCR α chain polypeptide; b) an interchain disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; c) an interchain disulfide bond between a cysteine ​​residue substitute at position 122 of TRAC and a cysteine ​​residue substitute at position 12 of TRBC; d) an interchain disulfide bond between a cysteine ​​residue substitute at position 124 of TRAC and a cysteine ​​residue substitute at position 11 of TRBC; e) an interchain disulfide bond between a cysteine ​​residue substitute at position 125 of TRAC and a cysteine ​​residue substitute at position 11 of TRBC; f) an interchain disulfide bond between a cysteine ​​residue substitute at position 126 of TRAC and a cysteine ​​residue substitute at position 11 of TRBC; g) an interchain disulfide bond between a cysteine ​​residue substitute at position 127 of TRAC and a cysteine ​​residue substitute at position 11 of TRBC; h) an interchain disulfide bond between a cysteine ​​residue substitute at position 128 of TRAC and a cysteine ​​residue substitute at position 11 of TRBC; i) an intrachain disulfide bond between a cysteine ​​residue substitute at position 39 of TRAC and a cysteine ​​residue substitute at position 85 of TRAC; j) an intrachain disulfide bond between the cysteine ​​residue substituent at TRAC position 26 and the cysteine ​​residue substituent at TRAC position 85.1; k) an amino acid substitution at position 4 of TRAC from Val to Ala, Thr, Ile, Leu or Met; l) an amino acid substitution at position 26 of TRAC from Thr to Ala, Val, Ile, Leu or Met; m) an amino acid substitution at position 39 of TRAC from Val to Ala, Thr, Ile, Leu or Met; n) an amino acid substitution at position 85 of TRAC from Ala to Ser, Thr, Val, Ile or Met; o) an amino acid substitution at position 105 of TRAC from Ala to Ser, Thr, Glu, Gln, Asp, Asn, His, Lys or Arg; p) an amino acid substitution at position 120 of TRAC from Phe to Tyr or His; q) an amino acid substitution at TRBC position 6 from Val to Ala, Thr, Ile, Leu or Met; r) an amino acid substitution at TRBC position 36 from His to Phe, Tyr or Trp; s) an amino acid substitution at TRBC position 86 from Ser to Ala or Thr; t) an amino acid substitution at position TRBC45.3 from Val to Ser, Thr, Glu, Gln, Asp, Asn, His, Lys or Arg; u) a deletion of 1 to 4 consecutive amino acids in the DE loop of the Cβ domain of the TCR construct; and v) Replacement of the amino acids at TRBC positions 84.4 to 85.4 with an amino acid sequence consisting of 2 to 4 amino acids, which allows the formation of a β-turn. is selected from the amino acid numbering is the IMGT numbering, the TCR construct has an elevated TCR melting temperature (Tm) relative to a corresponding TCR construct containing only the first disulfide bond; The TCR construct.

2. The first interchain disulfide bond is selected from the group consisting of: a) an interchain disulfide bond formed between i) a cysteine ​​residue contained in an amino acid stretch of 1 to about 10 amino acids in length at the C-terminus of the Cβ domain of the TCR β chain polypeptide, and ii) a cysteine ​​residue at TRAC position 128 of the Cα domain of the TCR α chain polypeptide; b) an interchain disulfide bond between a cysteine ​​residue substitute at position 84 of TRAC and a cysteine ​​residue substitute at position 79 of TRBC; c) an interchain disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; d) an interchain disulfide bond between the cysteine ​​residue substituent at position 122 of TRAC and the cysteine ​​residue substituent at position 12 of TRBC; e) an interchain disulfide bond between a cysteine ​​residue substitute at position 124 of TRAC and a cysteine ​​residue substitute at position 11 of TRBC; f) an interchain disulfide bond between a cysteine ​​residue substitute at position 125 of TRAC and a cysteine ​​residue substitute at position 11 of TRBC; g) an interchain disulfide bond between a cysteine ​​residue substitute at position 126 of TRAC and a cysteine ​​residue substitute at position 11 of TRBC; h) an interchain disulfide bond between a cysteine ​​residue substitute at position 127 of TRAC and a cysteine ​​residue substitute at position 11 of TRBC, and i) An interchain disulfide bond between the cysteine ​​residue substitute at position 128 of TRAC and the cysteine ​​residue substitute at position 11 of TRBC is selected from the first interchain disulfide bond is distinct from any additional interchain disulfide bonds; The TCR construct of claim 1.

3. The first interchain disulfide bond is selected from the group consisting of: a) an interchain disulfide bond formed between i) a cysteine ​​residue contained in an amino acid stretch of 1 to about 10 amino acids in length at the C-terminus of the Cβ domain of the TCR β chain polypeptide, and ii) a cysteine ​​residue at TRAC position 128 of the Cα domain of the TCR α chain polypeptide; b) an interchain disulfide bond between a cysteine ​​residue substitute at position 84 of TRAC and a cysteine ​​residue substitute at position 79 of TRBC; and c) An interchain disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC is selected from the first interchain disulfide bond is distinct from any additional interchain disulfide bonds; The TCR construct of claim 1.

4. The first interchain disulfide bond is selected from the group consisting of: a) an interchain disulfide bond formed between i) a cysteine ​​residue contained in an amino acid stretch of 1 to about 10 amino acids in length at the C-terminus of the Cβ domain of the TCR β chain polypeptide, and ii) a cysteine ​​residue at TRAC position 128 of the Cα domain of the TCR α chain polypeptide; and b) An interchain disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC is selected from the first interchain disulfide bond is distinct from any additional interchain disulfide bonds; The TCR construct of claim 1.

5. The one or more additional stabilizing mutations are selected from the group consisting of: a) an interchain disulfide bond formed between i) a cysteine ​​residue contained in an amino acid stretch of 1 to about 10 amino acids in length at the C-terminus of the Cβ domain of the TCR β chain polypeptide, and ii) a cysteine ​​residue at TRAC position 128 of the Cα domain of the TCR α chain polypeptide; b) an interchain disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; c) a disulfide bond between a cysteine ​​residue substitute at position 122 of TRAC and a cysteine ​​residue substitute at position 12 of TRBC; d) a disulfide bond between a cysteine ​​residue substitute at position 124 of TRAC and a cysteine ​​residue substitute at position 11 of TRBC; e) a disulfide bond between a cysteine ​​residue substitute at position 125 of TRAC and a cysteine ​​residue substitute at position 11 of TRBC; f) a disulfide bond between a cysteine ​​residue substitute at position 126 of TRAC and a cysteine ​​residue substitute at position 11 of TRBC; g) a disulfide bond between a cysteine ​​residue substitute at position 127 of TRAC and a cysteine ​​residue substitute at position 11 of TRBC, and h) A disulfide bond between the cysteine ​​residue substituent at position 128 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC The TCR construct of any one of claims 1 to 4, comprising an additional interchain disulfide bond selected from:

6. 2. The TCR construct of claim 1, wherein the first interchain disulfide bond is a disulfide bond between a cysteine ​​residue substitute at TRAC position 84.2 and a cysteine ​​residue substitute at TRBC position 79.

7. The one or more additional stabilizing mutations are selected from the group consisting of: a) an interchain disulfide bond formed between i) a cysteine ​​residue contained in an amino acid stretch of 1 to about 10 amino acids in length at the C-terminus of the Cβ domain of the TCR β chain polypeptide, and ii) a cysteine ​​residue at TRAC position 128 of the Cα domain of the TCR α chain polypeptide; b) a disulfide bond between a cysteine ​​residue substitute at position 122 of TRAC and a cysteine ​​residue substitute at position 12 of TRBC; c) a disulfide bond between a cysteine ​​residue substitute at position 124 of TRAC and a cysteine ​​residue substitute at position 11 of TRBC; d) a disulfide bond between a cysteine ​​residue substitute at position 125 of TRAC and a cysteine ​​residue substitute at position 11 of TRBC; e) a disulfide bond between a cysteine ​​residue substitute at position 126 of TRAC and a cysteine ​​residue substitute at position 11 of TRBC; f) a disulfide bond between a cysteine ​​residue substitute at position 127 of TRAC and a cysteine ​​residue substitute at position 11 of TRBC, and g) A disulfide bond between the cysteine ​​residue substituent at position 128 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC The TCR construct of claim 6, comprising an additional interchain disulfide bond selected from:

8. The TCR construct according to any one of claims 1 to 7, wherein the amino acid extension at the C-terminus of the Cβ domain of the TCR β chain polypeptide is 5 amino acids or less in length.

9. A TCR construct according to any one of claims 1 to 8, wherein the amino acid extension at the C-terminus of the Cβ domain of the TCR β chain polypeptide comprises all or part of the sequence of the IgG1 upper hinge region.

10. 10. The TCR construct of claim 9, wherein the amino acid extension comprises the sequence: EPKSC [SEQ ID NO: 19] or EPKSCDKTHT [SEQ ID NO: 16].

11. The one or more additional stabilizing mutations are selected from the group consisting of: an intrachain disulfide bond between a cysteine ​​residue substitute at position 39 of TRAC and a cysteine ​​residue substitute at position 85 of TRAC; and Intrachain disulfide bond between the cysteine ​​residue substitute at position 26 of TRAC and the cysteine ​​residue substitute at position 85.1 of TRAC The TCR construct of any one of claims 1 to 10, comprising an intrachain disulfide bond selected from:

12. The one or more additional stabilizing mutations are selected from the group consisting of: a) an amino acid substitution at position 26 of TRAC from Thr to Ala, Val, Ile, Leu or Met; b) an amino acid substitution at position 85 of TRAC from Ala to Ser, Thr, Val, Ile or Met; c) an amino acid substitution at TRBC position 6 from Val to Ala, Thr, Ile, Leu or Met, and d) Amino acid substitution at TRBC position 36 from His to Phe, Tyr or Trp The TCR construct of any one of claims 1 to 11, comprising one or more of the following:

13. a) the amino acid substitution at position 4 of TRAC is an amino acid substitution from Val to Ile; b) the amino acid substitution at position 26 of TRAC is a Thr to Ile amino acid substitution; c) the amino acid substitution at position 39 of TRAC is a Val to Ile amino acid substitution; d) the amino acid substitution at position 85 of TRAC is an amino acid substitution from Ala to Val; e) the amino acid substitution at position 105 of TRAC is an amino acid substitution from Ala to Ser; f) the amino acid substitution at position 120 of TRAC is a Phe to Tyr amino acid substitution; g) the amino acid substitution at TRBC position 6 is a Val to Ile or Leu amino acid substitution; h) the amino acid substitution at TRBC position 36 is a His to Phe amino acid substitution; i) the amino acid substitution at TRBC position 86 is a Ser to Thr amino acid substitution, and j) the amino acid substitution at position TRBC45.3 is a Val to Thr amino acid substitution; A TCR construct according to any one of claims 1 to 12.

14. a) the amino acid substitution at position 26 of TRAC is an amino acid substitution from Thr to Ile; b) the amino acid substitution at position 85 of TRAC is an amino acid substitution from Ala to Val; c) the amino acid substitution at TRBC position 6 is a Val to Ile or Leu amino acid substitution; d) the amino acid substitution at TRBC position 36 is a His to Phe amino acid substitution; The TCR construct of claim 12.

15. The method of claim 1, wherein the Cα domain and the Cβ domain are a) (i) an interchain disulfide bond formed between a cysteine ​​residue contained in an amino acid stretch comprising the sequence EPKSCDKTHT [SEQ ID NO: 16] at the C-terminus of the Cβ domain of a TCRβ chain polypeptide and (ii) a cysteine ​​residue at TRAC position 128 in the Cα domain of a TCRα chain polypeptide; an interchain disulfide bond between a cysteine ​​residue substitute at TRAC position 84.2 and a cysteine ​​residue substitute at TRBC position 79; an intrachain disulfide bond between a cysteine ​​residue substitute at TRAC position 39 and a cysteine ​​residue substitute at TRAC position 85; an amino acid substitution from Val to Ile at TRAC position 6; and an amino acid substitution from His to Phe at TRAC position 36 (v33048); b) an interchain disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; and an interchain disulfide bond between the cysteine ​​residue substituent at position 127 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC (v33049); c) an interchain disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; an interchain disulfide bond between the cysteine ​​residue substituent at position 127 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; an intrachain disulfide bond between the cysteine ​​residue substituent at position 39 of TRAC and the cysteine ​​residue substituent at position 85 of TRAC; an amino acid substitution at position 6 of TRAC from Val to Ile; and an amino acid substitution at position 36 of TRAC from His to Phe (v33050); d) an interchain disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; an interchain disulfide bond between the cysteine ​​residue substituent at position 126 of TRAC and the cysteine ​​residue substituent at position 11 of TRBC; an intrachain disulfide bond between the cysteine ​​residue substituent at position 39 of TRAC and the cysteine ​​residue substituent at position 85 of TRAC; an amino acid substitution from Val to Ile at position 6 of TRAC; and an amino acid substitution from His to Phe at position 36 of TRAC (v33053); and e) an interchain disulfide bond between the cysteine ​​residue substituent at position 84.2 of TRAC and the cysteine ​​residue substituent at position 79 of TRBC; an interchain disulfide bond between the cysteine ​​residue substituent at position 122 of TRAC and the cysteine ​​residue substituent at position 12 of TRBC; an intrachain disulfide bond between the cysteine ​​residue substituent at position 39 of TRAC and the cysteine ​​residue substituent at position 85 of TRAC; an amino acid substitution at position 6 of TRAC from Val to Ile; and an amino acid substitution at position 36 of TRAC from His to Phe (v33056).

2. The TCR construct of claim 1, comprising a stabilizing mutation selected from:

16. 1. A TCR construct comprising a TCR alpha chain polypeptide and a TCR beta chain polypeptide, the TCR α chain polypeptide comprises a variable α (Vα) domain and a constant α (Cα) domain, and the TCR β chain polypeptide comprises a variable β (Vβ) domain and a constant β (Cβ) domain; The TCR construct comprises: (a) a combination of amino acid mutations selected from the combinations of amino acid mutations described for any one of the variants shown in Table 2; or (b) a combination of amino acid mutations selected from the combinations of amino acid mutations described for any one of the variants shown in Table 3. Including, The amino acid numbering is the IMGT numbering. The TCR construct.

17. The TCR construct of any one of claims 1 to 16, wherein the TCR alpha chain polypeptide or the TCR beta chain polypeptide is fused to an immunoglobulin (Ig) Fc region.

18. The TCR construct of claim 17 , wherein the Ig Fc region is an IgG Fc region.

19. The TCR construct of claim 18 , wherein the IgG Fc region is an IgG1 Fc region.

20. The TCR construct of any one of claims 17 to 19, wherein the TCR β chain polypeptide is fused to the Ig Fc region.

21. 17. A TCR fusion protein comprising one or more of the TCR constructs of any one of claims 1 to 16 and a scaffold, wherein at least one of the TCR constructs is fused to the scaffold.

22. 22. The TCR fusion protein of claim 21 , wherein the scaffold comprises an immunoglobulin (Ig) Fc region.

23. 23. The TCR fusion protein of claim 22, wherein the Ig Fc region is an IgG Fc region.

24. 24. The TCR fusion protein of claim 23, wherein the IgG Fc region is an IgG1 Fc region.

25. 25. The TCR fusion protein of any one of claims 21 to 24, comprising one, two, three or four TCR constructs.

26. 25. The TCR fusion protein of any one of claims 21 to 24, comprising two or more TCR constructs, wherein two of the TCR constructs are fused in tandem.

27. 27. The TCR fusion protein of any one of claims 21 to 26, wherein at least one of the TCR constructs is fused to the scaffold via the TCR β chain polypeptide.

28. 28. The TCR fusion protein of any one of claims 21 to 27, further comprising one or more additional biologically active moieties.

29. 30. The TCR fusion protein of claim 28, wherein the one or more additional biologically active moieties comprises an antigen binding domain.

30. 30. The TCR fusion protein of claim 29, wherein the antigen binding domain is an scFv or a Fab.

31. 31. The TCR fusion protein of any one of claims 28 to 30, wherein at least one of the biologically active moieties is fused to the scaffold.

32. A pharmaceutical composition comprising the TCR construct of any one of claims 1 to 20, or the TCR fusion protein of any one of claims 21 to 31, and a pharma- ceutically acceptable carrier or diluent.

33. A polynucleotide or set of polynucleotides encoding the TCR construct of any one of claims 1 to 20, or the TCR fusion protein of any one of claims 21 to 31.

34. A method for preparing a TCR construct according to any one of claims 1 to 20, or a TCR fusion protein according to any one of claims 21 to 31, comprising transfecting a cell with a polynucleotide or set of polynucleotides according to claim 33, and culturing the cell under conditions suitable for expression of the TCR construct or TCR fusion protein.

35. 35. The method of claim 34, wherein the cells are cultured at a temperature between 30°C and 35°C.

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