Protein containing t cell receptor constant region and medical use thereof

Abstract

The present disclosure relates to a protein containing a T cell receptor constant region and the medical use thereof. Specifically, the present disclosure relates to a modified T cell receptor (TCR) constant region, a protein containing the TCR constant region, a coding nucleic acid, a vector, a cell, a pharmaceutical composition, and the use thereof.

Description

Protein containing T cell receptor constant region and its medical use

[0001] This disclosure claims priority to Chinese patent application No. 202410441976.6 filed on April 12, 2024. Technical Field

[0002] The present disclosure relates to proteins, engineered soluble TCRs, polynucleotides, vectors, cells, compositions comprising engineered T cell receptor (TCR) constant regions having one or more mutations, and methods of making and using the same. Background Art

[0003] TCRs are tools used by the adaptive immune system to recognize and eliminate "non-self" intracellular antigens. Natural TCRs are composed of two polypeptide chains, either α / β or γ / δ. The two forms are structurally similar but expressed on different T cells. The extracellular domain of α / β TCRs consists of an α chain and a β chain, each containing a variable region (Vα / Vβ) and a constant region (Cα / Cβ). Each constant region and variable region contains a pair of intrachain disulfide bonds. α / β TCRs are expressed on CD8+ effector T cells and CD4+ helper T cells. When complexed with HLA / MHC, they recognize foreign antigens that are degraded by proteases on the surface of infected or cancerous cells (Davis, et al., Nature, 1988. 334(6181):395-402; Heemels, et al., Annu Rev Biochem, 1995. 64:463-91).

[0004] There are many methods to apply the exquisite recognition properties of TCR for therapeutic purposes. In T cells, these T cells are redirected to tumor-associated antigens (Riley, et al., Nat Rev Drug Discov, 2019. 18(3): 175-196; Parkhurst, et al., Clin Cancer Res, 2017. 23(10): 2491-2505). In addition, the soluble extracellular domain of TCRs can be fused with a single-chain antibody (scFv) that binds to the activating T cell receptor (usually CD3) to redirect endogenous T cells to attack tumor cells. Unlike bispecific antibodies that directly recognize overexpressed antigens on the cell surface, TCRs or TCR bispecific molecules can recognize a large number of intracellular antigens and have a wide range of application potential (Liddy, et al., Nat Med, 2012. 18(6): 980-7).

[0005] However, expression of TCRs is extremely challenging. The common method to produce soluble TCRs is to express in E. coli, followed by formation of inclusion bodies, solubilization, refolding, assembly, and finally purification with low yield (van Boxel, et al., J Immunol Methods, 2009. 350(1-2): 14-21). To simplify the production process, some researchers have tried to use single-chain versions of TCR variable regions (scTvs) targeting specific HLA / petide complexes similar to antibody scFvs (Stone, et al., Methods Enzymol, 2012. 503: 189-222). However, scFvs are unstable, prone to aggregation, and have low solubility, while scTvs exhibit even worse expression and stability. There have been numerous studies to optimize scTvs proteins for therapy and diagnosis (Stone, et al., Methods Enzymol, 2012. 503: 189-222). However, the diversity of TCR variable regions is much higher than that of antibody variable regions, and these stability mutations designed in the variable regions can only be targeted to specific Va / Vp subunits, making it difficult to find stability mutations that are universally applicable to different TCRs.

[0006] Given that TCR extracellular domains are glycosylated and have complex disulfide pairing, mammalian expression can be used to produce soluble TCRs. Industrial antibody production has mainly shifted to mammalian expression systems. However, the assembly ability of a / b TCRs is lower than that of antibodies, and the expression level is poor when expressed in the commonly used Chinese hamster ovary (CHO) cell system. The bispecific molecule form related to soluble a / b TCRs can require TCRs to be expressed at antibody levels for proper molecular assembly, which is a major obstacle to its production. Recombinant fusion of bispecific ImmTac structures of soluble TCRs and antibody scFvs in bacteria is usually expressed in an insoluble inclusion body form in bacteria, which requires solubilization, refolding, assembly, and low yield. In addition, these structures have a rapid serum clearance due to the lack of recycling mechanisms.

[0007] For all these reasons, there is increasing interest in modifying the TCR constant region to improve its expression. There have been some reports of improving the expression and stability of soluble TCRs by introducing disulfide bonds and / or point mutations in the constant region.

[0008] Because TCRs have the ability to recognize a large number of intracellular antigens, soluble TCRs have important and wide-ranging uses in therapy and diagnosis. There is still a need for more abundant, general engineering techniques for TCRs targeting different antigens to improve their expression and stability, simplify production, and reduce the cost of large-scale preparation. In view of this, the present disclosure is proposed. The present disclosure relates to proteins containing one or more stability mutations of T cell receptors (TCRs), nucleic acids encoding such proteins, and methods of making and using such proteins. SUMMARY

[0009] The present disclosure provides engineered T cell receptor (TCR) constant regions containing one or more expression-enhancing mutations and / or stability mutations, proteins, engineered soluble TCRs, polynucleotides, vectors, cells, pharmaceutical compositions, methods of making, uses, and methods of treatment thereof.

[0010] T cell receptor (TCR) constant region

[0011] The present disclosure provides a T cell receptor (TCR) constant region comprising a TCR constant region alpha chain (Ca) and / or a TCR constant region beta chain (Cp), wherein the TCR Ca comprises one or more amino acid mutations at a position selected from the group consisting of 165, 166, 168, 170, 182, 198, and the TCR Cp comprises one or more amino acid mutations at a position selected from the group consisting of 132, 176, 181, 196, 197, 241.

[0012] The present disclosure provides a T cell receptor (TCR) constant region comprising a TCR constant region alpha chain (Ca) and / or a TCR constant region beta chain (Cp), wherein the TCR Ca comprises one or more amino acid mutations at a position selected from the group consisting of 134, 165, 166, 168, 170, 182, 198, and the TCR Cp comprises one or more amino acid mutations at a position selected from the group consisting of 132, 150, 176, 181, 196, 197, 241.

[0013] The present disclosure provides a T cell receptor (TCR) constant region comprising a TCR constant region alpha chain (Ca) and / or a TCR constant region beta chain (Cp), wherein the TCR Ca comprises one or more amino acid mutations at a position selected from the group consisting of 165, 166, 167, 168, 169, 170, 182, 198, and the TCR Cp comprises one or more amino acid mutations at a position selected from the group consisting of 132, 176, 181, 196, 197, 241.

[0014] The present disclosure provides a T cell receptor (TCR) constant region, comprising a TCR constant region alpha chain (Cα) and / or a TCR constant region beta chain (Cβ), wherein the TCR Cα comprises one or more amino acid mutations at a position selected from the group consisting of 134, 165, 166, 167, 168, 169, 170, 182, 198, and the TCR Cβ comprises one or more amino acid mutations at a position selected from the group consisting of 132, 150, 176, 181, 196, 197, 241.

[0015] In some embodiments, the amino acid mutations increase the expression level and / or the stability of the TCR constant region.

[0016] In some embodiments, the TCR constant region, the TCR Cα comprises one or more amino acid mutations selected from the group consisting of 134K, 165D, 166P / E, 167E, 168E / T, 169N, 170E, 182P, 198K; and the TCR Cβ comprises one or more amino acid mutations selected from the group consisting of 132M, 150A, 176L, 181K, 196E, 197T / V, 241K.

[0017] In some embodiments, the TCR Cα comprises one or more amino acid mutations selected from the group consisting of S134K, M165D, R166P / E, S167E, M168E / T, D169N, F170E, S182P, D198K; and the TCR Cβ comprises one or more amino acid mutations selected from the group consisting of I132M, D150A, E176L, N181K, T196E, F197T / V, D241K.

[0018] In some embodiments, the TCR constant region comprises a combination of amino acid mutations selected from the group consisting of:

[0019] (1-1) position 134, 165, 166, 167, 168, 169, 170, 182, 198 of the TCR Cα, and position 176 of the TCR Cβ;

[0020] (1-1-1) any one of position 134, 165, 166, 167, 168, 169, 170, 182, 198 of the TCR Cα, and position 176 of the TCR Cβ;

[0021] (1-1-2) position 165 of the TCR Cα, and position 176 of the TCR Cβ;

[0022] (1-1-3) position 166 of the TCR Cα, and position 176 of the TCR Cβ;

[0023] (1-1-4) Position 167 of TCR Cα, and position 176 of TCR Cβ;

[0024] (1-1-5) Position 168 of TCR Cα, and position 176 of TCR Cβ;

[0025] (1-1-6) Position 169 of TCR Cα, and position 176 of TCR Cβ;

[0026] (1-1-7) Position 170 of TCR Cα, and position 176 of TCR Cβ;

[0027] (1-1-8) Position 182 of TCR Cα, and position 176 of TCR Cβ;

[0028] (1-1-9) Position 198 of TCR Cα, and position 176 of TCR Cβ;

[0029] (1-1-10) Position 134 of TCR Cα, and position 176 of TCR Cβ;

[0030] (1-2) Positions 134, 165, 166, 167, 168, 169, 170, 182, 198 of TCR Cα, and position 241 of TCR Cβ;

[0031] (1-2-1) Any one of positions 134, 165, 166, 167, 168, 169, 170, 182, 198 of TCR Cα, and position 241 of TCR Cβ;

[0032] (1-2-2) Position 165 of TCR Cα, and position 241 of TCR Cβ;

[0033] (1-2-3) Position 166 of TCR Cα, and position 241 of TCR Cβ;

[0034] (1-2-4) Position 167 of TCR Cα, and position 241 of TCR Cβ;

[0035] (1-2-5) Position 168 of TCR Cα, and position 241 of TCR Cβ;

[0036] (1-2-6) Position 169 of TCR Cα, and position 241 of TCR Cβ;

[0037] (1-2-7) Position 170 of TCR Cα, and position 241 of TCR Cβ;

[0038] (1-2-8) Position 182 of TCR Cα, and position 241 of TCR Cβ;

[0039] (1-2-9) Position 198 of TCR C a, and position 241 of TCR C b;

[0040] (1-2-10) Position 134 of TCR C a, and position 241 of TCR C b;

[0041] (1-3) Position 132, 150, 196, 197, 241 of TCR C b, and position 176 of TCR C b;

[0042] (1-3-1) Any one of positions 132, 150, 196, 197, 241 of TCR C b, and position 176 of TCR C b;

[0043] (1-3-2) Position 132 of TCR C b, and position 176 of TCR C b;

[0044] (1-3-3) Position 150 of TCR C b, and position 176 of TCR C b;

[0045] (1-3-4) Position 196 of TCR C b, and position 176 of TCR C b;

[0046] (1-3-5) Position 197 of TCR C b, and position 176 of TCR C b;

[0047] (1-3-6) Position 241 of TCR C b, and position 176 of TCR C b.

[0048] In some embodiments, the TCR constant region comprises a combination of amino acid mutations selected from the group consisting of:

[0049] (2-1) 134K, 165D, 166P / E, 167E, 168E, 169N, 170E, 182P, 198K of TCR C a, and 176L of TCR C b;

[0050] (2-1-1) Any one of 134K, 165D, 166P / E, 167E, 168E, 169N, 170E, 182P, 198K of TCR C a, and 176L of TCR C b;

[0051] (2-1-2) 165D of TCR C a, and 176L of TCR C b;

[0052] (2-1-3) 166P / E of TCR C a, and 176L of TCR C b;

[0053] (2-1-4) 167E of TCR C a, and 176L of TCR C b;

[0054] (2-1-5) 168E of TCR Ca, and 176L of TCR Cp;

[0055] (2-1-6) 169N of TCR Ca, and 176L of TCR Cp;

[0056] (2-1-7) 170E of TCR Ca, and 176L of TCR Cp;

[0057] (2-1-8) 182P of TCR Ca, and 176L of TCR Cp;

[0058] (2-1-9) 198K of TCR Ca, and 176L of TCR Cp;

[0059] (2-1-10) 134K of TCR Ca, and 176L of TCR Cp;

[0060] (2-2) 134K, 165D, 166P / E, 167E, 168E, 169N, 170E, 182P, 198K of TCR Ca, and 241K of TCR Cp;

[0061] (2-2-1) any of 134K, 165D, 166P / E, 167E, 168E, 169N, 170E, 182P, 198K of TCR Ca, and 241K of TCR Cp;

[0062] (2-2-2) 165D of TCR Ca, and 241K of TCR Cp;

[0063] (2-2-3) 166P / E of TCR Ca, and 241K of TCR Cp;

[0064] (2-2-4) 167E of TCR Ca, and 241K of TCR Cp;

[0065] (2-2-5) 168E of TCR Ca, and 241K of TCR Cp;

[0066] (2-2-6) 169N of TCR Ca, and 241K of TCR Cp;

[0067] (2-2-7) 170E of TCR Ca, and 241K of TCR Cp;

[0068] (2-2-8) 182P of TCR Ca, and 241K of TCR Cp;

[0069] (2-2-9) 198K of TCR Ca, and 241K of TCR Cp;

[0070] (2-2-10) 134K of TCR C a, and 241K of TCR C b;

[0071] (2-3) 132M, 150A, 196E, 197T / V of TCR C b, and 176L of TCR C b;

[0072] (2-3-1) any one of 132M, 150A, 196E, 197T / V of TCR C b, and 176L of TCR C b;

[0073] (2-3-2) 132M of TCR C b, and 176L of TCR C b;

[0074] (2-3-3) 150A of TCR C b, and 176L of TCR C b;

[0075] (2-3-4) 196E of TCR C b, and 176L of TCR C b;

[0076] (2-3-5) 197T / V of TCR C b, and 176L of TCR C b;

[0077] (2-3-6) 241K of TCR C b, and 176L of TCR C b.

[0078] In some embodiments, the TCR constant region comprises a combination of amino acid mutations selected from:

[0079] (3-1) S134K, M165D, R166P / E, S167E, M168E, D169N, F170E, S182P, D198K of TCR C a, and E176L of TCR C b;

[0080] (3-1-1) any one of S134K, M165D, R166P / E, S167E, M168E, D169N, F170E, S182P, D198K of TCR C a, and E176L of TCR C b;

[0081] (3-1-2) M165D of TCR C a, and E176L of TCR C b;

[0082] (3-1-3) R166P / E of TCR C a, and E176L of TCR C b;

[0083] (3-1-4) S167E of TCR C a, and E176L of TCR C b;

[0084] (3-1-5) M168E of TCR C a, and E176L of TCR C b;

[0085] (3-1-6) D169N of TCR C a, and E176L of TCR C b;

[0086] (3-1-7) F170E of TCR C a, and E176L of TCR C b;

[0087] (3-1-8) S182P of TCR C a, and E176L of TCR C b;

[0088] (3-1-9) D198K of TCR C a, and E176L of TCR C b;

[0089] (3-1-10) S134K of TCR C a, and E176L of TCR C b;

[0090] (3-2) S134K, M165D, R166P / E, S167E, M168E, D169N, F170E, S182P, D198K of TCR C a, and D241K of TCR C b;

[0091] (3-2-1) any one of S134K, M165D, R166P / E, S167E, M168E, D169N, F170E, S182P, D198K of TCR C a, and D241K of TCR C b;

[0092] (3-2-2) M165D of TCR C a, and D241K of TCR C b;

[0093] (3-2-3) R166P / E of TCR C a, and D241K of TCR C b;

[0094] (3-2-4) S167E of TCR C a, and D241K of TCR C b;

[0095] (3-2-5) M168E of TCR C a, and D241K of TCR C b;

[0096] (3-2-6) D169N of TCR C a, and D241K of TCR C b;

[0097] (3-2-7) F170E of TCR C a, and D241K of TCR C b;

[0098] (3-2-8) S182P of TCR C a, and D241K of TCR C b;

[0099] (3-2-9) D198K of TCR Ca, and D241K of TCR C;

[0100] (3-2-10) S134K of TCR Ca, and D241K of TCR C;

[0101] (3-3) any one or a combination of I132M, D150A, T196E, F197T / V, D241K of TCR C, and E176L of TCR C;

[0102] (3-3-1) any one of I132M, D150A, T196E, F197T / V, D241K of TCR C, and E176L of TCR C;

[0103] (3-3-2) I132M of TCR C, and E176L of TCR C;

[0104] (3-3-3) D150A of TCR C, and E176L of TCR C;

[0105] (3-3-4) T196E of TCR C, and E176L of TCR C;

[0106] (3-3-5) F197T / V of TCR C, and E176L of TCR C;

[0107] (3-3-6) D241K of TCR C, and E176L of TCR C.

[0108] In some embodiments, the TCR constant region comprises a combination of amino acid mutations selected from:

[0109] (4-1) position 134 of TCR Ca, and position 176 of TCR C;

[0110] (4-2) positions 134, 165 of TCR Ca, and positions 176, 241 of TCR C;

[0111] (4-3) positions 165, 166, 168 of TCR Ca, and positions 176, 241 of TCR C;

[0112] (4-4) positions 165, 166, 168 of TCR Ca, and positions 176, 197, 241 of TCR C;

[0113] (4-5) positions 165, 166, 168 of TCR Ca, and positions 176, 196, 197, 241 of TCR C;

[0114] (4-6) 134, 165, 166, 168 of TCR Ca, and 176, 197, 241 of TCR Cp;

[0115] (4-7) 134, 165, 166, 168 of TCR Ca, and 176, 196, 197, 241 of TCR Cp;

[0116] (4-8) 134, 165, 166, 168, 182 of TCR Ca, and 176, 196, 197, 241 of TCR Cp;

[0117] (4-9) 134, 165, 166, 168 of TCR Ca, and 176, 181, 196, 197, 241 of TCR Cp;

[0118] (4-10) 134, 165, 166, 168, 182 of TCR Ca, and 176, 181, 196, 197, 241 of TCR Cp.

[0119] In some embodiments, the TCR constant region comprises a combination of amino acid mutations selected from:

[0120] (5-1) 134K of TCR Ca, and 176L of TCR Cp;

[0121] (5-2) 134K, 165D of TCR Ca, and 176L, 241K of TCR Cp;

[0122] (5-3) 165D, 166P, 168E of TCR Ca, and 176L, 241K of TCR Cp;

[0123] (5-4) 165D, 166P, 168E of TCR Ca, and 176L, 197T / V, 241K of TCR Cp;

[0124] (5-5) 165D, 166P, 168E of TCR Ca, and 176L, 196E, 197T / V, 241K of TCR Cp;

[0125] (5-6) 134K, 165D, 166P, 168E of TCR Ca, and 176L, 197T / V, 241K of TCR Cp;

[0126] (5-7) 134K, 165D, 166P, 168E of TCR Ca, and 176L, 196E, 197T / V, 241K of TCR Cp;

[0127] (5-8) S134K, M165D, R166P, M168E of TCR C a, and E176L, F197T / V, D241K of TCR C ;

[0128] (5-9) S134K, M165D, R166P, M168E of TCR C a, and E176L, F197T / V, D241K of TCR C ;

[0129] (5-10) S134K, M165D, R166P, M168E of TCR C a, and E176L, F197T / V, D241K of TCR C ;

[0130] (5-11) S134K, M165D, R166P, M168E of TCR C a, and E176L, F197T / V, D241K of TCR C.

[0131] In some embodiments, the TCR constant region comprises a combination of amino acid mutations selected from:

[0132] (6-1) S134K of TCR C a, and E176L of TCR C ;

[0133] (6-2) S134K, M165D of TCR C a, and E176L, D241K of TCR C ;

[0134] (6-3) M165D, R166P, M168E of TCR C a, and E176L, D241K of TCR C ;

[0135] (6-4) M165D, R166P, M168E of TCR C a, and E176L, F197T / V, D241K of TCR C ;

[0136] (6-5) M165D, R166P, M168E of TCR C a, and E176L, F197T / V, D241K of TCR C ;

[0137] (6-6) S134K, M165D, R166P, M168E of TCR C a, and E176L, F197T / V, D241K of TCR C ;

[0138] (6-7) S134K, M165D, R166P, M168E of TCR C a, and E176L, F197T / V, D241K of TCR C ;

[0139] (6-8) S134K, M165D, R166P, M168E, S182P in TCR Ca, and E176L, T196E, F197T / V, D241K in TCR Cβ;

[0140] (6-9) S134K, M165D, R166P, M168E in TCR Ca, and E176L, N181K, T196E, F197T / V, D241K in TCR Cβ;

[0141] (6-10) S134K, M165D, R166P, M168E, S182P in TCR Ca, and E176L, N181K, T196E, F197T, D241K in TCR Cβ;

[0142] (6-11) S134K, M165D, R166P, M168E, S182P in TCR Ca, and E176L, N181K, T196E, F197V, D241K in TCR Cβ.

[0143] In some embodiments, the TCR constant region comprises an amino acid mutation contained in any one of SEQ ID NOs: 5-37.

[0144] In some embodiments, the TCR Ca comprises a sequence as set forth in any one of SEQ ID NOs: 5-15, 30-33.

[0145] In some embodiments, the TCR Cβ comprises a sequence as set forth in any one of SEQ ID NOs: 16-29, 34-37.

[0146] In some embodiments, the TCR constant region, TCR Ca and / or TCR Cβ comprises an amino acid mutation that removes glycosylation.

[0147] In some embodiments, the amino acid mutation that removes glycosylation in the TCR Ca is at one or more positions selected from the group consisting of: position 146, 182, 191.

[0148] In some embodiments, the amino acid mutation that removes glycosylation in the TCR Ca comprises one or more amino acid residues selected from the group consisting of: 146Q, 182P, 191Q.

[0149] In some embodiments, the amino acid mutation that removes glycosylation in the TCR Ca comprises one or more amino acid residues selected from the group consisting of: N146Q, S182P, N191Q.

[0150] In some embodiments, the amino acid mutation that removes glycosylation in the TCR Cβ is at position 181.

[0151] In some embodiments, the amino acid mutation that removes glycosylation in the TCR Cβ is 181K.

[0152] In some embodiments, the amino acid mutation that removes glycosylation in the TCR Cβ is N181K.

[0153] The present disclosure provides a TCR constant region having one or more amino acid mutations in the DE loop of TCR Cα that increase expression and / or stability of the TCR constant region.

[0154] In some embodiments, the DE loop is amino acids 165 to 170 of TCR Cα.

[0155] In some embodiments, the DE loop mutation comprises one or more amino acid residues selected from the group consisting of: 165D, 166P / E, 167E, 168E / T, 169N, 170E.

[0156] In some embodiments, the mutation comprises one or more amino acid residues selected from the group consisting of: M165D, R166P / E, S167E, M168E / T, D169N, F170E.

[0157] In some embodiments, the mutation comprises one or more amino acid residues selected from the group consisting of: M165D, R166P, M168E.

[0158] In some embodiments, the mutation comprises one or more amino acid residues selected from the group consisting of: M165D, R166P / E, S167E, M168E / T, D169N.

[0159] In some embodiments, the mutation comprises one or more amino acid residues selected from the group consisting of: M165D, R166P, S167E, M168E, D169N.

[0160] In some embodiments, the mutation comprises one or more amino acid residues selected from the group consisting of: M165D, R166 P / E, S167E, M168E / T, D169N, F170E.

[0161] In some embodiments, the TCR constant region comprises the amino acid mutations contained in any one of SEQ ID NOs: 5-12.

[0162] In some embodiments, the TCR Cα comprises a sequence as set forth in any one of SEQ ID NOs: 5-12.

[0163] In some embodiments, the TCR Cα comprises an amino acid mutation that removes glycosylation.

[0164] In some embodiments, the amino acid mutation that removes glycosylation is located at one or more positions selected from the group consisting of: position 146, 182, 191.

[0165] In some embodiments, the amino acid mutation that removes glycosylation comprises one or more amino acid residues selected from the group consisting of: 146Q, 182P, and 191Q.

[0166] In some embodiments, the amino acid mutation that removes glycosylation comprises one or more amino acid residues selected from the group consisting of: N146Q, S182P, and N191Q.

[0167] In some embodiments, the TCR constant region further comprises a TCR Cβ.

[0168] In some embodiments, the TCR Cα and the TCR Cβ of the TCR constant region are linked by an interchain disulfide bond.

[0169] In some embodiments, the TCR constant region can be engineered.

[0170] Protein

[0171] In some embodiments, the present disclosure provides a protein comprising a first polypeptide and / or a second polypeptide, wherein the first polypeptide comprises a TCR Cα as defined in any of the preceding embodiments; and the second polypeptide comprises a TCR Cβ as defined in any of the preceding embodiments.

[0172] In some embodiments, the protein comprises an amino acid mutation contained in any one of SEQ ID NOs: 5-37.

[0173] In some embodiments, the TCR Cα comprises a sequence as set forth in any one of SEQ ID NOs: 5-15, 30-33.

[0174] In some embodiments, the TCR Cβ comprises a sequence as set forth in any one of SEQ ID NOs: 16-29, 34-37.

[0175] In some embodiments, the protein, the first polypeptide and / or the second polypeptide comprises at least one amino acid mutation that removes glycosylation.

[0176] In some embodiments, the amino acid mutation that removes glycosylation in the first polypeptide is located at one or more positions selected from the group consisting of: position 146, 182, 191.

[0177] In some embodiments, the amino acid mutation that removes glycosylation in the first polypeptide comprises one or more amino acid residues selected from the group consisting of: 146Q, 182P, 191Q.

[0178] In some embodiments, the amino acid mutation that removes glycosylation in the first polypeptide comprises one or more amino acid residues selected from the group consisting of: N146Q, S182P, N191Q.

[0179] In some embodiments, the amino acid mutation that removes glycosylation in the second polypeptide is at position 181.

[0180] In some embodiments, the amino acid mutation that removes glycosylation in the second polypeptide is 181K.

[0181] In some embodiments, the amino acid mutation that removes glycosylation in the second polypeptide is N181K.

[0182] In some embodiments, there is no natural glycosylation site in the TCR Cα and / or TCR Cβ.

[0183] In some embodiments, the protein, the first polypeptide and / or second polypeptide further comprises an amino acid residue that can form an interchain disulfide bond.

[0184] In some embodiments, the interchain disulfide bond is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more.

[0185] In some embodiments, the amino acid residue that can form an interchain disulfide bond in the first polypeptide is at one or more positions selected from the group consisting of: 123, 128, 158, 161, 166, 202.

[0186] In some embodiments, the amino acid residue that can form an interchain disulfide bond in the second polypeptide is at one or more positions selected from the group consisting of: 126, 128, 130, 131, 165, 168, 170, 188.

[0187] In some embodiments, the protein, the first polypeptide is linked to the second polypeptide by a natural and / or non-natural interchain disulfide bond.

[0188] In some embodiments, the amino acid residue that can form an interchain disulfide bond (a natural cysteine residue or substituted with a cysteine residue) is at one or more positions selected from the group consisting of:

[0189] position 161 of the TCR constant region alpha chain and position 168 of the TCR constant region beta chain;

[0190] position 158 of the TCR constant region alpha chain and position 188 of the TCR constant region beta chain;

[0191] position 123 of the TCR constant region alpha chain and position 128 of the TCR constant region beta chain;

[0192] position 158 of the TCR constant region alpha chain and position 170 of the TCR constant region beta chain;

[0193] position 128 of the TCR constant region alpha chain and position 126 of the TCR constant region beta chain;

[0194] position 166 of the TCR constant region alpha chain and position 165 of the TCR constant region beta chain;

[0195] position 202 of the TCR constant region alpha chain and position 130 of the TCR constant region beta chain;

[0196] position 123 of the TCR constant region alpha chain and position 131 of the TCR constant region beta chain.

[0197] In some embodiments, the interchain disulfide bond-forming amino acid residue is located at one or more positions selected from the group consisting of:

[0198] T161 of the TCR constant region alpha chain and S168 of the TCR constant region beta chain;

[0199] T158 of the TCR constant region alpha chain and S188 of the TCR constant region beta chain;

[0200] Y123 of the TCR constant region alpha chain and S128 of the TCR constant region beta chain;

[0201] T158 of the TCR constant region alpha chain and D170 of the TCR constant region beta chain;

[0202] S128 of the TCR constant region alpha chain and E126 of the TCR constant region beta chain;

[0203] R166 of the TCR constant region alpha chain and S165 of the TCR constant region beta chain;

[0204] P202 of the TCR constant region alpha chain and A130 of the TCR constant region beta chain;

[0205] Y123 of the TCR constant region alpha chain and E131 of the TCR constant region beta chain.

[0206] When the in situ residue is not Cys, the interchain disulfide bond-forming amino acid residue is a Cys resulting from substitution.

[0207] In some embodiments, the protein further comprises an antigen binding domain.

[0208] In some embodiments, the antigen binding domain comprises a tumor antigen binding domain, and / or, an immune cell antigen binding domain.

[0209] In some embodiments, the immune cell antigen binding domain is operably linked to other portions of the protein.

[0210] In some embodiments, the immune cell antigen binding domain is linked to other portions of the protein directly or through a linker.

[0211] In some embodiments, the immune cell antigen binding domain is linked to TCR Cα and / or TCR Cβ directly or through a linker.

[0212] In some embodiments, the immune cell antigen binding domain further comprises an immunoglobulin Fc region.

[0213] In some embodiments, the immune cell is selected from the group consisting of T cells, NK cells, NKT cells.

[0214] In some embodiments, the immune cell antigen binding domain is a CD3 binding domain.

[0215] In some embodiments, the tumor cell antigen comprises a tumor cell surface antigen and an MHC presented antigen.

[0216] In some embodiments, the protein comprises a tumor antigen binding domain and a CD3 binding domain.

[0217] Illustratively, a "tumor antigen" can be understood as those antigens presented on tumor cells. Endogenous antigens (such as viral antigens or tumor neoantigens) are degraded into amino acid peptides in the cytoplasm by proteasomes, enter the endoplasmic reticulum through the transporter associated with antigen processing (TAP), bind to MHC class I molecules, and are transported to the cell surface through the Golgi for recognition by CD8 + TCR of T cells. Extracellular antigens (such as bacterial toxins, tumor secreted proteins) are phagocytosed by antigen presenting cells (APCs), degraded into amino acid peptides in the endosome-lysosome structure, bind to MHC class II molecules and are transported to the surface of APCs for recognition by CD4 + TCR of T cells. Non-limiting examples of tumor antigens used herein are gp100, WT1, mesothelin, NY-ESO-1, MAGE-A4, AFP, MAGE-A4, MAGE-A8, PRAME, MART-1, KRAS, TP53, HPV E7, EBV, HERV-E, etc.

[0218] In some embodiments, the protein comprises a non-tumor antigen binding domain and a CD3 binding domain.

[0219] In some embodiments, the CD3 binding domain is linked to TCR Cα and / or TCR Cβ via an immunoglobulin Fc region.

[0220] In some embodiments, the protein comprises an amino acid mutation as contained in any one of SEQ ID NOs: 5-37.

[0221] In some embodiments, the protein comprises any one of SEQ ID NOs: 5-37.

[0222] In some embodiments, the protein is conjugated to other molecules, such as a labeling molecule for detection or a pharmaceutically active molecule for disease treatment, etc.

[0223] Soluble TCR

[0224] In some embodiments, the present disclosure provides a soluble TCR comprising a first polypeptide and / or a second polypeptide, wherein the first polypeptide comprises or is a TCR Cα as defined in any one of the preceding embodiments; and the second polypeptide comprises or is a TCR Cβ as defined in any one of the preceding embodiments.

[0225] In some embodiments, the present disclosure provides a soluble TCR comprising a first polypeptide, wherein the first polypeptide comprises a TCR Cα as defined in any one of the preceding embodiments.

[0226] In some embodiments, the soluble TCR further comprises a second polypeptide, wherein the second polypeptide comprises a TCR Cβ as defined in any one of the preceding embodiments.

[0227] In some embodiments, the soluble TCR comprises an amino acid mutation as contained in any one of SEQ ID NOs: 5-37.

[0228] In some embodiments, the TCR Cα comprises a sequence as set forth in any one of SEQ ID NOs: 5-15, 30-33.

[0229] In some embodiments, the TCR Cβ comprises a sequence as set forth in any one of SEQ ID NOs: 16-29, 34-37.

[0230] In some embodiments, the first polypeptide and / or the second polypeptide comprises a variable region alpha chain (Vα) and / or a beta chain (Vβ).

[0231] In some embodiments, the Vα and Vβ are a TCR Vα and a TCR Vβ.

[0232] In some embodiments, the TCR Va and the TCR Vp form a tumor antigen binding domain.

[0233] In some embodiments, the sequence of the TCR Ca is as set forth in any one of SEQ ID NOs: 5-15, 30-33.

[0234] In some embodiments, the sequence of the TCR Ca portion of the TCR constant region, protein is as set forth in any one of SEQ ID NOs: 5-15, 30-33.

[0235] In some embodiments, the sequence of the TCR Cp portion of the TCR constant region, protein is as set forth in any one of SEQ ID NOs: 16-29, 34-37.

[0236] In some embodiments, the TCR constant region can be engineered.

[0237] In some embodiments, the TCR constant region according to any one of the preceding, the protein according to any one of the preceding, or the soluble TCR according to any one of the preceding, wherein the amino acid position is defined according to the 2F53 pdb crystal structure.

[0238] 2F53 pdb crystal structure numbering is described in detail in STEVEN M. DUNN et al., Protein Science (2006), 15:710-21; https: / / files.rcsb.org / view / 2F53.pdb, as exemplified in FIG. 1-2. For example, the correspondence between the numbered TCR Ca amino acid residues and the naturally numbered amino acid residues of the sequences set forth in SEQ ID NOs: 1, 2 is as follows: the positions 134, 161, 165, 166, 167, 168, 169, 170, 182, 198 of the TCR Ca numbering of the disclosure correspond to the positions 22, 49, 53, 54, 55, 56, 57, 58, 70, 86 of SEQ ID NOs: 1, 2, respectively. For example, the correspondence between the numbered TCR Cp amino acid residues and the naturally numbered amino acid residues of the sequences set forth in SEQ ID NOs: 3, 4 is as follows: the positions 132, 150, 168, 176, 181, 196, 197, 241 of the TCR Cp numbering of the disclosure correspond to the positions 21, 39, 57, 65, 70, 85, 86, 130 of SEQ ID NO: 2 (Cp), respectively.

[0239] In some embodiments, the stability and / or expression level of the mutant containing the amino acid mutation is improved compared to the wild-type sequence.

[0240] In some embodiments, the T cell receptor (TCR) constant region, protein or soluble TCR mutant has improved stability and / or expression compared to the wild type sequence when the amino acid mutation is present in the TCR Cα.

[0241] In some embodiments, the T cell receptor (TCR) constant region, protein or soluble TCR mutant has improved stability and / or expression compared to the wild type sequence when the amino acid mutation is present in the TCR Cβ.

[0242] In some embodiments, the wild type sequence is TCR Cαwt, TCR Cβwt without the amino acid mutation for improved stability and / or expression.

[0243] In some embodiments, the wild type sequences of the TCR Cα and Cβ are SEQ ID NO. 1 and 3, respectively.

[0244] In some embodiments, the wild type sequence can also contain T161Cα, S168Cβ, which constitute TCR Cαwt(T161C), TCR Cβwt(S168C), with sequences of SEQ ID NO. 2, 4, respectively.

[0245] In some embodiments, the stability refers to thermal stability.

[0246] In some embodiments, the stability is evaluated by Tm value detection.

[0247] In some embodiments, the evaluation of stability and Tm value is described in the detection method of Example 1, for example.

[0248] In some embodiments, the expression is increased by at least 10%, at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, at least 2000%, at least 10000%, at least 20000%, at least 50000%, at least 100000%.

[0249] In some embodiments, the Tm value is increased by at least 0.5°C, at least 1°C, at least 5°C, at least 10°C, at least 15°C, at least 20°C, at least 25°C, at least 30°C, at least 35°C, at least 40°C.

[0250] In some embodiments, the stability and / or expression level is improved without affecting or with no significant decrease in the binding affinity of the TCR constant region, protein or soluble TCR to its ligand or receptor.

[0251] In addition to the above (1)-(7) (Note: (1) is an example, including (1-1)-(1-3), also including (1-1-1)-(1-1-9), etc.), the present disclosure also provides variants of the TCR constant region, protein or soluble TCR having any combination of the mutation sites, mutation types in the above (1)-(36), including but not limited to (7-1)-(7-68) selected from the following:

[0252] (7-1) S134K of TCR Cα;

[0253] (7-2) M165D of TCR Cα;

[0254] (7-3) R166P / E of TCR Cα;

[0255] (7-4) S167E of TCR Cα;

[0256] (7-5) M168E / T of TCR Cα;

[0257] (7-6) D169N of TCR Cα;

[0258] (7-7) F170E of TCR Cα;

[0259] (7-8) S182P of TCR Cα;

[0260] (7-9) D198K of TCR Cα;

[0261] (7-10) I132M of TCR Cβ;

[0262] (7-11) D150A of TCR Cβ;

[0263] (7-12) E176L of TCR Cβ;

[0264] (7-13) N181K of TCR Cβ;

[0265] (7-14) T196E of TCR Cβ;

[0266] (7-15) F197T / V of TCR Cβ;

[0267] (7-16) D241K of TCR Cβ;

[0268] (7-17) S134K of TCR C alpha, and E176L of TCR C beta;

[0269] (7-18) M165D of TCR C alpha, and E176L of TCR C beta;

[0270] (7-19) R166P / E of TCR C alpha, and E176L of TCR C beta;

[0271] (7-20) S167E of TCR C alpha, and E176L of TCR C beta;

[0272] (7-21) M168E / T of TCR C alpha, and E176L of TCR C beta;

[0273] (7-22) D169N of TCR C alpha, and E176L of TCR C beta;

[0274] (7-23) F170E of TCR C alpha, and E176L of TCR C beta;

[0275] (7-24) S182P of TCR C alpha, and E176L of TCR C beta;

[0276] (7-25) D198K of TCR C alpha, and E176L of TCR C beta;

[0277] (7-26) S134K of TCR C alpha, and D241K of TCR C beta;

[0278] (7-27) M165D of TCR C alpha, and D241K of TCR C beta;

[0279] (7-28) R166P / E of TCR C alpha, and D241K of TCR C beta;

[0280] (7-29) S167E of TCR C alpha, and D241K of TCR C beta;

[0281] (7-30) M168E / T of TCR C alpha, and D241K of TCR C beta;

[0282] (7-31) D169N of TCR C alpha, and D241K of TCR C beta;

[0283] (7-32) F170E of TCR C alpha, and D241K of TCR C beta;

[0284] (7-33) S182P of TCR C alpha, and D241K of TCR C beta;

[0285] (7-34) D198K of TCR C alpha, and D241K of TCR C beta;

[0286] (7-35) I132M of TCR C beta, and E176L of TCR C beta;

[0287] (7-36) D150A of TCR C beta, and E176L of TCR C beta;

[0288] (7-37) E176L of TCR C beta, and E176L of TCR C beta;

[0289] (7-38) N181K of TCR C beta, and E176L of TCR C beta;

[0290] (7-39) T196E of TCR C beta, and E176L of TCR C beta;

[0291] (7-40) F197T / V of TCR C beta, and E176L of TCR C beta;

[0292] (7-41) D241K of TCR C beta, and E176L of TCR C beta;

[0293] (7-42) S134K of TCR C alpha, and D241K, E176L of TCR C beta;

[0294] (7-43) M165D of TCR C alpha, and D241K, E176L of TCR C beta;

[0295] (7-44) R166P / E of TCR C alpha, and D241K, E176L of TCR C beta;

[0296] (7-45) S167E of TCR C alpha, and D241K, E176L of TCR C beta;

[0297] (7-46) M168E / T of TCR C alpha, and D241K, E176L of TCR C beta;

[0298] (7-47) D169N of TCR C alpha, and D241K, E176L of TCR C beta;

[0299] (7-48) F170E of TCR C alpha, and D241K, E176L of TCR C beta;

[0300] (7-49) S182P of TCR C alpha, and D241K, E176L of TCR C beta;

[0301] (7-50) M165D, S134K of TCR C a, and E176L, D241K of TCR C ;

[0302] (7-51) M165D, S134K of TCR C a, and E176L, D241K, F197T of TCR C ;

[0303] (7-52) M165D, S134K of TCR C a, and E176L, D241K, F197T, T196E of TCR C ;

[0304] (7-53) M165D, S134K of TCR C a, and E176L, D241K, F197T, T196E, N181K of TCR C ;

[0305] (7-54) M165D, R166P, M168E of TCR C a, and E176L, D241K of TCR C ;

[0306] (7-55) M165D, R166P, M168E of TCR C a, and E176L, D241K, F197T of TCR C ;

[0307] (7-56) M165D, R166P, M168E of TCR C a, and E176L, D241K, F197T, T196E of TCR C ;

[0308] (7-57) M165D, R166P, M168E of TCR C a, and E176L, D241K, F197T, T196E, N181K of TCR C ;

[0309] (7-58) M165D, R166P, M168E, S134K of TCR C a, and E176L, D241K of TCR C ;

[0310] (7-59) M165D, R166P, M168E, S134K of TCR C a, and E176L, D241K, F197T of TCR C ;

[0311] (7-60) M165D, R166P, M168E, S134K of TCR C a, and E176L, D241K, F197T, T196E of TCR C ;

[0312] (7-61) M165D, R166P, M168E, S134K of TCR Ca, and E176L, D241K, F197T, T196E, N181K of TCR C;

[0313] (7-62) M165D, R166P, M168E, S134K, S182P of TCR Ca, and E176L, D241K of TCR C;

[0314] (7-63) M165D, R166P, M168E, S134K, S182P of TCR Ca, and E176L, D241K, F197T of TCR C;

[0315] (7-64) M165D, R166P, M168E, S134K, S182P of TCR Ca, and E176L, D241K, F197T, T196E of TCR C;

[0316] (7-65) M165D, R166P, M168E, S134K, S182P of TCR Ca, and E176L, D241K, F197T, T196E, N181K of TCR C;

[0317] (7-66) M165D, S134K of TCR Ca, and E176L, D241K, F197V, T196E, N181K of TCR C;

[0318] (7-67) M165D, R166P, M168E of TCR Ca, and E176L, D241K, F197V, T196E, N181K of TCR C;

[0319] (7-68) M165D, R166P, M168E, S134K of TCR Ca, and E176L, D241K, F197V, T196E, N181K of TCR C;

[0320] (7-68) M165D, R166P, M168E, S134K, S182P of TCR Ca, and E176L, D241K, F197V, T196E, N181K of TCR C.

[0321] Polynucleotide

[0322] In some embodiments, the present disclosure provides a polynucleotide encoding any one of the foregoing T cell receptor (TCR) constant regions, proteins, or soluble TCRs.

[0323] In some embodiments, the polynucleotide of the present disclosure can be RNA, DNA, or cDNA. In some embodiments, the polynucleotide can be an isolated polynucleotide. In some embodiments, the polynucleotide can be codon-optimized.

[0324] In some embodiments, the present disclosure provides a vector containing the polynucleotide of any of the preceding embodiments. In some embodiments, the vector can be a eukaryotic vector, a prokaryotic vector, such as a plasmid, cosmid, phage, and the like. The vector can be, inter alia, an expression vector, i.e., a vector that can provide for expression of a T cell receptor (TCR) constant region, protein, or soluble TCR in vitro and / or in vivo, i.e., in a suitable host cell, host organism, and / or expression system. The expression vector typically comprises at least one nucleic acid of the present disclosure operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes, and the like). Selection of the elements and their sequences for expression in a particular host is within the ordinary skill in the art.

[0325] The polynucleotide of the present disclosure can be prepared or obtained by known means, e.g., by automated DNA synthesis and / or recombinant DNA techniques, based on information of the amino acid sequence of the TCR constant region, protein, or soluble TCR of any of the preceding embodiments, and / or can be isolated from a suitable natural source.

[0326] In some embodiments, the polynucleotide and vector of the present disclosure can be used to prepare the TCR constant region, protein, or soluble TCR of any of the preceding embodiments. In some embodiments, the polynucleotide and vector of the present disclosure are used to express the TCR constant region, protein, or soluble TCR of any of the preceding embodiments in vitro or in vivo, for different purposes of detection, diagnosis, treatment, modulation, and the like.

[0327] Cells

[0328] The present disclosure provides a cell comprising the polynucleotide, vector of any of the preceding embodiments; or expressing the TCR constant region, protein, or soluble TCR of any of the preceding embodiments.

[0329] In some embodiments, the cell is a host cell.

[0330] In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0331] Bacterial cells, for example, include cells of Gram-negative bacterial strains such as Escherichia coli strains, Proteus strains, and Pseudomonas strains, and Gram-positive bacterial strains such as Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains.

[0332] Fungal cells, for example, include cells of species of Trichoderma, Neurospora, and Aspergillus; or of species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0333] Mammalian cells, for example, include HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0334] However, the present disclosure can also use amphibian cells, insect cells, plant cells, and any other cells used in the art for expression of heterologous proteins.

[0335] The cells of the present disclosure are not capable of developing into a complete plant or animal individual.

[0336] Pharmaceutical compositions

[0337] The present disclosure provides a pharmaceutical composition comprising the TCR constant region, protein, soluble TCR, polynucleotide, or vector of any one of the preceding.

[0338] In some embodiments, the present disclosure provides a pharmaceutical composition containing an amount of the TCR constant region, protein, soluble TCR, polynucleotide, or vector of any one of the preceding effective to treat, alleviate or prevent a disease or condition, and one or more pharmaceutically acceptable excipients, diluents or carriers.

[0339] In some embodiments, the pharmaceutical composition unit dose can contain 0.01 to 99% by weight of the TCR constant region, protein, soluble TCR, polynucleotide, vector, or pharmaceutical composition unit dose can contain the T cell receptor (TCR) constant region, protein, soluble TCR, polynucleotide, or vector of any one of the preceding embodiments in an amount of 0.1-2000 mg. In some specific embodiments, 1-1000 mg.

[0340] In some embodiments, a product or kit is provided containing at least one container, each independently comprising a TCR constant region, protein, soluble TCR, polynucleotide, or vector of any one of the preceding embodiments. Optionally, the kit comprises a container and a label. The container, for example, a bottle, syringe, and test tube. The container holds a composition that is effective for treating a disorder. The label on, or associated with, the container indicates that the composition is used for treating the selected disorder. The composition comprises a TCR constant region, protein, soluble TCR, polynucleotide, or vector of any one of the preceding embodiments.

[0341] In some embodiments, a pharmaceutical composition is provided comprising a TCR constant region, protein, soluble TCR, polynucleotide, or vector of any one of the preceding embodiments. The TCR constant region, protein, soluble TCR, polynucleotide, or vector of any one of the preceding embodiments can be present in an amount effective to treat or ameliorate a disease (e.g., cancer), and the pharmaceutical composition can further comprise at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0342] Methods of manufacture

[0343] The present disclosure provides a method for manufacturing a TCR constant region, protein, soluble TCR, polynucleotide, vector, cell, or pharmaceutical composition of any one of the preceding embodiments.

[0344] In some embodiments, a method of manufacturing a cell is provided. In some embodiments, the method of manufacturing a cell comprises transforming or transfecting a host cell with the polynucleotide, vector. In some embodiments, the method further comprises steps of expanding, screening, identifying, and preserving the cell after transfecting or transducing the cell. Methods of cell expansion, screening, identification, transfection or transformation, culturing, and preservation are well known to those skilled in the art.

[0345] In some embodiments, the method of making any of the foregoing TCR constant regions, proteins, or soluble TCRs comprises: transforming, expressing a polynucleotide or vector encoding the TCR constant region, protein, or soluble TCR in a host cell as previously described; culturing the host cell of the disclosure under conditions that allow expression of the TCR constant region, protein, or soluble TCR of the disclosure; and, if desired, further comprising isolating the TCR constant region, protein, or soluble TCR from the culture of the host cell; and, if desired, further comprising purifying and / or modifying the TCR constant region, protein, or soluble TCR of the disclosure. Methods of cell transformation, culturing, and expression, isolation, purification, modification, preservation, etc. of the foregoing molecules are well known to those skilled in the art.

[0346] However, the TCR constant regions, proteins, soluble TCRs, polynucleotides, vectors of the disclosure can also be obtained by other methods known in the art for producing TCR constant regions, proteins, soluble TCRs, polynucleotides, vectors, such as chemical synthesis, including solid phase or liquid phase synthesis.

[0347] Uses and methods of treatment

[0348] The disclosure provides uses of the amino acid mutations of any of the foregoing for improving expression and / or stability of TCR constant regions.

[0349] In some embodiments, the amino acid mutations are amino acid mutations of TCR Ca and / or TCR C.

[0350] In some embodiments, the amino acid mutations of TCR Ca are as previously described in the amino acid mutation combinations (1)-(7) of the disclosure (Note: taking (1) as an example, comprising (1-1)-(1-3), also comprising (1-1-1)-(1-1-9), etc.).

[0351] In some embodiments, the amino acid mutations of TCR C are as previously described in the amino acid mutation combinations (1)-(7) of the disclosure (Note: taking (1) as an example, comprising (1-1)-(1-3), also comprising (1-1-1)-(1-1-9), etc.).

[0352] In some embodiments, the amino acid mutations are applied to any of the foregoing TCR constant regions, proteins, soluble TCRs, polynucleotides, vectors, cells, or pharmaceutical compositions.

[0353] The disclosure provides methods of using the TCR constant regions, proteins, soluble TCRs, polynucleotides, vectors, cells, or pharmaceutical compositions of any of the foregoing for treating, alleviating, preventing, or diagnosing a disease or disorder.

[0354] In some embodiments, the TCR constant region, protein, soluble TCR, polynucleotide, vector, cell, or pharmaceutical composition of any one of the preceding is provided for use in treating, ameliorating, preventing, or diagnosing a subject having a tumor or cancer.

[0355] In some embodiments, the TCR constant region, protein, soluble TCR, polynucleotide, vector, cell, or pharmaceutical composition of any one of the preceding is provided for use in treating, ameliorating, preventing, or diagnosing a disease or disorder.

[0356] In some embodiments, the TCR constant region, protein, soluble TCR, polynucleotide, vector, cell, or pharmaceutical composition of any one of the preceding is provided for use in treating, ameliorating, preventing, or diagnosing a subject having a tumor or cancer.

[0357] In some embodiments, a method of treating, ameliorating, alleviating, or preventing a disease or disorder is provided, comprising administering to a subject a TCR constant region, protein, soluble TCR, polynucleotide, vector, cell, or pharmaceutical composition of any one of the preceding.

[0358] In some embodiments, a method of ameliorating, alleviating, treating, or preventing a disease or disorder is provided, comprising administering to a subject a TCR constant region, protein, soluble TCR, polynucleotide, vector, cell, or pharmaceutical composition of any one of the preceding in an ameliorating, alleviating, treating, or preventing effective amount.

[0359] The present disclosure provides use of a TCR constant region, protein, soluble TCR, polynucleotide, vector, cell, or pharmaceutical composition of any one of the preceding in the manufacture of a medicament for ameliorating, alleviating, treating, or preventing a disease or disorder.

[0360] In some embodiments, use of a TCR constant region, protein, soluble TCR, polynucleotide, vector, cell, or pharmaceutical composition of any one of the preceding of the present disclosure in the manufacture of a medicament for ameliorating, alleviating, treating, or preventing a disease or disorder is provided.

[0361] In some embodiments, the disease or disorder is a proliferative disease or any other disease or disorder characterized by uncontrolled cell growth (e.g., cancer, in the present disclosure, cancer and tumor can be used interchangeably).

[0362] In some embodiments, the disease or disorder is a tumor or cancer.

[0363] The amino acid mutations provided by the present disclosure, whether DE loop mutations or other single point mutations in the Cα / β region of TCR, can significantly improve the stability and / or expression level of the TCR constant region and the entire extracellular protein. More surprisingly, double mutations, combined mutations, and deglycosylation mutations all have a significant cumulative effect. When applied to TCR bispecific antibodies and other products, the stability and / or expression level can be improved while maintaining the biological activity such as binding affinity to the target. BRIEF DESCRIPTION OF DRAWINGS

[0364] Figure 1 shows the numbering of amino acid residues in the constant region of the alpha chain (Cα) of TCR.

[0365] Figure 2 shows the numbering of amino acid residues in the constant region of the beta chain (Cβ) of TCR. DETAILED DESCRIPTION

[0366] DEFINITIONS

[0367] For the purposes of the present disclosure, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used in the present disclosure have the meanings that are commonly understood by one of ordinary skill in the art in the field of the present disclosure.

[0368] The three-letter and one-letter codes for amino acids used in the present disclosure are as described in J. Biol. Chem., 243, p. 3558 (1968).

[0369] The terms "TCR", "TCR molecule", and "T cell receptor" are used interchangeably in the present disclosure. A native TCR is a glycoprotein on the surface of a cell that is present in a heterodimeric form of an alpha / beta chain or a gamma / delta chain. In 95% of T cells, the TCR heterodimer is composed of an alpha and a beta chain, while 5% of T cells have a TCR composed of a gamma and a delta chain. The extracellular region of the TCR alpha and beta chains are generally considered to each have two "domains", a variable region (Vα, Vβ) and a constant region (Cα, Cβ). In addition, the alpha and beta chains of a native full-length TCR also contain a transmembrane region and a cytoplasmic region. The "TCR molecule" of the present disclosure includes, but is not limited to, a full-length TCR, as well as a soluble TCR, a single-chain TCR composed of fragments thereof.

[0370] The term “TCR variable region” refers to the variable domain of the T cell receptor extracellular region. Taking the TCR extracellular region alpha / beta chain as an example, the TCR variable region is located at the distal membrane end of the alpha / beta chain, comprising a TCR Va domain and a TCR Vb domain, and is the distal membrane structure of the cytoplasmic region, and the antigen binding domain composed of Va and Vb determines the antigen specificity of the TCR. Both TCR Va and Vb chains comprise three hypervariable or complementarity determining regions (CDR1a / b, CDR2a / b and CDR3a / b) surrounded by framework (FR) regions, and CDR3 is the main determinant of antigen recognition and specificity (i.e. the ability to recognize and interact with specific antigens), while CDR1 and CDR2 mainly interact with MHC molecules presenting antigenic peptides.

[0371] The term “TCR constant region” refers to the proximal membrane structure of the TCR extracellular region encoded by the TRAC gene, the TRBC gene, the TRDC gene, the TRGC gene, excluding the transmembrane region and the intracellular region of the full-length TCR sequence. It is encoded by the TRAC gene (for TCR alpha chain), the TRBC1 or TRBC2 gene (for TCR beta chain), the TRDC gene (for TCR delta chain), or the TRGC1 or TRGC2 gene (for TCR gamma chain). The sequences of TCR Ca (TRAC) and TCR Cb (TRBC) can be found in the public database of the International Immunogenetics Information System (IMGT), such as the constant domain sequence of the alpha chain of the TCR molecule “TRAC*01”, and the constant domain sequence of the beta chain of the TCR molecule “TRBC1*01” or “TRBC2*01”. The “TCR constant region” of the present disclosure encompasses natural TCR constant regions and variants thereof from different species, characterized by having all or part of the endogenous functions.

[0372] The term “C alpha domain” refers to the T cell receptor alpha constant region, which is used interchangeably with “TRAC”, “TCR C alpha” in the present disclosure. The C alpha domain of the human TCR constant region comprises AB loop, C chain, DE loop and F chain in turn, but can also be included in different orders. The DE loop is, for example, a structure composed of the amino acid sequence from 165 to 170 of the constant region.

[0373] The term “C beta domain” refers to the T cell receptor beta constant region, which is used interchangeably with “TRBC”, “TCR C beta” in the present disclosure. The C beta domain comprises: T cell receptor beta constant region 1 (TRBC1), T cell receptor beta constant region 2 (TRBC2). TRBC1 and TRBC2 have high homology, and the sequences of the two differ only in 3 amino acid residues.

[0374] The term “full-length TCR” refers to a TCR molecule comprising an extracellular region, a transmembrane region, a cytoplasmic region, and fragments thereof.

[0375] The term "soluble TCR" refers to a TCR molecule comprising the extracellular domain of a TCR, such as the variable region and / or the constant region, and fragments thereof, lacking the transmembrane region and the cytoplasmic region.

[0376] The term "antibody" is used in the broadest context to encompass a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies and antibody fragments (or antigen-binding fragments, or antigen-binding portions), so long as they exhibit the desired antigen-binding activity. An antibody can refer to an immunoglobulin, which is a four polypeptide chain structure connected by interchain disulfide bonds, consisting of two heavy chains and two light chains. The amino acid composition and the order of arrangement of the heavy chain constant region of immunoglobulin are different, so its antigenicity is also different. Accordingly, immunoglobulin can be divided into five categories, or called isotypes of immunoglobulin, namely IgM, IgD, IgG, IgA and IgE, and the corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain, respectively. The same class of Ig can be divided into different subclasses according to the difference in amino acid composition of the hinge region, the number and position of heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, IgG4. The light chain is divided into κ chain or λ chain through the constant region. Each of the five types of Ig can have κ chain or λ chain.

[0377] The term "antigen binding fragment" includes single chain antibodies (i.e. heavy or light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g. VH or VL or VHH), scFv, bi- or tri- or tetra-valent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies and epitope binding fragments of any of the above (see e.g. Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods of generating and making these antibody fragments are well known in the art (see e.g. Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). The Fab-Fv format was first disclosed in WO2009 / 040562 and the disulfide stabilized version of this, Fab-dsFv, was first disclosed in WO2010 / 035012. The antigen binding fragments of the present disclosure also include the Fab and Fab' fragments described in WO2005 / 003169, WO2005 / 003170 and WO2005 / 003171. Multivalent antibodies can comprise multispecificity, e.g. bispecificity or monospecificity (see e.g. WO92 / 22583 and WO05 / 113605).

[0378] The sequences of the heavy and light chains of an antibody, close to the N-terminus, are highly variable and form the variable region (V region); the remaining sequences, close to the C-terminus, are relatively stable and form the constant region (C region). The variable region comprises three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody and are also known as the complementarity determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) is composed of three CDR regions and four FR regions, arranged in the order from the amino-terminus to the carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0379] The term "Fc fragment" refers to the carboxy-terminal portion of a human immunoglobulin chain constant region, particularly an immunoglobulin heavy chain constant region, or a portion thereof, which is devoid of antigen binding activity and is the site of interaction of antibody molecules with effector molecules and cells. For example, an immunoglobulin Fc region can comprise two or more domains of a heavy chain CHI, CH2, CH3, CH4 in combination with an immunoglobulin hinge region. Depending on the amino acid sequence of the heavy chain constant region, immunoglobulins can be assigned to different classes, there being mainly five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM. Some of these can be further divided into subclasses (isotypes), e.g., IgG-I, IgG-2, IgG-3, IgG-4; IgA-I and IgA-2 and different genotypes.

[0380] The term "Fc fragment" preferably comprises at least one immunoglobulin hinge region, and CH2 and CH3 regions of IgG. More preferably it comprises one CH2 domain, one CH3 domain and one immunoglobulin hinge region of IgGl, the hinge region starting amino acid position can vary.

[0381] The term "specifically binds" generally indicates that a TCR binds more readily to its intended antigenic target via its antigen binding site than to random, unrelated non-target antigens. In particular, the term "specifically binds" means that the binding specificity of a TCR for its target antigen is at least 5-fold, preferably 10-fold, more preferably 25-fold, even more preferably 50-fold, most preferably 100-fold or more higher than its binding specificity for a non-target antigen.

[0382] The term "immune cell" refers to any cell of the immune system having one or more effector functions. Immune cells typically comprise cells that play a role in an immune response, and they usually have hematopoietic origin. The term "effector function" refers to a specialized function of an immune cell, e.g., a function or response that enhances or promotes an immune attack on a target cell, e.g., killing of the target cell, or inhibition of its growth or proliferation. For example, an effector function of a T cell can be cytolytic activity or activity involving helper or cytokine secretion. Examples of immune cells include T cells (e.g., alpha / beta T cells and gamma / delta T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived macrophages, among others.

[0383] The immune cells of the present disclosure can be self / autologous ("self") or non-self ("non-self," e.g., allogeneic, syngeneic, or xenogeneic). As used herein, "self" refers to a cell from the same subject. "Allogeneic" refers to a cell of the same species as compared to the comparison cell but not identical in genetic makeup. "Syngeneic" refers to a cell from a different subject that is identical in genetic makeup as compared to the comparison cell. "Xenogeneic" refers to a cell from a different species as compared to the comparison cell. In certain embodiments, the immune cells of the present disclosure are autologous or allogeneic.

[0384] The term "stabilizing mutation," "stability mutation," or "stability-enhancing mutation" refers to a mutation that can result in an improved thermal stability of a TCR ectodomain constant region of the present disclosure as compared to a corresponding TCR ectodomain constant region that does not comprise the mutation. The result of improved thermal stability can be determined by routine methods in the art, such as determining the melting temperature (Tm) of the TCR ectodomain constant region. A TCR ectodomain constant region of the present disclosure has an increased Tm value as compared to a corresponding TCR ectodomain constant region that does not comprise the stabilizing mutation. The expression format of a TCR ectodomain constant region determined to have improved stability includes, but is not limited to, soluble expression, inclusion body expression; location of stable distribution after expression includes, but is not limited to, cytosol, partial or complete chimerization to cell membrane surface, extracellular. The result of stabilization is exhibited in a TCR ectodomain constant region of the present disclosure after expression in a cell and optionally after isolation and purification or further engineering.

[0385] The term "expression-enhancing mutation" or "expression mutation" refers to a mutation that can result in a cell containing a polynucleotide encoding a TCR ectodomain constant region of the present disclosure expressing more of the TCR ectodomain constant region of the present disclosure as compared to a corresponding TCR ectodomain constant region that does not comprise the mutation. The result of improved expression can be determined by routine methods in the art, and the expression format of a TCR ectodomain constant region determined to have improved expression includes, but is not limited to, soluble expression, inclusion body expression; distribution after expression includes, but is not limited to, cytosol, partial or complete chimerization to cell membrane surface, extracellular.

[0386] The term "glycosylation mutation", "deglycosylation mutation", "reduced glycosylation mutation" or "reduced level of glycosylation mutation" refers to a mutation that can result in a reduced level of glycosylation of a TCR extracellular constant region of the disclosure as compared to a corresponding TCR extracellular constant region that does not comprise the mutation. The level of glycosylation can be determined by routine methods in the art. The TCR extracellular constant region of the disclosure has an increased Tm value as compared to a corresponding TCR extracellular constant region that does not comprise the stabilizing mutation. The expression format of the TCR extracellular constant region with reduced level of glycosylation includes, but is not limited to, soluble expression, inclusion body expression; the location of stable distribution after expression includes, but is not limited to, cytoplasm, partially or completely chimerized on the cell membrane surface, extracellular; the result of the reduced level of glycosylation is manifested in the TCR extracellular constant region of the disclosure after cell expression and optionally after isolation and purification or further modification.

[0387] The term "amino acid mutation" includes amino acid substitutions, deletions, insertions, modifications, and any combination thereof, to achieve a final construct such that the final construct possesses a desired property, such as enhanced stability. Amino acid sequence deletions and insertions include amino and / or carboxyl terminal deletions and amino acid insertions. A preferred amino acid mutation is an amino acid substitution. To alter, for example, the binding properties of a TCR constant region, a non-conservative amino acid substitution, i.e., the replacement of one amino acid by another with different structural and / or chemical properties, can be made. Amino acid substitutions include replacement by non-naturally occurring amino acids or by naturally occurring derivatives of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5- hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art, including site-directed mutagenesis, PCR, gene synthesis, chemical modification, and the like.

[0388] The term "glycosylation site" refers to an amino acid residue having a side chain to which a carbohydrate moiety (e.g., an oligosaccharide structure) can be attached. Glycosylation of polypeptides, such as antibodies, is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue, e.g., in the tripeptide sequences Asn-X-Ser and Asn-X-Thr, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyl amino acid, most commonly to serine or threonine. Removal of natural glycosylation sites can be conveniently accomplished by altering the amino acid sequence such that one or more of the above tripeptide sequences (for N-linked glycosylation sites) or one or more serine or threonine residues (for O-linked glycosylation sites) are replaced.

[0389] The term "disulfide bond" refers to a covalent bond formed between sulfur atoms in the structure R-S-S-R'. Amino acid cysteine contains a thiol group that can form a disulfide bond with a second thiol group, for example with the thiol group of another cysteine residue. A disulfide bond can be formed between the thiol groups of two cysteine residues located on two polypeptide chains, respectively, thereby forming an interchain bridge or interchain bond.

[0390] The terms "variant," "mutant" can refer to an entity that has some homology to a wild-type amino acid sequence or wild-type nucleotide sequence, that substantially retains at least one endogenous function. By way of example with a protein, a variant sequence can be obtained by adding, deleting, substituting, modifying, replacing and / or changing at least one amino acid residue in a naturally occurring protein. A variant sequence can comprise an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the subject sequence, preferably at least 90% identical.

[0391] The term "fragment" can also be considered a type of variant, which term generally refers to a selected region of a polypeptide or polynucleotide of interest functionally. Thus, a "fragment" refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[0392] The terms "conservative substitution," "conservative substitution," or "conservative sequence modification" are used interchangeably and apply to both amino acid and nucleotide sequences. With respect to a particular nucleotide sequence, a conservative modification results in a change in the nucleic acid that does not result in a change in the amino acid sequence of the protein encoded by the nucleic acid, or in a change in the nucleic acid that does not substantially change the sequence. With respect to amino acid sequences, "conservative modifications" result in substitution of amino acids with other amino acids having similar chemical properties (e.g., charge, size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.) such that the protein behaves in substantially the same manner. One skilled in the art would know that, in general, single amino acid substitutions in nonessential regions of a polypeptide do not substantially change biological activity (see, e.g., Watson et al. (1987) Molecμlar Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th ed.)).

[0393] The term "binding affinity" or "affinity" is used in the present disclosure as a measure of the strength of a noncovalent interaction between two molecules, e.g., a TCR or portion thereof and an antigen. The binding affinity between two molecules can be quantified by determining the dissociation constant (KD). KD can be determined by measuring the kinetics of complex formation and dissociation using, e.g., surface plasmon resonance (SPR) methods (Biacore). The rate constants corresponding to the association and dissociation of a monovalent complex are referred to as the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. KD is related to ka and kd by the equation KD = kd / ka. The value of the dissociation constant can be determined directly by well-known methods, and can even be calculated for complex mixtures by, e.g., those methods described in Caceci et al. (1984, Byte 9:340-362). For example, KD can be determined using double-filter nitrocellulose filter binding assays such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432). Other standard assays for assessing the binding capacity of an antibody to a target antigen are known in the art, including, e.g., ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays exemplified elsewhere in the present disclosure. The binding kinetics and binding affinity of an antibody can also be evaluated by standard assays known in the art, e.g., surface plasmon resonance (SPR), e.g., by using a Biacore™ system or KinExA. Binding affinities associated with interactions with different molecules, e.g., comparisons of the binding affinities of different antibodies for a given antigen, can be compared by comparing the KD values of the respective antibody / antigen complexes. Similarly, the specificity of an interaction can be evaluated by determining and comparing the KD values of the specific interaction of interest (e.g., between an antibody and an antigen) with the KD values of a non-specific interaction of interest (e.g., a control antibody known not to bind IGF-1R or TRGV9).

[0394] The terms "homology" and "identity" refer to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if every position in the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100. For example, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences match or are homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in the two sequences match or are homologous, then the two sequences are 95% homologous. Typically, when aligning two sequences, the comparison is performed to give the maximum percentage homology. For example, the comparison can be performed by the BLAST algorithm, where the parameters of the algorithm are selected to give the maximum match between each sequence over the entire length of each reference sequence. The following references relate to the BLAST algorithm commonly used for sequence analysis: BLAST ALGORITHMS: Altschul, SF et al., (1990) J. Mol. Biol. 215: 403-410; Gish, W. et al., (1993) Nature Genet. 3: 266-272; Madden, TL et al., (1996) Meth. Enzymol. 266: 131-141; Altschul, SF et al., (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J. et al., (1997) Genome Res. 7: 649-656. Other conventional BLAST algorithms, such as those provided by NCBI BLAST, are also well known to those skilled in the art.

[0395] The terms "nucleic acid" or "polynucleotide" are used interchangeably in this disclosure to refer to any DNA or RNA molecule, whether single-stranded or double-stranded, and, in the case of single-stranded, its complementary sequence, preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. A polynucleotide can also comprise a polynucleotide group.

[0396] The term "codon optimization" or "controlling species codon bias" refers to the preferred codon usage of a particular host cell. As will be appreciated by one of ordinary skill in the art, it is advantageous to modify a coding sequence to enhance its expression in a particular host. The codons most commonly used in a species are referred to as optimal codons, and those codons that are not commonly used are classified as rare or low-usage codons.

[0397] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Vectors disclosed herein are capable of replicating in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) or can integrate into the genome of the host

[0398] The term "cell" includes individual cells or cell cultures, which can be or have been the recipient of a vector for incorporation of a polynucleotide insert. By way of example, a "host cell" is primarily used to express a TCR constant region, TCR C alpha, protein or soluble TCR of the present disclosure to facilitate subsequent isolation and purification of the TCR constant region, TCR C alpha, protein or soluble TCR of the present disclosure. Host cells include the progeny of the parent cell, and therefore can not necessarily be completely identical to the original parent cell (in morphology or in genomic DNA complement) due to natural, accidental, or deliberate mutation. A host cell includes cells transfected and / or transformed in vivo with a polynucleotide of the present disclosure. "Cell," "cell line," and "cell culture" can be used interchangeably and all such designations include their progeny. It is also understood that all progeny can not necessarily be identical to the parental cell since there can be, e.g., mutations that occur during replication. A "mutant" includes progeny that have the same function or biological activity as the polynucleotide screened in the originally transformed cell. Host cells can include bacterial, microbial, plant, or animal cells. Bacteria that are readily transformed include members of the enterobacteria, e.g., strains of Escherichia coli or Salmonella; Bacillus, e.g., Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microbial hosts include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line), 293 cells, and NSO cells.

[0399] The terms "T cell" or "T lymphocyte" are art-recognized and are intended to encompass thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. The T cell can be a T helper (Th) cell, e.g., a T helper 1 (Thl) or T helper 2 (Th2) cell. The T cell can be a helper T cell (HTL; CD4 + T cell) CD4 + cell, a cytotoxic T cell (CTL; CD8 + T cell), CD4 + CD8 + T cell, CD4 - CD8 - T cell, or any other T cell subset. Other illustrative T cell populations suitable for use in particular embodiments include naive T cells (T N ), T memory stem cells (T SCM ), central memory T cells (T CM ), effector memory T cells (T EM ), and effector T cells (T EFF ).

[0400] The terms "polypeptide," "peptide," or "protein" are used interchangeably herein to refer to a polymer of amino acid residues, or a collection of polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are synthetic chemical mimics of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. A polypeptide sequence is typically depicted with its amino-terminal (N-terminal, N-terminal end) on the left and its carboxy-terminal (C-terminal, C-terminal end) on the right.

[0401] The term "operably linked" or "operable linkage" or "operable connection" refers to the linkage of two or more biological sequences in a manner such that they function in a desired manner, whether or not a spacer region (also known as a linker, a connector, a connecting sequence) is present. When used in reference to a polypeptide, the term denotes linkage of polypeptide sequences in a manner which allows the product of the linkage to have a desired biological function, with or without a spacer region between the two sequences. For example, an antibody variable region can be operably linked to a constant region to form a stable product having antigen binding activity. The term can also be used in reference to polynucleotides. For example, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., a promoter, an enhancer, a silencer sequence, etc.), the term denotes linkage of the polynucleotide sequence in a manner which allows the polypeptide to be expressed from the polynucleotide under conditions in which the regulatory sequence is allowed to direct expression.

[0402] The terms "inhibit" or "block" are used interchangeably and encompass both partial and total inhibition / blockade. "Inhibiting growth" (e.g., with respect to a cell) is intended to encompass any measurable decrease in cell growth.

[0403] The terms "administering," "administered," and "treatment" when applied to an animal, human, test subject, cell, tissue, organ, or biological fluid, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, test subject, cell, tissue, organ, or biological fluid, e.g., therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contact of the agent with the cell, as well as contact of the agent with a fluid that is in contact with the cell. "Administering," "administered," and "treatment" also mean treatment by an agent, diagnostic, binding composition, or by another cell in vitro and ex vivo, e.g., a cell. When applied to a human, veterinary, or research subject, it refers to therapeutic treatment, prophylaxis or preventative measures, research, and diagnostic applications.

[0404] The term "treatment" means the administration of a therapeutic agent to a subject, e.g., a TCR constant region, TCR Ca, protein, or soluble TCR of the disclosure or a pharmaceutical composition thereof as a therapeutic agent, to a subject already suffering from, suspected of suffering from, predisposed to suffering from, or symptoms of one or more proliferative diseases, where the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated subject or population, whether by inducing regression of such symptoms or inhibiting progression of such symptoms to any clinically measurable extent. The amount of therapeutic agent effective to alleviate any particular symptom of the disease (also referred to as the "therapeutically effective amount") can vary according to factors such as the disease state, age, and weight of the subject, and the ability of the drug to elicit a desired effect in the subject. Whether a disease symptom has been alleviated can be assessed by any clinical detection method typically used by a physician or other professional health care provider to assess the severity or progression status of the symptom. While an embodiment of the disclosure (e.g., a method of treatment or article of manufacture) can not be effective in alleviating a target disease symptom in a certain subject, it should be determined that the target disease symptom should be alleviated in a statistically significant number of subjects according to any statistical test known in the art, such as the Student t-test, the Chi-square test, the U-test according to Mann and Whitney, the Kruskal-Wallis test (H-test), the Jonckheere-Terpstra test, and the Wilcoxon test.

[0405] The term "pharmaceutical composition" means a mixture of one or more of the compounds described herein, or a physiologically / pharmaceutically acceptable salt or prodrug thereof, with other chemical components, such as physiologically / pharmaceutically acceptable carriers and / or excipients. The purpose of a pharmaceutical composition is to facilitate administration of the active ingredient to a subject, to facilitate absorption, and thereby facilitate biological activity.

[0406] The term "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical disorder. An effective amount also means an amount that is sufficient to allow or facilitate diagnosis. An effective amount for a subject can vary depending on factors such as the condition to be treated, the overall health status of the subject, the method route and dose of administration, and the severity of side effects. An effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxic effects. A subject of the present disclosure can be an animal or human subject.

[0407] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivering a TCR constant region, TCR Ca, protein, or soluble TCR. The carrier can be an anti-adherent, binder, coating, disintegrant, filler or diluent, preservative (such as an antioxidant, antibacterial, or antifungal agent), sweetener, absorption delaying agent, wetting agent, emulsifying agent, buffer, and the like. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), dextrose, vegetable oils (e.g., olive oil), saline, buffers, buffered saline, and isotonic agents such as sugars, polyalcohols, sorbitol, and sodium chloride.

[0408] The term "excipient" is an additional substance in a pharmaceutical formulation other than the main drug, which can also be called an auxiliary material. For example, preservatives, antioxidants, flavoring agents, fragrances, co-solvents, emulsifiers, solubilizers, osmotic pressure adjusting agents, coloring agents, and the like in a liquid formulation can be called excipients.

[0409] The term "optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, and the description includes instances where the event or circumstance occurs and instances where it does not. "And / or" should be considered as specific disclosure of each specified feature or component conjunctively with and without the other. Thus, as used in the phrase "A and / or B" in this disclosure, the term "and / or" covers A and B, A or B, A (alone), and B (alone). Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise," "comprising," "include," "including," and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to." The words "a" and "an" are taken to mean one or more.

[0410] The term "about" refers to a variation of approximately + / - 10% from a given value. It should be understood that such variations are always included in any given value provided herein, whether or not specifically referred to.

[0411] The terms "comprise", "have", "comprising", "including" and "containing" and grammatical variations thereof are inclusive or open-ended and do not exclude additional, unrecited elements and / or method steps.

[0412] The term "subject", "patient" of the present disclosure means a mammal, especially a primate, and particularly a human.

[0413] The following examples are intended to further describe the present disclosure, but these examples do not limit the scope of the present disclosure. The experimental methods in the examples of the present disclosure, unless otherwise specified, are generally performed according to conventional conditions, such as Antibody Techniques in Cold Spring Harbor Laboratory, Manual of Molecular Cloning; or according to the conditions recommended by the manufacturer of the raw materials or commercial products. Reagents not specified for a specific source are commercially available reagents.

[0414] Examples

[0415] Example 1. Mutations of TCR constant region Cα DE loop

[0416] The DE loop of TCR α chain plays an important role in the assembly of TCR-CD3 complex, which can be inserted into the groove of CD3δ chain. The DE loop is located on the interchain interface of TCR constant region, and we speculate that the DE loop may be a potential region affecting the self-stability of TCR constant region in our research of the present disclosure, and point mutations of the DE loop are studied to determine its actual role.

[0417] 1. Construction and expression of mutants

[0418] Different sequences of TCR extracellular constant region Cα / Cβ are synthesized by the method of full gene synthesis, and the sequences are as follows. When expressed, a His tag (HHHHHHHHHGS) (SEQ ID NO: 55) can be fused to the N terminus of the α chain, and a Flag tag (GSGDYKDDDDK) (SEQ ID NO: 56) can be fused to the C terminus of the β chain for easy purification. In addition to the natural disulfide bond containing the stalk region between the Cα chain and the Cβ chain, the T161Cα-S168Cβ disulfide bond is also connected.

[0419] >TCR Cαwt

[0420] >TCR Cαwt(T161C)

[0421] >TCR Cβwt

[0422] >TCR Cβwt(S168C)

[0423] Based on TCR Cαwt(T161C) (SEQ ID NO: 2), the amino acid residues in the DE loop region were respectively subjected to point mutation (see Table 1).

[0424] Table 1. Mutations of TCR constant region Cα DE loop

[0425] Based on each point mutation in Table 1, the TCR constant region Cα variant sequences are as follows:

[0426] >TCR Cα(M165D)

[0427] >TCR Cα(R166P)

[0428] >TCR Cα(R166E)

[0429] >TCR Cα(S167E)

[0430] >TCR Cα(M168E)

[0431] >TCR Cα(M168T)

[0432] >TCR Cα(D169N)

[0433] >TCR Cα(F170E)

[0434] The above amino acid sequences are codon-optimized according to the expression host to determine the nucleotide sequence of the encoding nucleic acid, and then the nucleotide sequences encoding the above TCR extracellular constant regions are respectively cloned into the pTT5 vector to synthesize expression vectors, which are sequenced and verified. The vector is transfected into ExpiCHO-S cells, the cells are cultured, and the protein is expressed, separated, and purified. After detection, the target protein is obtained.

[0435] 2. Performance characterization of mutants

[0436] 2-1. Tm value

[0437] The Tm value of the TCR protein is determined by DSF detection method, which is used to determine the influence of each mutation of TCR constant region Cα DE loop on the stability of TCR constant region Cα.

[0438] The method for detecting Tm value of DSF is as follows: freshly prepare a dilution of SYPRO Orange dye (5000x), dilute it to 50x, and then dilute the SYPRO Orange dye (50x) to 4x. Dilute the dye with PBS buffer. Add 20 μL of TCR protein solution (1-2 mg / mL) to a MicroAmp optical 96-well reaction plate, and then add 20 μL of SYPRO Orange dye (4x) and mix well. Set three replicates for each sample. Place the 96-well plate in a PCR instrument. Use the real-time PCR instrument software (Design & Analysis Software 2.5.0) to run the temperature scan.

[0439] 2-2, TCR expression level

[0440] The expression level is also an important factor for measuring the developability of TCR related products. The disclosure combines the theoretical molar extinction coefficient (ProtParam) to determine the concentration of purified TCR molecules by ultraviolet spectrophotometry (280 nm), and then calculates the expression level of TCR molecules. The Tm value and expression level detection results are shown in Table 2. Compared with the wild type TCR extracellular constant region molecule (TCR Cα / Cβ (S-S) wt) containing 161α-168β disulfide bond, the thermal stability (Tm) of the other TCR extracellular constant region DE loop single point mutants is generally improved, and the Tm value is improved by 0.6-5.5℃. Surprisingly, the expression level of all mutants is improved, with the lowest improvement of 20% and the highest improvement of even 130%. This reflects the importance of DE loop to the stability and expression level of TCR constant region, and all site mutations at least in one aspect can reflect the good product developability potential.

[0441] Table 2. Tm value and expression level of TCR constant region Cα DE loop single point mutant

[0442] Example 2. TCR constant region Cα / β mutation

[0443] In view of the good performance of DE loop mutation in improving the stability and expression level of TCR constant region, we try to use various algorithms to obtain more beneficial mutations in the TCR constant region. Based on TCR Cαwt(T161C) (SEQ ID NO: 2) and TCR Cβwt(S168C) (SEQ ID NO: 4), respectively, point mutations are made to TCR Cα and Cβ, respectively. Some beneficial mutations are selected and shown in Table 3 and Table 4.

[0444] Table 3. TCR Cα constant region mutation

[0445] Table 4. TCR beta chain constant region mutations

[0446] The sequences of some of the mutants are as follows:

[0447] > TCR C alpha (S134K)

[0448] > TCR C alpha (S182P)

[0449] > TCR C alpha (D198K)

[0450] > TCR C beta (I132M)

[0451] > TCR C beta (D150A)

[0452] > TCR C beta (E176L)

[0453] > TCR C beta (N181K)

[0454] > TCR C beta (T196E)

[0455] > TCR C beta (F197V)

[0456] > TCR C beta (F197T)

[0457] > TCR C beta (D241K)

[0458] The nucleotide optimization, plasmid synthesis, protein expression and purification steps and Tm determination methods for the above TCR constant region mutants are the same as in Example 1.

[0459] The effect of single-point mutation is shown in Table 5. Compared with the wild-type TCR extracellular constant region molecule containing 161a-168b disulfide bond (TCR Cα / Cβ (S-S) wt), the thermal stability (Tm) of all single-point mutants is improved, with the Tm value improved by 0.3-4.1°C. In terms of expression level, the expression level of CαD198K, CβI132M and CβD150A is slightly reduced, the expression level of CβF197V remains unchanged, and the expression level of other mutants is significantly improved, with the lowest improvement of 40% and the highest improvement of 200%. Among the single-point mutations, Ms-9 (CαS134K) and Ms-14 (CβE176L) have the most obvious effect on the expression level of TCR constant region.

[0460] Table 5. Tm value and expression level of TCR extracellular constant region single-point mutant

[0461] Example 3. TCR constant region Cα / β double mutation

[0462] Based on the experimental results of the above single-point mutation, Cα / β double mutation is designed (see Table 6).

[0463] Table 6. TCR extracellular constant region double mutation and sequence

[0464] The sequences of some mutants are as follows:

[0465] >Cβ (E176L / I132M)

[0466] >Cβ (E176L / D150A)

[0467] >Cβ (E176L / T196E)

[0468] >Cβ (E176L / F197T)

[0469] >Cβ (E176L / F197V)

[0470] >Cβ (E176L / D241K)

[0471] The steps of nucleotide optimization, plasmid synthesis, protein expression and purification of the mutants, and the Tm determination method are the same as those in Example 1.

[0472] As shown in Table 7, the expression level and thermal stability (Tm) of the TCR extracellular constant region double mutant molecules formed by fixing the beneficial mutation CβE176L in combination with other mutation types shown in Table 1, Table 3, Table 4 are further improved compared with Ms-14 (CβE176L single point mutation). In terms of thermal stability, the Tm values of other double mutants are improved, with an improvement range of 0.3-6.8°C, except for Md-8 (CαD169N / CβE176L). In terms of expression level, the expression levels of other double mutants are improved, with a minimum improvement of 10% and a maximum improvement of 110%, except for Md-9 (CαF170E / CβE176L).

[0473] Table 7. Tm values and expression levels of TCR extracellular constant region double point mutants based on E176L

[0474] Example 4. Combination mutations of TCR constant regions Cα / β

[0475] The combination of double mutations shows more excellent stability and expression level, and based on this, the advantageous mutations found in the double mutations are further combined to obtain 4-10 site combination mutants of TCR extracellular constant region (see Table 8).

[0476] Table 8. Combination mutations of TCR constant regions

[0477] The sequences of the mutants are as follows:

[0478] Cα (S134K / M165D)

[0479] Cα (M165D / R166P / M168E)

[0480] Cα (S134K / M165D / R166P / M168E)

[0481] Cα (S134K / M165D / R166P / M168E / S182P)

[0482] Cβ (E176L / F197T / D241K)

[0483] Cβ (E176L / T196E / F197T / D241K)

[0484] > Cβ (E176L / N181K / T196E / F197T / D241K)

[0485] > Cβ (E176L / N181K / T196E / F197V / D241K)

[0486] Mutant encoding nucleotide optimization, plasmid synthesis, protein expression and purification steps and Tm determination method are the same as

[0487] Example 1.

[0488] Table 9 shows that the molecular developability of the combined mutants is significantly improved: the expression level is as high as 194.1 mg / mL, which is 7.5 times that of the wild-type extracellular TCR constant region molecule (TCR Cα / Cβ (S-S) wt), and the Tm value is also increased by 6.5 to 13.1 °C.

[0489] Table 9. Expression amount and Tm value of TCR extracellular constant region combined mutants

[0490] Example 5. Preparation and expression amount, Tm detection of HIV T36-5 TCR extracellular domain full-length protein targeting HIV pMHC

[0491] To evaluate the effect of constant region mutation on the extracellular full-length protein of soluble TCR, we prepared TCR extracellular full-length protein targeting HLA-A24 / HIV-1 Nef134-10 (2F) pMHC target (sequence derived from PDB: 3VXT). The sequences of different TCR extracellular full-length proteins were synthesized by whole gene synthesis. Among them, the N-terminal of the α chain was fused with His tag, and the C-terminal of the β chain was fused with Flag tag. In addition to the natural disulfide bond, the α / β constant region chains can also be connected by 161α-168β disulfide bond. The plasmid synthesis, protein expression and purification steps and Tm determination method are the same as Example 1.

[0492] The TCR ectodomain full-length proteins of Table 10 were prepared. Among them, the V region of HIV T36-5 (S-S) wt and HIV T36-5 TCR (S-S) Mm-1 to Mm-6 targets HLA-A24 / HIV-1 Nef134-10 (2F) pMHC, and the constant region corresponds to the combined mutations of Mm-1 to Mm-6, respectively. Taking HIV T36-5 (S-S) wt as an example, the Vα sequence is SEQ ID NO: 38, the Cα sequence is SEQ ID NO: 2, the α chain is the fusion protein of the C-terminal of SEQ ID NO: 38 and SEQ ID NO: 2, which is shown as SEQ ID NO: 39; the Vβ sequence is SEQ ID NO: 40, the Cβ sequence is SEQ ID NO: 4, and the β chain is the fusion protein of the C-terminal of SEQ ID NO: 40 and SEQ ID NO: 4, which is shown as SEQ ID NO: 41. In this way, the α chain and β chain full-length of HIV T36-5 TCR (S-S) Mm-1 to Mm-6 can be obtained.

[0493] The sequences are as follows, and the underlined part is the constant region.

[0494] >HIV T36-5 (S-S) wt Vα

[0495] >HIV T36-5 (S-S) wt α chain

[0496] >HIV T36-5 (S-S) wt Vβ

[0497] >HIV T36-5 (S-S) wt β chain

[0498] >Cα (S134K / M165D / R166P / M168E / S182P / C161T)

[0499] >Cβ (E176L / T196E / F197T / D241K / C168S)

[0500] Table 10. TCR ectodomain full-length sequence

[0501] The results are shown in Table 11. The expression of the HIV T36-5 TCR molecule (HIV T36-5 TCR (S-S) wt) with the 161a-168b disulfide bond can be significantly improved to 27.9 mg / L, relative to the HIV T36-5 TCR wt. On this basis, the combination mutations in Table 8 further improve the expression of the TCR molecule by 4.1-5.3 times, with the highest being 147.4 mg / L. As shown in Table 11, the Tm1 and Tm2 values of the soluble TCR determined by DSC are determined by the variable region and the constant region, respectively. For HIV T36-5 TCR (S-S) wt and HIV T36-5 TCR (S-S) Mm-1, Tm1 and Tm2 are relatively close, and the DSC curve is partially superimposed, so only one peak is presented. The point mutations introduced in the present disclosure can improve Tm2 by 4-12°C (relative to Tm1 of HIV T36-5 TCR (S-S) wt), with the highest being 67.9°C. Even without the 161a-168b disulfide bond, the Mm-4 combination mutation can improve the expression of the HIV T36-5 TCR to 13.6 times that of the wild type. The above results show that the point mutations designed in the present disclosure can significantly improve the expression and thermal stability of the wild type or TCR protein containing the 161a-168b disulfide bond.

[0502] Table 11. Expression and Tm values of TCR extracellular domain full-length mutants

[0503] *Note: Because Tm1 and Tm2 of the wild type protein are relatively close, DSC only presents one superimposed single peak defined as Tm1; after introducing mutations, the stability of the constant region is improved, the peak shape is right-shifted, and a double peak appears.

[0504] Example 6. Construction, expression, expression and Tm value detection of HIV T36-5 TCR extracellular domain deglycosylation mutant

[0505] The wild type TCR extracellular constant region a chain and b chain have 3 and 1 potential N-glycosylation sites, respectively. To improve the purity of the TCR protein and reduce the production difficulty, we introduced point mutations to remove N sugar at the glycosylation sites of the TCR constant region. The point mutations S182P a and N181K b introduced above have removed part of the glycosylation sites, and further mutations (N146Q a and N191Q a) are made to the other two N sugar sites. The full-length sequence of the TCR extracellular domain is synthesized by full gene synthesis. Among them, the a chain is fused with His tag at the N terminal, the b chain is fused with Flag tag at the C terminal, and the a / b chain is connected by 161a-168b disulfide bond. The plasmid synthesis, protein expression, purification steps and Tm detection method are the same as in Example 1.

[0506] The sequences of the alpha chain and beta chain of HIV T36-5 TCR(S-S)wt are SEQ ID NO: 39 and 41. The sequence of HIV T36-5 TCR(S-S)Mm-5_degly is as follows:

[0507] > HIV T36-5 TCR(S-S)Mm-5_degly alpha chain

[0508] > HIV T36-5 TCR(S-S)Mm-5_degly beta chain

[0509] The results are shown in Table 12. After removing the N-glycosylation site, the TCR extracellular full-length expression amount is 165.8 mg / L, and the Tm2 is 67.6°C, which is significantly higher than that of the TCR extracellular full-length molecule before mutation.

[0510] Table 12. Expression amount and Tm value of TCR full-length deglycosylation

[0511] Example 7. Preparation and expression amount, Tm detection of TCR extracellular domain full-length protein targeting AFP pMHC

[0512] To further evaluate the effect of constant region mutation on the soluble TCR extracellular full-length protein, we prepared the TCR ST08A01 extracellular full-length protein targeting HLA-A*02:03 / AFP158-166 pMHC target (the sequence is derived from CN116496411A, SEQ ID NO: 7, 10). The sequences of different TCR extracellular full-length proteins were synthesized by whole gene synthesis method (Table 13), and the protein preparation and characterization methods were the same as those in Example 5 and Example 6.

[0513] Among them, the TCR alpha chain constant region is the constant region sequence after all glycosylation sites are removed, and the AFP158-166 TCR(S-S) sequence is as follows:

[0514] > C alpha (S134K / M165D / R166P / M168E / S182P / N146Q / N191Q)

[0515] > AFP158-166 TCR(S-S)wt V alpha

[0516] > AFP158-166 TCR(S-S)wt alpha chain

[0517] > AFP158-166 TCR(S-S)wt V beta

[0518] >AFP158-166 TCR(S-S) wt beta chain

[0519] Table 13. AFP158-166 TCR ectodomain full-length sequence

[0520] Table 14. Expression and Tm value of AFP158-166 TCR ectodomain full-length mutant

[0521] The results are shown in Table 14. The expression of AFP158-166 TCR molecule (AFP158-166 TCR (S-S) wt) with 161a-168b disulfide bond was 68.8 mg / L. On this basis, the introduction of the combination mutations in Table 8 further increased the expression of TCR molecules by 1.3 to 2.74 times, with the highest being 188.5 mg / mL. The point mutations introduced by the disclosure also significantly improved the Tm value. The above results show that the point mutations designed by the disclosure can significantly improve the expression and thermal stability. After removing the N-glycosylation site, the expression of the TCR ectodomain full-length was slightly improved compared to before N-glycosylation, with the highest being 193.6 mg / L, which was significantly higher than the TCR ectodomain full-length molecule before mutation.

[0522] Example 8. Construction of soluble TCR / CD3 bispecific antibody using TCR ectodomain mutant to redirect T cell targeting target cells

[0523] Taking AFP158-166 TCR (S-S) Mm-5_degly as an example, we designed a 1+1 asymmetric bispecific antibody of TCR x CD3, which used Fc to prolong the half-life of bispecific antibody, and Knobs-into-Holes (KiH) to avoid heavy chain mismatch. Among them, the TCR arm preferentially guiding the target cells is placed on the Hole, and the CD3 antibody or other functional molecule arm is placed on the Knob. The plasmid synthesis and protein expression method are the same as in Example 1. The harvested cell culture fluid is purified using a Protein A affinity column (MabSelect SuRe, GE), and the bound antibody is eluted using glycine. After neutralizing the eluate with 1M Tris, desalt.

[0524] The sequences are as follows:

[0525] >AFP158-166 TCR (S-S) Mm-5_degly alpha chain Hole LL234AA mutation (Fc silent)

[0526] >AFP158-166 TCR (S-S) Mm-5_degly beta chain

[0527] >CD3 HC Knob LL234AA mutation (Fc silent) I2C S107E

[0528] >CD3 LC Knob LL234AA mutation (Fc silent) I2C S107E

[0529] Note: Underlined shows constant region.

[0530] Soluble TCR / CD3 bispecifics show good expression level and thermal stability, and TCR constant region mutation does not significantly affect the affinity between TCR full-length protein and target.

[0531] Although the specific embodiments of the present disclosure are described above, those skilled in the art should understand that these are only illustrative, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present disclosure. Therefore, the protection scope of the present disclosure is defined by the appended claims.

Claims

1. An engineered T cell receptor (TCR) constant region comprising: TCR constant region α chain (Cα), and / or TCR constant region β chain (Cβ), in, The TCR Cα comprises one or more amino acid mutations selected from the group consisting of 165D, 168E / T, 134K, 166P / E, 167E, 169N, 170E, 182P, and 198K. The TCR Cβ comprises one or more amino acid mutations selected from the group consisting of: 176L, 241K, 132M, 150A, 181K, 196E, and 197T / V.

2. An engineered TCR constant region comprising a combination of amino acid mutations selected from the group consisting of: (1) TCR Cα comprises one or more amino acid mutations at positions selected from 134, 165, 166, 167, 168, 169, 170, 182, and 198; (2) TCR Cβ contains one or more amino acid mutations at positions selected from 132, 150, 176, 181, 196, 197, and 241; (1-1) positions 134, 165, 166, 167, 168, 169, 170, 182, and 198 of TCR Cα, and position 176 of TCR Cβ; (1-2) positions 134, 165, 166, 167, 168, 169, 170, 182, 198 of TCR Cα, and position 241 of TCR Cβ; or, (1-3) any one of positions 132, 150, 181, 196, 197, 241 of TCR Cβ or a combination thereof, and position 176 of TCR Cβ; Preferably, selected from: (2-1) 134K, 165D, 166P / E, 167E, 168E / T, 169N, 170E, 182P, 198K of TCR Cα, and 176L of TCR Cβ; (2-2) 134K, 165D, 166P / E, 167E, 168E / T, 169N, 170E, 182P, 198K of TCR Cα, and 241K of TCR Cβ; or (2-3) any one of 132M, 150A, 181K, 196E, 197T / V, and 241K of TCR Cβ or a combination thereof, and 176L of TCR Cβ; More preferably, selected from: (3-1) S134K, M165D, R166P / E, S167E, M168E / T, D169N, F170E, S182P, D198K of TCR Cα, and E176L of TCR Cβ; (3-2) S134K, M165D, R166P / E, S167E, M168E / T, D169N, F170E, S182P, D198K of TCR Cα, and D241K of TCR Cβ; or (3-3) Any one of I132M, D150A, N181K, T196E, F197T / V, and D241K of TCR Cβ or a combination thereof, and E176L of TCR Cβ.

3. The engineered TCR constant region according to claim 2, wherein The mutation is selected from the following combinations: (4-1) position 134 of TCR Cα and position 176 of TCR Cβ; (4-2) positions 134 and 165 of TCR Cα, and positions 176 and 241 of TCR Cβ; (4-3) positions 165, 166, and 168 of TCR Cα, and positions 176 and 241 of TCR Cβ; (4-4) positions 165, 166, and 168 of TCR Cα, and positions 176, 197, and 241 of TCR Cβ; (4-5) positions 165, 166, and 168 of TCR Cα, and positions 176, 196, 197, and 241 of TCR Cβ; (4-6) positions 134, 165, 166, and 168 of TCR Cα, and positions 176, 197, and 241 of TCR Cβ; (4-7) positions 134, 165, 166, and 168 of TCR Cα, and positions 176, 196, 197, and 241 of TCR Cβ; (4-8) positions 134, 165, 166, 168, and 182 of TCR Cα, and positions 176, 196, 197, and 241 of TCR Cβ; (4-9) positions 134, 165, 166, and 168 of TCR Cα, and positions 176, 181, 196, 197, and 241 of TCR Cβ; (4-10) positions 134, 165, 166, 168, and 182 of TCR Cα, and positions 176, 181, 196, 197, and 241 of TCR Cβ; Preferably, the mutation is selected from the following combinations: (5-1) 134K for TCR Cα and 176L for TCR Cβ; (5-2) 134K, 165D of TCR Cα, and 176L, 241K of TCR Cβ; (5-3) 165D, 166P, 168E of TCR Cα, and 176L, 241K of TCR Cβ; (5-4) 165D, 166P, 168E of TCR Cα, and 176L, 197T / V, 241K of TCR Cβ; (5-5) 165D, 166P, 168E of TCR Cα, and 176L, 196E, 197T / V, 241K of TCR Cβ; (5-6) 134K, 165D, 166P, 168E of TCR Cα, and 176L, 197T / V, 241K of TCR Cβ; (5-7) 134K, 165D, 166P, 168E of TCR Cα, and 176L, 196E, 197T / V, 241K of TCR Cβ; (5-8) 134K, 165D, 166P, 168E, 182P of TCR Cα, and 176L, 196E, 197T / V, 241K of TCR Cβ; (5-9) 134K, 165D, 166P, 168E of TCR Cα, and 176L, 181K, 196E, 197T / V, 241K of TCR Cβ; (5-10) 134K, 165D, 166P, 168E, 182P of TCR Cα, and 176L, 181K, 196E, 197T, 241K of TCR Cβ; (5-11) 134K, 165D, 166P, 168E, 182P of TCR Cα, and 176L, 181K, 196E, 197V, 241K of TCR Cβ; More preferably, the mutation is selected from the following combinations: (6-1) S134K of TCR Cα, and E176L of TCR Cβ; (6-2) S134K and M165D of TCR Cα, and E176L and D241K of TCR Cβ; (6-3) M165D, R166P, and M168E of TCR Cα, and E176L and D241K of TCR Cβ; (6-4) M165D, R166P, M168E of TCR Cα, and E176L, F197T / V, D241K of TCR Cβ; (6-5) M165D, R166P, M168E of TCR Cα, and E176L, T196E, F197T / V, D241K of TCR Cβ; (6-6) S134K, M165D, R166P, M168E of TCR Cα, and E176L, F197T / V, D241K of TCR Cβ; (6-7) S134K, M165D, R166P, M168E of TCR Cα, and E176L, T196E, F197T / V, D241K of TCR Cβ; (6-8) S134K, M165D, R166P, M168E, S182P of TCR Cα, and E176L, T196E, F197T / V, D241K of TCR Cβ; (6-9) S134K, M165D, R166P, M168E of TCR Cα, and E176L, N181K, T196E, F197T / V, D241K of TCR Cβ; (6-10) S134K, M165D, R166P, M168E, S182P of TCR Cα, and E176L, N181K, T196E, F197T, D241K of TCR Cβ; (6-11) S134K, M165D, R166P, M168E, S182P of TCR Cα, and E176L, N181K, T196E, F197V, D241K of TCR Cβ.

4. The engineered TCR constant region according to any one of the preceding claims, wherein TCR Cα and / or TCR Cβ contain amino acid mutations that abrogate glycosylation, Preferably, the amino acid mutations that remove glycosylation in TCR Cα comprise amino acid residues located at one or more positions selected from the group consisting of: 146, 182, 191; more preferably 146Q, 182P, 191Q; most preferably N146Q, S182P, N191Q; Preferably, the amino acid mutation for deglycosylation in TCR Cβ comprises an amino acid residue selected from the group consisting of: position 181; more preferably 181K; most preferably N181K.

5. An engineered TCR constant region having one or more amino acid mutations in the DE loop of TCR Cα, wherein: The DE loop is from amino acids 165 to 170 of TCR Cα.

6. The engineered TCR constant region according to claim 5, wherein The mutation comprises one or more amino acid residues selected from the group consisting of: 165D, 166P / E, 167E, 168E / T, 169N, 170E; Preferably, the mutation comprises one or more amino acid residues selected from the group consisting of: M165D, R166P / E, S167E, M168E / T, D169N, F170E.

7. The engineered TCR constant region according to any one of claims 5 or 6, wherein It contains amino acid mutations that remove glycosylation on TCR Cα. Preferably, the amino acid mutation that removes glycosylation comprises amino acid residues located at one or more positions selected from the group consisting of: 146, 182, 191; preferably 146Q, 182P and 191Q; more preferably N146Q, S182P and N191Q.

8. The engineered TCR constant region according to any one of claims 1 to 7, wherein The amino acid mutation site is a site defined according to the 2F53 pdb crystal structure; the modified TCR constant region has improved expression and / or stability compared to the wild-type TCR constant region.

9. A protein comprising a first polypeptide and / or a second polypeptide, wherein: The first polypeptide comprises a TCR Cα as defined in any one of claims 1 to 8; The second polypeptide comprises TCR Cβ as defined in any one of claims 1 to 8.

10. The protein according to claim 9, wherein The first polypeptide and / or the second polypeptide further comprises amino acid residues capable of forming interchain disulfide bonds; Preferably, the amino acid residues capable of forming an interchain disulfide bond in the first polypeptide are located at one or more positions selected from the following: positions 123, 128, 158, 161, 166, 202; Preferably, the amino acid residue capable of forming an interchain disulfide bond in the second polypeptide is located at one or more positions selected from the following: positions 126, 128, 130, 131, 165, 168, 170, and 188.

11. The protein according to claim 9 or 10, wherein The first polypeptide is linked to the second polypeptide via a natural and / or non-natural interchain disulfide bond; Preferably, the amino acid residues capable of forming interchain disulfide bonds are located at one or more sites selected from the following groups: 161 of the TCR constant region α chain and 168 of the TCR constant region β chain; position 158 of the TCR constant region α chain and position 188 of the TCR constant region β chain; position 123 of the TCR constant region α chain and position 128 of the TCR constant region β chain; position 158 of the TCR constant region α chain and position 170 of the TCR constant region β chain; position 128 of the TCR constant region α chain and position 126 of the TCR constant region β chain; position 166 of the TCR constant region α chain and position 165 of the TCR constant region β chain; position 202 of the TCR constant region α chain and position 130 of the TCR constant region β chain; position 123 of the TCR constant region α chain and position 131 of the TCR constant region β chain; More preferably, the amino acid residues capable of forming interchain disulfide bonds are located at one or more sites selected from the group consisting of: T161 of the TCR constant region α chain and S168 of the TCR constant region β chain; T158 of the TCR constant region α chain and S188 of the TCR constant region β chain; Y123 of the TCR constant region α chain and S128 of the TCR constant region β chain; T158 of the TCR constant region α chain and D170 of the TCR constant region β chain; S128 of the TCR constant region α chain and E126 of the TCR constant region β chain; R166 of the TCR constant region α chain and S165 of the TCR constant region β chain; P202 of the TCR constant region α chain and A130 of the TCR constant region β chain; Y123 of the TCR constant region α chain and E131 of the TCR constant region β chain.

12. The protein according to any one of claims 9 to 11, wherein The protein further comprises an antigen binding domain; Preferably, the antigen binding domain comprises a tumor antigen binding domain and / or an immune cell antigen binding domain; More preferably, the immune cells are selected from T cells, NK cells, and NKT cells; More preferably, the immune cell antigen binding domain is a CD3 binding domain.

13. The protein according to any one of claims 9 to 12, wherein The protein comprises a tumor antigen binding domain and a CD3 binding domain; Preferably, the CD3 binding domain further comprises an immunoglobulin Fc region; More preferably, the CD3 binding domain is linked to TCR Cα and / or TCR Cβ via the immunoglobulin Fc region.

14. An engineered soluble TCR comprising a first polypeptide and / or a second polypeptide, wherein: The first polypeptide comprises a TCR Cα as defined in any one of claims 1 to 8; The second polypeptide comprises TCR Cβ as defined in any one of claims 1 to 8.

15. The engineered soluble TCR according to claim 14, wherein The first polypeptide and / or the second polypeptide comprises a variable region α chain (Vα) or β chain (Vβ); Preferably, the Vα and Vβ are TCR Vα and TCR Vβ; More preferably, the TCR Vα and TCR Vβ form a tumor antigen binding domain.

16. The engineered soluble TCR according to any one of claims 14-15, wherein: The TCR Cα comprises a sequence selected from any one of the following: SEQ ID NOs: 5-15, 30-33; The TCR Cβ comprises a sequence selected from any one of the following: SEQ ID NOs: 16-29, 34-37.

17. A polynucleotide encoding: The modified TCR constant region according to any one of claims 1 to 8, The protein according to any one of claims 9 to 13, or The engineered soluble TCR according to any one of claims 14 to 16.

18. A vector comprising the polynucleotide according to claim 17, which is a eukaryotic expression vector or a prokaryotic expression vector.

19. A cell comprising or expressing the polynucleotide of claim 17 or the vector of claim 18; Preferably, the cell is a host cell; More preferably, the host cell is selected from bacteria, yeast, and mammalian cells.

20. A pharmaceutical composition comprising: The engineered TCR constant region of any one of claims 1 to 8, the protein of any one of claims 9 to 13, the engineered soluble TCR of any one of claims 14 to 16, the polynucleotide of claim 17, or the vector of claim 18; and one or more pharmaceutically acceptable excipients, diluents or carriers.

21. A preparation method, wherein the preparation method is selected from any one of the following: The TCR constant region of any one of claims 1 to 8, the protein of any one of claims 9 to 13, or the engineered soluble TCR of any one of claims 14 to 16; The method comprises the following steps: Cultivating the cells of claim 19, and then purifying and recovering the engineered TCR constant region, protein, or engineered soluble TCR.

22. Use of amino acid mutations of TCR Cα and / or TCR Cβ to increase the expression level and / or stability of TCR constant regions, wherein: The amino acid mutation of TCR Cα is as defined in any one of claims 1 to 8; The amino acid mutation of TCR Cβ is as defined in any one of claims 1 to 8.

23. A method of treating a disease or condition in a subject in need thereof, comprising: Administering to a subject an effective amount of the engineered TCR constant region according to any one of claims 1 to 8, the protein according to any one of claims 9 to 13, the engineered soluble TCR according to any one of claims 14 to 16, the polynucleotide according to claim 17, the vector according to claim 18, or the pharmaceutical composition according to claim 20; Preferably, the disease or disorder is a tumor or cancer.

Images