Soluble heterodimeric T cell receptor, and preparation method and use thereof
Provided is a high-stability T cell receptor (TCR). The TCR comprises (i) the whole or a part of TCRα chain except a transmembrane domain thereof, and (ii) the whole or a part of TCRβ chain except a transmembrane domain thereof, both the (i) and the (ii) comprising a functional variable domain and at least a part of a constant domain of a TCR chain. An artificial interchain disulfide bond links the constant domains of the TCRα and β chains, and a Tm value of the T cell receptor is greater than or equal to 45° C.
1. A T cell receptor (TCR) which has an artificial interchain disulfide bond formed by introducing a cysteine residue into TCR α chain and β chain constant region, wherein the TCR having an artificial interchain disulfide bond has a Tm≥45° C.;
wherein the cysteine residues that form an artificial interchain disulfide bond are at a substitution position selected from the group consisting of:
R53C in Exon 1 of TRAC*01, and S54C in Exon 1 of TRBC1*01 or TRBC2*01; and
Y1OC in Exon 1 of TRAC*01, and E20C in Exon 1 of TRBC1*01 or TRBC2*01;
wherein the TCR comprises: (i) all or part of the TCR α chain other than its transmembrane domain, and (ii) all or part of the TCR β chain other than its transmembrane domain, wherein (i) and (ii) each comprises a functional variable domain and at least a portion of a constant domain of the TCR chain, wherein a conjugate is linked to the C- or N-terminus of the TCR α and/or β chains, and wherein the conjugate is a detectable marker or a therapeutic agent.
2. The TCR of claim 1 , wherein the TCR is soluble.
3. The TCR of claim 1 , wherein the TCR does not have any natural interchain disulfide bond.
4. The TCR of claim 3 , wherein the C-terminus of the native TCR is truncated in the TCR so that a cysteine residue for forming a natural interchain disulfide bond is removed.
5. The TCR of claim 3 , wherein a cysteine residue for forming a natural interchain disulfide bond is substituted with another residue.
6. The TCR of claim 1 , wherein the TCR β chain constant region has no unpaired cysteine residue.
7. The TCR of claim 1 , wherein the therapeutic agent bound with the TCR is an antibody against CD3 which is linked at C- or N- terminal of the TCR α and/or β chains.
8. A nucleic acid molecule comprising a sequence encoding an α chain and/or a β chain of the TCR according to claim 1 , or its complementary sequence.
9. A vector comprising a nucleic acid molecule of claim 8 .
10. A host cell or a genetically engineered cell which comprises a vector comprising a nucleic acid molecule of claim 8 or in which an exogenous nucleic acid molecule of claim 8 is integrated in a chromosome.
11. An isolated cell which expresses a TCR, wherein the cell is a bacteria and the TCR has an artificial interchain disulfide bond formed by introducing a cysteine residue into TCR a chain and b chain constant region, wherein the TCR having an artificial interchain disulfide bond has a Tm≥45° C.;
wherein the cysteine residues that form an artificial interchain disulfide bond are at a substitution position selected from the group consisting of:
R53C in Exon 1 of TRAC*01, and S54C in Exon 1 of TRBC1*01 or TRBC2*01; and
Y10C in Exon 1 of TRAC*01, and E20C in Exon 1 of TRBC1*01 or TRBC2*01;
wherein the TCR comprises: (i) all or part of the TCR α chain other than its transmembrane domain, and (ii) all or part of the TCR β chain other than its transmembrane domain, wherein (i) and (ii) each comprises a functional variable domain and at least a portion of a constant domain of the TCR chain.
12. A method for preparing a TCR, which comprises:
(i) culturing the host cell of claim 10 , thereby expressing an α chain and/or β chain of the TCR; and
(ii) isolating or purifying the α chain and/or β chain;
(iii) refolding the α chain and/or β chain, thereby obtaining the TCR.
13. A TCR complex comprising one or more TCR molecules of claim 1 .
14. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR of claim 1 , or a TCR complex comprising one or more said TCR.