Soluble T cell receptors
View Patent ↗The present invention provides a soluble T cell receptor (sTCR), which comprises (i) all or part of a TCR α chain, except the transmembrane domain thereof, and (ii) all or part of a TCR β chain, except the transmembrane domain thereof. (i) and (ii) each comprise a functional variable domain and at least a part of the constant domain of the TCR chain, and are linked by a disulphide bond between constant domain residues which is not present in native TCR.
1. An isolated nucleic acid molecule comprising a sequence encoding all or part of a TCR α chain, except the transmembrane domain thereof,
wherein the all or part of the TCR α chain comprises a functional variable domain and at least a part of the constant domain of the TCR α chain, and wherein the all or part of the TCR α chain comprises a cysteine residue substituted for an amino acid selected from the group consisting of:
(a) Thr 48 of exon 1 of TRAC*01 (amino acid residue 10 of SEQ ID NO:1);
(b) Thr 45 of exon 1 of TRAC*01 (amino acid residue 10 of SEQ ID NO:2);
(c) Tyr 10 of exon 1 of TRAC*01 (amino acid residue 10 of SEQ ID NO:3); and
(d) Ser 15 of exon 1 of TRAC*01 (amino acid residue 10 of SEQ ID NO:4).
2. The nucleic acid molecule of claim 1 which encodes all of the extracellular constant Ig domain of the TCR α chain.
3. A vector comprising a nucleic acid molecule comprising a sequence encoding all or part of a TCR α chain, except the transmembrane domain thereof, wherein the all or part of the TCR α chain comprises a functional variable domain and at least a part of the constant domain of the TCR α chain, and wherein the all or part of the TCR α chain comprises a cysteine residue substituted for an amino acid selected from the group consisting of
(a) Thr 48 of exon 1 of TRAC*01 (amino acid residue 10 of SEQ ID NO:1);
(b) Thr 45 of exon 1 of TRAC*01 (amino acid residue 10 of SEQ ID NO:2);
(c) Tyr 10 of exon 1 of TRAC*01 (amino acid residue 10 of SEQ ID NO:3); and
(d) Ser 15 of exon 1 of TRAC*01 (amino acid residue 10 of SEQ ID NO:4).
4. An isolated host cell comprising the vector of claim 3 .
5. A method for producing a soluble TCR, said method comprising:
(i) transforming a first host cell with a nucleic acid molecule encoding a TCR α chain comprising a non-native cysteine residue in a first position of the constant domain thereof;
(ii) transforming a second host cell with a nucleic acid molecule encoding a TCR β chain comprising a non-native cysteine residue in a second position of the constant domain thereof;
(iii) incubating said host cells under conditions causing expression of the TCR chains;
(iv) refolding the TCR chains in a refolding buffer; and
(v) isolating the refolded soluble TCR,
wherein the non-native cysteine residues are substituted for native residues whose β carbon atoms are 0.6 nm or less apart in the native TCR structure and wherein the refolded soluble TCR comprises a disulfide bond formed from cysteine residues substituted for amino acid residues selected from the group consisting of:
(a) Thr 48 of exon 1 of TRAC*01 (amino acid residue 10 of SEQ ID NO:1) and Ser 57 of exon 1 of TRBC1*01 or TRBC2*01 (amino acid residue 10 of SEQ ID NO:5);
(b) Thr 45 of exon 1 of TRAC*01 (amino acid residue 10 of SEQ ID NO:2) and Ser 77 of exon 1 of TRBC1*01 or TRBC2*01 (amino acid residue 10 of SEQ ID NO:6);
(c) Tyr 10 of exon 1 of TRAC*01 (amino acid residue 10 of SEQ ID NO:3) and Ser 17 of exon 1 of TRBC1*01 or TRBC2*01 (amino acid residue 10 of SEQ ID NO:7);
(d) Thr 45 of exon 1 of TRAC*01 (amino acid residue 10 of SEQ ID NO:2) and Asp 59 of exon 1 of TRBC1*01 or TRBC2*01 (amino acid residue 10 of SEQ ID NO:8); and
(e) Ser 15 of exon 1 of TRAC*01 (amino acid residue 10 of SEQ ID NO:4) and Glu 15 of exon 1 of TRBC1*01 or TRBC2*01 (amino acid residue 10 of SEQ ID NO:9).
6. An isolated nucleic acid molecule comprising a sequence encoding all or part of a TCR β chain, except the transmembrane domain thereof, wherein the all or part of the TCR β chain comprises a functional variable domain and at least a part of the constant domain of the TCR β chain, and wherein the all or part of the TCR β chain comprises a cysteine residue substituted for an amino acid selected from the group consisting of:
(a) Ser 57 of exon 1 of TRBC1*01 or TRBC2*01 (amino acid residue 10 of SEQ ID NO:5);
(b) Ser 77 of exon 1 of TRBC1*01 or TRBC2*01 (amino acid residue 10 of SEQ ID NO:6);
(c) Ser 17 of exon 1 of TRBC1*01 or TRBC2*01 (amino acid residue 10 of SEQ ID NO:7);
(d) Asp 59 of exon 1 of TRBC1*01 or TRBC2*01 (amino acid residue 10 of SEQ ID NO:8); and
(e) Glu 15 of exon 1 of TRBC1*01 or TRBC2*01 (amino acid residue 10 of SEQ ID NO:9).
7. The nucleic acid molecule of claim 6 which encodes all of the extracellular constant Ig domain of the TCR β chain.
8. A vector comprising comprising a sequence encoding all or part of a TCR β chain, except the transmembrane domain thereof, wherein the all or part of the TCR β chain comprises a functional variable domain and at least a part of the constant domain of the TCR β chain, and wherein the all or part of the TCR β chain comprises a cysteine residue substituted for an amino acid selected from the group consisting of:
(a) Ser 57 of exon 1 of TRBC1*01 or TRBC2*01 (amino acid residue 10 of SEQ ID NO:5);
(b) Ser 77 of exon 1 of TRBC1*01 or TRBC2*01 (amino acid residue 10 of SEQ ID NO:6);
(c) Ser 17 of exon 1 of TRBC1*01 or TRBC2*01 (amino acid residue 10 of SEQ ID NO:7);
(d) Asp 59 of exon 1 of TRBC1*01 or TRBC2*01 (amino acid residue 10 of SEQ ID NO:8); and
(e) Glu 15 of exon 1 of TRBC1*01 or TRBC2*01 (amino acid residue 10 of SEQ ID NO:9).
9. An isolated host cell comprising the vector of claim 8 .