High affinity T cell receptor and use thereof
The present invention is directed to a high affinity T cell receptor (TCR) against a tumor-associated antigen, an isolated nucleic acid molecule encoding same, a T cell expressing said TCR, and a pharmaceutical composition for use in the treatment of diseases involving malignant cells expressing said tumor-associated antigen.
1. A functional TCR α and/or β-chain fusion protein, comprising:
a) at least one epitope-tag, and
b) the amino acid sequence of an α and/or β-chain of a TCR, wherein the TCR binds to a tyrosinase peptide comprising the amino acid sequence of YMDGTMSQV (SEQ ID NO: 9), wherein the amino acid sequence of the α and/or β-chain of the TCR is encoded by:
i) a nucleic acid molecule coding for the V(D)J regions of a TCR that recognizes tyrosinase and comprises the nucleic acid sequence of SEQ ID NO: 1 coding for the α-chain and/or the nucleic acid sequence of SEQ ID NO: 2 coding for the β-chain of said TCR;
ii) a derivative of the nucleic acid molecule of i), coding for the α- or β-chain, wherein the chain has been altered by one or more additions, deletions, or conservative substitutions of from 1-15 amino acids, the additions, deletions, or conservative substitutions being outside the CDR3 region of each chain; or
iii) a nucleic acid molecule coding for the V(D)J regions of a TCR that recognizes tyrosinase and comprising a CDR3 region encoded by the nucleic acid sequence of SEQ ID NO: 3 or 4 or having the amino acid sequence of SEQ ID NO: 5 or 6,
wherein said epitope-tag is selected from
i) an epitope-tag added to the N- and/or C-terminus of said α and/or β chain, or added into the α and/or β chain sequence, but outside the CDR3 region,
ii) an epitope-tag inserted into a constant region of said α and/or β chain, and
iii) an epitope-tag replacing a number of amino acids in a constant region of said α and/or β-chain.
2. The functional TCR α and/or β-chain fusion protein of claim 1 , wherein the amino acid sequence of the α and/or β-chain of the TCR is encoded by a nucleic acid molecule coding for the V(D)J regions of a TCR that recognizes tyrosinase and comprising the nucleic acid sequence of SEQ ID NO: 1 coding for the α-chain and/or the nucleic acid sequence of SEQ ID NO: 2 coding for the β-chain of said TCR.
3. The functional TCR α and/or β-chain fusion protein of claim 1 , wherein the epitope-tag is selected from the group consisting of CD20-tag, Her2/neu-tag, myc-tag, FLAG-tag, T7-tag, HA-tag, His-tag, S-tag, GST-tag, and GFP-tag.
4. The functional TCR α and/or β-chain fusion protein of claim 1 , wherein the amino acid sequence of the α and/or β-chain of the TCR is encoded by a nucleic acid molecule coding for the V(D)J regions of a TCR that recognizes tyrosinase and comprising a CDR3 region encoded by the nucleic acid sequence of SEQ ID NO: 3 or 4 or having the amino acid sequence of SEQ ID NO: 5 or 6.