Epitope-tag surface expressed proteins and nucleic acids thereof
The present invention relates to a method for selecting a host cell population expressing a functional fusion protein comprising at least one epitope-providing amino acid sequence (epitope-tag) and the amino acid sequence of a protein that is expressed on the surface of said host cell. Preferably, the amino acid sequence comprises an alpha or beta chain of a T-cell receptor. The present invention further relates to uses of said functional T-cell receptor (TCR) alpha or beta chain fusion protein in medicine, in particular in adoptive transfer.
1. A human or mouse T-cell receptor (TCR) alpha chain fusion protein expressed on the surface of a T-cell, comprising:
a) at least one epitope-providing amino acid sequence (epitope-tag), and
b) the amino acid sequence of a human or mouse alpha chain of a TCR that is known to bind a specific antigen,
wherein said epitope-tag is selected from
a) an epitope-tag added to the N- terminus of said alpha chain,
b) an epitope-tag inserted into a constant region of said alpha chain, and
c) an epitope-tag replacing a number of amino acids in a constant region of said alpha chain.
2. The T-cell receptor (TCR) alpha chain fusion protein according to claim 1 , wherein said epitope-tag has a length of between 6 to 15 amino acids.
3. The T-cell receptor (TCR) alpha chain fusion protein according to claim 1 , wherein said fusion protein comprises two or more epitope-tags, either spaced apart or directly in tandem.
4. The T-cell receptor (TCR) alpha chain fusion protein according to claim 1 , wherein said epitope-tag is selected from the group consisting of: a myc-tag, FLAG-tag, T7-tag, HA (hemagglutinin)-tag, His-tag, S-tag, GST-tag, and GFP-tag.
5. The T-cell receptor (TCR) alpha chain fusion protein according to claim 1 , wherein said fusion protein has two myc-tag sequences that are attached to the N-terminus of an alpha TCR-chain.
6. A method for producing a fusion protein according to claim 1 , comprising a chemical synthesis of said protein.
7. An isolated nucleic acid molecule encoding the fusion protein according to claim 1 .
8. The nucleic acid molecule according to claim 7 , wherein said molecule is selected from DNA, RNA, PNA, CNA, mRNA or mixtures thereof.
9. A vector comprising an isolated nucleic acid molecule encoding the fusion protein of claim 1 .
10. An isolated host cell, transfected with a vector according to claim 9 , wherein the host cell is a T-cell or a T-cell-precursor cell or a non-pluripotent stem cell.
11. The isolated host cell according to claim 10 , wherein said host cell expresses on its surface
(i) a human or mouse T-cell receptor (TCR) alpha chain fusion protein, comprising:
a) at least one epitope-providing amino acid sequence (epitope-tag), and
b) the amino acid sequence of a human or mouse alpha chain of a TCR that is known to bind a specific antigen,
wherein said epitope-tag is selected from
I) an epitope-tag added to the N- terminus of said alpha chain,
II) an epitope-tag inserted into a constant region of said alpha chain, and
III) an epitope-tag replacing a number of amino acids in a constant region of said alpha chain, or
(ii) a TCR comprising the TCR alpha chain fusion protein of (i).
12. A pharmaceutical composition, comprising a fusion protein according to claim 1 , a nucleic acid molecule according to claim 7 , a vector according to claim 9 or an isolated host T-cell according to claim 11 , together with a pharmaceutically acceptable carrier.