Marker-suicide gene useful in adoptive cell therapy
The present invention provides a polypeptide having the formula: St-R1-S1-Q-S2-R2 wherein St is a stalk sequence which, when the polypeptide is expressed at the surface of a target cell, causes the R and Q epitopes to be projected from the cell surface; R1 and R2 are a Rituximab-binding epitopes each having the an amino acid sequence selected from the group consisting of SEQ ID No. 1, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16 or a variant thereof which retains Rituximab-binding activity; S1 and S2 are optional spacer sequences, which may be the same or different; and Q is a QBEnd1O-binding epitope having the amino acid sequence shown as SEQ ID No. 2 or a variant thereof which QBEnd1O-binding activity. The invention also provides a nucleic acid sequence encoding such a polypeptide and uses thereof in adoptive cell transfer.
1. A polypeptide having the formula:
St-R1-S1-Q-S2-R2
wherein
R1 and R2 are a Rituximab-binding epitopes each having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16;
S1 and S2 are spacer sequences, which may be the same or different;
Q is a QBEnd10-binding epitope having the amino acid sequence shown as SEQ ID NO: 2;
St is a stalk sequence which, when the polypeptide is expressed at the surface of a target cell, causes the R and Q epitopes to be projected from the cell surface; and
wherein the spacer sequences S1 and S2 have a combined length of at least about 10 amino acids, such that the distance between R1 and R2 is too long for the polypeptide to bind both antigen binding sites of Rituximab simultaneously.
2. A polypeptide according to claim 1 , wherein the distance between R1 and R2 is more than 76.57 Å.
3. A polypeptide according to claim 1 , wherein the stalk sequence is derivable from CD8alpha.
4. A polypeptide according to claim 1 which comprises the sequence shown as SEQ ID NO: 4.
5. A vector which comprises a nucleic acid sequence encoding a polypeptide having the formula:
St-R1-S1-Q-S2-R2
wherein
R1 and R2 are a Rituximab-binding epitopes each having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16;
S1 and S2 are spacer sequences, which may be the same or different;
Q is a QBEnd10-binding epitope having the amino acid sequence shown as SEQ ID NO: 2;
St is a stalk sequence which, when the polypeptide is expressed at the surface of a target cell, causes the R and Q epitopes to be projected from the cell surface; and
wherein the spacer sequences S1 and S2 have a combined length of at least about 10 amino acids, such that the distance between R1 and R2 is too long for the polypeptide to bind both antigen binding sites of Rituximab simultaneously.
6. A vector according to claim 5 , which also comprises a transgene of interest.
7. A vector according to claim 6 , wherein the transgene of interest encodes a chimeric antigen receptor or a T-cell receptor, such that when the vector is used to transduce a target cell, the target cell co-expresses the polypeptide and the chimeric antigen receptor or T-cell receptor.
8. A cell which expresses a polypeptide according to claim 1 .
9. A cell according to claim 8 which co-expresses the polypeptide and a protein of interest (POI) at the cell surface.
10. A cell which comprises a nucleic acid sequence encoding a polypeptide having the formula:
St-R1-S1-Q-S2-R2
wherein
R1 and R2 are a Rituximab-binding epitopes each having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16;
S1 and S2 are spacer sequences, which may be the same or different;
Q is a QBEnd10-binding epitope having the amino acid sequence shown as SEQ ID NO: 2;
St is a stalk sequence which, when the polypeptide is expressed at the surface of a target cell, causes the R and Q epitopes to be projected from the cell surface; and
wherein the spacer sequences S1 and S2 have a combined length of at least about 10 amino acids, such that the distance between R1 and R2 is too long for the polypeptide to bind both antigen binding sites of Rituximab simultaneously.
11. A cell according to claim 8 , which is a T cell.
12. A polypeptide according to claim 3 , wherein the stalk sequence comprises the amino acid sequence shown in SEQ ID NO: 3.