Fusion protein for use in the treatment of HvG disease
The invention provides a fusion protein for use in the treatment of HvG disease in a patient having received a transplant, for use in suppressing the host's immune response directed against the transplant. The fusion protein is adapted for use in suppressing the immune rejection of a transplant which contains or expresses HLA-A*02 or SLA-01*0401 in a recipient patient who is negative for HLA-A*02 or SLA-01*0401, i.e. the patient prior to transplantation does not express HLA-A*02 or SLA-01*0401. The fusion protein is a chimeric antigen receptor (CAR), which upon expression in regulatory T-cells (T reg ) causes a specific suppressor activity of the regulatory T-cells in the presence of HLA-A*02 or SLA-01*0401.
1. Fusion protein comprising a single-chain variable fragment antibody domain (scFv), a hinge, a transmembrane domain, an intracellular hCD28 signalling domain and an intracellular hCD3ζ (hCD3 zeta) signalling domain forming a chimeric antigen receptor having specificity for HLA-A*02 (CAR-A*02) or having specificity for SLA-01*0401 (CAR-SLA-01*0401) for use in the treatment of HvG disease in a patient, the single-chain variable fragment antibody domain (scFv) having an amino acid sequence which is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 12 or SEQ ID NO: 16 to SEQ ID NO: 19.
2. Fusion protein according to claim 1 , wherein the hinge and the transmembrane domain have an amino acid sequence of SEQ ID NO: 13, the hCD28 signalling domain has an amino acid sequence of SEQ ID NO: 14, and the hCD3ζ (hCD3 zeta) signalling domain has an amino acid sequence of SEQ ID NO: 15.
3. Fusion protein according to claim 1 , wherein the hinge is a hΔFc IgG domain having an amino acid sequence of SEQ ID NO: 20.
4. Fusion protein according to claim 1 , wherein the hinge and the transmembrane domain, which is a CD8 transmembrane domain, have an amino acid sequence of SEO ID NO: 13, the hCD28 signalling domain has an amino acid sequence of SEQ ID NO: 14, and the hCD3 ζ domain has an amino acid sequence of SEQ ID NO: 15, or the hCD28 signalling domain including the hCD3ζ signalling domain have an amino acid sequence of SEQ ID NO: 21.
5. Fusion protein according to claim 1 , expressed in a CD4 + CD25 + CD127 low HLA-A*02 negative human regulatory T (Treg) cell.
6. Fusion protein according to claim 1 , comprising or consisting of, from N-terminal to C-terminal, one scFv domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 12, a hinge and a transmembrane domain having an amino acid sequence of SEQ ID NO: 13, a hCD28 signalling domain having an amino acid sequence of SEQ ID NO: 14, and a hCD3ζ (hCD3 zeta) signalling domain having an amino acid sequence of SEQ ID NO: 15.
7. Fusion protein according to claim 1 , comprising or consisting of, from N-terminal to C-terminal, one scFv domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 16 to SEQ ID NO: 19, a hΔFc IgG domain as a hinge having an amino acid sequence of SEQ ID NO: 20, a hCD28 transmembrane domain and a hCD28/hCD3 signalling domain having an amino acid sequence of SEQ ID NO: 21.
8. Fusion protein according to claim 1 , expressed from a nucleic acid sequence encoding the fusion protein with optionally an additional N-terminal secretory leader peptide.
9. Fusion protein according to claim 1 , expressed from a nucleic acid sequence encoding the fusion protein with an additional N-terminal secretory leader peptide and an additional C-terminal P2A-hFOXP3 having an amino acid sequence of SEQ ID NO: 22.
10. Fusion protein according to claim 8 , wherein the leader peptide has an amino acid sequence selected from SEQ ID NO: 24 and SEQ ID NO: 25.
11. Fusion protein according to claim 1 , wherein the fusion protein provides suppressor activity to a CD4 + CD25 + CD127 low HLA-A*02 negative human regulatory T (Treg) cell in the presence of HLA-A*02 positive solid tissue or in the presence of SLA-01*0401 positive solid tissue.
12. Fusion protein according to claim 10 , wherein the fusion protein provides for homing capability to secondary lymphoid organs in a CD4 + CD25 + CD127 low HLA-A*02 negative human regulatory T (Treg) cell.
13. Process for providing a human regulatory T (Treg) cell having suppressor activity in the presence of HLA-A*02 positive solid tissue or in the presence of SLA-01*0401 positive solid tissue, comprising the steps of
a. isolating from a blood sample CD4 + CD25 + CD127 low human regulatory T (Treg) cells to produce isolated Treg cells,
b. introducing a nucleic acid sequence encoding and expressing a fusion protein according to one of claims 1 to 12 into the isolated Treg cells to produce Treg cells expressing the fusion protein, wherein the Treg cells expressing the fusion protein are not expanded in in vitro culture.
14. Process according to claim 13 , wherein isolating the human regulatory T cells is isolating HLA-A*02 negative human regulatory T cells.
15. Process for providing a human regulatory T (Treg) cell having suppressor activity in the presence of SLA-01*0401 positive solid tissue, comprising the steps of
a. isolating from a blood sample CD4 + CD25 + CD127 low human regulatory T (Treg) cells to produce isolated Treg cells,
b. introducing a nucleic acid sequence encoding and expressing a fusion protein according to one of claims 1 to 12 into the isolated Treg cells to produce Treg cells expressing the fusion protein, wherein the Treg cells expressing the fusion protein are not expanded in in vitro culture.
16. Process according to claim 13 , wherein the nucleic acid sequence is comprised in a retroviral vector that is packaged in a retroviral particle and is introduced into the isolated Treg cells by transduction.
17. Process according to claim 13 , wherein following step b., the Treg cells are kept in culture for 24 h, followed by isolating Treg cells expressing the fusion protein.
18. Process according to claim 17 , wherein the Treg cells are kept in culture in a medium containing low dose IL-2, which medium does not contain an agent stimulating expansion of Treg cells.