CAR for use in the treatment of HvG disease
The invention provides an optimized and far potent chimeric antigen receptor for its use in the treatment of HvG disease in a patient having received a transplant, for use in suppressing the hosts 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 in a recipient patient who is negative for HLA-A*02, i.e. the patient prior to transplantation does not express HLA-A*02. 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.
1 . A 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) for use in the treatment of HvG disease in a patient, wherein the single-chain variable fragment antibody domain (scFv) has an amino acid sequence which is SEQ ID NO: 9.
2 . The fusion protein according to claim 1 , wherein the intracellular signalling domain comprises a hCD28 signalling domain and an intracellular hCD3ζ (hCD3 zeta) signalling domain.
3 . The fusion protein according to claim 1 , wherein the hinge and the transmembrane domain have the amino acid sequence of SEQ ID NO: 1, the hCD28 signalling domain has the amino acid sequence of SEQ ID NO: 2, and the hCD3ζ (hCD3 zeta) signalling domain has the amino acid sequence of SEQ ID NO: 3.
4 . The fusion protein according to claim 2 , wherein the hinge is a hΔFc IgG domain having the amino acid sequence of SEQ ID NO: 4.
5 . The fusion protein according to claim 1 , wherein the hinge and the transmembrane domain, which is a CD8 hinge and a CD8 transmembrane domain, have the amino acid sequence of SEQ ID NO: 1, the hCD28 signalling domain has the amino acid sequence of SEQ ID NO: 2, and the hCD3ζ domain has the amino acid sequence of SEQ ID NO: 3, or the hCD28 signalling domain including the hCD3ζ signalling domain have the amino acid sequence of SEQ ID NO: 5.
6 . A CD4 + CD25 + CD127 low HLA-A*02 negative human regulatory T (Treg) cell expressing the fusion protein according to claim 1 .
7 . The fusion protein according to claim 1 , wherein the patient is HLA-A*02 negative and in that the patient contains or is intended to contain a solid tissue transplant which is HLA-A*02 positive.
8 . The fusion protein according to claim 1 , wherein a signal peptide of SEQ ID NO: 10 is linked at the N-terminus of SEQ ID NO: 9.
9 . The fusion protein according to claim 1 , comprising or consisting of, from N-terminus to C-terminus, one scFv domain having the amino acid sequence of SEQ ID NO: 9, a hinge and a transmembrane domain having the amino acid sequence of SEQ ID NO: 1, a hCD28 signalling domain having the amino acid sequence of SEQ ID NO: 2, and a hCD3ζ (hCD3 zeta) signalling domain having the amino acid sequence of SEQ ID NO: 3, and a signal peptide of SEQ ID NO: 10 at the N-terminus.
10 . The fusion protein according to claim 1 , comprising or consisting of, from N-terminal to C-terminal, one scFv domain having the amino acid sequence of SEQ ID NO: 9, a hΔFc IgG domain as a hinge having the amino acid sequence of SEQ ID NO: 4, a hCD28 transmembrane domain and a hCD28/hCD3 signalling domain having the amino acid sequence of SEQ ID NO: 5, and a signal peptide of SEQ ID NO: 10 at the N-terminus.
11 . The 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.
12 . The 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 the amino acid sequence of SEQ ID NO: 6.
13 . The fusion protein according to claim 11 , wherein the leader peptide has the amino acid sequence of SEQ ID NO: 8.
14 . The fusion protein according to claim 1 , wherein when the fusion protein is expressed in a CD4 + CD25 + CD127 low HLA-A*02 negative human regulatory T (Treg) cell in the presence of HLA-A*02 positive solid tissue, the Treg cell has suppressor activity.
15 . The fusion protein according to claim 1 , wherein when the fusion protein is expressed in a CD4 + CD25 + CD127 low HLA-A*02 negative human regulatory T (Treg) cell, the Treg cell has homing capability to secondary lymphoid organs.
16 . A process for providing a human regulatory T (Treg) cell having suppressor activity in the presence of HLA-A*02 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 claim 1 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 an in vitro culture.
17 . The process according to claim 16 , wherein isolating the human regulatory T cells is isolating HLA-A*02 negative human regulatory T cells.
18 . The process according to claim 16 , 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.
19 . The process according to claim 16 , wherein following step b., the Treg cells are kept in culture for 24 h, followed by isolating Treg cells expressing the fusion protein.
20 . The process according to claim 19 , 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.
21 . A Treg cell containing a nucleic acid construct encoding a fusion protein according to claim 1 .
22 . A method of treating HvG disease in a patient, said method comprising administering to said patient the Treg cell of claim 6 .