Chimeric antigen receptors with an optimized hinge region
The present invention relates to multi-functional proteins which comprise (i) a signal peptide, (ii) a target specific recognition domain, (iii) a linker region, connecting domain (ii) and domain (iv) which comprises a specific modified hinge region of the human CD8 alpha-chain, and (iv) an effector domain. The present invention furthermore relates to nucleic acids encoding the proteins, expression constructs for expressing the protein in a host cell and host cells. The proteins of the invention are chimeric antigen receptors with an optimized linker or hinge region that are suitable for generating target-specific effector cells, for use as a medicament, in particular in the treatment of cancer and in adoptive, target-cell specific immunotherapy.
1. An isolated polynucleotide encoding a protein comprising:
(i) a signal peptide;
(ii) a target specific recognition domain;
(iii) a linker region, connecting the target specific recognition domain and an effector domain, wherein the linker region does not contain cysteine residue(s) and consists of:
a) the amino acid sequence of SEQ ID NO: 2, or
b) an amino acid sequence that differs in one, two or three amino acid residues from the amino acid sequence of SEQ ID NO. 2; and
(iv) the effector domain comprises:
a) a transmembrane domain and an intracellular domain of human costimulatory CD28 receptor fused to an intracellular domain of a zeta-chain of a human CD3 complex of a T-cell receptor, or
b) a zeta chain of a human CD3 complex of a T-cell receptor encoded by a nucleic acid sequence of SEQ ID NO:9.
2. The polynucleotide of claim 1 , comprising:
a) a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2,
b) a nucleic acid sequence of SEQ ID NO: 8,
c) a nucleic acid sequence complementary to the sequence of a) or b), or
d) a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 8.
3. The polynucleotide of claim 1 , comprising:
a) a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 5,
b) a nucleic acid sequence of SEQ ID NO: 10,
c) a nucleic acid sequence complementary to the sequence of a) or b),
d) a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 10,
e) a nucleic acid sequence of SEQ ID NO: 9,
f) a nucleic acid sequence complementary to SEQ ID NO: 9, or
g) a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 9.
4. The polynucleotide of claim 1 , comprising:
a) a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 7,
b) a nucleic acid sequence of SEQ ID NO: 12,
c) a nucleic acid sequence complementary to the sequence of a) or b),
d) a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 12,
e) a nucleic acid encoding the amino acid sequence of SEQ ID NO: 6,
f) a nucleic acid sequence of SEQ ID NO: 11,
g) a nucleic acid sequence complementary to the sequence of e) or f), or
h) a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 11.
5. An expression construct comprising the polynucleotide of claim 1 .
6. A host cell comprising the polynucleotide of claim 1 or expressing the protein encoded by said polynucleotide of claim 1 , wherein the host cell is selected from effector cells of the immune system, and wherein the effector cells of the immune system are natural killer (NK) cells, natural killer T (NKT) cells, or a lymphocyte preparation containing NK cells and NKT cells.
7. A method for generating target-specific effector cells wherein said method comprises introducing the polynucleotide of claim 1 into a cell and expressing the protein encoded by said polynucleotide.
8. A composition comprising the polynucleotide of claim 1 or the protein encoded by said polynucleotide and a pharmaceutical carrier.
9. The polynucleotide of claim 1 , wherein the target specific recognition domain (ii) binds an antigen, receptor, peptide ligand or protein ligand.
10. The polynucleotide of claim 9 , wherein the antigen is a tumor-associated surface antigen, a lineage-specific or tissue-specific surface antigen or a virus-specific surface antigen.
11. The polynucleotide of claim 1 , wherein the target specific recognition domain (ii) is an antibody or an antigen-binding fragment of said antibody.
12. The polynucleotide of claim 11 , wherein the antibody or the antigen-binding antibody fragment is a single chain Fv (scFv) fragment, an Fab fragment, a diabody, a variable domain of the antibody heavy chain or an antibody light chain.
13. The polynucleotide of claim 1 , wherein the nucleic acid sequence encodes the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 7 under the proviso that amino acid residue no. 308 is a serine.
14. The polynucleotide of claim 1 , wherein the nucleic acid sequence encodes the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 6 under the proviso that amino acid residue no. 308 is a serine.