Disulfide-linked multivalent MHC class I comprising multi-function proteins
Herein is reported a disulfide-linked multivalent multi-function protein, characterized in that it comprises two or more antigen presenting domains, exactly one antibody Fc-region, and at least one antigen binding site, wherein the antigen presenting domain comprises in N- to C-terminal direction either (i) a β2-microglobulin, and (ii) the extracellular domains α1, α2, and α3 of a class I MHC molecule with a relative frequency of less than 1%, or (i) a T-cell response eliciting peptide, (ii) a β2-microglobulin, and (iii) the extracellular domains α1, α2, and α3 of a class I MHC molecule with a relative frequency of 1% or more, wherein the antigen binding site binds to a cancer cell surface antigen or a virus-infected cell surface antigen and wherein the antigen presenting domain has at least two non-naturally occurring cysteine residues which form an intrachain/interdomain disulfide bond.
1. A disulfide-linked multivalent multi-function protein, comprising:
two antigen presenting domains,
one antibody Fc-region, and
one or more antigen binding sites,
wherein the antigen presenting domain comprises in an N- to C-terminal direction either:
(i) a T-cell response eliciting peptide,
(ii) a β2-microglobulin, and
(iii) extracellular domains α1, α2, and α3 of a class I MHC molecule with a relative frequency of 1% or more,
or
(i) a T-cell response eliciting peptide,
(ii) extracellular domains α1, α2, and α3 of a class I MHC molecule with a relative frequency of 1% or more, and
(iii) a β2-microglobulin,
wherein the antigen binding site binds to a cancer cell surface antigen, and
wherein the antigen presenting domain has at least two non-naturally occurring cysteine residues that form an intrachain/interdomain disulfide bond, wherein one non-naturally occurring cysteine residue in the antigen presenting domain is in a linker between the T-cell response eliciting peptide and either the β2-microglobulin or the α1 domain of the class I MHC molecule, and the other non-naturally occurring cysteine residue is in one of the extracellular domains α1, α2, and α3 of the class I MHC molecule and wherein the at least two non-naturally occuring cysteine residues are at position 11 and at position 227 or at position 11 and at position 108 .
2. The disulfide-linked multivalent multi-function protein according to claim 1 , wherein the antibody Fc-region comprises a first and second disulfide-linked Fc-region polypeptide, whereby one of the antigen binding sites is linked to the first Fc-region polypeptide and the second antigen binding site is linked to the second Fc-region polypeptide.
3. The disulfide-linked multivalent multi-function protein according to claim 1 , wherein the antigen binding site comprises; i) an antibody heavy chain and an antibody light chain, or ii) a scFv fusion polypeptide comprising in an N- to C-terminal direction: a scFv antibody fragment and said antibody Fc-region polypeptide, or iii) a scFab fusion polypeptide comprising in N- to C-terminal direction: a scFab and said antibody Fc-region polypeptide, or the antigen binding sites comprise independently of each other i) an antibody heavy chain and an antibody light chain, whereby the individual chains can be wild-type chains or modified chains, ii) a scFv fusion polypeptide comprising in N- to C-terminal direction: a scFv antibody fragment and said antibody Fc-region polypeptide, or iii) a scFab fusion polypeptide comprising in N- to C terminal direction: a scFab and said antibody Fc-region polypeptide.
4. The disulfide-linked multivalent multi-function protein according to claim 1 , wherein the T-cell response eliciting peptide is a human cytomegalovirus-derived peptide.
5. The disulfide-linked multivalent multi-function protein according to claim 4 , wherein the human cytomegalovirus-derived peptide has the amino acid sequence of SEQ ID NO: 01.
6. The disulfide-linked multivalent multi-function protein according to claim 1 , wherein the class I MHC molecule with a relative frequency of 1% or more is selected from the group consisting of HLA-A*0201, HLA-A*1101, HLA-A*2402, HLA-A*340101, HLA C*0304, HLA-C*0401, and HLA-C*0702.
7. The disulfide-linked multivalent multi-function protein according to claim 1 , wherein the two antigen presenting domains comprise:
(i) a virus-derived peptide,
(ii) a β2-microglobulin,
(iii) a soluble HLA-A allele A*0201, and
(iv) cysteine residues at least at position 11 and at position 227 and the cysteine residues at position 11 and position 227 form a disulfide bond.
8. A pharmaceutical formulation comprising the disulfide-linked multivalent multi-function protein according to claim 1 , and a pharmaceutically acceptable carrier.
9. The disulfide-linked multivalent multi-function protein according to claim 1 , wherein the antigen presenting domain comprises in an N- to C-terminal direction:
(i) a virus-derived peptide having an amino acid sequence of SEQ ID NO: 01,
(ii) a first linker peptide having an amino acid sequence of SEQ ID NO: 139,
(iii) a β2-microglobulin having an amino acid sequence of SEQ ID NO: 71,
(iv) a second linker peptide having an amino acid sequence of SEQ ID NO: 83,
(v) an extracellular domains α1, α2, and α3 of a class I MHC molecule with a relative frequency of 1% or more having an amino acid sequence of SEQ ID NO: 140,
(vi) a third linker peptide having an amino acid sequence of SEQ ID NO: 136, and
(vii) cysteine residues at least at position 11 and at position 227 and the cysteine residues at position 11 and position 227 form a disulfide bond.