Mutant forms of EtxB and CtxB and their use as carriers
The present invention describes the use of a mutant form of EtxB or CtxB to deliver an agent to a target cell wherein the mutant has GM-1 binding activity; but wherein the mutant has a reduced immunogenic and immunomodulatory activity relative to the wild type form of EtxB or CtxB.
1. A method of delivering a peptide to a MHC class I antigen processing pathway of an antigen presenting cell in a mammal, wherein the method comprises:
providing an antigen presenting cell;
contacting the cell with a mutant of E. coli heat labile enterotoxin B (EtxB; SEQ ID NO: 15) or Vibrio cholera cholera toxin B (CtxB; SEQ. ID NO. 12) covalently linked to the peptide, wherein said mutant comprises at least one of the following point mutations within a region spanning amino acid residues E51 to I58 of the β4-α2 loop of EtxB or CtxB: CtxB (E51A) (CtxB with E51A point mutation: SEQ ID NO: 17), CtxB (Q56A) (CtxB with Q56AA point mutation: SEQ ID NO: 18), CtxB (H57A) (CtxB with H57A point mutation: SEQ ID NO: 13), and EtxB (H57S) (EtxB with H57S point mutation: SEQ ID NO: 19), and has GM-1 binding activity, but reduced immunogenic and immunomodulatory activity relative to the corresponding wild type form of EtxB or CtxB.
2. The method of claim 1 wherein the mutant comprises a point mutation at H57A or H57S.
3. The method of claim 1 , further comprising the step of monitoring the elicitation of a cytotoxic T lymphocyte (CTL) response in said mammal.
4. The method of claim 1 wherein the covalently linked peptide is derived from a protein of interest (POI) or an antigen.
5. The method of claim 4 wherein the antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a parasitic antigen; and a tumor associated antigen (TAA).
6. The method of claim 1 , further comprising monitoring peptide presentation by the MHC class 1 antigen processing pathway in the antigen presenting cell.
7. An in vivo method of delivering a peptide to a MHC class I antigen processing pathway of an antigen presenting cell in a mammal, wherein the method comprises:
contacting an antigen presenting cell in a mammal with a mutant of E. coli heat labile enterotoxin B (EtxB; SEQ ID NO: 15) or Vibrio cholera cholera toxin B (CtxB; SEQ. ID NO. 12) covalently linked to the peptide, wherein said mutant comprises at least one of the following point mutations within a region spanning amino acid residues E51 to I58 of the β4-α2 loop of EtxB or CtxB: CtxB (E51A) (CtxB with E51A point mutation: SEQ ID NO: 17), CtxB (Q56A) (CtxB with Q56AA point mutation: SEQ ID NO: 18), CtxB (H57A) (CtxB with H57A point mutation: SEQ ID NO: 13), and EtxB (H57S) (EtxB with H57S point mutation: SEQ ID NO: 19), and has GM-1 binding activity, but reduced immunogenic and immunomodulatory activity relative to the corresponding wild type form of EtxB or CtxB.
8. The method of claim 7 wherein the mutant comprises a point mutation at H57A or H57S.
9. The method of claim 7 , further comprising the step of monitoring the elicitation of a cytotoxic T lymphocyte (CTL) response in said mammal.
10. The method of claim 7 wherein the covalently linked peptide is derivable derived from a protein of interest (POI) or an antigen.
11. The method of claim 10 wherein the antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a parasitic antigen; and a tumor associated antigen (TAA).
12. The method of claim 7 , further comprising monitoring peptide presentation by the MHC class 1 antigen processing pathway in the antigen presenting cell.