Epidermal growth factor fusion proteins with mutant cholera toxin B subunits
The present disclosure provides for a synthetic immunogenic protein for use as an immuno-modulatory agent to enhance mammalian immune reactions towards conjugated protein or peptide containing antigens that are otherwise poorly immunogenic, including but not limited to self-antigens. The chimeric immunogenic proteins of the present disclosure can be used in the treatment of many illnesses, including but not limited to cancers, infectious disease, autoimmune disease, allergies and any clinical indication involving or affected by the immune response of a mammalian host.
1. A recombinant synthetic protein, comprising:
a monomeric polypeptide sequence able to assemble into stable homo-pentamers, wherein the monomeric polypeptide sequence includes
a synthetic β subunit of an A1B5 group of bacterial holotoxins derived from a Cholera Toxin B (CTB) subunit that includes at least one mutation relative to a wildtype CTB amino acid sequence (SEQ ID NO: 18) selected from the group consisting of T1F, P2T, Q3D, N4I, M37I, A38I, I39L, I40V, T41N, T78S, E79N, A80S, A95S, A102V, and N103R,
a peptide spacer, and
a polypeptide including a full length Epidermal Growth Factor (EGF),
wherein the polypeptide is separated from the synthetic β subunit by the peptide spacer.
2. The recombinant protein according to claim 1 , wherein the synthetic β subunit of an A1B5 group of bacterial holotoxins is SEQ ID NO: 18.
3. The recombinant protein according to claim 1 , wherein the at least one mutation relative to a wildtype CTB amino acid sequence (SEQ ID NO: 18) further includes a mutation selected from the group consisting of L8I, A10G, F25L, A32V, N44G, V82I, A95S, and combinations thereof.
4. The recombinant protein according to claim 1 , wherein the synthetic β subunit is IQDGITDLCAEYHNTQIHTLNDRIFSYTESLAGKREBLVNFKNGATFQVEVPGSFVSTLQ AAAIERMKDTLRIAYLTGAKVEKLCVWNNKTPHAIAAISMSG (SEQ ID NO: 3).
5. The recombinant protein according to claim 1 , wherein the polypeptide further includes a full length of a second growth factor selected from the group consisting of IGF-1, IGF-2, FGF1, FGF2, TGF-α, TGF-β, VEGF-A, VEGF-B, VEGF-C, VEGF-D, PDGF, NGF, EGF, HGF, BMP's, PDL1 and IL's 1-6.
6. The recombinant protein according to claim 1 , wherein the polypeptide includes a full length of one or more growth factors as a single domain or as two or more multiple repeats.
7. The recombinant protein according to claim 1 , wherein the polypeptide spacer is selected from the group consisting of SSG (SEQ ID NO:5), SSGGG (SEQ ID NO:6), SGG (SEQ ID NO:7), GGSGG (SEQ ID NO:8), GGGGS (SEQ ID NO:9), SSGGGSGGSSG (SEQ ID NO:10), GGSGGTSGGGSG (SEQ ID NO:11), SGGTSGGGGSGG (SEQ ID NO:12), GGSGGTSGGGGSGG (SEQ ID NO:13), SSGGGSGGSSG (SEQ ID NO:14), SSGGGGSGGGSSG (SEQ ID NO:15), SSGGGSGGSSGGG (SEQ ID NO:16), and SSGGGGSGGGSSGGG (SEQ ID NO:17).
8. The recombinant protein according to claim 1 , wherein the polypeptide spacer includes an amino acid sequence of a growth factor.
9. The recombinant protein according to claim 1 , wherein the polypeptide spacer includes one or more host T-cell epitopes.