Self-assembling synthetic proteins comprising cholera toxin beta subunit
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 sequence that is able to assemble into stable pentamers, including
a Cholera Toxin β (CTB) subunit TPONITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQ HIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN (SEQ ID NO: 18) having mutations TIF, P2T, Q3D, N4I, M37I, A381, 139L, 140V, T41N, T78S, E79N, A80S, A95S, A102V, and N103R;
a peptide spacer; and
a polypeptide including substantially a full-length 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-1, IL-2, IL-3, IL-4, IL-5, and IL-6, wherein the part thereof includes a neutralizing domain of the growth factor,
wherein the polypeptide is separated from the CTB subunit by the peptide spacer, which prevents the polypeptide from sterically inhibiting assembly of the pentamers by the CTB subunit.
2. The recombinant protein according to claim 1 , wherein the monomeric sequence further comprises one or more additional polypeptide sequences.
3. The recombinant protein according to claim 2 , wherein the one or more additional polypeptide sequences include a full-length growth factor, or part thereof, 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-1, IL-2, IL-3, IL-4, IL-5, and IL-6.
4. The recombinant protein according to claim 1 , wherein the growth factor is TGF-α, TGF-β, or EGF.
5. The recombinant protein according to claim 3 , wherein the part thereof includes a neutralizing domain of the growth factor, optionally wherein the part thereof includes a full length or neutralizing domain of one or more growth factors in the protein as a single domain or as two or more multiple repeats.
6. The recombinant protein according claim 1 , wherein the spacer comprises, in part, a growth factor or neutralizing domain thereof, or is selected from the group consisting of SSG, SSGGG (SEQ ID NO: 6), SGG, 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) or includes one or more host T-cell epitopes.
7. A process of preparing a stable homo-pentamer complex comprising assembling monomeric sub-units of claim 1 to form one or more stable homo-pentamer complexes.
8. A process of preparing a multivalent vaccine formulation comprising mixing one or more single monomeric sub-units of claim 1 to form a multivalent vaccine.
9. A process for treating a patient comprising administering an immunogenic dose of the multivalent vaccine formulation of claim 8 to the patient during a treatment period.