Self-assembling protein nanocage decorated with antibodies (SAPNA) and parts thereof
The present invention provides for a protein cage polypeptide (or scaffolding protein) useful or capable of forming a hollow tetrahedral pyramid structure, and a “self-assembling protein nanoparticle decorated with antibodies” (SAPNA) which is a chimeric protein assembly comprising: (a) one or more antibodies and (b) the protein cage polypeptide that provides a scaffold upon which to array the antibodies. In some embodiments, the antibody is capable of binding specifically to a pathogenic biological agent, or part thereof.
1 . A protein cage polypeptide, or scaffolding protein, capable of forming a hollow tetrahedral pyramid structure, wherein the protein cage polypeptide, or scaffolding protein; comprises an amino acid sequence that specifically binds to an antibody or part thereof, wherein the protein cage polypeptide, or scaffolding protein, comprises the following structure: Polypeptide 1-AHL-Polypeptide 2-INSERT A-Polypeptide 3-INSERT B-Polypeptide 4; wherein AHL is an “alpha helical linker” and wherein INSERT A and/or INSERT B each independently comprise the amino acid sequence DCAWHLGELVWCT (SEQ ID NO:41) or GCDCAWHLGELVWCTCG (SEQ ID NO:42) and the protein cage polypeptide comprises an amino acid sequence that comprises any one of SEQ ID Nos:20-39.
2 . The protein cage polypeptide, or scaffolding protein, of claim 1 , wherein INSERT A has a length of about 17 to about 25 amino acids and/or INSERT B has a length of about 28 to about 85 amino acids.
3 . The protein cage polypeptide of claim 1 , wherein Polypeptide 1 comprises an amino acid sequence comprising amino acid identity to the amino acid sequence from the N-terminus to up to the AQEAQKQK sequence of any one of SEQ ID Nos:20-39.
4 . The protein cage polypeptide of claim 1 , wherein Polypeptide 1 comprises an amino acid sequence comprising the following: YGTAR, TDD, LXENLGTR, IDV, TGXRT, and/or SA; wherein X is any charged amino acid residue.
5 . The protein cage polypeptide of claim 1 , wherein Polypeptide 1 comprises about 278 to about 303 amino acid residues.
6 . The protein cage polypeptide of claim 1 , wherein AHL comprises an amino acid sequence comprising: AQEAQKQK.
7 . The protein cage polypeptide of claim 1 , wherein AHL comprises about 5, 6, 7, 8, 9, 10, or 11 amino acid residues.
8 . The protein cage polypeptide of claim 1 , wherein Polypeptide 2 comprises an amino acid sequence comprising amino acid identity to the amino acid sequence from the C-end of the AQEAQKQK sequence to the N-end of INSERT A of any one of SEQ ID Nos:20-39.
9 . The protein cage polypeptide of claim 1 , wherein Polypeptide 2 comprises an amino acid sequence comprising the following: LTEVETYVLS (SEQ ID NO:43).
10 . The protein cage polypeptide of claim 1 , wherein Polypeptide 2 comprises about 30 to about 36 amino acid residues.
11 . The protein cage polypeptide of claim 1 , wherein Polypeptide 3 comprises an amino acid sequence comprising amino acid identity to the amino acid sequence from the C-end of INSERT A to the N-end of INSERT B of any one of SEQ ID Nos:20-39.
12 . The protein cage polypeptide of claim 1 , wherein Polypeptide 3 comprises an amino acid sequence comprising the following: FTLTVPSERGLQR (SEQ ID NO:44) and/or CATCEQIAD (SEQ ID NO:45).
13 . The protein cage polypeptide of claim 1 , wherein Polypeptide 3 comprises about 110 to about 130 amino acid residues.
14 . The protein cage polypeptide of claim 1 , wherein Polypeptide 3 comprises 121 amino acid residues.
15 . The protein cage polypeptide of claim 1 , wherein Polypeptide 4 comprises an amino acid sequence comprising amino acid identity to the amino acid sequence from the C-end of INSERT B to an amino acid sequence comprising: EHHHHHH of any one of SEQ ID Nos:20-35 or 37-39.
16 . The protein cage polypeptide of claim 1 , wherein Polypeptide 4 comprises about 5 to about 13 amino acid residues.
17 . The protein cage polypeptide of claim 1 , wherein Polypeptide 4 comprises 8 amino acid residues.
18 . The protein cage polypeptide of claim 1 , wherein the protein cage polypeptide comprises the amino acid sequence of any one of SEQ ID Nos:20-39.
19 . The protein cage polypeptide of claim 18 , wherein the protein cage polypeptide comprises an amino acid sequence comprising any one or more, or all, charged amino acids that are bolded and underlined as follows
(SEQ ID NO: 40)
MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDA
GYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSM
GTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQ E FFDGIV
AAVKADRYAFYTGFFNDFYNL D ENLGTRISEEAVRNSWNTAASGGFFAAA
AAPTTWYTDFRADIPRIDVPALILHGTG D RTLPI E NTARVFHKALPSAEY
VEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIP
SGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTL
TVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEIS
LSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLE
HHHHHH.
20 . The protein cage polypeptide of claim 1 , wherein the protein cage polypeptide comprises a polypeptide of from about 400 to about 700 amino acid residues.
21 . The protein cage polypeptide of claim 20 , wherein the protein cage polypeptide comprises a polypeptide of from about 450 to about 650 amino acid residues.
22 . The protein cage polypeptide of claim 1 , wherein the antibody is an IgG antibody.
23 . The protein cage polypeptide of claim 1 , wherein the part of the antibody is a Fc region of an IgG antibody.
24 . The protein cage polypeptide of claim 22 , wherein the IgG antibody is a human IgG antibody.
25 . The protein cage polypeptide of claim 23 , wherein the Fc region of an IgG antibody is part of an Fc chimeric protein.
26 . The protein cage polypeptide, or scaffolding protein, of claim 1 , wherein the binding affinity K a of the protein cage polypeptide, or scaffolding protein, to the antibody or part thereof, is equal to or greater than 10 7 M −1 .
27 . The protein cage polypeptide, or scaffolding protein, of claim 1 , wherein the protein cage polypeptide, or scaffolding protein, specifically binds to the antibody or part thereof, or any chimeric protein, molecule or compound comprising the antibody, or part thereof.
28 . A hollow tetrahedral pyramid structure comprising twelve protein cage polypeptides of claim 1 assembled as the tetrahedral pyramid structure.
29 . A “self-assembling protein nanoparticle decorated with antibodies” (SAPNA) which is a chimeric protein assembly comprising: (a) one or more antibodies and (b) a protein cage polypeptide of claim 1 that provides a scaffold upon which to array the antibodies, wherein the one or more antibodies are bound to the INSERT A and/or INSERT B of the protein cage polypeptide.
30 . A “self-assembling protein nanoparticle decorated with antibodies” (SAPNA) structure comprising: (1) one protein cage polypeptide or scaffolding protein of claim 1 , or a plurality thereof assembled into a 3-dimensional assembly, (2) optionally one or more human or rabbit IgG antibodies, (3) optionally an IgG binding loop, and (4) optionally, when the plurality of polypeptides or scaffolding proteins (or engineered protein cage proteins (PCs)) are assembled into a 3-dimensional assembly, a cargo of interest confined or enclosed by the 3-dimensional assembly.
31 . A method for detecting or isolating a pathogenic biological agent, or part thereof, the method comprising: a) providing a “self-assembling protein nanoparticle decorated with antibody” (SAPNA) of claim 30 wherein the antibody is capable of binding specifically to a pathogenic biological agent, or part thereof, (b) contacting the SAPNA with a sample comprising the pathogenic biological agent, or part thereof, such that the SAPNA binds the pathogenic biological agent, or part thereof; (c) detecting the SAPNA pathogenic biological agent, or part thereof via detection, and/or separating the SAPNA bound pathogenic biological agent, or part thereof, from the rest of the sample; and (d) determining the abundance of the pathogenic biological agent, or part thereof.