Methods of making anti-GP41 antibody-specific capture agents
The present application provides stable peptide-based antibody capture agents and methods of use as detection and diagnosis agents. The application further provides methods of manufacturing antibody capture agents using iterative on-bead in situ click chemistry.
1. A method of producing a synthetic capture agent, the method comprising:
(a) providing:
(i) an antibody of interest, wherein the antibody of interest is specific for an epitope,
(ii) a compound comprising a peptide corresponding to the epitope and an azido group or an alkynyl group, and
(iii) a library of candidate peptides, wherein the candidate peptides each comprise an amino acid sequence comprising the formula X1-X2-X3-X4-X5, wherein X1, X2, X3, X4 and X5, each represent glycine or a D-amino acid other than cysteine and methionine, wherein the candidate peptides each further comprise an azido group if the compound comprises an alkynyl group or an alkynyl group if the compound comprises an azido group; and
(b) contacting the antibody of interest, the compound, and the library of candidate peptides, whereby a disubstituted 1,2,3-triazole linkage is formed between the compound and one of the candidate peptides when the azido group and alkynyl group of the compound and one of the candidate peptides are brought in close proximity upon binding of the compound and a candidate peptide to the antibody of interest, whereby the candidate peptide linked to the compound is identified as the candidate peptide to be used in the capture agent,
wherein:
(i) the capture agent is the linked candidate peptide and compound formed in step (b) or
(ii) the capture agent is formed by covalently linking together a separate instance of the peptide of the compound and a separate instance of the candidate peptide identified in step (b), thereby producing the capture agent, wherein the peptide of the compound and identified candidate peptide are linked together via a 1,4-substituted-1,2,3-triazole residue (Tz4) or via a 1,5-substituted-1,2,3-triazole residue (Tz5), wherein the capture agent specifically binds the antibody of interest.
2. The method of claim 1 , wherein the compound comprises an amino acid sequence 80 to 100% identical to IWCGSGKLICTTA (SEQ ID NO:1).
3. The method of claim 2 , wherein the compound comprises the amino acid sequence of IWCGSGKLICTTA (SEQ ID NO:1).
4. The method of claim 2 , wherein the candidate peptide identified in step (b) comprises an amino acid sequence 80 to 100% identical to an amino acid sequence selected from the group consisting of dkGlp (SEQ ID NO: 8); vadpa (SEQ ID NO: 9); nidnG (SEG ID NO:2); pGvtf (SEQ ID NO:10); srrs (SEQ ID NO: 11); dq/eGaf (SEQ ID NO: 12); ywdyn (SEQ ID NO. 13); hllyq (SEQ ID NO: 14); sGaqs (SEQ ID NO:15); ddwai (SEQ ID NO: 16); qidlr (SEQ ID NO: 17); tfLqs (SEQ ID NO: 18); hnpfk (SEQ ID NO. 3); wGehp (SEQ ID NO: 19); qndwK (SEQ ID NO: 20); ltsrY (SEQ ID NO: 21); diksp (SEQ ID NO: 22); eihny (SEQ ID NO. 4); qpidq (SEQ ID NO. 23); kidry (SEQ ID NO. 24); qsp/dwl (SEQ ID NO: 25); and eqtfd (SEQ ID NO: 26).
5. The method of claim 4 , wherein the candidate peptide identified in step (b) comprises an amino acid sequence 80 to 100% identical to an amino acid sequence selected from the group consisting of nidnG (SEQ ID NO:2), hnpfk (SEQ ID NO:3) and eihny (SEQ ID NO:4).
6. The method of claim 5 , wherein the candidate peptide identified in step (b) comprises an amino acid sequence of nidnG (SEQ ID NO:2).
7. The method of claim 5 , wherein the candidate peptide identified in step (b) comprises an amino acid sequence of hnpfk (SEQ ID NO:3).
8. The method of claim 5 , wherein the candidate peptide identified in step (b) comprises the amino acid sequence of eihny (SEQ ID NO:4).
9. The method of claim 1 , wherein the compound and candidate peptide identified in step (b) are linked together via a 1,4-substituted-1,2,3-triazole residue (Tz4) or via a 1,5-substituted-1,2,3-triazole residue (Tz5).
10. The method of claim 1 , wherein the capture agent is labeled with a label selected from the group consisting of biotin, copper-DOTA, biotin-PEG3, aminooxyacetate, 19 FB, 18 FB and FITC-PEG3.
11. The method of claim 1 , wherein the capture agent is labeled with a detectable moiety selected from the group consisting of 64 Cu DOTA, 68 Ga DOTA, 68 Ga NOTA, 18 F, Al 18 F NOTA, 64 Cu, 68 Ga, 89 Zr, 124 I, 86 Y, 94m Tc, 110m In, 11 C and 76 Br.
12. The method of claim 1 , wherein the capture agent has the structure:
13. The method of claim 1 , wherein the capture agent has the structure:
14. The method of claim 1 , wherein the capture agent has the structure: