Systematic discovery, maturation and extension of peptide binders to proteins

Patent metadata

Field	Value
Publication number	US-10976317-B2
Application number	US-201816191407-A
Country	US
Kind code	B2
Filing date	Nov 14, 2018
Priority date	Dec 27, 2013
Publication date	Apr 13, 2021
Grant date	Apr 13, 2021

Abstract

The invention comprises systems, methods and arrays for identification and optimization of novel peptide binders to protein targets. Embodiments include steps of peptide binder discovery, core peptide maturation, N-terminal and C-terminal extension and kinetics analysis of the final peptide binder.

First claim

We claim:

1. A method of identifying a peptide binder comprising the steps of:
(a) exposing a protein target of interest to a first peptide microarray produced by maskless photolithography comprising a first population of peptide binders, whereby the protein target binds to peptide binders of the first population of peptide binders;
(b) identifying overlap in sequences of the peptide binders of the first population of peptide binders which bind the protein target of interest, whereby a core binder sequence of the first population of peptide binders is determined;
(c) performing at least one alteration to each core binder sequence of the first population of peptide binders wherein the alteration is selected from a single amino acid substitution, a double amino acid substitution, an amino acid insertion, and an amino acid deletion, whereby a second population of peptide core binder sequences is generated; and
(d) exposing the protein target of interest to a second peptide microarray produced by maskless photolithography comprising the second population of peptide core binder sequences generated in step (c), whereby the protein target binds to at least one of the altered peptide core binder sequences of the second population of peptide core binder sequences; and
(e) identifying one or more sequences of the second population of peptide core binder sequences demonstrating strong binding properties to the protein target, whereby a mature core binding sequence is determined;
wherein:
the first peptide microarray comprises at least 1.0×10 6 peptides; and
the first population of peptide binders comprise all 5-mer peptides comprising all 20 natural amino acid excluding cysteine and optionally excluding methionine and the 5-mer peptides have the amino acid sequence -M 1 M 2 M 3 M 4 M 5 -.

2. The method of claim 1 , wherein the alteration in step (c) comprises the single amino acid substitution or the double amino acid substitution and the single amino acid substitution or the double amino acid substitution occurs at each of M 1 , M 2 , M 3 , M 4 , and M 5 using each of the other 18 natural amino acids.

3. The method of claim 1 , wherein the alteration in step (c) comprises the amino acid insertion and the amino acid insertion occurs before or after one of M 1 , M 2 , M 3 , M 4 , and M 5 .

4. The method of claim 1 , wherein the alteration in step (c) comprises the amino acid deletion and the amino acid deletion occurs at one of M 1 , M 2 , M 3 , M 4 , and M 5 to form 4-mer peptides.

5. The method of claim 4 , wherein the 4-mer peptides are further altered and the alteration is selected from a single amino acid substitution, a double amino acid substitution, an amino acid insertion.

6. The method of claim 1 , wherein the alteration in step (c) to each core binder sequence of the first population of peptide binders comprises the single amino acid substitution and the single amino acid substitution occurs at one of M 1 , M 2 , M 3 , M 4 , and M 5 using each of the other 18 natural amino acids; the single amino acid insertion and the single amino acid insertion occurs before or after one of M 1 , M 2 , M 3 , M 4 , and M 5 ; and/or the single amino acid deletion and the single amino acid deletion occurs at one of M 1 , M 2 , M 3 , M 4 , and M 5 to form 4-mer peptides.

7. The method of claim 6 , wherein the 4-mer peptides are further altered and the alteration is selected from a single amino acid substitution, a double amino acid substitution, an amino acid insertion.

8. The method of claim 1 , wherein the second peptide microarray comprises at least 1.0×10 6 peptides.

9. The method of claim 6 , wherein the second peptide microarray comprises at least 1.2×10 6 peptides.

10. The method of claim 9 , wherein the second peptide microarray comprises at least 1.8×10 6 peptides.

11. The method of claim 1 , wherein the first peptide microarray comprises at least 1.2×10 6 peptides.

12. The method of claim 11 , wherein the first peptide microarray comprises at least 1.8×10 6 peptides.

13. The method of claim 1 , wherein the second population of peptide binders comprise natural amino acids, non-natural amino acids, or combinations thereof.

14. The method of claim 1 , wherein the second population of peptide binders comprise linear peptides, cyclic peptides, constrained peptides, or combinations thereof.

15. The method of claim 1 further comprising:
(f) performing at least one of N-terminal and C-terminal extension of the mature core binding sequence determined in step (e), whereby a third population of peptide extended mature core binder sequences is generated; and
(g) exposing the protein target of interest to a third peptide microarray produced by maskless photolithography comprising the third population of peptide extended mature core binder sequences generated in step (f), whereby the protein target binds to at least one of the extended peptide mature core binder sequences of the third population of peptide extended mature core binder sequences; and
(h) identifying overlap in the N-terminal or C-terminal peptide binder sequences of the peptides comprising the population of extended mature core binders, whereby an extended, matured core binder sequence is determined.

16. The method of claim 15 , wherein the third peptide microarray comprises at least 1.0×10 6 peptides.

Assignees

• Roche Sequencing Solutions Inc

Inventors

• Patel Jigar
• Lyamichev Victor
• Albert Thomas

Classifications

• G01N33/57555

  of the prostate · CPC title

• C07K1/047 Primary

  Simultaneous synthesis of different peptide species; Peptide libraries · CPC title

• C40B30/04

  by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding · CPC title

• G01N33/6845

  Methods of identifying protein-protein interactions in protein mixtures · CPC title

• G01N2333/36

  from Actinomyces; from Streptomyces (G) · CPC title