Method for generating an affinity reagent library

Patent metadata

Field	Value
Publication number	US-10300451-B2
Application number	US-201414899880-A
Country	US
Kind code	B2
Filing date	Jun 19, 2014
Priority date	Jun 20, 2013
Publication date	May 28, 2019
Grant date	May 28, 2019

Abstract

The present disclosure provides a method for generating an affinity reagent library against a target protein which interacts with a ligand, which comprises the following steps; •i) determining one or more structural element(s) of the ligand which are involved in ligand: target protein interaction; •ii) producing a library of peptides which retain these structural element(s); and •iii) grafting each peptide from the library of peptides into a portion of the affinity reagent molecule such that it may interact with the target protein, in order to produce an affinity reagent library.

First claim

The invention claimed is:

1. A method for generating an affinity reagent library against a target protein which interacts with a ligand, wherein the target protein is αVβ6, and wherein the method comprises:
i) generating a library of peptides which comprise:
a hairpin which contains, at its turn, an RGD motif;
an α-helix or a 3 10 -helix located C-terminal to the hairpin; and
a LQEKGI (SEQ. ID NO: 34)C-terminal capping motif; and

ii) grafting each peptide from the library of peptides into a portion of the affinity reagent molecule such that it may interact with the target protein, in order to produce an affinity reagent library.

2. A method according to claim 1 , wherein the affinity reagent is an antibody or a functional fragment thereof.

3. A method according to claim 2 , wherein in step (ii) the peptide is grafted in the position of a complementarity determining region (CDR) to produce an antibody library.

4. A method according to claim 3 , wherein the CDR is CDR3.

5. A method according to claim 1 , wherein the affinity reagent is an antibody mimetic.

6. A method according to claim 5 , wherein the antibody mimetic is an Affibody, a DARPin, an Anticalin, an Avimer, a Versabody or a Duocalin.

7. A method according to claim 1 , wherein the peptide further comprises an EP N-terminal capping motif.

8. A method according to claim 1 , wherein the peptide comprises the sequence:

(SEQ ID NO: 1)

E 1 P 2 R 3 G 4 D 5 L 6 X 7 X 8 L 9 A 10 A 11 R 12 Z 13 K 14 R 15 Z 16 F 17 N 18 E 19 Z 20 L 21 A 22 Z 23

L 24 Q 25 E 26 K 27 G 28 I 29

wherein X and Z are random amino acid residues.

9. A method according to claim 1 , wherein the peptide comprises the sequence:

(SEQ ID NO: 2)

E 1 P 2 R 3 G 4 D 5 L 6 X 7 X 8 L 9 A 10 A 11 Z 12 L 13 K 14 Z 15 E 16 F 17 Z 18 E 19 N 20 Z 21 L 22 A 23

Z 24 L 25 Q 26 E 27 K 28 G 29 I 30

wherein X and Z are random amino acid residues.

10. A method for selecting an affinity reagent capable of binding to an αVβ6 protein, which comprises the step of making an affinity reagent library against the αVβ6 protein by a method according to claim 1 and screening the library for binding to the αVβ6 protein.

11. A peptide comprising the amino acid sequence shown as SEQ ID NO: 23 or SEQ ID NO: 31.

12. An αVβ6 antibody comprising a V H -CDR3 sequence comprising a peptide according to claim 11 .

13. A method of diagnosing or treating a disease comprising administering to a subject an antibody according to claim 12 .

14. A method according to claim 13 , wherein the disease is cancer.

15. An antibody according to claim 12 , wherein the antibody is conjugated to a detectable and/or therapeutic agent.

16. A method according to claim 13 , wherein the antibody is conjugated to a detectable and/or therapeutic agent.

17. A method according to claim 1 , wherein the affinity reagent is a single chain fragment variable (scFv).

Assignees

• Univ London Queen Mary
• Univ Of Kent

Inventors

• Nissim Ahuva
• Marshall John
• Howard Mark

Classifications

• C07K16/2839

  against the integrin superfamily · CPC title

• C07K2317/565

  Complementarity determining region [CDR] · CPC title

• B01J2219/00725

  Peptides · CPC title

• C07K16/18

  against material from animals or humans · CPC title

• B01J19/0046 Primary

  Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays (synthesis methods per se C40B50/00) · CPC title