Direct affinity measurement of human IgG1 binding multimeric antigens

Patent metadata

Field	Value
Publication number	US-12287335-B2
Application number	US-202017062279-A
Country	US
Kind code	B2
Filing date	Oct 2, 2020
Priority date	Dec 9, 2015
Publication date	Apr 29, 2025
Grant date	Apr 29, 2025

Abstract

Herein is reported a method for determining the binding affinity of the binding sites of a bivalent full length antibody of the human IgG1 subclass to a homo-multimeric antigen comprising the steps of i) incubating a mixture comprising the antibody and a polypeptide that is derived from lysine-gingipain of Porphyromonas gingivalis at a pH of from pH 7.5 to pH 8.5, in the presence of a reducing agent, at a temperature of from 30° C. to 42° C., for time of from 10 min. to 240 min. to cleave the antibody into Fabs and Fc-region, and ii) determining the binding affinity of the Fabs of the antibody for its antigen using a surface plasmon resonance method by directly applying the incubated reaction mixture obtained in the previous step in the surface plasmon resonance method and therewith determining the binding affinity of the binding sites of the bivalent full length antibody of the human IgG1 subclass.

First claim

What is claimed is:

1. A method for determining the binding affinity of the binding sites of a bivalent full length antibody of the human immunoglobulin G type 1 (IgG1) isotype to a homo-multimeric antigen comprising the following steps:
incubating a mixture comprising the antibody and lysine-gingipain of Porphyromonas gingivalis at a pH of 7.5 to 8.5, in the presence of a reducing agent, at a temperature of 30° C. to 42° C., for a time of 10 minutes to 240 minutes to cleave the antibody into antigen-binding fragments (Fabs) and C-terminal fragment, crystallizing fragment-(Fc)-region, and
determining the binding affinity of the Fabs of the antibody for its antigen using surface plasmon resonance by directly applying the incubated reaction mixture obtained in the previous step in the surface plasmon resonance method,
thereby determining the binding affinity of the binding sites of the bivalent full length antibody of the human IgG1 isotype.

2. The method according to claim 1 , wherein the lysine-gingipain of Porphyromonas gingivalis has the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

3. The method according to claim 1 , wherein the lysine-gingipain of Porphyromonas gingivalis has an amino acid sequence that comprises at least residues 230 to 739 of SEQ ID NO: 1.

4. The method according to claim 1 , wherein the reducing agent is selected from the group consisting of 2-mercaptoethanol, cysteine, and dithiothreitol.

5. The method according to claim 4 , wherein the reducing agent is cysteine.

6. The method according to claim 1 , wherein the reducing agent is cysteine at a concentration of 0.5 mM to 10 mM.

7. The method according to claim 1 , wherein the pH value is about pH 8.

8. The method according to claim 1 , wherein the incubating is at a temperature of 35° C. to 38° C.

9. The method according to claim 1 , wherein the incubating is for a time of about 60 minutes.

10. The method according to claim 1 , wherein the antibody comprises in the Fc-region the mutations P329G, L234A and L235A in both heavy chain polypeptides.

11. A method for selecting an antibody specifically binding to a homo-multimeric antigen comprising the following steps:
providing a plurality of bivalent full length antibodies of the human IgG1 isotype binding to the same homo-multimeric antigen,
determining the binding affinity of each of the antibodies of the plurality of antibodies to its antigen with a method according to claim 1 , and
selecting one or more antibodies based on the binding affinity determined in the previous step.

12. The method according to claim 1 , wherein the incubated mixture is used for the determination of the binding affinity without intermediate purification.

13. The method according to claim 2 , wherein the antibody comprises in the Fc-region the mutations P329G, L234A and L235A in both heavy chain polypeptides.

14. The method according to claim 13 , wherein the incubated mixture is used for the determination of the binding affinity without intermediate purification.

15. The method according to claim 14 , wherein the reducing agent is selected from the group consisting of 2-mercaptoethanol, cysteine, and dithiothreitol.

16. The method according to claim 15 , wherein the reducing agent is cysteine.

17. The method according to claim 15 , wherein the reducing agent is cysteine at a concentration of 0.5 mM to 10 mM.

18. The method according to claim 15 , wherein the pH value is about pH 8.

19. The method according to claim 18 , wherein the incubating is at a temperature of 35° C. to 38° C.

20. The method according to claim 19 , wherein the incubating is for a time of about 60 minutes.

21. The method of claim 1 , wherein the homomultimeric antigen is vascular endothelial growth factor-A (VEGF-A), carcinoembryonic antigen (CEA), angiopoietin-2 (ANG2), or fibroblast activation protein (FAP).

22. The method of claim 11 , wherein the homomultimeric antigen is vascular endothelial growth factor-A (VEGF-A), carcinoembryonic antigen (CEA), angiopoietin-2 (ANG2), or fibroblast activation protein (FAP).

Assignees

• Hoffmann La Roche

Inventors

• Molhoj Michael
• Gassner Christian
• Moelleken Joerg
• Endesfelder Manuel

Classifications

• G01N33/56911

  Bacteria · CPC title

• G01N33/566

  using specific carrier or receptor proteins as ligand binding reagents {where possible specific carrier or receptor proteins are classified with their target compounds} · CPC title

• G01N33/5375

  by changing the physical or chemical properties of the medium or immunochemicals, e.g. temperature, density, pH, partitioning · CPC title

• G01N21/553

  and using surface plasmons (fluorescence excitation G01N21/648; enhanced Raman G01N21/658) · CPC title

• C07K2317/92

  Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value · CPC title