Method for the detection of free binding partner of a multispecific binder

Patent metadata

Field	Value
Publication number	US-9671394-B2
Application number	US-201414308539-A
Country	US
Kind code	B2
Filing date	Jun 18, 2014
Priority date	Dec 19, 2011
Publication date	Jun 6, 2017
Grant date	Jun 6, 2017

Abstract

Herein is reported a method for the detection of free antigen of a multispecific antibody in a sample, whereby the antigen to be detected can be specifically bound by a first binding site of the multispecific antibody, comprising the step of incubating a sample comprising free antigen and multispecific antibody with an anti-idiotypic antibody that specifically binds to a second binding site of the multispecific antibody, which is different from the first binding site, and thereby depleting the multispecific antibody from the sample.

First claim

The invention claimed is:

1. An in vitro method for the determination of the presence and/or amount of an antigen of a multispecific antibody in a sample, whereby the antigen to be detected can be specifically bound by a first binding specificity of the multispecific antibody, comprising the steps of:
incubating the sample comprising the multispecific antibody, multispecific antibody bound antigen and free antigen with an anti-idiotypic antibody that specifically binds to a second binding specificity of the multispecific antibody, which is different from the first binding specificity,
depleting the anti-idiotypic antibody-multispecific antibody-complex and anti-idiotypic antibody-multispecific antibody-antigen complex from the sample prior to the determination of the presence and/or the amount of free antigen, and
determining the presence and/or the amount of free antigen in the multispecific antibody-depleted sample.

2. The method according to claim 1 , wherein the binding specificity is a binding site or a pair of an antibody heavy chain variable domain and an antibody light chain variable domain.

3. The method according to claim 1 , wherein the determining the presence and/or the amount of the antigen comprises the following steps:
incubating the multispecific antibody-depleted sample with a capture antibody that specifically binds to the antigen to form a capture antibody-antigen complex, and
correlating the formed capture antibody-antigen complex to the amount of the antigen in the sample.

4. The method according to claim 1 , wherein the determining of the amount of the antigen comprises the following steps:
incubating the multispecific antibody-depleted sample with a capture antibody that specifically binds to the antigen to form a capture antibody-antigen complex,
incubating the capture antibody-antigen complex with a tracer antibody, whereby the capture antibody and the tracer antibody bind to non-overlapping epitope on the antigen, and
correlating the formed capture antibody-antigen-tracer antibody complex to the amount of the antigen in the sample.

5. The method according to claim 1 , wherein the determining of the amount of the antigen comprises the following steps:
incubating the multispecific antibody-depleted sample with a capture antibody that specifically binds to the antigen to form a capture antibody-antigen complex,
incubating the capture antibody-antigen complex with a tracer antibody, whereby the capture antibody and the tracer antibody bind to non-overlapping epitope on the antigen,
incubating the capture antibody-antigen-tracer antibody complex with a detection antibody comprising a detectable label, whereby the detection antibody specifically binds to the tracer antibody at an epitope outside the variable domains of the tracer antibody, and
correlating the formed capture antibody-antigen-tracer antibody complex to the amount of the antigen in the sample.

6. The method according to claim 1 , wherein the multispecific antibody is a bispecific antibody, or a trispecific antibody, or a tetraspecific antibody, or a pentaspecific antibody, or a hexaspecific antibody.

7. The method according to claim 1 , wherein the multispecific antibody is a bispecific antibody.

8. The method according to claim 1 , wherein the multispecific antibody is a bispecific antibody that has a first binding specificity that specifically binds to a first antigen or first epitope on an antigen and that has a second binding specificity that specifically binds to a second antigen or to a second epitope on the antigen.

9. The method according to claim 1 , wherein the method comprises the following steps:
incubating the sample comprising antigen and multispecific antibody with an anti-idiotypic antibody that specifically binds to a second binding specificity of the multispecific antibody which is different from the first binding specificity, to form an anti-idiotypic antibody-multispecific antibody complex,
removing the anti-idiotypic antibody-multispecific antibody complex from the sample, and
determining the amount of the antigen in the multispecific-antibody depleted sample.

10. The method according to claim 1 , wherein the anti-idiotypic antibody has an association constant k a of 10 5 l/mol/s or more to the second binding specificity of the multispecific antibody.

11. The method according to claim 1 , wherein the anti-idiotypic antibody has a K D value of 5*10 −8 mol/l or less for the binding to the second binding specificity of the multispecific antibody.

12. The method according to claim 1 , wherein the incubating with the anti-idiotypic antibody is for about 10 minutes to about 36 hours.

13. The method according to claim 1 , wherein the sample is adjusted to a multispecific antibody concentration of about 2 μg/ml to about 15 μg/ml.

14. The method according to claim 1 , wherein the sample is adjusted to a total antigen concentration of from about 1 ng/ml to about 250 ng/ml.

15. The method according to claim 1 , wherein the anti-idiotypic antibody is bound or conjugated to a solid phase.

16. The method according to claim 1 , wherein the anti-idiotypic antibody is conjugated (immobilized) via a specific binding pair.

17. The method according to claim 16 , wherein the binding pair (first component/second component) is selected from streptavidin or avidin/biotin, antibody/antigen, lectin/polysaccharide, steroid/steroid binding protein, hormone/hormone receptor, enzyme/substrate, IgG/Protein A and/or G.

18. The method according to claim 16 , wherein the anti-idiotypic antibody is conjugated to biotin and immobilization is performed via immobilized avidin or streptavidin.

19. The method according to claim 1 , wherein the anti-idiotypic antibody is biotinylated and the solid phase is streptavidin coated.

20. The method according to claim 1 , wherein the anti-idiotypic antibody is a biotinylated anti-idiotypic antibody against the multispecific antibody and is conjugated to a solid phase via streptavidin.

21. The method according to claim 1 , wherein the solid phase is a streptavidin coated paramagnetic bead or a streptavidin coated sepharose bead.

22. The method according to claim 1 , wherein the anti-idiotypic antibody is a mixture comprising at least two anti-idiotypic antibodies that differ in the antibody site at which they are conjugated to the solid phase.

23. The method according to claim 1 , wherein the anti-idiotypic antibody mixture comprises the anti-idiotypic antibody conjugated via at least two different amino groups to the solid phase.

24. The method according to claim 1 , wherein the conjugation of the anti-idiotypic antibody to its conjugation partner is performed by chemically binding via N-terminal and/or ε-amino groups (lysine), and/or ε-amino groups of different lysines, carboxy-, sulfhydryl-, hydroxyl- and/or phenolic functional groups of the amino acid backbone of the drug antibody, and/or sugar alcohol groups of the carbohydrate structure of the drug antibody.

25. The method according to claim 15 , wherein the anti-idiotypic antibody is conjugated to the solid phase by passive adsorption.

26. The method according to claim 1 , wherein the sample comprises multispecific antibody, free antigen and multispecific antibody-antigen complexes and the detection is of free antigen of the multi specific antibody.

Assignees

• Hoffmann La Roche

Inventors

• Stubenrauch Kay-Gunnar
• Wessels Uwe

Classifications

• G01N33/53 Primary

  Immunoassay; Biospecific binding assay; Materials therefor · CPC title

• G01N33/54306 Primary

  Solid-phase reaction mechanisms · CPC title

• G01N33/5306

  Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding · CPC title

• G01N33/54393

  Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding · CPC title

• G01N33/543 Primary

  with an insoluble carrier for immobilising immunochemicals · CPC title