Competitive ligand binding assay for detecting neutralizing antibodies

Patent metadata

Field	Value
Publication number	US-10261094-B2
Application number	US-201415031126-A
Country	US
Kind code	B2
Filing date	Oct 30, 2014
Priority date	Oct 31, 2013
Publication date	Apr 16, 2019
Grant date	Apr 16, 2019

Abstract

A immunogenicity assay for detecting the presence of neutralizing antibodies to a biotherapeutic protein wherein the biotherapeutic protein has been administered to a patient in need, comprising the steps of (a) obtaining a sample from the patient; (b) incubating the sample in the presence of a capture reagent; and (c) adding a detecting reagent, wherein a decreased signal relative to a control sample indicates the presence of a neutralizing antibody to the biotherapeutic agent.

First claim

What is claimed is:

1. A method for detecting the presence of neutralizing antibodies to a biotherapeutic protein, wherein the biotherapeutic protein has been administered to a patient in need, comprising the steps of (a) decreasing the pH of the patient sample to an acidic pH, (b) combining the patient sample with an antibody that binds to a target of the biotherapeutic protein and a capture reagent comprising a labeled biotherapeutic protein, wherein the label on the biotherapeutic protein facilitating binding of the biotherapeutic protein to a surface, and (c) adding a detecting reagent comprising a labeled target of the biotherapeutic protein, wherein a decreased signal relative to a control sample indicates the presence of a neutralizing antibody to the biotherapeutic protein.

2. The method of claim 1 , wherein the protein biotherapeutic is a monoclonal antibody.

3. The method of claim 2 , wherein the protein biotherapeutic is an anti-interleukin-6 receptor α (IL-6Rα) monoclonal antibody.

4. The method of claim 3 , wherein the protein biotherapeutic is sarilumab or tocilizumab.

5. The method of claim 4 , wherein the protein biotherapeutic is sarilumab.

6. The method of claim 1 , wherein the patient is treated for an IL-6-dependent disease.

7. The method of claim 6 , wherein the IL-6-dependent disease is one of rheumatoid arthritis, diabetes, atherosclerosis, Alzheimer's disease, systemic lupus erythematosus, multiple myeloma, connective tissue disorders, Castleman's disease and prostate cancer.

8. The method of claim 7 , wherein the IL-6-dependent disease is rheumatoid arthritis.

9. The method of claim 1 , wherein the capture reagent is labeled sarilumab.

10. The method of claim 9 , wherein the labeled sarilumab is biotinylated sarilumab.

11. The method of claim 10 , wherein the biotinylated sarilumab is bound to an avidin-coated plate.

12. The method of claim 1 , wherein the target is soluble IL-6 receptor α.

13. The method of claim 1 , wherein the labeled target is ruthenium labeled soluble IL-6 receptor α.

14. The method of claim 1 , wherein the pH of the patient sample is decreased to an acidic pH by treatment with an acidic solution.

15. The method of claim 1 , further comprising increasing the pH of the patient sample to a neutral pH following the decrease of the patient sample to an acidic pH.

16. The method of claim 1 , wherein the method exhibits a sensitivity tolerance of about 150 ng/mL, a drug tolerance of about 500 ng/mL, and a target interference tolerance of about 1 μg/mL.

17. A method for detecting the presence of neutralizing antibodies to sarilumab, wherein sarilumab has been administered to a patient in need, comprising the steps of (a) decreasing the pH of a serum sample to an acidic pH; (b) combining a patient serum sample with a capture reagent comprising biotinylated sarilumab; (c) increasing the pH of the combined serum sample and capture reagent to a neutral pH; and (d) adding a detecting reagent, wherein the detecting reagent comprises labeled soluble IL-6 receptor α, wherein a decreased signal relative to a control sample indicates the presence of a neutralizing antibody to sarilumab.

18. The method of claim 17 , wherein the detecting reagent is ruthenium labeled soluble IL-6 receptor α.

19. The method of claim 17 , wherein the patient is suffering from rheumatoid arthritis.

20. The method of claim 17 , wherein the method exhibits a sensitivity tolerance of about 150 ng/mL, a drug tolerance of about 500 ng/mL, and a target interference tolerance of about 1 μg/mL.

21. The method of claim 14 , wherein the acidic solution is acetic acid.

22. The method of claim 15 , wherein the pH is increased to a neutral pH by treatment with a basic solution.

23. The method of claim 22 , wherein the basic solution is a Tris base.

24. The method of claim 17 , wherein the pH is decreased to an acidic pH by treatment with an acid solution.

25. The method of claim 24 , wherein the acidic solution is acetic acid.

26. The method of claim 17 , wherein the pH is increased to a neutral pH by treatment with a basic solution.

27. The method of claim 26 , wherein the basic solution is a Tris base.

28. The method of claim 1 , wherein the label of the labeled biotherapeutic-protein is biotin.

29. The method of claim 1 , wherein the antibody binds free target of the biotherapeutic protein released by the low pH treatment and mitigates target interference.

Assignees

• Regeneron Pharma

Inventors

• Rajadhyaksha Manoj
• Partridge Michael
• Torri Albert

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61P37/02

  Immunomodulators · CPC title

• A61P9/10

  for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis · CPC title

• A61P3/10

  for hyperglycaemia, e.g. antidiabetics · CPC title

• A61P35/00

  Antineoplastic agents · CPC title