# US 2016/0252520 A1 · Competitive ligand binding assay for detecting neutralizing antibodies

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- Number: US 2016/0252520 A1
- Document ID: US20160252520A1
- Status: Published application
- Kind: A1
- Application number: 15/031,126
- Filed: 2014-10-30
- Published: 2016-09-01
- Earliest claimed: 2013-10-31
- Assignees: Regeneron Pharmaceuticals, Inc.
- Applicants: Regeneron Pharmaceuticals, Inc.
- Inventors: Manoj RAJADHYAKSHA; Gary SHYU; Camille GEORGAROS; Michael PARTRIDGE; Albert TORRI
- Web page: https://patentgrep.com/patent/US20160252520A1

## CPC

- G01N 33/6854: {Immunoglobulins}
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 13/08: of the prostate
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 35/00: Antineoplastic agents
- A61P 37/02: Immunomodulators
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- G01N 33/5306: {Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding}
- G01N 2333/705: Assays involving receptors, cell surface antigens or cell surface determinants
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## 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.

## Claims

### Claim 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) combining a patient sample with a capture reagent, and (b) adding a detecting reagent, wherein a decreased signal relative to a control sample indicates the presence of a neutralizing antibody to the biotherapeutic agent.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1, 2)

The method of claim 1 or 2, wherein the protein biotherapeutic is an anti-interleukin-6 receptor α (IL-6Rα) monoclonal antibody.

### Claim 4 (depends on 1, 2, 3)

The method of any one of claim 1-3, wherein the protein biotherapeutic is sarilumab or tocilizumab.

### Claim 5 (depends on 4)

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

### Claim 6 (depends on 5)

The method of claim 5, wherein the patient is treated for an IL-6-dependent disease.

### Claim 7 (depends on 6)

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

### Claim 8 (depends on 7)

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

### Claim 9

The method of any one of the above claims, wherein the capture reagent comprises the labeled biotherapeutic protein.

### Claim 10

The method of any one of the above claims, wherein the capture reagent is labeled sarilumab.

### Claim 11 (depends on 10)

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

### Claim 12 (depends on 11)

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

### Claim 13

The method of any of the above claims, wherein the detection reagent is labeled soluble IL-6 receptor α.

### Claim 14 (depends on 13)

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

### Claim 15

The method of any of the above claims wherein the patient sample is subjected to treatment with low pH prior to step (b).

### Claim 16 (depends on 15)

The method of claim 15, wherein the patient sample is subjected to treatment with acetic acid prior to step (b).

### Claim 17 (depends on 15, 16)

The method of claim 15 or 16, wherein the treatment with low pH is followed by neutralization.

### Claim 18

The method of any of the above claims, wherein the assay 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.

### Claim 19

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) combining a patient serum sample with a capture reagent comprising biotinylated sarilumab, (b) subjecting the combined serum sample and capture reagent to treatment with low pH, followed by a neutralizing step; 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 the biotherapeutic agent.

### Claim 20 (depends on 19)

The method of claim 19, wherein the detection reagent is ruthenium labeled soluble IL-6 receptor α.

### Claim 21 (depends on 19, 20)

The method of claims 19-20, wherein the patient is suffering from rheumatoid arthritis.

### Claim 22 (depends on 19, 20, 21)

The method of any of claims 19-21, wherein the assay 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.

### Claim 23

A kit, comprising the capture and detection reagents of any of the above claims and instructions for use.