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Methods for detecting neutralizing antibodies to parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHRP) analog

An analog, antibody technology, applied in chemical instruments and methods, anti-hormone immunoglobulin, anti-animal/human immunoglobulin, etc., can solve the problems of sensitivity level, specificity level and long duration

Active Publication Date: 2021-08-20
RADIUS HEALTH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, current assays suffer from a number of disadvantages, including the level of sensitivity, level of specificity, and long duration of the assay

Method used

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  • Methods for detecting neutralizing antibodies to parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHRP) analog
  • Methods for detecting neutralizing antibodies to parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHRP) analog
  • Methods for detecting neutralizing antibodies to parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHRP) analog

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] A study was performed to validate a cell-based assay to detect neutralizing antibodies (NAbs) to PTH peptide in postmenopausal women. The assay involves treatment of the rat epithelial cell line UMR-106 in the presence of human serum that may or may not contain neutralizing antibodies (NAbs), followed by detection of PTH-induced cellular cyclic adenosine monophosphate (cAMP) by a competitive immunoassay ability. Detection of cAMP was performed using a competitive electrochemiluminescence assay, in which neutralizing antibodies to PTH resulted in decreased induction of cAMP by PTH and increased detection signal.

[0052] Material

[0053] Reagents used for PTH assay validation are shown in Tables 1 and 2 below.

[0054] Table 1

[0055]

[0056] *Please note that the passage number label on the WCB vial is incorrect, P4 should be read as "passage atthaw".

[0057] ** Concentration determined by quantitative ELISA

[0058] Table 2

[0059]

[0060]

[0061]...

Embodiment 2

[0107] Example 2. Verification and Calibration of Anti-PTHrP Detection Method

[0108] A study was performed to validate a cell-based assay to detect neutralizing antibodies (NAbs) to PTHrP peptides in postmenopausal women. The assay involves treatment of the rat epithelial cell line UMR-106 in the presence of human serum that may or may not contain neutralizing antibodies (NAbs), followed by a competitive immunoassay for the ability of PTHrP to induce cellular cyclic adenosine monophosphate (cAMP) . Detection of cAMP was performed using a competitive electrochemiluminescence assay, in which a neutralizing antibody to PTHrP resulted in a decrease in the induction of cAMP by PTHrP and an increase in the detection signal.

[0109] Material

[0110] Reagents used for PTH assay validation are shown in Tables 6 and 7 below.

[0111] Table 6

[0112]

[0113] *Please note that the passage number label on the WCB vial is incorrect, P4 should be read as "passage atthaw".

[...

Embodiment 3

[0150] Example 3. Determination of Anti-PTH and Anti-PTHrP Concentrations

[0151] Concentrations of anti-PTH or anti-PTHrP IgG were determined in two rabbit polyclonal antibody reagents.

[0152] Reagents and materials:

[0153] Greiner Bio-One high binding microplate 96-well (Greiner bio-one high bindingmicroplate 96-well), prod#655061, L / N E16093KS

[0154] • Anti-PTH (Ab) (1-34) (Human) - Phoenix Pharmaceuticals, cat#G-055-08, L / N 01553-4.

[0155] • Anti-PTHrP (Ab) (1-34) (Human, Rat, Mouse) - Phoenix Pharmaceuticals, cat#H-056-04, L / N 01736-1.

[0156] · PTH(1-34) (Human) Peptide - Phoenix Pharmaceuticals cat#055-03, L / N430926

[0157] PTHrP(1-34) (Human, Rat, Mouse) Peptide - Phoenix Pharmaceuticals cat#056-04, L / N432088

[0158] Rabbit IgG biotin-Rockland, cat#011-0602, L / N36734

[0159] ·Donkey anti-rabbit IgG(H+L)HRP, prod#711-035-152, L / N 125015

[0160] Washing buffer: 1X PBS+0.05% tween 20 (Fisher, L / N 160170) Covance internal buffer L / N 170320-1, Exp: 17S...

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Abstract

The present disclosure is directed to methods (e.g., in vitro methods) for detecting the presence of neutralizing antibodies to PTH or PTHrP analog in a sample. The in vitro method comprises the steps of obtaining a sample from a subject; contacting the sample with a cell; measuring cyclic adenosine monophosphate (cAMP) levels; and detecting the presence of neutralizing antibodies when cAMP levels are reduced relative to a negative control sample without neutralizing antibodies. An in vitro method of detecting the presence of neutralizing antibodies in a sample from a subject treated with Abaloparatide, is also provided. Further provided herein is a kit for carrying out the methods described herein comprising components required to carry out the obtaining, contacting, measuring and detecting steps and instructions for use.

Description

[0001] related application [0002] This application claims priority to U.S. Provisional Patent Application 62 / 791,267, filed January 11, 2019, the entire contents of which are expressly incorporated herein by reference. Background technique [0003] Despite these benefits, immunogenicity can still be produced by protein therapeutics such as abaloparatide. Abaloparatide is a parathyroid hormone-related peptide (PTHrP) (1-34) analog that acts as a PTH1 receptor (PTH1R) agonist. The activity of PTH1R activates the cyclic adenosine monophosphate (cAMP) signaling pathway in target cells, resulting in increased bone density and bone mineral content. (Abaloparatide) Injection Product Labeling (April 28, 2017). [0004] Among patients receiving abaloparatide (Abalopparide) for 18 months, 49% developed anti-Abaloparatide antibodies (anti-Abaloparatide antibodies), of which 68% developed abaloparatide neutralizing antibodies. Of those tested for cross-reactivity, 2.3% and 0% develo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/26G01N33/50
CPCC07K16/26C07K2317/76G01N33/5032G01N2333/635
Inventor H·K·钱德勒
Owner RADIUS HEALTH INC
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