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Improved competitive ligand binding assays

An antibody and specific technology, applied in the field of determination of anti-drug antibodies, can solve the problem of undetectable neutralizing antibody reaction

Pending Publication Date: 2021-12-10
REGENERON PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, some neutralizing antibody responses may not be detectable in NAb assays due to interference from drugs in the sample

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0074] Example 1: Competitive Ligand Binding NAb Assay: Format and Drug Resistance

[0075] Materials and methods

[0076] Materials and Reagents

[0077] All solutions were prepared in assay buffer (1% BSA in 1XPBS) unless otherwise stated. Read buffer T (4X) was from Meso Scale Discovery (MSD, Gaithersburg, MD). Glacial acetic acid (17.4M) was from Thermo Fisher Scientific (Waltham, MA). Human and monkey sera were from BioIVT (Westbury, NY). Fully human monoclonal antibody drug, fully human competitive anti-target A antibody, recombinant human target, monoclonal neutralizing anti-drug antibody used as a positive control, and anti-human monoclonal antibody were purchased from Regeneron (Barretta Village) , New York State) production. Antibodies and targets were labeled with biotin using EZ-link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific) and ruthenium NHS ester (MSD) according to the manufacturer's instructions. DyNAbeads TM Antibody conjugation kits were from The...

example II

[0097] Example II: Residue of bead-coupled proteins in the NAb assay step

[0098] Materials and methods

[0099] See Example 1.

[0100] result

[0101] Initial experiments used biotin-drug bound to streptavidin beads to extract ADA (and NAb) from samples. However, in the CLB NAb assay, low labeled drug concentrations are necessary to achieve maximum sensitivity (Hu, J. et al., Journal of Immunological Methods 419:1-8 (2015); Wu, B.W., et al., " A Competitive Ligand-Binding Assay for the Detection of Neutralizing Antibodies," Detection and Quantification of Antibodies and Biopharmaceuticals: Practical and Applied Considerations, Michael G. Tovey (ed.), John Wiley & Sons, Hoboken, NJ , USA (2011)). Therefore, any biotin-drug transferred from the enrichment step to the assay step may interfere. Figure 2A The data in showed that the biotinylated drug used in the NAb enrichment step would carry over to the NAb assay step, resulting in an approximately 5-fold increase in...

example III

[0103] Example III: Minimizing Interference from Drug-Trapping Proteins

[0104] Materials and methods

[0105] See Example 1.

[0106] result

[0107] Attempts to reduce the amount of coupled protein or increase wash steps did not adequately minimize interference from these proteins after their transfer to the NAb assay. To mitigate residual interferences, different methods were developed for each NAb assay.

[0108] When the target is used as a capture reagent in the drug removal step, the protein remains and inhibits the NAb assay signal ( Figure 2B , 2C and 3A). However, in a drug capture assay format, the addition of an anti-target antibody resolves the problem caused by protein carryover. In the presence of anti-target mAb, positive and negative control samples were subjected to a drug removal step, and target-conjugated beads produced assay signals that were nearly identical to control samples without a drug removal step ( Figure 3A ).

[0109] In target c...

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Abstract

Improved assays for the detection and optionally the quantification of anti-drug antibodies (ADAs) in a sample are provided. The disclosed assays include a protein drug capture assay format and a protein drug target capture assay format, each of which have certain advantages over existing assays. In some embodiments the assays are designed so that drug:anti-drug complexes are washed away before adding to the target coated plate. Target interference can potentially be eliminated or minimized with a competing target blocking reagent in the sample incubation step. In an exemplary target capture assay format, a mild acid approach is used to minimize free target interference.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 846,872, filed May 13, 2019, and U.S. Provisional Patent Application No. 62 / 859,914, filed June 11, 2019, both adopted in their entirety Incorporated herein by reference. technical field [0003] Aspects of the invention relate generally to assays for detecting drug interactions, in particular assays for detecting anti-drug antibodies in a sample. Background technique [0004] Administration of biotherapeutics to patients can induce undesired immunogenic responses in patients, which can lead to the development of anti-drug antibodies (ADA) (Mire-Sluis, A.R., et al., J Immunol Methods. "289(1):1-16(2004)). Neutralizing antibodies (NAbs) are a subset of ADAs that inhibit the binding of a drug to its target, rendering the drug biologically inactive. By definition, NAbs neutralize drug effects and may reduce clinical activity...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/543G01N33/533G01N33/50
CPCG01N33/94G01N33/6854G01N33/54306G01N33/533G01N33/54326G01N33/5047G01N2458/40G01N33/532
Inventor M·A·帕特里奇G·O·萨姆纳E·K·卡拉尤苏弗
Owner REGENERON PHARM INC
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