(M1430-01-03) Achieving Drug Tolerance in a Multiplexed Neutralizing Antibody Assay (nAb) Using Competition and Heat Solid Phase Extraction (SPE) Conditions

Purpose: Drug interference in nAb assays is common and acid dissociation followed by SPE is typically needed to achieve drug tolerance (1–3). NGM707 is an antagonistic humanized monoclonal antibody (mAb) that binds to two targets, immunoglobulin-like transcript 2 (ILT2) and 4 (ILT4), which has been developed for solid tumor indications. Drug tolerance of 100 µg/mL is required for the current nAb assay, and interference was observed during assay development. However, standard SPE approaches using acid dissociation, as well as dissociation tests with pH, salt, or detergent failed to dissociate NGM707 from the nAb positive control (PC) (2, 4, 5). Therefore, we hypothesized a competitive SPE procedure in combination with additional treatments would be necessary to dissociate NGM707 for removal prior to nAb assessment. As NGM707 binds and inhibits multiple targets, a multiplexed ligand binding assay (LBA) approach was selected for nAb assay development (6).

Methods: A flow cytometry assay was used to test competition SPE dissociation conditions using SPE reagents including streptavidin (SA) magnetic beads loaded with biotin-drug and nAb PC. For competition optimization studies the nAb PC was labeled with digoxygenin (DIG). Loaded SA magnetic beads were treated with multiple dissociation conditions with 1- to 100-fold excess of competing antibody for various times. Subsequently, beads were stained with an anti-DIG antibody followed by fluorescently labeled species-specific secondary antibodies prior to flow cytometry analysis. The optimized solid phase extraction method was performed by loading streptavidin (SA) magnetic beads with a biotinylated anti-idiotypic antibody (α-ID) to remove NGM707 from samples. Following SPE, supernatant containing the PC was measured in MSD U-Plex assay plate coated with biotin-ILT2 or ILT4. Samples were then analyzed for nAB by incubating at 48°C and ruthenylated NGM707 was used for detection.

Results: Flow cytometry results demonstrated maximal dissociation of NGM707 from the nAb PC was achieved with 100-fold excess competing antibody and heating to 48°C. In addition, sample dilution to the assay minimum required dilution (MRD) prior to competition and treatment for at least 18 hours were necessary. Following translation of the optimized conditions to an SPE method preliminary cut point factors of 1.06 for ILT2 and 1.11 for ILT4 were determined for nAb assay optimization. Further nAb assay optimization demonstrated that blocking for 30 minutes, labeling challenge ratios of 5:1 for biotinylated ILT2 and ILT4, and sample incubation at 48°C for 2.5 hours were necessary for optimal assay performance. Sensitivity was shown to be < 100 ng/mL for both ILT2 and ILT4. The assay demonstrated acceptable accuracy and precision achieving > 80% of PC spiked individual samples at ± 30% relative error, and < 20% CV, after the competitive SPE procedure. Finally, > 100 µg/mL drug tolerance at 300 ng/mL PC was achieved for both ILT2 and ILT4 when the competitive SPE method with heat was used. Conversely, without SPE, drug tolerance values of 0.28 µg/mL for ILT2 and 0.12 µg/mL for ILT4 at 300 ng/mL PC were observed.

Conclusion: A multiplexed LBA nAb assay utilizing competition and heat SPE conditions to achieve sufficient drug tolerance was developed for NGM707, despite the inability of acid to dissociate NGM707 from nAbs. Optimal dissociation conditions identified by flow cytometry screening were translatable to the SPE method, providing an approach for future molecules where standard dissociation methods are ineffective.

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