(W1130-02-11) Having Appropriate Critical Reagents Can Make Bi-Specific Drug Anti-Drug Antibody Assays Less Challenging and Easy to Specifically Quantify

Purpose: Here we describe the development of an Anti-Drug Antibody (ADA) Assay to measure ADA against a Bi-Specific Drug (Drug-1). Bispecific antibodies (BsAbs) are antibodies with two binding sites directed at two different antigens or two different epitopes on the same antigen. Drug-1 is a BsAb which has two arms that recognize completely different cytokine targets. The variable region Arms of Drug-1 will be referred to as Arm 1 & Arm 2 (Figure 1). Drug-2 is a monoclonal antibody (mAb) that has the same target as Arm 1 of Drug-1. Drug-3 is a mAb that has the same target as Arm 2 of Drug-1. Therefore, Drug-1 is a Bi-Specific mAb that is a combination of Drug-2 and Drug-3 using one arm from each.

Methods: Screening ADA Assay: samples/PCs were diluted 1:10 in assay diluent, further diluted 1:2 and acid dissociated by adding 200 mM HCL, mixed on a plate shaker table at RT for 30 minutes, then diluted again 1:2 in a neutralizing Master Mix containing biotinylated and Ruthinylated Drug-1 and mixed by shaking at RT for 90 minutes. 50 µL of each sample was then transferred to a pre-washed MSD-Gold streptavidin plate, and mixed shaking at RT for 60 minutes. Plates were then washed, blotted, 2X MSD Read Buffer added and plates read on MSD Imager reader. Electro chemiluminescence (ECL) values were converted to relative ECL (rECL) by dividing the Sample/PC ECL by the Negative Control ECL. See Figure 1. Confirmation ADA assay: an excess of unlabeled Drug is introduced to the samples to out-compete the labeled Drug for the PC. Unlabeled Drug bound to PC does not bind to the plate, so is washed away, reducing ECL signal by up to 100% (100% inhibition). With the bi-specific Drug-1 or with a mixture of the two mono-specific Drugs, nearly 100% inhibition is achieved. When the mono-specific Drug-2 or Drug-3 are run in the confirmation assay alone, only about 50% inhibition is reached, as 100% of the free arm is still available for the biotinylated- and Ruthinylated-drug to bind.

Results: Table 1: Eleven Antibodies were tested (5 mAb and 1 pAb against Arm 1 of Drug-1, and 4 mAb and 1 pAb against the Arm 2 of Drug-1). Although one of the mAbs PCs looked very good, the two polyclonal antibody PCs both gave a high ECL signal that were very similar in strength at the same concentrations, so were chosen to move forward. Figure 1: depicts how the ADA assay works and how the signals may vary depending on the ADAs/PCs in the sample. Figure 2: shows that we can determine if positive ADAs are against Arm-1, Arm-2 or both Arms of Drug-1. Running samples in the standard screening assay and adding separate standard curves of each PC as well as the two combined will allow quantification of the ADAs against each arm of the BsAb. Adding the signals together from samples containing the two individual PCs (PC-1 & PC-2) is equal to the signal from the samples where the two PCs are combined and is consistent at all concentrations tested. Figure 3: shows that we can determine if confirmed positive ADAs are against either arm of the BsAb Drug-1 or both by measuring the percent inhibition. Figures 4 & 5: show that in a sample spiked with unequal amounts of PC-1 & PC-2 we can determine which arm/s of the BsAb Drug-1 the ADAs are attaching to by measuring the percent inhibition caused by adding excess unlabeled Drug-1, Drug-2 or Drug-3. Figure 6: Sensitivity curve of the combined PCs full range 0-5,000 ng/mL and low range 0-2.5 ng/mL to better see Cut-point. With a sample derived estimated Cut-point of 1.33 rECL, the Sensitivity of the assay was about 700 pg/mL. Figure 7A: shows that the Drug tolerance (tolerance for unlabeled Drug-1 in the sample) for the assay was greater than 1 mg/mL. Figure 7B: Target tolerance (the highest spiked concentration at which the NC has not crossed above the cut-point) was 3,125 pg/mL.

Conclusion: As Biologics, BsAb drugs require monitoring for ADAs in the serum of the subjects receiving them. Here we have developed an assay that can measure total ADAs and quantify the relative levels of ADAs directed at each arm of the BsAb Drug-1 in both the Screening and Confirmation assays. Having the appropriate critical reagents like the two PCs against the specific arms of Drug-1 allow quantification of PCs or ADAs against each arm of Drug-1 as well as Total PC/ADA in the Screening Assay. Having the appropriate critical reagents like Drug-2 & Drug-3 in addition to Drug-1 allow quantification in the Confirmation Assay by measuring the percent inhibition of each arm of Drug-1 compared to total inhibition which is achieved by an excess of unlabeled Drug-1, or an excess of a combination of unlabeled Drug-2 & Drug 3.

References: Ma, J., Mo, Y., Tang, M., Shen, J., Qi, Y., Zhao, W., Huang, Y., Xu, Y. and Qian, C., 2021. Bispecific antibodies: from research to clinical application. Frontiers in immunology, 12, p.626616.
Kontermann, R.E. and Brinkmann, U., 2015. Bispecific antibodies. Drug discovery today, 20(7), pp.838-847.
Fan, G., Wang, Z., Hao, M. and Li, J., 2015. Bispecific antibodies and their applications. Journal of hematology & oncology, 8, pp.1-14.

Acknowledgements: Disclosures: All authors are employees of GSK PLC. Some authors are owners of GSK PLC shares. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was used in this manuscript's production.

Table 1, Figure 1, Figure 2

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Figure 6, Figure 7