(W1530-02-09) Development of Non-Cell Based Potency Assays for Bispecific Antibodies

Purpose: In recent years, the development of Bispecific Antibodies (BsAb) has become an increasingly explored approach in cancer immunotherapy. Unlike traditional chemotherapeutics which indiscriminately kill all rapidly dividing cells, BsAb immunotherapy is a targeted approach to cancer treatment. Since 2014, nine BsAbs have been approved by the FDA, including 2 approvals in 2023 for the treatment of B-cell lymphoma. Potency assays are essential in the development of most biologics, including BsAbs. During the development of BsAbs, potency assays measuring the ability of the BsAbs to bind to their intended targets are critical. However, the development of potency assays for BsAbs is often complicated by the necessity to effectively assess interactions between a BsAb and both target antigens. In this study, two potency assays were developed for a Phase I BsAb, henceforth referred to as Antibody X. Antibody X was designed to bridge effector T-cells and target tumor cells to promote T-cell activation and the subsequent lysis of tumor cells. Antibody X contains antigen binding sites targeting a cell surface protein X (CSPX) on T-cells, and a cell surface CSPY (CSPY) on certain tumor cells (Figure 1). CSPX plays a vital role in T-cell activation while CSPY is a tumor-associated antigen expressed in various solid tumor types. To align with the mechanism of action (MOA) of Antibody X, two potency assays were developed to measure the binding of Antibody X to each of the target antigens independently.

Methods: Capture protein, either CSPX or CSPY, was immobilized on standard Meso Scale Discovery (MSD) multi-array plates. Serial dilutions of Antibody X (analyte) and a negative control were added to the plates. Ruthenylated secondary antibody was bound to Antibody X and used to generate an electrochemiluminescence response directly proportional to the amount of analyte detected in each well. The ECL units were measured on an MSD plate reader (MESO Sector S 600). Mean, R², %CV, and Relative Potency (REP) were calculated using a 4-parameter nonlinear logistic (4PL) curve using Gen5 Secure, version 3.02 (Agilent Technologies).

Results: Two potency assays were developed for Antibody X: a CSPX-based binding assay and a CSPY-based binding assay. During the development of these potency assays, capture protein concentration and ratio of capture protein to primary antibody were explored and optimized. In order to achieve optimal 4PL curves with sufficient data points in the upper and lower asymptotes for each potency assay, the Antibody X dose concentration ranges were optimized independently. For the CSPX-based potency assay, 10,000ng/mL was determined to be optimal for the highest concentration of the reference standards, and 1.6-fold serial dilution was used to generate an 11-point reference curve. For the Protein X-based potency assay, 4,000ng/mL was determined to be optimal for the highest concentration of the reference standards, and 1.5-fold serial dilution was used to generate a 9-point reference curve. For both potency assays, relative potency at 50%, 100% and 150% nominal drug concentrations (NDCs) for the test samples was measured. Representative 4PL curves for the CSPX-based potency assay with test samples at 50%, 100% and 150% NDC are shown in Figure 2. Following assay development, validation was initiated for both the CSPX- and CSPY-based potency assays and facilitated the continuation of product development. Test sample linearity during assay validation of the CSPX-based potency assay is shown in Figure 3.

Conclusion: Potency assays are a critical component during the development of many BsAbs with binding interactions representing a necessary component essential to their MOA. The development of potency assays for BsAbs has unique challenges, including the requirement of successful interactions between the BsAb and both target antigens. Here, the development of two potency assays to measure Antibody X binding CSPX and CSPY are described. The use of two independent binding assays for potency determination represents a valuable approach to gaining essential insights into antigen binding of BsAbs during the early phases of drug development.

References: 1. Food and Drug Administration. (2023). Bispecific Antibodies: An Area of Research and Clinical Applications.
2. FDA-2019-D-0621. Bispecific Antibody Development Programs Guidance for Industry. May 2021, Pharmaceutical Quality/CMC.
3. FDA-2023-D-4299. Potency Assurance for Cellular and Gene Therapy Products. December 2023.

Figure 1. Antibody X bridges T-cells and tumor cells through interactions with CSPX and CSPY.

Figure 2. Dose-response curves at 50%, 100% and 150% NDC

Figure 3. Sample linearity for CSPX-based potency assay