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Discovery and Basic Research

(M1530-06-36) Platform Approach for Identifying the Most Functional and Developable Bispecific T Cell Engager

Monday, October 21, 2024
3:30 PM – 4:30 PM MT
Purpose: The purpose of this study was to develop a platform to identify the most functional and developable bispecific T cell engager. The screening strategy places a strong focus on intelligent bispecific design which considers avidity, spacing and formatting of Fab and scFv arms. Coupled with our in-house analytics and bioassay suites, we were able to express and rapidly test all designs to determine the optimal bispecific T cell engager.  

Methods: The identification of a functional T cell engager focuses on the combination of anti-CD19 and anti-CD3. Designs focused on the formatting of both arms as either a Fab or a scFv as initially it was unclear which format would be stable.  To identify the best configuration for functionality, several parameters were also investigated. Different arm ratios were trialed including a 2:2, 1:2, 2:1 and 1:1 format. Both symmetrical and non-symmetrical designs were also explored using Knob into hole technology. Lastly, the spatial distance between the two arms was evaluated to determine if this had an impact on activity (Figure 1). All designs were transiently expressed using ExpiCHOTM cells using the ExpifectamineTM CHO transfection method. Various heavy and light chain ratios were tested during transfection. Each design was purified by protein A resin, and the monomericity was determined via ASEC-HPLC. Purified designs were assessed for thermal stability using the UNcleTM system. Each design was screened for their ability to activate T cells using a bioluminescent assay containing an engineered Jurkat T cell that expresses a luciferase reporter driven by an NFAT-response element.

Results: A board range of expression levels were seen across designs as well as monomericity post protein A purification (Figure 2), a critical first step for judging design success. Interrogation of the designs using a T cell activation bioassay showed a clear trend, all CD19 binders formatted to a scFV showed limited activation of T cells and were subsequently discarded from the panel. Of the designs that did show binding, one design showed the highest activity and was significantly higher than the others (Figure 3). This breadth in outcomes across the designs highlights the need to screen early to both maximize function and ensure developability.

Conclusion: Using our platform approach we demonstrated an integrated expression and analysis system that allows for the focused screening of bispecific designs to quickly identify the best format as early as possible in the development process.

References: Chen, W. et al., 2020. One size does not fit all: navigating the multi-dimensional space to optimize T-cell engaging protein therapeutics. MABS VOL. 13 NO.1

Figure 1. Schematic of all designs screened in this study (Purple = anti CD19, red = anti CD3)

Figure 2. Monomericity of each design after protein A affinity purification. Graph shows the relative abundance of each form of the antibody broken down into higher molecular weight species (HMWS), antibody monomer (Monomer) and lower molecular weight species (LMWS).

Figure 3. T cell activation bioassay responses. (Left) designs that show activation, (right) designs that show limited or no activation. Blincyto was used as a positive control in each case.