Background CD3 T-cell engagers (TCEs) represent a promising approach in cancer immunotherapy, yet challenges in efficacy and safety have hindered clinical development. To address these barriers, we developed a TCE platform comprised of novel CD3-binding antibodies, bispecific engineering technology, and a high-throughput process for identifying pairs of CD3- and tumor-binding antibodies with desired properties. By assessing hundreds of CD3-binding antibodies in bispecific formats, we identified rare CD3-binders that can be used to create TCEs that show decoupling of cell killing and cytokine release in vitro. In addition, we have generated antibodies targeting costimulatory receptors CD28 and 4-1BB. Here, we present strategies to combine optimized TCEs with costimulation, which has the potential to increase efficacy while mitigating cytokine release-associated toxicities.

Methods We generated 180 PSMA x CD3 TCEs and used high-throughput T-cell-dependent cellular cytotoxicity (TDCC) and cytokine release assays to identify molecules with desired functional profiles. To explore drivers of high potency/low cytokine release TCE phenotypes, we performed TDCC and cytokine release assays. Functional results were used to assess the impact of TCE design features, including CD3 binding affinity and kinetics, epitope recognition, and tumor-binding antibody properties. Using single B-cell screening, we identified additional TCE building blocks, including γδ-, CD28-, and 4-1BB-binding antibodies, with costimulatory potential assessed using T-cell activation assays.

Results In our studies, TCEs with high potency and low cytokine release are derived from three clonally-related groups of novel CD3-binding antibodies that do not compete with the commonly used CD3-binder, SP34-2, in epitope binning experiments. In contrast, TCEs derived from a large number of CD3-binders with a range of affinities and binding kinetics do not exhibit this property. These results suggest that CD3 binding epitope is a key contributor to high tumor-cell killing and low cytokine release, and that this property cannot be achieved by affinity-tuning alone. We further explore the potential to enhance TCE potency with costimulation strategies using CD28- and 4-1BB-binding antibodies.

Conclusions Building on previous work, we define parameters that influence TCE function and provide mechanistic insights into the high potency/low cytokine release profile. We also demonstrate potential strategies to enhance TCE activity for challenging targets and indications. These data provide important insights for the design of TCEs that enhance the potency, durability, and specificity of T-cell responses.