Background T-cell activation requires appropriate strength of stimulation and costimulation. Blocking coinhibitory pathways prevents T-cell exhaustion and escape. Here, we present the discovery of diverse panels of lead-quality antibodies, including antibodies against CD3 and PD-L1, ready for reformatting into advanced modalities for the development of finely tuned multi-drug approaches against a variety of tumor targets.
Methods AlivaMab Discovery Services (ADS) enabled the discovery of diverse CD3 (and PDL-1) antibody panels from immunized AlivaMab® Mice. These antibodies were characterized for binding properties and engineered into scFv. For direct comparison, each anti-CD3 scFv arm was combined with the same anti-TAA (tumor associated antigen) arm to create bispecific molecules with a silenced Fc using a standard knob-in-hole. scFvs in the context of bispecific molecules were tested for binding to CD3 in the context of T-Cell Receptor (TCR), and for functionality by Redirected T-Cell Cytotoxicity (RTCC) as well as cytokine production. PBMCs from four different donors were evaluated in functional assays.
Results We demonstrate that in bispecific contexts CD3 scFvs retain binding to CD3 in the context of TCR and have favorable biophysical properties. Moreover, bispecific molecules demonstrated different levels of in vitro potency as measured by both RTCC and cytokine production readouts. In addition, we were able to identify differential donor-dependent cytotoxicity and cytokine release. PBMC phenotyping from different donors revealed differences in percentages of T-cell populations and the activation status of T-cells. Importantly, we were able to identify bispecific antibodies, that demonstrated cytotoxic activity, but very low cytokine release across all tested donor samples.
Conclusions CD3 scFvs T-cell engagers that balance potency and cytokine release were identified. Our most recent findings provide evidence that the biological activity of CD3-T cell engagers correlates with the PBMC phenotypes highlighting the importance of functional validation of these molecules using PBMCs from multiple donors.
Acknowledgements Larry Green, Lippy Lippincott, Stacey Borders, Mohammad Abu Odeh, Soneela Ramesh, Crystal Bantados, Ankita Srivastava
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