Abstract A020: Engineering developability of anti-CD3 scFv-based multispecific T-cell engagers
TL;DR
Stabilized anti-CD3 scFv not only improve properties of two clinical CD20 Fab-based multispecifics but rescue assembly of a third clinical CD20 molecule, which did not produce with the parental, unmodified scFv or with the industry standard engineered disulfide bond VH44-VK100.
Abstract
Multispecific T-cell engager antibody assembly requires solutions to avoid non-cognate chain-pairing. The single-chain fragment variable (scFv) format solves the LC pairing problem, however biophysical properties are usually significantly inferior to Fab-based molecules. Here we engineer an anti-CD3 antibody in the scFv format to significantly improve aggregation and stability, including low pH stress-induced aggregation resistance, and hydrophobicity. We describe the impact of design parameters, such as varying valency (e.g. 1 + 1 and 2 + 2), topology, and the sequence of the anti-tumor associated antigen Fv as fragment antigen-binding (Fab) format. We observe that stabilized anti-CD3 scFv not only improve properties of two clinical CD20 Fab-based multispecifics but rescue assembly of a third clinical CD20 molecule, which did not produce with the parental, unmodified scFv or with the industry standard engineered disulfide bond VH44-VK100. Lastly, we describe the ability of these stabilizing mutations to improve biophysical properties of scFv regardless of VH and VK germline, and CDR H3 and L3 sequence. Our findings are expected to be broadly applicable and to significantly improve outcomes of scFv-based multispecific production. Robert Pejchal, Kevin Schutz, Michael Battles, John Bukowski, Olivia Scheideler, Allie LeMay. Engineering developability of anti-CD3 scFv-based multispecific T-cell engagers [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr A020.