T cell engager (TCE) multispecifics can utilize scFv-based anti-CD3 arms to avoid light-chain mispairing, but scFvs typically have higher aggregation propensity at low pH. An engineered disulfide bond between VH44–Vκ100 has been shown to stabilize the format but typically compromises initial product quality. This poster describes how engineering a medium-affinity anti-CD3 scFv in Adimab's yeast platform led to the development of stabilizing mutation sets that performed well across a range of formats and developability assays.

Approach and outcomes 

• Thermal pressuring of anti-CD3 scFv ADI-26909 in Adimab's yeast platform yielded three sets of stabilizing mutations on a G93A backbone. ADI Set 1 (VH-L45P, VL-F98W) and ADI Set 3 (V37L, R44P, F98W, G100P) were characterized across a range of formats and tumor-associated antigen (TAA) pairings. Comparative analysis of these stabilizing mutations across developability assays in the 1+1 scFv-Fc + Fab-Fc format showed improved monomer percentage, low-pH stability, and maintained HIC and CD3 binding affinity.
• For the obinutuzumab 2+2 butterfly format, both the parental scFv and the disulfide scFv failed to express. ADI Set 3 rescued both yield and monomer purity, increasing titer from 2.8 to 141.6 mg/L (50-fold over parental) and ProA SEC monomer purity from 29% to 92%.
• In low-pH stress assays (one hour hold at pH 3.5), ADI Set 3 fully rescued low-pH stability in the 1+1 format. In 2+2 formats, the degree of rescue depended on the identity of the anti-CD20 (Fab) arm. Combining ADI Set 3 with a novel disulfide (DS1) stabilized the 2+2 butterfly format for both concentration and exchange into neutral phosphate buffer and low-pH stress.

• T cell-dependent cellular cytotoxicity (TDCC) profiling obinutuzumab, mosunetuzumab, and odronextamab anti-CD20 arms showed that ADI Set 3 maintained or improved potency, with the obinutuzumab arm EC50 improving from 44 to 9.9 pM.
• ADI Set 1 (VH-L45P, VL-F98W) was evaluated across human Vκ and Vλ germlines. Vκ median ProA SEC monomer improved from 92.3% to 95.7%.

Why it matters 

scFv-based anti-CD3 arms offer a route to avoid light-chain mispairing in TCE multispecifics, but their manufacturability challenges have limited their application. The mutation sets identified through thermal pressuring in Adimab's yeast platform address these challenges directly: ADI Set 3 surpasses the established disulfide benchmark on initial (post-ProA) monomer percentageand low-pH stability in the 1+1 format; rescues assembly of a 2+2 butterfly format that fails with both the parental scFv and established disulfide,and improves functional potency across multiple anti-CD20 arms. Broad transferability across Vκ germlines is being explored further.

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