Adimab scientists describe an integrated toolkit that includes chain-pairing control, high-throughput Chain Exchange (ChEx), curated anti-CD3 and anti-CD28 panels, and analytical/functional assays to enable fast assembly and evaluation of multispecific T cell engagers (TCEs). Our work focuses on reliable subunit pairing, predictable chromatography behavior, tunable CD3 potency, and adjustable CD28 costimulation. 

Approach and outcomes

• Controlled assembly, verified analytically: IgG-like multispecifics were specifically assembled using engineered HC–HC and HC–LC pairing mutations (CH3; CH1/Cκ). A therapeutic TCE that uses these interfaces has been evaluated in clinical trials, and analytical ion-exchange (aIEX) plus Fab LC-MS confirmed correct assembly from equimolar transient CHO transfections. 
• High-throughput paneling with predictable chromatography: Using ChEx, we converted 24 monospecific inputs into as many as 144 multispecifics, and the aIEX retention time of each output matched the mean of its inputs, indicating predictable biophysical behavior. 
• CD3 panel with relevant biology: The anti-CD3 lineage spanned a broad affinity range, was human–cynomolgus cross-reactive, and bound the N-terminal portion of CD3ε. 
• CD28 panel with tunable costimulation: Anti-CD28 antibodies were isolated from diverse library types and showed format flexibility. CD28 costimulation enhanced CD3×HER2 RTCC and cytokine secretion under the defined assay conditions, and lineage optimization (CDRH1/H2) yielded “strong/weak” variants that shifted IL-2 EC50 values. 

These results demonstrate a practical route to build large, developable TCE panels quickly. Subunit pairing was enforced, chromatography signatures were predictable, CD3 activity was selectable, and CD28 costimulation was tunable. The toolkit addresses key bottlenecks in assembly, throughput, and functional tuning in multispecific antibody development. 

View poster