mAbs | TriMab Platform Enables Conditional Dual-Target T-Cell Engager Design

This study developed a conditional dual-target T-cell engager based on TriMab technology, significantly improving therapeutic index through affinity regulation and immune synapse formation mechanisms, offering novel solutions for solid tumor therapy.

 

Literature Overview
The article titled 'Improving dual targeting selectivity in T-cell engagers via synapse-gated and affinity-tuned trispecific antibody design' published in mAbs journal reviews strategies to enhance solid tumor treatment selectivity through synapse-gated and affinity-optimized trispecific antibody (TriMab) design. It highlights that conventional T-cell engagers (TCEs) face limitations in solid tumor therapy due to insufficient tumor specificity and off-target toxicity. The research demonstrates an AND-gated targeting mechanism by incorporating a non-activating anchor arm (e.g., anti-ROR1) with affinity-modulated activating arm (e.g., anti-EGFR) and anti-CD3 structure, achieving selective immune synapse formation exclusively on dual-positive cells.

Background Knowledge
T-cell engagers represent critical immunotherapeutic agents redirecting patient T-cells to attack cancer cells. However, solid tumor treatments often encounter toxicities toward normal tissues due to lack of tumor-specific antigens. The research team developed TriMab technology through dual-target recognition mechanisms while maintaining monoclonal antibody (mAb)-like pharmacokinetic properties. The design incorporates affinity-optimized anchor arms (e.g., anti-ROR1) and activating arms (e.g., anti-EGFR[v1-v3]) to ensure T-cell activation exclusively on dual-positive cells, implementing AND-gated logic. Validated across multiple target combinations including HER2/EGFR TriMab, the study demonstrates broad applicability. This systematic engineering strategy establishes a foundation for improving safety and efficacy in solid tumor immunotherapy.

 

 

Research Methods and Experiments
The research team constructed TriMab molecules using the DuetMab platform containing three distinct antigen-binding fragments (Fabs): a high-affinity anchor arm (e.g., anti-ROR1), affinity-reduced activation arm (e.g., anti-EGFR[v1-v3]), and T-cell recruiting arm (anti-CD3). Correct heavy-light chain pairing was facilitated through knob-into-hole (KiH) mutations and electrostatic steering modifications, while Fc-mediated effector functions were minimized by introducing L234F, L235E, P331S mutations in the CH3 domain. Molecular binding properties and immune synapse formation were evaluated using flow cytometry, bio-layer interferometry (BLI), and confocal imaging. Anti-tumor activity and pharmacokinetics were tested in mouse xenograft models.

Key Conclusions and Perspectives

• The TriMab design achieves AND-gated T-cell activation through high-affinity anchor and low-affinity activation arms, inducing immune synapse formation exclusively on dual-positive cells.
• In vitro experiments demonstrated ROR1/EGFR[v3] TriMab exhibited 500-fold enhanced target selectivity against NCI-H358 cells while showing negligible toxicity to ROR1 knockout cells.
• In vivo xenograft models confirmed potent anti-tumor activity with minimal toxicity to healthy tissues, validating the platform's selectivity.
• TriMab molecules exhibited mAb-like pharmacokinetics with 4.2–5.8 days half-life in humanized FcRn mice, indicating favorable developability.
• Platform universality was further validated through HER2-targeted anchor arm substitution, demonstrating applicability across diverse dual-target combinations.

Research Significance and Prospects
This study establishes a universal design framework for dual-target T-cell engagers to improve solid tumor treatment safety. Future applications should expand target combinations and validate safety/efficacy in preclinical models. The TriMab engineering strategy provides a paradigm for next-generation TCE development with particular potential in personalized cancer immunotherapy.

 

 

Conclusion
This study successfully developed and validated the TriMab technology for T-cell engagers, achieving dual-target-dependent activation through combined affinity modulation and immune synapse control. The approach significantly enhances solid tumor targeting selectivity while minimizing toxicity. Results demonstrate favorable pharmacokinetics and developability across multiple tumor antigen combinations. This platform holds promise for establishing safer and more effective TCE design paradigms, particularly for solid tumors lacking tumor-specific antigens.

 

Peng Zhao, John Schardt, Chi-I Chiang, Even Walseng, and Yariv Mazor. Improving dual targeting selectivity in T-cell engagers via synapse-gated and affinity-tuned trispecific antibody design. mAbs.