Comparative evaluation of bispecific antibody formats targeting PD-1 and LAG-3 for dual checkpoint blockade in cancer immunotherapy

Bispecific antibodies (BsAbs) targeting PD-1 and LAG-3 offer a promising strategy in cancer immunotherapy by enhancing antitumor immunity and overcoming resistance to PD-1 blockade. Despite the growing interest in PD-1/LAG-3 BsAbs, a systematic comparison of different BsAbs formats remains lacking, leaving a gap in the rational design of optimized therapeutics. In this study, we systematically compared three BsAb formats-YG-003D1 (Ab-ScFv format), YG-003D2 (DVD format), and YG-003D3 (Knob-into-Hole (KIH) format)-to evaluate their structural, functional, and pharmacokinetic properties, providing critical insights into their therapeutic potential. YG-003D1 exhibited the strongest binding and blocking activity due to its tetravalent Ab-ScFv structure, which facilitated dual-target engagement with minimal steric hindrance. However, it had relatively low expression yields and a tendency to form aggregates, which could impact manufacturability and long-term stability. YG-003D2, utilizing a DVD format, exhibited mild steric hindrance in dual-target engagement, leading to a moderate reduction in blocking efficiency, particularly in LAG-3 inhibition. Nonetheless, its bivalency for both PD-1 and LAG-3 may provide advantages in specific therapeutic contexts. In contrast, YG-003D3, with its asymmetric KIH format, demonstrated the most favorable balance of manufacturability, stability, and pharmacokinetics. It had high expression yields, minimal aggregation, and a half-life comparable to IgG, making it the most promising candidate for clinical development. However, its monovalent binding per target resulted in slightly reduced blocking potency compared to YG-003D1. While YG-003D3 demonstrated the best overall balance of properties, alternative formats such as YG-003D1 could be refined through Fc engineering or linker optimization to enhance manufacturability and reduce aggregation. Similarly, YG-003D2's steric hindrance could be mitigated by introducing flexible linkers to improve dual-target engagement. Further modifications to YG-003D3, such as affinity tuning or Fc engineering, could enhance its blocking potency while retaining its favorable pharmacokinetics. These insights not only provide a rational framework for PD-1/LAG-3 bispecific inhibitor design but also serve as a reference for broader applications of BsAbs in immunotherapy.