In vitro characterization of fully human antagonistic anti-BTLA monoclonal antibodies

Introduction: B and T lymphocyte attenuator (BTLA) is an immune checkpoint receptor essential for immune homeostasis and tolerance. Unlike PD-1 or CTLA-4, BTLA is broadly expressed on lymphoid and some myeloid cells, including T cells, B cells, dendritic cells, and macrophages. Engagement with its ligand HVEM (herpesvirus entry mediator) transmits inhibitory signals that suppress immune activation. Within tumors, BTLA contributes to immune evasion by dampening anti-tumor responses. Given its unique expression profile and regulatory function, we developed monoclonal antibodies (mAbs) targeting BTLA as an immunotherapeutic strategy to enhance anti-tumor immunity. Methods: Fully human anti-BTLA mAbs identified by hybridoma screening were reformatted as huIgG4 (S228P/L235A) and transiently expressed using Curia's TunaCHO® platform. The S228P mutation prevents Fab-arm exchange, stabilizing mAb structure, while L235A minimizes FcgammaR binding and effector functions. Binding potency was determined by ELISA; kinetics by Octet® biolayer interferometry (BLI) against human and cynomolgus BTLA. BLI also assessed FcgammaR, FcRn (at pH 6.0 vs 7.2), and C1q binding to evaluate recycling potential and complement activation. Neutralization of HVEM binding to BTLA was quantified by modified ELISA and cell-based reporter assays. Results: Six fully human anti-BTLA mAbs and a clinical candidate comparator JS004 displayed similar ~1 nM binding potencies (IC 50 ) values to human and cyno BTLA by ELISA. Clone 13-F7-A exhibited the highest affinity (K d < 1 nM) for both human and cyno BTLA among all 7 mAbs tested. Four mAbs and the comparator JS004 were high affinity binders to human and cyno BTLA (K d 1-32 nM). All six fully human mAbs exhibited reduced FcgammaR binding relative to JS004. Each bound FcRn at pH 6.0 but not at 7.2, consistent with normal endosomal recycling. None bound C1q, whereas a positive-control IgG1 did bind to C1q as expected. In an ELISA-based biofunctional assay, all mAbs blocked HVEM binding to BTLA with similar IC 50 values (~1 nM). Conclusions: We generated potent, high affinity, human/cyno cross-reactive, fully human antagonistic anti-BTLA mAbs. Targeting BTLA offers promising opportunities for cancer immunotherapy and may demonstrate strong synergy when combined with other checkpoint antagonists, potentially overcoming resistance mechanisms and improving clinical outcomes.