A novel anti-PD-1/VEGF bispecific fusion protein with enhanced VEGF binding capacity and developability for synergistic immunotherapy.

Combination therapy targeting PD-1 and VEGF has demonstrated significant clinical benefits in multiple cancers. However, developing a single-molecule bispecific agent capable of simultaneously blocking both pathways with enhanced VEGF-binding capacity remains challenging. Here, we indicate the design, production, and comprehensive characterization of PKVBP, a novel anti-PD-1/VEGF bispecific fusion protein. PKVBP features a novel design that represents the first report of a bispecific fusion protein incorporating tandem VEGFR1 D2 domains at the C-terminus of an Fc-silenced human IgG4 backbone, enabling tetravalent VEGF engagement. Through systematic optimization of mammalian cell expression and a robust purification process, high-purity PKVBP was obtained with excellent recovery. Structural analysis confirmed proper assembly, domain integrity, and favorable thermal stability. Functional assays demonstrated simultaneous binding to PD-1 and VEGF, with substantially enhanced VEGF affinity and binding capacity compared to bevacizumab. Preliminary stability studies revealed that PKVBP maintains remarkable physicochemical and functional integrity under both long-term storage and stress conditions, with minimal oxidation and sustained target engagement. These findings highlight PKVBP as a promising bispecific candidate with superior VEGF-blocking capability and favorable developability, supporting its further investigation for synergistic cancer immunotherapy.