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Background Immune checkpoint inhibitors, like PD-1 or PD-L1 inhibitors, have changed the standard of care in multiple tumors, but durable responses have only been observed in a subset of patients. Overexpression of VEGF is frequently found in various tumors and plays critical roles in tumor angiogenesis and suppression of anti-tumor immune response. Bispecific antibodies targeting PD-(L)1 and VEGF have shown promising efficacy with a favorable safety profile in clinical trials. Specifically, ivonescimab, a cooperative PD-1 x VEGF bispecific antibody, demonstrated superiority to anti-PD-1 in randomized Phase 3 trial. 1 2 CR-001 is a tetravalent bispecific antibody targeting PD-1 and VEGF. It consists of a bivalent, Fc-silenced anti-VEGF-A IgG1 antibody, with two anti-PD-1 single chain variable fragments fused to its C-terminus. Here, we report preclinical studies characterizing the cooperative pharmacology of CR-001 in vitro, anti-tumor activity in vivo, and single dose pharmacokinetic (PK) study in cynomolgus monkeys.Methods The antigen binding affinities of CR-001 with PD-1 and VEGF were measured by surface plasmon resonance. To evaluate the cooperative pharmacology in vitro, the binding activity of CR-001 to PD-1 was evaluated in PD-1 positive cells by flow cytometry in the presence of VEGF. The blockade of PD-1/PD-L1 signaling pathway was determined by NFAT luciferase reporter assay. SCID mice implanted with tumor cells were used to evaluate in vivo anti-tumor activity. The PK and receptor occupancy (RO) of CR-001 were evaluated in cynomolgus monkeys after a single intravenous administration.Results CR-001 bound with high affinity to human PD-1 and VEGF-A. In cell-based reporter assays, CR-001 inhibited PD-1/PD-L1 signaling and VEGF-A/VEGFR2 signaling with sub-nanomolar EC 50 values. The PD-1 binding of CR-001 was increased in the presence of VEGF, leading to higher potency in PD-1/PD-L1 signaling blockade relative to CR-001 alone. Furthermore, CR-001 with VEGF showed increased IL-2 secretion compared to CR-001 alone in T cell activation assay using human PBMCs co-cultured with PD-L1 positive tumor cells. In vivo, CR-001 demonstrated potent anti-tumor activity, and was well-tolerated in cynomolgus monkeys after a single intravenous dose with peak PD-1 RO > 80%.Conclusions CR-001 demonstrated cooperative pharmacology with increased binding to PD-1 in the presence of VEGF, augmenting PD-1/PD-L1 signaling blockade and enhancing T cell activation in vitro, consistent with preclinical evaluation of ivonescimab. 1 In xenograft models, CR-001 demonstrated potent anti-tumor activity. CR-001 was well-tolerated in the cynomolgus monkey after a single dose. These data support clinical development of CR-001.References Zhong, et al. Design of a fragment crystallizable-engineered tetravalent bispecific antibody targeting programmed cell death-1 and vascular endothelial growth factor with cooperative biological effects. iScience. 2025;28(3):111722.Xiong, et al. Ivonescimab versus pembrolizimab for PD-L1-positive non-small cell lung cancer (HARMONi-2): a randomized, double-blind, phase 3 study in China. Lancet. 2025;405(10481):839–849.Ethics Approval All animal protocols were reviewed and approved by the IACUC institution of the respective CROs. Further animals were housed, provided enrichment and monitored in accordance with the institution’s SOPs and protocols.