A novel anti-PD-1×CTLA-4×VEGF tri-specific antibody enables tumor-selective immune checkpoint blockade with potent anti-tumor activity

Combination strategies integrating immune checkpoint blockade and anti-angiogenic therapy by co-targeting PD-1 and VEGF pathways have demonstrated meaningful clinical benefit across multiple tumor types. However, the incorporation of CTLA-4 blockade remains limited by systemic immune-related toxicities. To address these challenges, we engineered a novel tri-specific antibody designed to concurrently inhibit PD-1, CTLA-4, and VEGF while enabling tumor-selective, PD1-dependent CTLA-4 engagement. This tri-specific antibody demonstrated high-affinity PD-1 binding and checkpoint blockade activity comparable to pembrolizumab, along with potent VEGF neutralization consistent with clinically validated anti-VEGF antibodies. Importantly, CTLA-4 blockade by the tri-specific antibody was minimal in CTLA-4 single-positive reporter systems, but was markedly enhanced in PD-1/CTLA-4 dual-expressing cells. This avidity-dependent mechanism preferentially enables CTLA-4 inhibition in activated T cells within the tumor microenvironment, thereby providing a potential strategy to improve the therapeutic index relative to conventional systemic CTLA-4 blockade. In ex vivo primary human immune cell assays, the tri-specific antibody induced strong IL2 release, supporting enhanced functional immune modulation. In vivo efficacy was further evaluated across complementary tumor models. In the MC38 syngeneic mouse model, the tri-specific antibody produced superior tumor growth inhibition relative to pembrolizumab or ipilimumab monotherapy, indicating cooperative immune checkpoint engagement. In the COLO205 xenograft model, anti-tumor activity was comparable to bevacizumab, confirming preserved VEGF pathway blockade and anti-angiogenic function. Moreover, non-human primate studies demonstrated favorable tolerability, pharmacokinetic behavior, and developability characteristics of the tri-specific antibodies, with no unexpected safety findings, supporting further translational development. In summary, this novel PD-1 × CTLA-4 × VEGF tri-specific antibody integrates immune checkpoint inhibition with angiogenesis blockade through a rationally designed, avidity-based mechanism that conditionally enhances CTLA-4 engagement in tumor-relevant immune contexts. These preclinical findings support its potential as a next-generation immuno-oncology therapy with improved balance between efficacy and safety, warranting further clinical investigation.