A first-in-class PD-L1/B7-H3/VEGF tri-specific ADC achieves enhanced preclinical antitumor efficacy through direct cytotoxicity, immune checkpoint blockade and VEGF inhibition

PD-L1 is an established immunotherapy target highly expressed in various solid tumors, while B7-H3 is another extensively investigated target overexpressed on a wide range of tumor cells. ADCs targeting PD-L1 or B7-H3 have entered clinical development and shown promising efficacy. PD-L1-directed ADCs may combine direct cytotoxicity and immunotherapy, demonstrating antitumor activity even in PD-L1-negative patients. However, ADC-related toxicity may limit their clinical dosing and thus restrict the full immune-mediated effects. Meanwhile, VEGF/PD-(L)1 bispecific antibodies are emerging as next-generation immunotherapies, with multiple trials exploring their combination with ADCs. Here, we developed ALK208, a trispecific ADC targeting PD-L1, B7-H3, and VEGF, conjugated to a topoisomerase I inhibitor payload. It is designed to integrate direct tumor cell killing, immune checkpoint blockade, and anti-angiogenic activity for maximal antitumor efficacy. Through optimized linker-payload design and DAR, ALK208 exhibited an improved safety profile, enabling higher dose levels to support robust PD-L1 blockade and VEGF neutralization. In preclinical studies, ALK208 simultaneously bound B7-H3, PD-L1, and VEGF with affinity comparable to monospecific antibodies. It effectively blocked VEGF/VEGFR and PD-1/PD-L1 interactions in reporter assays. Concurrent binding of B7-H3 and PD-L1 enhanced cellular binding and internalization in B7-H3/PD-L1 double-positive tumor cells, leading to more potent cytotoxicity than monospecific ADCs. VEGF-mediated crosslinking further strengthened binding, internalization, and cell-killing activity compared to a PD-L1/B7-H3 bispecific ADC. In the presence of VEGF homodimer, ALK208 also showed enhanced PD-L1 blockade in functional assays. Importantly, it did not kill activated T cells or APCs in vitro, suggesting a favorable immune safety profile. In xenograft models, ALK208 outperformed clinical-stage PD-L1 and PD-L1/B7-H3 ADCs. In summary, ALK208 is a first-in-class PD-L1/B7-H3/VEGF trispecific ADC that integrates multiple complementary mechanisms to maximize antitumor efficacy while maintaining a promising safety profile.