A Multispecific Checkpoint Inhibitor Nanofitin with a Fast Tumor Accumulation Property and Anti-Tumor Activity in Immune Competent Mice

Immune checkpoint inhibitors have revolutionized cancer treatment but remain limited by on-target/off-tumor effects that narrow their therapeutic window. Although PD-L1 is mainly expressed by tumor cells, these effects could reduce bloodstream availability and tumor accumulation of PD-L1 inhibitors. Enhancing tumor specificity through bispecific proteins targeting two tumor-associated antigens offers a promising strategy. This study evaluated a bispecific Nanofitin, B10-B11, targeting PD-L1 and EGFR. In vitro, B10-B11 efficiently bound to human A431 and murine CT26 cell lines, validating these models for in vivo studies. Nanofitins' accumulation in tumors and their anti-tumor efficacy were assessed, respectively, in A431 xenograft and CT26 immunocompetent mouse models. In both experiments, B10-B11 was compared with its albumin binding fused counterpart (B10-B11-ABNF). This study showed that the dual-targeting approach with the bispecific Nanofitin enhanced in vitro PD-L1 neutralization compared to the monomeric form and led to in vivo anti-tumor activity evidenced by reduced tumor growth and increased CD3+ T cells and F4/80+ macrophages in tumors. This activity was further correlated with Nanofitin's tumor accumulation at 7 h post-injection, which was highest for the B10-B11-ABNF. This study highlights the potential of bispecific Nanofitins, particularly with albumin binding to enable rapid and uniform tumor accumulation of effective PD-L1 immunotherapy.