Anti-TGF-beta/PD-L1 bispecific antibody promotes T cell infiltration and exhibits enhanced antitumor activity in triple-negative breast cancer

Background Agents blocking programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) have been approved for triple-negative breast cancer (TNBC). However, the response rate of anti-PD-1/PD-L1 is still unsatisfactory, partly due to immunosuppressive factors such as transforming growth factor-beta (TGF-beta). In our previous pilot study, the bispecific antibody targeting TGF-beta and murine PD-L1 (termed YM101) showed potent antitumor effect. In this work, we constructed a bispecific antibody targeting TGF-beta and human PD-L1 (termed BiTP) and explored the antitumor effect of BiTP in TNBC. Methods BiTP was developed using Check-BODYTM bispecific platform. The binding affinity of BiTP was measured by surface plasmon resonance, ELISA, and flow cytometry. The bioactivity was assessed by Smad and NFAT luciferase reporter assays, immunofluorescence, western blotting, and superantigen stimulation assays. The antitumor activity of BiTP was explored in humanized epithelial-mesenchymal transition-6-hPDL1 and 4T1-hPDL1 murine TNBC models. Immunohistochemical staining, flow cytometry, and bulk RNA-seq were used to investigate the effect of BiTP on immune cell infiltration. Results BiTP exhibited high binding affinity to dual targets. In vitro experiments verified that BiTP effectively counteracted TGF-beta-Smad and PD-L1-PD-1-NFAT signaling. In vivo animal experiments demonstrated that BiTP had superior antitumor activity relative to anti-PD-L1 and anti-TGF-beta monotherapy. Mechanistically, BiTP decreased collagen deposition, enhanced CD8(+) T cell penetration, and increased tumor-infiltrating lymphocytes. This improved tumor microenvironment contributed to the potent antitumor activity of BiTP. Conclusion BiTP retains parent antibodies' binding affinity and bioactivity, with superior antitumor activity to parent antibodies in TNBC. Our data suggest that BiTP might be a promising agent for TNBC treatment.