Blocking the interaction of programmed cell death protein 1 (PD-1) and its ligand PD-L1 is known as a promising immunotherapy for treatment of a variety of tumors expressing PD-L1 on their cell surface. In the last decade, several antibodies against the PD-1/PD-L1 interaction have been approved, while there are few reports of small molecule inhibitors against PD-1/PD-L1 axis. Due to many advantages of cancer treatment with small molecules over antibodies, we developed several peptidic PD-L1 antagonists using computational peptide design methods, and evaluated them both in vitro and in vivo. Importantly, among six peptides with best affinity to PD-L1, four peptides exhibited significant potency to block PD-1/PD-L1 axis at molecular level. Moreover, the PD-L1 expression in nine human colorectal cancer cell lines stimulated with interferon-γ was compared and LoVo cells with the highest expression were selected for further experiments. The peptides could also restore the function of activated Jurkat T cells, which had been suppressed by stimulated LoVo cells. A blockade assay in tumor-bearing mice experiments indicated that peptides HS5 and HS6 consisting of a D-amino acid in their structures, could also effectively reduce tumor growth in vivo, without induction of any observable liver or renal toxicity, tissue damages and loss of body weight. As new designed peptides showed no toxicity against murine colon cancer cells in vitro, the observed anti-tumor results in mice are most probably due to disrupting the PD-1/PD-L1 interaction. Thus, peptides described in this study can be considered as proper low molecular weight candidates for immunotherapy of cancer.

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