Blockade of the immunoinhibitory PD-1/PD-L1 pathway using monoclonal antibodies has shown impressive results with durable clinical antitumor responses. Anti-PD-1 and anti-PD-L1 antibodies have now been approved for the treatment of a number of tumor types, whereas the development of small molecules targeting immune checkpoints lags far behind. We characterized two classes of macrocyclic-peptide inhibitors directed at the PD-1/PD-L1 pathway. We show that these macrocyclic compounds act by directly binding to PD-L1 and that they are capable of antagonizing PD-L1 signaling and, similarly to antibodies, can restore the function of T-cells. We also provide the crystal structures of two of these small-molecule inhibitors bound to PD-L1. The structures provide a rationale for the checkpoint inhibition by these small molecules, and a description of their small molecule/PD-L1 interfaces provides a blueprint for the design of small-molecule inhibitors of the PD-1/PD-L1 pathway.

The PD-1/PD-L1 interaction has emerged as a significant target in cancer immunotherapy. Current medications include monoclonal antibodies, which have shown impressive clinical results in the treatment of several types of tumors. The cocrystal structure of human PD-1 and PD-L1 is expected to be a valuable starting point for the design of novel inhibitors, along with the recent crystal structures with monoclonal antibodies, small molecules, and macrocycles.

The protein-protein interaction PD1/PD-L1 is an important immune checkpoint and several recently approved monoclonal antibodies show promising anti cancer activities in the clinical practice. However, only a small percentage of cancer patients benefit from PD1/PD-L1 directed mAbs. Moreover, some patients experience immune related side effects upon treatment with these mAbs. Recently, several atomic-resolution structures of human PD1/PD-L1, and small molecules, peptides and mAbs with PD-L1 and PD1 open the field for structure based drug design. Small molecules and peptides targeting PD1/PD-L1 promise to enhance tumor activity while showing less immune related side effects. Areas covered: We reviewed the small molecules classes and peptides targeting PD1/PD-L1. Expert opinion: Currently approved PD1/PD-L1 directed therapeutics show room for improvement. Three classes of non mAb small molecule classes have been discovered so far: (cyclic) peptides as direct competitive PD1/PD-L1 antagonists; small molecules disrupting PD1/PD-L1 and inducing a PD-L1 dimerization; and a small molecule class of unknown mode-of-action. An example of the later group CA-170 is currently investigated in a Phase 1 trial in patients with advanced solid tumors and lymphomas. Potential advantages of small molecules over mAbs include high distribution and better tumor penetration, improved PK/PD, less side effects and oral bioavailability.

CA-170 is currently the only small-molecule modulator in clinical trials targeting PD-L1 and VISTA proteins – important negative checkpoint regulators of immune activation. The reported therapeutic results to some extent mimic those of FDA-approved monoclonal antibodies overcoming the limitations of the high production costs and adverse effects of the latter. However, no conclusive biophysical evidence proving the binding to hPD-L1 has ever been presented. Using well-known in vitro methods: NMR binding assay, HTRF and cell-based activation assays, we clearly show that there is no direct binding between CA-170 and PD-L1. To strengthen our reasoning, we performed control experiments on AUNP-12 – a 29-mer peptide, which is a precursor of CA-170. Positive controls consisted of the well-documented small-molecule PD-L1 inhibitors: BMS-1166 and peptide-57.

A series of C 2 -symmetric inhibitors was designed and evaluated for inhibitory activity against the programmed cell death-1/programmed death-ligand 1(PD-1/PD-L1) protein−protein interaction (PPI) in a homogenous time-resolved fluorescence (HTRF) assay and PD-1 signaling in cell-based coculture assays. C 2 -symmetric inhibitors 2a (LH1306) and 2b (LH1307) exhibited IC 50 values of 25 and 3.0 nM, respectively, in the HTRF assay. While 2a was ∼3.8-fold more potent than previously reported inhibitor 1a, 2b could not be differentiated from 1b due to their high potency and the limit of our HTRF assay conditions. In one cell-based coculture PD-1 signaling assay, 2a and 2b were 8.2-and 2.8-fold more potent in inhibiting PD-1 signaling than 1a and 1b, respectively. NMR and X-ray cocrystal structural studies provided more structural insights into the interaction between 2b and PD-L1; 2b binds to PD-L1 at the PD-1 binding site and induces the formation of a more symmetrically arranged PD-L1 homodimer than that previously reported for other inhibitors.