Monoclonal antibodies (mAbs) blocking immune checkpoint molecules, especially programmed cell death 1 (PD-1) and its ligands programmed cell death 1 ligand 1 (PD-L1) and ligand 2 (PD-L2), are currently been investigated for treatment of various tumors [1-3]. PD-L1 and PD-L2 are usually upregulated on the surface of multiple tumor cells to mediate immune tolerance through the interaction with inhibitory PD-1 molecule [4]. Thus, blocking PD-1/PD-Ls interaction has brought promising future for tumor immunotherapy. To date, several PD-1/PD-L1 blockade antibodies have been approved for clinical use or under phase III clinical trials (eg, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab and BMS-936559, etc.)[4]. The PD-1 targeting therapeutic antibodies block the PD-1/PD-L1 or PD-1/PDL2 interaction to restore tumor-specific T cell reactivity, without mediating antibody-dependent cell-mediated cytotoxicity (ADCC). Recently, the structural basis of hPD-1/pembrolizumab (a PD-1 targeting therapeutic antibody developed by Merck & Co., Inc., USA) has been revealed, providing a molecular insight into blocking PD-1-mediated immune suppression by antibody [5]. PD-L1 targeting therapeutic antibodies possess PD-1/PD-L1 blockade activity with or without ADCC activity. As one of the PD-L1 targeting antibodies, avelumab is a human IgG1 antibody with ADCC activity developed by Merck (Darmstadt, Germany) and Pfizer, which is now in multiple phase III clinical trials against non-small cell lung cancer (NCT02395172), advanced renal cell cancer (NCT02684006) and gastric cancer (NCT02625610)[6]. The crystal structures of PD-L1 couplexed with its receptor PD-1 have been extensively studied, including human PD-L1 (hPD-L1) alone, mouse PD-1 (mPD-1) complexed with hPD-L1 and human PD-1 (hPD-1) complexed with hPD-L1 [7-9]. Though the complex structure of hPD-1 with a commercial mAb pembrolizumab has been solved very recently [5], hPD-L1/mAb complex structure has not been investigated. In this study, we expressed the single chain Fv fragment (scFv) of avelumab and hPD-L1 with two immunoglobulin (Ig) domains as inclusion bodies in E. coli. Then we applied in vitro refolding method to obtain soluble proteins, and the two refolded proteins can survive well in gel filtration (Supplementary information, Figure S1A). The binding kinetics of avelumab-scFv/hPD-L1 was analyzed by surface plasmon resonance (SPR). The binding avidity was determined by calculating dissociation constant (Kd) which was 42.1 pM for avelumab-scFv (Supplementary information, Figure S1B). Subsequently, we performed crystal screen with the avelumab-scFv/hPD-L1 complex protein, and obtained well-diffractable crystals in 0.2 M magnesium chloride hexahydrate, 0.1 M HEPES-Na, pH 7.5, 30% v/v isopropanol (see more details in Supplementary information, Data S1). The crystal structure of the hPD-Ll complexed with avelumab scFv was determined by molecular replacement at a resolution of 3.2 Å (Supplementary information, Table S1A). The PD-L1 consists of two Ig domains, the N-terminal IgV domain and the C-terminal IgC domain. The overall complex structure reveals that avelumab utilizes both heavy chain (VH) and light chain (VL) to bind to the IgV domain of PD-L1 on the side (Figure 1A and Supplementary information, Figure S1C). The interaction with hPD-L1 involves five of the six complementarity-determining regions (CDRs) of both VH and VL with a buried area of~ 1 856 Å2. The VH of avelumab dominates the binding to hPD-L1 by all three CDR loops, and VL contributes partial contacts by CDR1 and CDR3 loop (Figure …