Fig 2: Interaction between HLX10 and hPD-1.(a) HLX10 Fab adopts a typical immunoglobulin fold structure. Heavy chain and light chain are colored in lime and deep teal, respectively. Intra-molecular disulfide bonds are shown in magenta sticks. (b) Side view of binding interface between HLX10 and hPD-1. hPD-1 is represented as surface in white, with BC loop, C’D loop and FG loop in light orange, light blue and light green, respectively. HCDR1, HCDR2 and HCDR3 loops are colored in yellow, orange, and red, respectively, while LCDR1, LCDR2 and LCDR3 loops are colored in blue, green, and magenta, respectively. (c, d) Interaction within interface of CDRs loop of heavy chain and hPD-1. Arg86 of hPD-1 is involved in several hydrogen bonds and salt bridges with HLX10.

Fig 3: Comparison of binding epitope of HLX10 with Pembrolizumab and Nivolumab.(a) Binding of Nivolumab is mainly located on the top (N-terminal extension, BC loop, and FG loop) of h-PD-1, whereas Pembrolizumab and HLX10 is located on CC’FG sheets of h-PD-1. hPD-1 is represented as surface in grey. PD-L1, Pembrolizumab, Nivolumab, and HLX10 are colored in slate, deep teal, lime and magenta ribbon, respectively (b) Residues of hPD-1 contribute to the interactions with different binders. hPD-1 is represented as white surface with binding epitope colored in slate. The residues which are involved in hydrogen bond, salt bridge and hydrophobic interaction are colored in blue, red and black, respectively.

Fig 4: Binding epitope of HLX10 overlaps with PD-L1 and PD-L2.(a-c) Comparison of binding interface of PD-L1, PD-L2 and HLX10. hPD-1 is represented as white surface with binding interface of binders (PD-L1, PD-L2 and HLX10) colored in slate. The C’D loop of hPD-1 is missing in hPD-1/hPD-L1 (PDB: 4ZQK) and hPD-1/hPD-L2 structures (PDB: 6UMT) because of structure flexibility. (d) rearrangement of CC’ loop of hPD-1 for HLX10 binding. Cα atom of Gln75 displays an 8 Å movement. Except C’C loop of hPD-1, there is no significant conformational change between apo and bound form of hPD-1 structures (overall backbone RMSD = 0.351Å). The C’D loop is missing (D85-D92) in apo form hPD-1 structure (PDB: 3RRQ) because of structure flexibility.

Fig 5: Binding and PD-L1 ligand blocking of clG4 and Nivolumab antibodies.ELISA assay comparing the binding of clG4 and Nivolumab reference to human PD-1 (a). ELISA assay comparing the ability of clG4 and Nivolumab reference to block binding of human PD-Ll to human PD-1 (b). Data points are means ± SD.