Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), an immune checkpoint blocker, has achieved great success in immunotherapy over the last many years. Ipilimumab, an antibody targeting CTLA-4, was the first to receive regulatory approval for clinical use. However, the clinical usage of these CTLA-4 antibodies is often associated with severe immunotherapy-related adverse events (irAEs). Recently, professor Liu’s group reported a new generation of anti-CTLA-4 mAb that cause much less irAEs but showed even higher efficacy in inducing rejection of large established tumors and T_reg depletion in tumor tissue than both ipilimumab and treme-IgG1. In order to delineate how the new anti-CTLA-4 antibodies differ from ipilimumab and tremelimumab in interacting with their shared target, CTLA-4, Professor Dou’s group from the College of Life Sciences, Beijing Normal University, Professor Liu’s group from University of Maryland and Professor Zhou’s group from School of Medicine, Shanghai Jiaotong University worked together to explore the structural mechanism of the antibody (HL32) complexed with CTLA-4.

They solved a tetramer structure containing a human CTLA-4 homodimer and two copies of the HL32-Fab molecule at 3.05 Å resolution. HL32-Fab mainly binds strands A and G of the front β-sheet and the FG loop of CTLA-4 through its heavy chain (VH) with its heavy chain complementarity determining region 3 (HCDR3). This region forms a long extended β-hairpin and anneals to the side of the front β-sheet of CTLA-4. The binding interface involves extensive hydrophobic interactions formed by a cluster of aromatic residues, which is much different with Ipilimumab. The binding surface analysis showed that the Fab of HL32 binds the right-bottom corner of the front beta-sheet of CTLA-4 which significantly overlap with the B7 binding position. In addition, an important advance in understanding the physiological function of CTLA-4 is that CTLA-4 undergoes constitutive recycling and mediates transendocytosis of B7. Genetic inactivation of this process leads to autoimmune diseases in human. In this context, it is plausible that antibodies binding on CTLA-4 could have different effects on these processes, ultimately leading to different toxicity. Some of them demonstrated that ipilimumab and treme-IgG1 interfered with CTLA-4 recycling causing lysosomal degradation of CTLA-4, while HL32 preserves CTLA-4 recycling as it rapidly dissociates from CTLA-4 after endocytosis due to its sensitivity to late endosomal and lysosomal pH. Detailed structural analysis showed that there are four histidine residues near the binding interface of CTLA-4 and HL32. Changes of pH could affect the charge state of imidazole rings of these histidine residues leading to altered local interactions, thereby perturb the binding interactions between HL32 and CTLA-4.

The research titled "Structure of CTLA-4 complexed with a pH-sensitive cancer immunotherapeutic antibody" was published online on November 3, 2020 in Cell Discovery. Gaohan, a Ph.D. student from the College of Life Sciences, Beijing Normal University, is the first author. Professor Dou, Liu and Zhou are the co-corresponding authors.

Original link is https://www.nature.com/articles/s41421-020-00202-9