Specifically, the HCDR3 loop of VH domain (L100, Y101, S102 and Y104) formed five hydrogen bonds with residues from BC loop (P63, K65) and FG loop (P136) of CTLA-4, indicating a dominant role of HCDR3 in the interaction with CTLA-4 (Figure 3b; supplementary Table S3)

Specifically, the HCDR3 loop of VH domain (L100, Y101, S102 and Y104) formed five hydrogen bonds with residues from BC loop (P63, K65) and FG loop (P136) of CTLA-4, indicating a dominant role of HCDR3 in the interaction with CTLA-4 (Figure 3b; supplementary Table S3). wedge-into-hole binding mode is unique for JS007 and may be responsible for the high-affinity binding to CTLA-4. These findings have provided an important molecular understanding of the high-affinity CTLA-4 blockade mAbs and shed light on future development of agents targeting CTLA-4. KEYWORDS: CTLA-4, antibody, high affinity, structure, JS007 Introduction Immune checkpoint therapy (ICT), or immune checkpoint blockade, takes advantage of the restoration of preexisting anti-tumor T cell immunity and has reshaped the scenario of clinical therapy for tumors.1C3 Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), BRD9185 first discovered in 1987, competes the common ligands B7-1/B7-2 with stimulatory CD28 to negatively regulate T cell reactivity and serves as a critical inhibitory checkpoint molecule.4 Studies demonstrated that CTLA-4 is mainly expressed in regulatory T cells (Treg) and controls immune tolerance by enabling Tregs to rip B7-1/B7-2 molecules off the antigen-presenting cells.5 The binding of CTLA-4 can induce the endocytosis of B7-1/B7-2 molecules into CTLA-4-expressing cells, and then result in B7 degradation. Monoclonal antibodies (mAbs) that target CTLA-4 to block the interaction between CTLA-4 and its ligands have been proven to be beneficial in activating anti-tumor immunity, and been approved in clinical treatment of multiple tumors. Currently, two CTLA-4 targeting mAbs, ipilimumab (Bristol-Myers Squibb, IgG1 subtype) and tremelimumab (AstraZeneca, IgG2 subtype) are extensively investigated in clinical trials.6,7 Ipilimumab became the first ICT mAb to be commercialized when it was granted an approval by the US Food and Drug Administration (FDA) in 2011.8 To date, tremelimumab has not been approved as monotherapy, but it was approved by FDA in 2022 in combination with Imfinzi (anti-PD-L1 durvalumab) for hepatocellular carcinoma.9 Combination therapy of ipilimumab with anti-PD-1 mAbs can substantially improve survival BRD9185 of cancer patients and has gained particular interest in recent years.10C13 A large-scale Phase 3 clinical trial in patients with advanced melanoma revealed that the ipilimumab and nivolumab combination arm had a median survival of more than 60?months, whereas the ipilimumab-only arm had a median survival of 19.9?months.14 However, high rates of severe (grades 3C4) immunotherapy-related adverse effects (irAEs) were also observed with ipilimumab, which is higher than that with anti-PD-1 nivolumab (“type”:”clinical-trial”,”attrs”:”text”:”NCT03048136″,”term_id”:”NCT03048136″NCT03048136, “type”:”clinical-trial”,”attrs”:”text”:”NCT03048136″,”term_id”:”NCT03048136″NCT03048136).15,16 The pH-sensitive mAb HL32, which dissociates from CTLA-4 after endocytosis and allows CTLA-4 recycling, has been shown to decrease immunotherapy-related adverse effects in mouse model.17C19 The structures of these mAbs with CTLA-4 have been reported and the binding and blocking mechanisms defined.20,21 Ipilimumab and tremelimumab bind to the same region on CTLA-4 and Rabbit Polyclonal to HTR2B mainly locate on the front -sheet, which is also occupied by its ligands. The complex structure of HL32 and CTLA-4 reveals that multiple histidines are observed within the interface and may be BRD9185 responsible for the pH-sensitive binding, which is also observed in the structure of pH-sensitive mAbs targeting the PD-L1.22 Here, we identified a CTLA-4 specific humanized mAb JS007 with superior binding affinity and tumor suppression efficacy over ipilimumab. Structural studies of the interaction between JS007 and CTLA-4 revealed that JS007 adopts a unique wedge-into-hole binding mode to engage the CTLA-4, which may be responsible for the high-binding affinity. These findings will greatly enhance our understanding of high-binding affinity CTLA-4 blockade antibody and provide a structural basis for further engineering modification. Results Superior binding, blocking and T cell activating reactivity of JS007 By using serial dilutions of JS007 proteins to stain Chinese hamster ovary (CHO) cells stably expressing CTLA-4 (CHO-CTLA-4), a flow cytometry-based binding assay was performed to investigate the dose-dependent binding of JS007. The binding of ipilimumab was analyzed in parallel as a positive control. The results reveal that the concentration for 50% of maximal binding (EC50) of JS007 (0.22?g/mL) is about 6 times lower than that of ipilimumab (EC50?=?1.37?g/mL), indicating JS007 has a higher binding affinity.