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Programmed cell death protein 1 (PD‐1), an immune checkpoint receptor expressed by activated T, B, and NK cells, is a well‐known target for cancer immunotherapy. Tislelizumab (BGB‐A317) is an anti‐PD‐1 antibody that has recently been approved for treatment of Hodgkin's lymphoma and urothelial carcinoma. Here, we show that tislelizumab displayed remarkable antitumor efficacy in a B16F10/GM‐CSF mouse model. Structural biology and Surface plasmon resonance (SPR) analyses revealed unique epitopes of tislelizumab, and demonstrated that the CC′ loop of PD‐1, a region considered to be essential for binding to PD‐1 ligand 1 (PD‐L1) but not reported as targeted by other therapeutic antibodies, significantly contributes to the binding of tislelizumab. The binding surface of tislelizumab on PD‐1 overlaps largely with that of the PD‐L1. SPR analysis revealed the extremely slow dissociation rate of tislelizumab from PD‐1. Both structural and functional analyses align with the observed ability of tislelizumab to completely block PD‐1/PD‐L1 interaction, broadening our understanding of the mechanism of action of anti‐PD‐1 antibodies., We evaluated the antitumor efficacy of tislelizumab (BGB‐A317), an approved anti‐PD‐1 antibody. Its critical epitopes were investigated by both structural biology and SPR studies, and the CC′ loop of PD‐1 was discovered to be a novel targetable region for anti‐PD‐1 antibodies. The unique epitopes and slow dissociation rate of tislelizumab contribute to its complete blocking activity to PD‐L1.

Poly (ADP-ribose) polymerase (PARP) plays a significant role in DNA repair responses; therefore, this enzyme is targeted by PARP inhibitors in cancer therapy. Here we have developed a number of fused tetra- or pentacyclic dihydrodiazepinoindolone derivatives with excellent PARP enzymatic and cellular PARylation inhibition activities. These efforts led to the identification of pamiparib (BGB-290, 139), which displays excellent PARP-1 and PARP-2 inhibition with IC50 of 1.3 and 0.9 nM, respectively. In a cellular PARylation assay, this compound inhibits PARP activity with IC50 = 0.2 nM. Cocrystal of pamiparib shows similar binding sites with PARP with other PARP inhibitors, but pamiparib is not a P-gp substrate and shows excellent drug metabolism and pharmacokinetics (DMPK) properties with significant brain penetration (17-19%, mice). The compound is currently being investigated in phase III clinical trials as a maintenance therapy in platinum-sensitive ovarian cancer and gastric cancer.

Background OX40 is a member of the tumor necrosis factor receptor super family (TNFRSF) primarily expressed on activated CD4+ and CD8+ T cells, as well as natural killer (NK) T and NK cells. It is an immune costimulatory receptor which binds to its ligand OX40L and activates downstream NF-κB pathway to induce immune cell activation, proliferation, and survival.1–3 Current agonistic anti-OX40 antibodies in clinic, which are mostly ligand-competitive antibodies, showed limited clinical responses, mainly at lower doses. Blockade of OX40-OX40L interaction might limit the efficacy of these ligand-competitive antibodies at higher doses, as OX40-OX40L interaction is essential for enhancing effective anti-tumor immunity. Here we report pre-clinical data of BGB-A445, which is a ligand non-blocking agonistic anti-OX40 humanized antibody. Methods Cell-based flow cytometry assay was established to determine whether BGB-A445 interferes with OX40-OX40L interaction. Co-crystal structure of OX40/BGB-A445 Fab was solved to study the molecular binding mechanism. A mixed lymphocyte reaction (MLR) assay was set up to investigate the ability of BGB-A445 to activate CD4+ T-cells. The anti-tumor efficacy of BGB-A445 was evaluated in MC38 colon cancer and CT26WT colon cancer models either as a single agent or in combination with anti-PD-1 antibody. Results The flow cytometry study showed that BGB-A445 did not interfere with the binding of OX40 to OX40L even at high concentrations. In contrast, MOXR0916, an anti-OX40 agonistic antibody developed by Genentech, completely blocked OX40 binding to OX40L. Additionally, the co-crystal structure of OX40/BGB-A445 Fab complex indicated that BGB-A445 interacts with the CRD4 region of OX40 which is distant from OX40L binding region. In the MLR assay, combined with an anti-PD-1 antibody, BGB-A445 co-stimulated CD4+ T-cells to secrete IL-2 dose-dependently, while MOXR0916 did not. In the MC38 colon cancer model in human OX40 knock-in mice, BGB-A445 demonstrated remarkable anti-tumor efficacy in a dose-dependent manner, while MOXR0916 showed a ‘hook effect’ in the same setting. In addition, BGB-A445 exhibited significant anti-tumor activity in the PAN02 pancreatic model which is resistant to anti-PD-1 treatment. Besides, BGB-A445 revealed significant combination effects with anti-PD-1 therapy in both MC38 and CT26WT models. Conclusions In conclusion, differentiated from current clinical stage anti-OX40 antibodies, BGB-A445 is an agonistic antibody that does not block the OX40-OX40L interaction. Both in vitro and in vivo results demonstrated that BGB-A445 has remarkable immune stimulating effect and anti-tumor efficacy either as a single agent or in combination with anti-PD-1 therapy, thus warranting further clinical investigation. References Croft M. Control of immunity by the TNFR-related molecule OX40 (CD134). Annu Rev Immunol 2010;28:57–78. Gramaglia I, et al. Ox-40 ligand: a potent costimulatory molecule for sustaining primary CD4 T cell responses. J Immunol 1998;161:6510–6517. Song J, So T, Croft M. Activation of NF-kappaB1 by OX40 contributes to antigen-driven T cell expansion and survival. J Immunol 2008;180:7240–7248.

Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor expressed by activated T, B, and NK cells, which interacts with its ligand PD-L1/L2 to inhibit T-cell proliferation and effector functions such as tumor cell killing and cytokine production. Two anti-PD-1 antibodies approved by the FDA, pembrolizumab and nivolumab, have shown efficacy in many cancer types, nevertheless there are some indications where limited efficacy is observed. Tislelizumab (BGB-A317), an investigational anti-PD-1 antibody, has demonstrated significant clinical activity (85.7% ORR, including 61.4% CR) in relapsed/refractory classical Hodgkin’s lymphoma (R/R cHL). Additionally, tislelizumab is being studied in global pivotal trials in a number of malignancies, including non-small cell lung cancer, hepatocellular carcinoma, and esophageal squamous cell carcinoma. However, how tislelizumab binds to PD-1 has yet to be shown, particularly in comparison to pembrolizumab and nivolumab. Here we report the co-crystal structure of PD-1 extracellular domain and the Fab of tislelizumab. Tislelizumab interacts with IgV-like domain of PD-1 with an interface area of 1112 Å2. Structure-guided mutagenesis of PD-1 and surface plasmon resonance were performed to compare the binding of tislelizumab, pembrolizumab and nivolumab to mutant and wild type PD-1. The dissociation rate (kd) of tislelizumab from wild type PD-1 is about 100-fold and 50-fold slower than that of pembrolizumab and nivolumab, respectively. Gln75, Thr76, Asp77 and Arg86 on PD-1 are critical epitopes for tislelizumab, but mutation of them showed little effect on binding of PD-1 to pembrolizumab and nivolumab. Both the co-crystal structure and mutagenesis study identified the unique epitopes of tislelizumab that correlate to the extremely slow-off property of tislelizumab after binding to PD-1. In conclusion, we observed that tislelizumab is differentiated from pembrolizumab and nivolumab by its unique binding epitopes as well as binding kinetics. Citation Format: Yingcai Feng, Yuan Hong, Hanzi Sun, Bo Zhang, Hongfu Wu, Kang Li, Xuesong (Mike) Liu, Ye Liu. The molecular binding mechanism of tislelizumab, an investigational anti-PD-1 antibody, is differentiated from pembrolizumab and nivolumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2383.

SummaryThe obligate intracellular pathogen Chlamydia trachomatis is the most common cause of sexually transmitted bacterial disease. It secretes a protease known as chlamydial protease/proteasome-like activity factor (CPAF) that degrades many host molecules and plays a major role in Chlamydia pathogenesis. Here, we show that mature CPAF is a homodimer of the catalytic domains, each of which comprises two distinct subunits. Dormancy of the CPAF zymogen is maintained by an internal inhibitory segment that binds the CPAF active site and blocks its homodimerization. CPAF activation is initiated by trans-autocatalytic cleavage, which induces homodimerization and conformational changes that assemble the catalytic triad. This assembly leads to two autocatalytic cleavages and removal of the inhibitory segment, enabling full CPAF activity. CPAF is covalently bound and inhibited by the proteasome inhibitor lactacystin. These results reveal the activation mechanism of the CPAF serine protease and suggest new opportunities for anti-Chlamydia drug development.