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.

The ability to evade apoptosis is a hallmark of cancer. B-cell lymphoma-2 (Bcl-2), an anti-apoptosis protein, is overexpressed and leads to oncogenesis or drug resistance in various tumor types, including lymphoma and leukemia. Bcl-2 is a well-validated target for B cell malignancies as demonstrated by a Bcl-2 inhibitor venetoclax which was recently approved for the treatment of chronic lymphocytic leukemia (CLL) and is currently in phase III clinical development for other hematologic malignancies. With longer term treatment, recurrent mutation G101V in Bcl-2 has been reported to mediate resistance to venetoclax in patients with CLL. Herein, we report the pharmacological properties of BGB-11417, a highly potent and selective BCL-2 inhibitor, in preclinical models. BGB-11417 potently inhibited both wildtype and G101V-mutated Bcl-2 in SPR binding assay with IC50 of 0.035 and 0.28 nM, respectively. BGB-11417 was a more potent Bcl-2 inhibitor than venatoclax in both enzymatic and cellular assays. In a binding assay for BH3 peptide and Bcl-2, BGB-11417 and venatoclax showed an IC50 of 0.014 nM and 0.2 nM, respectively. In addition, BGB-11417 potently inhibited the proliferation of a Bcl-2 dependent acute lymphoblastic leukemias (ALL) cell line RS4;11 with an IC50 of 0.81 nM, but not a Bcl-xL dependent T-ALL cell line Molt-4. Moreover, BGB-11417 exhibited potent cell killing activity against a variety of lymphoma and leukemia cell lines, including follicular lymphomas (FL), mantle cell lymphomas (MCL), diffuse large B-cell lymphomas (DLBCL) and acute myeloid leukemias (AML). BGB-11417 was also highly selective, showing ≥2000 folds selectivity to Bcl-xL, BCL-W, MCL-1 and BCL2A1. In pharmacokinetics (PK) and pharmacodynamics (PD) studies, oral administration of BGB-11417 displayed a clear PK and PD correlation in RS4;11 ALL xenografts as shown by the increase of cleaved caspase 3 level with the increase of BGB-11417 concentration in tumor tissue. Furthermore, BGB-11417 demonstrated significantly greater efficacy than venetoclax in human ALL, MCL and DLBCL xenograft models without body weight loss. Collectively, BGB-11417 is a potent and highly selective Bcl-2 inhibitor with superior anti-tumor activities compared with venetoclax in preclinical studies. The phase I study of BGB-11417 for treatment of hematological cancers is ongoing. Citation Format: Nan Hu, Yunhang Guo, Hai Xue, Ye Liu, Yin Guo, Fan Wang, Xiaomin Song, Ying Guo, Shuaishuai Chen, Haipeng Xu, Taichang Zhang, Yanwen Ma, Xuebing Sun, Yuan Hong, Yutong Zhu, Aiying Xu, Zhenzhen Cheng, Haimei Xing, Zhiwei Wang, Xuesong Liu, Lai Wang. Preclinical characterization of BGB-11417, a potent and selective Bcl-2 inhibitor with superior antitumor activities in haematological tumor models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3077.

BackgroundOX40 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.MethodsCell-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.ResultsThe 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.ConclusionsIn 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.ReferencesCroft 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.

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CD4 T cells are known to help the induction, migration, and effector functions of CD8 antitumor responses. Here, we reported an additional layer of CD4 helper function, i.e. activation of endogenous CD4 T cells could modulate the tumor milieu. We engineered a lentivector to express a nominal fusion Ag composed of hepatitis B surface protein (HBsAg) and IgG2a Fc domain (HBS-Fc-lv) that induced potent CD8 as well as robust CD4 response. Remarkably, immunization with this vector caused regression of established tumors. Immunological analysis revealed that functional CD8 and CD4 T effector (Teff) was markedly increased while the Treg ratio was substantially decreased in the regressing tumors. HBS-Fc-lv immunization also shifted the suppressive tumor lesion microenvironment to one that is immune stimulating. Mechanistically, the decrease of tumor Treg ratio and the improvement of antitumor effect of HBS-Fc-lv immunization were dependent on CD4 activation and IFNg production. CD4 Teff cells were preferentially expanded in the vaccine draining lymph nodes and selectively recruited into tumor lesions as the chemokines for Teff recruitment were significantly increased in the tumor lesions after HBS-Fc-lv immunization. These data indicate that immunization strategies capable of activating both CD8 and CD4 T cells can improve the tumor milieu and generate an enhanced antitumor effect.