Bispecific antibodies, while showing great therapeutic potential, pose formidable challenges with respect to their assembly, stability, immunogenicity, and pharmacodynamics. Here we describe a novel class of bispecific antibodies with native human immunoglobulin format. The design exploits differences in the affinities of the immunoglobulin isotypes for Protein A, allowing efficient large-scale purification. Using this format, we generated a bispecific antibody, REGN1979, targeting the B cell marker, CD20, and the CD3 component of the T cell receptor, which triggers redirected killing of B cells. In mice, this antibody prevented growth of B cell tumors and also caused regression of large established tumors. In cynomolgus monkeys, low doses of REGN1979 caused prolonged depletion of B cells in peripheral blood with a serum half-life of approximately 14 days. Further, the antibody induced a deeper depletion of B cells in lymphoid organs than rituximab. This format has broad applicability for development of clinical bispecific antibodies.
Abstract T-cell-redirecting bispecific antibodies have emerged as a new class of therapeutic agents designed to simultaneously bind to T cells via CD3 and to tumor cells via tumor-cell-specific antigens (TSA), inducing T-cell-mediated killing of tumor cells. The promising preclinical and clinical efficacy of TSAxCD3 antibodies is often accompanied by toxicities such as cytokine release syndrome due to T-cell activation. How the efficacy and toxicity profile of the TSAxCD3 bispecific antibodies depends on the binding affinity to CD3 remains unclear. Here, we evaluate bispecific antibodies that were engineered to have a range of CD3 affinities, while retaining the same binding affinity for the selected tumor antigen. These agents were tested for their ability to kill tumor cells in vitro, and their biodistribution, serum half-life, and anti-tumor activity in vivo. Remarkably, by altering the binding affinity for CD3 alone, we can generate bispecific antibodies that maintain potent killing of TSA + tumor cells but display differential patterns of cytokine release, pharmacokinetics, and biodistribution. Therefore, tuning CD3 affinity is a promising method to improve the therapeutic index of T-cell-engaging bispecific antibodies.
Abstract T-cell redirecting bispecific antibodies are an emerging class of therapeutic agents designed to simultaneously bind to T cells (via CD3) and tumor cell specific antigens (TSA), with the goal of inducing T cell-mediated killing of tumor cells. Despite promising pre-clinical and clinical efficacy from TSAxCD3 antibodies, these agents have associated toxicities that remain challenging. Here we present findings from a series of bispecific antibodies that were engineered to have a range of CD3 affinities, but which retained the same binding affinity to the selected tumor antigen. These agents were tested for killing of tumor cells in vitro, and for biodistribution, serum half-life, and anti-tumor activity in vivo. By altering the binding strength for CD3, bispecifics were generated that maintained potent killing of TSA+ cells but displayed differential profiles of cytokine release, pharmacokinetics, and biodistribution. Our results suggest that tuning CD3 affinity is a promising method to improve the therapeutic window of T cell-engaging bispecific antibodies. Citation Format: Lauric Haber, Kara Olson, Robert Babb, Marcus Kelly, Alison Crawford, Marc Retter, David DiLillo, Erica Ullman, Jennifer Finney, Lauren Canova, Arpita Pawashe, Danica Chiu, Kristin Vazzana, Priyanka Ram, Katja Mohrs, Amanda Dorvilliers, Jenny Xiao, Sosina Makonnen, Carlos Hickey, Cody Arnold, Jason Giurleo, Supriya Patel, Richard Tavare, Ya Ping Chen, Gang Chen, William Olson, Gavin Thurston, John Chia-Yang Lin, Aynur Hermann, Jessica Kirshner, Eric J. Smith. Selection of CD3 affinity allows generation of T-cell redirecting bispecific antibodies with unique pharmacokinetic and biodistribution properties [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 4555.
Abstract Advanced ovarian cancer has a high rate of recurrence, thus there is a need for therapies that can produce durable responses and extend overall survival. Infiltrating CD3+ T cells are reported to correlate with improved clinical outcome in stage III/IV ovarian cancer. Bispecific antibodies that engage T cells via CD3 and a tumor antigen on ovarian tumor cells may be able to exploit these tumor-infiltrating T cells and have drawn interest as a novel strategy of antitumor immunotherapy. Mucin16 (MUC16) is a well described antigen highly expressed in ovarian cancer and several other tumors. We generated a human bispecific antibody (REGN4018) that binds MUC16 on tumor cells and CD3, bridging MUC16-expressing cells with CD3+ T cells. REGN4018 demonstrates MUC16-directed T cell activation and polyclonal T cell killing of MUC16-expressing tumor cells in vitro and in vivo. Binding and cytotoxicity are minimally affected by high concentrations of CA-125, the shed form of MUC16 that is used as a biomarker for ovarian cancer. Several murine tumor models were developed to determine the in vivo anti-tumor effects of REGN4018. As a xenogenic tumor model, human OVCAR-3 cells were grown as ascites in immunodeficient mice [NOD-SCID-IL2Rgamma deficient (NSG)] pre-implanted with human PBMC. Significant tumor inhibition by REGN4018 was observed at doses of ≥ 0.1 mg/kg. To enable investigation of clinical bispecific antibodies in tumor-bearing immune competent mice, mice were genetically engineered to humanize both CD3 and a part of MUC16 covering the antibody binding region. ImmunoPET imaging demonstrated localization of REGN4018 in both T cell-rich organs such as the spleen and lymph nodes as well as in MUC16-expressing tumors. REGN4018 inhibited growth of murine tumors transfected with human MUC16 in syngeneic tumor models at doses ≥ 0.05 mg/kg. The safety and tolerability of REGN4018 were evaluated in cynomolgous monkey studies. REGN4018 administration resulted in a minimal and transient increase in serum cytokines and C-reactive protein with no overt toxicity. Collectively, these data show the potent anti-tumor activity and tolerability of REGN4018 and provide strong support for the clinical testing of REGN4018 in patients with MUC16-expressing ovarian cancers. Citation Format: Alison Crawford, Lauric Haber, Kristin Vazzana, Lauren Canova, Priyanka Ram, Jennifer Principio, Arpita Pawashe, Curtis Colleton, Marcus Kelly, Sosina Makkonen, Carlos Hickey, Paurene Duramad, Stephen Godin, John Lin, Eric Smith, Gavin Thurston, Jessica R. Kirshner. REGN4018, a novel MUC16xCD3 bispecific T-cell engager for the treatment of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1777.
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