Abstract 3270: Mechanism of action of a novel agonist TNFR2 antibody that induces co-stimulation of T cells and promotes robust anti-tumor immunity

Abstract

Abstract TNFR2 is a member of the TNF receptor superfamily that is upregulated upon T cell activation and is highly expressed by tumor-infiltrating effector and regulatory T cells. We investigated TNFR2 levels on T cells in syngeneic mouse tumor models and in secondary lymphoid tissues by flow cytometry. Non-regulatory T cells in the spleen and lymph nodes expressed little TNFR2 whereas Tregs constitutively expressed intermediate levels. In contrast, tumor-infiltrating effector T cells expressed high levels of TNFR2 with Tregs expressing the highest levels. To investigate TNFR2 as a therapeutic target, we generated a novel monoclonal antibody specific to murine TNFR2 and investigated its mechanism of action. Antibodies against murine TNFR2 were generated by screening a human antibody-display library or by rabbit immunization. Antibodies were assessed for affinity, ability to compete with TNFα and for developability. A select number of antibodies were expressed as murine IgG2a and evaluated for activity in multiple syngeneic mouse tumor models. The mechanism of action of the most active clone, Y9, was investigated further. In vitro, Y9 stimulation of purified T cells from healthy mice caused increased proliferation and effector function, indicating that Y9 acts as an agonist and can provide co-stimulation. In vivo, Y9 treatment of mice with established tumors resulted in complete tumor clearance across a variety of models. Using CRISPR knockout cell lines, we showed that Y9 activity did not depend on TNFR2 expression on tumor cells. However, it required T cells as it showed no activity in nude mice. The activity of Y9 on immune cells was further confirmed by its decreased activity in mice depleted of NK or CD8+ T cells. Unlike the proposed Treg-depletion mechanism for other co-stimulatory therapeutic antibodies, depletion of Tregs is not the primary mechanism of action of Y9 treatment. Instead, decreased TNFR2 and other co-inhibitory receptor surface expression was observed following treatment. Y9 activity depended on FcγR binding as demonstrated by the lack of activity of an antibody variant with mutations preventing FcγR binding. We showed further that FcγR binding facilitated enhanced agonist activity by comparing activity of Y9 variants with different Fc isotypes and in FcγR knockout mice. We present a novel anti-TNFR2 antibody that exhibited pronounced anti-tumor in vivo activity in our mouse models with co-stimulation of tumor-specific T cells as its dominant mechanism of action. A corresponding human anti-TNFR2 antibody (MM-401) has been identified and is being developed as a potential novel treatment option for cancer patients. Citation Format: Ross B. Fulton, Adam Camblin, James F. Sampson, Jennifer Richards, Christina Wong, Alexander Koshkaryev, Lia Luus, Yang Jiao, Lihui Xu, Violette Paragas, Maja Razlog, Marco Muda, Eric M. Tam, Daryl C. Drummond, Andreas Raue. Mechanism of action of a novel agonist TNFR2 antibody that induces co-stimulation of T cells and promotes robust anti-tumor immunity [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 3270.