Generation and characterization of novel co-stimulatory anti-mouse TNFR2 antibodies.

Tumor necrosis factor receptor 2 (TNFR2) has gained much research interest in recent years because of its potential pivotal role in autoimmune disease and cancer. However, its function in regulating different immune cells is not well understood. There is a need for well-characterized reagents to selectively modulate TNFR2 function, thereby enabling definition of TNFR2-dependent biology in human and mouse surrogate models. Here, we describe the generation, production, purification, and characterization of a panel of novel antibodies targeting mouse TNFR2. The antibodies display functional differences in binding affinity and potency to block TNFα. Furthermore, epitope binding showed that the anti-mTNFR2 antibodies target different domains on the TNFR2 protein, associated with varying capacity to enhance CD8+ T-cell activation and costimulation. Moreover, the anti-TNFR2 antibodies demonstrate binding to isolated splenic mouse Tregs ex vivo and activated CD8+ cells, reinforcing their potential use to establish TNFR2-dependent immune modulation in translational models of autoimmunity and cancer.

Functional characterization of the selective pan-allele anti-SIRPα antibody ADU-1805 that blocks the SIRPα-CD47 innate immune checkpoint.

BACKGROUND: Accumulating preclinical data indicate that targeting the SIRPα/CD47 axis alone or in combination with existing targeted therapies or immune checkpoint inhibitors enhances tumor rejection. Although several CD47-targeting agents are currently in phase I clinical trials and demonstrate activity in combination therapy, high and frequent dosing was required and safety signals (acute anemia, thrombocytopenia) were recorded frequently as adverse events. Based on the restricted expression pattern of SIRPα we hypothesized that antibodies targeting SIRPα might avoid some of the concerns noted for CD47-targeting agents. METHODS: SIRPα-targeting antibodies were generated and characterized for binding to human SIRPα alleles and blockade of the interaction with CD47. Functional activity was established in vitro using human macrophages or neutrophils co-cultured with human Burkitt's lymphoma cell lines. The effect of SIRPα versus CD47 targeting on human T-cell activation was studied using an allogeneic mixed lymphocyte reaction and a Staphylococcus enterotoxin B-induced T-cell proliferation assay. Potential safety concerns of the selected SIRPα-targeting antibody were addressed in vitro using a hemagglutination assay and a whole blood cytokine release assay, and in vivo in a single-dose toxicity study in cynomolgus monkeys. RESULTS: The humanized monoclonal IgG2 antibody ADU-1805 binds to all known human SIRPα alleles, showing minimal binding to SIRPß1, while cross-reacting with SIRPγ, and potently blocking the interaction of SIRPα with CD47. Reduced FcγR binding proved critical to retaining its function towards phagocyte activation. In vitro characterization demonstrated that ADU-1805 promotes macrophage phagocytosis, with similar potency to anti-CD47 antibodies, and enhances neutrophil trogocytosis. Unlike CD47-targeting agents, ADU-1805 does not interfere with T-cell activation and is not expected to require frequent and extensive dosing due to the restricted expression of SIRPα to cells of the myeloid lineage. ADU-1805 is cross-reactive to cynomolgus monkey SIRPα and upon single-dose intravenous administration in these non-human primates (NHPs) did not show any signs of anemia, thrombocytopenia or other toxicities. CONCLUSIONS: Blocking the SIRPα-CD47 interaction via SIRPα, while similarly efficacious in vitro, differentiates ADU-1805 from CD47-targeting agents with respect to safety and absence of inhibition of T-cell activation. The data presented herein support further advancement of ADU-1805 towards clinical development.