1166 Avidity engineered multifunctional antibodies that strongly activate and expand anti-tumor immunity in situ, turning cold patient tumor tissue hot and exerting durable tumor control in mice

Background Antibodies and ADCs are mainstays in the treatment of cancer. However, given difficulties in achieving a deep and sustained response, significant improvements are desirable. We report on first in class ‘Booster’ molecules, based on clinically validated ADCC-competent antibodies, equipped with two immunomodulatory domains that uniquely combine co-stimulation and cytokine signaling, and are affinity engineered to be functional only when in contact with a tumor cell. We see strong and durable expansion without exhaustion and increased cytotoxicity of immune cells in the presence of cancer cells in vitro, broad activation of anti-tumor immune cell types in patient tumors ex vivo, and durable reduction of tumor burden in vivo, with significantly better activity than adoptive cell therapy or control antibody without fusion domains.Methods NRG mice were engrafted with luciferase expressing tumor cells via intraperitoneal injection 7 days prior to treatment. Mice received 1 million NK cells freshly isolated from healthy donors. Compounds were administered biweekly, and low dose IL2 thrice weekly. NK cell expansion was quantified in blood and tumor burden monitored monitored via bioluminescence. ex vivo: in situ activation of tumor-resident immune populations was evaluated by nanostring in freshly isolated tumor tissue. in vitro: long-term cytotoxicity was measured by quantifying live tumor cells using automated microscopy with regular co-culture re-sets. Expansion was performed by stimulating NK cells weekly with tumor cells that were opsonized with Booster or antibody.Results Mice treated with Her2, TROP2, or EGFR Boosters demonstrated superior tumor control than trastuzumab, sacituzumab, or cetuximab analogue treated mice respectively. Boosters induced superior and sustained NK cell expansion in mice, with up to 5000x higher NK numbers compared to the relevant control antibody analogue. Our Boosters reprogram the full immune microenvironment ex vivo in freshly isolated patient tumor tissue, transforming a cold tumor into a hot tumor. They activate multiple cytotoxic pathways in three different tumor types. In separate in vitro assays we saw sustained expansion and enhanced long-term cytotoxicity of NK cells for at least 6 weeks.Conclusions In correlation with extensive in vitro and ex vivo data, we observe a prolonged and significant improvement in tumor control in mice treated with Boosters compared to mice treated with parent antibody analogues. Work is ongoing to develop these molecules, with preparations ongoing to bring them to the clinic.

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