T cell engagers display meaningful, but limited, clinical activity often associated with toxicity. Activating innate immune cells is considered a safer, yet efficacious, alternative to T cells. We report here the preclinical development of AFVT-2101, a novel bispecific innate cell engager designed to induce tumor killing by high avidity binding to CD16A on innate cells and folate receptor alpha (FRα), a tumor-associated antigen with upregulated expression on certain solid tumor cells. Binding of AFVT-2101 to CD16A and FRα was assessed by ELISA and flow cytometry in the presence of physiological levels of IgG. The antitumor activity of the innate engager was evaluated by antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in vitro assays, using purified NK cells or differentiated macrophages as effector cells against tumor cell lines with varying levels of FRα expression. In vivo studies were conducted in transgenic mice expressing human CD16A on myeloid cells and transplanted with FRα-expressing cancer cell lines. AFVT-2101 binds to CD16A and FRα with an apparent avidity of 0.1 nM and 0.05 nM, respectively. Physiological levels of human IgG do not alter binding kinetics. AFVT-2101 induces potent and selective ADCC and ADCP, even on FRαlow cancer cells. Further, AFVT-2101 is more efficacious and potent in both ADCC and ADCP assays than farletuzumab, an Fc-competent monoclonal antibody targeting FRα, which shares the same VH/VL sequence as AFVT-2101. In in vivo experiments, AFVT-2101 is active and significantly inhibits the growth of FRα+ tumors. Ex vivo flow cytometry analyses show a repolarization of the tumor microenvironment (TME) with reduction of the frequency of tumor-associated macrophages upon treatment with AFVT-2101. A reduction of FRα+ tumor cells in groups receiving therapy was also observed. In conclusion, AFVT-2101 binds selectively to CD16A and FRα, even in the presence of physiological levels of IgG, resulting in a potent and selective elimination of FRα+ tumor cells through the engagement of NK cell cytotoxicity (ADCC) and macrophage phagocytosis (ADCP). The high avidity for CD16A likely imparts increased potency and efficacy compared to the FRα-specific antibody farletuzumab. Moreover, the innate immune engager significantly restrains in vivo tumor growth and induces pharmacodynamic changes in line with TME repolarization. AFVT-2101 engages innate immune cells in a target-restricted manner and potentially imparts an efficacious clinical profile with favorable tolerability. Citation Format: Nicolo' Rigamonti, Jan Schmollinger, Peter Sandy, Michael Tomaszowski, Josef Caslavsky, Ahmad Trad, Daniel O’Shannessy, Jana Siegler, Eric J. Gaukel, Daniela Penston, Uwe Reusch, Zoë Johnson. AFVT-2101: A FRα × CD16A bispecific innate cell engager for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1811.