The novel CD16A/anti-CD3 bifunctional protein, eCD16A/anti-CD3-BFP, redirects T cell cytotoxicity toward antibody-bound target cells

Monoclonal antibodies enhance innate immunity, while bispecific T cell engager antibodies redirect adaptive T cell immunity. To stimulate both innate and adaptive mechanisms, we created a bifunctional eCD16A/anti-CD3-BFP adapter protein for combined use with clinically approved monoclonal IgG1 antibodies. The adaptor protein contains the extracellular domain of the human CD16A high-affinity variant, which binds the Fc domain of IgG1 antibodies, and an anti-human CD3 single-chain variable fragment that redirects T cell cytotoxicity. Functional characterization of eCD16A/anti-CD3-BFP was performed using in vitro, ex vivo, and in vivo animal assays. Combination treatments of eCD16A/anti-CD3-BFP with rituximab (anti-CD20), cetuximab (anti-EGFR), trastuzumab (anti-HER2) or anti-PD-L1 IgG1 led to specific killing of antigen-expressing tumor cells. In an ex vivo assay, eCD16A/anti-CD3-BFP combined with rituximab effectively eliminated B cells within peripheral blood samples from healthy donors and cancer patients; the effectiveness of this treatment was further enhanced by addition of either autologous or allogeneic γ9δ2 T cells. In mouse xenograft studies, the combination of eCD16A/anti-CD3-BFP and rituximab led to rapid depletion of tumor cells. The presence of non-targeting competing serum immunoglobulins interfered with efficacy, but this interference could be overcome by eCD16A/anti-CD3-BFP dose escalation or by using a Fc-glycoengineered version of rituximab. Moreover, combining eCD16A/anti-CD3-BFP with anti-Epstein Barr virus (EBV) antibodies directed T cell cytotoxicity toward EBV-infected cells. These findings support further development of eCD16A/anti-CD3-BFP as a novel anti-cancer therapeutic to stimulate both innate and adaptive immunity.