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Background A need remains to effectively overcome the immunosuppressive tumor microenvironment (TME) of solid tumors using novel immunotherapies, without eliciting harmful systemic toxicities. Herein we describe a novel trispecific antibody targeting the tumor-associated antigen (TAA) Nectin-4, with CD3 and CD40 as vital players in the activation and costimulatory signaling of inflammatory immune cells with the intention of bolstering the immune response to overcome TME suppression. Nectin-4 is over expressed in many tumors including lung, breast, colon and ovarian cancers.Methods Using the ADAPTIR-FLEX® platform, APVO451 was designed to have bivalent binding to Nectin-4 and monovalent binding to CD40 and CD3. Binding affinities to each domain were assessed using SPR and flow cytometry. Various in vitro coculture assays were utilized to evaluate T cell and innate cell proliferation, activation, and cytokine production using flow cytometry or multiplex bead assays. Luciferase-based reporter assays triggered via either CD3 or CD40 were used to evaluate functionality and requirement for Nectin-4-crosslinking. Live-cell imaging measured T cell-mediated tumor cell killing, mimicking the TME with the addition of immature dendritic cells (iDC) to the culture.Results As designed, APVO451 binds at a high affinity to the Nectin-4 protein but at a much lower affinity to CD3 and CD40 proteins. Based on activation and proliferation results, APVO451 augments immune cell effector phenotype compared to a CD3 T-cell engager control. As demonstrated by reporter and in vitro cell culture assays, Nectin-4-crosslinking is required for either CD3 or CD40 functionality, as neither T cells nor myeloid cells were activated in the absence of Nectin-4-expressing tumor. CD40 incorporation is critical for inducing IL-12 production and upregulating CD80/CD86 on antigen presenting cells (APCs), as demonstrated in phenotyping and cytokine release assays, resulting in enhanced T cell effector function. These results were demonstrated in imaging assays, whereby APVO451 was able to ablate the human NSCLC tumor line H2122 despite the addition of the immuno-suppressive iDCs.Conclusions The trispecific APVO451 activated T cells and converted iDCs to immuno-costimulatory cells in a Nectin-4-dependent manner overcoming the immunosuppressive environment better than a traditional T-cell engager. The CD40-directed arm of APVO451 led to the induction of IL-12 secretion and upregulation of CD80/CD86, which may play an important role in overcoming the suppression of MDSC in the TME. This trispecific antibody is a promising anti-cancer therapeutic for the treatment of various solid tumors with suppressive TME via costimulation of APC while simultaneously providing signal 1 to tumor-infiltrating T cells.