BackgroundTumorous heterogeneity is a hallmark of tumor evolution and cancer progression, being a longstanding challenge to targeted immunotherapy. Ex vivo armed T cells (EATs) using IgG-(L)-scFv bispecific antibodies (BsAbs) are potent tumor-specific cytotoxic effectors. To improve the anti-tumor efficacy of EATs against heterogeneous solid tumors, we explored multi-antigen targeting approaches.MethodsEx vivo expanded T cells were armed with BsAbs built on the IgG-(L)-scFv platform, where an anti-CD3 (huOKT3) scFv was attached to the carboxyl end of both light chains of a tumor specific IgG. Multispecificity was created by combining monospecific EATs, combining BsAbs on the same T cell, or combining specificities on the same antibody. Three multi-antigens targeting EAT strategies were tested: (1) pooled-EATs (EATs each with unique specificity administered simultaneously) or alternate-EATs (EATs each with unique specificity administered in an alternating schedule), (2) dual-EATs or multi-EATs (T cells simultaneously armed with ≥2 BsAbs), and (3) TriAb-EATs (T cells armed with BsAb specific for two targets besides CD3 (TriAb)). The properties and efficiencies of these three strategies were evaluated by flow cytometry, in vitro cytotoxicity, cytokine release assays, and in vivo studies performed in BALB-Rag2^-/-IL-2R-γc-KO (BRG) mice xenografted with cancer cell line (CDX) or patient-derived tumor (PDX).ResultsMulti-EATs retained target antigen specificity and anti-tumor potency. Cytokine release with multi-EATs in the presence of tumor cells was substantially less than when multiple BsAbs were mixed with unarmed T cells. When tested against CDXs or PDXs, dual-EATs or multi-EATs effectively suppressed tumor growth without clinical toxicities. Most importantly, dual-EATs or multi-EATs were highly efficient in preventing clonal escape while mono-EATs or TriAb- EATs were not as effective.ConclusionsMulti-EATs have the potential to increase potency, reduce toxicity, and overcome tumor heterogeneity without excessive cytokine release. Arming T cells with multiple BsAbs deserves further exploration to prevent or to treat cancer resistance.