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Background The clinical efficacy of bispecific T cell engagers for treatment of solid tumors remains limited due to poor immune cell infiltration, lack of truly tumor-specific antigens, and insufficient costimulatory signaling required for sustained T cell activation. To address these challenges, we developed a combinatorial approach utilizing two functionally interrelated bispecific antibodies (bsAbs): one construct activates T cells by CD3 engagement, while the other provides costimulatory signaling via CD28. The two bsAbs target different antigens expressed on tumor cells and tumor vessels/stroma.Methods We engineered a bsAb targeting fibroblast activation protein (FAP) and CD28, designated BiCo-3. This molecule is combined with our CD276xCD3 bsAb CC-3, which is presently undergoing clinical investigation as single agent ( NCT05999396). FAP is predominantly expressed in tumor-associated fibroblasts within the stroma, whereas CD276 is broadly expressed on tumor cells and neovascularization. T cell activation, proliferation, cytokine production, and tumor cell killing upon combinatorial versus single agent treatment was assessed in meaningful in vitro assays. Efficacy and safety in vivo were tested using xenogeneic tumor models with human immune cell engraftment as amongst others by using surrogate bsAbs targeting murine FAP.Results BiCo-3 provided CD28-mediated costimulation strictly dependent on binding to FAP-expressing stromal cells, while CC-3 facilitated CD3-dependent activation of T cells against CD276-positive tumor cells. In co-culture assays using PBMC and target cell populations, the combinatorial treatment induced profoundly more sustained T cell anti-tumor immunity compared to CD3 bsAb alone. BiCo-3 effectively reinforced T cell function over a broad range of tumor-to-stroma cell ratios. Experimental settings in which either antigen was expressed exclusively on one target cell population, i.e. CD276-positive/FAP-negative tumor cells and CD276-negative/FAP-positive fibroblasts, documented that the combination therapy elicits robust T cell proliferation and tumor cell lysis. In a LNCaP (CD276-positive/FAP-negative) xenograft model, a murine surrogate version of BiCo-3 which binds to murine FAP reinforced T cell reactivity induced by the crossreactive CD276xCD3 bsAb CC-3. This resulted in eradication of established tumors, documenting the efficacy of BiCo-3 and CC-3 upon targeting the tumor microenvironment.Conclusions Combining bsAbs stimulating CD3 and CD28 enables robust T cell activation and anti-tumor responses even when targeting two different antigens expressed on distinct tumor and stromal cell compartments. BiCo-3 enhances efficacy of CD3-directed bsAbs across a broad range of concentrations and tumor-to-stroma ratios while preserving specificity and safety. Our dual-signal approach presents a promising strategy to overcome key limitations of CD3-directed bsAb therapy in solid tumors.Ethics Approval The study was approved by the IRB (Ethics Committee of the Medical Faculty of the Eberhard Karls Universitaet Tuebingen) at the University Hospital Tuebingen and was conducted in accordance with the Declaration of Helsinki; reference number 184/2023BO. Human material was collected after informed consent was obtained.