Intratumoral B7H3:CD3 Bispecific T-cell Engager Drives Localized T-cell Accumulation in Canine Sarcoma Patients

Abstract

BackgroundBispecific T-cell Engagers (TCEs) targeting B7H3 (CD276) show promise for solid tumors but are limited by systemic toxicities and poor tumor penetration. Intratumoral (IT) delivery is proposed as a solution, but the safety and spatial pharmacodynamics (PD) remain poorly defined in these malignancies. Spontaneous canine tumors serve as a highly translatable model for human therapeutic development due to its clinical, genetic, and immunological similarities to human patients. This study evaluates the feasibility of an IT-delivered B7H3:CD3 TCE in a trial that enrolls companion dogs with solid tumors. MethodsWe engineered a canine B7H3:CD3 TCE and validated its ability to induce T-cell activation and T-cell mediated cytotoxicity in vitro on several B7H3-expressing canine tumor cell lines. Two STS canine patients received intratumoral columnar injections of the TCE and saline (internal control) at fixed distance of 1.5cm using a custom-engineered multi-needle assembly. Safety was evaluated by physical examinations and hematological and biochemical changes in peripheral blood. PD response was analyzed by H&E and immunohistochemistry. ResultsIn vitro assays validated the cytotoxicity of the B7H3:CD3 TCE on B7H3+ canine tumor cell lines. TCE IT administration (7.83 g / 148.2 pmol) was well tolerated with no adverse events greater than Grade 1 and no evidence of systemic cytokine release or organ toxicity. Immunohistochemistry of tumors collected 7 days after TCE administration revealed a significant five-fold increase in CD3+ T-cell density at the TCE injection site (within 0.5 cm radius) compared to internal saline controls. ConclusionsThis study demonstrated the feasibility of evaluating pharmacodynamic response to IT delivery of B7H3:CD3 TCE, namely local T-cell accumulation. T-cell localization around the TCE injection site supports our hypothesis that effective IT immunotherapy might require enhanced volumetric coverage using multi-needle injections and/or co-stimulatory strategies to convert T-cell localization into a robust, sustained anti-tumor response.