Brasel, K. | bioRxiv Science

bioRxiv2026

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

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.

immunology↗

bioRxiv2025

Efficacy of DNA Methyltransferase Inhibitor Immune Priming Therapy in Combination with PD-1 Inhibitors to Treat High-Risk Pediatric Brain Tumors

BackgroundDespite intensive therapies, outcomes for high-risk pediatric brain tumors (PBTs) remain dismal, prompting the search for novel treatments. DNA methyltransferase inhibitors (DNMTi) have been shown to prime tumors to improve response to checkpoint inhibition. The aim of this study was to investigate the potential of decitabine (DAC), in combination with a PD-1 inhibitor, to improve survival in pediatric high-risk brain tumor models. MethodsAnalysis of human PBT datasets was performed to determine gene expression levels of immune cell associated markers. Tumor response to DAC, with or without a PD-1 inhibitor, was tested in murine models representing H3-wildtype diffuse intrinsic pontine glioma (DIPG), H3K27-mutant diffuse midline glioma (DMG), atypical teratoid rhabdoid tumor (ATRT), and medulloblastoma (MB). CyTOF analysis of allograft tumors was performed to characterize changes within the tumor microenvironment. ResultsAnalysis of PBT subtypes revealed heterogeneous expression of immune cell markers, checkpoint receptors, and MHC molecules. DAC treatment decreased DNA methylation and increased neoantigen expression in human and mouse tumor cells. DAC alone or in combination with a PD-1 inhibitor resulted in prolonged survival in syngeneic mouse models of DIPG and ATRT but not DMG and MB models. CyTOF analysis of mouse tumors revealed changes in local immune cell infiltration upon combination treatment. ConclusionsDAC in combination with a PD-1 inhibitor can alter the immune microenvironment in mouse tumor models. Changes were observed in H3-wildtype DIPG and ATRT models, suggesting that certain tumor subtypes may respond to checkpoint blockade after immune augmentation with DNMTi. Key PointsO_LIPBTs show heterogenous expression of immune cell infiltrates C_LIO_LIDAC or DAC plus a PD-1 inhibitor shows extension of survival in H3-wildtype DIPG and ATRT mouse models C_LIO_LIMyeloid-derived suppressor cell abundance could be a major contributing factor to treatment response C_LI Importance of the StudyChildren with high-risk PBTs face dismal outcomes. Immune checkpoint inhibitor (ICI) successes have been demonstrated in a variety of adult malignancies; however, such beneficial outcomes have not been realized in PBTs. Here we investigate single and combination treatment of DNMTi and PD-1 checkpoint inhibition in syngeneic mouse models of high-risk PBTs. Our results suggest that some H3-wildtype DIPG and ATRT tumor types may be responsive to checkpoint therapy post immunomodulation and warrant further investigation.

immunology↗