Moore, P. F. | 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↗

bioRxiv2024

Single cell RNA-sequencing of feline peripheral immune cells with V(D)J repertoire and cross species analysis of T lymphocytes

IntroductionThe domestic cat (Felis catus) is a valued companion animal and a model for virally induced cancers and immunodeficiencies. However, species-specific limitations such as a scarcity of immune cell markers constrain our ability to resolve immune cell subsets at sufficient detail. The goal of this study was to characterize circulating feline T cells and other leukocytes based on their transcriptomic landscape and T-cell receptor repertoire using single cell RNA-sequencing. MethodsPeripheral blood from 4 healthy cats was enriched for T cells by flow cytometry cell sorting using a mouse anti-feline CD5 monoclonal antibody. Libraries for whole transcriptome, alpha/beta T cell receptor transcripts and gamma/delta T cell receptor transcripts were constructed using the 10x Genomics Chromium Next GEM Single Cell 5 reagent kit and the Chromium Single Cell V(D)J Enrichment Kit with custom reverse primers for the feline orthologs. ResultsUnsupervised clustering of whole transcriptome data revealed 7 major cell populations - T cells, neutrophils, monocytic cells, B cells, plasmacytoid dendritic cells, mast cells and platelets. Sub cluster analysis of T cells resolved naive (CD4+ and CD8+), CD4+ effector T cells, CD8+ cytotoxic T cells and gamma/delta T cells. Cross species analysis revealed a high conservation of T cell subsets along an effector gradient with equitable representation of veterinary species (horse, dog, pig) and humans with the cat. Our V(D)J repertoire analysis demonstrated a skewed T-cell receptor alpha gene usage and a restricted T-cell receptor gamma junctional length in CD8+ cytotoxic T cells compared to other alpha/beta T cell subsets. Among myeloid cells, we resolved three clusters of classical monocytes with polarization into pro- and anti-inflammatory phenotypes in addition to a cluster of conventional dendritic cells. Lastly, our neutrophil sub clustering revealed a larger mature neutrophil cluster and a smaller exhausted/activated cluster. DiscussionOur study is the first to characterize subsets of circulating T cells utilizing an integrative approach of single cell RNA-sequencing, V(D)J repertoire analysis and cross species analysis. In addition, we characterize the transcriptome of several myeloid cell subsets and demonstrate immune cell relatedness across different species.

immunology↗