Abstract PR002: T cell engager therapy affects the spatial distribution and phenotype of T cells in the tumor microenvironment
Authors: Tomaszewski Billy , Curtis Matthew , Foo Conrad , Zhang Gu , Chang Patrick , Clark Robyn , Nguyen Thao , Webster Joshua , Rost Sandra , Jesudason Raj , Piskol Robert , Totpal Klara , McGinnis Lisa , Walsh Kevin , Ye Weilan 
Published: February 2025

Purpose: The purpose of this study is to determine how T cell engagers (TCEs) impact the localization and phenotype of key T-cell subsets in preclinical solid tumor models. Background: TCEs are a class of immunotherapy that have demonstrated substantial benefit to patients with hematologic cancers and have shown evidence of clinical activity in solid tumor malignancies. Mechanisms of response to TCEs, particularly in solid tumors, are not well understood. Identification of diagnostic strategies to stratify patients that are more likely to respond is expected to enhance the clinical benefit of TCEs. Friedrich et al. (Cancer Cell 2023) found that effector CD8 T cell subsets significantly expanded in TCE responsive but not refractory patients in multiple myeloma using single cell RNA sequencing (scRNAseq). Furthermore, the increased abundance of exhausted T cells at baseline was associated with a worse prognosis. We hypothesize that analogous populations exist in preclinical solid tumor models, which have distinct localization and cellular interactions within the TCE treated tumor microenvironment. Methods: Either hCD3 transgenic C57BL/6J or wildtype FVB mice were implanted with either ID8-LyPD1 or Fo5-HER2 tumors. Mice were grouped at a tumor volume of ∼ 300mm^3 and treated with either 10mpk of LyPD1 TCE, 5mpk of HER2 TCE, or vehicle. scRNAseq data was produced at day 1 and 4 post-treatment according to the 10X gene expression kit protocol and was processed in R using Seurat. Flow cytometry data was generated using commercially available antibodies, acquired on a BD FACSymphony and analyzed using FlowJo. Xenium data was generated using a custom probe panel according to the 10X Xenium protocol and was processed in either Python or R using the Squidpy or Seurat packages. Results: In the ID8-LyPD1 TCE model, we identified proliferative effector (CD8.pe) and terminally differentiated (CD8.Tex) CD8 populations by scRNAseq. The abundance of CD8.pe increased 3-fold and CD8.Tex expanded 2-fold upon TCE treatment. We then validated the expansion of these CD8 subsets by flow cytometry. Notably, increases in the number of CD8.pe per gram of tumor significantly correlated with reductions in tumor volume. Due to the impact of TCE treatment on these CD8 subsets and the importance of CD8.pe in tumor outcomes, we profiled the localization and cellular interactions of CD8s in preclinical solid tumors. Cell-cell communication inference analysis suggested that CD8s were more frequently targets of cellular interactions in TCE treated tumors. Utilizing Xenium spatial transcriptomics we verified that CD8.pe and CD8.Tex were more abundant in TCE treated samples and that their interactions with other key cell subsets were enhanced by TCE treatment. Conclusions: In summary we found that key CD8 subsets were expanded by TCE treatment in preclinical solid tumor models, that the abundance of CD8.pe correlates with reduced tumor burden, and that TCE treatment promotes cellular interactions among CD8s and other key cell subsets in the tumor microenvironment.

Citation Format: Billy Tomaszewski, Matthew Curtis, Conrad Foo, Gu Zhang, Patrick Chang, Robyn Clark, Thao Nguyen, Joshua Webster, Sandra Rost, Raj Jesudason, Robert Piskol, Klara Totpal, Lisa McGinnis, Kevin Walsh, Weilan Ye. T cell engager therapy affects the spatial distribution and phenotype of T cells in the tumor microenvironment [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr PR002.