Abstract PR12: Assessing in vivo mechanisms regulating the therapeutic activity of bi-specific T-cell engager (BiTE®) molecules in immunocompetent mice expressing a chimeric human/mouse CD3ϵ receptor
Authors: Belmontes Brian , Tan Hong , Zhong Wendy , Chui Danny , Cook Kevin , O'Brien Sarah , Martin Melissa , Alexander Brandy , Eng Jason , Harrold John , Aeffner Famke , Chun Matthew , Bakker Alice , Balazs Mercedesz , DeVoss Jason , Coxon Angela , Canon Jude , Egen Jackson , Nolan-Stevaux Olivier 
Published: June 2021

Abstract
The development and success of Blincyto®, a Bi-Specific T cell Engager (BiTE®), to treat acute lymphoblastic leukemia has expanded the class of anticancer immunotherapy agents. BiTE® molecules consist of a single-chain Fc antibody containing tandem single-chain variable fragments (scFv) recognizing the CD3 receptor on T lymphocytes and a tumor-associated antigen and induce redirected T-cell cytotoxicity and tumor cell lysis. Despite the clinically validated efficacy of BiTE® molecules, there has been little elucidation of the parameters governing their in vivo activity. This is due, in part, to a lack of immunocompetent murine tumor models that allow for engagement of endogenous T cells in the context of an intact immune system, which more accurately mimic the human therapeutic setting. We have developed a genetically engineered mouse model (huCD3 mouse) in which a human/mouse chimeric CD3ϵ receptor recognized by BiTE® molecules was engineered into the mouse CD3ϵ genomic locus. Here, we characterize the immune cell distribution of the huCD3 mouse and utilize BiTE® molecules and syngeneic tumor models to elucidate pharmacokinetic, pharmacodynamic, and efficacy relationships in vivo. The peripheral B and T cell distribution profile of huCD3 mice and WT littermates are comparable, but T cells isolated from huCD3 mice express lower levels of. In T cell-dependent cytotoxic (TDCC) assays, an anti-huEpCAM BiTE® molecule exhibited similar potency against MC38 mouse cells expressing human EpCAM co-cultured with T cells from the huCD3 mice or a human donor, if mouse T-cells were preactivated in vitro. In vivo, MC38-huEpCAM tumors grew robustly and anti-huEpCAM BiTEÂ treatment resulted in dose-dependent tumor growth inhibition, with several tumor-free animals at higher doses. Flow cytometry analysis of disaggregated tumors and serum cytokine analysis indicated that BiTE® treatment resulted in T-cell activation and a cytokine response at doses that correlate with tumor regression. In vivo studies were also performed using B16F10-huEPCAM cells, which generate tumors with reduced T-cell infiltration compared to MC38-huEpCAM. Additionally, we used WT mice reconstituted with different ratios of huCD3e and WT bone marrow to investigate the T-cell number requirement for optimal BiTE®-mediated efficacy. We also evaluated the efficacy of an anti-mouse CD19 BiTE® molecule against MC38-CD19 tumor cells to compare the requirements for antitumor efficacy versus normal B-cell depletion in the blood and in lymphoid organs. Finally, we performed a rational combination study using BiTE® molecules and a PD1 inhibitor, demonstrating that this combination can lead to the eradication of poorly T cell-infiltrated solid tumors. These data begin to elucidate the in vivo mechanisms regulating the efficacy of BiTE® molecules under physiologically relevant conditions.
This abstract is also being presented as Poster A29.
Citation Format: Brian Belmontes, Hong Tan, Wendy Zhong, Danny Chui, Kevin Cook, Sarah O'Brien, Melissa Martin, Brandy Alexander, Jason Eng, John Harrold, Famke Aeffner, Matthew Chun, Alice Bakker, Mercedesz Balazs, Jason DeVoss, Angela Coxon, Jude Canon, Jackson Egen, Olivier Nolan-Stevaux. Assessing in vivo mechanisms regulating the therapeutic activity of bi-specific T-cell engager (BiTE®) molecules in immunocompetent mice expressing a chimeric human/mouse CD3ϵ receptor [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR12.