Abstract 4168: BALB/c-CD3EDG HuGEMMTM mouse model for evaluation of CD3-targeted tumor immunotherapy

Abstract

Abstract Introduction: T cell immune response plays critical roles in cancer immunotherapy. CD3 complex, composed of CD3ε, CD3δ and CD3γ chains, is an essential component of TCR-CD3 complex mediating TCR signaling. Targeting CD3 complex has therefore become a popular strategy for manipulation of T cell function. Clinically relevant animal models for proper evaluation of human CD3 antibodies is in great demand in the field. Currently, adoptive transfer of human CD3+ T cells or transplantation of human hematopoietic stem/precursor cells to immunodeficient mice are usually employed. However, these mouse models are not fully immunocompetent. In this study we have evaluated in vitro CD3 EDG functionality and in vivo therapeutic efficacy, in BALB/c-hCD3EDG knock-in mouse in which all the three components of the CD3 complex — Cd3ε, Cd3δ, and Cd3γ— are replaced by their human counterparts, CD3E, CD3D, and CD3G. Methods: A humanized CD3EDG mouse model (HuGEMM) on BALB/c background was generated by replacing the entire mouse CD3EDG with its human counterpart through ES cell-based gene targeting. The cell surface expression of CD3E and intracellular expression of CD3D and CD3G were confirmed by FACS. Naïve CD3+ T cells were isolated from the splenocytes of homozygous CD3EDG HuGEMM and activated by anti-hCD3 antibody. T cell proliferation was demonstrated by CFSE dilution assay. To validate the TCR functionality of humanized CD3EDG in vivo, we evaluated bispecific antibody anti-hCD3/hEpCAM5 in BALB/c-CD3EDG HuGEMM bearing CT26-hEpCAM5. Results: The expression of human CD3E, CD3D and CD3G were confirmed by flow cytometry. hEpCAM5 antigen expression was confirmed in CT26 cell line in vitro and tumor in vivo by flow cytometry. The TCR functionality was detected by in vitro T cell activation assays using anti-hCD3 antibody. In vivo efficacy was demonstrated using a bispecific antibody that simultaneously binds to human CD3 and human tumor associated antigen EpCAM5 in human CD3 EDG knock-in mice engrafted with CT26-hEpCAM5 tumor. The therapeutic efficacy of the bispecific antibody showed 32% tumor growth inhibition compared to control treatment. No significant body weight loss was recorded in any of the animals. Conclusion: This study showed the successful establishment of humanized CD3 knock-in BALB/c mouse models providing a full immunocompetent model for human CD3-targeted tumor immunotherapies evaluation. Citation Format: Demi Xiaojuan Liu, Kaixia Lian, Xuefei Yan, Jie Lin, Lei Zheng, Annie Xiaoyu An, Xiaoxi Xu, Ludovic Bourre, Jingjing Wang. BALB/c-CD3EDG HuGEMMTM mouse model for evaluation of CD3-targeted tumor immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4168.