Abstract 7254: Mouse models for alleviating GvHD and assessing on-target off-tumor toxicity of bispecific T-cell engagers

Abstract Bispecific T cell engagers (TCEs), which simultaneously target CD3 on the surface of T cells and a tumor-associated antigen (TAA) located on cancer cells, have shown great promise in tumor treatment. However, multiple factors including targets, indications, dosage and immunogenicity influence the efficacy and safety of TCEs. Furthermore, the intricate mechanism of action may limit the translatability from preclinical study to clinical application. To improve the translation of early research findings into effective therapeutic strategies, we introduced two optimized mouse models for preclinical evaluation of TCEs. Due to poor cross-species reactivity of anti-CD3 and anti-TAA antibodies, mice that express wild type murine CD3 molecules are not suitable for the evaluation of TCEs. Severe immunodeficient animals engrafted with both human T cells and cancer cells represent an ideal model. However, graft-versus-host disease (GvHD) is often observed, which can make data interpretation challenging. To this end, the NCG-MHC-dKO mouse model was developed by knocking out both murine MHC class I and II molecules in the NCG background. Compared to conventional NCG mice, NCG-MHC-dKO mice alleviated the GvHD response and prolonged the life span of PBMC-engrafted animals, thereby extending the therapeutic window. We have verified the efficacy of talquetamab, a GPRC5D-directed T cell engager, in huPBMC-NCG-MHC-dKO mice inoculated with NCI-H929 cell line. Talquetamab could significantly inhibit tumor growth in NCG-MHC-dKO mice, with the animals maintaining stable body weight and healthy condition throughout the treatment period. These findings highlighted NCG-MHC-dKO mice as an ideal platform for evaluating the efficacy of TCEs. Although the huPBMC-NCG-MHC-dKO model is very powerful for the efficacy evaluation of TCEs, it cannot be used to evaluate on-target off-tumor toxicity due to the absence of human TAA in mouse tissues with compromised immune systems. To this end, the BALB/c-hCD3EDG mouse model was developed by inserting the entire gene regions including regulatory sequences of the human CD3E, CD3D, and CD3G genes into the mouse genome, while simultaneously deleting the endogenous mouse CD3e, CD3d, and CD3g genes. Replacing the murine CD3 molecules with human counterparts did not impact the immune system of BALB/c-hCD3EDG mice. In addition, we have developed multiple human TAA knock-in mouse models in the BALB/c-hCD3EDG background. The expression of TAAs in these mice resembled that in humans. Together, these models can be used not only to evaluate the efficacy, but also on-target off-tumor toxicity of TCEs. In conclusion, our mouse models provide valuable tools to increase the translatability of preclinical models to evaluate TCEs. Citation Format: Hongyan Sun, Bei Zhang, Yunlong Jiang, Jianming Xu, Xiang Gao, Jun Wang. Mouse models for alleviating GvHD and assessing on-target off-tumor toxicity of bispecific T-cell engagers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7254.