Abstract 1814: Generation and validation of a novel humanized TNFR2 mouse model for the evaluation of therapeutic TNFR2 antibody
Authors: Zhao Xiaofeng , Pang Ran , Liu Xiaoqing , Li Wenjing , He Xianfei , Ouyang Xuesong , Cao Zhuoxiao , Tang Renhong , Yang Wenqing 
Published: July 2021

Abstract
Tumor necrosis factor receptor-2 (TNFR2) is a TNF receptor superfamily member which, unlike the well-characterized TNFR1, has been reported to have restricted expression in the immune system especially on suppressive regulatory T cell (Treg). Therapeutically targeting TNFR2 has recently been demonstrated; however, one of the limitations in the development of TNFR2 targeting biologics is the lack of preclinical animal models with competent immune system due to their limited cross-reactivity with murine TNFR2. To overcome this limitation, we developed and validated potentially the first humanized TNFR2 mouse line in Balb/c background allowing the evaluation of anti-TNFR2 biologics in various models of cancer and autoimmunity. The genes encoding the extracellular domain of mouse Tnfrsf1b were replaced by their human counterparts while leaving the cytoplasmic domain untouched. Blood immune cell phenotyping verified the expression of hTNFR2 on both myeloid and lymphoid cells in these TNFR2 humanized mice, and the hTNFR2 expression levels are comparable with that of mTNFR2 in wildtype Balb/c mice. In vitro functional study was performed using Tregs freshly isolated from the spleen of both TNFR2 humanized and wildtype Balb/c mice. Upon co-incubation with both mouse IL-2 and mouse/human TNFα either with or without anti-CD3/CD28 Dynabeads stimulation, the Tregs isolated from the spleen of TNFR2 humanized mice were more proliferative compared to either mouse IL-2 or mouse TNFα single treated Tregs, indicating the TNFα-TNFR2 signalling pathway is maintained and functional after the genetic modification. In the meantime, we also showed that mouse TNFα can bind and signal through human TNFR2 while human TNFα can only bind and signal through human TNFR2 but not mouse counterpart, which confirmed the necessity of humanizing only the receptor TNFR2 but not the ligand TNFα. The observations of proliferation of the TNFR2 humanized mice derived Tregs can be only reduced by the ligand blocking anti-human TNFR2 antibody but not the ligand blocking anti-mouse TNFR2 antibody further support the usefulness of this model for in vivo efficacy study. Indeed, consistent with the above-mentioned in vitro functional results, our lead anti-human TNFR2 antibodies can significantly suppress growth of allograft tumors established on the humanized TNFR2 mice. Taken together, our data support the use of this novel Balb/c humanized TNFR2 model for in vivo evaluation of therapeutics targeting human TNFR2.
Citation Format: Xiaofeng Zhao, Ran Pang, Xiaoqing Liu, Wenjing Li, Xianfei He, Xuesong Ouyang, Zhuoxiao Cao, Renhong Tang, Wenqing Yang. Generation and validation of a novel humanized TNFR2 mouse model for the evaluation of therapeutic TNFR2 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1814.