In recent years, immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment. One of the potential therapeutic targets that seems increasingly attractive is LAG-3 (Lymphocyte activation gene 3, CD223), a human protein encoded by the LAG3 gene. LAG-3 can be found on activated T cells, NK cells, B cells and plasmacytoid DCs and serves as a negative regulator of immunity. Inhibition of LAG-3 enhances the antitumor effect of specific CD8+ T cells, which is an observation pursued by several pharmaceutical companies with their products in pre-clinical or early clinical development. Therefore, it is clear there is a need for suitable pre-clinical models able to accurately reflect the relevant physiological processes to evaluate the safety and efficacy of LAG3-targeted therapeutics. Biocytogen has generated a humanized LAG-3 mouse model for both in vitro functional validation and in vivo efficacy evaluation of anti-hLAG-3 antibodies. The targeting strategy is that exons 2~7 of mouse Lag3 gene which encode the extracellular domain are replaced by human LAG3 exons 2~7. After an introduction of this modification, human LAG-3 was detectable on Tregs in the mouse spleen and the anti-hLAG-3 antibodies and recombinant human FGL-1 protein associated well to the mouse splenocytes. Basal leukocyte subpopulations, including T/B/NK cells, DC, granulocytes, and monocytes/macrophages, were similar between humanized and wild-type mice. Additionally, anti-human LAG-3 antibodies partially blocked LAG-3/FGL-1 binding. Furthermore, using the MC38 tumor model, we show robust efficacy of anti-human LAG-3 antibodies in inhibiting tumor growth in vivo.In summary, LAG-3 humanized mice are a useful tool for in vivo efficacy evaluation of anti-human LAG-3 antibodies. Moreover, Biocytogen has also generated PD-1/PD-L1/LAG3 multi-gene humanized mice, in which blood cell composition, morphology and the levels of ALT, AST and other indicators were comparable to wild-type mice. This model is ideal to assess an increased therapeutic benefit in cancer treatment provided by combination therapies. Indeed, in our hands using the MC38 tumor model, the combination of anti-LAG-3 antibodies with anti-PD-1 (or anti-PD-L1) antibodies shows synergistic inhibitory effects , demonstrating that the PD-1/PD-L1/LAG3 humanized mice represent a powerful tool for preclinical assessment of in vivo efficacy of future therapeutics.

Citation Format: Huilin Li, Xiaofei Zhou, Dirui Li, Veronika Chromikova, Qingcong Lin. Improved LAG-3 humanized knock-in mouse model for assessment of mono- and combination therapy strategies for cancer treatment [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 2942.