Abstract Background The preclinical evaluation of novel immune therapies require mouse models with a humanized immune system. We previously demonstrated that either peripheral blood mononuclear cells (PBMC), subsets of PBMCs like T and NK cells or CD34+ hematopoietic stem cells (HSC) can be used to establish such models. With the development of next-generation NOG mice, lineage-specific differentiation of immune cell sub-populations of interest can be supported and Fc gamma receptor knock-out mice can exclude false positive or false negative results in studies with antibody-based therapies. Methods HSC-humanized mice were generated by i.v. transplantation of CD34+ stem cells using single donors or mixed HSC donor pools. HSC were transplanted to immunodeficient NOG mice and next-generation NOG strains as: NOG-EXL, hIL-2 NOG, hIL-6 NOG, and FcResolv™ NOG. Engraftment and lineage-specific differentiation were compared between the strains. Long-term survival was monitored and human immune cells counts in blood were analyzed by FACS every four weeks. Breast cancer PDX were established on humanized FcResolv™ NOG mice and treated with different targeted antibodies, like Herceptin or check point inhibitors. Response to treatment was compared with PDX growing on NOG mice to evaluate whether murine FcγRs are confounding the study, leading to false positive or negative results. Results Humanized hIL-2 NOG mice showed significantly decreased survival after HSC transplantation in comparison to the other mouse strains. Mice had to be sacrificed within 6-8 weeks after HSC transplantation. In the other mouse strains, transplanted HSCs engrafted and differentiated mainly into B and T cells. NOG-EXL mice displayed the highest engraftment, with up to 80% of human cells in the blood, including a higher portion of myeloid cells after 8 to 12 weeks. Humanized NOG, hIL-6 NOG and FcResolv™ NOG mice showed the longest survival rate with over 400 days. We have shown that breast cancer PDX models engraft on humanized FcResolv™ NOG and FcResolv™ NOG-EXL and removing murine FcγRs can improve accuracy for efficacy assessment of antibody-based therapies which include an Fc domain. Conclusions Next-generation NOG mouse strains, transgenic for human cytokines, can further improve the humanization of mouse models by inducing a lineage-specific differentiation of transplanted hematopoietic stem cells. Engraftment of human tumor cells or PDX seems not to be impaired on mice transgenic for human cytokines. These improved human tumor-immune cell models allow more predictive preclinical translational studies on tumor immune biology as well as evaluation of new therapies. Citation Format: Maria Stecklum, Philip Dube, Ditte Olsen, Jens Hoffmann. Humanized mouse models 2.0 - Improved preclinical evaluation of novel immune cell therapies, check point inhibitors, and immune cell engagers by excluding false positive or negative results abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3385.
Stecklum et al. (Fri,) studied this question.