Humanized immune system mice are revolutionary research tools for the development of novel immune cell-targeting oncology therapeutics, but the use of first-generation models such as the huNOG (NOG mice engrafted with human CD34+ hematopoietic stem cells) has been limited for some applications by impaired human myeloid cell differentiation.
Several investigators sought to understand the mechanistic bottleneck and identified evolutionary disparities between mouse cytokine growth factors and human cytokine receptors necessary for myeloid lineage differentiation1.
With immuno-oncology scientists investigating the role of myeloid cells such as myeloid-derived suppressor cells and tumor-associated macrophages, a model with successful human myeloid cell engraftment is critical.
Humanized Mice with Myeloid Engraftment
Several approaches have been explored to generate humanized immune system mice with myeloid cell engraftment, with the leading approach the generation of human cytokine transgenic models on super immunodeficient background strains. The three most commonly cited models are the NOG-EXL, NSG™-SGM3, and the MISTRG.
NOG-EXL
The NOG-EXL (hGM-CSF/hIL3-NOG) expresses human GM-CSF and IL-3 under control of the SV40 promoter on the NOG background, with cytokine expression levels in the range of ~35 pg/ml for hGM-CSF and ~80 pg/ml for hIL-32. It is commercially available pre-engrafted with human CD34+ stem cells as the huNOG-EXL. Both models are based on the CIEA NOG mouse® and available from Taconic Biosciences.
NSG™-SGM3
The NSG™-SGM3, also referred to as the NSGS, expresses three different human cytokines, GM-CSF, IL-3 and KITLG, on the NSG™ background. The transgene uses the relatively stronger viral CMV promoter and results in cytokine expression levels at supraphysiologic levels of ~2000-4000 pg/ml1,3. It is commercially available pre-engrafted with human immune cells from the Jackson Laboratory.
MISTRG
The MISTRG model is more complex than the first two. The base model is a Rag2/Il2rg double knockout on a mixed BALB/c x 12924 background. Because this strain background (as opposed to the NOD strain) is not optimally permissive to engraftment of human stem cells, a transgene expressing human SIRPα is added to improve hematopoietic stem cell (HSC) engraftment. Additionally, the model expresses human GM-CSF, IL-3, M-CSF, and TPO. These cytokines are all under the endogenous mouse promoters except for hGM-CSF, which is under the human promoter4. While this model is reported to engraft myeloid cells most successfully, it has been largely inaccessible by the humanized mouse community. The model was designed by Richard Flavell of Yale and made in collaboration with Regeneron Pharmaceuticals4. The Jackson Laboratory imported the strain in 2015 in anticipation of distribution, but has since discontinued production of MISTRG mice.
Fortunately for the research community, both the NSG™-SGM3 and NOG-EXL are readily available to both academic and pharma/biotech researchers worldwide.
Short Lifespan of Humanized NSG™-SGM3 Can Be Challenging for Immuno-Oncology Studies
huNSG and huNOG mice can survive one year or more after engraftment, which is more than sufficient time for immuno-oncology studies. In contrast, the early-onset anemia and graft exhaustion seen in humanized NSG™-SGM3 mice is a challenge, as it limits the useable study window. Wunderlich et al. reported that “Analysis of a large number of hu-NSGS showed that 100% of mice died between 10 and 27 weeks of engraftment, while all nonengrafted control mice remained alive and well.”5
huNOG-EXL Have Extended Lifespan Compared to huNSG™-SGM3 and are Suitable for Immuno-Oncology Studies
Figure: Comparison of survival between huNOG-EXL and huNSG™-SGM3 (internal Taconic data and reference 5).
According to long-term data from Taconic Biosciences' production colony, huNOG-EXL survival times far exceed those reported for humanized NSG™-SGM3 mice. By 16 weeks post engraftment, approximately 50% of humanized NSG™-SGM3 mice are expected to succumb to anemia, whereas huNOG-EXL viability remains >90%. By 30 weeks post engraftment, well after all humanized NSG™-SGM3 mice reportedly succumb to anemia, huNOG-EXL viability still exceeds 30%5.
Scientists from Tesaro demonstrated significant anti-tumor activity with a novel anti-PD-1 antibody in huNOG-EXL mice implanted with A549 human lung cancer cells. Tumor infiltrating lymphocytes were increased in tumors of mice treated with the therapeutic as compared to an isotype control. In another study by Tesaro and Charles River, three different tumor types were implanted into huNOG-EXL mice, and various human lymphoid and myeloid cells were identified in those tumors.
These studies demonstrate that huNOG-EXL mice can successfully engraft key human immune cell types and support immuno-oncology studies with sufficient timelines for tumor growth and treatment.
Learn more about the huNOG-EXL at Taconic's website.
References:
1. Nicolini, F. E.; Cashman, J. D.; Hogge, D. E.; et al. Leukemia 2003, 18 (2), 341-347.
2. Ito, R.; Takahashi, T.; Katano, I.; et al. The Journal of Immunology 2013, 191 (6), 2890-2899.
3. https://www.jax.org/strain/013062
4. Rongvaux, A.; Willinger, T.; Martinek, J.; et al. Nature Biotechnology 2014, 32 (4), 364-372.
5. Wunderlich, M.; Stockman, C.; Devarajan, M.; et al. JCI Insight 2016, 1 (15).