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Designing HSC Humanized Model Efficacy Studies

by Emilie T.E. Gross, PhD, February 14, 2019 at 01:00 PM | Tags

Factors to consider when planning immuno-oncology agent efficacy studies in humanized mice

Factors to consider when planning immuno-oncology agent efficacy studies in humanized miceReview some key considerations in humanized model study design for successful preclinical oncology research.

Humanized Models for Immunotherapy Assessment

HuCD34+ HSC humanized models are highly useful tools in immunotherapy drug development. They allow the long-term assessment of human-specific immunotherapies by recapitulating, to a certain extent, human immune responses during preclinical testing.

We’ve previously covered model uses and common misconceptions. This post looks at some of the key factors you need to consider to enable a successful, huCD34+ HSC humanized model preclinical I/O efficacy testing study.

Which Immune Compartments are Needed for Your Agent’s Mechanism of Action?

There are different types of huCD34+ HSC humanized mouse available, which we reviewed in detail last year. Their immune cell reconstitution and performance vary, however, so you need to consider the mechanism of action of the immuno-oncology agent you’re studying.

Myeloid Cell Involvement

For example, do you need myeloid cell reconstitution for your agent to function effectively? If yes, then you may need to look past the commonly used huNSG™ and huNOG® models on to the next generation of humanized mice.

Super immunodeficient murine backgrounds such as NSG-SGM3 and NOG-EXL support greater human myeloid cell reconstitution than the NSG and NOG. HuNSG-SGM3 expresses human SCF, GM-CSF, IL-3, and has higher engraftment of monocytes, macrophages, and dendritic cells compared with the huNSG model.

The huNOG-EXL mouse expresses human GM-CSF and IL-3, with higher levels of myeloid cell differentiation following human HSC engraftment compared with huNOG.

Tumor Promoting Immune Cells

Targeting tumor-promoting immune cells (e.g. M2 macrophages and MDSC) requires the recreation of the immune suppressive tumor environment in which these cells thrive. This can be achieved with the CD34+ huNOG-IL6 model. Human IL-6 is expressed systemically in this strain, which mediates the generation of an immune suppressive tumor microenvironment.

Selecting a tumor model known to express high levels of IL-6 and transplanting to a humanized murine model favoring myeloid differentiation (such as the huNOG-EXL or huNSG-SGM3) can be an alternative strategy to recapitulate a tumor promoting immune environment.

T Cell Activation

Finally, if you need to test adoptive T cell therapy strategies (e.g. CAR-T cells) recent evidence shows that the NOG-IL-2 model might be best. This particular model doesn’t actually fall into the category of human CD34+ engrafted murine models due to its moribundity, caused by graft versus host disease onset.

By extension, you can also speculate that the NOG-IL-2 model could be the experimental set up of choice for adoptive NK therapies, or a variety of T cell therapy efficacy definitions (e.g. bi-specific antibodies with T cell engager moiety, tumor vaccines), which would be investigated in an adoptive T cell transfer context.

Overall, carefully considering and choosing the right humanized mouse is a key component to successfully designing a humanized study. This should be tailored to create the best immune and cytokinic environment for your specific agent mechanism.

Is Your Tumor Model Capable of Growing in a Humanized Environment?

There’s actually very little data published on the potential for even the most common xenograft models to grow in the reconstituted, allogenic, human immune environment found within humanized mice.

This makes it highly important to perform a pilot study with any conventional xenograft or patient-derived xenograft (PDX) tumor model that you’d like to use. This pilot study should trial at least two CD34+ HSC donors.

While most human cell line derived, conventional xenografts and PDX will grow in immunodeficient models, their immunogenicity can be revealed in humanized mice. This phenomenon is further emphasized in an allogenic context, and may lead to rejection or absence of growth of the tumor model in the absence of therapy.

Consider Including at Least Five HSC Donors

Some immunotherapies may only respond with a certain population of HSC donors. Therefore, for successful efficacy testing, it’s important to use multiple donors per study. We suggest using at least five donors as a minimum, which allows you to delineate the potential for efficacy in humanized studies and obtain statistically significant results.

In addition, this allows you to test a variety of HLA types within a reconstituted hematopoietic system, since most humanized studies are run with a partial HLA mismatch.

Finally, mouse selection is performed at twelve weeks post-engraftment to choose animals with a minimum of 50% of human CD45+ cells and 15% of human CD3+ cells in peripheral blood.

Consider T Cell Therapy Evaluation Model Selection Parameters

When testing T cell therapies in humanized mice, there are a couple of points which need to be determined during model selection.

First, you need to assess the tumor cell surface level expression of MHC class I. Positive MHC class I expression is required to induce a full T cell response. Tumors can evolve to downregulate their MHC class I expression through a variety of mechanisms in order to evade T cell responses in the host.

MHC class I expression can be versatile, based on the immune environment where the tumor develops (e.g. IFN-γ secretion by T cells upregulates MHC class I expression). Therefore, expression analysis should be included in the model establishment phase before running an efficacy study.

Second, the tumor mutational burden, if available, is a parameter that needs to be considered. Mutational burden is predictive of response to immunotherapies such as anti-PD-1 agents. A high mutational burden augments tumor antigenicity. In other words, a high mutational burden increases the likelihood of tumors bearing mutated antigens at their surface, which are detected by CD8+ CTL cells, in conjunction with MHC class I.

Depending on the cancer type you wish to target, choosing a model with a high mutational burden combined with positive MHC class I expression can be a great first screen to assess the potency of T cell therapies.


HuCD34+ HSC humanized models provide a wealth of data on human-specific immunotherapy efficacy, to progress immuno-oncology drug development programs. Carefully planning and assessing key factors before starting efficacy studies provides the greatest likelihood of preclinical success.


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