The human immune system is a powerful, intricate defense system that is effective against a wide range of microorganisms and other invading contaminants. However, one’s own immune system can pose a serious threat when it responds incorrectly or overreacts to stimuli such as an infectious disease or therapeutic drug. Well-known adverse immune responses include severe allergic reactions, rheumatoid arthritis, and type 1 diabetes. Across the fields of drug development, infectious disease research, and clinical practice, many experts are concerned with a less-familiar phenomenon: cytokine release syndrome (CRS). White blood cells can become overactivated, rapidly releasing cytokine signaling proteins that then activate more white blood cells. This escalating cycle can cause symptoms ranging from mild fever and inflammation to lethal organ failure.
In 1999, 17-year-old clinical trial participant Jesse Gelsinger tragically died of a cytokine storm after receiving a gene therapy injection; this high-profile case stalled gene therapy research for many years. In 2006, six healthy clinical trial participants developed CRS that led to organ failure shortly after receiving a monoclonal antibody drug called TGN1412. This example left the scientific community especially rattled because preclinical trials in rats, monkeys, and in vitro human cells had given no warning of CRS risk for that therapeutic. To prevent incidents like these and satisfy regulators, pharmaceutical researchers need preclinical tools that accurately model potential human immune responses without unnecessarily endangering patients.
The solution: humanized mice. Engrafting human immune systems into mice yields a versatile preclinical platform for generating reproducible, translationally-relevant data about the safety and efficacy of novel therapeutics. With this tool, scientists can evaluate CRS risk, perform dose ranging studies, and observe downstream organ effects of immune responses in a diversified population, giving better predictions of clinical outcomes.
Comparing Humanized Mice CRS Studies to Other Preclinical Approaches
Up to 97 percent of immuno-oncology therapeutics fail in clinical trials, with 50 percent of failures resulting from safety or efficacy problems.1 Current preclinical assays do not present fast, cost-effective methods for de-risking drug development. To avoid losing precious time and money, pharmaceutical developers should only move the most promising candidates beyond lead selection. The more information that can be gathered preclinically, the better scientists will be able to select the correct compounds.
In Vitro Assays Only Show Part of the Picture
In vitro cytokine release assays involve culturing human peripheral blood mononuclear cells (PBMCs)—including T cells, B cells, and natural killer cells—with the drug of interest in a closed, controlled environment. This approach allows scientists to measure cytokine production, cytotoxicity, and targeted efficacy against cancer cells. These assays are especially useful for drug target validation because they are generally quick and inexpensive.
However, CRS is impossible to measure completely or accurately in vitro because it is a systemic phenomenon that affects far more than just the immune cells and cancer cells that interact with the substance in the blood. Studies have also shown that in vitro assay results can include false positives or negatives, and can also vary significantly by drug mechanism of action (MoA). For example, in vitro assessments of the TGN1412 monoclonal antibody yielded false negatives, failing to predict the strong, dangerous CRS response that the human trial participants experienced.2
How In Vivo CRS Evaluation Studies Capture Systemic Information
Scientists create humanized mice by grafting adult human PBMCs into immunodeficient mice, creating a functioning human immune system circulating through and interacting with mouse vasculature and organs. The grafted immune system is established after 6 days, at which point researchers can inject a drug to study its effects.3 Monitoring for CRS over the following 1-7 days involves serially measuring body weight and temperature, quantifying human cytokines at key time points, and performing flow cytometry and other assays.
In addition to evaluating CRS, researchers can use serum analysis and histology to investigate additional effects such as downstream organ infiltration and toxicity. It is also possible to engraft tumors to evaluate on-target efficacy. These assays are more predictive because they capture human-specific immune responses within a complex biological system. Furthermore, direct comparisons have shown that in vivo CRS assays deliver more higher-quality data than in vitro assays.3
Decreasing Reliance on Non-Human Primate Trials
Non-human primates were previously the animal model that gave the closest preclinical approximation of human physiology and disease progression for drug safety and efficacy testing. However, studies in these animals are extremely expensive, time-consuming, and ethically weighty, and the approximation may not be as close as once thought. More recent studies of TGN1412 concluded that the CRS response in clinical trial participants had a direct connection to the CD28 receptor on human CD4+ effector memory T cells. None of the animals used for preclinical tests express this receptor, which is why the risk of CRS went undetected.4
Using Humanized Mice to Gather New Data Types Preclinically
To maximize the potential of in vivo CRS evaluation studies, researchers should not limit themselves to the same investigations performed with in vitro assays and non-human primate models. With more robust preclinical assessments, patients can receive safer, more effective therapeutics even in early phases of clinical trials.
Early Dose Ranging
By engrafting tumors into humanized mice and measuring tumor shrinkage following therapeutic administration, researchers can compare efficacy and safety not only of different drugs but also of different doses of the same drug. Using a single PBMC donor to avoid confounding variables, scientists can perform dose ranging studies that reveal the therapies and dosages that strike the ideal balance of maximal tumor shrinkage with minimal toxicity. These preclinical studies will allow for more strategic investments and have the potential to accelerate final dose determination processes once the therapeutics begin moving through clinical trials.
Modeling Diversity for More Accurate Safety and Efficacy Predictions
Different individuals can have radically different responses to the same therapeutic, even at the same dose. This diversity extends to both efficacy and toxicities like CRS. The more diverse the study population, the more likely a drug is to be accessible and beneficial to the population as a whole. Fortunately, researchers have found that humanized mice show reproducible, representative variation in cytokine release levels depending on the PBMC donor used to generate their human immune systems.3
Modeling the diversity of a patient population through pre-characterized PBMC donors provides drug developers with preclinical results that may increase the chances of success for new therapies. Some individuals may be highly sensitive and more likely to experience toxic CRS from a given therapeutic. The inclusion of such individuals in early clinical trials might cause the drug’s maximum tolerated dose to be set at a level below that which would be required to produce efficacy in a broader population. With a representative in vivo preclinical study, pharmaceutical developers can pinpoint factors that increase sensitivity and risk of toxicity and use that information to pre-screen high-risk individuals—who are unlikely to benefit from the positive effects of the drug—out of the clinical trial. The trial can then move forward and potentially yield a higher maximum tolerated dose, increasing potential efficacy.
Ultimately, in vivo CRS evaluation studies with humanized mice could enable a precision medicine approach to therapeutic selection. Scientists could humanize mice using an individual patient’s PBMCs and perform testing that would allow them to predict in a matter of weeks what treatments will be safest and most effective for that patient.
Testing Multiple Modalities in Humanized Mice
Many substances are capable of triggering CRS, but drugs with a direct influence on the immune system receive the greatest scrutiny when it comes to immunotoxicity. It can be particularly difficult to predict how newer immunomodulatory modalities such as bispecific antibodies and cell therapies will affect patients after being administered. An in vivo CRS evaluation study enables scientists and drug developers to map out potential downstream biological reactions and fine-tune dose planning.
Bispecific Antibodies
Bispecific monoclonal antibodies, also known as bispecifics, simultaneously bind two unique antigens or two separate epitopes of the same antigen. While this multifaceted approach expands these drugs’ arsenal of strategies for recruiting the immune system to fight cancer, it also expands the array of potential off-target effects. As a result, it can be more difficult to predict how these therapeutics will affect cytokine levels and other systems.
Testing bispecifics preclinically in humanized mice with multiple PBMC donors enables scientists to study biodistribution, organ infiltration, and other things that cannot be observed in vitro, especially not at the same time. In validation studies, researchers have observed significant donor-specific variation in cytokine release levels following injection of bispecific antibody drugs.
Furthermore, these mice can be used for preclinical drug interaction studies that offer robust predictions of whether each drug combination will affect cytokine release levels. These studies not only yield greater amounts of systemic data but also provide more accurate and translatable results than standard experiments because they are more likely to capture any effects resulting from features unique to human immune cells.
CAR-T Cell Therapies
CAR-T cell therapies involve injecting a patient with T cells that have been modified to express a chimeric antigen receptor (CAR) protein that primes them to hunt down and attack cancer cells. While these immunotherapies demonstrate great potential for treating a wide range of difficult cancers, they also pose a high risk of CRS. The modified T cells—efficient killing machines—are difficult to control once they’ve been introduced into a patient. Studies show that 37-93 percent of lymphoma patients and 77-93 percent of leukemia patients experience CRS after receiving CAR-T cell therapy, and approximately 50 percent of patients involved in early CAR-T cell therapy clinical trials have required intensive care management.5 Yet other patients barely develop a mild fever. Preclinical studies in a diverse humanized mouse population can help scientists pinpoint factors that make some patients’ negative responses stronger than others. In vivo evaluation studies allow scientists to observe cascading cytokine release effects and systemic interactions between modified CAR-T cells and other tissues, rather than just the cancerous cells’ targets. Gaining a better understanding of CRS and a holistic data set resulting from CAR-T cell treatment will not only de-risk clinical trials but could also help medical professionals pinpoint more effective interventions for patients who do experience this toxicity.
Conclusion
In vivo CRS assays in humanized mice may potentially keep clinical trial participants safer while helping bring more effective therapies more quickly to patients in need. Pharmaceutical developers can develop potentially safer efficacious drugs and save resources that can be poured into optimizing and delivering the most promising candidates.
This humanized mouse platform also has broader applications for helping scientists understand human immunity on a deeper level. For example, future studies could reveal underlying biological factors that make some people more likely to develop CRS in response to specific infectious diseases. Researchers may also uncover broader trends in factors that affect variations in patients’ responses to different treatment categories—such as monoclonal antibodies compared to cell therapies—when it comes to both safety and efficacy profiles.
Leveraging humanized mouse studies effectively will help the pharmaceutical and healthcare industries be more informed and effective going forward.
References
- Fogel DB. Factors associated with clinical trials that fail and opportunities for improving the likelihood of success: A review. Contemp Clin Trials Commun. 2018;11:156-164.
- Grimaldi C, Finco D, Fort MM et al. Cytokine release: A workshop proceedings on the state-of-the-science, current challenges and Future Directions. Cytokine. 2016;85:101-108.
- Ye C, Yang H, Cheng M, et al. A rapid, sensitive, and reproducible in vivo PBMC humanized murine model for determining therapeutic‐related cytokine release syndrome. The FASEB Journal. 2020;34(9):12963-12975.
- Eastwood D, Findlay L, Poole S, et al. Monoclonal antibody TGN1412 trial failure explained by species differences in CD28 expression on CD4+ effector memory T cells. Br J Pharmacol. 2010;161(3):512-526.
- Santomasso B, Bachier C, Westin J, Rezvani K, Shpall EJ. The other side of CAR T-cell therapy: Cytokine release syndrome, neurologic toxicity, and financial burden. American Society of Clinical Oncology Educational Book. 2019;(39):433-444
About the Author
Dr. Keck is the Senior Director for Innovation and Product Development at The Jackson Laboratory and a recent recipient of JAX’s Presidential Innovation Fellow Award. In this role, he is responsible for developing and driving cutting-edge immuno-oncology and autoimmune platforms and services to empower preclinical drug developers across the globe, as well overseeing internal and external collaborations that may lead to new JAX product offerings.
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