the Jackson Laboratory

Adriano Flora

The extreme specificity of therapeutic antibodies is arguably the most valuable characteristic of this class of drugs. Opposite to small molecules, which tend to interact promiscuously with many proteins and cause unexpected side effects, antibodies show an unparalleled degree of selectivity and, in most cases, bind only their specific antigen. The downside of this exquisite specificity is that therapeutic antibody candidates usually do not recognize the ortholog of their target proteins in the most commonly used animal models, namely rats and mice, depriving drug developers of valuable in vivo tools. Accordingly, preclinical researchers have widely adopted non-human primates (NHPs) as the go-to in vivo model due to their sequence conservation with humans. However, because of ethical implications and financial and regulatory hurdles linked to NHP use, drug developers rely increasingly upon in vitro systems, missing the complex systemic response typical of in vivo models.

"Although the complete replacement of monkeys in developing therapies is still unrealistic, the reduction of their use is inevitable. The adoption of humanized mice as predictive tools in preclinical research is growing and plays an increasingly important role in the path of leading drug candidates to the clinic"

The lack of relevant in vivo data in the early phases of preclinical development can significantly impact the clinical success of therapeutic antibody candidates. If researchers do not have access to translationally relevant in vivo models during the lead identification and selection process, they risk focusing their effort on therapeutic candidates who may turn out unstable or exceedingly toxic. If these liabilities are revealed during the NHP study or in the clinical phase, the cost could be very high and put the entire program at risk. Scientists at the Jackson Laboratory (JAX) have leveraged their knowledge of mouse genetics to create a new generation of models that mimic crucial aspects of human physiology relevant for therapeutic antibody development. JAX’s models for antibody developers fall into two categories: genetically humanized mice and immune-system humanized mice.

Genetically humanized mice for antibody drug development carry genomic modifications designed to replicate human-specific aspects of the response to therapeutic antibodies. The most characterized models are mice expressing the human neonatal Fc receptor (FcRn), a membrane protein involved in recycling IgGs and regulating their serum half-life. Opposite to WT mice, which interact with human antibodies in a non-physiological fashion, FcRn humanized mice are fully predictive of the stability of human antibodies. Antibody-based therapeutics come in over sixty formats and are tailored to modulate specific aspects of the immune system response. The extensive engineering required to generate these molecules affects all the characteristics of the lead candidates, starting with their in vivo stability. Therefore, evaluating the half-life of different drug formats in the initial phases of development is essential to identify the most promising candidates and assess the effect of functional mutation on their serum half-life.

The Antibody Evaluation services at JAX and some of the leading pharmaceutical companies routinely use these FcRn humanized mice to compare the stability of antibody-based leads and predict their half-life in patients. In this way, researchers can eliminate liabilities early in the pipeline and select the molecules with the desired half-life without testing multiple candidates in NHPs

Immune-system humanized mice are very different from the genetically humanized models described above. These models are based on highly immunodeficient mice transplanted with human hematopoietic stem cells or human peripheral blood mononucleate cells (hPBMCs). All of the models developed at JAX derive from the NSG™, a mouse strain generated in the laboratory of Dr. Lenny Schultz. Thanks to its impaired immune function, the NSG™ strain and its derivatives provide the optimal environment for human cell engraftment. The access to human immune cells in an in vivo setting provides preclinical drug developers with a new set of tools that are, arguably, even more, relevant than NHPs for efficacy and toxicity testing. To begin with, the target proteins are fully human. Since many of the new therapeutic candidate antibodies are so specific that they barely cross-react with NHP proteins, the presence of functional human immune effector cells is crucial to providing meaningful efficacy and toxicity data. Secondly, the profile of expression of the target proteins is faithfully recapitulated in these models, especially in hPBMC mice, where mature, fully differentiated immune cells are engrafted into the mouse. Finally, using different donors in a single experiment mirrors the range of responses usually observed in the clinic and captures the complexity expected in a patient population. The laboratory of Dr. Jim Keck at JAX has spent the past few years developing and characterizing immune system-humanized mice as tools for drug developers. One of the latest innovations introduced by Dr. Keck’s group is a system based on hPBMC mice to predict Cytokine Release Syndrome (CRS) associated with immuno-stimulatory drugs. The use of this assay allows preclinical researchers to interrogate the immune system on different aspects of the cytokine release induced by their drug candidates, identify the least toxic candidates, define the therapeutic window where a drug is still effective and not yet harmful, and explore potential strategies to alleviate the toxicity.

Using a combination of genetic and immune-system humanized mice is rapidly becoming a standard approach to verify the stability, efficacy, and toxicity of many advanced and antibody-based therapies. Researchers are taking full advantage of these new tools to generate robust and predictive data to support preclinical development, in some cases even replacing the use of NHPs. Although the complete replacement of monkeys in developing these therapies is still unrealistic, the use of humanized in preclinical research mice is constantly growing and plays an increasingly important role in the path of leading drug candidates to the clinic.