Due to advancements in genetic engineering, small animal models are continuing to change in response to market needs. Model developers strive to better reflect the role of this important asset in both research and translational science through increased humanization of specific genes.

Immuno-oncology, biologic drugs, and cell and gene therapies are some of the most important drivers of the humanization trend. As these models incorporate more human traits, they facilitate therapeutic drug development and provide more human-like responses. This in turn drives the market to request even more complex models, solidifying the humanization trend.

GEN spoke with five leading small animal model developers to learn more about how they are responding to the market and regulatory forces.

Why humanize?

Biocytogen Pharmaceuticals, a developer of novel antibody-based drugs, also provides animal models and preclinical pharmacology services. For example, the company’s Biocytogen Boston division was established in 2018 to provide models, antibody discovery services, and preclinical pharmacology services to clients in the United States and abroad.

When GEN spoke with Qingcong Lin, PhD, the CEO of Biocytogen Boston, he noted that his company emphasized three animal model categories. The first category includes individual in situ targeted knockout/knockin humanized models that can simulate the expression of single human genes with endogenous mouse promoters.

The second category includes fully human antibody models in which the entire human antibody gene repertoire is knocked into the corresponding murine region in situ. Models of this type at Biocytogen include the RenMab, RenLite, and RenNano models. Models of the RenNano type, launched late last year, possess full human heavy chain variable regions and are capable of producing heavy-chain-only antibodies, which are also known as single-domain antibodies (sdAbs) or nanobodies.

Nanobodies have smaller molecular weights than traditional antibodies and offer improved tissue penetration, so they are effective at infiltrating tumors and crossing blood-brain barriers. Additionally, nanobodies have a longer CDR3 region, so they are better equipped to recognize hidden epitopes. With their simpler structure, nanobodies are stable and easy to produce and engineer as building blocks for complex multispecific modalities.

“The complexity and diversity of the highly demanded RenNano models are very rich,” Lin asserted. “Humanization gives nanobodies high affinity, high specificity, and good druggability due to in vivo selection and maturation.”

The third category includes immune cell humanization models. The second-
generation immunodeficient B-NDG mice not only lack mature T cells, B cells, and functional natural killer (NK) cells, and they have select human cytokine signaling by knockout/knockin of human cytokine genes, making these mice suitable for engraftment and growth of human hematopoietic stem cells, peripheral blood mononuclear cells, and human tumor cells/tissues. Several methods can be used to introduce human cytokines to support human myeloid cells, NK cells, and dendritic cell development and proliferation.

“We pioneered in situ knockout/knockin humanized mice models,” Lin said. “The original focus was on immune checkpoints, then we moved to cytokines and cytokine receptors, and now we are expanding our cohorts to an anti-GPCR antibody platform and a TCR-mimic antibody platform to target intracellular antigens. To date, we have about 400 individual gene knockout/knockin models, and many additional double, triple, and quadruple combinations.”

“If we have more relevant models for research and drug discovery and better clinical translation, you can reduce animal usage as well as the number of clinical trials due to failures,” Lin stressed. He added that Biocytogen has a large program for TCR humanization.

Longitudinal models

For the development of clinically relevant patient-derived xenograft (PDX) models, Crown Bioscience has access to tissue from clinical trials incorporating the latest targeted therapies and immunotherapies. A particular focus is on establishing models with mechanisms of drug resistance, including longitudinal PDX models from individual patients—from early to late disease, before and after treatment, and during resistance and relapse—to help researchers understand these mechanisms.

“We are sourcing more tissues from predictive patients, mostly from targeted therapies in multiple indications (such as colorectal, breast, lung, prostate, ovarian, brain, and leukemic cancers),” said Ludovic Bourré, PhD, vice president of research and innovation, Crown Bioscience. “For some patients, we also have tissue from the primary and metastatic tumors.” These models are deeply characterized using advanced genomics and proteomics technology. Standard-of-care data are added as well to ensure that the models match what has been clinically shown.


overview of Crown's mouse models
Crown Bioscience, a contract research organization, provides preclinical and translational platforms to help its customers advance their research and development in oncology, immuno-oncology, and immune-mediated inflammatory diseases. In this image, Crown gives an overview of its mouse models. The company emphasizes that it has an extensive collection of patient-derived xenograft models and cell line–derived xenograft models.

Crown Bioscience recently acquired Indivumed Services, gaining more access to biospecimens and reinforcing existing biomarker discovery capabilities. “We can incorporate these assets to establish more clinically relevant PDX models,” Bourré remarked. PDX models can be used for mouse preclinical trials as well as biomarker identification and patient stratification.

Bourré added, “We have a robust humanized model platform (HuGEMM) composed of single and multiple knockin models to test combination therapies with the major clinically approved immune checkpoints (that is, antiPD-1, antiPD-L1d, and anti-CTLA4), as well as models for the most recent immuno-
oncology targets. We have also developed immune humanized models to aid myeloid and NK cell targeting therapy.”

For safety testing, a model based on the CD34 humanized mouse model is available. It can be used to evaluate cytokine release—a key concern with biologics and cell therapies—and to test counteracting antibodies. Models are also available to support research into inflammatory diseases and autoimmune diseases such as irritable bowel disease, lupus, rheumatoid arthritis, type 1 diabetes, and multiple sclerosis.

“We need to learn from the clinic to establish new preclinical models,” Bourré concluded. “If you conduct immuno-
oncology, inflammatory, and autoimmune disease research, you need a humanized system, even if it is a mouse model. Humanized models are increasingly tailored to test new targets and proof of concept.”

Driving market needs

With the waning of the COVID-19 pandemic, laboratories are becoming fully operational again. “The increase in the start of new projects is driving the need for new and innovative research animal models,” said Steve Festin, PhD, senior director of scientific and commercial development, Charles River Laboratories. “Additionally, an enhanced focus is on the development of improved translational models.”

The growing development of cell and gene therapies requires models that reflect human conditions. To create such models, developers are employing endonuclease technology that can introduce human-like mutations to selected genes.

With the increase of cell-mediated therapeutics in the discovery pipeline, interest is growing in human immune cell–engrafted models, including mobilized adult stem cell sources, as well as in predictable and reliable peripheral blood mononuclear cell models with less graft-versus-host disease. And more models are being tailored with a specific focus on personalized medicine and precision therapeutics to address specific mutations behind rare diseases.

Charles River has been expanding its selection of triple-immunodeficient models. These are models that are capable of hosting xenograft cells, tissue, and human immune system components. One of the new models is the SRG rat. It is licensed from Hera BioLabs, and it is designed for tumor biology, oncology, immunology, and xenograft transplant research. Charles River has also added two new mouse strains to its NCG Plus portfolio. The first, NCG-X, is designed to allow for humanization without myeloablation. The second, NCG-M, is designed to have higher levels of human myeloid cells. It can also serve as a mouse model of acute myeloid leukemia.

Festin cited a recent study (Rowe et al. Cancer Res. 2023; 83(7_Suppl.): 39) in which NCG models and adult stem cells “were used to conduct large, single-donor studies of tumor growth modulation.” He also referred to a study (Smutova et al. Int. J. Toxicol. 2023; 42(3): 232–253) in which the NCG mouse model “was studied as a test system for toxicity, engraftment, and tumorigenicity assessments of cell therapies.”

Festin also maintained that expansion of the NCG Plus portfolio will support easier humanization with diverse immune populations and bespoke models to support oncology research. “New models and applications have been driven by an increased preclinical need to evaluate novel therapeutic targets and modalities,” he elaborated. “[There is a] growing need to support cell and gene therapies that target multiple therapeutic indications.”

More precise models

Besides supporting basic research and therapeutic development, the Jackson Laboratory (JAX) is committed to the “three R’s,” that is, to the replacement, reduction, and refinement of animal models. “As biological research expands, so does the need for more precise models to support that research,” said Sierra Kent, PhD, business unit manager, JAX. “Ideally, we will create better translational models that will more accurately predict clinical outcomes and reduce the quantity of animals used for scientific research.”

JAX seeks out cutting-edge models that have been thoroughly characterized both genotypically and phenotypically. Among the new strains are models that can be used as tools or reporters to control gene and protein expression in specific cells and tissues.

Two major therapeutic areas drive the research organization’s mouse model development: immuno-modulation and gene therapy. “Modern cancer therapeutics, such as bispecific T-cell engagers, work by interacting with specific components of the patient’s immune system, meaning that those specific components are required for efficacy testing,” Kent explained. “To address the need for these components, we generate models that have different ratios and quantities of specific human immune cell subtypes.”

For example, when NSG-SGM3-IL15 mice are engrafted with human hematopoietic stem cells, they generate a robust quantity of differentiated T cells and functional NK cells. This model enables studies where multiple components of the human immune system are necessary to create the appropriate in vivo response to a tumor or therapeutic.

Similarly, the new NSG “FLT3” (NSG Flt3KO hFLT3LG Tg) mouse can generate two major sets of functional dendritic cells in addition to T and NK cells. “These new strains and our service portfolio enable researchers to study oncology, graft-versus-host disease, and cytokine release syndrome, reducing development risk,” Kent asserted.

As gene therapy advances, the need increases for highly specialized models that replicate human rare diseases or indications that could be reversed by gene therapy. Kent said that many therapeutic developers look to JAX for model generation to navigate the genome and create models to recapitulate human indications.

The impact of biological drugs

“The rapid rise of biological drugs is driving innovation across the drug discovery spectrum,” said Louise Baskin, senior director, Taconic Biosciences. “These drugs require a different type of test subject than small-molecule drugs. Improvements in standard methods can help accelerate bringing these therapies to market.”

Taconic’s most recent launches aim to improve the accuracy of study results for antibody-based therapeutics. The company’s FcResolv NOG portfolio comprises super-immunodeficient mice with functional knockout of Fc gamma receptors.

Humanized immune system models from Taconic Biosciences
Humanized immune system models from Taconic Biosciences include the Standard Access (SA) and Early Access (EA) models in the huNOG-EXL portfolio. Taconic indicates that both the SA and EA models support superior levels of human cell engraftment and the differentiation of both human myeloid and lymphoid cells. SA animals are shipped following human immune cell reconstitution and are best suited for shorter experiments. EA models are shipped prior to human immune cell reconstitution and are suitable for engraftment of slow-growing tumors, longer treatment paradigms, or various study customizations.

These receptors are known to interact with therapeutics containing an Fc domain, which is conserved across most antibody-based drugs. Elimination of the mouse version of this receptor has been shown to reduce false positives and false negatives, helping investigators gain more accurate assessments of drug efficacy.

Another newly launched model is a variation of the widely used huNOG-EXL humanized immune system model that supports both human lymphoid and myeloid cells. “Immuno-oncology drugs have driven the popularity of this model, particularly those that go beyond manipulation of T cells to target other human immune cell types,” Baskin noted.

The rapid rise in biologic drugs has challenged the market. One need is for improved animal models for discovery and preclinical assessment. Genetic modifications that “humanize” key genes are essential to mouse models that are used to assess physiological responses to biologics. Another approach is target humanization through human cell or tissue engraftment.

Baskin emphasized that when animal models provide a more biologically appropriate response, they can contribute to reductions in the usage of animal models. Less ambiguous and more consistent results mean fewer repeated studies. And “lower” animals that provide more human-like responses could substitute for “higher” animals. A six-month assay with the rasH2 transgenic mouse has gained regulatory approval as a replacement to the standard two-year wild-type mouse carcinogenicity assay, contributing to a massive improvement in animal welfare and a major reduction in animal use.

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