Humanized mice, defined as animals carrying human genes or cells, are relative newcomers to the drug development field. While regulatory agencies routinely accept IND filings with data generated in humanized mice, they still fall short of recommending their use to replace more traditional preclinical models due to their novelty. Moreover, despite efforts to harmonize the use of these models, the field is still fragmented, and there is no consensus on when or how to use them for IND-enabling experiments. Considering the complex regulatory picture and the technical intricacies associated with humanized mice, why shouldn't a drug developer stick with the old familiar tools already available, such as in vitro methods, WT mice, rats, and non-human primates (NHPs)?
The answer is straightforward: humanized mice can predict how drug candidates will behave in the clinic and allow drug developers to fine-tune their lead molecules to reduce their toxicity and increase their efficacy before they are tested in humans. Leading pharmaceutical companies have extensively validated these tools and are applying them to their development pipeline, as testified by many peer-reviewed articles and seminar presentations at scientific meetings1,2,3. In the next paragraph, we will see a few examples of how these drug developers have used humanized models to advance their development programs.
First-in-human
Preclinical scientists working on innovative formats face a daunting challenge: predicting the clinical PK of molecules that have never been injected into human beings. To add to the complexity of the issue, most large molecules are eliminated from the blood using active mechanisms that rely on species-specific protein interactions. Two well-known examples are IgG-based and albumin-conjugated molecules, which are recycled through their interaction with the neonatal Fc receptor (FcRn). Because of evolutionary divergence, the relative affinity of these compounds to FcRn widely differs between species. To overcome this problem, translational scientists have exploited the possibility of modifying the mouse genome, replacing the mouse FcRn with its human counterpart4,5.
Sanofi is a company at the forefront of developing engineered molecules that exploit the FcRn recycling mechanism to increase their half-life. The DMPK group of Sanofi (among other leading pharmaceutical companies) has shown that FcRn transgenic mouse models can be successfully used to predict the PK of Fc-silenced monoclonal, bispecific, and tri-specific antibodies6,7, paving the way to a rational selection of the dose selection in first-in-human trials.
The double-edged sword of specificity
The more specific an antibody, the less likely it will have off-target liabilities. However, high specificity toward a human epitope usually means a lack of cross-reactivity with homologous proteins in the commonly used preclinical species. The absence of in vivo systems to test toxicity and efficacy leads to a very complex approach to the clinical trial due to the need for a more reliable estimation of the therapeutic window. An example is RG6333, a CD19xCD28 bispecific antibody8. Because of TGN1412, a CD28-targeting antibody that almost killed six people in a clinical trial, CD28 is considered a high-risk target. Since the CD28 arm of RG6333 is not cross-reactive with rodents or NHPs, the developers decided to test its potential toxicity in humanized mice, demonstrating its safety compared with TGN1412. With this approach, the preclinical group could meet the regulatory requirements and suggest a rationale for designing the clinical regimen.
Combination is the future
Few clinicians doubt that only by combining drugs against different aspects of tumor biology we will be able to develop efficacious treatments. However, the simultaneous administrations of two or more drugs pose immense challenges to clinical scientists who need to find the best therapeutic window. Researchers at Roche have used humanized mice to test the combination of bispecific antibodies with small molecules, checkpoint inhibitors, and other bispecific antibodies9,10. Using humanized models, the preclinical scientists could evaluate the efficacy of the different combinations at various doses and inform clinical researchers about the best dosing for the optimal therapeutic windows.
First: do no harm
The primary mandate of clinicians is to avoid toxic effects that nullify the benefits of treatment. Since most immunotherapeutic drugs activate the immune system, they risk triggering an overactive immune response, usually by inducing an excessive cytokine release. Iatrogenic cytokine- release syndrome (CRS) is recognized as one of the most common and dangerous side effects of almost all immunotherapies currently in development. Drug developers face two significant hurdles when predicting CRS: the lack of cross-reactivity with non-human species and large patient variability. Humanized mice have been proven to be the optimal solution to address these issues. By expressing human targets, they overcome the specificity issue, and using multiple donors for the humanization procedure mirrors the variability seen in the clinic11. Several articles show how preclinical researchers have used similar tools to identify new approaches to manage drug-induced CRS without reducing the efficacy of the treatments, paving the way for novel clinical strategies to address acute immunotoxicity12,13.
The value of being relevant
The rational use of humanized mice provides invaluable information to optimize clinical trial protocols and maximize the likelihood of success of drug development efforts. The knowledge accumulated in the past few years allowed scientists to introduce additional genetic modifications in the original mouse strains to improve the performance of these models and mimic relevant aspects of the human immune system with increased fidelity, enhancing their translational value14.
While regulatory agencies have recently recognized the lack of translational value for most preclinical species15, the need for robust in vivo data is still widely recognized to ensure the safety and efficacy of new immuno-oncology molecular entities entering clinical trials. With the only species still considered translationally relevant, NHPs, in short supply16, humanized mouse models are becoming the only viable choice to generate reliable translational data and ensure that the new molecular entities entering the clinic are safe and efficacious.
References
- Pasquiers, B.; Benamara, S.; Felices, M.; Nguyen, L.; Declèves, X. Review of the Existing Translational Pharmacokinetics Modeling Approaches Specific to Monoclonal Antibodies (mAbs) to Support the First-In-Human (FIH) Dose Selection. Int. J. Mol. Sci. 2022, 23, 12754.
https://doi.org/10.3390/ijms232112754 - Novartis Compiles Mouse Avatar "Encyclopedia". Cancer Discov (2016) 6 (1): 5-6.
https://doi.org/10.1158/2159-8290.CD-NB2015-168 - Cristina C. et al. Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy. Science Translational Medicine 12 Jun 2019 Vol 11, Issue 496
https://doi.org/10.1126/scitranslmed.aav5989 - Proetzel G. and Roopenian D. Humanized FcRn mouse models for evaluating pharmacokinetics of human IgG antibodies. Methods 2014 Volume 65, Issue 1 Pages 148-153.
https://doi.org/10.1016/j.ymeth.2013.07.005 - Avery B.L., et al. Utility of a human FcRn transgenic mouse model in drug discovery for early assessment and prediction of human pharmacokinetics of monoclonal antibodies. 2016 mAbs, 8:6, 1064-1078,
https://doi.org/10.1080/19420862.2016.1193660 - Mackness B.C., et al. Antibody Fc engineering for enhanced neonatal Fc receptor binding and prolonged circulation half-life, mAbs, 2019 11:7, 1276-1288,
https://doi.org/10.1080/19420862.2019.1633883 - Valente D. et al. Pharmacokinetics of novel Fc-engineered monoclonal and multispecific antibodies in cynomolgus monkeys and humanized FcRn transgenic mouse models. mAbs Volume 12, 2020 -Issue 1
https://doi.org/10.1080/19420862.2020.1829337 - Johannes Sam, Thomas Hofer, Christine Kuettel, Christina Claus, Sylvia Herter, Guy Georges, Jenny Tosca Thom, Leo Kunz, Samuel Gebhardt, Florian Limani, Stefanie Briner, Silvia Jenni, Anne Schönle, Marine Le Clech, Ahmet Varol, Esther Bommer, Birte Appelt, Sara Colombetti, Stephan Gasser, Marina Bacac, Christian Klein, Pablo Umana; RG6333 (CD19-CD28), a CD19-Targeted Affinity-Optimized CD28 Bispecific Antibody, Enhances and Prolongs the Anti-Tumor Activity of Glofitamab (CD20-TCB) in Preclinical Models. Blood 2022; 140 (Supplement 1): 3142–3143. doi:
https://doi.org/10.1182/blood-2022-159941 - Johannes Sam, Sara Colombetti, Tanja Fauti, Andreas Roller, Marlene Biehl, Linda Fahrni, Valeria Nicolini, Mario Perro, Tapan Nayak, Esther Bommer, Anne Schoenle, Maria Karagianni, Marine Le Clech, Nathalie Steinhof, Christian Klein, Pablo Umaña and Marina Bacac Combination of T-Cell Bispecific Antibodies With PD-L1 Checkpoint Inhibition Elicits Superior Anti-Tumor Activity Front. Oncol., 30 November 2020 Sec. Cancer Immunity and Immunotherapy Volume 10 - 2020
https://doi.org/10.3389/fonc.2020.575737 - Christian Klein, et al. Targeting Intracellular WT1 in AML Utilizing a T Cell Bispecific Antibody Construct: Augmenting Efficacy through Combination with Lenalidomide. 2019 Blood, Volume 134, Supplement 1, Page 4450, ISSN 0006-4971,
https://doi.org/10.1182/blood-2019-130121 - Ye C., Yang H., Cheng M., Shultz L.D., Greiner D.L., Brehm M.A., Keck J.G. A rapid, sensitive, and reproducible in vivo PBMC humanized murine model for determining therapeutic-related cytokine release syndrome. 2020 FASEB J. Sep;34(9):12963-12975. doi:
https://doi.org/10.1096%2Ffj.202001203R - Gabrielle Leclercq, Nathalie Steinhoff, Hélène Haegel, Donata De Marco, Marina Bacac & Christian Klein Novel strategies for the mitigation of cytokine release syndrome induced by T cell engaging therapies with a focus on the use of kinase inhibitors. 2022 OncoImmunology, 11:1,
https://doi.org/10.1080/2162402X.2022.2083479 - Gabrielle Leclercq-cohen, Marina Bacac & Christian Klein Rationale for combining tyrosine kinase inhibitors and T cell redirecting antibodies to mitigate cytokine release syndrome (CRS). 2023 Expert Opinion on Biological Therapy, 23:3, 223-225, DOI:
https://doi.org/10.1080/14712598.2023.2166786 - Cogels M.M., et al. Humanized Mice as a Valuable Pre-Clinical Model for Cancer Immunotherapy Research. 2021 Front Oncol. Nov 18;11:784947. doi:
https://doi.org/10.3389/fonc.2021.784947 PMID: 34869042; PMCID: PMC8636317. - https://www.science.org/content/article/fda-no-longer-needs-require-animal-tests-human-drug-trials
- https://www.science.org/content/article/supply-monkeys-research-crisis-point-u-s-government-report-concludes