By Courtney Ferrebee, PhD, Field Applications Scientist, Taconic Biosciences.
Although immunotherapy has revolutionised cancer therapy, the effectiveness of currently available immunotherapies varies significantly across patients and treatment types. As a result, researchers are striving to continually improve immuno-oncology approaches using the patient’s own immune cells to combat tumours. Humanised immune system (HIS) mouse models that express human cytokines and support a broad range of human immune cell types have become integral tools to investigate and advance oncology drug discovery focused on cell-based therapies, which make up a growing proportion of cancer treatments. Increasing evidence of the clinical translatability of HIS mouse models is proving essential to advancing cell-based immunotherapies that may overcome the challenges of first-generation immuno-oncology therapeutics.
Going beyond the limits of immune checkpoint inhibitors (ICIs)
As the onco-therapeutic field makes more strides with immunotherapies, research efforts are being directed toward determining their efficacy and mechanisms of action and improving treatment options to encompass a wider variety of tumour indications. Immunotherapies have become more popular due to their ability to trigger and train the patient’s immune system to recognise and attack tumour cells, provide strong immune responses, and reduce the severity of adverse side effects generally associated with chemotherapies. Immunotherapy efficacy depends on the treatment type, patient health (including co-morbidities), tumour molecular and cellular characteristics, and tumour indications.
Immune checkpoint inhibitors (ICIs) like pembrolizumab (Keytruda) have numerous benefits and have become a standard of treatment for many patients. For example, in randomised clinical trials of patients with non-small cell lung cancer (NSCLC), pembrolizumab has been shown to be effective in slowing down disease progression and improving survival rates1.
However, adverse effects such as cytokine release syndrome (CRS) and neurotoxicity still occur. To mitigate these risks, clinicians are turning to other immunomodulators, such as cell-based therapies, bispecific antibodies, and cytokine treatments. With the approval of Carvykti and other chimeric antigen T cell (CAR-T) therapies, there is an emergence of cell-based strategies using a more personalised treatment approach. In the area of adoptiveT cell transfer (ACT), which includes tumour infiltrating lymphocytes (TIL), CAR-T and T cell receptor therapy (TCR), clinical data has shown that T cell function is a strong predictive marker of immunotherapy response and patient health2. This suggests there is value in focusing on how to utilise a targeted cytotoxic approach with T cells for better patient outcomes. In vivo models, particularly HIS models, allow preliminary investigation of cell-based therapeutic strategies—often in combination with other therapeutic modalities—to overcome the limitations and challenges of T cell-based immuno-oncology treatments and identify effective options for clinical investigation.
Using hIL2 Immunodeficient Mice to investigate ACT/TIL, TCR, and CAR-T cell therapies
HIS mouse models are immunodeficient mice humanised through the engraftment of human immune cells. They also may express transgenic human cytokines to facilitate reconstitution and function of specific immune cell subsets needed to study cell therapy.
After approval of many well-known ICIs, including Keytruda and nivolumab (Opdivo), there was a push to show therapeutic efficacy pre-clinically in mice. Though results have been mixed, there is no doubt that HIS mice are an effective tool to study ICIs3. Many studies have shown HIS and humanised transgenic models can support T cell functionality, with researchers using hIL2 transgenic models that express transgenic human IL2 (hIL2) cytokines to evaluate T cell therapies such as ACT/TIL,CAR-T, and TCR therapies. Expression of human IL2 is critical for the function of adoptively transferred TILs and CAR-T in humanised mice. Jespersen et al. demonstrated the ability to model clinical responses to ACT in autologous HIS mice4. Patient-derived xenografts (PDX) of melanoma were grafted into hIL2 mice, and TILs derived from the same melanoma patient were reactivated and expanded in vitro with IL2 and used to treat the PDX engrafted in the mice. Researchers discovered that the IL2 expressed in these mice allowed these TILs to eradicate the tumours and metastases present, similar to ACT therapy inpatients5. Immunodeficient mice that did not express hIL2 did not benefit from treatment and showed little or no reduction in tumour volume.
Studies have also shown that combining cell-based treatments with ICIs can enhance responsiveness and efficacy, similar to what’s seen in the clinic, providing a foundation for more directed or targeted T cell therapies against tumours based on the expression of specific antigens6-8. Forsberget al. treated uveal melanoma with HER2-directed CAR-Ts in hIL2 immunodeficient mice9. Compared to controls, CAR-T cells engineered to target HER2 expressing melanoma cells significantly reduced tumour volumes in hIL2 immunodeficient mice. Researchers also have used this strategy to target neoantigens derived from tumour mutations to effectively treat and eradicate tumours in hIL2 immunodeficient mice10.
An emerging area in the immuno-oncology field is a precision medicine approach in which HIS mice could be used to predict patient outcomes and determine if patient-derived TIL or ICIs are sufficient to eradicate tumours11. In recent studies, IOV-4001, a TIL cell PDCD-1 knockout TIL cell therapy in clinical trials as of 2022, showed improved efficacy in hIL2 immunodeficient mice12. Additionally, the IOV-4001 combination therapy strategy with TILs eliminated the adverse effects typically experienced with ICIs. Besides demonstrating that these cells retain their functionality, the study also demonstrates a scalable way to manufacture this therapeutic. Further research using hIL2 immunodeficient mice could involve investigating whether ACT is solely mediated by CD8-mediated cytotoxicity or if additional T cells such as CD4 and T regs are involved in anti-tumour immunity. According to emerging theories, the process of tumour eradication is more complex than simply activating cytotoxic CD8 T cells. Among the ways CD4 T cells may contribute to anti-tumour immunity, CD8 T cells may be provided with effector cytokines such as IFNγ and TNFα13. It is likely these T cells could play a much bigger role in immunotherapy, and preclinical studies would be needed to help understand the mechanisms involved. Regulatory T cells also have become an important part of the story. Using their function and their ability to control inflammation has been an avenue of immunotherapy for autoimmune disease, with increasing evidence that they can play a role in tumour escape and evasion14,15.
Using hIL15 Immunodeficient Mice to Investigate CAR-NK and NK cell Therapies
CAR-NK and NK cell therapies also are being considered as options in the clinic, with the FDA clearing FT536 in early 2022 for solid tumour treatment. The anti-tumour effects of adoptive NK cell therapy have been demonstrated in ovarian and colon cancer xenograft models, although clinical results are mixed and highly influenced by the tumour indication16,17. These results indicate a need for more preclinical studies for feasibility and translatability purposes.
hIL15 immunodeficient mice are emerging as potential models for investigating these therapies based on IL15’s involvement with NK cell development18. Several studies have shown that hIL15 immunodeficient mice support stable engraftment and function of peripheral blood NK cells19-21. Additional studies have demonstrated that NK cells in this model have cytotoxic killing properties and can reduce tumour volumes similar to activated T cells in the hIL2 model22. Further, these studies and models demonstrate the potential of CAR-NK therapies, such as CAR-NK92, in treating a wide variety of cancer indications23,24. CAR-NK92, for example, has been considered for various tumour indications in the clinic. Early in vivo studies in immunodeficient mice with orthotopic xenografts resulted in mixed outcomes. Improving these preclinical studies could be as straightforward as providing a better platform or model for NK cell functionality with hIL15 expression.
The future of HIS mice for preclinical investigation of cell-based therapies
In what ways do HIS mice fit into the narrative of cell-based therapies in the future? Researchers are beginning to answer this question by extending the utility of current models and developing more clinically relevant models to test therapies. NK and CAR-NK cells can mediate similar mechanisms of cytotoxicity through antibody-dependent cell cytotoxicity (ADCC) and act through the Fcγ receptor25. Since aportion of the murine immune system remains present, it can be difficult to isolate these mechanisms in mouse models. Is it possible to improve these models to investigate whether CAR-NK and NK cell therapies can elicit human ADCC mechanisms by removing background murine signalling? Researchers are now exploring this approach in murine Fc-gamma receptor deficient hIL15 immunodeficient mice26.
Additionally, myeloid supportive HIS models that express hIL3 and human granulocyte macrophage colony stimulating factor (hGM-CSF) can be repurposed to explore current and theoretical therapies on the clinical market, such as dendritic cell (DC) and macrophage-based therapies. DCs stimulated by anti-LAG3 and CTLA4 ICI therapies can induce innate immunity against tumours and current clinical strategies use the patient’s own DC to develop an oncological therapeutic vaccine27-29. DCs harvested from patients could be loaded with tumour antigens ex-vivo and then injected into the patient for presentation and activation. However, this therapy faces several challenges, including the variability of patient immunity and the practicality of ex vivo cultures. The future could involve creating genetically engineered DCs or “CAR-DC” cells using patient-derived neoantigen-loaded cells to improve clinical outcomes30. Preclinical studies in HIS models with strong DC lineages could support evaluating these theoretical scenarios prior to the clinic.
ICI studies also have demonstrated that modulating macrophages can enhance immunotherapy effectiveness, with CAR macrophages (CAR-M) proposed as a treatment strategy. This therapy also would produce cytokines and chemokines to activate effector T cells 31,32. CAR-Ms demonstrate tumour trafficking, tumour clearance, and immunosuppression resistance. The potential for this therapy is endless and suggests macrophages could be used to target ‘cold’ tumours that immune systems are unable to detect or remove33. Clinical trials show feasibility in studying and treating solid tumours. Furthermore, CAR-M strategies could be applied to therapeutic vaccines in the same way as DC-based therapies. Mesenchymal stem cell (MSC) therapy, amongst other stem cell therapies, are innovative tools currently being explored for cancer treatment. MSCs have been traditionally used for regenerative medicine. However, some studies have shown that MSCs, while previously thought to contribute to cancer pathogenesis, could help to suppress cancer growth34,35. Because these cells are so heavily involved with the tumour microenvironment they could be used as a vehicle to deliver nanoparticles or drugs to solid tumours. Additional strategies suggest using the cells to inhibit angiogenesis and stop tumour growth and metastases. Other stem cell-based approaches look to induce tumour cell death through mechanisms, including secretion of therapeutic proteins, enhancing innate immunity, and enhancing efficacy of therapeutic payload36.
The next line of thinking would be to create models that support multiple subsets of immune cells, allowing researchers to combine different cell therapies or examine the effects of specific cell therapies on other immune cell types. NK cells, for example, have been shown to enhance CAR-T efficacy in vivo37. By extending these studies to HIS models, investigators could gain a wealth of clinically relevant information. Such combination studies raise the question of whether to combine other immunotherapy strategies to improve patient outcomes; for instance, oncolytic virotherapy and CAR-T/NK appear to offer good patient outcomes38,39. As IO preclinical medicine evolves, with the promise of new and increasingly viable therapies, humanised preclinical models will remain essential to testing these scientific theories and therapeutic strategies. The ability to evaluate these therapies for efficacy and treatment in the clinic is crucial to innovation. By evaluating cell-based therapies and immunotherapy strategies in HIS translatable models, investigators will progress and advance the field and bring safer, more efficacious cancer treatments to the market.
Volume 23 – Issue 3, Summer 2022
About the author
Courtney Ferrebee, PhD, is a field applications scientist at Taconic Biosciences. Ferrebee possesses over a decade of biomedical research experience. After earning a B.A. in biology from Agnes Scott College and a Ph.D. in molecular medicine from Wake Forest School of Medicine, she completed postdoctoral research on the development of novel, protein-based HIV immunogens for an HIV immunisation regimen.
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