Unlock the Potential of Your Preclinical Oncology Studies

Unlock the full potential of your preclinical oncology studies with a humanized mouse model Table of Contents Introduction 3 1. What mouse models are available? 4 2. Humanized mouse models in immuno-oncology 5 BioVolume: Insightful preclinical oncology research 6 Uterine leiomyosarcomas 7 Acute Myeloid leukemia (AML) 8 3. What can humanized mouse models be used for? 9 ICP 9 Cytokine-based immunotherapies 10 ADCC studies 10 CAR-T studies 10 Oncolytic virus efficacy 11 4. Select the right humanized mouse model 12 1. Cell source 12 2. Cell quality 12 3. Mouse strain 12 4. Boosted vs. non-boosted 12 5. Enhance your studies with a partner 13 Benefit 1: Assistance in selecting the most appropriate model 14 Benefit 2: Access to cutting-edge technologies to support your studies 15 Benefit 3: Access to dedicated teams of experts 16 Beyond insights: Welfare, speed, and experience 16 6. Unlock the full potential of your preclinical studies 17 7. References 18 transcurebioservices.com 2 The landscape of drug development is notoriously tricky to navigate. Approximately 90% of drugs fail across all clinical trials — rising as high as 95% for oncology — despite showing efficacy in preclinical studies (Figure 1).1 The low success rate indicates that significant barriers still exist when it comes to translating findings from preclinical investigations to in-human trials. One such challenge arises from the limitations of preclinical models, as many lack the relevancy needed to accurately represent the human system under study. These issues are especially pertinent for treatments that target or harness the human immune system (HIS) to combat cancer, inflammation, or infection. Traditional preclinical models struggle to accurately recapitulate the complex interactions between the immune system and the disease as they occur in humans. Consequently, the data generated doesn’t provide the critical insight needed into factors that influence treatment efficacy, such as dosage, pharmacokinetic (PK), and pharmacodynamic (PD) data. Humanized mouse models can be the key to more efficacious treatments. By providing a more precise representation of important interactions, the models allow researchers to gain deeper insights into how a disease responds to treatment. In this guide, we explore how humanized mouse models can help accelerate the development of novel, safe, and effective treatments for oncology. We examine: • The various types of mouse model available • The role and application of humanized mouse models in immuno-oncology research • How to select the most appropriate model for your specific needs Finally, we look at the benefits of partnering with a preclinical CRO specializing in humanized mouse models to further enhance your oncology research efforts. Figure 1: Likelihood of approval (LOA) of a drug across different indications from Phase I. [Source: Mullard A, Parsing clinical success rates, Nature Reviews Drug Discovery (2016).1 ] Infectious disease Haematology LOA from Phase 1 (%) Opthalmology Other Metabolic Gastroenterology Allergy Endocrine Respiratory Urology Autoimmune All indications Neurology Cardiovascular Psychiatry Oncology 0 5 10 15 20 25 30 26.1 19.1 17.1 16.3 15.3 15.1 14.7 13.2 12.8 11.4 11.1 8.4 6.6 6.2 5.1 9.6 Introduction transcurebioservices.com 3 Several mouse models exist for conducting oncology research (Figure 2), each with its own advantages and limitations: • Syngeneic mouse models enable researchers to study antitumor responses in immunocompetent models, shedding light on the interactions between immune and tumor targets. However, these models are limited to the use of mouse tumor cells, which means that the heterogeneity of human tumors and the nuances of the human immune system are not accounted for • Immunodeficient animals offer an alternative approach by allowing scientists to observe the effects of a drug on human tumors directly. However, these models fall short when it comes to evaluating immuno-oncology therapeutic approaches, which have proven to be highly effective against various forms of cancer • Transgenic mouse models, which express specific human targets, can only assess a limited number of targets — constraining the scope of the research The CD34+ humanized mouse model has emerged as the most clinically relevant option, as it reconstitutes all human immune targets and can accurately predict clinical immune response triggers. What’s more, the model achieves a fully functional human immune system (HIS) within 14 weeks. Humanized mouse models offer several advantages for oncology research, including: • A deeper understanding of the immune system, which can lead to the identification of more promising treatment candidates, and ultimately, reduced development costs and timelines • Avoidance of graft versus host disease (GvHD) — a common drawback in peripheral blood mononuclear cells (PBMC) engrafted mouse models — in which the grafted immune system attacks mouse cells. GvHD can compromise animal welfare and introduce variability in the data. In contrast, CD34+ HIS mouse models do not develop GvHD, resulting in higher quality results • Stable humanization throughout the animal’s lifetime Owing to the advantages of humanized mouse models, it is no wonder that many researchers are opting to include them in their preclinical studies. So, how can humanized mice be used? Figure 2: A summary of the benefits and weaknesses of different mouse models. Syngeneic mouse model Study of anti-tumor responses Limited to mouse tumor cells Does not account for the heterogeneity of tumors and nuances of the human immune system Immunodeficient mouse model Observe drug effects directly on human tumors Cannot evaluate immunooncology therapeutic approaches Transgenic mouse model Expresses specific human targets Can only assess a limited number of targets CD34+ humanized mouse model Presence of all immune cell types (myeloid cells, T cells, B cells, NK cells) No GvHD Stable humanization 1. What mouse models are available? transcurebioservices.com 4 Implanting a mouse with the tumor, followed by treatment administration via intraperitoneal (IP) injection, intravenous (IV) injection, or daily gavage Sacrificing the animal when the ethical limit is reached, and if required, sending the tissues back to the lab for further analysis Continual monitoring of the tumor growth, with researchers conducting assessments two to three times a week to obtain readouts such as tumor growth kinetics, tumor engraftment, and PK/PD data Humanized mice are extensively used in immuno-oncology studies as they are robust and can handle various treatments effectively. What’s more, they can provide valuable insights into tumor growth and treatment response through tumor measurement. Although tumor growth has traditionally been measured using calipers, advanced techniques that enable non-invasive monitoring of tumor progression, such as BioVolume, are becoming increasingly important tools for more accurate measurements. The typical use of humanized mice in immuno-oncology studies involves: 2.Humanized mouse models in immuno-oncology transcurebioservices.com 5 One of the key benefits of humanized mouse models is the ability of human immune (HI) cells to interact with the tumor, allowing researchers to observe more representative antitumor or pro-tumoral effects of a treatment. For instance, glioblastoma tumors were found to grow faster in humanized mice due to an abundance of associated macrophages that promote tumor growth. Conversely, melanoma cells are highly immunogenic and exhibit slower growth in humanized mice compared to non-humanized mice. BioVolume: Insightful preclinical oncology research BioVolume 3D imaging, a world first, enables the measurement and visualization of tumor growth in preclinical research, oncology, and drug development animal models. The non-invasive solution works by reconstructing tumors from 3D, RGB, and thermal imaging, then uses a machine learning algorithm to automatically calculate subcutaneous tumor length, height, and width. Working with a CRO partner that provides BioVolume imaging can bring multiple benefits to your research, including: • Increased measurement accuracy • Enhanced experiment reproducibility • Improved animal welfare Figure 3: Illustration showing how BioVolume measures tumor volume accurately and reproducibly. transcurebioservices.com 6 Hot tumors vs. cold tumors In a bid to deliver a tumor model that will unlock the most insight from your research, TransCure bioServices has examined more than 50 different cell-line derived xenograft (CDX)-human tumor models, including: • B-lymphoma • Acute myeloid leukemia • Multiple lymphoma • Melanoma • Colon carcinoma • Lung cancer • Ovarian cancer We have developed a “hot vs. cold” classification to determine the level of infiltration. Looking at A549 (lung carcinoma), we can see that the tumor is highly infiltrated — a “hot” tumor. On the other hand, RKO (colon carcinoma) is much less so, corresponding to a “cold” tumor. TransCure bioServices can use this classification, tumor immune infiltration data, and the mechanism of action to help you choose the most appropriate tumor model for your studies. Uterine leiomyosarcomas Humanized mice can be used to test treatments in various cancer types such as uterine leiomyosarcomas (uLMS), classified by aggressive tumors with poor prognosis. In uLMS, the tumors are classed as cold tumors — that is, they have minimal T cell infiltration and are typically unresponsive to immunotherapy.2 Despite being prime targets for immune checkpoint blockade (ICB), studies proved unsuccessful owing to T cell exclusion from the tumor microenvironment (TME), which is a major mechanism of intrinsic resistance to ICB. To prime tumors for ICB, research has started to focus on identifying approaches to inflame them. In a recent study, PI3K/mTOR inhibitors were shown in humanized mice to induce clonal T cell expansion, eliciting an anti-tumor response and significant tumor growth reduction.3 Here, the treatment was shown to convert a cold tumor into a hot tumor response — a response that cannot be observed in traditional immunodeficient mouse models. hCD45 RKO Colon Carcinoma 103 hCD45/g of tumor Raji B Lymphoma 105 hCD45/g of tumor HCT-116 Colon Carcinoma 105 hCD45/g of tumor A549 Lung carcinoma 106 hCD45/g of tumor A375 Melanoma 105 hCD45/g of tumor U87-MG Glioblastoma 105 hCD45/g of tumor 22RV1 Prostate Carcinoma 103 hCD45/g of tumor Figure 4: Tumor classification scale ranging from highly infiltrated (hot) on the left to much less infiltrated (cold) on the right. transcurebioservices.com 7 Acute Myeloid leukemia (AML) Another area where CD34+ humanized mice offer deeper insights in is identifying potential treatments for cancers that have liquid tumors, such as those found in AML. So, how does a typical study look? Engraftment and tumor induction To begin, the mouse undergoes engraftment of human CD34+ hematopoietic stem cells and complete hematopoiesis reconstitution. Leukemic cells are then administered by intravenous injection to induce the development of leukemia. The cells can be sourced from patient samples, established tumor cell lines or genetically engineered cells. Genetically engineered tumor cells with reporter genes as luciferase, mCherry or green fluorescent protein (GFP) are particularly useful as they allow the easy tracking of the tumor burden and leukemia spread in the mouse’s body (Figure 5). Treatment and analysis Once leukemia is established in the mice, various treatments can be administered to study treatment efficacy: • Chemotherapy • Targeted therapy • Immunotherapy • A combination of treatments Throughout the study, pathological analysis — including survival analysis, flow cytometry, histology, and molecular analysis — can be performed to monitor the progression of the disease. Figure 5: Fluorescence imaging of humanized mice with AML (left) and radiance data after engraftment (right). [Source: AML-Luc TransCure bioServices internal data.] transcurebioservices.com 8 Humanized mouse models can be employed for a wide range of testing, including the investigation of different mechanisms of action in cancer treatments. We explore five important applications here: immune checkpoint inhibitors (ICP), cytokinebased immunotherapy studies, antibody-dependent cellular cytotoxicity (ADCC) studies, chimeric antigen receptor (CAR)-T studies, and oncolytic virus efficacy. ICP ICP immunotherapies involve drugs that block checkpoint proteins made up of immune system cells, such as T cells, that prevent the immune system from attacking cancer cells. Using humanized mouse models for ICP studies allows researchers to study both the efficacy and mechanism of action in a single experiment, while also enabling tumor-infiltrating leukocyte (TIL) analysis after tumor dissociation. Case study: Malignant melanoma Malignant melanoma is an aggressive form of cancer. Patients with advanced melanoma have a poor prognosis, with only a 20% 5-year survival rate. Humanized mice model engrafted with an aggressive form of the melanoma tumor cell line showed significant tumor volume inhibition after treatment with an oncolytic virus. An even greater tumor growth inhibition effect could be observed in combination with pembrolizumab — pembrolizumab alone had no effect.4 The data demonstrates that humanized mice could be ideal to evaluate checkpoint inhibitors strategies — in particular anti-PD1 or anti-CTLA4 approaches, and even combination strategies with additional immunotherapies that further enhance the immune response. 3. What can humanized mouse models be used for? transcurebioservices.com 9 CAR-T studies In CAR-T cell therapy, T cells are isolated from the spleens of humanized mice and used to produce CAR-T cells with the same behavior as normal human T cells. The CAR-T cells can then be re-infused into animals from the same CD34+ donors to investigate their ability to treat cancers. Modelling CAR-T therapies in humanized mice enables researchers to investigate the persistence, efficacy and immunosafety of CAR-T treatments in the context of a complete human immune system for deeper insights. ADCC studies ADCC is a mechanism of cell-mediated immune defense where an effector cell lyses target cells. ADCC actively involves NK cells activation, and so humanized mice with NK-cell boosting are an ideal model for ADCC studies. In an internal study performed by TransCure bioServices, investigators injected A431 melanoma cells subcutaneously into hIL15-boosted mice. Bi-specific antibodies were directed against the tumor target and injected at different doses. Notably, NK cells caused full tumor regression at a high dose. Cytokine-based immunotherapies Humanized mice also show great potential in evaluating targeted immunotherapies, such as those that are cytokinebased. A recent study demonstrated the therapeutic efficacy of an antibody IL-12 fusion protein (NHS-IL-12) in a CD34+ humanized mouse model inoculated with human rhabdomyosarcoma cells.5 Therapy significantly improved the survival rate and decreased tumor volume. In addition, the treatment increased tumor mononuclear infiltration, inducing a high amount of NK cells and macrophages. transcurebioservices.com 10 Case study: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) treatment CD123 is an IL-3 receptor alpha chain that is overexpressed in BPDCN. A recent study used autologous CD34+ humanized mice to evaluate the immune functions and safety of CD28/4-1BB CAR-T cells that target CD123.6 Using the mice, researchers were able to investigate potential toxicity effects by re-infusing CAR-T cells. A low on-target/off-tumor toxicity against various subsets of normal cells was found, suggesting that CD28/4-1BB CAR-T cells have promising therapeutic potential for treating BPDCN with minimal off-target effects. Oncolytic virus efficacy Oncolytic viruses are viruses that infect and directly kill cancer cells by replicating in the targeted cells and inducing cell lysis. When the virus kills the cells, the cancer cells release cancerous materials — such as tumor associated antigens — which stimulate the immune response. In addition, oncolytic viruses can now be genetically modified to deliver therapeutic payloads that will further boost the immunity. Humanized mouse models can be used in studies investigating oncolytic virus efficacy, as demonstrated in the earlier case study. Additionally, TransCure bioServices has performed internal studies using humanized mice engrafted with tumor cells to investigate the tumor growth inhibition effect of a genetically modified oncolytic virus. Contact us to learn more about these studies. transcurebioservices.com 11 4. Boosted vs. non-boosted Non-boosted models often display lower amounts of myeloid/ dendritic cells and NK cells, with a majority of T and B cells present. While these are valuable and effective tools for many studies, some may require better distinction between myeloid/ dendritic and NK cells. In such cases, mice can undergo boosting to exhibit enhanced differentiation between cell types (Figure 6). Boosted mouse models are formed through hydrodynamic plasmid DNA delivery, which allows for more customized immune system development. Researchers are subsequently able to tailor their model to their specific needs, as the mice can be differentiated into the required cell types. It is crucial to recognize that not all humanized mouse models are created equal, and choosing a high-quality model is vital for the success of your research. There are four factors to consider when selecting the right humanized mouse model — the cell source, cell quality, the mouse strain, and boosting. Natural killer (NK) cells Myeloid cells Dendritic cells (DCs) 1. Cell source The origin of the cells used in the model plays a significant role in its performance: • Donor blood, as in PBMC mice, which include differentiated lymphocytes, monocytes, and dendritic cells. Proportions vary depending on the donor, and these mice often develop GvHD and show poor development of other immune populations. • Cord blood, which is rich in human hematopoietic stem cells (HSC) and serves as a source of CD34+ cells used to reconstitute the human immune system. Cord blood offers better engraftment properties compared to other sources. 2. Cell quality High-purity CD34+ cells are crucial for high-quality results. Contamination with other cell types, such as CD4 or CD8 cells could induce GvHD, lowering animal welfare and interfering with your findings. 3. Mouse strain Numerous highly immunodeficient mouse strains are available, including NSG, NRG, NOG, and BRGS. However, many of these strains lack human cytokines, limiting the development of the human innate immune system.7 Some strains, such as NOG, are genetically modified to express human cytokines to overcome this limitation. Figure 6: Plasmid boosting activates CD34+ development into a customizable human immune system, with the most appropriate proportions of cells. 4.Select the right humanized mouse model transcurebioservices.com 12 There are many considerations involved when working with humanized mice, such as selecting an appropriate model and determining the most suitable studies for your research. It is important to note, however, that most suppliers will only provide the mice and not offer any additional services. As a result, researchers can be left feeling unsupported for their studies in four significant ways: 1. You must possess a wide range of knowledge across various fields 2. Labs need to be equipped with an extensive selection of top-of-the-range instruments for analysis 3. A supplier will not support you in understanding and interpreting the data generated 4. Humanized mice are more challenging to work with compared to syngeneic or immunodeficient models, and a certain level of expertise is necessary to maximize the benefits of your experiments To overcome these challenges, you should consider collaborating with a partner rather than simply relying on a supplier. A partner can actively support you throughout all stages of your experiment, from initial discussions and customized protocol development to executing state-of-theart experiments, data analysis, and comprehensive reporting. Working with a partner can offer three main benefits, which we explore in more detail here. 5. Enhance your studies with a partner transcurebioservices.com 13 Benefit 1: Assistance in selecting the most appropriate model A valuable partner can provide crucial support in choosing the most suitable humanized mouse model for your research. They can perform a comprehensive tumor infiltration analysis of the humanized mouse by dissecting the tumor and preparing a single cell suspension, then use flow cytometry to obtain a complete picture of the tumor’s behavior. Subsequently, an appropriate model can be selected, helping you gain more relevant insights from your studies. TransCure bioServices also has access to detailed information about the infiltration quality in your humanized mouse model. Given that each tumor has unique characteristics in recruiting human immune cells, they can help you identify a profile relevant to your study, ensuring that your research benefits from the most appropriate model that helps you generate the data you need. transcurebioservices.com 14 Benefit 2: Access to cutting-edge technologies to support your studies A reliable partner will possess a wide range of advanced technologies to facilitate your research. For instance, TransCure bioServices offers state-of-the-art services including flow cytometry and immune profiling, immunohistochemistry (IHC) histology platforms, in vivo imaging, and in vitro assays. Flow cytometry and immune profiling TransCure bioServices uses the highest-quality instrumentation with 14 channels each for detecting complex human immune cell panels. Using the equipment for flow cytometry and immune profiling: • Enables analysis of immune phenotypes from various mouse tissues • Provides insight into biomarkers expressed by tissues/tumors • Enhances understanding of drug mechanisms of action and therapeutic efficacy Immunohistochemistry (IHC) histology platforms TransCure bioServices can conduct morphology studies using standard or specific labeling/staining techniques, enabling the investigation of biomarker expression or immune infiltration through chromogenic and fluorescent IHC. In vivo imaging Using in vivo imaging, a partner such as TransCure bioServices can offer non-invasive detection and quantification of tumors from their onset to full development. Orthotopic, metastatic dissemination, and subcutaneous tumor progression can all be monitored using this technique. In vitro assays TransCure bioServices provides a deeper understanding of drug mechanisms of action through T/B cell stimulation assays or oncological in vitro assays. transcurebioservices.com 15 Experience The experience of your partner should also be considered. A partner with extensive experience in the field, such as TransCure bioServices with their ten years of expertise, can help you better contextualize your data for greater guidance in your research. Speed Keep in mind that not all mice are immediately available. Humanized mice, for instance, take 14 weeks to generate. Limited availability can hinder your studies, reduce flexibility, and complicate your planning. However, some partners, like TransCure bioServices, have CD34+ HIS mice ready for use in studies. Their state-of-the-art facilities can house up to 9,600 mice, and they can supply over 1,000 humanized mice per month, enabling you to start your experiments and generate data more quickly. Welfare Seek a partner that prioritizes animal welfare. A partner that does so will be Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited, signifying that they are dedicated to maintaining the highest standards of animal welfare. Benefit 3: Access to dedicated teams of experts Working with a partner means gaining access to a diverse group of specialists with expertise across various fields. A collaboration, therefore, allows you to tap into the knowledge you need to design your study and interpret your results effectively. By leveraging the experience of these dedicated teams, you can optimize your research outcomes and make more informed preclinical decisions. Beyond insights: Welfare, speed, and experience Although, ultimately, a partner can offer advice, technology and expertise, there are other considerations that can make them stand out amongst the rest: transcurebioservices.com 16 Humanized mouse models provide unparalleled insights into preclinical studies, increasing the likelihood of successful outcomes in clinical trials and — ultimately — the development of safe and effective treatments. In particular, humanized models are especially valuable for immuno-oncology research, supporting studies for: However, to select the right preclinical model you must consider several factors, such as the method of generation and cell source. Additionally, although HIS mouse models offer many advantages, they suffer from limited differentiation of cells. This limitation can be overcome, however, by choosing a boosted mouse that contains the right proportion of cell types relevant to your study. To truly unlock the value of your experiments, you should seek a partner, rather than a supplier. By partnering with an organization like TransCure bioServices, your lab can access a wealth of expertise, cutting-edge equipment, and comprehensive support from the inception of your experiment to data analysis. With these resources at your disposal, your lab can make better informed decisions that enable you to advance only the most promising drug candidates and accelerate your product’s journey to market. As an AAALAC-accredited organization with over a decade of experience, TransCure bioServices is dedicated to providing top-quality humanized mouse models of the highest welfare to enhance your research and drive innovation in immuno-oncology treatments. To gain deeper insight from your oncological preclinical studies, get in touch with our team today. • ICP treatments • Cytokine-based therapies • Bi-specific antibodies • ADCC • CAR-T • Cell therapies • Gene-based therapies • Oncolytic viruses 6. Unlock the full potential of your preclinical studies transcurebioservices.com 17 Or visit transcurebioservices.com 1 Mullard, A., (2016). Parsing clinical success rates. Nat. Rev. Drug Discov., 15, 447. Available at https://doi.org/10.1038/nrd.2016.136 2 De Wispelaere, W., et al., (2021). Resistance to Immune Checkpoint Blockade in Uterine Leiomyosarcoma: What Can We Learn from Other Cancer Types? Cancers (Basel). 2021 Apr 23;13(9):2040. Available at https://doi.org/10.3390/cancers13092040 3 De Wispelaere, W., et al., (2022). Exploiting the Immune-modulatory Effects of PI3K/mTOR inhibitors to Remodel the Tumor Microenvironment in Uterine Leiomyosarcoma. [Poster]. 28th Congress of the European Association for Cancer Research, 2022 4 Kuryk, L., Møller, A-S W., Jaderberg, M., (2018). Combination of immunogenic oncolytic adenovirus ONCOS-102 with anti-PD-1 pembrolizumab exhibits synergistic antitumor effect in humanized A2058 melanoma huNOG mouse model, OncoImmunology, 8(2):e1532763. Available at https://doi.org/10.1080/2162402x.2018.1532763 5 Schilbach, K., et al., (2015). Cancer-targeted IL-12 controls human rhabdomyosarcoma by senescence induction and myogenic differentiation, OncoImmunology, 4:7. Available at https://doi.org/10.1080/2162402X.2015.1014760 6 Bôle-Richard, E., et al., (2020). CD28/4-1BB CD123 CAR T cells in blastic plasmacytoid dendritic cell neoplasm. Leukemia, Dec;34(12):3228-3241. Available at https://doi.org/10.1038/s41375-020-0777-1 7 Chuprin, J., et al., (2023). Humanized mouse models for immuno-oncology research. Nat. Rev. Clin. Onc., pp. 192–206. Available at https://doi.org/10.1038/s41571-022-00721-2 References Contact us here