CAR Cell Therapy Research Models: Conversation with The Experts

Chimeric Antigen Receptors (CARs), also known as chimeric immunoreceptors, are able to recognize and bind to specific antigens on the surface of cancerous cells. Genetically engineering immune cells, such as T cells or natural killer (NK) cells, with cancer-targeting CARs can serve as a powerful cell therapy tool. Cancer cell immunotherapy represents one of the most exciting scientific and medical breakthroughs in recent decades.

Brief Background on CAR Cell Therapy

The CAR structure includes 3 regional domains: extracellular domain, transmembrane domain, and an intracellular signal domain. The extracellular domain contains antibody single-chain variable (VH-linker-VL, scFv) and hinge regions that target the recognition of tumor target antigens. CAR-T cells are T cells armed with CAR: the scFv segment of the CAR molecule can give T cells specific targets, and the intracellular signal domain aims to replicate the events to activate T cells. Therefore, compared with T cells, the combat effectiveness of CAR-T cells is significantly enhanced.

Although CAR T cell therapies have proven particularly effective in patients with blood cancers like leukemia and lymphoma, they cannot perfectly apply to all kinds of diseases. Researchers are trying different solutions to get over these limitations, such as generating universal CAR-T cells by removing TCR and CD52, in order to avoid graft versus host disease (GVDH). This approach can be used to broaden the application range for manufacturing and allogeneic, or universally-applicable, CAR-T cell therapy treatments. At the same time, many researchers have focused their attention on researching the potential applications of CAR on relatives of T cells’- our natural killer (NK) cells. Given that CAR-NK cells are suitable for allogeneic cell therapy and offer unique advantages over CAR-T cells, CAR-NK cell therapies have become a rapidly growing area of research.

Q&A with Cell Therapy Model Expert: Dr. Shawn Zhou

We spoke with Dr. Shawn Zhou about the various types of mouse models used in current cell therapy research, including how next-generation immunodeficient mouse models such as our proprietary C-NKG mice have several features that are advantageous for cancer immunotherapy studies.

                                                                                         Shawn Zhou, Ph.D

Biography:

Earned molecular genetics Ph.D from Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences in 2012 and conducted postdoc in Biomedical Engineering Department of Tufts University from 2013 to 2014. Worked as Senior Scientist and Principal Project manager in GenScript Biotechnology Company from 2015 to 2020. Have been working in Cyagen as R&D director from 2021. Highly experienced in antibody drug, gene and cell therapy preclinical research.

1. Which types of mouse models can be used in drug efficacy, toxicity, pharmacodynamics and pharmacokinetics studies in immunotherapy for cancer?

Animal models play an important role in the preclinical evaluation of cancer immunotherapy. The establishment of appropriate animal models provides a powerful tool for studying the mechanisms of tumorigenesis and metastasis, even validating the efficacy of anti-tumor drugs.

Immunodeficient mice refer to the mice with defects in one or more immune components (such as T, B, NK cells) in the immune system. This type of mouse is widely used in the research of oncology because it allows human tumors to grow in the mice and is currently the best model to establish human cancer cell line-derived xenografts (CDX) and patient-derived tumor xenografts (PDX).

Different types of models may be used in different types of immunotherapies.

For immune checkpoint inhibitors and immune system modulators/activators, two types of mouse models can be used. One is syngeneic models with the humanized target gene; the other CDX or PDX models in immunodeficient mice engrafted with the human immune system (HIS). In a syngeneic humanized model, we are studying the mouse tumor with the mouse immune system, only the target gene has been humanized, whereas in a CDX/PDX HIS model, we are studying the human tumor with the human immune system.

For immune cell therapy (e.g., CAR-T or CAR-NK), syngeneic models or CDX/PDX models in immunodeficient mice can be used, but CDX/PDX models are preferred over syngeneic models because it allows us to study human CAR-T cells’ anti-tumor activity against human tumors, without the concerns of the high cost and technical challenges in making human immune system-engrafted mice.

2. What are the advantages and limitations of the human immune system engrafted-mouse models you mentioned earlier?

Human immune system engrafted-mouse models generally contain human PBMCs and/or human stem cells (e.g., CD34+ hematopoietic stem cell (HSC) humanized mice). They are better than partially humanized models (e.g., genetically engineered hPD1 expressing mouse model) in terms of better mimicking the human immune system, and like I have mentioned earlier, allowing us to study the human tumor in an intact human immune system. However, there are still a few limitations:

(1)  The PBMC-engrafted model develops a lethal human anti-mouse graft versus host disease (GvHD);

(2)  Experimental results may vary due to heterogeneity in different human immune cell donors;

(3)  Human immune system engrafted-mouse models are generally more expensive than genetically engineered humanization (partially humanized) mouse models.

3. In your view, what are the most important recent advances in mouse models for cancer immunotherapy studies?

Based on existing immunodeficient mouse models (e.g., C-NKG or NSG), next-generation immunodeficient mouse models that express cytokines (e.g., IL6, I15, etc.) are being developed in order to regulate T, B or NK cell function or proliferation.

C-NKG mice are a severe immunodeficiency model independently developed by Cyagen using the NOD-Scid background strain to knock out the Il2rg gene. The C-NKG mouse model is recognized as having high degree of immunodeficiency and has good performances in studies of tumors, immunity, autoimmune diseases, immunotherapy vaccine, graft-versus-host disease (GVHD), safety evaluations, and more. For example, human IL2 transgenic C-NKG mice can be used for Tumor Infiltrating Lymphocyte (TILs) therapy efficacy evaluation; human IL15 transgenic C-NKG mice can be used for CAR-NK therapy efficacy evaluation. C-NKG mice have prolonged life spans compared with the NOD SCID mice, which have a high tendency of developing thymic lymphoma. The C-NKG model is compatible with transplanted hematopoietic stem cells (HSCs), peripheral blood mononuclear cells (PBMCs), CDXs, PDXs, and adult stem cells and tissues.

Next-generation immunodeficient mouse models such as C-NKG mice have several features that are advantageous for cancer immunotherapy studies:

● Lack mature T, B, and natural killer (NK) immune cells

● Decreased complement activity

● Dysfunction of macrophages and dendritic cells

● Extremely low incidence of T and B cell leakage with age

● Extremely low incidence of lymphoma (different from NOD SCID model)

● Can be used in both long-term and short-term experiments

● Does not develop diabetes

Conclusions: Mouse Models for Cancer Immunotherapy

The ideal non-human models are able to recreate various aspects of the human tumor microenvironment and simulate the human immune system — such HIS models have been serving the rapid advancement of cancer immunotherapies. Dr. Zhou spoke with Caitlin Smith (on behalf of Biocompare) about the importance of selecting the appropriate HIS animal model systems when testing cancer immunotherapies.

“Human immune system mouse models are better than partially humanized models in theory, because the models mimic the human in vivo conditions as much as possible,” says Shawn Zhou, R&D director at Cyagen Biomodels. But there are a few limitations. For example, the PBMC-based model can develop graft versus host disease, experiments using immune cells from different human donors can suffer from repeatability problems, and xenografted models are more expensive.

The efficacy of immune checkpoint inhibitor drugs can be evaluated in humanized mouse models expressing human immune checkpoint target (e.g. PD1), or in the human immune system (HIS) reconstructed mouse model “in which severe combined immunodeficient (SCID) mice are reconstituted with functional human peripheral blood mononuclear cells (PBMCs), a class of immune cells that includes lymphocytes (T, B, and NK cells), monocytes, and dendritic cells,” says Dr. Zhou.

Mouse models are also used to develop many other cancer therapy tools. “For monoclonal antibodies (e.g., anti-claudin18.2 antibody drugs) and immune system modulators (e.g., cytokine IL-6 drugs), the appropriate mouse models can be generated by transplanting human cancer cell line-derived xenograft (CDX) or patient-derived xenograft (PDX) along with human PBMC (e.g., hPBMC C-NKG) or human hematopoietic stem cell (e.g., hCD34 C-NKG) into severe immunodeficient mice,” says Dr. Zhou. “For treatment vaccines, (e.g., neoantigen drugs), the appropriate mouse models of cancer cell line-derived xenograft (CDX) or patient-derived xenograft (PDX) are generally generated by transplanting both human PBMCs and human antigen-presenting cells (APCs) (e.g., dendritic cells),” says Dr. Zhou.

Innovation in mouse models for cancer immunotherapy research continues with state-of-the-art models. For example, Cyagen’s C-NKG SCID mouse model shows higher survival rates than other SCID mouse  after transplantation of human cells or tissues, and with implantation can receive a greater proportion of human cells or tissues. “The C-NKG model is compatible with transplanted hematopoietic stem cells (HSCs), peripheral blood mononuclear cells (PBMCs), PDXs, CDXs, or adult stem cells and tissues,” says Dr. Zhou. Newer immunodeficient mouse models such as C-NKG with specific immune system modulator, providing a more suitable to help regulate T, B, or NK cells. “For example, human IL2 transgenic C-NKG mice can be used for tumor infiltrating lymphocyte therapy efficacy evaluation, and human IL15 transgenic C-NKG mice can be used for CAR-NK therapy efficacy evaluation,” says Zhou.

Cyagen's Mouse Models for Cancer Immunotherapy Research

Animal models of disease are an indispensable tool to study the mechanisms of human disease development, as well as for drug screening and efficacy evaluations. To provide the highest-quality models to researchers, Cyagen continuously optimizes our gene editing technologies and recruits leading specialists to support your scientific innovation. Cyagen has established an animal platform for drug development, screening, or evaluation covering a wide range of disease studies, including cancer, immunity, endocrine, cardiovascular, neurological, and infectious diseases.

Cyagen can provide you with a variety of drug evaluation models along with phenotype analysis services – delivering reliable and expedient data reporting for your project.

Humanized Mouse Models

A humanized mouse is typically defined as a mouse that has been engrafted with something from a human. This could be a short strand of human DNA, human tissue, a human tumor, a humanized immune system, or parts of the human microbiome. Humanized mice are widely used small animal models for studies of cancer, human development, immuno-oncology, and infectious disease research.

Humanized Mouse Model Catalog:

When your research requires humanized animal models, Cyagen offers multiple options. Please contact us if your preferred model is not found in the catalog list.

Immunodeficient Mice

Cyagen's drug screening and evaluation mouse model platform can provide you with multiple types of immunodeficiency mouse models such as C-NKG, Rag1 knockout (KO), Rag2 KO, Tcra KO, etc., to assist in the research and development of new drugs.

Tumor Models

Cyagen’s animal model platform for drug research and development can provide various custom tumor models, tumor cell line transplantation models, and human tumor xenotransplantation models (CDX) according to your research needs. We can develop all kinds of tumor models – including subcutaneous, in situ or metastatic – and provide highly customized pharmacodynamic services for the corresponding models to meet your research needs.