Sophia Ma1,2
1Crystal Springs Uplands School, Hillsborough, CA, USA
2Cambridge Centre for International Research, Cambridge, UK

Abstract
Type 1 diabetes (T1D) is an autoimmune disorder characterized by a loss of insulin-producing β-cells, causing hyperglycemia. Current treatments, such as insulin therapy, aim to combat symptoms but not β-cell loss. Chimeric antigen receptor (CAR) T-cell and CAR regulatory T cells (Tregs) are emerging therapies that target autoreactive immune cells and protect β-cells. This review discusses the mechanisms of CAR technology, its application to T1D, findings from preclinical studies, and challenges to clinical translation. Advancements in CAR therapies may transform treatments for T1D by addressing the autoimmunity of the disease.

Keywords: type 1 diabetes, chimeric antigen receptor, CAR T-cell, CAR-Treg, autoimmune

Introduction
Type 1 diabetes (T1D) is the destruction of β-cells by autoreactive immune cells in the islets of Langerhans. The incidence of T1D is escalating worldwide, especially among children and adolescents, making it an increasing public health concern1. This disease is caused by a combination of genetic and environmental factors. Insulin is a hormone that helps to regulate glucose levels in the blood. It binds to insulin receptors on the cell membrane, triggering signals that promote cellular responses, causing glucose transporters to allow glucose to enter the cell and be used as energy or stored as glycogen2. With insufficient amounts of insulin due to β-cells being attacked by the immune system, people with diabetes have hyperglycemia3. Common symptoms of diabetes include fatigue, hunger, polydipsia, and polyuria. Additionally, T1D can cause organ damage due to prolonged hyperglycemia. Currently, the most common treatment for diabetes is insulin therapy, which can be delivered through injections and pumps. Insulin must be administered frequently in diabetes patients to effectively regulate blood glucose levels. However, taking too much insulin can lead to hypoglycemia, which causes shaking, hunger, and sweating. While insulin therapy does generally ameliorate symptoms, it does not address the autoimmune destruction of β-cells4. There has been a rise in the development of cell-based therapies for T1D to combat the autoimmune part of this disease.

CAR technology is an emerging area of research for T1D. CAR T-cells are genetically modified T cells that are able to recognize and destroy certain cells when injected into patients. Their ability to bind to specific antigens has allowed for their success as a form of cancer treatment, motivating new research to use CAR technology for autoimmune disorders5. CAR technology holds potential to be used to treat T1D through creating specific CAR T-cells and CAR-Tregs that can protect β-cells and increase insulin production by destroying and suppressing autoreactive immune cells.

Discussion

Pathogenesis of T1D
In T1D, pancreatic β-cells located in the islets of Langerhans are mistakenly attacked by the immune system. The human leukocyte antigen (HLA) region, located on chromosome 6, plays a crucial role in the regulation of immune responses. The HLA region has different classes of genes that encode major histocompatibility complex (MHC) proteins to present antigens on the surface of cells, helping the body distinguish between self and non-self6. Individuals with diabetes have certain HLA Class II proteins that present self-antigens, such as insulin or insulin-derived peptides, to CD4+ helper T cells in ways that trigger the immune system7. Improper presentation of antigens in people with diabetes leads to autoimmune destruction; these CD4+ T cells then stimulate cytotoxic T cells, which trigger apoptosis in the β-cells of the pancreas. The HLA Class II region has the genes HLA-DR, HLA-DQ, and HLA-DP, which encode proteins that present antigens to CD4+ helper T cells8. Certain HLA Class II haplotypes, most commonly DR4-DQ8 and DR3-DQ2, are associated with increased risk of developing T1D9. While there is no cure for diabetes, advancements in CAR technologies have provided the ability to modify T cells and regulatory T cells to help treat T1D from an immune standpoint.

Structure of Chimeric Antigen Receptors
Chimeric antigen receptors are synthetic transmembrane receptors. Their structure is made of four regions: the antigen receptor domain, hinge region, transmembrane domain, and intracellular signal transduction domain, all in tandem10. The antigen receptor domain helps the CAR find and bind to its target cells. It is composed of a signal peptide and a single-chain variable fragment (scFv). The signal peptide helps the newly made CAR protein enter the T cell’s or regulatory T cell’s endoplasmic reticulum11. This ensures that the CAR in CAR T-cells and CAR-Tregs is properly made, transported, and expressed on the surface of the cell, allowing it to recognize its specific antigens. A scFv is the antigen recognition part of the receptor. It is made by using a flexible linker—a short peptide sequence—to connect variable portions of heavy and light chains of an antibody, allowing the scFv to remain antigen specific while being smaller than an antibody10. The hinge region spaces up the antigen-binding region to the transmembrane domain12. The complex is typically derived from the constant domain of an antibody, and it provides flexibility for the scFv, allowing the CAR to reach and bind to antigens more effectively13. The length and design of a hinge region are important to optimize the activity of a CAR T-cell. Longer hinges provide greater flexibility and the development to reach epitopes close to the cell membrane. Shorter hinges have less flexibility and can reach epitopes further from the cell membrane14. Typically, a hydrophobic alpha helix structure, the transmembrane domain connects the antigen receptor domain to the T cell membrane, and is often derived from CD28, which is a protein expressed on T cells10. The transmembrane domain affects the expression levels of CAR on the surface of the cell membrane13. One or more intracellular T cell signaling domains are present in the endodomain of a CAR, and they are the functional part of the synthetic protein. They send activation signals to T cells after the CAR binds to its target, triggering cytokine release and apoptosis in the target cells. The primary signal transduction domains are commonly derived from CD3ζ, while the co-stimulatory domains—which strengthen T cell response—are often derived from CD2810. The structure of CARs allows for specific recognition of antigens and the activation of an immune response. When the scFv binds to its target antigen, intracellular signaling is triggered, and the T cell is activated. The form of a CAR is critical, and it impacts the cell’s function. CAR generations are classified by the number of intracellular signaling domains present. First generation (1G) CAR cells have a CD3ζ signaling domain and do not possess any costimulatory domains15. With limited signaling, reduced proliferation, insufficient cytokine release, and restricted in vivo persistence, 1G CAR cells are antiquated and generally no longer used14. Second generation (2G) CAR cells combine a CD3ζ signal and a costimulatory domain16. An additional intracellular signal provides improved CAR cell activation and proliferation17, although persistence and relapse remain challenges14. Third generation (3G) CAR cells are composed of CD3ζ and two costimulatory domains, which allow for increased proliferation and persistence18. However, due to more signaling, side effects and CAR cell exhaustion can be more severe19. Fourth generation (4G) CAR cells are also known as T cells redirected for universal cytokine-mediated killing (TRUCK), universal CAR (UniCAR-T), or armored CAR-T-cells14,20. In addition to CD3ζ and two costimulatory domains, 4G CAR cells secrete transgenic proteins (like cytokines)21. 4G CAR cells have an improved anti-tumor response, but are limited by off-target responses14. Fifth generation CAR cells incorporate cytokine-inducing signaling to enhance activation, persistence, and proliferation14. CAR generations continue to be developed and improved for the treatment of autoimmune diseases.

CAR T-cells and CAR-Tregs for Type 1 Diabetes
CAR technology can be used as a treatment in T1D patients through CAR T-cells and CAR-Tregs. CAR T-cells are engineered to kill T lymphocytes that mistakenly attack insulin-producing β-cells. Additionally, regulatory T cells can be modified to express CARs that bind to β-cells, resulting in the release of immunosuppressant factors to limit continued autoimmune destruction. CAR T-cells target autoreactive T cells that are killing pancreatic β-cells or B cells that produce autoantibodies, which aids in the destruction of β-cells. T cells are isolated from the patient and genetically modified to express CARs that target markers or specific receptors expressed on autoreactive immune cells21. For example, CD19 is a marker on all B cells, so CD19-specific CAR T-cells aim at depleting the overall number of B cells in the body22. Autoreactive B cells have receptors that recognize self-antigens such as insulin; engineering a CAR that is able to recognize those receptors results in decreased production of autoantibodies and antigen presentation that contributes to the destruction of β-cells23. Autoreactive cytotoxic T cells may have defective T cell receptors (TCR), making certain TCRs another plausible target for CARs23. Additionally, autoreactive helper T cells are a possible target because they activate B cells and cytotoxic T cells. Once the T cells are engineered to express a certain CAR and have been successfully expanded, the CAR T-cells can be infused back into the diabetic patient to begin their function21. CAR-T cells engineered to target MHC class II peptides on autoreactive B cells are able to delay the onset of diabetes in non-obese diabetic (NOD) mice24.

Instead of triggering apoptosis in other cells, Tregs suppress their activity without killing them. Tregs also help to maintain homeostasis by restraining undesired immune responses25. This ability allows for their potential to be used in autoimmune disorders, including T1D. CAR-Tregs are genetically modified regulatory T cells that release immunosuppressive factors in response to certain antigens23. Tregs are only 5-10% of CD4+ T cells, making them more challenging to isolate than cytotoxic T cells26. However, FoxP3 is a Treg-specific transcription factor and marker, and it can be used to help identify Tregs26,27. Similar to CAR T-cells, once they are isolated, Tregs are engineered to express CARs specific to antigens of the islets of Langerhans. In this way, CAR-Tregs are able to locally suppress autoreactive immune cells in the pancreas. Overall, in individuals with T1D, CAR-Tregs aim to inhibit or suppress the function of autoreactive cells and protect β-cells from further destruction, thereby increasing insulin production. CAR-Tregs can suppress autoreactive T cells in the presence of insulin in NOD mice28.

Challenges with CAR Technology for Type 1 Diabetes
Although the prospect of CAR therapies for T1D is promising, there are many challenges that must be addressed before they can be clinically used. One risk associated with CAR T-cells is the development of cytokine release syndrome (CRS), a condition that occurs when the immune system is overstimulated because of persistent or overly aggressive CAR T-cell activation29. Another challenge with CAR T-cells is identifying specific T and B cell receptors that are unique to autoreactive immune cells. Identification of specific receptors is crucial to avoid off-target effects, but it remains difficult due to the variety of different receptors12. CRS typically cannot be developed with CAR-Tregs because their function is to suppress the immune system. However, CAR-Tregs could cause overall immune suppression if they act outside of the islets of Langerhans. Further research on dosing strategy is needed to minimize these adverse effects while optimizing the efficacy of this therapy, which ensures that the CAR T-cells or Tregs remain stable and functional over time. Cost is a significant limitation for both CAR T-cells and CAR-Tregs, especially since CAR therapy is personalized and uses autologous cells12. For this reason, there is ongoing research aiming to use allogeneic cells as a way to reduce both time and cost of manufacturing. However, the use of allogeneic cells also has many challenges, including the possibility of immune rejection.

Conclusion
CAR T-cells and CAR-Tregs show great promise as therapies for T1D since it is an autoimmune disease. Both aim to protect insulin-producing β-cells from further destruction, albeit in different ways. CAR T-cells eliminate autoreactive immune cells, whereas CAR-Tregs suppress their activity. Future work should focus on improving antigen specificity to avoid off-target effects, increasing CAR cell longevity and stability, refining dosing strategies to optimize safety and efficacy, advancing clinical trials, and reducing overall cost to make CAR therapies for T1D more accessible. With recent advancements in this field of research, CAR therapies may be able to address the autoimmune destruction that characterizes T1D and greatly improve the lives of individuals with the disease.

Acknowledgements
The author would like to thank Drs Hazal Gezmis and Hakan Coskun for their guidance and feedback during the writing process, and acknowledge the support of CCIR.

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