CAR T Cell Therapy
CAR T Cell Therapy
Scientists very first began to experiment with chimeric antigen receptor (CAR) T cells in the late 1980’s, when a trio of researchers genetically engineered T cells to express an antibody recognizing a hapten and redirecting their specificity. 1,Two The authors of this probe were the very first to suggest the use of chimeric T cell receptors in combating tumors. Years later, in 2011, Carl June and colleagues reported a case explore where a chronic lymphoid leukemia (CLL) patient treated with autologous anti-CD19 CAR T cells (CART19) displayed a finish response. Three This finding fueled the fire in immuno-oncology research, and CAR T cell immunotherapy has since brought fresh hope to many leukemia patients who are seemingly out of treatment options.
Adoptive T cell immunotherapy has been at the forefront of the immunology research. As fresh articles are being published daily, describing fresh methods to design or develop CAR T cells and proposing fresh ways to improve their efficacy in the clinic, it’s not an effortless task to stay up to date in this fast-paced field of research. Here, we review our selection of key latest findings in CAR T cell research and discuss their implications for taking T cell therapy from bench to bedside.
Prior to being used as therapy in the clinic, CAR T cells are developed and manufactured on the bench. Primary human T cells isolated from the patients are activated, expanded and genetically modified to express CARs recognizing the selected antigen ex vivo. Many factors are taken to consideration at this stage, including target selection and method of genetic modification.
1. Target Selection
CARs are synthetic transmembrane receptors containing the extracellular scFv domain of an antibody, which confers antigen specificity, and intracellular components that typically include CD3ζ and co-stimulatory (ex. CD28, 4-1BB, OX-40) signaling domains to mimic T cell signalling (Figure 1). Four,Five Upon antigen recognition, CAR T cells are activated and initiate their effector functions such as cytokine production and cytotoxicity, which ultimately result in the elimination of any cells voicing target antigen. As tumor antigens are typically also voiced on non-malignant cells, target selection poses a challenge in CAR T cell design.
Figure 1. Structure of a Chimeric Antigen Receptor (CAR)
Adapted from the Production of CAR T Cell wallchart, produced by Nature Protocols and supported by STEMCELL Technologies.
The most basic of CAR T cells target a single tumor-specific antigen for blood cancer therapy. Examples include engineered T cells targeting CD19, which is voiced on B cell lymphomas and lymphocytic leukemias, 6,7,8 and CD123 (IL3RA), which is overexpressed in acute myeloid leukemia (AML) and other hematologic malignancies. 9,Ten
Targeting AFP-MHC sophisticated with CAR T cell therapy for liver cancer 11
Clinical Cancer Research, 2017
Researchers are now beginning to explore the use of CAR T cell therapy for solid tumors, including hepatocellular carcinoma (HCC, liver cancer). 11,12 A large proportion of tumor-specific antigens are intracellular or secreted, and thus are largely considered “undruggable” by antibody therapy. On the basis that all secreted proteins are processed into peptides and introduced on cell surface by MHC I, Liu et al. sought to develop CAR T cells targeting a secreted HCC-specific secreted antigen, alpha-fetoprotein (AFP). The result of their work is a CAR T cell targeting AFP peptide complexed with MHC I that can specifically lyse HCC cells while sparing others.
An NCR1-based chimeric receptor endows T-cells with numerous anti-tumor specificities 13
Rather than specificity, few studies have suggested an alternative treatment to broadly target numerous tumors at the same time. One particular research group developed CAR T cells voicing natural cytotoxic receptor one (NCR1), a receptor naturally voiced on natural killer (NK) cells that partly mediates recognition of a broad range of tumor cells, including carcinomas, neuroblastomas and leukemias. The precise identification of tumor-associated ligands that are recognized by NCR1 remains a challenge. Tal et al. have developed CAR T cells that express the extracellular domain of NCR1, resulting in genetically modified T cells that can recognize a diverse range of tumors.
Precision tumor recognition by T cells with combinatorial antigen-sensing circuits 14
The challenge in target selection remains that such tumor-specific antigens are uncommon, and thus CAR T cell activation in bystander tissues can occur. As a result, researchers are exploring ways to increase the specificity of CAR T cells by using combinations of target antigens. Roybal et al. developed a dual receptor system, where antigen A is required to induce the expression of CAR, and antigen B is required for the activation of CAR T cells. As a result, these engineered T cells are activated only when both antigens are present in a localized environment. This novel system not only improves the precision of tumor targeting by CAR T cells, but also expands the potential for CAR T cell use with the more common tumor-associated antigens rather than tumor-specific antigens.
Systemic immunity is required for effective cancer immunotherapy 15
Cancer immunotherapy for solid tumors generally aims to boost specific, local immune responses in the tumor microenvironment as described in the probe above. Recently, an article published in Cell by the Engleman lab turned this concept upside down. Using organism-wide analysis and mass cytometry, Spitzer et al found that systemic immunity is in fact required for effective immunotherapy leading to tumor rejection. A subset of peripheral CD4+ T cells was particularly found to be the cell type responsible for the anti-tumor immunity in response to immunotherapy.
Perhaps this unexpected observation will influence how scientists develop and evaluate CAR T cells in preclinical research, as the right balance inbetween local and systemic immune responses may be required. Still, targeting malignant cells while sparing normal, healthy cells is key reducing long term side effects of CAR T cell therapy.
Two. Genetic Modification
T cells are redirected to a particular target by genetically modifying them to express CARs. Presently, the most common method to genetically modify T cells is using viral (ex. gammaretroviral or lentiviral) vectors that has been engineered to carry the CAR construct. Sixteen In brief, T cells are activated and then transduced with virus particles that release their cargo into the cell, leading to injection of the CAR construct in the genome of transduced T cells (Figure Two). Li et al recently published a utter protocol on gammaretroviral production and T cell transduction seventeen using T cells isolated with EasySep™.
Figure Two. Retroviral Transduction of T Cells
Adapted from the Production of CAR T Cell wallchart, produced by Nature Protocols and supported by STEMCELL Technologies.
Genome-wide profiling exposes remarkable parallels inbetween injection site selection properties of the MLV retrovirus and the piggyBac transposon in primary human CD4+ T cells Eighteen
Molecular Therapy, 2016
There are a number of variables that accompany genetic modification. In this probe, the authors showcased that different vectors and transposons can have varying biases for genomic injection sites, which can influence the risk of insertional mutagenesis. MLV retrovirus and piggyBac transposon in particular choose to integrate at transcriptional embark sites. In addition to vector selection, other variables such as that timing of transduction have been shown to influence the quality of the final cell therapy product. Nineteen
Evaluation of TCR gene editing achieved by TALENs, CRISPR/Cas9, and megaTAL nucleases 20
Molecular Therapy, 2016
Albeit viral transduction remains the most efficient method of genetically engineering human T cells, there are viral-free methods that are presently being explored, including CRISPR/Cas9, TALENs and megaTAL nucleases. 20,21,22 In this particular article, the Blazar lab demonstrated that megaTAL and CRISPR/Cas9 can be used to disrupt endogenous T cell receptor in primary human T cells for the generation of CAR T cells. These methods need to be further optimized to be considered efficient alternatives for CAR T cell production.
Three. Fresh Concepts
Cutting edge research on the bench continually proposes fresh and arousing concepts in CAR T cell design. Of particular interest is delivery: how to efficiently produce T cells into tumors, and what engineered T cells can supply to tumors in vivo.
Biopolymer implants enhance the efficacy of adoptive T-cell therapy 23
Nature Biotechnology, 2015
In this article, Stephan et al. described a method to improve the delivery of T cells to solid tumors in vivo using bioengineered polymer matrices. When implanted near solid tumors or at resection sites, biopolymer implants are capable of delivering and expanding tumor-specific T cells. This mode of delivery results in significant enhancement of tumor regression for inoperable tumors and prevention of relapse after surgery compared to conventional injections.
Engineering T cells with customized therapeutic response programs using synthetic Notch receptors 24
In this investigate, Roybal et al. used synthetic Notch receptors to engineer T cells that can produce any therapeutic payloads in response to a given antigen in vivo. Upon ligand trussing, the synthetic Notch receptor initiates a transcriptional program leading to the expression of a chosen factor such as secreted cytokines (ex. IL-12), cytotoxic agents (ex. TRAIL), adjuvants (ex. flagellin), transcription factors (ex. Tbet) and therapeutic antibodies (ex. anti-PD-1). This finding permits scientists to dictate the effector response of engineered T cells in vivo, which can be customized depending on the state of the patient.
These fresh concepts can be applied to improve therapeutic efficacy, but some may also increase the cost of therapy. With the already exorbitant cost of T cell therapy, clinical trials will be needed to determine whether any benefits of translating these novel concepts from bench to bedside are significant enough.
View the next tab to read about combinations of CAR T cells with other forms of therapy
There is presently a significant number of existing cancer treatments available for “bedside” use in the clinics, each with its own strengths and weaknesses. Scientists are exploring whether combining T cell therapy with other forms of treatment may have beneficial effects for cancer patients. Here we discuss the potential of combining CAR T cell therapy with chemotherapy or with treatments designed to counteract immunosuppression in the tumor microenvironment.
1. Chemotherapy
Patients receiving CAR T cell therapy may have also received other types of treatment. The most common of these treatments is chemotherapy. The following studies suggest that some forms of chemotherapy and T cell therapy can complement each other:
Enhancement of adoptive T cell transfer with single low dose pretreatment of doxorubicin or paclitaxel in mice 25
Hsu et al. displayed that a single low dose of preconditioning with chemotherapy (doxorubicin or paclitaxel) can enhance the activation and longevity of adoptively transferred CD8+ T cells, leading to enhanced therapeutic efficacy. This preconditioning can also reduce the number of immunosuppressive regulatory T cells and myeloid derived suppressor cells in the tumor microenvironment.
Effector T cells abrogate stroma-mediated chemoresistance in ovarian cancer 26
T cell therapy may also enhance the effectiveness of chemotherapy. Fibroblasts represent a major component of the tumor microenvironment, and can promote resistance to platinum-based chemotherapy drugs such as cisplatin. In this explore, Wang et al. displayed that effector T cells counteract the effect of fibroblasts on chemoresistance. As a result, T cell therapies may not only directly eliminate tumor cells, but also enhance their sensitivity to conventional chemotherapeutic agents.
Two. Lowering Immunosuppression
Numerous studies suggest that reducing the levels of immunosuppressive factors can increase the effectiveness of CAR T cell therapy in patients. For example, PD-1 blockade and STAT3 inhibitors have both been shown to reduce immunosuppressive factors in the tumor microenvironment and enhance T cell responses. 27,28 Interestingly, TLR8 ligands and chromatin remodelling drug can also lower the barriers against effective T cell therapy:
TLR8 signaling enhances tumor immunity by preventing tumor‐induced T‐cell senescence 29
EMBO MOlecular Medicine, 2014
Tumors can produce cyclic adenosine monophosphate (cAMP) to induce T cell senescence. In this investigate, Ye et al showcased that the administration of TLR8 ligands to activate TLR8 signalling in tumor cells can block the induction of and switch roles T cell senescence, thereby enhancing anti-tumor effects.
Histone deacetylase inhibitor sensitizes apoptosis-resistant melanomas to cytotoxic human T lymphocytes through regulation of TRAIL/DR5 pathway 30
Journal of Immunology, 2014
CD8+ T cells use TNF-related apoptosis-inducing ligand (TRAIL) to induce apoptosis of tumor cells. TRAIL recognize death receptors such as DR5 on target cells, but some tumors have down-regulated DR5 expression, resulting in diminished sensitivity to cytotoxic T cell killing. In this probe, Jazirehi et al discovered that the administration of histone deacetylase inhibitor SAHA upregulates DR5 expression and increases the expression of pro-apoptotic molecules in apoptosis-resistant melanomas.
Research describing combinatorial effects of CAR T cell therapy with other forms of treatments will present options for personalized approaches of T cell therapy catered to specific patient needs. Combinatorial therapy may additionally permit the use of lower doses of T cells to achieve the required effectiveness, which may help to reduce potential side effects.
Closing Remarks
CAR T cell immunotherapy has brought a tremendous excitement in the world of research. Scientists are antsy to seize the chance to accomplish what they had set out to do by doing research: make a difference in the lives of patients. Hundreds of research groups are in a race to see who can make it from bench to bedside very first. Despite the competitive nature of the immunotherapy field, collaborations are quickly forming with the hope to make T cell therapy more accessible, much like our collaboration with GE Healthcare.
Production of CAR T Cells Wallchart
This free Nature Protocols Wallchart summarizes the processes involved in producing CAR T cells for therapy, including cell enrichment, activation and expansion.
T Cell Therapy Reagents
Our collaboration with GE Healthcare aims to develop cGMP-grade T cell isolation, activation and expansion reagents for commercial-scale cell therapy production.
