Authors: Laura Castelletti & John E.J. Rasko
The dramatic success achieved using chimeric antigen receptor (CAR) T cells in haematological malignancies has prompted concerted efforts to apply the same technology in solid tumours. CAR T cells offer a form of cancer immuno-gene therapy that is achieved by genetic modification of a patients’ autologous T cells to express CAR molecules on their surface. CARs have a modular design commonly comprising a single-chain variable fragment (scFv) which binds to antigens, a hinge, a transmembrane domain and intracellular signalling domains such as CD3ζ, CD28 and 4-1BB. Thanks to this engineering, CAR T cells have the ability to selectively bind and kill antigen-expressing cancer cells in an HLA-independent manner. CAR T cells can also persist in the patients’ bodies and reactivate upon renewed antigen encounter, thus assuring a long-term protection against cancer.
CAR T cells have demonstrated high success rates in eradicating haematological malignancies of diverse B cell origin. Based on this compelling achievement, CAR T cell therapy is being actively pursued against solid tumours. CARs have the ability to bind to any molecular type – be it protein, lipid or sugar/carbohydrate. Target tumour-associated antigens (TAA) need to be carefully chosen based on their expression level, heterogeneity, essentiality and accessibility. Common target antigens been tested in CAR T cell trials in solid tumours include Mesothelin, human epidermal growth factor receptor 2 (HER2), Mucin 1 (MUC1), Carcinoembryonic antigen (CEA), Epidermal Growth Factor Receptor (EGFR), the ganglioside GD2, Interleukin 13 Receptor (IL13Rα2) and Prostate-specific membrane antigen (PSMA). The common feature of these targets is their low expression in normal tissues and high expression on the surface of cancer cells. This is to avoid on target/off-tumour toxicities, which can cause CAR T cells to attack cells in healthy tissues. Such on target/off-tumour toxicities have led to deaths in clinical trials. Ideally, the target should also be essential for cancer cell survival, in order to avoid antigen escape, as well as easily accessible and expressed homogeneously in the tumour.
To expand the pool of TAA, intracellular proteins can be targeted by genetically modifying T cells to express a recombinant T cell receptor (TCR). TCR T cells bind to peptides derived from intracellular TAA presented on a Human Leukocyte Antigen (HLA or MHC) molecule. As with CAR T cells designed to target surface molecules, ideal intracellular target TAA are peptides from proteins exclusively expressed by cancer cells or with a high differential in expression between cancer and normal tissues. Common targets of TCR T cell therapy currently in clinical trials are New York oesophageal squamous cell carcinoma (NY-ESO) and melanoma-associated antigen (MAGE)-A3. Although TCR T cells allow for a larger variety of TAA to be targeted, they are limited by HLA-restriction and the fact that targets must be proteins. This cell therapy strategy is further limited by ‘defence’ mechanisms present in cancer cells, such as reduced expression of HLA and antigen-processing molecules.
CAR T cell therapy toxicities
It is anticipated that CAR T cell therapies developed against solid tumour antigens will demonstrate toxicities similar to those observed in haematological malignancies. Lymphodepletion is commonly used prior to CAR T cell injection and carries its own toxicities, such as transient cytopenia including neutropenia, lymphopenia as well as a prolonged decrease in CD4 + T cells. A small number of patients can also develop infections, diarrhoea, hyperbilirubinemia and fludarabine-induced neurotoxicity. The main toxicities arising from CAR T cell therapy relate to Cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS). CRS in its mildest form consists of a flu-like disorder with fever, malaise, headache, tachycardia, and myalgia but at worst it can be life-threatening. Its severity correlates with tumour burden and infused dose of CAR T cells. Most CRS-related toxicities are low grade and manageable, usually through supportive care, anti-IL6 receptor monoclonal antibody treatment (tocilizumab) and steroids. ICANS manifests as decreased attention and concentration, dyspraxia, encephalopathy, lethargy, agitation or delirium, headache, and aphasia. In the majority of cases, symptoms resolve within 4 weeks. More severe cases present with cerebral oedema, seizures, focal deficits, and diminished consciousness including coma. Treatment is symptom dependent.
Despite a large number of clinical trials using CAR and TCR T cells in solid tumours, the success rate so far has been disappointingly low, with only a small number of patients achieving complete responses. This is likely the result of multiple challenges: (A) heterogeneous expression of target TAA; (B) inability of CAR T cells to successfully traffic and home into the tumour; (C) immunosuppressive tumour microenvironment (TME). A plethora of strategies are being explored to address these challenges (Figure 1)
Figure 1: Barriers to CAR T cell activity in solid tumours (in red) and possible strategies to overcome them (in green). A) Low tumor infiltration caused by the physical barrier of the stroma could be overcome by loco-regional delivery of CAR T cells or by expressing the CCR2b chemokine receptor on CAR T cells, which attracts them to the tumor. Combination with oncolytic viruses expressing chemokines may also increase CAR T cell trafficking to the tumor. B) Reactive Oxygen Species (ROS) andsolubleimmunosuppression mediators prostanglandin E (PGE2), adenosine and TGF-β contribute to the reduction of anti-tumor activity of CAR T cells. Strategies that successfully rescued anti-MSLN CAR T cell anti-tumor activity include the knock out (KO) of receptors for TGFβ (TGFBR2) and adenosine (A2AR) in CAR T cells as well as catalase expression against ROS. The combination of anti-MSLN CAR T cells with TGF-β-targeting oncolytic viruses and TNFα-IL2-producing oncolytic viruses has also helped enhance their efficacy. C) Exhaustion due to PD-1/PD-L1 signaling can be counteracted via knock out of PD-1 or by inserting a PD-1 Dominant-Negative Receptor (DNR) or a PD-1/CD28 switch receptor. Combination with checkpoint inhibitors against PD-1, TIM3 or LAG3 as well as the engineering of CAR T cells to secrete anti-PD-1 antibodies have also been studied. D) CAR T cells with improved CAR design have been shown to have increased persistence and efficacy in mouse models. FAS DNR makes CAR T cells more resilient to apoptosis. E) CAR T safety can be improved by limiting ‘on target/off tumor’ toxicities through the introduction of a suicide switch or the use of mRNA to have transient CAR expression. (Image modified from Castelletti et al., 2021 – made with BioRender)
One approach to overcome TAA heterogeneity is tandem CARs, which recognise and are activated by two targets. Another approach explores the use of universal adaptor CARs, which consist of a CAR binding to a soluble adaptor which in turn conveys specificity against a TAA. Locoregional delivery instead of systemic infusion is a common approach to circumvent the challenge of CAR T cell trafficking to the tumour. Another tactic is to engineer CAR T cells to express chemokine receptors which attract them and enhance their homing to the tumour site.
The TME varies depending on the solid tumour type, however the main immunosuppressive features are the induction of exhaustion (through receptor such as PD-1, TIM3 and LAG3) and apoptosis in CAR T cells as well as the suppression of CAR T cell activity by myeloid-derived suppressor cells, tumour-associated macrophages and regulatory T cells. These tumour-resident immune cells produce reactive oxygen species (ROS), lactate, indoleamine 2,3-dioxygenase (IDO), and prostaglandin E2 (PGE2), as well as adenosine, which impact negatively on the anti-tumour functions and persistence of CAR T cells. In response to these challenges, CAR T cells have been engineered to be TME-resistant through the introduction of dominant-negative receptors (e.g. FAS receptor, PD-1 receptor and TGF-β receptor) as well as switch receptors (PD-1-CD28). CAR T cells have also been modified to secrete antibodies (e.g. anti-PD-1 or PDL-1 antibodies) and cytokines (IL-12, IL-15, IL-18, IL-21 and IL-7R). Novel strategies seek to overcome metabolic-driven dysfunction (e.g. co-expression of catalase against ROS). Combination therapy of CAR T cells with other anti-cancer treatments such as chemotherapy, anti-angiogenesis drugs, radiation therapy as well as anti-PD-1 immunotherapy have also shown evidence of anti-tumour activity. New manufacturing methods are also being explored to enhance CAR T cell fitness, survival and persistence.
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Bio: Laura Castalettia,c is a research scientist specialised in microbiology and immuno-oncology working at the University of Sydney’s Li Ka Shing Cell & Gene Therapy Program. Her current projects focus on the research parallel to a clinical trial using mesothelin-targeted CAR T cell therapy against pancreatic cancer delivered intra-tumourally. She has previous experience as a senior scientist in oncology cell therapy in GSK, where she progressed many CAR and TCR T cell therapy projects from early development to clinical trials.
Bio: Professor John Raskoa,b,c is an Australian pioneer in the application of stem cells and genetic therapies. For over two decades he has directed the Department of Cell and Molecular Therapies at Royal Prince Alfred Hospital and the Gene and Stem Cell Therapy Program at the Centenary Institute, University of Sydney. As a clinical haematologist, pathologist and scientist he has contributed to the understanding of stem cells and blood cell development, gene therapies, cancer causation and treatment, human genetic diseases and molecular biology.
John frequently appears on radio and television to discuss biomedical research, medical tourism, and scientific fraud. He delivered the prestigious 2018 Boyer Lecture series for the Australian Broadcasting Commission. Service to national and international professional societies, as well as a series of prestigious awards, recognise his commitment to excellence in medical research, including appointment as an Officer of the Order of Australia.
aLi Ka Shing Cell and Gene Therapy Program, Faculty of Medicine & Health, The University of Sydney, Sydney, NSW, 2006, Australia
bGene and Stem Cell Therapy Program Centenary Institute, The University of Sydney, Camperdown, NSW, 2050, Australia
cCell and Molecular Therapies, Royal Prince Alfred Hospital, Camperdown, NSW, 2050, Australia