Image Courtesy of Malia Kuo.
Emily Whitehead’s entire world turned upside down in 2010 when, at the age of five, she was diagnosed with B-cell acute lymphocytic leukemia (ALL), the most common childhood cancer. After two years of unsuccessful chemotherapy, all hope seemed to be lost. Her doctors recommended she be taken home to Philipsburg, Pennsylvania, to die peacefully with her family around.
Fortune was on the Whiteheads’ side, however. A combination of persistence and perfect timing led their treatment search to the Children’s Hospital of Philadelphia, which had just received approval for treating their first refractory ALL patient using a novel, yet promising approach: CAR-T cell therapy.
Chimeric antigen receptor (CAR)-T cells are an engineered variant of T cells—white blood cells in our bodies integral to the immune response against infection. The term “chimeric” refers to how the antigen-recognizing part of the receptors derives from extracellular antibodies. These cells are equipped with T cell receptors (TCRs) on their surfaces, allowing them to recognize virus- or bacteria-infected cells. Similarly, cancer cells can display tumor-specific antigens (markers) that enable their recognition and destruction by this mechanism. A specific subset of these T cells, called CD8+ T cells, which are distinguished by expressing CD8 molecules on their surfaces, are referred to as “killer” or “cytotoxic” T lymphocytes (CTLs) because they can trigger immunecell-mediated death of target cells.
Could CTLs then serve as “living drugs” against cancer? Over the past few decades, medical researchers have grown keen to answer this question, hoping to improve the rather lackluster survival rates of prevalent forms of cancer treatment such as surgery, chemotherapy, and radiation.
Israeli immunologist Zelig Eshhar was the first to produce CD8+ T cells with genetically engineered TCRs modified to recognize the antigens of choice. These were the first-generation CAR-T cells.
CAR-T cells thus offered an exciting opportunity to harness the existing machinery of the immune system to target and kill cancer cells simply by engineering TCRs to specifically target antigens displayed by tumor cells. University of Pennsylvania immunologist Carl June pioneered the development of second-generation CAR-T cells, engineered to kill leukemia cells in mouse malignancy models. At this time, he encountered a hospice-referred Emily Whitehead.
CAR-T Cell Therapy—The Contemporary Landscape
To the delight of everyone involved, Emily’s cancer went into remission immediately following the administration of June’s CAR-T therapy. In June 2012, she was released from the hospital, and today, she remains cancer-free. This event marked a pivotal point in cancer immunotherapy: a later clinical trial showed that ninety percent of refractory ALL patients receiving CAR-T achieved complete remission for a disease that would have otherwise proven fatal.
To date, six CAR-T cell therapies have been FDA-approved for treating various blood cancers. The treatment process is similar for each type of CAR-T therapy. A patient’s blood is first removed to isolate CD8+ T cells. These cells are then genetically engineered, often using CRISPR-Cas9 and/or lentiviral gene-editing technology, to remove the natural TCR and insert a CAR directed towards the antigen of choice. Other modifications, such as removing inhibitory molecules and introducing co-stimulatory molecules, are often performed. These CAR-T cells are then cultured in a laboratory setting to grow millions of anti-cancer T cells, which are finally re-infused into the patient to target and kill malignant cells.
While CAR-T remission and survival rates have at times considerably exceeded other therapy options, access remains poor. Due to the complexity of the procedure, a singular infusion can cost up to $500,000, excluding other costs such as hospital stay and follow-up protocols. Additionally, geographic barriers can limit patients’ access to this life-saving treatment as fewer than two hundred centers in the U.S. are authorized to administer the treatment.
Moreover, several limitations have prevented widespread adoption and greater efficacy. While all currently approved CAR-T cell therapies target blood cancers, their effectiveness in solid tumors has been lackluster. A major challenge is improving the persistence and proliferation of CAR-T cells post-infusion since they often do not survive long enough to mediate long-term cancer control. Additionally, identifying and targeting “neoantigens,” antigens that are specifically expressed or overexpressed by tumor cells, has been challenging for CAR-T cells.
CRISPR Engineering and the Next Generation
To further improve the function and specificity of CAR-T cells, researchers have experimented with the ability to boost T cell function via genetic engineering: whether by knocking out (deleting) inhibitory receptors or inserting co-stimulatory genes. The innovation of CRISPR-Cas9 gene-editing technologies vastly improved this capability. By using RNA as a guide to direct DNA cleavage at specific regions in the genome, CRISPR-Cas9-mediated editing tends to be more precise than previously used techniques, resulting in fewer off-target side effects.
Over the past few years, researchers have mostly used CRISPR-Cas9 gene-editing technologies to screen the genome of CD8+ T cells for knockout targets by identifying genes that, when silenced, augment antitumor efficacy.
Associate Professor Sidi Chen, Associate Research Scientist Lupeng Ye, and their team at the Yale School of Medicine recently published a paper in Cell Metabolism outlining their development of a CRISPR activation (CRISPRa) gain-of-function (GOF) screen in CD8+ T cells. While previous CRISPR screens only focused on identifying knockout targets, a GOF screen would identify genes that, when overexpressed (“knocked-in”), would enhance CAR-T function.
Thus, this novel platform helps identify a new class of gene-editing targets that can be harnessed as functional boosters for CAR-T cell therapy optimization. “CRISPRa screen is very, very new in immune cells and completely different from prior knockout screens. We try to find several targets that can reprogram T cells and use CRISPR engineering to ‘knock-in’ these targets into engineered T cells so the CAR-T can have boosted function when killing cancer,” Ye said.
Identifying Useful Targets for CAR-T Optimization
With a primary gain of function screening method, the researchers sought to identify genes that, when activated, would enhance the “degranulation” ability of CD8+ T cells. Degranulation, the process of releasing cytotoxic molecules from internal secretory vesicles, is one of the primary mechanisms by which CTLs mediate the killing of their target cells.
A key characteristic of CRISPRa is that it uses truncated “dead-guide” RNA (dgRNA) instead of traditional “single guide” RNA (sgRNA). Whereas sgRNA binds to Cas9 and cuts a specific target sequence, “dgRNA […] can also complex with Cas9 and bind to targets, but cannot cleave DNA,” Ye explained. Instead, the dgRNA is designed to contain two special loop structures that recruit proteins involved in DNA transcription, ultimately resulting in overexpression of the genes the dgRNAs are meant to target.
The authors of this study began by designing a mouse genome-scale dgRNA library targeting more than 22,000 genes, which will be delivered to CD8+ T cells isolated from Cas9-transgenic mice to conduct CRISPRa. Using an immunogenic mouse tumor model, researchers co-cultured dgRNA-transduced tumor-targeting CD8+ T cells with their target tumor cells. After four hours, the authors measured the levels of CD107a, a molecule expressed after degranulation. Then, the researchers used fluorescence-activated cell sorting to isolate CD8+ T cells with CD107a.
Genetic sequencing revealed which dgRNAs were most significantly enriched in the CD107a+ population. “If the gene is highly enriched, the signal will be really strong. We picked the targets with the strongest signals to do our initial validation,” Ye said. One of the screen’s top hits, the PRODH2 gene, led to increased degranulation and more rapid proliferation in CD8+ T when overexpressed compared to control cells. Could PRODH2 serve as a functional booster for human CAR-T cells?
Metabolic Reprogramming Supercharges CAR-T Cells
Indeed, Chen’s team confirmed that PRODH2 overexpression in human CAR-T cells, either by CRISPR knock-in or traditional lentiviral delivery, enhanced tumor killing and proliferation. These findings were validated in three in vitro cellular models: leukemia, multiple myeloma, and breast cancer. These effects were replicated in vivo, using human tumor xenograft models for the same three cancers in mice. PRODH2 overexpression led to reduced tumor growth and greater survival in CAR-T cell therapy.
But why? The authors performed various profiling techniques to gain insights into the mechanism underlying how PRODH2 overexpression enhances CAR-T cell antitumor efficacy. mRNA sequence analyses showed that PRODH2 knock-in significantly altered gene expression of the cell cycle, activation/effector function, and metabolism-related programs in CAR-T cells.
PRODH2’s effects on CAR-T cell antitumor efficacy seemed to be driven by metabolic reprogramming related to proline, an amino acid building block. “If we overexpress PRODH2, then proline metabolism will be reprogrammed,” Ye said. Metabolomics data of PRODH2-overexpressing CAR-T cells revealed increased biochemical activity of the pathway and alterations in other intersecting metabolic pathways, such as the metabolism of arginine, another amino acid. In fact, the cancer-killing ability was improved when direct substrates of PRODH2 were supplied to PRODH2-knockin CAR-T cells, but not in control CAR-T that normally lack the enzyme. This confirmed that the metabolic activity of PRODH2 was responsible for enhanced cytotoxic activity.
Hope for the Future
Chen’s team established a novel, genome-wide GOF screening technique in primary CD8+ T cells that can identify desperately-needed functional boosters in a robust and unbiased manner. The beauty of the screen is its versatility. “This doesn’t have to be T-cell or cancer-specific—ours is a flexible and broad platform that can be utilized to perform screens on virtually any other type of immune cells,” Chen said. “This platform can be a broadly enabling technology for us and everyone else in the world to utilize GOF screens in various systems, including stem cells, NK cells, macrophages, and even other cells relevant to other diseases.” In the future, the authors wish to validate the other targets identified in their screen. They hope to ultimately translate their work into clinical practice by improving the anti-cancer efficacy of CAR-T therapies.