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Keyword: act

"Juicing" Th17 cells with FDA-approved small molecule beta-catenin and p110 delta inhibitors during in vitro expansion for adoptive T cell therapy (ACT) profoundly improves their therapeutic properties, report investigators at the Medical University of South Carolina (MUSC) in an article published online ahead of print on April 20, 2017 by JCI Insight.

MUSC cancer immunologist Dr. Chrystal M. PaulosACT involves harvesting T cells, rapidly amplifying and/or modifying them in the laboratory to boost their cancer-fighting ability, and then reinfusing them back to the patient to boost anticancer immunity. One challenge for ACT has been that the rapid expansion of T cells in the laboratory can cause them to age and wear out, decreasing their longevity after reinfusion.

"Juicing" Th17 cells with the FDA-approved small molecules enhanced their potency, function and stem-like (less differentiated) quality, suggesting that they would persist better after reinfusion into patients, and also reduced regulatory T cells in the tumor microenvironment, which can blunt the immune response. These findings highlight novel investigative avenues for next-generation immunotherapies, including vaccines, checkpoint modulators, and ACT.

"This is exciting because we might be able to overcome some of the delays and disadvantages of rapid expansion in the laboratory," explains senior author Chrystal M. Paulos, Ph.D., associate professor of immunology and Endowed Peng Chair of Dermatology at MUSC and a member of the MUSC Hollings Cancer Center. "We might be able to use fewer cells (for ACT) because we can pharmaceutically 'juice' these T cells to make them more fit in the oppressive tumor microenvironment."

Building upon their previous findings that ICOS costimulation is critical for generating human Th17 cells and for enhancing their antitumor activity, an MUSC research team led by Paulos and including postdoctoral fellow Kinga Majchrzak report for the first time that repurposing FDA-approved small molecule drugs that inhibit two ICOS-induced pathways greatly enhances the antitumor potency of T cells.

Several biologic properties of the Wnt/ beta-catenin and P13K delta pathways led the team to suspect that they supported the antitumor activities of Th17 cells. For example, these pathways are active in both regulating T cell cytokine production during the immune response and in promoting self-renewal of hematopoietic stem cells (HSCs) and sustaining HSCs in an undifferentiated state. So, they designed a series of experiments to determine whether these two pathways were also active in enhancing Th17 antitumor memory and effectiveness.

To test this idea, they pharmaceutically inhibited PI3K delta and beta-catenin in Th17 cells (using idelalisib [CAL-101] to block the PI3K delta pathway and indomethacin [Indo] to inhibit beta-catenin)-anticipating that this would weaken Th17 cells' antitumor activity. To their surprise, the exact opposite occurred. ICOS-stimulated Th17 cells that were treated in vitro with CAL-101 plus Indo elicited a more potent antitumor response against melanoma in mice.

"My post-doc student came to me and said, 'I think I made a mistake because the data are going in the opposite direction to what we originally predicted!" says Paulos. "So, she repeated the experiment several times but we kept getting the same result. The data showed that using drugs to inhibit these pathways actually made the Th17 cells even better at killing tumors."

The team found that Th17 cells treated with CAL-101 express less FoxP3, suggesting that the drug suppresses Treg conversion while sustaining central memory-like Th17 cells. This finding is highly important because the phenotypic plasticity of Th17 cells in vivo allows their conversion to Tregs or Th1 cells with weak antitumor properties. These data suggest that treatment with CAL-101 can halt the development of these poorly therapeutic phenotypes and, thus, enhance the T cells' antitumor activity.

While the findings were initially counterintuitive and perplexing from a mechanistic perspective, in retrospect Paulos sees that they make sense. "Essentially, the T cells are younger," explains Paulos. "We know that T cells used for ACT age and wear out over time. Somehow these drugs sustain their youth and function. They're able to keep all the properties of their youth-they expand better and they're more functional and handle the oppressive tumor microenvironment better."

The discovery that existing FDA-approved drugs that block p110 delta and beta-catenin can make T cells more efficient tumor killers in vivo is an exciting prospect for Paulos' team. "From a clinical standpoint, this finding indicates that the therapeutic effectiveness of ACT could be improved by simple treatments with readily available drugs. It opens a lot of new investigative avenues for next-generation immunotherapy trials," she says.

"This research offers tremendous promise for the treatment of patients with serious forms of skin cancer," says Dirk M. Elston, M.D., chair of the Department of Dermatology and Dermatologic Surgery at MUSC.

Paulos has a patent on ICOS signaling in adoptive T cell transfer therapy (US 9133436), and Paulos, Majchrzak, and J.S. Bowers have a patent on pharmaceutical drug combinations or genetic strategies that instill durable antitumor T cell memory and activity (patent application P1685).

Summary: Boosting the immune system’s cancer-fighting ability is the aim of adoptive T cell immunotherapy, in which the patient’s T cells are harvested, expanded, and then reinfused. Ensuring T cell persistence after reinfusion has been a challenge. In the October 15, 2016 Cancer Research, investigators at the Medical University of South Carolina and Loyola University report that culturing T cells in N-acetyl cysteine (NAC) significantly improves persistence. Adding NAC to melanoma immunotherapy protocols could improve outcomes.

A collaborative team of investigators at the Medical University of South Carolina (MUSC) and Loyola University have demonstrated for the first time that culturing T cells in N-acetyl cysteine (NAC) before they are infused as immunotherapy improves effectiveness and outcomes in a preclinical model of melanoma. These findings were reported in the October 15, 2016 issue of Cancer Research.

Both incidence and mortality rates for metastatic melanoma continue to rise. Only about 15% of Stage IV melanoma patients receiving standard treatment can expect to survive for five years. By contrast, clinical trial data show that up to 40% of Stage IV melanoma patients survive for five years when treated with adoptive cell therapy (ACT), a form of immunotherapy that calls for infusion of autologous, melanoma-specific T cells.

ACT aims to boost a patient’s own immune responses against the cancer. To do this, the patient’s own T cells are harvested, genetically modified with a therapeutic T cell receptor, activated, and then rapidly expanded to generate large numbers of T cells for therapeutic re-infusion. Unfortunately, patient responses vary. Better outcomes are positively correlated with persistence of the transferred cells. The rapid expansion of harvested T cells before reinfusion increases their susceptibility to activation-induced cell death (AICD), prompting the authors to hypothesize that AICD reduces ACT’s overall effectiveness.

Researchers have long known that factors limiting T cell persistence also limit ACT efficacy but, until now, no one knew that something as simple as changing the culture condition by supplementing with NAC could improve survival of the reinfused T cells. The research team showed that adding NAC to the in vitro T cell expansion culture prevents increases in the DNA damage marker ?H2AX and significantly improves T cell persistence and immunotherapy outcomes, including reduced tumor growth and enhanced survival.  

The team found that nearly 40% of NAC-cultured T cells were detectable in tumors after transfer compared to approximately 1.2% of standard-culture T cells. They also found that mice receiving NAC-cultured cells experienced significantly delayed tumor growth compared to mice receiving standard-culture cells (P<.0001).

"We were really surprised by the number of adoptively transferred T cells we saw in the tumor,” said Christina Voelkel-Johnson, Ph.D., Associate Professor of Microbiology and Immunology at MUSC's Hollings Cancer Center and the senior author on the article. “Given the harsh environment T cells encounter within tumors, we did not expect that the number of NAC-cultured T cells would be 33-fold higher than T cells not cultured in NAC.”

Adding NAC to existing protocols should pose little risk to patients, according to Voelkel-Johnson, because NAC is already in clinical use for many indications and the culturing of T cells in NAC would occur outside of the patient. “The only difference would be a change to the cell culture protocol in an effort to generate cells with an improved phenotype," said Voelkel-Johnson.

The road to discovering the protective role of NAC began with the MUSC/Loyola research team hypothesizing that preventing T cells from becoming susceptible to AICD during in vitro expansion might improve their persistence and effectiveness upon reinfusion.

The team started by examining the role of p53, which is crucial for coordinating cellular stress responses and determining the fates of damaged cells (i.e., whether they will be repaired or allowed to die). While it is known that inhibiting p53 protects T cells from AICD, the molecular-level mechanisms that provide this protection were unknown. The investigators created conditions similar to those that occur during in vitro T cell expansion by re-stimulating the T cell receptors (TCR) to assess p53 status and cell death. They found that AICD following TCR re-stimulation is accompanied by phosphorylation of p53 on Ser15 and accumulation of p53 in the cell nucleus.

The next set of experiments clarified that the PI3K-like serine kinase, ataxia telangiectasia mutated (ATM), is necessary for the phosphorylation of p53 on Ser15 after TCR restimulation. They also found that inhibiting ATM almost completely prevents cell death (99%) after TCR restimulation. Thus, ATM appears to be a required upstream factor for AICD onset.

While activation of ATM and p53 are parts of a known DNA damage response pathway, ATM also responds to oxidative stress or hypoxia. So, the team needed to determine whether the changes in ATM and p53 were being caused by DNA damage or oxidative stress/hypoxia. They looked at two established DNA damage markers, gH2AX and p-SMC-1, and found that, within 15 minutes of TCR restimulation, both gH2AX and p-SMC-1 increased three-fold. This suggested that DNA damage and ATM activation occur in parallel, leading the authors to conclude that TCR restimulation causes DNA damage, which then triggers the ATM/p53 DNA damage response pathway.

Because it is known that AICD depends on the generation of reactive oxygen species (ROS), the investigators focused on ROS as a potential cause of DNA damage. They incubated some T cells with the antioxidant NAC prior to restimulation and compared them to T cells that were expanded using a standard protocol. Results showed that ROS levels were significantly lower in the NAC cultures than in the standard cultures. In addition, pretreatment with NAC reduced activation of the DNA damage markers, ?H2AX and p-ATM, by 94% and 69%, respectively. These results confirmed that ROS generated during TCR restimulation play a central role in damaging the DNA.

This is the first study to show that expanding therapeutic T cells in the presence of NAC prior to adoptive transfer improves their ability to resist AICD. Most importantly, using these "AICD-resistant" T cells improves therapeutic outcomes in a preclinical model by enhancing T cell persistence, increasing tumor control, and improving survival.

"Now we are looking at studies to help us understand exactly how NAC changes the phenotype of T cells,” said Voelkel-Johnson. “How does it make these cells survive? How is trafficking to tumors improved? There may be benefits to culturing T cells in NAC aside from generating AICD resistance that we haven't yet recognized.”

It is now accepted that our immune system is capable of mounting an attack against cancer. However, tumors have devised ways to elude detection and to render tumor reactive or “effector” T cells indolent. Cancer immunotherapies such as adoptive T cell transfer (ACT) seek to reinvigorate and reinforce tumor-reactive cells so that they can effectively target tumor.

In ACT therapy, exhausted T cells in the vicinity of a tumor are harvested; expanded, conditioned, and sometimes genetically reengineered to better recognize and target the tumor; and then reinfused. T cell growth factors, such as interleukin (IL) 2 and IL-15, are often administered to promote proliferation of the reinfused T cells that have been trained to target the patient’s tumor. IL-15 is a more recently discovered cytokine and seems to promise some advantages over IL-2, which is toxic at therapeutic doses and which can stimulate regulatory T cells that blunt the effect of effector T cells.  One limitation of translating this therapy to the clinic is that preconditioning with chemotherapy or radiation is required for best results. Chemotherapy and radiation are expensive, associated with substantial adverse effects, and require hospital admission.T cells expressing IL-2 receptor alpha outcompete host cells for IL-2

In an article published in the October 28, 2015 issue of Science Translational Medicine, senior author Mark P. Rubinstein, Ph.D., and his colleagues in the Department of Surgery and the Department of Microbiology and Immunology at the Medical University of South Carolina and his collaborators at the University of California San Diego report the surprising finding that curative responses were achieved with ACT in a mouse model of melanoma without lymphodepletion when IL-2 but not IL-15 was co-administered. These findings are important because they suggest that low-dose IL-2 could be used as an alternative to chemotherapy and radiation as a preconditioning regimen for ACT therapy.

Host cells are thought to outcompete the reinfused or “donor” cells for T cell growth factors such as IL-2 and IL-15. Chemotherapy and radiation knock down the number of host cells so that the donor cells can more effectively compete for IL-2 or IL-15.  Rubinstein and his colleagues found that curative responses were achieved with IL-2 without lymphodepletion when the effector T cells were engineered to express elevated levels of IL-2 receptor alpha (IL-2R?). The presence of IL-2R? on the surface of the effector T cells enabled them to outcompete host cells for IL-2. This suggests that, if the effector T cells harvested from patients are engineered to express high levels of IL-2R?, then low doses of IL-2 may be adequate to achieve significant clinical response, making preconditioning with chemotherapy or radiation unnecessary.

The study authors also describe a novel mechanism that helps account for the ability of IL-2 to mediate curative responses in the absence of chemotherapy or radiation. They found that, in T cells expressing IL-2R?, IL-2 does not degrade as expected after being taken up by the effector T cell. Instead, IL-2R? rescues IL-2 from being degraded, thereby enabling IL-2 to be recycled so it can continue to optimize effector T cell response. “The ability of IL-2R? to sustain IL-2 signaling provides a molecular mechanism to explain how IL-2 therapy may be particularly useful clinically,” says Rubinstein. “This mechanism could provide a novel way to enhance the tumor-killing potential of T cells transferred to patients in the absence of prior chemotherapy or radiation.”

 The lead researchers in the Rubinstein laboratory for this study were Ee Wern Su, Ph.D.,and Caitiln Moore. Ms. Moore is currently in medical school at MUSC. For a complete list of authors, please view the article's abstract.

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