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NCCN Shares Latest Expert Recommendations for Prostate Cancer in Spanish and Portuguese
Reporter: Stephen J. Williams, Ph.D.
Currently many biomedical texts and US government agency guidelines are only offered in English or only offered in different languages upon request. However Spanish is spoken in a majority of countries worldwide and medical text in that language would serve as an under-served need. In addition, Portuguese is the main language in the largest country in South America, Brazil.
The LPBI Group and others have noticed this need for medical translation to other languages. Currently LPBI Group is translating their medical e-book offerings into Spanish (for more details see https://pharmaceuticalintelligence.com/vision/)
Below is an article on The National Comprehensive Cancer Network’s decision to offer their cancer treatment guidelines in Spanish and Portuguese.
PLYMOUTH MEETING, PA [8 September, 2021] — The National Comprehensive Cancer Network® (NCCN®)—a nonprofit alliance of leading cancer centers in the United States—announces recently-updated versions of evidence- and expert consensus-based guidelines for treating prostate cancer, translated into Spanish and Portuguese. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) feature frequently updated cancer treatment recommendations from multidisciplinary panels of experts across NCCN Member Institutions. Independent studies have repeatedly found that following these recommendations correlates with better outcomes and longer survival.
“Everyone with prostate cancer should have access to care that is based on current and reliable evidence,” said Robert W. Carlson, MD, Chief Executive Officer, NCCN. “These updated translations—along with all of our other translated and adapted resources—help us to define and advance high-quality, high-value, patient-centered cancer care globally, so patients everywhere can live better lives.”
Prostate cancer is the second most commonly occurring cancer in men, impacting more than a million people worldwide every year.[1] In 2020, the NCCN Guidelines® for Prostate Cancer were downloaded more than 200,000 times by people outside of the United States. Approximately 47 percent of registered users for NCCN.org are located outside the U.S., with Brazil, Spain, and Mexico among the top ten countries represented.
“NCCN Guidelines are incredibly helpful resources in the work we do to ensure cancer care across Latin America meets the highest standards,” said Diogo Bastos, MD, and Andrey Soares, MD, Chair and Scientific Director of the Genitourinary Group of The Latin American Cooperative Oncology Group (LACOG). The organization has worked with NCCN in the past to develop Latin American editions of the NCCN Guidelines for Breast Cancer, Colon Cancer, Non-Small Cell Lung Cancer, Prostate Cancer, Multiple Myeloma, and Rectal Cancer, and co-hosted a webinar on “Management of Prostate Cancer for Latin America” earlier this year. “We appreciate all of NCCN’s efforts to make sure these gold-standard recommendations are accessible to non-English speakers and applicable for varying circumstances.”
NCCN also publishes NCCN Guidelines for Patients®, containing the same treatment information in non-medical terms, intended for patients and caregivers. The NCCN Guidelines for Patients: Prostate Cancer were found to be among the most trustworthy sources of information online according to a recent international study. These patient guidelines have been divided into two books, covering early and advanced prostate cancer; both have been translated into Spanish and Portuguese as well.
NCCN collaborates with organizations across the globe on resources based on the NCCN Guidelines that account for local accessibility, consideration of metabolic differences in populations, and regional regulatory variation. They can be downloaded free-of-charge for non-commercial use at NCCN.org/global or via the Virtual Library of NCCN Guidelines App. Learn more and join the conversation with the hashtag #NCCNGlobal.
[1] Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global Cancer Statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin, in press. The online GLOBOCAN 2018 database is accessible at http://gco.iarc.fr/, as part of IARC’s Global Cancer Observatory.
About the National Comprehensive Cancer Network
The National Comprehensive Cancer Network® (NCCN®) is a not-for-profit alliance of leading cancer centers devoted to patient care, research, and education. NCCN is dedicated to improving and facilitating quality, effective, efficient, and accessible cancer care so patients can live better lives. The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) provide transparent, evidence-based, expert consensus recommendations for cancer treatment, prevention, and supportive services; they are the recognized standard for clinical direction and policy in cancer management and the most thorough and frequently-updated clinical practice guidelines available in any area of medicine. The NCCN Guidelines for Patients® provide expert cancer treatment information to inform and empower patients and caregivers, through support from the NCCN Foundation®. NCCN also advances continuing education, global initiatives, policy, and research collaboration and publication in oncology. Visit NCCN.org for more information and follow NCCN on Facebook @NCCNorg, Instagram @NCCNorg, and Twitter @NCCN.
Please see LPBI Group’s efforts in medical text translation and Natural Language Processing of Medical Text at
Multiple factors related to initial trial design may predict low patient accrual for cancer clinical trials
Reporter: Stephen J. Williams, Ph.D.
UPDATED 5/15/2019
A recently published paper in JCNI highlights results determining factors which may affect cancer trial patient accrual and the development of a predictive model of accrual issues based on those factors.
Nearly one in four publicly sponsored cancer clinical trials fail to enroll enough participants to draw valid conclusions about treatments or techniques. Such trials represent a waste of scarce human and economic resources and contribute little to medical knowledge. Although many studies have investigated the perceived barriers to accrual from the patient or provider perspective, very few have taken a trial-level view and asked why certain trials are able to accrue patients faster than expected while others fail to attract even a fraction of the intended number of participants. According to a study published December 29 in the JNCI: Journal of the National Cancer Institute, a number of measurable trial characteristics are predictive of low patient accrual.
Caroline S. Bennette, M.P.H., Ph.D., of the Pharmaceutical Outcomes Research and Policy Program, University of Washington, Seattle, and colleagues from the University of Washington and the Fred Hutchinson Cancer Research Center analyzed information on 787 phase II/III clinical trials sponsored by the National Clinical Trials Network (NCTN; formerly the Cooperative Group Program) launched between 2000 and 2011. After excluding trials that closed because of toxicity or interim results, Bennette et al. found that 145 (18%) of NCTN trials closed with low accrual or were accruing at less than 50% of target accrual 3 years or more after opening.
The authors identified potential risk factors from the literature and interviews with clinical trial experts and found multiple trial-level factors that were associated with poor accrual to NCTN trials, such as increased competition for patients from currently ongoing trials, planning to enroll a higher proportion of the available patient population, and not evaluating a new investigational agent or targeted therapy. Bennette et al. then developed a multivariable prediction model of low accrual using 12 trial-level risk factors, which they reported had good agreement between predicted and observed risks of low accrual in a preliminary validation using 46 trials opened between 2012 and 2013.
The researchers conclude that “Systematically considering the overall influence of these factors could aid in the design and prioritization of future clinical trials…” and that this research provides a response to the recent directive from the Institute of Medicine to “improve selection, support, and completion of publicly funded cancer clinical trials.”
In an accompanying editorial, Derek Raghavan, M.D., Levine Cancer Institute, writes that the focus needs to be on getting more patients involved in trials, saying, “we should strive to improve trial enrollment, giving the associated potential for improved results. Whether the basis is incidental, because of case selection bias, or reflects the support available to trial patients has not been determined, but the fact remains that outcomes are better.”
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Contact info:
Article: Caroline S. Bennette, M.P.H., Ph.D., cb11@u.washington.edu
Other investigators also feel that initial trial design is of UTMOST importance for other reasons, especially in the era of “precision” or “personalized” medicine and why the “basket trial” or one size fits all trial strategy is not always feasible.
Dr. Maurie Markman, MD gives insight into why the inital setup of a trial and the multi-center basket type of accrual can be a problematic factor in obtaining meaningful cohorts of patients with the correct mutational spectrum.
The anticancer clinical research paradigm has rapidly evolved so that subject selection is increasingly based on the presence or absence of a particular molecular biomarker in the individual patient’s malignancy. Even where eligibility does not mandate the presence of specific biological features, tumor samples are commonly collected and an attempt is subsequently made to relate a particular outcome (eg, complete or partial objective response rate; progression-free or overall survival) to the individual cancer’s molecular characteristics.
One important result of this effort has been the recognition that there are an increasing number of patient subsets within what was previously—and incorrectly—considered a much larger homogenous patient population; for example, non–small cell lung cancer (NSCLC) versus EGFR-mutation–positive NSCLC. And, while it may still be possible to conduct phase III randomized trials involving a relatively limited percentage of patients within a large malignant entity, extensive and quite expensive effort may be required to complete this task. For example, the industry-sponsored phase III trial comparing first-line crizotinib with chemotherapy (pemetrexed plus either carboplatin or cisplatin) in ALK-rearrangement–positive NSCLC, which constitutes 3% to 5% of NSCLCs, required an international multicenter effort lasting 2.5 years to accrue the required number of research subjects.1
But what if an investigator, research team, or biotech company desired to examine the clinical utility of an antineoplastic in a patient population representing an even smaller proportion of patients with NSCLC such as in the 1% of the patient population with ROS1 abnormalities,2 or in a larger percentage of patients representing 4%-6% of patients with a less common tumor type such as ovarian cancer? How realistic is it that such a randomized trial could ever be conducted?
Further, considering the resources required to initiate and successfully conduct a multicenter international phase III registration study, it is more than likely that in the near future only the largest pharmaceutical companies will be in a position to definitively test the clinical utility of an antineoplastic in a given clinical situation.
One proposal to begin to explore the benefits of targeted antineoplastics in the setting of specific molecular abnormalities has been to develop a socalled “basket trial” where patients with different types of cancers with varying treatment histories may be permitted entry, assuming a well-defined molecular target is present within their cancer. Of interest, several pharmaceutical companies have initiated such clinical research efforts.
Yet although basket trials represent an important research advance, they may not provide the answer to the molecular complexities of cancer that many investigators believe they will. The research establishment will have to take another step toward innovation to “N-of-1” designs that truly explore the unique nature of each individual’s cancer.
Trial Illustrates Weaknesses
A recent report of the results of one multicenter basket trial focused on thoracic cancers demonstrates both the strengths but also a major fundamental weakness of the basket trial approach.3
However, the investigators were forced to conclude that despite accrual of more than 600 patients onto a study conducted at two centers over a period of approximately 2 years, “this basket trial design was not feasible for many of the arms with rare mutations.”3
They concluded that they needed a larger number of participating institutions and the ability to adapt the design for different drugs and mutations. So the question to be asked is as follows: Is the basket-type approach the only alternative to evaluate the clinical relevance of a targeted antineoplastic in the presence of a specific molecular abnormality?
Of course, the correct answer to this question is surely: No!
The following is a video on the website ClinicalTrials.gov which is a one-stop service called EveryClinicalTrial to easily register new clinical trials and streamline the process:
UPDATED 5/15/2019
Another possible roadblock to patient accrual has always been the fragmentation of information concerning the availability of clinical trails and coordinating access among the various trial centers, as well as performing analytics on trial data to direct new therapeutic directions. The NIH has attempted to circumvent this problem with the cancer trials webpage trials.gov however going through the vast number of trials, patient accrual requirements, and finding contact information is a daunting task. However certain clinical trial marketplaces are now being developed which may ease access problems to clinical trials as well as data analytic issues, as highlighted by the Scientist.com article below:
Scientist.com Launches Trial Insights, A Transformative Clinical Trials Data Analytics Solution
The world’s largest online marketplace rolls out first original service, empowering researchers with on demand insights into clinical trials to help drive therapeutic decisions
SAN DIEGO–(BUSINESS WIRE)–Scientist.com, the online marketplace for outsourced research, announced today the launch of Trial Insights, a digital reporting solution that simplifies data produced through clinical trial, biomarker and medical diagnostic studies into an intuitive and user-friendly dashboard. The first of its kind, Trial Insights curates publicly available data nightly from information hubs such as clinicaltrials.gov and customizes it to fit a researcher or research organization’s specific project needs.
Trial Insights, new clinical trial reporting solution, allows researchers to keep track of the evolving landscape of drugs, diseases, sponsors, investigators and medical devices important to their work.
“Trial Insights offers researchers an easy way to navigate the complexity of clinical trials information,” said Ron Ranauro, Founder of Incite Advisors. “Since Trial Insights’ content is digitally curated, researchers can continuously keep track of the evolving landscape of drugs, diseases, sponsors, investigators and medical devices important to their work.”
As the velocity, variety and veracity of data available on sites like clinicaltrials.gov continues to increase, the ability to curate it becomes more valuable to different audiences. With the advancement of personalized medicine, it is important to make the data accessible to the health care and patient communities. Information found on the Trial Insights platform can help guide decision making across the pharmaceutical, biotechnology and contract research organization industries as clinical trial data is a primary information source for competitive intelligence, research planning and clinical study planning.
“We are extremely excited to launch the first Scientist.com exclusive, original service offering to our clients in the life sciences,” said Mark Herbert, Scientist.com Chief Business Officer. “Our goal at Scientist.com is to help cure all diseases by 2050, and we believe solutions like Trial Insights, which greatly simplifies access to and reporting of clinical trial data, will get us one step closer to reaching that goal.”
NIH Considers Guidelines for CAR-T therapy: Report from Recombinant DNA Advisory Committee
Reporter: Stephen J. Williams, Ph.D.
UPDATED 5/10/2022
In the mid to late 1970’s a public debate (and related hysteria) had emerged surrounding two emerging advances in recombinant DNA technology;
the development of vectors useful for cloning pieces of DNA (the first vector named pBR322) and
the discovery of bacterial strains useful in propagating such vectors
As discussed by D. S, Fredrickson of NIH’s Dept. of Education and Welfare in his historical review” A HISTORY OF THE RECOMBINANT DNA GUIDELINES IN THE UNITED STATES” this international concern of the biological safety issues of this new molecular biology tool led the National Institute of Health to coordinate a committee (the NIH Recombinant DNA Advisory Committee) to develop guidelines for the ethical use, safe development, and safe handling of such vectors and host bacterium. The first conversations started in 1974 and, by 1978, initial guidelines had been developed. In fact, as Dr. Fredrickson notes, public relief was voiced even by religious organizations (who had the greatest ethical concerns)
On December 16, 1978, a telegram purporting to be from the Vatican was hand delivered to the office of Joseph A. Califano, Jr., Secretary of Health, Education,
and Welfare. “Habemus regimen recombinatum,” it proclaimed, in celebration of the
The overall Committee resulted in guidelines (2013 version) which assured the worldwide community that
organisms used in such procedures would have limited pathogenicity in humans
vectors would be developed in a manner which would eliminate their ability to replicate in humans and have defined antibiotic sensitivity
So great was the success and acceptance of this committee and guidelines, the NIH felt the Recombinant DNA Advisory Committee should meet regularly to discuss and develop ethical guidelines and clinical regulations concerning DNA-based therapeutics and technologies.
A PowerPoint Slideshow: Introduction toNIH OBA and the History of Recombinant DNA Oversight can be viewed at the following link:
Please see the following link for a video discussion between Dr. Paul Berg, who pioneered DNA recombinant technology, and Dr. James Watson (Commemorating 50 Years of DNA Science):
The Recombinant DNA Advisory Committee has met numerous times to discuss new DNA-based technologies and their biosafety and clinical implication including:
A recent Symposium was held in the summer of 2010 to discuss ethical and safety concerns and discuss potential clinical guidelines for use of an emerging immunotherapy technology, the Chimeric Antigen Receptor T-Cells (CART), which at that time had just been started to be used in clinical trials.
Considerations for the Clinical Application of Chimeric Antigen Receptor T Cells: Observations from a Recombinant DNA Advisory Committee Symposium Held June 15, 2010[1]
Contributors to the Symposium discussing opinions regarding CAR-T protocol design included some of the prominent members in the field including:
Drs. Hildegund C.J. Ertl, John Zaia, Steven A. Rosenberg, Carl H. June, Gianpietro Dotti, Jeffrey Kahn, Laurence J. N. Cooper, Jacqueline Corrigan-Curay, And Scott E. Strome.
The discussions from the Symposium, reported in Cancer Research[1]. were presented in three parts:
Summary of the Evolution of the CAR therapy
Points for Future Consideration including adverse event reporting
Considerations for Design and Implementation of Trials including mitigating toxicities and risks
1. Evolution of Chimeric Antigen Receptors
Early evidence had suggested that adoptive transfer of tumor-infiltrating lymphocytes, after depletion of circulating lymphocytes, could result in a clinical response in some tumor patients however developments showed autologous T-cells (obtained from same patient) could be engineered to express tumor-associated antigens (TAA) and replace the TILS in the clinical setting.
However there were some problems noticed.
Problem: HLA restriction of T-cells. Solution: genetically engineer T-cells to redirect T-cell specificity to surface TAAs
Problem: 1st generation vectors designed to engineer T-cells to recognize surface epitopes but engineered cells had limited survival in patients. Solution: development of 2nd generation vectors with co-stimulatory molecules such as CD28, CD19 to improve survival and proliferation in patients
A summary table of limitations of the two types of genetically-modified T-cell therapies were given and given (in modified form) below
Type of Gene-modified T-Cell
Limitations
aβ TCR
CAR
Affected by loss or decrease of HLA on tumor cells
yes
no
Affected by altered tumor cell antigen processing?
yes
no
Need to have defined tumor target antigen?
no
yes
Vector recombination with endogenous TCR
yes
no
A brief history of construction of 2nd and 3rd generation CAR-T cells given by cancer.gov:
Differences between second- and third-generation chimeric antigen receptor T cells. (Adapted by permission from the American Association for Cancer Research: Lee, DW et al. The Future Is Now: Chimeric Antigen Receptors as New Targeted Therapies for Childhood Cancer. Clin Cancer Res; 2012;18(10); 2780–90. doi:10.1158/1078-0432.CCR-11-1920)
The first efforts to engineer T cells to be used as a cancer treatment began in the early 1990s. Since then, researchers have learned how to produce T cells that express chimeric antigen receptors (CARs) that recognize specific targets on cancer cells.
The T cells are genetically modified to produce these receptors. To do this, researchers use viral vectors that are stripped of their ability to cause illness but that retain the capacity to integrate into cells’ DNA to deliver the genetic material needed to produce the T-cell receptors.
The second- and third-generation CARs typically consist of a piece of monoclonal antibody, called a single-chain variable fragment (scFv), that resides on the outside of the T-cell membrane and is linked to stimulatory molecules (Co-stim 1 and Co-stim 2) inside the T cell. The scFv portion guides the cell to its target antigen. Once the T cell binds to its target antigen, the stimulatory molecules provide the necessary signals for the T cell to become fully active. In this fully active state, the T cells can more effectively proliferate and attack cancer cells.
2. Adverse Event Reporting and Protocol Considerations
The symposium had been organized mainly in response to two reported deaths of patients enrolled in a CART trial, so that clinical investigators could discuss and formulate best practices for the proper conduct and analysis of such trials. One issue raised was lack of pharmacovigilence procedures (adverse event reporting). Although no pharmacovigilence procedures (either intra or inter-institutional) were devised from meeting proceedings, it was stressed that each institution should address this issue as well as better clinical outcome reporting.
Analysis of non T and T-cell subsets, e.g. natural killer cells and CD*8 cells
3. Consideration for Design of Trials and Mitigating Toxicities
Early Toxic effects– Cytokine Release Syndrome– The effectiveness of CART therapy has been manifested by release of high levels of cytokines resulting in fever and inflammatory sequelae. One such cytokine, interleukin 6, has been attributed to this side effect and investigators have successfully used an IL6 receptor antagonist, tocilizumab (Acterma™), to alleviate symptoms of cytokine release syndrome (see review Adoptive T-cell therapy: adverse events and safety switches by Siok-Keen Tey).
Below is a video form Dr. Renier Brentjens, M.D., Ph.D. for Memorial Sloan Kettering concerning the finding he made that the adverse event from cytokine release syndrome may be a function of the tumor cell load, and if they treat the patient with CAR-T right after salvage chemotherapy the adverse events are alleviated..
Early Toxic effects – Over-activation of CAR T-cells; mitigation by dose escalation strategy (as authors in reference [3] proposed). Most trials give billions of genetically modified cells to a patient.
Late Toxic Effects – long-term depletion of B-cells . For example CART directing against CD19 or CD20 on B cells may deplete the normal population of CD19 or CD20 B-cells over time; possibly managed by IgG supplementation
Below is a curation of various examples of the need for developing a Pharmacovigilence Framework for Engineered T-Cell Therapies
As shown above the first reported side effects from engineered T-cell or CAR-T therapies stemmed from the first human trial occuring at University of Pennsylvania, the developers of the first CAR-T therapy. The clinical investigators however anticipated the issue of a potential cytokine storm and had developed ideas in the pre-trial phase of how to ameliorate such toxicity using anti-cytokine antibodies. However, until the trial was underway they were unsure of which cytokines would be prominent in causing a cytokine storm effect from the CAR-T therapy. Fortunately, the investigators were able to save patient 1 (described here in other posts) using anti-IL1 and 10 antibodies.
Over the years, however, multiple trials had to be discontinued as shown below in the following posts:
The NIH has put a crimp in the clinical trial work of Steven Rosenberg, Kite Pharma’s star collaborator at the National Cancer Institute. The feds slammed the brakes on the production of experimental drugs at two of its facilities–including cell therapies that Rosenberg works with–after an internal inspection found they weren’t in compliance with safety and quality regulations.
In this instance Kite was being cited for manufacturing issues, apparantly fungal contamination in their cell therapy manufacturing facility. However shortly after other CAR-T developers were having tragic deaths in their initial phase 1 safety studies.
Juno Halts Cancer Trial Using Gene-Altered Cells After 3 Deaths
Juno halts its immunotherapy trial for cancer after three patient deaths
Juno Therapeutics said Tuesday it will restart one of its most prominent clinical trials after the Food and Drug Administration lifted a hold that had been placed last week on the trial.
The FDA halted Juno’s “Rocket” clinical trial after the company reported thattwo patients undergoing treatment had died. Juno determined the deaths resulted from swelling in the brain caused by a new drug that had been added to the treatment.
he trial seeks to treat patients with relapsed acute lymphoblastic leukemia by using engineered T-cells to attack cancer cells.
Juno added the chemotherapy drug fludarabine to the treatment plan as part of an early step that gets the patient’s body ready for the T-cell injection. Previously, Juno was using only cyclophosphamide in that part of the treatment.
Under an agreement with the FDA, Juno will continue the trial without fludarabine, using only cyclophosphamide instead.
So What Did this all mean for the CAR-T world? Is it end for CAR-T Therapies?
NO!
This, as I have posted before is a matter of pharmacovigilence, the part of drug development and premarketing trials and postmarketing analysis that deals with adverse events and safety
The JUNO trial called for co-treatment with the drug fludarabine, and antimetabolite known, in some cases to promote a cytotoxic lysis syndrome and central nervous system complications. GRANTED these two side effects were deemed RARE however it appears that the addition of fludarabine pre CART therapy aggravated either the side effect of fludarabine pretreatment or a cytoxic lysis syndrome from CAR-T (an adverse event from CAR-T therapy as I had posted here INCLUDING REPORTS OF TWO DEATHS DURING THE MSK CAR-t TRIAL
Certainly with so many issues there would seem to be more rigorous work to either establish a pharmacovigilence framework or to develop alternative engineered T cells with a safer profile
However here we went again
New paper sheds fresh light on Tmunity’s high-profile CAR-T deaths
Jason Mast
Editor
The industry-wide effort to push CAR-T therapies — wildly effective in several blood cancers — into solid tumors took a hit last year when Tmunity, a biotech founded by CAR-T pioneer Carl June and backed by several blue-chip VCs, announced it shut down its lead program for prostate cancer after two patients died.
On a personal note this trial was announced in a Bio International meeting here in Philadelphia a few years ago in 2019
and the indication was for prostate cancer, in particular hormone resistant castration resistant. Another one was planned for pancreatic cancer from the same group and the early indications were favorable.
Tmunity Therapeutics, a clinical-stage biotherapeutics company, has halted the development of its lead CAR T-cell product following the deaths of 2 patients who were enrolled to a trial investigating its use in solid tumors.1
The patients reportedly died from immune effector cell-associated neurotoxicity syndrome (ICANS), which is a known adverse effect associated with CAR T-cell therapies.
“What we are discovering is that the cytokine profiles we see in solid tumors are completely different from hematologic cancers,” Oz Azam, co-founder of Tmunity said in an interview with Endpoints News. “We observed ICANS. And we had 2 patient deaths as a result of that. We navigated the first event and obviously saw the second event, and as a result of that we have shut down the version one of that program and pivoted quickly to our second generation.”
Previously, with first-generation CAR T-cell therapies in patients with blood cancers, investigators were presented with the challenge of overcoming cytokine release syndrome. Now ICANS, or macrophage activation, is proving to have deadly effects in the realm of solid tumors. Carl June, the other co-founder of Tmunity, noted that investigators will now need to dedicate their efforts to engineering around this, as had been done with tocilizumab (Actemra) in 2012.
The company is dedicated to the development of novel approaches that produce best-in-class control over T-cell activation and direction in the body.2 The product examined in the trial was developed to utilize engineered patient cells to target prostate-specific membrane antigen; it was also designed to use a dominant TGFβ receptor to block an important checkpoint involved in cancer.
Twenty-four patients were recruited for the dose-escalating study and the company plans to release data from high-dose cohorts later in 2021.
“We are going to present all of this in a peer-reviewed publication because we want to share this with the field,” Azam said. “Because everything we’ve encountered, no matter what…people are going to encounter this when they get into the clinic, and I don’t think they’ve really understood yet because so many are preclinical companies that are not in the clinic with solid tumors. And the rubber meets the road when you get in the clinic, because the ultimate in vivo model is the human model.”
Azam added that the company plans to develop a new investigational new drug for version 2, which they hope will result in a safer product.
References
Carroll J. Exclusive: Carl June’s Tmunity encounters a lethal roadblock as 2 patient deaths derail lead trial, raise red flag forcing rethink of CAR-T for solid tumors. Endpoints News. June 2, 2021. Accessed June 3, 2021. https://bit.ly/3wPYWm0
Research and Development. Tmunity Therapeutics website. Accessed June 3, 2021. https://bit.ly/3fOH3OR
Forward to 2022
Reprogramming a new type of T cell to go after cancers with less side effects, longer impact
A Sloan Kettering Institute research team thinks new, killer, innate-like T cells could make promising candidates to treat cancers that so far haven’t responded to immunotherapy treatments. (koto_feja)
Immunotherapy is one of the more appealing and effective kinds of cancer treatment when it works, but the relatively new approach is still fairly limited in the kinds of cancer it can be used for. Researchers at the Sloan Kettering Institute have discovered a new kind of immune cell and how it could be used to expand the reach of immunotherapy treatments to a much wider pool of patients.
The cells in question are called killer innate-like T cells, a threatening name for a potentially lifesaving innovation. Unlike normal killer T cells, killer innate-like T cells stay active much longer and can burrow further into potentially cancerous tissue to attack tumors. The research team first reported these cells in 2016, but it’s only recently that they were able to properly understand and identify them.
“We think these killer innate-like T cells could be targeted or genetically engineered for cancer therapy,” said the study’s lead author, Ming Li, Ph.D., in a press release. “They may be better at reaching and killing solid tumors than conventional T cells.”
Below is the referenced paper from Pubmed:
Evaluation of the safety and efficacy of humanized anti-CD19 chimeric antigen receptor T-cell therapy in older patients with relapsed/refractory diffuse large B-cell lymphoma based on the comprehensive geriatric assessment system
Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy has led to unprecedented results to date in relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL), yet its clinical application in elderly patients with R/R DLBCL remains somewhat limited. In this study, a total of 31 R/R DLBCL patients older than 65 years of age were enrolled and received humanized anti-CD19 CAR T-cell therapy. Patients were stratified into a fit, unfit, or frail group according to the comprehensive geriatric assessment (CGA). The fit group had a higher objective response (OR) rate (ORR) and complete response (CR) rate than that of the unfit/frail group, but there was no difference in the part response (PR) rate between the groups. The unfit/frail group was more likely to experience AEs than the fit group. The peak proportion of anti-CD19 CAR T-cells in the fit group was significantly higher than that of the unfit/frail group. The CGA can be used to effectively predict the treatment response, adverse events, and long-term survival.
Introduction
Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL), accounting for 30–40% of cases, with the median age of onset being older than 65 years [1]. Although the five-year survival rate for patients with DLBCL has risen to more than 60% with the application of standardized treatments and hematopoietic stem cell transplantation, nearly half of patients progress to relapsed/refractory (R/R) DLBCL. Patients with R/R DLBCL, especially elderly individuals, have a poor prognosis [2,3], so new treatments are needed to prolong survival and improve the prognosis of this population.
As a revolutionary immunotherapy therapy, anti-CD19 chimeric antigen receptor (CAR) T-cell therapy has achieved unprecedented results in hematological tumors [4]. As CD19 is expressed on the surface of most B-cell malignant tumors but not on pluripotent bone marrow stem cells, CD19 has been used as a target for B-cell malignancies, including B-cell acute lymphoblastic leukemia, NHL, multiple myeloma, and chronic lymphocytic leukemia [5]. Despite the wide application and high efficacy of anti-CD19 CAR T-cell therapy, reports of adverse events (AEs) such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxic syndrome (ICANS) have influenced its use [6]. Especially in elderly patients, AEs associated with anti-CD19 CAR T-cell therapy might be more obvious.
Although anti-CD19 CAR T-cell therapy has been reported in the treatment of NHL, including R/R DLBCL, few studies to date have assessed the safety of anti-CD19 CAR T-cell therapy in elderly R/R DLBCL patients, and its clinical application in the elderly R/R DLBCL population is limited. In ZUMA-1 [7] to R/R DLBCL patients who received CAR T-cell therapy, the CR rate in patients ≥65 years was higher than that of in patients <65 years (75% vs. 53%). Lin et al. [8] reported 49 R/R DLBCL patients (24 patients >65 years, 25 patients <65 years) who received CAR T-cell therapy with a median follow-up of 179 days. The CR rate at 100 days was 51%, while the 6-month progression-free survival (PFS) and overall survival (OS) were 48% and 71%, respectively. Neither of the two studies carried out a comprehensive geriatric assessment (CGA) of fit, unfit, and frail groups of R/R DLBCL patients over 65 years of age and further analyzed the differences in efficacy and side effects in the three groups. The CGA is an effective system designed to evaluate the prognosis and improve the survival of elderly patients with cancer. The CGA system includes age, activities of daily living (ADL), instrumental ADL (IADL), and the Cumulative Illness Rating Score for Geriatrics (CIRS-G) [9].
In this study, elderly R/R DLBCL patients were grouped according to their CGA results (fit vs. unfit/frail) before receiving humanized anti-CD19 CAR T-cell therapy. We then analyzed the efficacy and AEs of anti-CD19 CAR T-cell therapy and compared findings between these groups.
Well it appears that the discriminator was only fitness going into the trial a bit odd that the whole field appears to be lacking in development of Safety Biomarkers.
However Genentech (subsidiary of Roche) may now be using some data to develop therapies which may combat resistance to CART therapies which may provide at least, for now, a toxicokinetic approach to reducing AEs by lowering the amount of CARTs needed to be administered.
Roche’s Genentech is exploring inhibiting ESCRT as an anticancer strategy, said Ira Mellman, Ph.D., Genentech’s vice president of cancer immunology. (Roche)
Cancer cells deploy various tactics to avoid being targeted and killed by the immune system. A research team led by Roche’s Genentech has now identified one such method that cancer cells use to resist T-cell assault by repairing damage.
To destroy their targets, cancer-killing T cells known as cytotoxic T lymphocytes (CTLs) secrete the toxin perforin to form little pores in the target cells’ surface. Another type of toxin called granzymes are delivered directly into the cells through those portals to induce cell death.
By using high-res imaging in live cells, the Genentech-led team found that the membrane damage caused by perforin could trigger a repair response. The tumor cells could recruit endosomal sorting complexes required for transport (ESCRT) proteins to remove the lesions, thereby preventing granzymes from entering, the team showed in a new study published in Science.
Killer T cells destroy virus-infected and cancer cells by secreting two protein toxins that act as a powerful one-two punch. Pore-forming toxins, perforins, form holes in the plasma membrane of the target cell. Cytotoxic proteins released by T cells then pass through these portals, inducing target cell death. Ritter et al. combined high-resolution imaging data with functional analysis to demonstrate that tumor-derived cells fight back (see the Perspective by Andrews). Protein complexes of the ESCRT family were able to repair perforin holes in target cells, thereby delaying or preventing T cell–induced killing. ESCRT-mediated membrane repair may thus provide a mechanism of resistance to immune attack. —SMH
Abstract
Cytotoxic T lymphocytes (CTLs) and natural killer cells kill virus-infected and tumor cells through the polarized release of perforin and granzymes. Perforin is a pore-forming toxin that creates a lesion in the plasma membrane of the target cell through which granzymes enter the cytosol and initiate apoptosis. Endosomal sorting complexes required for transport (ESCRT) proteins are involved in the repair of small membrane wounds. We found that ESCRT proteins were precisely recruited in target cells to sites of CTL engagement immediately after perforin release. Inhibition of ESCRT machinery in cancer-derived cells enhanced their susceptibility to CTL-mediated killing. Thus, repair of perforin pores by ESCRT machinery limits granzyme entry into the cytosol, potentially enabling target cells to resist cytolytic attack.
Cytotoxic lymphocytes, including cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, are responsible for identifying and destroying virus-infected or tumorigenic cells. To kill their targets, CTLs and NK cells secrete a pore-forming toxin called perforin through which apoptosis-inducing serine proteases (granzymes) are delivered directly into the cytosol. Successful killing of target cells often requires multiple hits from single or multiple T cells (1). This has led to the idea that cytotoxicity is additive, often requiring multiple rounds of sublethal lytic granule secretion events before a sufficient threshold of cytosolic granzyme activity is reached to initiate apoptosis in the target (2).
Loss of plasma membrane integrity induced by cytolytic proteins or mechanical damage leads to a membrane repair response. Damage results in an influx of extracellular Ca2+, which has been proposed to lead to the removal of the membrane lesion by endocytosis, resealing of the lesions by lysosomal secretion, or budding into extracellular vesicles (3). Perforin pore formation was initially reported to enhance endocytosis of perforin (4), but subsequent work has challenged this claim (5). Endosomal sorting complexes required for transport (ESCRT) proteins can repair small wounds and pores in the plasma membrane caused by bacterial pore-forming toxins, mechanical wounding, and laser ablation (6, 7). ESCRT proteins are transiently recruited to sites of membrane damage in a Ca2+-dependent fashion, where they assemble budding structures that shed to eliminate the wound and restore plasma membrane integrity. ESCRT-dependent membrane repair has been implicated in the resealing of endogenous pore-mediated plasma membrane damage during necroptosis (8) and pyroptosis (9).
Localization of target-derived ESCRT proteins to the cytolytic synapse
To investigate whether ESCRT-mediated membrane repair might be involved in the removal of perforin pores during T cell killing, we first determined whether ESCRT proteins in cancer-derived cells were recruited to sites of CTL engagement after perforin secretion. We used CTLs from OT-I mice that express a high-affinity T cell receptor (TCR) that recognizes the ovalbumin peptide SIINFEKL (OVA257-264) bound to the major histocompatibility complex (MHC) allele H-2Kb (10). We performed live-cell microscopy of OT-I CTLs engaging SIINFEKL-pulsed target cells that express enhanced green fluorescent protein (EGFP)–tagged versions of Tsg101 or Chmp4b, two ESCRT proteins implicated in membrane repair (6). To correlate recruitment of ESCRT proteins with perforin exposure in time, we monitored CTL-target interaction in media with a high concentration of propidium iodide (PI), a cell-impermeable fluorogenic dye that can rapidly diffuse through perforin pores to bind and illuminate nucleic acids in the cytosol and nucleus of the target (5). EGFP-tagged ESCRT proteins were consistently recruited to the site of CTL engagement within 30 to 60 s after PI influx (Fig. 1, A and B). EGFP-Tsg101 and EGFP-Chmp4b in target cells accumulated at the cytolytic synapse after PI influx in 25 of 27 (92.6%) and 31 of 33 (93.9%) of conjugates monitored, respectively, compared with a cytosolic EGFP control, which was not recruited (Fig. 1C and movies S1 to S3). Notably, ESCRT-laden material, presumably membrane fragments, frequently detached from the target cell and adhered to the surface of the CTL (Fig. 1, D and E, and movie S2). We observed this phenomenon in ~60% of conjugates imaged in which targets expressed EGFP-Tsg101 or EGFP-Chmp4b (17 of 27 and 20 of 33 conjugates, respectively; Fig. 1D). Shedding of ESCRT-positive membrane from the cell after repair occurs after laser-induced plasma membrane wounding (6, 7). Plasma membrane fragments shed from the target cell into the synaptic cleft likely contain ligands for CTL-resident receptors. Target cell death would separate the CTL and target, revealing target-derived material on the CTL surface.
FIG. 1. Fluorescently tagged ESCRT proteins in targets localize to site of CTL killing after perforin secretion.
(A) Live-cell spinning disk confocal imaging of a fluorescently labeled OT-I CTL (magenta) engaging an MC38 cancer cell expressing EGFP-Tsg101 (green) in media containing 100 μM PI (red). Yellow arrowheads highlight ESCRT recruitment. T-0:00 is the first frame of PI influx into the target cell (time in minutes:seconds). Scale bar, 10 μm. (B) Graph of EGFP-Tsg101 and PI fluorescence intensity at the IS within the target over time, from example in (A). AU, arbitrary units. (C and D) Quantification of CTL-target conjugates exhibiting accumulation of EGFP at the synapse after PI influx (C) or detectable EGFP-labeled material associated with CTL after target interaction (D) (EGFP condition: N = 22 conjugates in seven independent experiments; EGFP-Tsg101 condition: N = 27 conjugates in nine independent experiments; EGFP-Chmp4b condition: N = 33 conjugates in 24 independent experiments). (E) Live-cell spinning disk confocal imaging of OT-I CTL (magenta) killing MC38 expressing EGFP-Chmp4b (green), demonstrating the presence of target-derived EGFP-Chmp4b material (yellow arrowheads) associated with CTL membrane after a productive target encounter. T-0:00 is the first frame of PI influx into the target cell. Scale bar, 10 μm.
3D cryo-SIM and FIB-SEM imaging of CTLs caught in the act of killing target cells
Although live-cell imaging indicated that ESCRT complexes were rapidly recruited at sites of T cell–target cell contact, light microscopy alone is of insufficient resolution to establish that this event occurred at the immunological synapse (IS). We thus sought to capture a comprehensive view of the IS in the moments immediately after secretion of lytic granules. We used cryo–fluorescence imaging followed by correlative focused ion beam–scanning electron microscopy (FIB-SEM), which can achieve isotropic three-dimensional (3D) imaging of whole cells at 8-nm resolution or better (11–13). To capture the immediate response of target cells after perforin exposure, we developed a strategy whereby cryo-fixed CTL-target conjugates were selected shortly after perforation, indicated by the presence of a PI gradient in the target (fig. S1A). In live-cell imaging experiments, PI fluorescence across the nucleus of SIINFEKL-pulsed ID8 target cells began as a gradient and became homogeneous 158 ± 64 s, on average, after initial PI influx (N = 31 conjugates; fig. S1, B and C, and movie S4). Thus, fixed CTL-target conjugates that exhibited a gradient of PI across the nucleus would have been captured within ~3 min of perforin exposure.
Coverslips of CTL-target conjugates underwent high-pressure freezing and were subsequently imaged with wide-field cryogenic fluorescence microscopy followed by 3D cryo–structured illumination microscopy (3D cryo-SIM) performed in a customized optical cryostat (14). We selected candidate conjugates for FIB-SEM imaging on the basis of whether a gradient of PI fluorescence was observed across the nucleus of the target emanating from an attached CTL (movie S5). FIB-SEM imaging of the CTL-target conjugate at 8-nm isotropic voxels resulted in a stack of >10,000 individual electron microscopy (EM) images. The image stack was then annotated using a human-assisted machine learning–computer vision platform to segment the plasma membranes of each cell along with cell nuclei and various organelles (https://ariadne.ai/).
We captured four isotropic 3D 8-nm-resolution EM datasets of CTLs killing cancer cells moments after the secretion of lytic granule contents (Fig. 2A and movie S6). Semiautomated segmentation of the cell membranes, nuclei, lytic granules, Golgi apparatus, mitochondria, and centrosomes of the T cells allow for easier visualization and analysis of the 3D EM data. All FIB-SEM datasets and segmentations can be explored online at https://openorganelle.janelia.org (see links in the supplementary materials). Reconstructed views of the segmented data clearly demonstrate the polarization of the centrosome, Golgi apparatus, and lytic granules to the IS—all of which are hallmarks of CTL killing [Fig. 2A, i to iii, and movie S6, time stamp (TS) 1:33] (15, 16). On the target cell side, we noted cytoplasmic alterations consistent with cell damage including enhanced electron density of mitochondria adjacent to the IS (fig. S2A). Close visual scanning of the postsynaptic target cell membrane in the raw EM data failed to reveal obvious perforin pores, which have diameters (16 to 22 nm) close to the limit of resolution for this technique (17).
FIG. 2. Eight-nm-resolution 3D FIB-SEM imaging of whole CTL-target conjugate.
(A) 3D rendering of segmented plasma membrane predictions derived from isotropic 8-nm-resolution FIB-SEM imaging of a high-pressure frozen OT-I CTL (red) captured moments after secretion of lytic granules toward a peptide-pulsed ID8 ovarian cancer cell (blue). (i) Side-on sliced view corresponding to the gray horizontal line within the inset box in (A). Seen here are 3D renderings of the segmented plasma membrane of the cancer cell (blue) as well as the CTL plasma membrane (red), centrosome (gold), Golgi apparatus (cyan), lytic granules (purple), mitochondria (green), and nucleus (gray). (ii and iii) A zoomed-in view from the dashed white box in (i) shows the details of the IS (ii) and a single corresponding FIB-SEM slice docked onto the segmented data (iii). (B) Single top-down FIB-SEM slice showing overlaid target cell (blue) and CTL (red) segmentation. (i) Zoomed-in view from dashed white box in (B) details the intercellular material (IM) (gray) between the CTL and target at the IS. (C) Zoomed-in image of a 3D rendering of the surface of the target cell plasma membrane (white) opposite the intercellular material (IM) at the IS. Yellow arrowheads mark plasma membrane buds protruding into the synaptic cleft. (i and ii) Accompanying images demonstrate the orientation of the view in (C) with the rendering of the CTL (red) present (i) and removed (ii), and the dashed yellow box in (ii) indicates the area of detail shown in (C).
The segmentation of the two cells illustrates the detailed topography of the plasma membrane of the CTL and target at the IS (fig. S2B). The raw EM and segmentation data reveal a dense accumulation of particles, vesicles, and multilamellar membranous materials, which crowd the synaptic cleft between the CTL and the target (Fig. 2B and movie S6, TS 0:40 to 0:50). The source of this intercellular material (IM) was likely in part the lytic granules because close inspection revealed similar particles and dense vesicles located within as-yet-unreleased granules (fig. S2C). To determine whether some of the membranous material within the intercellular space might also have been derived from the target cell, we examined the surface topology of the postsynaptic target cell. We noted multiple tubular and bud-like protrusions of the target cell membrane that extended into the synaptic space; thus, at least some of the membrane structures observed were still in continuity with the target cell (Fig. 2C and movie S6, TS 0:58 to 1:11). ESCRT proteins have been shown to generate budding structures in the context of plasma membrane repair (6), which led us to next assess where target-derived ESCRT proteins are distributed in the context of the postsecretion IS.
To map the localization of target-derived ESCRT proteins onto a high-resolution landscape of the IS, we captured three FIB-SEM datasets that have associated 3D cryo-SIM fluorescence data for mEmerald-Chmp4b localization (Fig. 3A, fig. S3, and movie S7). This correlative light and electron microscopy (CLEM) revealed that mEmerald-Chmp4b expressed in the target cell was specifically recruited to the target plasma membrane opposite the secreted IM (Fig. 3, B and C). The topography of the plasma membrane at the site of ESCRT recruitment was markedly convoluted, exhibiting many bud-like projections (movie S7, TS 0:37 to 0:40). mEmerald-Chmp4b fluorescence also overlapped with some vesicular structures in the intercellular synaptic space (Fig. 3C). Together, the live-cell imaging and the 3D cryo-SIM and FIB-SEM CLEM demonstrate the localization of ESCRT proteins at the synapse that was the definitive site of CTL killing and was thus spatially and temporally correlated to perforin secretion. These data implicate the ESCRT complex in the repair of perforin pores.
FIG. 3. Correlative 3D cryo-SIM and FIB-SEM reveal localization of target-derived ESCRT within the cytolytic IS.
(A) Three example datasets showing correlative 3D cryo-SIM and FIB-SEM imaging of OT-I CTLs (red) captured moments after secretion of lytic granules toward peptide-pulsed ID8 cancer cells (blue) expressing mEmerald-Chmp4b (green fluorescence). (B and C) Single FIB-SEM slices corresponding to the orange boxes in (A), overlaid with CTL and cancer cell segmentation (B) or correlative cryo-SIM fluorescence of mEmerald-Chmp4b derived from the target cell (C).
Function of ESCRT proteins in repair of perforin pores
We next investigated whether ESCRT inhibition could enhance the susceptibility of target cells to CTL-mediated killing. Prolonged inactivation of the ESCRT pathway is itself cytotoxic (9). We thus developed strategies to ablate ESCRT function that would allow us a window of time to assess CTL killing (fig. S4). We used two approaches to block ESCRT function: CRISPR knockout of the Chmp4b gene or overexpression of VPS4aE228Q (E228Q, Glu228 → Gln), a dominant-negative kinase allele that impairs ESCRT function (fig. S4, A to C) (10). We took care to complete our assessment of target killing well in advance of spontaneous target cell death (fig. S4D).
We tested the capacity of OT-I CTLs to kill targets presenting one of four previously characterized peptides that demonstrate a range of potencies at stimulating the OT-I TCR: SIINFEKL (N4), the cognate peptide, and three separate variants (in order of highest to lowest affinity), SIITFEKL (T4), SIIQFEHL (Q4H7), and SIIGFEKL (G4) (18, 19). Target cells were pulsed with peptide, washed, transferred to 96-well plates, and allowed to adhere before the addition of OT-I CTLs. Killing was assessed by monitoring the uptake of a fluorogenic caspase 3/7 indicator (Fig. 4, A to D, and fig. S5A). Killing was significantly more efficient in ESCRT-inhibited target cells for both CRISPR depletion of Chmp4b (Fig. 4, A to C) and expression of the dominant-negative VPS4aE228Q (Fig. 4D). The difference in killing between the ESCRT-inhibited and control cells was greater when the lower-potency T4, Q4H7, and G4 peptides were used. Nevertheless, ESCRT inhibition moderately improved killing efficiency even in the case of the high-potency SIINFEKL peptide. ESCRT inhibition had no effect on MHC class I expression on the surface of target cells (fig. S5B). Thus, ESCRT inhibition could sensitize target cells to perforin- and granzyme-mediated killing, especially at physiologically relevant TCR-peptide MHC affinities.
FIG. 4. ESCRT inhibition enhances susceptibility of cancer cells to CTL killing and recombinant lytic proteins.
(A) Representative time-lapse data of killing of peptide-pulsed Chmp4b knockout (KO) or control B16-F10 cells by OT-I CTLs. Affinity of the pulsed peptide to OT-I TCR decreases from left to right. Error bars indicate SDs. (B) Images extracted from T4 medium-affinity peptide condition show software-detected caspase 3/7+ events in control and Chmp4b KO conditions. (C and D) Data representing the 4-hour time point of assays measuring OT-I T cells killing either Chmp4b KO (C) or VPS4 dominant-negative (D) target cells with matched controls. Error bars indicate SDs of data. Data are representative of at least three independent experimental replicates. pMHC, peptide-MHC; HA, hemagglutinin. (E and F) Determination of sublytic dose of Prf. B16-F10 cells expressing VPS4a (WT or E228Q) were exposed to increasing concentrations of Prf. Cell viability was determined by morphological gating (E). FSC, forward scatter; SSC, side scatter. (G and H) B16-F10 cells expressing VPS4a (WT or E228Q) were exposed to a sublytic dose of Prf in combination with increasing concentrations of recombinant GZMB (rGZMB). Cell death was determined by Annexin V–allophycocyanin (APC) staining (G). Controls include a condition with no perforin and 5000 ng/ml rGZMB and sublytic perforin with no rGZMB. Graphs in (F) and (H) represent the means of three experiments, and error bars indicate SDs. Statistical significance was determined by multiple unpaired t tests with alpha = 0.05. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.
We next directly tested the effects of ESCRT inhibition when target cells were exposed to both recombinant perforin (Prf) and granzyme B (GZMB), the most potently proapoptotic granzyme in humans and mice (20). Prf alone at high concentrations can lyse cells (4), so we first determined a sublytic Prf concentration that would temporarily permeabilize the plasma membrane but permit the cells to recover. B16-F10 cells expressing either VPS4aWT (WT, wild-type) or VPS4aE228Q were exposed to a range of Prf concentrations in the presence of PI, and cell viability and PI uptake were assessed using flow cytometry. Cells that expressed dominant-negative VPS4aE228Q were more sensitive to Prf alone than ESCRT-competent cells (Fig. 4, E and F). At 160 ng/ml Prf, there was no significant difference in cell viability for either condition. Cells in the live gate that were PI+ had been permeabilized by Prf but recovered. Although the percentage of PI+ live cells was similar under both sets of conditions, the mean fluorescence intensity of PI was higher in live ESCRT-inhibited cells (fig. S6). A delay in plasma membrane resealing could account for this difference.
We reasoned that delaying perforin pore repair might also enhance GZMB uptake into the target. ESCRT-inhibited cells were more sensitive to combined perforin-GZMB when cell death was measured by Annexin V staining (Fig. 4, G and H). Similar results were observed when these experiments were repeated with a murine lymphoma cancer cell line (fig. S7). The observation that ESCRT-inhibited target cells are more sensitive to both CTL-secreted and Prf-GZMB supports the hypothesis that the ESCRT pathway contributes to membrane repair after Prf exposure.
Escaping cell death is one of the hallmarks of cancer. Our findings suggest that ESCRT-mediated membrane repair of perforin pores may restrict accessibility of the target cytosol to CTL-secreted granzyme, thus promoting survival of cancer-derived cells under cytolytic attack. Although other factors may contribute to setting the threshold for target susceptibility to killing, the role of active repair of perforin pores must now be considered as a clear contributing factor.
Acknowledgments
We thank members of the Mellman laboratory for advice, discussion, and reagents; B. Haley for assistance with plasmid construct design; the Genentech FACS Core Facility for technical assistance; S. Van Engelenburg of Denver University for invaluable discussions and guidance; A. Wanner, S. Spaar, and the Ariande AI AG (https://ariadne.ai/) for assistance with FIB-SEM segmentation, CLEM coregistration, data presentation, and rendering; D. Bennett of the Janelia Research Campus for assisting with data upload to https://openorganelle.janelia.org; and the Genentech Postdoctoral Program for support.
Funding: A.T.R. and I.M. are funded by Genentech/Roche. C.S.X., G.S., A.W., D.A., N.I., and H.F.H. are funded by the Howard Hughes Medical Institute (HHMI).
Please look for a Followup Post concerning “Developing a Pharmacovigilence Framework for Engineered T-Cell Therapies”
References
Ertl HC, Zaia J, Rosenberg SA, June CH, Dotti G, Kahn J, Cooper LJ, Corrigan-Curay J, Strome SE: Considerations for the clinical application of chimeric antigen receptor T cells: observations from a recombinant DNA Advisory Committee Symposium held June 15, 2010. Cancer research 2011, 71(9):3175-3181.
Kandalaft LE, Powell DJ, Jr., Coukos G: A phase I clinical trial of adoptive transfer of folate receptor-alpha redirected autologous T cells for recurrent ovarian cancer. Journal of translational medicine 2012, 10:157.
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