Posts Tagged ‘Adverse event’

Curation of Recently Halted Oncology Trials Due to Serious Adverse Events – 2015

Curator: Stephen J. Williams, Ph.D.

The following is reports of oncology clinical trials in 2015 which have been halted for Serious Adverse Events (SAE), in most instances of an idiopathic nature. For comparison I have listed (as of this writing) the oncology drug approvals (8) for 2015. (from CenterWatch.com)

Oncology Drugs Approved in 2015

Farydak (panobinostat); Novartis; For the treatment of multiple myeloma, Approved February 2015

Ibrance (palbociclib); Pfizer; For the treatment of ER-positive, HER2-negative breast cancer, Approved February 2015

Lenvima (lenvatinib); Eisai; For the treatment of thyroid cancer, Approved February 2015

Lonsurf (trifluridine and tipiracil); Taiho Oncology; For the treatment of metastatic colorectal cancer , Approved September 2015

Odomzo (sonidegib); Novartis; For the treatment of locally advanced basal cell carcinoma, July 2015

Opdivo (nivolumab); Bristol-Myers Squibb; For the treatment of metastatic squamous non-small cell lung cancer, Approved March 2015

Unituxin (dinutuximab); United Therapeutics; For the treatment of pediatrics with high-risk neuroblastoma, Approved March 2015

Varubi (rolapitant); Tesaro; For the prevention of delayed nausea and vomiting associated with chemotherapy, Approved September 2015

Death Forces FDA to Place Clinical Hold on Advaxis (ADXS) Cancer Drug

from Biospace News

October 7, 2015
By Alex Keown, BioSpace.com Breaking News Staff

PRINCETON, N.J. – Following the death of a patient, the U.S. Food and Drug Administration (FDA) placed a hold on Advaxis (ADXS)’s experimental cancer treatment axalimogene filolisbac, which is currently in mid-stage trials.

In a statement issued this morning, Advaxis maintains the patient’s death was a result of the severity of her cancer and not due to the company’s experimental cancer treatment. It is seeking proof from the FDA that the drug was not a factor in the death. Still, the hold on the experimental cancer drug will cause the company to halt four clinical trials, Advaxis said. Other clinical trials, including those with the experimental ADXS-PSA and ADXS-HER2, are not affected by this hold. The company said it will continue to actively enroll and dose patients.

The FDA placed a hold on the drug on Oct. 2 after the company submitted a safety report to the regulatory agency that week. The drug is being developed to treat patients with persistent or recurrent metastatic (squamous or non-squamous cell) carcinoma of the cervix (PRmCC) who have progressed on at least one prior line of systemic therapy. Phase I trials released at the end of September showed treatment with axalimogene filolisbac resulted in a 38.5 percent 12-month overall survival rate in 26 patients. Patients typically fighting PRmCC who have failed at least one line of therapy have a typical survival rate of four to seven months.

Read full story here

FDA Halts Trial of Halozyme’s PEGPH20 for Pancreatic Cancer

Apr 9, 2014 Alex Philippidis

Halozyme Therapeutics acknowledged today that the FDA placed a formal clinical hold on its troubled Study 202 assessing its experimental drug PEGPH20 in patients with pancreatic cancer—less than a week after the company temporarily halted enrolling and dosing patients in the ongoing Phase II trial.

The agency told Halozyme it placed the clinical hold following the company’s pause in study activity. The trial’s independent data monitoring committee is evaluating data from the trial to learn why patients treated with PEGPH20 as well as nab-paclitaxel and gemcitabine saw a higher rate of blood clots and other thromboembolic events compared with patients treated with nab-paclitaxel and gemcitabine alone.

“We will be providing this information to the data monitoring committee and the FDA in parallel so they can complete their respective assessments,” Helen Torley, M.B. Ch.B., M.R.C.P., Halozyme’s president and CEO, said in a statement.

“Pancreatic cancer has one of the lowest survival rates of any cancer. We remain committed to evaluating PEGPH20 as a possible therapy to address this devastating disease,” Dr. Torley added.

As with Halozyme’s statement last week, the company’s latest remarks did not indicate when Halozyme expects to resume enrolling and dosing patients in Study 202, or how many patients had been enrolled and dosed when the temporary halt occurred.

The trial was envisioned as having 124 subjects, divided evenly between a treatment arm of PEGPH20 and nab-paclitaxel, and a gemcitabine arm, preceded by eight subject “run-in” phase assessing safety and tolerability, according to Study 202’s page on ClinicalTrials.gov (NCT01839487), last updated on January 27.

The study is one of two Phase II trials for PEGPH20; the other, SWOG, also aims to assess the drug for pancreatic cancer.

PEGPH20 is an investigational PEGylated form of Halozyme’s FDA-approved recombinant human hyaluronidase rHuPH20 (marketed as Hylenex®), designed to dramatically increases the half-life of the compound in the blood and allow for intravenous administration.

The temporary halt for Study 202 came two months after Halozyme publicly cited “potential acceleration of the PEGPH20 program” among several R&D programs for which it raised funds through a public offering of common stock that closed in February and generated approximately $107.8 million in net proceeds.

Read more at GenNEWS

FDA orders CytRx to halt patient enrollment after death of a cancer patient

CytRx ($CYTR) has run into an unexpected roadblock with its cancer drug conjugate aldoxorubicin, slamming the brakes on new patient recruitment in all their clinical trials after the FDA dropped a partial clinical hold on the program. According to the biotech the hold was forced by the death of a patient who was given the drug through a compassionate use program.

LA-based CytRx execs say that patients already enrolled in the studies will continue to receive the therapy as investigators added new safety measures, retooling trial protocols to include an “appropriate inclusion/exclusion criteria, an additional patient screening assessment and an evaluation of serum electrolytes prior to aldoxorubicin administration.” The patient who died, they added, had not qualified for any of its studies.

As it stands now, the biotech doesn’t know exactly how long the partial hold will last, but their announcement sought to calm jumpy investors, saying they expected to resolve the FDA’s demands “expeditiously” and can stick to their current timelines. CytRx says it expects to report preliminary results from their mid-stage study of Kaposi’s sarcoma in the second quarter of 2015 and preliminary results from the ongoing Phase II clinical trial of aldoxorubicin in glioblastoma multiforme in the first half of 2015. The company added that it is committed to completing enrollment in their Phase III trial by the end of next year.

hat reassurance appears to have helped with investors, who seemed to count this as more of a temporary setback than a catastrophe. Shares for CytRx were down about 9% in mid-morning trading.

Aldoxorubicin uses a linker molecule to attach to albumin in the blood and concentrate in tumors, where the acidic environment releases the chemotherapy doxorubicin in doses up to four times higher than what’s used now. Late last year their stock soared after their drug scored promising results for progression-free survival in a Phase IIb trial.

This case illustrates one reason why biotechs often quietly squirm under the pressure of compassionate use programs. They can be expensive to operate, time-consuming and raise fresh concerns when a patient dies or experiences a setback. On the other hand, if regulators take action like this following the death of an advanced stage cancer patient, there may have been something about the case that triggered broader concerns for the entire patient population

Clot risk in Lilly lung-cancer drug raises FDA concerns

July 7, 2015

Eli Lilly and Co.’s experimental lung cancer drug has raised concerns with U.S. regulators that it may increase patients’ risk of suffering potentially deadly blood clots.

The drug, known as necitumumab, improved patients’ overall chances of survival, yet people taking the medicine also experienced more risk, Food and Drug Administration staff said in a report Tuesday. Indianapolis-based Lilly is seeking to sell the medicine to treat a subset of the most common type of lung cancer.

FDA advisers will meet Thursday to discuss the risks and benefits of necitumumab for patients with advanced squamous non-small cell lung cancer, in combination with chemotherapy. The FDA is expected to decide if Lilly can sell the drug by the end of the year.

While the safety of necitumumab reflects that of similar drugs, the increased danger of clotting “in this already high risk population is of concern,” FDA staff wrote.

One study showed that out of 538 patients taking necitumumab and chemotherapy, 9 percent experienced a serious clot, compared with 5 percent of 541 patients given only chemotherapy, according to the staff report.

Squamous lung cancer accounts for 25 percent to 30 percent of all lung cancer, according to the American Cancer Society.

Patients in a clinical trial who took necitumumab lived a median of 11.5 months, 1.6 months longer than those who got only chemotherapy, the FDA staff report said.

Opdivo Side Effects Center (as seen on Rxlist.com) (NOTE:TRIAL NOT HALTED)

Last reviewed on RxList 10/05/2015

Opdivo (nivolumab) is a human monoclonal antibody used to treat patients with unresectable or metastatic melanoma and disease progression following ipilimumab and, if BRAF V600 mutation positive, a BRAF inhibitor; and to treat metastatic squamous non-small cell lung cancer with progression on or after platinum-based chemotherapy. Common side effects of Opdivo include fatigue, rash, itching, cough, upper respiratory tract infection, swelling of the extremities, shortness of breath, muscle pain, decreased appetite, nausea, vomiting, constipation, diarrhea, weakness, swelling, fever, abdominal pain, chest pain, joint pain, and weight loss.

Opdivo FDA Prescribing Information: Side Effects
(Adverse Reactions)

Clinical Trials Experience

Because clinical trials are conducted under widely varying conditions, adverse reaction rates observed in the clinical trials of a drug cannot be directly compared to rates in the clinical trials of another drug and may not reflect the rates observed in clinical practice.

The data described in the WARNINGS AND PRECAUTIONS section and below reflect exposure to OPDIVO in Trial 1, a randomized trial in patients with unresectable or metastatic melanoma and in Trial 3, a single-arm trial in patients with metastatic squamous non-small cell lung cancer (NSCLC).

Clinically significant adverse reactions were evaluated in a total of 691 patients enrolled in Trials 1, 3, or an additional dose finding study (n=306) administering OPDIVO at doses of 0.1 to 10 mg/kg every 2 weeks [see WARNINGS AND PRECAUTIONS].

Unresectable or Metastatic Melanoma

The safety of OPDIVO was evaluated in Trial 1, a randomized, open-label trial in which 370 patients with unresectable or metastatic melanoma received OPDIVO 3 mg/kg every 2 weeks (n=268) or investigator’s choice of chemotherapy (n=102), either dacarbazine 1000 mg/m² every 3 weeks or the combination of carboplatin AUC 6 every 3 weeks plus paclitaxel 175 mg/m² every 3 weeks [see Clinical Studies]. The median duration of exposure was 5.3 months (range: 1 day to 13.8+ months) with a median of eight doses (range: 1 to 31) in OPDIVO-treated patients and was 2 months (range: 1 day to 9.6+ months) in chemotherapy treated patients. In this ongoing trial, 24% of patients received OPDIVO for greater than 6 months and 3% of patients received OPDIVO for greater than 1 year.

In Trial 1, patients had documented disease progression following treatment with ipilimumab and, if BRAF V600 mutation positive, a BRAF inhibitor. The trial excluded patients with autoimmune disease, prior ipilimumab-related Grade 4 adverse reactions (except for endocrinopathies) or Grade 3 ipilimumab-related adverse reactions that had not resolved or were inadequately controlled within 12 weeks of the initiating event, patients with a condition requiring chronic systemic treatment with corticosteroids ( > 10 mg daily prednisone equivalent) or other immunosuppressive medications, a positive test for hepatitis B or C, and a history of HIV.

The study population characteristics in the OPDIVO group and the chemotherapy group were similar: 66% male, median age 59.5 years, 98% white, baseline ECOG performance status 0 (59%) or 1 (41%), 74% with M1c stage disease, 73% with cutaneous melanoma, 11% with mucosal melanoma, 73% received two or more prior therapies for advanced or metastatic disease, and 18% had brain metastasis. There were more patients in the OPDIVO group with elevated LDH at baseline (51% vs. 38%).

OPDIVO was discontinued for adverse reactions in 9% of patients. Twenty-six percent of patients receiving OPDIVO had a drug delay for an adverse reaction. Serious adverse reactions occurred in 41% of patients receiving OPDIVO. Grade 3 and 4 adverse reactions occurred in 42% of patients receiving OPDIVO. The most frequent Grade 3 and 4 adverse reactions reported in 2% to less than 5% of patients receiving OPDIVO were abdominal pain, hyponatremia, increased aspartate aminotransferase, and increased lipase.

FDA Approves Eisai’s LENVIMA™ (lenvatinib) for the Treatment of Patients with Locally Recurrent or Metastatic, Progressive, Radioactive Iodine-Refractory Differentiated Thyroid Cancer

– Press release from Eisai (NOTE: TRIAL NOT HALTED)

Feb 13, 2015

WOODCLIFF LAKE, N.J., Feb. 13, 2015 /PRNewswire/ — Eisai Inc. announced today that the U.S. Food and Drug Administration (FDA) approved the company’s receptor tyrosine kinase inhibitor LENVIMA™ (lenvatinib) for the treatment of locally recurrent or metastatic, progressive, radioactive iodine-refractory differentiated thyroid cancer (RAI-R DTC). LENVIMA was approved following a priority review by the FDA, which is designated for drugs the FDA believes have the potential to provide a significant improvement in the treatment of a serious condition. LENVIMA demonstrated a statistically significant progression-free survival (PFS) prolongation and response rate in patients with progressive, differentiated thyroid cancer who had become refractory to radioactive iodine (RAI) therapy.

In the clinical trial, adverse events led to dose reductions in 68% of patients who received LENVIMA and 5% of patients who received placebo. Some patients will need to discontinue treatment for serious adverse reactions. In the trial, 18% of patients treated with LENVIMA and 5% who received placebo discontinued treatment. The most common adverse reactions (at least 10%) that resulted in dose reductions of LENVIMA were hypertension (13%), proteinuria (11%), decreased appetite (10%), and diarrhea (10%).

AstraZeneca halts a pair of lung cancer trials over a safety scare

From October 9, 2015 | By of FierceBiotech

“AstraZeneca ($AZN) is pressing pause on trials combining two of its most important pipeline cancer treatments after tracking reports of lung disease, halting enrollment as it gathers more information.

The company is testing a combination of AZD9291 and durvalumab, formerly MEDI4736, in two studies involving patients with non-small cell lung cancer. Late last month, AstraZeneca hit the brakes on enrollment in both trials due to an increase in reports of interstitial lung disease, which can lead to dangerous scarring and impaired pulmonary function. The pauses are temporary, the company stressed in an emailed statement, and patients already enrolled in the study will be given new consent forms to ensure they understand the risks before choosing whether keep getting treatment.”

Other posts on this site on Cytotoxicity and Cancer include

Novel Approaches to Cancer Therapy [11.1]

Misfolded Proteins – from Little Villains to Little Helpers… Against Cancer

Multiple Lung Cancer Genomic Projects Suggest New Targets, Research Directions for Non-Small Cell Lung Cancer

A Synthesis of the Beauty and Complexity of How We View Cancer

Good and Bad News Reported for Ovarian Cancer Therapy

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Twitter is Becoming a Powerful Tool in Science and Medicine

 Curator: Stephen J. Williams, Ph.D.

Updated 4/2016

Life-cycle of Science 2

A recent Science article (Who are the science stars of Twitter?; Sept. 19, 2014) reported the top 50 scientists followed on Twitter. However, the article tended to focus on the use of Twitter as a means to develop popularity, a sort of “Science Kardashian” as they coined it. So the writers at Science developed a “Kardashian Index (K-Index) to determine scientists following and popularity on Twitter.

Now as much buzz Kim Kardashian or a Perez Hilton get on social media, their purpose is solely for entertainment and publicity purposes, the Science sort of fell flat in that it focused mainly on the use of Twitter as a metric for either promotional or public outreach purposes. A notable scientist was mentioned in the article, using Twitter feed to gauge the receptiveness of his presentation. In addition, relying on Twitter for effective public discourse of science is problematic as:

  • Twitter feeds are rapidly updated and older feeds quickly get buried within the “Twittersphere” = LIMITED EXPOSURE TIMEFRAME
  • Short feeds may not provide the access to appropriate and understandable scientific information (The Science Communication Trap) which is explained in The Art of Communicating Science: traps, tips and tasks for the modern-day scientist. “The challenge of clearly communicating the intended scientific message to the public is not insurmountable but requires an understanding of what works and what does not work.” – from Heidi Roop, G.-Martinez-Mendez and K. Mills

However, as highlighted below, Twitter, and other social media platforms are being used in creative ways to enhance the research, medical, and bio investment collaborative, beyond a simple news-feed.  And the power of Twitter can be attributed to two simple features

  1. Ability to organize – through use of the hashtag (#) and handle (@), Twitter assists in the very important task of organizing, indexing, and ANNOTATING content and conversations. A very great article on Why the Hashtag in Probably the Most Powerful Tool on Twitter by Vanessa Doctor explains how hashtags and # search may be as popular as standard web-based browser search. Thorough annotation is crucial for any curation process, which are usually in the form of database tags or keywords. The use of # and @ allows curators to quickly find, index and relate disparate databases to link annotated information together. The discipline of scientific curation requires annotation to assist in the digital preservation, organization, indexing, and access of data and scientific & medical literature. For a description of scientific curation methodologies please see the following links:

Please read the following articles on CURATION

The Methodology of Curation for Scientific Research Findings

Power of Analogy: Curation in Music, Music Critique as a Curation and Curation of Medical Research Findings – A Comparison

Science and Curation: The New Practice of Web 2.0

  1. Information Analytics

Multiple analytic software packages have been made available to analyze information surrounding Twitter feeds, including Twitter feeds from #chat channels one can set up to cover a meeting, product launch etc.. Some of these tools include:

Twitter Analytics – measures metrics surrounding Tweets including retweets, impressions, engagement, follow rate, …

Twitter Analytics – Hashtags.org – determine most impactful # for your Tweets For example, meeting coverage of bioinvestment conferences or startup presentations using #startup generates automatic retweeting by Startup tweetbot @StartupTweetSF.


  1. Tweet Sentiment Analytics

Examples of Twitter Use

A. Scientific Meeting Coverage

In a paper entitled Twitter Use at a Family Medicine Conference: Analyzing #STFM13 authors Ranit Mishori, MD, Frendan Levy, MD, and Benjamin Donvan analyzed the public tweets from the 2013 Society of Teachers of Family Medicine (STFM) conference bearing the meeting-specific hashtag #STFM13. Thirteen percent of conference attendees (181 users) used the #STFM13 to share their thoughts on the meeting (1,818 total tweets) showing a desire for social media interaction at conferences but suggesting growth potential in this area. As we have also seen, the heaviest volume of conference-tweets originated from a small number of Twitter users however most tweets were related to session content.

However, as the authors note, although it is easy to measure common metrics such as number of tweets and retweets, determining quality of engagement from tweets would be important for gauging the value of Twitter-based social-media coverage of medical conferences.

Thea authors compared their results with similar analytics generated by the HealthCare Hashtag Project, a project and database of medically-related hashtag use, coordinated and maintained by the company Symplur.  Symplur’s database includes medical and scientific conference Twitter coverage but also Twitter usuage related to patient care. In this case the database was used to compare meeting tweets and hashtag use with the 2012 STFM conference.

These are some of the published journal articles that have employed Symplur (www.symplur.com) data in their research of Twitter usage in medical conferences.

B. Twitter Usage for Patient Care and Engagement

Although the desire of patients to use and interact with their physicians over social media is increasing, along with increasing health-related social media platforms and applications, there are certain obstacles to patient-health provider social media interaction, including lack of regulatory framework as well as database and security issues. Some of the successes and issues of social media and healthcare are discussed in the post Can Mobile Health Apps Improve Oral-Chemotherapy Adherence? The Benefit of Gamification.

However there is also a concern if social media truly engages the patient and improves patient education. In a study of Twitter communications by breast cancer patients Tweeting about breast cancer, authors noticed Tweeting was a singular event. The majority of tweets did not promote any specific preventive behavior. The authors concluded “Twitter is being used mostly as a one-way communication tool.” (Using Twitter for breast cancer prevention: an analysis of breast cancer awareness month. Thackeray R1, Burton SH, Giraud-Carrier C, Rollins S, Draper CR. BMC Cancer. 2013;13:508).

In addition a new poll by Harris Interactive and HealthDay shows one third of patients want some mobile interaction with their physicians.

Some papers cited in Symplur’s HealthCare Hashtag Project database on patient use of Twitter include:

C. Twitter Use in Pharmacovigilance to Monitor Adverse Events

Pharmacovigilance is the systematic detection, reporting, collecting, and monitoring of adverse events pre- and post-market of a therapeutic intervention (drug, device, modality e.g.). In a Cutting Edge Information Study, 56% of pharma companies databases are an adverse event channel and more companies are turning to social media to track adverse events (in Pharmacovigilance Teams Turn to Technology for Adverse Event Reporting Needs). In addition there have been many reports (see Digital Drug Safety Surveillance: Monitoring Pharmaceutical Products in Twitter) that show patients are frequently tweeting about their adverse events.

There have been concerns with using Twitter and social media to monitor for adverse events. For example FDA funded a study where a team of researchers from Harvard Medical School and other academic centers examined more than 60,000 tweets, of which 4,401 were manually categorized as resembling adverse events and compared with the FDA pharmacovigilance databases. Problems associated with such social media strategy were inability to obtain extra, needed information from patients and difficulty in separating the relevant Tweets from irrelevant chatter.  The UK has launched a similar program called WEB-RADR to determine if monitoring #drug_reaction could be useful for monitoring adverse events. Many researchers have found the adverse-event related tweets “noisy” due to varied language but had noticed many people do understand some principles of causation including when adverse event subsides after discontinuing the drug.

However Dr. Clark Freifeld, Ph.D., from Boston University and founder of the startup Epidemico, feels his company has the algorithms that can separate out the true adverse events from the junk. According to their web site, their algorithm has high accuracy when compared to the FDA database. Dr. Freifeld admits that Twitter use for pharmacovigilance purposes is probably a starting point for further follow-up, as each patient needs to fill out the four-page forms required for data entry into the FDA database.

D. Use of Twitter in Big Data Analytics

Published on Aug 28, 2012


Course: Information 290. Analyzing Big Data with Twitter
School of Information
UC Berkeley

Lecture 1: August 23, 2012

Course description:
How to store, process, analyze and make sense of Big Data is of increasing interest and importance to technology companies, a wide range of industries, and academic institutions. In this course, UC Berkeley professors and Twitter engineers will lecture on the most cutting-edge algorithms and software tools for data analytics as applied to Twitter microblog data. Topics will include applied natural language processing algorithms such as sentiment analysis, large scale anomaly detection, real-time search, information diffusion and outbreak detection, trend detection in social streams, recommendation algorithms, and advanced frameworks for distributed computing. Social science perspectives on analyzing social media will also be covered.

This is a hands-on project course in which students are expected to form teams to complete intensive programming and analytics projects using the real-world example of Twitter data and code bases. Engineers from Twitter will help advise student projects, and students will have the option of presenting their final project presentations to an audience of engineers at the headquarters of Twitter in San Francisco (in addition to on campus). Project topics include building on existing infrastructure tools, building Twitter apps, and analyzing Twitter data. Access to data will be provided.

Other posts on this site on USE OF SOCIAL MEDIA AND TWITTER IN HEALTHCARE and Conference Coverage include:

Methodology for Conference Coverage using Social Media: 2014 MassBio Annual Meeting 4/3 – 4/4 2014, Royal Sonesta Hotel, Cambridge, MA

Strategy for Event Joint Promotion: 14th ANNUAL BIOTECH IN EUROPE FORUM For Global Partnering & Investment 9/30 – 10/1/2014 • Congress Center Basel – SACHS Associates, London

REAL TIME Cancer Conference Coverage: A Novel Methodology for Authentic Reporting on Presentations and Discussions launched via Twitter.com @ The 2nd ANNUAL Sachs Cancer Bio Partnering & Investment Forum in Drug Development, 19th March 2014 • New York Academy of Sciences • USA

PCCI’s 7th Annual Roundtable “Crowdfunding for Life Sciences: A Bridge Over Troubled Waters?” May 12 2014 Embassy Suites Hotel, Chesterbrook PA 6:00-9:30 PM

CRISPR-Cas9 Discovery and Development of Programmable Genome Engineering – Gabbay Award Lectures in Biotechnology and Medicine – Hosted by Rosenstiel Basic Medical Sciences Research Center, 10/27/14 3:30PM Brandeis University, Gerstenzang 121

Tweeting on 14th ANNUAL BIOTECH IN EUROPE FORUM For Global Partnering & Investment 9/30 – 10/1/2014 • Congress Center Basel – SACHS Associates, London


Statistical Analysis of Tweet Feeds from the 14th ANNUAL BIOTECH IN EUROPE FORUM For Global Partnering & Investment 9/30 – 10/1/2014 • Congress Center Basel – SACHS Associates, London

1st Pitch Life Science- Philadelphia- What VCs Really Think of your Pitch

What VCs Think about Your Pitch? Panel Summary of 1st Pitch Life Science Philly

How Social Media, Mobile Are Playing a Bigger Part in Healthcare

Can Mobile Health Apps Improve Oral-Chemotherapy Adherence? The Benefit of Gamification.

Medical Applications and FDA regulation of Sensor-enabled Mobile Devices: Apple and the Digital Health Devices Market

E-Medical Records Get A Mobile, Open-Sourced Overhaul By White House Health Design Challenge Winners

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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;

  1. the development of vectors useful for cloning pieces of DNA (the first vector named pBR322) and
  2. 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

end of a long struggle to revise the NIH Guidelines for Research Involving

Recombinant DNA Molecules

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 to NIH 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:

  1. Summary of the Evolution of the CAR therapy
  2. Points for Future Consideration including adverse event reporting
  3. 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)

Constructing a CAR T Cell (from cancer.gov)

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.

Case Report of a Serious Adverse Event Following the Administration of T Cells Transduced With a Chimeric Antigen Receptor Recognizing ERBB2[2] had reported the death of a patient on trial.

In A phase I clinical trial of adoptive transfer of folate receptor-alpha redirected autologous T cells for recurrent ovarian cancer[3] authors: Lana E Kandalaft*, Daniel J Powell and George Coukos from University of Pennsylvania recorded adverse events in pilot studies using a CART modified to recognize the folate receptor, so it appears any adverse event reporting system is at the discretion of the primary investigator.

Other protocol considerations suggested by the symposium attendants included:

  • Plan for translational clinical lab for routine blood analysis
  • Subject screening for pulmonary and cardiac events
  • Determine possibility of insertional mutagenesis
  • Informed consent
  • 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 effectsCytokine 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..

Please see video below:

http link: https://www.youtube.com/watch?v=4Gg6elUMIVE

  • 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 Effectslong-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:

What does this mean for Immunotherapy? FDA put a temporary hold on Juno’s JCAR015, Three Death of Celebral Edema in CAR-T Clinical Trial and Kite Pharma announced Phase II portion of its CAR-T ZUMA-1 trial

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

By DAMIAN GARDE @damiangarde and MEGHANA KESHAVAN @megkesh

JULY 7, 2016

In Juno patient deaths, echoes seen of earlier failed company


JULY 8, 2016


After a deadly clinical trial, will immune therapies for cancer be a bust?

By DAMIAN GARDE @damiangarde

JULY 8, 2016

This led to warnings by FDA and alteration of their trials as well as the use of their CART as a monotherapy

Hours after Juno CAR-T study deaths announced, Kite enrolls CAR-T PhII

Well That Was Quick! FDA Lets Juno Restart Trial With a New Combination Chemotherapuetic

 at Seattle Times

FDA lets Juno restart cancer-treatment 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
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

see Live Conference Coverage on this site

eProceedings for BIO 2019 International Convention, June 3-6, 2019 Philadelphia Convention Center; Philadelphia PA, Real Time Coverage by Stephen J. Williams, PhD @StephenJWillia2

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.

From Onclive

Source: https://www.onclive.com/view/car-t-cell-therapy-trial-in-solid-tumors-halted-following-2-patient-deaths 

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.


  1. 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
  2. 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.


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.


Source: https://www.fiercebiotech.com/research/genentech-uncovers-how-cancer-cells-resist-t-cell-attack-potential-boon-immunotherapy

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.

The following is the Science paper

Membrane repair in target cell defenses

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


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 (67). 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 (67). 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 (1113). 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] (1516). 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) (1819). 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.


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”



  1. 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.
  2. Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA: Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Molecular therapy : the journal of the American Society of Gene Therapy 2010, 18(4):843-851.
  3. 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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FDA: Strengthening Our National System for Medical Device Post-market Surveillance

Reporter: Aviva Lev-Ari, PhD, RN


September 7, 2012 | By Damian Garde

The FDA wants industry feedback on a host of new post-market surveillance initiatives, designed to better track, analyze and report the performance of medical devices.

In a report released Thursday, the agency proposes a four-point plan to improve its post-market system, including the previously announced unique ID program and a modernization of MedWatch. The agency is taking any and all opinions from devicemakers and members of the healthcare community through its website, and the FDA plans to host public meetings on the plan this month.


Here’s a summary of the four points:


Establish a unique device ID system: In keeping with its July announcement, the FDA wants to require devicemakers to tag their products with an alphanumeric code, disclosing the device’s production information, serial number, manufacturing date and expiration date. The goal is to help the FDA and healthcare community to more accurately track and analyze device-related adverse events. Once rolled out, the ID system will cost the industry $65 million, the FDA has said.

Promote international device registries: The agency isn’t looking to found a huge, centralized registry, housing data on device uses and reactions. Instead, the FDA wants to help governments and private outfits set up and operationalize smaller registries, sharing data with one another to keep tabs on device performance. The agency plans to host a series of public workshops to educate would-be registry founders on the best way to move forward.

Modernize adverse event reporting: Currently, the FDA relies on spontaneous reporting for when devices go awry, primarily using its voluntary MedWatch system. That model is inherently limited, the agency says, and it wants to institute automated reporting systems in hospitals, encourage more electronic reporting, develop a mobile app for MedWatch and update the MAUDE adverse event database, which the FDA says is technologically outdated.

Develop new tools and methods for post-market surveillance: This is the catch-all part of the FDA’s plan, in which the agency discusses future innovations that could generate, synthesize and interpret post-market data to drive decision-making. For instance, the FDA wants to automate data analysis to identify spikes in adverse events across disparate data sources. The agency also proposes instituting quantitative decision analysis in its post-market deliberations, aiming to better standardize its methods.




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