April 18, 2019

Scientists advance creation of ‘artificial lymph node’ to fight cancer, other diseases

In a proof-of-principle study in mice, scientists at Johns Hopkins Medicine report the creation of a specialized gel that acts like a lymph node to successfully activate and multiply cancer-fighting immune system T-cells. The work puts scientists a step closer, they say, to injecting such artificial lymph nodes into people and sparking T-cells to fight disease.

In the past few years, a wave of discoveries has advanced new techniques to use T-cells — a type of white blood cell — in cancer treatment. To be successful, the cells must be primed, or taught, to spot and react to molecular flags that dot the surfaces of cancer cells. The job of educating T-cells this way typically happens in lymph nodes, small, bean-shaped glands found all over the body that house T-cells. But in patients with cancer and immune system disorders, that learning process is faulty, or doesn’t happen.

To address such defects, current T-cell booster therapy requires physicians to remove T-cells from the blood of a patient with cancer and inject the cells back into the patient after either genetically engineering or activating the cells in a laboratory so they recognize cancer-linked molecular flags.

One such treatment, called CAR-T therapy, is costly and available only at specialized centers with laboratories capable of the complicated task of engineering T-cells. In addition, it generally takes about six to eight weeks to culture the T-cells in laboratories and, once reintroduced into the body, the cells don’t last long in the patient’s body, so the effects of the treatment can be short-lived.

The new work, reported April 10 in the journal Advanced Materials, is a bid by Johns Hopkins scientists to find a more efficient way of engineering T-cells.

“We believe that a T-cell’s environment is very important. Biology doesn’t occur on plastic dishes; it happens in tissues,” says John Hickey, a Ph.D. candidate in biomedical engineering at the Johns Hopkins University School of Medicine and first author of the study report.

To make the engineered T-cells’ environment more biologically realistic, Hickey — working with his mentors Hai-Quan Mao, Ph.D., associate director of the Johns Hopkins Institute for NanoBioTechnology and Jonathan Schneck, M.D., Ph.D., professor of pathology, medicine and oncology at the Johns Hopkins University School of Medicine — tried using a jelly-like polymer, or hydrogel, as a platform for the T-cells. On the hydrogel, the scientists added two types of signals that stimulate and “teach” T-cells to hone in on foreign targets to destroy.

In their experiments, T-cells activated on hydrogels produced 50 percent more molecules called cytokines, a marker of activation, than T-cells kept on plastic culture dishes.

Because hydrogels can be made to order, the Johns Hopkins scientists created and tested a range of hydrogels, from the very soft feel of a single cell to the more rigid quality of a cell-packed lymph node.

“One of the surprising findings was that T-cells prefer a very soft environment, similar to interactions with individual cells, as opposed to a densely packed tissue,” says Schneck.

More than 80 percent of T-cells on the soft surface multiplied themselves, compared with none of the T-cells on the most firm type of hydrogel.

When the Johns Hopkins team put T-cells onto a soft hydrogel, they found that the T-cells multiplied from just a few cells to about 150,000 cells — plenty to use for cancer therapy — within seven days. By contrast, when the scientists used other conventional methods to stimulate and expand T-cells, they were able to culture only 20,000 cells within seven days.

In the next set of experiments, the scientists injected the T-cells engineered in either the soft hydrogels or the plastic culture dishes into mice implanted with melanoma, a lethal form of skin cancer. Tumors in mice with T-cells cultured on hydrogels remained stable in size, and some of the mice survived beyond 40 days. By contrast, tumors grew in most of the mice injected with T-cells cultured in plastic dishes, and none of these mice lived beyond 30 days.

“As we perfect the hydrogel and replicate the essential feature of the natural environment, including chemical growth factors that attract cancer-fighting T-cells and other signals, we will ultimately be able to design artificial lymph nodes for regenerative immunology-based therapy,” says Schneck, a member of the Johns Hopkins Kimmel Cancer Center.

April 15, 2019

Kubanda Cryotherapy, AssistENT Win Inaugural Bisciotti Student Prize

Two teams of young entrepreneurs — one with a novel way to treat cancer in pets, the other with a discreet device to improve breathing — won the inaugural Bisciotti Foundation Prize for Student Entrepreneurship.

Kubanda Cryotherapy was awarded the first-place prize of $30,000 in nondilutive funding and AssistENT received the $20,000 second prize. The winning teams were announced April 6 as part of the Brunch & Pitches event at FastForward U during Alumni Weekend.

FastForward U received 66 applications for the Bisciotti Student Prize. The goal of the prize is to help student startups grow their businesses as they graduate from FastForward U student entrepreneurship programs. All startups were required to have a founder or co-founder who is a current student or recent graduate of The Johns Hopkins University and one founder or co-founder committed to the company after graduation.

Applicants were also asked to demonstrate plans to grow and thrive in Baltimore, helping to meet a goal of both the Bisciotti Student Prize and Johns Hopkins Technology Ventures.

“We were blown away by the quantity and quality of applications received for our inaugural Bisciotti Student Prize,” says Kevin Carter, FastForward U’s student venture coordinator. “We’re excited to see how Kubanda and AssistENT use this funding to help move their ventures to market as well as establish their places in Baltimore’s growing entrepreneurial ecosystem.”

Applications for the 2019–20 Bisciotti Student Prize will open in January 2020.

Kubanda Cryotherapy

The team — all Whiting School of Engineering (WSE) students or alums: Bailey Surtees (’17), Yixin Hu (’17, M.S. ’19), Pascal Acree (’20), Grace Kuroki (’20), Evelyn McChesney (’20), Varun Kedia (’20), Andrea Niu (’22), Dennis Gong (’22) and Rebecca Yu (’22)

The pitch: affordable cancer care for pets through a novel, low-cost cryoablation device

More than 12 million pets are diagnosed with cancer each year, but only a quarter of their owners pursue treatment for nonbenign tumors. Owners who decide to treat the cancer must then find a specialist — less than 1 percent of veterinarians are licensed oncologists. Kubanda Cryotherapy’s device would allow a local veterinarian to freeze and kill cancerous masses in a single outpatient session at a cost of $1,200. Current cryoablation procedures done on pets were designed for human use, cost more than $20,000 and must be performed at a specialist center or research hospital. While focused on the veterinary market, the company ultimately wants to use its technology to treat breast cancer patients in low- and middle-income countries.

Kubanda is pursuing patent protection for its technology through Johns Hopkins Technology Ventures, the university’s commercialization arm. (The university owns the intellectual property behind Kubanda but his licensed it back out to the company.) Kubanda plans to use the grant money to build and produce a first-generation device.

AssistENT

The team: Clayton Andrews (WSE ’17) and Patrick Byrne, professor of otolaryngology — head and neck surgery and biomedical engineering, and director of the Division of Facial Plastic and Reconstructive Surgery

The pitch: N-Stent, a nasal breathing aid designed for everyday life

Chronically restricted nasal breathing affects more than 15 percent of Americans. About 125,000 people each year undergo nasal reconstructive surgery to permanently reopen their airways, but the procedure is invasive, the recovery is slow and one-fifth of patients report unimproved or worsened airways. External nasal strips have been found to improve airflow, but they are highly visible and their adhesive can damage the user’s skin. AssistENT’s N-Stent, by contrast, can be inserted and removed from the nose in seconds and worn all day. A clinical trial showed nasal airflow improved by 76 percent among 35 patients.

The company is pursuing patent protection for its technology and is aiming to have N-Stent on the market in January, pricing a one-month supply at $12. AssistENT plans to use the grant money in part to make a third-generation prototype and to develop its website, which will sell N-Stent.

April 11, 2019

After returning from space, astronaut has no lingering, major epigenetic differences from earthbound twin brother

In a landmark study, a group of U.S. scientists from Johns Hopkins, Stanford University and other institutions has found no long-lasting, major differences between the epigenomes of astronaut Scott Kelly, who spent a year in space aboard the International Space Station, and his twin brother, Mark, who remained on Earth.

What this study tells us about the perils of space travel on a person’s genome is not clear, say the scientists, but research on additional astronauts in space could eventually help scientists predict the types of medical risks they may face on long space journeys where people experience less gravity than on Earth, exposure to harmful ultraviolet rays and other risks to health.

“This is the dawn of human genomics in space,” says Andrew Feinberg, the Bloomberg Distinguished Professor of Medicine, Biomedical Engineering and Mental Health at The Johns Hopkins University. “We developed the methods for doing these types of human genomic studies, and we should be doing more research to draw conclusions about what happens to humans in space.”

Epigenetic changes involve chemical “tweaks” to DNA that can influence gene activity, but the changes don’t affect the underlying genetic code itself. The changes affect when and how a gene is read, or expressed, for its protein-encoding instructions. When epigenetic changes occur at the wrong time or place, the process can turn genes on or off at the wrong time and place, too.

Scientists have long monitored and studied the physiological effects of space travel on astronauts. However, most of these astronauts travel on spaceflight missions of six months or less, not the longer missions required to travel to Mars or elsewhere. More research is needed to understand the impact of long spaceflight missions on the human body, where there is more exposure to radiation, restricted diet, less exercise, lower gravity and disrupted sleep cycles.

Feinberg notes that studying identical twins — who, by nature, have the same genetic material — was an important and rare opportunity to compare physiological and genomic changes when one twin went into space and the other remained on Earth. “However, since we only have two people in our study, we can’t say that these changes are due to space travel itself,” says Feinberg. “We need more studies of astronauts to draw such conclusions.”

For the study, described in the April 12 issue of Science, scientists collected blood samples, physiological data and cognitive measurements from Scott and Mark Kelly at various time points over 27 months before, during and after Scott’s one-year space mission. The samples from Scott during the flight were collected on the space station when shipments from Earth arrived on a Soyuz rocket and, that same day, shipped back to Earth on the rocket so that the samples could be processed within 48 hours.

Feinberg and former postdoctoral student Lindsay Rizzardi, now a senior scientist at the HudsonAlpha Institute for Biotechnology, focused on epigenetic changes to Scott and Mark’s genomes.

Specifically, Feinberg and his team examined two types of white blood cells (CD4+ and CD8+) isolated from Mark and Scott’s blood. They focused on epigenetic marks consisting of chemical modifications called methyl groups that are added onto the DNA in a process called methylation.

Overall, they found that about just as many epigenetic changes occurred in earthbound Mark’s DNA as in his space-flying twin. There was a less than 5 percent difference in overall methylation between the twins during the mission. The largest difference occurred nine months into the mission when 79 percent of Scott’s DNA was methylated, compared with 83 percent of Mark’s.

The locations of methylation changes in the genome were different for each twin. For example, the scientists found methylation changes near genes involved in immune system responses in Scott during his time in space, but not in Mark. This correlated with data from other researchers involved in the current study who found increases in certain biochemical markers associated with inflammation in Scott but not Mark.

“It was encouraging to see that there was no massive disruption of the epigenome in either Mark or Scott,” says Rizzardi. “However, with only two people in the study, we’re limited in the conclusions we can draw about the effect of space travel on the genome. But the findings give us clues to what we should examine more closely in future studies of astronauts.”

In the current study, Scott’s biological samples were shipped back to Earth immediately, but in the future, astronauts may need to process and store samples on the spacecraft. Feinberg, Rizzardi and NASA scientist Brian Crucian developed detailed instructions for doing complicated experiments in microgravity. Feinberg and Rizzardi traveled for a week on the famed “Vomit Comet,” a plane that simulates weightlessness, to test their protocols for overcoming the challenges of collecting, purifying and storing blood samples aboard the space station.

Of the studies led by scientists at Stanford University, Colorado State University, Cornell University and others, some of the notable results included Scott’s in-flight lengthening of telomeres, the protective endcaps on chromosomes. The telomere lengthening, as previously reported, returned to normal when Scott returned to Earth.

In addition, more than 90 percent of genes that changed activity levels during Scott’s flight returned to normal six months after the flight. Yet, Feinberg notes, these changes are not indicative of space flight alone, nor do they differ from what might occur normally.

The scientists also found that the shape of Scott’s eyeball changed over the course of the flight, including a thicker retinal nerve and folds in the choroid layer that surrounds the eye. These changes typically affect visual acuity and, says Feinberg, have occurred in other male astronauts but not females. The scientists also observed cognitive changes and increased stress levels in Scott during the flight, which, again, may not be attributed to space flight alone.

Feinberg says this study lays the groundwork to make predictions about an astronaut’s gene-related and physiological function during a long-term mission: “If we know what to expect, we can anticipate health problems astronauts may encounter and ensure that medicines and other remedies are at hand during a mission.”

April 10, 2019

Experimental drug delivers one-two punch to vision loss

In studies with lab-grown human cells and in mice, Johns Hopkins Medicine researchers have found that an experimental drug may be twice as good at fighting vision loss as previously thought.


The new research shows that the compound, named AXT107, stops abnormal blood vessels in the eye from leaking vision-blocking fluids. These results build on previous research that showed the same compound stopped the growth of abnormal vessels in animal studies of the blinding disease diabetic macular edema and wet age-related macular degeneration.Diabetic macular edema and wet age-related macular degeneration are the leading causes of vision loss in the U.S.  Approximately 750,000 Americans age 40 and older have diabetic macular edema, and wet age-related macular degeneration affects over 1.6 million Americans age 50 and older. Both diseases can eventually cause blindness if untreated.

Current drugs for diabetic macular edema and wet age-related macular degeneration focus on halting the growth of these abnormal vessels to preserve what vision is left. The current standard of treatment is monthly injections directly into the eye to suppress new blood vessel growth. These frequent visits can be a burden for patients due to the discomfort, a small risk for each injection and, for some patients, difficulty getting to the appointment because their vision is not good enough to drive.

“Our findings give us a better understanding of how this potential treatment stops the disease from progressing and does it more quickly, efficiently, and has a longer duration than current drugs used in people with vision loss of this kind,” says Aleksander Popel, professor of biomedical engineering at the Johns Hopkins University School of Medicine.

The study was published in the Feb. 21 issue of the Journal of Clinical Investigation Insight.

In healthy eyes, the cells that make up blood vessels are bound together by proteins residing on the surface of the cell that are directed into place by Tie 2, another protein. Tie2 proteins pack tightly together where cells meet their neighbors and act like Velcro to create a fluid-tight connection between cells in the blood vessel’s wall. In diabetic macular edema, the Tie2 proteins disperse across the cell and no longer can maintain the fluid-tight barrier between the inside of a blood vessel and the outside. Gaps form between the cells, allowing fluids to permeate into the surrounding tissue.

To understand how the drug they developed could strengthen these connections, the researchers designed a series of experiments to explore how AXT107 affects the control of Tie2 and the Velcro-like proteins.

In their first experiment, the researchers used cells derived from human blood vessels grown in the lab that mimicked those seen in wet age-related macular degeneration. When they added the AXT107 drug to these cells, the researchers found that AXT107 initiated a series of changes to cellular proteins. Using a technique to measure protein changes, the researchers found that Tie2 proteins seemed to migrate across the cell. Groups of Tie2 proteins began to congregate where cells met their neighbors, and began rebuilding connections with other blood vessel cells.

The researchers note that when observed under a microscope, the cells went from jagged-looking around the edges to having smooth and continuous outer edges that could be better suited for one cell to fit snugly against another. “It was like zipping them up with a zipper,” says Popel.

The researchers further tested whether these smooth cells could create a watertight barrier, which would be necessary to create a blood vessel that doesn’t leak. So they grew the cells in a single layer and tested whether fluid could pass through by pouring a fluorescent liquid on top of the cells and checking to see if any of the glowing liquid ended up underneath. The researchers observed that cells treated with 100 µM of the AXT107 drug allowed 2.5 times less dye through the cell layer than control cells receiving no drug. This showed the researchers that the drug helped blood vessel cells create a watertight seal between them.

The researchers next wanted to see if the same effect could be achieved in living blood vessels. They used a fluorescent dye to observe the blood vessels in the eyes of normal mice and mice genetically engineered to mimic human macular degeneration. In the healthy mice, the researchers observed glowing blood vessels with crisp edges and very little fluorescence outside of the vessel. However, in the mice with macular degeneration, glowing liquids passed through the blood vessels, blurring the barrier between blood vessels and the surrounding tissues.

The researchers treated the engineered mice with leaky blood vessels, like those seen in macular degeneration, with injections of the AXT107 peptide into the animals’ eyes. After four days, the researchers found that in mice treated with AXT107, about half as much of the fluorescent dye leaked from their vessels as in animals that received saline injections containing no drug. These results, say researchers, show that the AXT107 drug was able to seal up leaking vessels and prevent vision-blocking fluids from permeating into the surrounding tissue.

Popel says previous studies of AXT107 in animal models showed the drug lasted longer than current treatments by forming a small clear gel of slow-releasing drug in the eye. If proved effective in humans, patients might need only one or two injections to the eye per year, instead of the monthly injections that are the current standard of care.

Popel says AXT107 provides a new therapeutic approach that targets two clinically validated pathways for retina diseases while the anti-VEGF agents only target one aspect of the disease. “In addition to potentially improving the response for patients, the longer duration of AXT107 may allow for less frequent dosing, thus reducing the treatment burden for patients,” says Popel.

The researchers say they plan to test the AXT107 peptide for safety and efficacy in clinical trials of people with diabetic macular edema next year.

Other researchers involved in this study include Adam Mirando, Jikui Shen, Raquel Lima e Silva, Zenny Chu, Nicholas Sass, Valeria Lorenc, Peter Campochiaro, Jordan Green and Niranjan Pandey of the Johns Hopkins University School of Medicine and AsclepiX Therapeutics.