Dr. Kastner brings people together to leverage complementary strengths and achieve a common goal.
Published October 1, 2019
By Marie Samanovic Golden, PhD
Daniel L. Kastner, MD, PhD, Scientific Director for the Intramural Research Program at the National Human Genome Research Institute (NHGRI), received the 2019 Ross Prize in Molecular Medicine — an honor established by The Feinstein Institutes for Medical Research and the Springer Nature journal Molecular Medicine — for his pioneering work on the genomics of auto-inflammatory diseases.
Dr. Daniel Kastner (right) with colleague Dr. David Beck (left)
“The Ross Prize is the most memorable, exciting, rewarding prize that I have ever received,” declared Kastner.
In the 1990s, Dr. Kastner led an international consortium that identified the gene responsible for familial Mediterranean fever (FMF), a rare inherited disorder characterized by recurrent fevers and severe inflammation.
What makes Dr. Kastner unique is that he is a master in bringing people together, helping them to leverage complementary strengths and achieve a common goal. This manifested in the international FMF consortium, comprising six groups with a total of 46 collaborators located in Israel, Australia and four centers around the United States.
“Ideal collaborations are win-win propositions,” said Kastner, and “trust is the currency of the realm.”
Advances in Autoinflammatory Disease Research
The endeavor was a resounding success. It also laid the groundwork for the identification of the tumor necrosis factor receptor-associated periodic syndrome (TRAPS), a second periodic fever syndrome beside FMF, which led to the novel concept of an emerging family of autoinflammatory diseases.
Inflammation is now thought to play an important role in a number of rare monogenic diseases akin to FMF and TRAPS, as well in more common and genetically complex diseases like gout.
Colleagues of Dr. Kastner, like Dr. Luke O’Neil from Trinity College Dublin, take the bold position that addressing inflammation could impact any number of ailments. Certainly it is the case that inflammation plays an important role in several common diseases such as atherosclerosis and cancer. However, “the inflammatory process is a double-edged sword” warned Kastner.
Indeed, dampening patients’ autoinflammatory diseases with anti-inflammatory agents brings them to a normal, base-level of immunity — and may even be protective against other inflammation-mediated disorders. But in most individuals, a blanket prescription of anti-inflammatories could prevent their immune systems from performing its most basic and necessary function: fighting off microbial infections.
Developing the Clinical Infrastructure
Looking ahead, Dr. Kastner developed a clinical infrastructure at the National Institutes of Health (NIH) to examine patients with undiagnosed inflammatory diseases, using genetics to identify the cause of rare diseases and autoinflammatory disorders. As of 2019, the inflammatory diseases section has seen over 2,000 patients, referred from around the world. This prolific program led to the identification of more than 15 new diseases, and over half of them now have effective therapies.
Treatments for these diseases, such as cytokine inhibitors or JAK-kinase inhibitors, target the molecular pathways involved, but are only effective for as long as patients take them. Thus, curative measures such as bone-marrow transplants, or potentially gene therapy, are attractive to patients and their families. But these are not without risk, advised Kastner.
For inflammatory diseases caused by mutations in white blood cells, bone marrow transplants are appealing and logical in lieu of a lifetime of treatment. However, depending on the clinical circumstances, this measure may come with a significant mortality rate, he explained.
Weighing the Risk-Benefits
It is difficult to justify such risk if patients are responding to effective drugs such as colchicine (for the control of FMF), with no reported long-term side effects in the last 50 years. Dr. Kastner is constantly working to weigh these risk-benefits with his patients.
Dr. Kastner shared that he owes a debt of gratitude to Dr. Robert Rich, his first research mentor at Baylor College of Medicine, who not only allowed him, but also expected him to follow his interests independently as a young scientist. Dr. Rich also urged him to go back to medical school after his PhD, to apply his new knowledge to the care of patients.
Kastner continues this tradition, constantly moving between the bench and the bedside in his continued quest to understand inflammatory disease.
Sam Parnia, a leading expert in resuscitation science research, explains how death is not an absolute, but a process, and what happens when patients experience death.
Sam Parnia MD, PhD
Published September 30, 2019
By Robert Birchard
Across time and cultures, people have been conditioned to view death as an endpoint to the experience of life. However, advances in resuscitation science and critical care medicine have challenged assumptions about the finality of death. Sam Parnia, Director of the Critical Care & Resuscitation Research Division of Pulmonary, Critical Care & Sleep Medicine at New York University Langone Medical Center, recently spoke to The New York Academy of Sciences about his resuscitation science research. Dr. Parnia’s work illuminates how death is not an absolute, but a process, and what happens when patients experience death — sharing insights from his research in his own words:
What is death?
Death occurs when the heart stops beating. We call this death by cardiopulmonary criteria and it is how death is defined for more than 95 percent of people. A person stops breathing and their brain shuts down, causing all life processes to cease. More recently with the birth of modern intensive care medicine and the ability to artificially keep people’s hearts beating, doctors like myself can keep a patient’s heart beating longer.
Where people may have suffered irreversible brain damage and brain death, this leads to a situation where the brain has died, but the person’s heart is still beating, so legally, they are declared dead based upon irreversible brain death, or death by brain death criteria. This happens in a small fraction of the cases where people are declared dead.
For millennia death was considered an irreversible event and nothing could restore life. During the last decade, we’ve realized it’s only after a person has died that the cells inside their body, including the brain, begin their own death process. We used to think that you had five or 10 minutes before brain cells died, from a lack of oxygen, but we now know that’s wrong.
You have hours, if not days, before the brain and other organs in the body are irreversibly damaged after death. It’s actually the restoration of oxygen and blood flow back into organs after a person’s heart stops, but is then resuscitated that paradoxically leads to accelerated cell death. So, this accelerated secondary injury process is what we need to combat in medicine now.
Why is the term “near-death” experience inaccurate?
The problem with this term is that it is inconsistent with what people actually experience. It is undefined and imprecise. If I said ‘an airplane was involved in a near-miss incident,’ what does that mean? Did you have another plane come in within an inch of another plane, or were they a mile away? The term is ill-defined, and, it doesn’t take into consideration the fact that a lot of people have biologically died and returned.
What is a death experience?
I call it an “experience of death” because that’s what it is. People report a unique cognitive experience in relation to death. They may have a perception of seeing their body and the doctors and nurses trying to revive them, yet feel very peaceful while observing. Some report a realization that they may have actually died.
Later they develop a perception or a sensation of being pulled towards a type of destination. During the experience, they review their life from birth, until death, and interestingly this review is based upon their humanity.
They don’t review their lives based on what people strive for, like a career, promotions, or an amazing vacation. Their perspective is focused on their humanity. They notice incidents where they lacked dignity, acted inappropriately towards others, or conversely, acted with humanity and kindness.
They re-experience and relive these moments, but also, what’s fascinating, which sort of blows me away because I can’t really explain it, is they also describe these experiences from the other person’s perspective.
If they caused pain, they experience the same pain that other person felt, even if they didn’t realize it at the time. They actually judge themselves. They suddenly realize why their actions were good or bad, and many claim to see the downstream consequences of their actions.
How do studies of cardiac arrest inform the debate on the nature of consciousness?
Traditionally, researchers had proposed that mind or consciousness – our self – is produced from organized brain activity. However, nobody has ever been able to show how brain cells, which produce proteins, can generate something so different i.e. thoughts or consciousness. Interestingly, there has never been a plausible biological mechanism proposed to account for this.
Recently some researchers have started to raise the question that maybe your mind, your consciousness, your psyche, the thing that makes you, may not be produced by the brain. The brain might be acting more like an intermediary. It’s not a brand new idea. They have argued that we have no evidence to show how brain cells or connections of brain cells could produce your thoughts, mind or consciousness.
The fact that people seem to have full consciousness, with lucid well-structured thought processes and memory formation from a time when their brains are highly dysfunctional or even nonfunctional is perplexing and paradoxical.
I do agree that this raises the possibility that the entity we call the mind or consciousness may not be produced by the brain. It’s certainly possible that maybe there’s another layer of reality that we haven’t yet discovered that’s essentially beyond what we know of the brain, and which determines our reality.
So, I believe it is possible for consciousness to be an as of yet undiscovered scientific entity that may not necessarily be produced by synaptic activity in the brain.
The human body regenerates itself constantly, replacing old, worn-out cells with a continuous supply of new ones in almost all tissues. The secret to this perpetual renewal is a small but persistent supply of stem cells, which multiply to replace themselves and also generate progeny that can differentiate into more specialized cell types. For decades, scientists have tried to isolate and modify stem cells to treat disease, but in recent years the field has accelerated dramatically.
A major breakthrough came in the early 21st century, when researchers in Japan figured out how to reverse the differentiation process, allowing them to derive induced pluripotent stem (iPS) cells from fully differentiated cells. Since then, iPS cells have become a cornerstone of regenerative medicine. Researchers can isolate cells from a patient, produce iPS cells, genetically modify them to repair any defects, then induce the cells to form the tissue the patient needs regenerated.
On April 26, 2019, the New York Academy of Sciences and Takeda Pharmaceuticals hosted the Frontiers in Regenerative Medicine Symposium to celebrate 2019 Innovators in Science Award winners and highlight the work of researchers pioneering techniques in regenerative medicine. Presentations and an interactive panel session covered exciting basic research findings and impressive clinical successes, revealing the immense potential of this rapidly developing field.
Symposium Highlights
New cell lines should reduce the time and cost of developing stem cell-derived therapies.
The body’s microbiome primes stem cells to respond to infections.
iPS cell-derived therapies have already treated a deadly genetic skin disease and age-related macular degeneration.
Polyvinyl alcohol is a superior substitute for albumin in stem cell culture media.
A newly isolated type of stem cell reveals the stepwise process driving early embryo organization.
Speakers
Shinya Yamanaka Kyoto University
Shruti Naik New York University
Michele De Luca University of Modena and Reggio Emilia
Masayo Takahashi RIKEN Center for Biosystems Dynamics Research
Hiromitsu Nakauchi Stanford University and University of Tokyo
Brigid L.M. Hogan Duke University School of Medicine
Emmanuelle Passegué Columbia University Irving Medical Center
Hans Schöler Max Planck Institute for Molecular Biomedicine
Austin Smith University of Cambridge
Moderator: Azim Surani University of Cambridge
Sponsors
Recent Progress in iPS Cell Research Application
Speakers
Shinya Yamanaka Kyoto University
Highlights
Current protocols for using induced pluripotent stem (iPS) cells clinically are slow and expensive.
HLA “superdonor” iPS cell lines can be used to treat multiple patients, reducing costs.
A unique academic-industry partnership is helping iPS cell therapies reach the clinic.
Faster, Cheaper, Better
Shinya Yamanaka of Kyoto University, gave the meeting’s keynote presentation, summarizing his laboratory’s recent work using induced pluripotent stem (iPS) cells for regenerative medicine. The first clinical trial using iPS cells to treat age-related macular degeneration started five years ago. In his clinical trial, physicians isolated somatic cells from a patient, then used developed culture techniques to derive iPS cells from them. iPS cells can proliferate and differentiate into any type of cell in the body, which can then be transplanted back into the patient. Experiments over the past five years have shown that this approach has the potential to treat diseases ranging from age-related macular degeneration to Parkinson’s disease.
However, this autologous transplantation strategy is slow and expensive. “It takes up to a year just evaluating one patient, [and] it costs us almost one million US dollars,” said Yamanaka. Before transplanting the differentiated cells, the researchers evaluated the entire iPS cell derivation and iPS cell differentiation processes, adding to time and cost. As another strategy, Yamanaka’s team is working on the iPS Cell Stock for Regenerative Medicine. Here, iPS cells are derived from blood cells of healthy donors, not the patients, and are stocked. The primary problem with this approach is the human leukocyte antigen (HLA) system, which encodes multiple cell surface proteins. Each person has a specific combination of HLA genes, or haplotype, defining the HLA proteins expressed on their own cells. The immune system recognizes and eliminates any cell expressing non-self HLA proteins. Because there are millions of potential HLA haplotypes, cells derived from one person will likely be rejected by another.
The homozygous “superdonor” cell line has limited immunological diversity, allowing it to match multiple patients.
The homozygous “superdonor” cell line has limited immunological diversity, allowing it to match multiple patients.
To address that, Yamanaka and his colleagues are collaborating with the Japanese Red Cross to develop “superdonor” iPS cells. These cells carry homozygous alleles for different human lymphocyte antigen (HLA) genes, limiting their immunological diversity and making them match multiple patients. So far, the team has created four “superdonor” cell lines, allowing them to generate cells compatible with 40% of the Japanese population. Those cells are now being used in clinical trials treating macular degeneration and Parkinson’s disease, with more indications planned.
“So far so good,” said Yamanaka, but he added that “in order to cover 90% of the Japanese population we would need 150 iPS cell lines, and in order to cover the entire world we would need over 1,000 lines.” It took the group about five years to generate the first four lines, so simply repeating the process that many more times isn’t practical.
Instead, Yamanaka hopes to take the HLA reduction a step further, knocking out most of the major HLA genes to generate cells that will survive in large swaths of the population. However, simply knocking out entire families of genes isn’t enough. Natural killer cells attack cells that are missing particular cell surface antigens, so the researchers had to leave specific markers in the cells, or reintroduce them transgenically. Natural killer and T cells from various donors ignore leukocytes derived from these highly engineered iPS cells, proving that the concept works. With this approach, just ten lines of iPS cells should yield a range of donor cells suitable for any human HLA combination. Yamanaka expects these gene-edited iPS cells to be available in 2020.
By 2025, Yamanaka hopes to announce “my iPS cell” technology. This technology will reduce the cost and time for autologous transplantation to about $10,000 and one month per patient.
While preclinical and early clinical trials on iPS cells have yielded promising results, the new therapies must still cross the “valley of death,” the pharmaceutical industry’s term for the unsuccessful transition and industrialization of innovative ideas identified in academia to routine clinical use. In an effort to make that process more reliable, Yamanaka and his colleagues have begun a unique collaboration with Takeda Pharmaceutical Company Limited, Japan’s largest drug maker. The effort involves 100 scientists, 50 each from the company and academic laboratories. The corporate researchers gain access to the latest basic science developments on iPS cell technology, while the academics can use the company’s cutting-edge R&D know-how equipment and vast chemical libraries.
In one project, the collaborators used iPS cells to derive pancreatic islet cells, and then encapsulated the cells in an implantable device to treat type 1 diabetes. The system successfully decreased blood glucose in a mouse model, and the team is now scaling up cell production to test it in humans in the future. Another effort identified chemicals in Takeda’s compound library that speed cardiomyocyte maturation, which the researchers are now using to improve iPS cell-derived treatments for heart failure. In a third project, the team has modified iPS cell-derived T cells to identify and attack tumors, again showing promising results in a mouse model.
Inflammation causes persistent changes in epithelial stem cells, priming them for subsequent immune responses.
Modified iPS cells can be used to cure a patient with a deadly genetic skin defect.
A small population of self-renewing stem cells maintains human skin cells.
Sparring Partners
Shruti Naik, Early-Career Scientist winner of the 2019 Innovators in Science Award, discussed her work on epithelial barriers. These barriers, which include skin and the linings of the gut, lungs, and urogenital tract, exhibit nuanced responses to the many microbes they encounter. Injuries and pathogenic infections trigger prompt inflammatory responses, but the millions of harmless commensal bacteria that live on these surfaces don’t. How does the epithelium know the difference?
To ask that question, Naik first studied germ-free mice, which lack all types of bacteria. These animals have defective immune responses against pathogens that affect epithelia, so commensal bacteria are clearly required for developing normal epithelial immunity. Naik inoculated the germ-free mice with bacterial strains found either on the skin or in the guts of normal mice, then assessed their immune responses in those two compartments.
“When you gave gut-tropic bacteria, you were essentially able to rescue immunity in the gut but not the skin, and conversely when you gave skin-tropic bacteria, you were able to rescue immunity in the skin and not the gut,” said Naik. Even though the commensal bacteria caused no inflammation, they did activate certain T cells in the epithelia they colonized, apparently preparing those tissues for subsequent attacks by pathogens.
Next, Naik took germ-free mice inoculated with Staphylococcus epidermidis, a normal skin commensal bacterium, and challenged them with an infection by Candida albicans, a pathogenic yeast. The bacterially primed mice produced a much more robust immune response against the yeast infection than control animals that hadn’t gotten S. epidermidis. Naik confirmed that this immune training effect operates through the T cell response she’d seen before. “You essentially develop an immune arsenal to your commensals that helps protect against pathogens,” Naik explained, adding that each epithelial barrier requires its own commensal bacteria to trigger this response.
Augmented wound repair in post-inflammation skin reveals that naive and inflammation-educated skin stem cells respond differently to subsequent stresses.
Augmented wound repair in post-inflammation skin reveals that naive and inflammation-educated skin stem cells respond differently to subsequent stresses.
The response to epithelial commensals is remarkably durable; Naik found that the skin T cells in the inoculated mice remained on alert a year after their initial activation. That led her to wonder whether non-hematopoietic cells, especially epithelial stem cells, contribute to immunological memory in the skin.
To probe that, Naik and a colleague used a mouse model in which the topical drug imiquimod induces a temporary psoriasis-like skin inflammation. By tracing the lineages of cells in the animals’ skin, the researchers found that epithelial stem cells expand during this inflammation, and then persist. Challenging the mice with a wound one month after the inflammation resolves leads to faster healing than if the mice hadn’t had the inflammation. Several other models of wound healing yielded the same result. The investigators concluded that naive and inflammation-educated skin stem cells respond differently to subsequent stresses.
Naik’s team found that inflammation causes persistent changes in skin stem cells’ chromatin organization. Using a clever reporter gene assay, they demonstrated that the initial inflammation leaves inflammatory gene loci more open in the chromatin, making them easier to activate after subsequent insults. “What was really surprising to us was that this change never fully resolved,” said Naik. Even six months after the acute inflammation, skin stem cells retained the distinct post-inflammatory chromatin structure and the ability to heal wounds quickly. This chronic ready-for-action state isn’t always beneficial, though. Naik noticed that the mice that had had the inflammatory treatment were more prone to developing tumors, for example.
In establishing her new laboratory, Naik has now turned her focus to another aspect of epithelial immunity: the link between immune responses and tissue regeneration. She looked first at a type of T cells found in abundance around hair follicles on skin. Mice lacking these cells exhibit severe delays in wound healing, apparently as a result of failing to vascularize the wound area. That implies a previously unknown role for inflammatory T cells in vascularization, which Naik and her lab are now probing.
Skin Deep
Michele De Luca, Senior Scientist winner of the 2019 Innovators in Science Award, has developed techniques for regenerating human skin from transgenic epidermal stem cells. Researchers first isolated holoclones, or cells derived from a single epidermal stem cell, over 30 years ago. These cells can be used to grow sheets of skin in culture for both research and clinical use, but scientists have only recently begun to elucidate how the process works.
The first stem cell-derived therapies tested in humans were for skin and eye burns, allowing doctors to regenerate and replace burned epidermal tissue from a patient’s own stem cells. That’s the basis of Holoclar, a stem cell-based treatment for severe eye burns approved in Europe in 2015.
Holoclar and similar procedures work well for injured patients with normal epithelia. “We wanted to genetically modify those cells in order to address one of the most important genetic diseases in the dermatology field, which is epidermolysis bullosa (EB), a devastating skin disease,” said De Luca. In EB, patients carry a genetic defect in cell adhesion that causes severe blisters all over their skin and prevents normal healing. A large number of EB patients die as children from the resulting infections, and those who survive seldom get beyond young adulthood before succumbing to squamous cell carcinomas.
De Luca developed a strategy to isolate stem cells from a skin biopsy, repair the genetic defect in these cells with a retroviral vector, and then grow new skin in culture that can be transplanted back to the patient, replacing their original skin with genetically repaired skin. In 2015, the researchers carried out the procedure on a young boy named Hassan, who had arrived in the burn unit of a German hospital with EB after fleeing Syria. The burn unit was only able to offer palliative care, and his prognosis was poor because of his constant blistering and infections. De Luca’s team received approval to perform their gene therapy on him.
The new strategy, which combines cell and gene therapy, resulted in the restoration of normal skin adhesion in Hassan.
After isolating and modifying epidermal stem cells from Hassan and growing new sheets of skin in culture, De Luca’s team re-skinned the patient’s arms and legs, then his abdomen and back. The complete procedure took about three months. The new skin resists blister formation even when rubbed and heals normally from minor wounds. In the ensuing three and a half years, Hassan has begun growing normally and living an ordinary, healthy life.
Detailed analysis of skin biopsies showed that Hassan’s epidermis has normal cellular adhesion machinery and revealed that his skin is now derived from a population of proliferating transgenic stem cells, with no single clone dominating. By tracing the lineages of cells carrying the introduced transgene, De Luca was able to identify self-renewing transgenic stem cells, intermediate progenitor cells, and fully differentiated stem cells, indicating normal skin growth and replacement.
Besides being good news for the patient, the results confirmed a longstanding theory of skin regeneration. “These data formally prove that the human epidermis is sustained only by a small population of long-lived stem cells that generates [short-lived epithelial] progenitors,” said De Luca, adding that “with this in mind, we’ve started doing other clinical trials.”
The researchers plan to continue targeting junctional as well as dystrophic forms of EB, both of which are genetically distinct from EB simplex. Initial experiments revealed that in these conditions, transplant recipients developed mosaic skin, where some areas continued to be produced from cells lacking the introduced genetic repair. The non-transgenic cells appeared to be out-competing the transgenic cells and supplanting them, undermining the treatment. De Luca and his colleagues developed a modified strategy that gave the transgenic cells a competitive advantage. This approach and additional advances should allow them to achieve complete transgenic skin coverage.
The Journal of Investigative Dermatology. 2017;137(4):836-44.
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Good for What Ails Us
Speakers
Masayo Takahashi RIKEN Center for Biosystems Dynamics Research
Hiromitsu Nakauchi Stanford University and University of Tokyo
Highlights
The first clinical use of iPS cells in humans replaced retinal cells in a patient with age-related macular degeneration.
“Superdonor” stem cells can evade immune rejection in multiple patients.
Culturing hematopoietic stem cells has been an ongoing challenge for immunologists.
Polyvinyl alcohol, used in making school glue, is a superior substitute for bovine serum albumin in stem cell culture media.
Large doses of hematopoietic stem cells may obviate the need for immunosuppression in stem cell therapy.
An iPS Cell for an Eye
Masayo Takahashi, of RIKEN Center for Biosystems Dynamics Research, began her talk with a brief description of the new Kobe Eye Center, a purpose-built facility designed to house a complete clinical development pipeline dedicated to curing eye diseases. “Not only cells, not only treatments, but a whole care system is needed to cure the patients,” said Takahashi. In keeping with that philosophy, the Center includes everything from research laboratories to a working eye hospital and a patient welfare facility.
Takahashi’s recent work has focused on treating age-related macular degeneration (AMD). In AMD, the retinal pigment epithelium that nourishes other retinal cells accumulates damage, leading to progressive vision loss. AMD is the most common cause of serious visual impairment in the elderly in the US and EU, and there is no definitive treatment. Fifteen years ago, Takahashi and her colleagues derived retinal pigment epithelial cells from monkey embryonic stem cells and successfully transplanted them into a rat model of AMD, treating the condition in the rodents. They were hesitant to extend the technique to humans, though, because it required suppressing the recipient’s immune response to prevent them from rejecting the monkey cells.
The advent of induced pluripotent stem (iPS) cell technology pointed Takahashi toward a new strategy, in which she took cells from a patient, derived iPS cells from them, and then prompted those cells to differentiate into retinal pigment epithelial cells that were perfectly compatible with the patient’s immune system. Her team then transplanted a sheet of these cells into the patient. That experiment, in 2014, was the first clinical use of iPS cells in humans. “The grafted cells were very stable,” said Takahashi, who has checked the graft in multiple ways in the ensuing years.
Having proven that iPS cell-derived retinal grafts can work, Takahashi and her colleagues sought to make the procedure cheaper and faster. Creating customized iPS cells from each patient is a huge undertaking, so instead the team investigated superdonor iPS cells that can be used for multiple patients. These cells, described by Shinya Yamanaka in his keynote address, express fewer types of human leukocyte antigens than most patients, making them immunologically compatible with large swaths of the population. Just four lines of superdonor iPS cells can be used to derive grafts for 40% of all Japanese people.
Transplantation of an iPS cell-derived sheet into the retina ultimately proved successful.
Transplantation of an iPS cell-derived sheet into the retina ultimately proved successful.
In the next clinical trial, Takahashi’s lab performed several tests to confirm that the patients’ immune cells would not react with the superdonor cells, before proceeding with the first retinal pigment epithelial graft. Nonetheless, after the graft the researchers saw a minuscule fluid pocket in the patient’s retina, apparently due to an immune reaction. Clinicians immediately gave the patient topical steroids in the eye to suppress the reaction. “Then after three weeks or so, the reaction ceased and the fluid was gone, so we could control the immune reaction to the HLA-matched cells,” said Takahashi. Four subsequent patients showed no reaction whatsoever to the iPS superdonor-derived grafts.
While the retinal grafts were successful, none of the patients have shown much improvement in visual acuity so far. Takahashi explained that subjects in the clinical trial all had very severe AMD and extensive loss of their eyes’ photoreceptors. “I think if we select the right patients, we could get good visual acuity if their photoreceptors still remain,” said Takahashi.
Takahashi finished with a brief overview of her other projects, including using aggregates of iPS cells and embryonic stem cells to form organoids, which can self-organize into a retina. She hopes to use this system to develop new therapies for retinitis pigmentosa, another major cause of vision loss. Finally, Takahashi described a project aimed at reducing the cost and increasing the efficacy of stem cell therapies even further by employing a sophisticated laboratory robot. The system, called Mahoro, is capable of learning techniques from the best laboratory technicians, then replicating them perfectly. That should make stem cell culturing procedures much more reproducible and significantly reduce the cost of deploying new therapies.
A Sticky Problem
Hiromitsu Nakauchi, of Stanford University and the University of Tokyo, described his group’s efforts to overcome a decades-old challenge in stem cell research. Scientists have known for over 25 years that all of the blood cells in a human are renewed from a tiny population of multipotent, self-renewing hematopoietic stem cells. In an animal that’s had all of its hematopoietic lineages eliminated by ionizing radiation, a single such cell can reconstitute the entire blood cell population. This protocol is the basis for several experimental models.
In theory, then, a single hematopoietic stem cell should also be able to multiply indefinitely in pure culture, allowing researchers to produce all types of blood cells on demand. In practice, cultured stem cells inevitably differentiate and die off after just a few generations in culture. Nakauchi and his colleagues have been trying to fix that problem. “After years of hard work, we decided to take the reductionist approach and try to define the components that we use to culture [hematopoietic stem cells],” said Nakauchi.
The team focused on the most undefined component of their culture media: bovine serum albumin (BSA). This substance, a crude extract from cow blood, has been considered an essential component of growth media since researchers first managed to culture mammalian cells. However, Nakauchi’s lab found tremendous variation between different lots of BSA, both in the types and quantities of various impurities in them and in their efficacy in keeping stem cells alive. Worse, factors that appeared to be helpful to the cells in some BSA lots were harmful when present in other lots. “So this is not science; depending on the BSA lot you use, you get totally different results,” said Nakauchi.
Next, the researchers switched to a recombinant serum albumin product made in genetically engineered yeast. That exhibited less variation between lots, and after optimizing their culture conditions they were able to grow and expand hematopoietic stem cells for nearly a month. Part of the protocol they developed was to change the medium every other day, which they found was required to remove inflammatory cytokines and chemokines being produced by the stem cells. That suggested the cells were still under stress, perhaps in response to some of the components of the recombinant serum albumin.
Polyvinyl alcohol can replace BSA in culture medium.
Polyvinyl alcohol can replace BSA in culture medium.
The ongoing problems with serum albumin products led Nakauchi to ask why albumin is even necessary in tissue culture. Scientists have known for decades that cells don’t grow well without it, but why not? While trying to figure out what the albumin was doing for the cells, Nakauchi’s lab tested it against the most inert polymer they could find: polyvinyl alcohol (PVA). Best known as the primary ingredient for making school glue, PVA is also used extensively in the food and pharmaceutical industries. To their surprise, hematopoietic stem cells grew better in PVA-spiked medium than in medium with BSA. The PVA-grown cells showed decreased senescence, lower levels of inflammatory cytokines, and better growth rates.
In long-term culture, Nakauchi and his colleagues were able to achieve more than 900-fold expansion of functional mouse hematopoietic stem cells. Transplanting these cells into irradiated mice confirmed that the cells were still fully capable of reconstituting all of the hematopoietic lineages. Further experiments determined that PVA-containing medium also works well for human hematopoietic stem cells.
Besides having immediate uses for basic research, the ability to grow such large numbers of hematopoietic stem cells could overcome a fundamental barrier to using these cells in the clinic. Current hematopoietic stem cell therapies require suppressing or destroying a patient’s existing immune system to allow the transplanted cells to become established, but this immunosuppression can lead to deadly infections. Transplanting a much larger population of stem cells can overcome the need for immunosuppression, but growing enough cells for this approach has been impractical. Using their new culture techniques, Nakauchi’s team can now produce enough hematopoietic stem cells to carry out successful transplants without immunosuppression in mice. They hope to take this approach into the clinic soon.
Brigid L.M. Hogan Duke University School of Medicine
Emmanuelle Passegué Columbia University Irving Medical Center
Hans Schöler Max Planck Institute for Molecular Biomedicine
Austin Smith University of Cambridge
Moderator: Azim Surani University of Cambridge
Highlights
A dramatic transition separates early embryonic stem cells from their descendants.
Newly isolated formative stem cells represent an intermediate step in development.
Organoids derived from iPS cells provide excellent models for studying human physiology and disease.
In the Beginning
Austin Smith, from the University of Cambridge, gave the final presentation, in which he discussed his studies on the progression of embryonic stem cells through development. In mammals, embryonic development begins with the formation of the blastocyst. In 1981, researchers isolated cells from murine blastocysts and demonstrated that each of them can grow into a complete embryo. Stem cells isolated after the embryo has implanted itself into the uterus, called epiblast stem cells, have lost that ability but gained the potential to differentiate into multiple cell lineages in culture. “So we have two different types of pluripotent stem cells in the mouse, and they’re different in just about every way you could imagine,” said Smith.
Work on other species, including human cells, suggests that this transition between two different types of stem cells is a common feature of mammalian development. The transition from the earlier to the later type of stem cell is called capacitation. To find the factors driving capacitation, Smith and his colleagues looked for differences in gene transcription patterns and chromatin organization during the process, in both human and murine cells. What they found was a global re-wiring of nearly every aspect of the cell’s physiology. “Together these things lead to the acquisition of both germline and somatic lineage competence, and at the same time decommission that extra-embryonic lineage potential,” Smith explained.
Having characterized the cells before and after capacitation, the researchers wanted to isolate cells from intermediate stages of the process to understand how it unfolds. To do that, they extracted cells from mouse embryos right after implantation, then grew them in culture conditions that minimized their exposure to signals that would direct them toward specific lineages. Detailed analyses of these cells, which Smith calls formative stem cells, shows that they have characteristics of both the naive embryonic stem cells and the later epiblast stem cells. Injecting these cells into mouse blastocysts yields chimeric mice carrying descendants of the injected cells in all their tissues. The formative stem cells can therefore function like true embryonic stem cells, albeit less efficiently.
The developmental sequence of pluripotent cells.
The developmental sequence of pluripotent cells.
Post-implantation human embryos aren’t available for research, but Smith’s team was able to culture naive stem cells and prompt them to develop into formative stem cells. These cells exhibit transcriptional profiles and other characteristics homologous to those seen in the murine formative stem cells.
Having found the intermediate cell type, Smith was now able to assemble a more detailed view of the steps in development. Returning to the mouse model, he compared the chromatin organization of naive embryonic, formative, and epiblast stem cells. The difference between the naive and formative cells’ chromatin was much more dramatic than between the formative and epiblast cells.
Based on the results, Smith proposes that naive embryonic stem cells begin as a “blank slate,” which then undergoes capacitation to become primed to respond to later differentiation signals. The capacitation process entails a dramatic change in the cell’s transcriptional and chromatin organization and occurs around the time of implantation. “We think we now have in culture … a cell that represents this intermediate stage and that has distinctive functional properties and distinctive molecular properties,” said Smith. After capacitation, the formative stem cells undergo a more gradual shift to become primed stem cells, which are the epiblast stem cells in mice.
Smith concedes that the human data are less detailed, but all of the experiments his team was able to do produced results consistent with the mouse model. Other work has also found corroborating results in non-human primate embryos, implying that the same developmental mechanisms are conserved across mammals.
Organoid Recitals
After the presentations, a panel consisting of members of the Innovators in Science Award’s Scientific Advisory Council and Jury took the stage to address a series of questions from the audience.
The panel first took up the question of how researchers can better study human stem cells, given the ethical challenges of working with embryos. Brigid Hogan described organoid cultures, in which researchers stimulate human iPS cells to grow into minuscule organ-like structures. “This is a way of looking at human development at a stage when it’s [otherwise] completely inaccessible,” said Hogan. Other speakers concurred, adding that implanting human organoids into mice provides an especially useful model.
Another audience member asked about the potential for human stem cell therapy in the brain. Hogan pointed to the use of fetal cells for treating Parkinson’s disease as an example, but panelist Hans Schöler suggested that that could be a unique case. Patients with Parkinson’s disease suffer from deficiency in dopamine-secreting neurons, so implanting cells that secrete dopamine in the correct brain region may provide some relief.
Panelists also addressed the use of stem cells in regenerative medicine, where researchers are targeting the nexus of aging, nutrition, and brain health. Emmanuelle Passegué explained that the body’s progressive failure to regenerate itself from its own stem cells is a hallmark of aging. “I think we are getting to an era where transplantation or engraftment [of cells] will not be the answer, it will really be trying to reawaken the normal properties of the [patient’s own] stem cells,” said Passegué.
As the meeting concluded, speakers and attendees seemed to agree that the field of stem cell research, like the cells themselves, is now poised to develop in a wide range of promising directions.
Motivated to help his son battle a rare genetic disorder known as NGLY1, this researcher has transition from the field of computer science to biology and drug development.
Matthew Might, Director, Hugh Kaul Precision Medicine Institute at the University of Alabama at Birmingham is a computer scientist by training but now works in the field of drug repositioning. His research is dedicated to finding new therapeutic purposes for already existing drugs. Recently he sat down to discuss his work.
How would you describe your work?
As a researcher I’m focused directly on scaling up the process of drug repositioning for specific rare and medically complex patients. I’m investing heavily in the creation of bioinformatics infrastructure that allows us to repurpose drugs and implement a workflow that can guide patients through the identification of a therapy.
What brought you to the field of drug repositioning?
My shift from computer science to medicine was motivated entirely by my son Bertrand. He is the first discovered case of an ultra-rare genetic disorder known as NGLY1 deficiency. The lack of any available treatments inspired me to look for existing medications that could be repurposed to treat his disease. Over time I was able to use a variety of techniques to find three different compounds with some therapeutic potential for his disorder.
How difficult was transitioning from computer science to medicine?
The transition was gradual. Initially, I learned about genetics, and then I began reading about glycobiology, which is an interesting way to learn biology. After learning enough glycobiology and about metabolism, I made some predictions about what Bertrand might be deficient in. That led to the discovery of the first natural product that serves as a therapy for his disorder.
Six years was the total time it took to go from computer science to having enough biology to make a reasonable therapy prediction. I wouldn’t have done this without the personal motivator, but I think this is not just the right time to have more computer science in biology. It’s the right time to have more computer scientists in biology.
When looking for drug repositioning candidates do you identify a disease or treatment first?
Matthew Might
We start with the disease. When a patient comes in with a rare monogenic disease, we ask four questions about the gene: ‘Is the gene overactive? Is it underactive? Is activity absent? Or has its activity become toxic?’ These answers tell us what direction to go.
The two most common directions are genes that have a gain of function or partial loss of function. We’re going to be manipulating the gene that has a variant. The tools we’ve developed are capable of answering questions like, ‘Given a gene, how do I modulate its activity up or down through any mechanism whatsoever?’ A lot of what we do is harvesting data sets that enable us to answer that specific question better.
What’s the biggest challenge in identifying drugs to be repositioned?
From a bioinformatics perspective the challenge we face is that only a small fraction of genetic targets have a candidate drug known to hit them. This isn’t a limitation of the drugs, it’s a limitation of our knowledge about them. If I could have the NIH fund one experiment, it would take all approved drugs and do an exhaustive transcription against many cell lines. That would show the impact of every drug on every cell type, on every gene. If we had that database, we could do much more drug repurposing.
How do you get these treatments to patients?
Once we’ve found a target, we will do whatever we can computationally to find a compound that might modulate the target. If we get lucky and we hit it computationally, we will end up generating a research report. We do this when we find an approved compound, or a natural product that’s already available.
The report is a factual summary of the information we’ve discovered about the compound and includes research papers that back this up. We turn that report over to the patient’s treating physician. At that point, it’s up to the treating physician to decide what to do.
While the development of vaccines against infectious diseases has had a profound impact on life expectancy, there remain many resistant and emerging infections for which no effective vaccines are available, such as malaria, HIV, and Zika. Recent advances in biotechnology and our understanding of human immunity hold great promise for conquering new diseases. For example, advances in structural biology allow for the discovery of new antigens that can target broad viral families, such as influenza, or complex parasites like malaria. Novel clinical trials for maternal immunizations have shown encouraging results for reducing dangerous diseases in newborn infants. Furthermore, recent progress in DNA- or RNA-based vaccines holds promise for inexpensive and fast production, which is especially favorable for responding to emerging epidemics. Learn more about recent breakthroughs in vaccine development in this summary of our May 20, 2019 symposium, which gathered the world’s leaders in vaccine development.
Symposium Highlights:
Emerging infectious diseases can be treated quickly with a passive vaccine containing human monoclonal antibodies isolated from the blood of an infected patient.
Targeting multiple stages of the malaria life cycle is a promising strategy for the development of a successful vaccine targeting this complex parasite.
Clinical trials show promise for maternal immunizations in protecting newborn infants from respiratory syncytial virus (RSV) and Group B streptococcus.
A vaccine containing the influenza hemagglutinin (HA) fusion protein without the head domain can elicit protection against a broad group of influenza viruses.
Synthetic DNA and mRNA vaccines are simple to manufacture and show promise for treating a wide range of diseases, including Ebola, HIV, Zika, influenza, and malaria.
A promising new adjuvant, AS01, has contributed to breakthrough vaccines for Malaria, tuberculosis, and shingles.
Speakers
James E. Crowe, Jr., MD Vanderbilt University Medical Center
New Approaches for Understanding the Immune System for Vaccine Development
Speaker
James E. Crowe, Jr. Vanderbilt University Medical Center
Human Antibodies and Repertoires for Emerging Infectious Diseases
James Crowe, of Vanderbilt University Medical Center, discussed his lab’s work developing treatments for emerging infectious diseases using monoclonal human antibodies. “Antibodies essentially are a passive vaccine,” explained Crowe. Currently, it takes about two years to develop a vaccine for an infectious disease agent, which is not quick enough for outbreak response. Therefore, Crowe argues that antibodies are the “most appropriate public health measure for most emerging infections.” Crowe’s group is working on two strategies for developing human antibody drugs: one focuses on speed, whereas the other aims to develop broad antibodies ahead of an outbreak.
The Rapid Rational Antibody Design and Delivery (RRADD) project uses ultra-fast techniques to respond to a specific outbreak in the moment. They recently used Zika as a test case. Starting with a blood sample from a surviving patient, their facility used single-cell RNA-sequencing to produce a list of antibody genes within a day. These antibodies were quickly produced and then tested in a high-throughput real-time cell culture system to assay for protection against Zika infection. Leading candidates were tested in mouse and primate models, leading to the discovery of protective antibodies within 78 days.
Illustrations of antibody (Ab)- antigen (Ag) complexes for human monoclonal antibodies (mABs) recently discovered in the AHEAD100 project.
The second strategy is the Advanced Human Epidemic Antibody Defenses (AHEAD100) project, a methodical approach that aims to develop antibodies for the 100 most likely infectious diseases ahead of any future outbreaks. Interestingly, they found broad antibodies that work across viruses of a related class, such as noroviruses, alphaviruses, and flu.
Taking on the Big Challenges Facing Novel Vaccine Development
Adrian Hill University of Oxford
Wayne Koff Human Vaccines Project
New Generation Malaria Vaccines
Adrian Hill from the University of Oxford presented his work on the development of a malaria vaccine. Malaria causes 500,000 deaths each year, but developing an effective vaccine is challenging. “Even if you get a good antigen, you need remarkably high immunogenicity,” Hill explained. Therefore, Hill’s group aims to develop a vaccine that targets multiple stages of the malaria parasite life cycle.
In the first stage, mosquitos introduce malaria sporozoites into a human host. Hill’s group and others have been developing vaccines that combine malaria antigens with virus-like particles to induce antibody production against sporozoites. Hill and colleagues are developing R21, a more potent version of the RTS,S vaccine currently in Phase III trials. In R21, 100% of the molecules encode the sporozoite antigen. Studies show that this formulation allows for a lower dose, as antibody titers are indistinguishable between a 10 µg dose of R21 and a 50 µg dose of RTS,S. Furthermore, R21 shows a more durable response, with higher titers at six months versus RTS,S. By 2020, they expect efficacy results from the first Phase IIB trial.
As the malaria life cycle progresses, sporozoites infect liver cells, where the parasite matures. “[To target] the liver stage, you need T-cells” said Hill. Inducing T-cells requires a viral vector approach. Research on mice and clinical studies from Hill’s group show that the ME-TRAP antigen viral vector can induce high levels of resident memory T-cells in the liver. There are ongoing field clinical trials for this vaccine.
The Future of Vaccine Development
Wayne Koff, the president and CEO of the Human Vaccines Project, described the nonprofit’s research decoding the human immune system. Vaccines for complex infectious and non-communicable diseases such as HIV, tuberculosis, and cancer have been difficult to develop. Koff believes that a better understanding of human immunity is essential for accelerating vaccine development for these diseases.
One strategy is to investigate why some people respond to vaccines and infections much better than others. “If we can understand this, we can get at the pathogens we haven’t been able to tackle,” said Koff. Recent developments in single cell multi-omics allow for an in-depth analysis of an individual’s immune system. A growing body of evidence suggests that immunity biomarkers at baseline can predict an individual’s response to immunization. Researchers performed single cell RNA-sequencing on innate immune cells before immunization and successfully identified biomarkers predictive of the response to the Hepatitis B vaccine. By integrating all of the pre-immunization data, investigators could build biostatistical models that accurately predicted final antibody titers, while revealing pathways that may be involved in the response mechanism.
This data suggests that “we all have an immune set point,” said Koff, which leads to the opportunity to modulate this set point before immunization to improve outcomes. Furthermore, smaller trials that account for individual variability and assay predictive signatures may be more effective than standard large vaccine efficacy trials.
Kathrin Jansen from Pfizer discussed recent advances in maternal immunization. Infants under six months are the most vulnerable to infection, but most vaccines are not available at this early stage of life. Furthermore, “20% of stillbirths seem to be associated with an infectious disease,” said Jansen. Active antibody transfer from mother to baby during pregnancy is an essential mechanism for protecting infants from infectious diseases. The goal of maternal immunization is to enhance maternal antibody levels to further protect newborns. Jansen explained that these vaccines could either “augment pre-existing antibody responses or induce a de novo response” to infections the mother has not yet been exposed to.
Jansen presented recent findings for maternal vaccines targeting respiratory syncytial virus (RSV) and Group B streptococcus bacteria, two infections that are especially deadly for newborn infants. In a recent Phase I/II trial, the Group B streptococcus vaccine induced high levels of antibody titers for up to six months in healthy adults, giving confidence to move forward for testing in pregnancy. Recent structural biology studies of RSV identified a metastable form of the viral fusion protein. With this form in mind, a screen for vaccine candidates revealed molecules that were 30 times more powerful than the current licensed prophylactic antibody in rodents. Data from a Phase I/II study will be available later this year.
Protecting Infants from RSV via Maternal Immunization
Greg Glenn, of Novavax, presented recent progress on the development of an RSV maternal vaccine. RSV is the leading cause of hospitalization of infants in the United States. While the Pfizer version of the vaccine, described by Kathrin Jansen, resembles the metastable prefusion form of the viral fusion protein, the Novavax version targets an earlier, stable form known as the prefusogenic form. This vaccine contains a near full-length fusion protein, but with deletions in a furin cleavage site. “These deletions fix the protein structure, and that allows it to be very stable,” Glenn explained. Through stabilizing the prefusogenic form, the virus is prevented from successfully infecting cells, which allows the vaccine to be produced in culture with higher yields. Furthermore, all antibodies that target the metastable prefusion form also target the prefusogenic form. Immunization with the Novavax vaccine induces antibodies to a variety of viral epitopes, which are also transferred to the infant.
Schematic showing the different forms of the fusion (F) protein of the RSV virus.
Currently, Novavax is running a worldwide Phase III randomized placebo-controlled trial to evaluate protection of infants against RSV with their maternal vaccine. The vaccine was given “to immunized mothers in third trimester, and we monitored infants intensely for six months,” explained Glenn. The trial showed a 40% reduction in their primary endpoint, which was medically significant RSV lower respiratory tract infection at 90 days old.
Next Generation Vaccines to Eliminate Congenital Cytomegalovirus: We are halfway there
Sallie Permar, of Duke University, shared her work developing an effective vaccine for congenital cytomegalovirus (CMV), which is the most common congenital infection and cause of birth defects worldwide. Developing a vaccine has been tricky, as it’s unknown exactly what maternal immune responses are protective against congenital CMV transmission. Permar’s group is investigating these questions with a novel, non-human primate model as well as data analysis from previous vaccine trials.
Permar and colleagues infected seronegative rhesus monkeys with CMV at the beginning of pregnancy. “We used a model of severe pathology with maternal CD4+ T-cell depletion followed by an intravenous inoculation to ask whether antibodies alone could be protective against congenital CMV transmission,” explained Permar. Data from a small group of animals suggests that treatment with passive antibodies from donor plasma prior to inoculation prevents fetal transmission. This result indicates that stimulating potent antibody responses could be a promising route to an effective maternal CMV vaccine.
Previous trials of a vaccine containing glycoprotein B, the main fusion protein of the virus, have shown partial effectiveness. Permar’s group probed the trial data to investigate what immune responses correlate with protection against CMV in infected versus uninfected vaccine recipients. “The ability of vaccine-elicited antibodies to bind to glycoprotein B-transfected cells was higher in uninfected vaccinees,” said Permar, suggesting that eliciting antibodies that bind to glycoproteins is a promising vaccine target. Furthermore, the infected group of vaccine recipients was still protected against specific CMV strains, suggesting that a broader immunogen might be more effective.
Guiding Vaccine Candidates: Antibodies That Can Neutralize Influenza and Malaria
Ian Wilson, from the Scripps Research Institute, shared his recent work investigating the structural biology of antibodies to guide vaccine candidates for influenza and malaria. Wilson’s group aims to “design immunogens or even small molecules from the structural information about how antibodies bind.”
Human antibodies that neutralize a broad range of flu subtypes have been characterized in the last ten years. Interestingly, the broadest antibodies bind to the less immunogenic “stem” domain of the influenza hemagglutinin (HA) fusion protein, rather than the “head” domain. “We are using this information to try to think of novel vaccines,” said Wilson. “If we chop off the immunogenic head, then we can target the response against the stem.” Indeed, a recently developed headless HA construct elicited protection against all influenza A group 1 antibodies in mice and monkeys.
Wilson’s group has also probed the structural biology of human antibodies elicited in recent RTS,S malaria vaccine trials. Cryo-EM revealed the structure of antibodies binding to the circumsporozoite protein (CSP) of malaria: the antibodies spiral all the way around the NANP peptide repeats of the protein. Furthermore, antibodies in the spiral bind in close proximity, and often, somatic mutations strengthen these homotypic contacts for a more stable spiral. Future work will explore the relevance of this spiral structure for vaccine purposes.
Synthetic DNA Approaches for Difficult Infectious Disease Targets
David Weiner, of the Wistar Institute, presented recent findings on the development and efficacy of synthetic DNA vaccines. DNA vaccines are “very consistent, very simple to manufacture, temperature stable,” and allow for local transfection without systemic expression, explained Weiner. Recent early stage clinical trials have shown promising results for using synthetic DNA vaccines as immunotherapy to treat human papillomavirus (HPV)-related cancers. Synthetic DNA is also promising for treating emerging infectious diseases. Wiener discussed three examples, Ebola, MERS, and Zika, where prophylactic treatment with synthetic DNA induced a 95%–100% response rate, and transmission into the clinic occurred in only 7–15 months.
Weiner also discussed his group’s work developing a DNA-encoded monoclonal antibody (dMAb) platform. Muscle or skin tissue “is transfected and becomes a factory for expression of the protein. The idea is getting [the antibody] secreted into the bloodstream at detectable levels,” said Weiner. They have developed dMAbs targeting Ebola, HIV, and Zika that induce robust antibody expression and viral protection in animal models. For HIV, multiple dMAbs can be delivered at one time, which has been shown to induce broad neutralizing titers against nine HIV subtypes in non-human primates.
Weiner and collaborators are also working to engineer DNA cassettes that encode self-assembling nanoparticles directly in vivo. Nanoparticles targeting HIV showed improved immune responses versus the monomeric form: “It’s dose sparing, it’s much faster seroconversion and much higher titers, and it elicits very good CD8+ T-Cells,” Weiner said.
mRNA Vaccines: A New Era in Vaccinology
Drew Weissman, of the University of Pennsylvania, discussed recent advances in the development of mRNA vaccines for infectious diseases. Why use RNA? In theory, the cost of mRNA production would be much less than that of protein, which requires large-scale cell culture followed by purification that differs for every protein. Weissman’s group developed a platform using nucleoside-modification and purification techniques to optimize mRNA structures that induce high and long-lived translation when delivered within lipid nanoparticles to peripheral sites.
Mice vaccinated with the A/Cal/7/2009 HA mRNA vaccine challenged with the distant flu virus H5N1 showed full protection. These results suggest immunization with HA mRNA could result in a universal flu vaccine.
Weissman discussed mRNA vaccines developed with their platform targeting influenza, HSV-2, HIV, and malaria, which have all shown promising results in animal models. For influenza, a single immunization with an mRNA vaccine coding for the hemagglutinin (HA) fusion protein in mice resulted in titers 50 times higher than the current FDA approved vaccine. As a mechanism of action, they found that the lipid nanoparticles used for vaccine delivery induce T- follicular helper cells, which drive long-term immune memory and are “critical in the induction of potent antibody responses,” explained Weissman. Furthermore, their mRNA vaccines induce responses to subdominant epitopes in the presence of dominant epitopes, which isn’t seen with whole proteins. This response is useful because subdominant epitopes, such as the HA stem domain, can be broadly cross-reactive across viral subtypes. Vaccinated mice challenged with distant flu viruses were fully protected, “suggesting that using a full HA could give you a universal vaccine,” said Weissman.
Transforming New Technologies into Vaccines: Genomics, Adjuvants and Self-Amplifying RNAs
Rino Rappuoli, of GlaxoSmithKline, shared how new technologies will allow us to conquer new diseases. Recent advances have allowed for major improvements in reverse vaccinology — using human genomics and structural biology to discover new antigens and instruct vaccine design. “Today we have the tools of synthetic biology,” said Rappuoli. At GSK, “we are using self-amplifying mRNA instead of simple mRNA. We use the replicon of the alphavirus to amplify the RNA and give a better response.” Nucleic acid vaccines work well in animal models, and the challenge now is testing whether it will work well in humans.
Rappuoli also discussed encouraging new advances in antigen delivery using nanoparticles or Generalized Modules for Membrane Antigens (GMMA). While self-assembling natural nanoparticles have been around for years, fully synthetic nanoparticles have only recently been designed. “We are going from mimicking nature to completely computationally designing vaccines,” explained Rappuoli. GMMAs consist of outer membrane vesicles from bacteria, which are engineered to release these vesicles in large quantities with the desired antigens. Rappuoli also highlighted recent developments in adjuvants, substances within vaccines that enhance the immune response to antigens. A promising new adjuvant, AS01, has contributed to breakthrough vaccines for Malaria, tuberculosis, and shingles. Moving forward, Rappuoli aims to use these new technologies to target vaccines for the elderly, emerging infections, and antimicrobial resistance.
“Optimized degrader molecules will have fast rates of degradation and relatively short exposure with therapeutic doses that result in complete elimination of the target protein, which can result in a more durable and deeper effect.”
Published July 23, 2019
By Robert Birchard
Eric Fischer, PhD
Around 80% of disease-causing proteins, including key drivers of many cancers and other serious neurological conditions like Alzheimer’s disease, cannot be targeted by currently available therapeutics. These so called “undruggable” proteins lack specific surface areas required for treatments such as small molecule inhibitors or antibodies to bind with disease causing proteins and modulate their function.
However, an alternative therapeutic strategy known as targeted protein degradation has shown the potential to remedy these “undruggable” proteins. Utilizing small molecules known as PROTACs, this strategy harnesses the cell’s waste disposal system to promote the destruction of disease-causing proteins. Dr. Eric Fischer, Assistant Professor of Biological Chemistry and Molecular Pharmacology at Harvard Medical School, recently sat down with us to help create this primer on PROTACs, and their potential implications for treating disease.
What are PROTACs?
PROteolysis TArgeting Chimeras, or PROTACS for short, are two separate molecules bound together to form a two headed molecule. One end binds to an ubiquitin ligase, while the other end binds to the “undruggable” protein targeted by pharmacologists. In illustrations, PROTACs are often depicted as dumbbells, but it may be more helpful to think of them as flexible harnesses.
How do PROTACs work?
PROTACs are designed to take advantage of the cell’s waste disposal system that removes unneeded proteins. This system, known as the proteasome, is important for the cell to remove unneeded or damaged proteins and recycle their building blocks to make new proteins. The proteasome plays critical roles in cell growth, management of cellular stress, and in the immune system. One end binds to the targeted proteins, while the other end of the molecule binds to the ubiquitin ligase, which then marks the targeted protein for destruction. This lets the cell’s proteasome know that this specific protein can be destroyed. In this way the body’s regularly occurring mechanisms are co-opted to destroy disease-causing proteins.
Optimized degrader molecules will have fast rates of degradation and relatively short exposure with therapeutic doses that result in complete elimination of the target protein, which can result in a more durable and deeper effect.”
Eric Fischer, PhD
What makes PROTACs so unique?
Most therapies are divided between small molecule inhibitors or therapeutic antibodies/biologics. However, “PROTACs are small molecules and as such not restricted to targeting surface proteins, however, in contrast to traditional small molecule inhibitors, PROTACs are fundamentally different,” explained Dr. Fischer, “While inhibitors need to achieve an almost perfect degree of target engagement over an extended period of time to exert their pharmacologic effect, PROTACs follow more of a hit and run strategy.”
“Optimized degrader molecules will have fast rates of degradation and relatively short exposure with therapeutic doses that result in complete elimination of the target protein, which can result in a more durable and deeper effect,” he explained. “More importantly, however, small molecule degraders completely eliminate the disease-causing protein and as such can target the non-catalytic activity of enzymes but also scaffolding proteins, and other non-enzymatic targets.”
When will PROTACs be more widely available?
While researchers have demonstrated the potential of PROTACs in the lab, the first clinical trials are just opening. Still Dr. Fischer is very optimistic, “The technology has rapidly spread, and we can expect to see many more programs entering clinical development. Due to the pioneering work of a growing academic community spearheading this field, the concepts underlying protein degradation are largely public domain and widely available.”
What is the future of PROTACs research?
“I believe the field of targeted protein degradation is here to stay and will significantly expand our repertoire of therapeutic modalities,” said Dr. Fischer. “I also believe it is still in its infancy and many challenges lie ahead of us to broadly deploy this to the more challenging targets.” PROTACs could potentially prove the impossible is possible by allowing scientists to destroy disease-causing proteins that were previously considered beyond their reach.
Sthuthi Satish is exploring her interest in a variety of STEM fields through her participation in the Junior Academy, but her first experience with the wonders of science started very close to home.
Published May 1, 2019
By Mandy Carr
Sthuthi Satish
Sthuthi Satish can’t remember when she started dreaming of being a doctor. What she does remember is being seven years old and looking at her mother’s medical charts showing stage two cancer. Her mom underwent surgery and beat cancer and Sthuthi’s love for surgery began.
The 15 year-old, who attends Bangalore International School in India, admits to not understanding the complications of surgery then, but saw the possibilities of it. Today, she hopes to become a neurosurgeon.
“My love for the brain is rather recent,” she said. “I am fascinated by the fact that the brain controls pretty much all conscious actions in the human body, and yet we know so little about it.”
Building Upon Previous STEM Experience
Sthuthi had few opportunities to join STEM activities before participating in the New York Academy of Sciences’ Junior Academy program. She worked on many challenges focusing on sustainability and aerospace. She worked with other high school students from across the globe as part of the winning team for the Human-Wildlife Challenge.
Sthuthi was concerned that no one was addressing the negative effects of solar panels on wild birds. Her team believes that infrared sensors and speakers producing beeping noises at 3 kHz can deter birds from landing on solar panels.
During her first year in the Junior Academy, she saw a posting on Launchpad, the Academy’s virtual collaboration platform, about getting involved in a Girls in Science panel at the third annual International Day of Women and Girls in Science event at the United Nations Headquarters in New York City. She stayed in touch with one of the organizers, HRH Princess Dr. Nisreen El-Hashemite, Executive Director of the Royal Academy of Science International Trust which lead to an invitation from Dr. El-Hashemite to chair a panel at the 2019 event.
Finding What Drives Her
This is also how she became a Girls in Science Advocate for the Royal Academy of Science. Additionally, Sthuthi is one of the administrators on the Girls in Science 4 SDGs International platform, a program Dr. El-Hashemite made possible. For Sthuthi it’s all about priorities.
“I always believe that if I have enough time to watch Netflix, then I definitely have time to work on something I love,” she said. Sthuthi hopes to attend college either in the United States or Sweden.
The next decade will be a pivotal one for the integration of AI and Big Data into healthcare, bringing both tremendous advantages as well as challenges.
Suchi Saria, PhD
Published May 1, 2019
By Sonya Dougal, PhD
One of the most common causes of death among hospital patients in the United States is also one of the most preventable — sepsis.
Sepsis symptoms can resemble other common conditions, making it notoriously challenging to identify, yet early diagnosis and intervention are critical to halting the disease’s rapid progress. In children, for each hour that sepsis treatment is delayed, the risk of death increases by as much as 50 percent.
Novel innovations, such as the one pioneered by Suchi Saria, director of the Machine Learning and Healthcare Lab and the John C. Malone Assistant Professor at Johns Hopkins University, are helping to reverse this trend. In 2013, Saria and a team of collaborators began testing a machine learning algorithm designed to improve early diagnosis and treatment of sepsis.
Using troves of current and historical patient data, Saria’s artificial intelligence (AI) system performs real-time analysis of dozens of inpatient measurements from electronic health records (EHRs) to monitor physiologic changes that can signal the onset of sepsis, then alert physicians in time to intervene.
“Some of the greatest therapeutic benefits we’re going to see in the future will be from computational tools that show us how to optimize and individualize medical care,” Saria said. She explained that the emergence of EHRs, along with the development of increasingly sophisticated AI algorithms that derive insights from patient data, will fuel a seismic shift in medicine — one that merges “what we are learning from the data, with what we already know from our best physicians and best practices.”
Nick Tatonetti, PhD
Electronic Health Records: A Gold Mine for Computer Scientists
EHRs have become a data gold mine for computer scientists and other researchers who are tapping them in ways designed to improve physician-patient encounters, inform and simplify treatment decisions, and reduce diagnostic errors. Like many other technological advances, though, there are those physicians who regard EHR systems with less enthusiasm.
A 2016 American Medical Association study revealed that physicians spend nearly twice as much time engaged in EHR tasks than they do in direct clinical encounters. Physician and author Atul Gawande recently lamented in The New Yorker that “a system that promised to increase my mastery over my work has, instead, increased my work’s mastery over me.”
Yet, data scientist Nicholas Tatonetti, the Herbert Irving Assistant Professor of Biomedical Informatics at Columbia University envisions a day when such AI algorithms will enable physicians to deepen their interaction with patients by freeing them from the demands of entering data into the EHR. Tatonetti has designed a system using natural language processing algorithms that takes accurate notes while physicians talk with patients about their symptoms. Like Saria’s AI system, Tatonetti’s takes advantage of the vast amount of data captured in EHRs to alert physicians in real time to potentially dangerous drug interactions or side effects.
Unknown Interactions
Anyone who has filled a prescription is familiar with the patient information leaflet that accompanies each medication, detailing potential side effects and known drug interactions. But what about the unknown interactions between medications?
Ajay Royyuru, PhD
Tatonetti has also developed an algorithm to analyze existing data in electronic health records, along with information in the FDA’s “adverse outcomes” database, to tease out previously unknown interactions between drugs. In 2016, he published a study showing that ceftriaxone, a common antibiotic, can interact with lansoprazole, an over-the-counter heartburn medication, increasing a patient’s risk of a potentially dangerous form of cardiac arrhythmia.
As data-driven AI techniques become more accessible to clinicians, the treatment of conditions both straightforward, like hypertension, and highly complex, such as cancer, will be transformed.
A Paradigm Shift in Physician-Patient Interactions
Ajay Royyuru, vice president of healthcare and life sciences research at IBM and an IBM Fellow, explained that, “when a practitioner makes a patient-specific decision, the longitudinal trail of information from thousands of other patients from that same clinic is often not empowering that physician to make that decision. The data is there, but it’s not yet being used to provide those insights.”
In the coming years, physicians and researchers will be able to aggregate and better utilize EHR data to guide treatment decisions and help set patients’ expectations.
The ability to draw on information from tens or even hundreds of thousands of patients, in addition to a physician’s own experience and expertise, could represent a paradigm shift in physician-patient interactions, according to Bethany Percha, assistant professor at the Icahn School of Medicine at Mount Sinai, and CTO of the Precision Health Enterprise, a team that turns AI research into tangible products for the health system.
“Big Data offers us the promise of using data to have a real dialogue with patients — if you’re newly diagnosed with cancer, it means giving people a realistic, data-driven assessment of what their future is likely to be,” she said.
Biases and Pitfalls
Despite the surge of interest and investment in AI over the past two decades, significant barriers to its widespread application and deployment in healthcare remain.
AI systems that tap current and historical patient health data risk reinforcing well-noted biases and embedded disparities. Medical research and clinical trials have long suffered from a lack of both ethnic and gender diversity, and EHR data may reflect patient outcomes and treatment decisions influenced by race, sex or socioeconomic status. AI systems that “learn” from datasets that include these biases will inherently share and perpetuate them.
Percha noted that greater transparency within the algorithms themselves — such as systems that learn which features an algorithm uses to make a prediction — could alert users to obvious examples of bias. Removing bias from AI algorithms is a work in progress, but the research community’s awareness of the issue and efforts to address it mirror a greater push to eliminate bias and decrease inequities in medicine overall. Optimistically, Percha noted that Big Data and AI may ultimately help create a more level playing field in healthcare delivery.
“Clinical decisions made on the basis of data have the potential to be much more standardized across different health facilities, so people who are in a rural area, for example, might have access to the same decision-making benefits as someone in a city,” she said.
Patient Data Privacy
Ensuring patient data privacy is another hot-button issue. Training artificial intelligence systems requires access to massive troves of patient data. Despite the fact that this information is anonymized, some patient advocates and bioethicists object to this access without explicit permission from the patients themselves.
Another privacy issue looms equally large: how to safely collect and protect the streams of potentially useful health data generated by wearable devices and in-home technologies without making patients and consumers feel, in Royyuru’s words, “like they are living their lives in front of a camera.” Studies have shown that data from smartphone apps can provide valuable information about the progression of certain diseases, such as Parkinson’s.
Wearables and in-home IoT devices can also extend the realm of clinical observation well beyond the doctor’s office, revealing, for example, important details about a Parkinson’s patient’s ability to complete the tasks of daily living. Yet Royyuru emphasizes that unless patients trust that their data will be kept private and ethically utilized, these technologies will fizzle long before they’re widely adopted.
Building Trust
The next decade will be a pivotal one for the integration of AI and Big Data into healthcare, bringing both tremendous advantages as well as challenges. Some applications of AI, such as image recognition, are already especially well-suited to healthcare — AI algorithms often match or even outperform radiologists in interpreting medical images — while others are far from ready for widespread use.
Saria, who has deployed her system successfully at multiple hospitals says, “physicians often greet news of AI breakthroughs with skepticism because they’re being over-promised results without clear data demonstrating this promise. True integration and adoption of AI requires not just careful attention to physician workflows, but transparency into exactly how and why an algorithm has arrived at a particular recommendation.”
Rather than replacing or challenging a physician’s place in the healthcare ecosystem, Saria believes that AI has the ability to lighten the load, and as algorithms improve, generate diagnostic and treatment recommendations that physicians and patients can both deem trustworthy.
“We are still figuring out how to make real-time information available so that it’s possible for physicians or expert decision-makers to understand, interpret and determine the right thing to do — and to do that in an error-free way, over and over again,” Saria said. “It’s a high-stakes scenario, and you want to get to a good outcome.”
Mark Shervey, Max Tomlinson, Matteo Danieletto, Sarah Cherng, Cindy Gao, Riccardo Miotto, and Bethany Percha, PhD, Mount Sinai Health System, Icahn School of Medicine at Mount Sinai.
The Honorees of the 2019 Innovators in Science Award are tapping the potential of stem cells.
Published May 1, 2019
By Hallie Kapner
Stem cells are the ultimate asset in the body’s efforts to heal damage and repair wounds. These powerhouses of regeneration are responsible for maintaining the integrity of skin, bone and other tissues. The 2019 Innovators in Science Award, sponsored by Takeda Pharmaceuticals, recognizes two outstanding researchers in the field of regenerative medicine. The Senior Scientist and Early-Career Scientist winners are advancing our understanding of the miraculous inner workings and remarkable healing powers of stem cells.
Turning Stem Cell Research into Life-Saving Therapies
Michele De Luca, MD
Michele De Luca, MD, first encountered epithelial stem cells in the 1980s, during a research fellowship at Harvard Medical School in the lab of stem cell therapy pioneer Howard Green.
“I fell in love with the concept, the cell type, and the system,” he said, describing how the thrall of regenerative medicine — then in its infancy — would come to dominate the next thirty years of his career.
De Luca, winner of the Senior Scientist Award and director of the Center for Regenerative Medicine “Stefano Ferrari” at the University of Modena and Reggio Emilia in Modena, Italy, has made fundamental discoveries in the molecular and genetic characteristics of epithelial stem cells, translating those findings into therapies that change and save patients’ lives.
De Luca’s earliest clinical triumphs in skin regeneration were in the treatment of burn patients. Using the patient’s own epidermal stem cells, De Luca grew skin grafts in culture, then successfully used them to repair large lesions. In collaboration with Graziella Pellegrini, professor of cell biology at the University of Modena and Reggio Emilia, De Luca went on to pioneer new stem cell culture and grafting techniques, ultimately developing the first corneal regenerative therapy, Holoclar, which utilizes limbal stem cells to generate healthy corneal tissue for patients who have sustained chemical burns or other ocular injuries. The technique, which can restore lost sight in some cases, was approved by the European Medical Agency as a commercial stem cell therapy in 2015.
Decades of research, experimentation, and clinical trials prepared De Luca well for the day (later that same year) when he first learned of a seven-year-old boy in Germany suffering from a debilitating and often fatal skin condition, junctional epidermolysis bullosa, which is caused by a genetic mutation. Working against the clock, De Luca and a team of collaborators in Modena and Germany attempted a highly experimental epithelial stem cell gene therapy.
The team used a retroviral vector to introduce a functional copy of the mutated gene into the patient’s stem cells, then rapidly grew healthy sheets of skin for transplantation. Three years later, the transgenic skin grafts remain symptom-free. De Luca noted that his case has provided critical insights into epidermal stem cell biology and the potential for using gene therapy for other skin conditions.
“To me, this is the essence of regenerative medicine, and this is the future,” he said.
Decoding the “Crosstalk” Between Epithelial Stem Cells and the Immune System
Shruti Naik, PhD
Shruti Naik, PhD, assistant professor in the departments of pathology, medicine, and dermatology at NYU School of Medicine and winner of the Early-Career Scientist Award, is exploring the interplay between immune cells, stem cells, and resident microbes in epithelial tissues.
By eavesdropping on what she describes as a “vital conversation” between these groups, Naik hopes to better understand how their interplay with each other — and with the external environment — facilitates healing and regeneration. Her work is also providing insight into the devastating conditions that can result when these systems break down, such as non-healing wounds and ulcers.
Naik’s work aims to systematically decode the dialogue among various cell communities within barrier tissues as they encounter and respond to external stimuli or injury, with a particular focus on the role of epithelial stem cells, which play pivotal yet poorly understood roles in the body’s defensive and regenerative processes. Naik’s research has revealed surprising sensitivities and attributes of these cells.
“Stem cells are actually exquisite sensors of inflammation, and we’ve discovered that they can even remember inflammation and change their behavior accordingly,” she said.
This cellular memory can promote healing by “tuning” the stem cells to respond and regenerate tissue more quickly.
Understanding which immune signals modulate the activity of stem cells, and how the microbial communities of the skin, lung, and gut can influence the process of tissue repair, may lead to new therapeutic approaches for chronic ulcers and other wounds.
“We’re really at the beginning of a new era of understanding how stem cells sense inflammatory and stress signals and incorporate them into generating new tissues,” Naik said.
From a focus on immunotherapy to better understanding intratumoural heterogeneity, these researchers are making significant advances in cancer treatment.
Published April 15, 2019
By Marie Gentile, Robert Birchard, and Mandy Carr
According to the American Cancer Society, one in every six deaths worldwide can be attributed to cancer, more than HIV/AIDS, tuberculosis, and malaria combined. While steady research progress has saved the lives of countless cancer patients—much work remains. Two cutting-edge, cancer researchers are challenging traditional research paradigms to better inform new therapies.
Translational medicine or “bench to bedside” is the near universal method for developing cancer treatments, but Padmanee Sharma, MD, PhD, a professor at The University of Texas MD Anderson Cancer Center is reversing this time-tested approach.
Instead of bringing newly devised treatments from the lab to patients enrolled in clinical trials, she is first bringing patients’ clinical data into the lab for researchers to study. “We’re collecting samples from patients in clinical trials, especially immunotherapy trials, and trying to understand how their immune system is evolving,” explained Dr. Sharma. “The immune response is a very dynamic process, which means that the anti-tumor immune response has been evolving and differentiating in some cases for years before a patient presents with a finding of cancer.”
A Focus on Immunotherapy
Padmanee Sharma, MD, PhD
“Although we have great mouse models, these models are not able to mimic the longitudinal and dynamic human immune response that develops over years,” she said. “The patient remains the best model.” With the patient’s clinical info, researchers try to understand the patient’s immune system and generate hypotheses that they think will help improve the patient’s treatment regimen.
Dr. Sharma’s research is focused on immunotherapy approaches which, “unleash the power of the immune system to reject cancer,” she explained. “Immunotherapy is my idea of precision medicine. Most people think of precision medicine as therapies against specific ’driver’ genetic mutations, which are mutations in the cancer cells that enable development and survival of cancer cells, but since cancer cells are constantly evolving and developing new mutations, it may be difficult to develop targeted treatments against each new mutation.”
“Immunotherapy is a match for cancer because it targets the immune system, not the cancer cells, and the immune response is constantly evolving in each patient’s body generating precise and specific T-cell responses against the mutations, regardless of whether the mutation is a ’driver’ or ’passenger’ mutation.”
“Immunotherapy can also be combined with other treatments such as surgery, radiation, chemotherapy, and targeted therapy because we can take advantage of the fact that these therapies are helping to kill some cancer cells, with subsequent recognition by the immune system, which can be enhanced with immunotherapy agents to drive additional anti-tumor responses as well as elicit development of memory immune responses to enable protection against cancer recurrence,” finished Dr. Sharma.
Tracking the Evolution of Tumors
Part of the need for Dr. Sharma’s approach stems from the complex cellular makeup of cancer, referred to as tumor heterogeneity. Within every patient’s tumor is an opportunity for Darwinian evolution, where specific cancer cell populations (subclones) are selected based on advantageous characteristics like metastatic ability, drug resistance, or immune escape.
Charles Swanton, FRCP, FRS, PhD
“This becomes a problem as the cell to cell variation, and clone to clone variation leads to variation in tumors, and is the ultimate challenge in developing effective cancer treatments,” said Charles Swanton, FRCP, FRS, PhD from The Francis Crick Institute. This is why there are few successful one-size-fits-all approaches to therapy in oncology.
Together with a team of leading researchers, Dr. Swanton spearheaded the TRACERx (TRAcking Cancer Evolution through therapy (Rx)) lung study, which hopes to transform our understanding of non-small cell lung cancer in particular and cancer in general by identifying the relationship between intratumoural heterogeneity in lung cancer tumors and poor clinical outcomes.
“Intratumoural heterogeneity is the difference in tumor subclones within the same patient, either within the primary site, between the primary metastatic sites, or between metastasis,” explained Dr. Swanton. “We’re trying to understand how a lethal metastatic subclone evolves from a heterogeneous primary tumor, and whether we can detect and monitor evolution in real time in patients’ blood to track evolution during the disease…And ultimately use all of that information to develop better diagnostic approaches, better clinical trial designs, and hopefully better therapies; ultimately, to control the disease for longer.”
Combining Lessons from Different Fields
With their powers combined, the approaches taken by Drs. Sharma and Swanton are critical for building a complete picture of the inner workings of cancer. Knowledge of the evolution and origins of different tumor clones will provide new targets to prime the host immune system, allowing it to destroy cancer cells and reduce the chances of disease recurrence.
In fact, work in this direction is already underway as Dr. Swanton is involved in setting up a company that will develop immune-based therapies targeting the mutations identified in the trunk of the tumour’s evolutionary tree—so-called—clonal mutations. Thus, as Dr. Sharma states “The new realm of cancer immunotherapies should be seen as one of hope for all patients.”
