January 1, 2015 (Vol. 35, No. 1)

Unlikely as it sounds, stem cell technology is evolving to occupy two very different niches—personalized medicine and industrial-scale manufacturing.

Stem cell-based technologies and the use of stem cells as research and discovery tools and as cell therapy products continue to advance.

As stem cell therapeutics progress through preclinical testing and into human studies, they face regulatory scrutiny, and manufacturers seek solutions to automate and improve the efficiency and cost-effectiveness of production at industrial scale. For disease modeling, drug screening, and predictive toxicology—and as tools for predicting drug response in personalized medicine paradigms—stem cells are playing an increasingly important role.

Stem cells, stem-cell-based products, and stem-cell applications are being developed by various companies including Capricor Therapeutics, Cellular Dynamics International, Athersys, and Advbiols. These companies are scheduled to deliver presentations at the Phacilitate Cell and Gene Therapy Forum, which will be held  January 26–28 in Washington, DC.

Capricor Therapeutics reminds us that it announced encouraging results at the 2014 annual meeting of the American Heart Association. At this event, the company presented safety and preliminary efficacy data from a Phase I study of its CAP-1002 allogeneic cardiosphere-derived stem cells. The study was an opportunity to evaluate the delivery of the stem cells via intracoronary injection to patients with left-ventricular dysfunction following an anterior myocardial infarction.

Capricor is developing CAP-1002 as an off-the-shelf cell therapy derived from donor heart tissue, and in addition to an ongoing Phase II study in this indication, the company will soon initiate a clinical study in patients with heart failure, and is also initiating a clinical development program with CAP-1002 in Duchenne Muscular Dystrophy cardiomyopathy.

The Phase I CAP-1002 met its primary safety endpoint. Initial efficacy data revealed improvements in cardiac structure and function, notably reductions in infarct scar size, and trends for improved ejection fractions. The company suggests interpreting these data with caution due to the small sample size and lack of a contemporaneous control group.

Patients who already had antibodies to the CAP-1002 cells on entry to the study did not have any safety issues, but they also “did not show the same degree of efficacy,” relates Rachel Smith, Ph.D., vice president of research and development at Capricor. As a result, the company designed the Phase II trial to exclude antibody-positive individuals from all but a small exploratory cohort.

During the past year or so, Capricor has begun developing another product: the exosomes secreted by the cardiosphere-derived stem cells. These exosomes “account for a lot of the benefit we see in preclinical models,” notes Dr. Smith. “They inherit regenerative properties from the stem cells,” in part via particular microRNAs that likely have an important role in their therapeutic capabilities.

The possibility of using exosomes as a therapeutic product—rather than cells—offers several hypothetical advantages, according to Dr. Smith, including the ability to administer higher single doses and the potential for repeat dosing. From a manufacturing, delivery, and storage perspective, exosomes should be stable at a range of temperatures and might even be amenable to lyophilization.

“We are now testing exosomes in preclinical studies in cardiac indications, such as cardiomyopathy and postmyocardial infarction, and are also exploring the use of exosomes in other, noncardiac indications,” adds Dr. Smith.

Capricor is currently developing a pilot manufacturing process for exosome production, as well as methods for the efficient extraction of exosomes from the culture media.

Human iPSC-derived RPE cells (right, micrograph) grown on a scaffold (left, artist rendered). The microvilli, or little projections on the surface of each RPE cell, communicate with the photoreceptors in the eye, an essential interaction for sight. [Nathan Hotaling (NIST); Vladimir Khristov and Kapil Bharti (National Eye Institute)]

Industrial-Scale Manufacturing

Cellular Dynamics International (CDI) has developed an automated, industrial-scale manufacturing system to produce large quantities of human induced pluripotent stem cells (iPSCs) and terminal tissue cells for drug discovery and development and therapeutic applications.

“The drug discovery field is moving away from a single-donor disease model to more of a panel-based approach that can replicate a lot of the data being generated in genome-wide association studies (GWAS),” says Chris Parker, vice president and chief commercial officer at CDI. “You need an industrial engine to do the large-scale reprogramming, develop any selection of cell lines in parallel, and manufacture the terminally differentiated cells so they can be put into a pharmaceutical drug discovery and drug development environment.”

Otherwise, investigators are working with such small amounts of material from individual patients “that they cannot really do interrogative research,” Parker observes. In addition to replicating the phenotype of a disease artificially in vitro, researchers also need to be able to compare different genotypes, study the cells of multiple individuals, and have sufficient material to be able to probe the biology and identify underlying disease mechanisms.

The CDI technology enables the company to scale up production and “make virtually unlimited quantities of cells from one iPSC line” and to “scale out,” expanding multiple iPSC lines from multiple individuals in parallel. The ability to look at individual differences in biology that lead to variations in disease phenotype can also be used to study predictive toxicology and drug response. Using patient-derived iPSCs to replicate an individuals’ biology in a dish, clinicians can begin to conduct “in vitro clinical trials,” adds Parker, and measure biomarkers indicative of potential toxicity and drug efficacy.

The cellular components of a kidney’s blood vessels were first removed by a detergent solution, leaving behind a “decellularized” organ (left). Red and blue polymers, representing the arterial and venous blood supply, were then injected to demonstrate that the blood supply infrastructure remained intact. This scaffold was then “recellularized” (right) using CDI’s iCell® Endothelial Cells, which were labelled with fluorescent green.These cells became incorporated into the blood supply infrastructure. [Copyright © 2014 John Wiley & Sons, Inc.]

In CDI’s first foray into cellular therapeutics, the National Eye Institute recently awarded the company $1.2 million in funding to produce autologous iPSCs and iPSC-derived human retinal pigment epithelial cells from individuals affected with dry age-related macular degeneration for use in preclinical studies in preparation for a clinical trial involving these patients.

Looking ahead, Parker notes that CDI is working to understand “how we can mature these cells to a more adult-like phenotype and put them into more three-dimensional, organoid, tissue engineering types of contexts.”

MultiStem® is an adult-derived, off-the-shelf allogeneic stem cell product platform developed by Athersys. The stem cells are derived from bone marrow and are distinct from mesenchymal stem cells (MSCs). They have demonstrated multiple mechanisms of action with therapeutic implications in various disease indications, including anti-inflammatory and immunomodulatory activity, according to John Harrington, Ph.D., executive vice president and CSO at Athersys. The company is initially targeting inflammatory, immune, neurological, and cardiovascular diseases in the clinical development of MultiStem.

An advantage of MultiStem is its lack of immunogenicity. It does not induce an antibody or T-cell mediated immune reaction in patients after single or repeat dosing. The cells’ capacity for immunomodulation “is one of their fundamental properties,” says Dr. Harrington. “As an added benefit, MultiStem administration does not require tissue matching, and the cells do not cause immune sensitization in patients receiving the product.”

The ability to manufacture MultiStem at industrial scale is another advantage of the product. According to Dr. Harrington, the platform produces cells that grow faster than MSCs and can be grown in culture through more population doublings to generate millions of doses from a single donor.

“We are able to take a more traditional manufacturing approach compared to MSCs, using a master cell bank and a working cell bank, similar to how recombinant proteins are manufactured,” asserts Dr. Harrington.

Athersys is in the process of transitioning its manufacturing platform to traditional stirred tank bioreactors using microcarriers. According to Dr. Harrington, the company is currently working at the 50 L scale and plans to scale up to a multithousand liter scale over the next couple of years.

MultiStem has advanced to Phase II clinical testing, including an ongoing study in ulcerative colitis with Pfizer, an ongoing trial in ischemic stroke, and a soon-to-be-initiated trial in acute myocardial infarction. Phase I studies have been completed in graft-versus-host disease and acute myocardial infarction.

Finding a Commercial Niche

Will one type of stem cell-based product dominate for therapeutic applications?

“It’s still too early to know,” says Christopher A. Bravery, Ph.D., principal consultant at Advbiols. Each type of product may solve a particular problem or help overcome a certain obstacle, but each also tends to present problems of its own. “I suspect the reality will be that everything will have its niche,” Dr. Bravery predicts.

Focusing on induced pluripotent stem cells, which have the potential to develop into any type of cell, the main challenge is whether the field currently has the potential “to develop them cost effectively into anything at all,” Dr. Bravery ventures. Pluripotent cells have to be differentiated into specific cell types before being given to patients, and reliable and efficient stem cell expansion and differentiation remain an issue as cell therapies move toward the clinic and commercial development.

Companies need to determine how much genetic drift is occurring in stem cell populations during expansion, and to identify approaches to maximize genetic stability. Understanding and documenting epigenetic changes may become part of the cell characterization process in the future as well. An efficient, cost-effective manufacturing process to produce an iPSC-derived cell therapy “will need very high differentiation rates and low amounts of the cells you don’t want,” to minimize safety concerns, offers Dr. Bravery. Some efforts to develop off-the-shelf allogeneic cellular therapeutics are banking on HLA-typed iPSC-lines.

“The idea of having tissue-matched banks of cells is unrealistic, in my view,” Dr. Bravery says. In an article that appeared online November 13, 2014 in Stem Cells and Development, he details the continuing technical challenges (including the need for a validated, reliable reprogramming method that yields highly consistent iPSC lines) and the manufacturing hurdles (the ability to produce cell therapy products with comparable quality, safety, and efficacy from an array of HLA-typed iPSC lines).

Dr. Bravery also believes that creating an autologous iPSC product will likely face insurmountable problems unless a much more efficient and less costly type of manufacturing strategy can be developed. Even if manufacturing hurdles can be overcome for autologous iPSC products, there remains the issue of how to minimize donor-to-donor variation and how to define “acceptable” variation to ensure therapeutic potency and likely efficacy.

“Regulators are looking for consistency in manufacturing,” insists Dr. Bravery, noting that what consistency even means for a cell product is not yet clearly understood. Manufacturers need to show regulators a consistent, well-defined manufacturing process, and the ability to undertake process/production changes while maintaining consistency and product quality. Changes in manufacturing will be inevitable throughout the life of a product to make the production process more cost-effective. Companies need to be able to demonstrate that a change will not affect the safety of the product and will not require additional (and costly) clinical studies in the future.

There is still much work to be done to develop optimal methods for characterizing cellular therapies and to identify the best analytical tools for product characterization, according to Dr. Bravery.

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