Tal Danino, PhD, and Rosa L. Vincent, PhD, at Columbia University’s department of biomedical engineering, and colleagues, reported on their developments in Science, in a paper titled “Probiotic-guided CAR-T cells for solid tumor targeting,” in which they concluded, “These findings highlight the potential of the ProCAR platform to address the roadblock of identifying suitable CAR targets by providing an antigen that is orthogonal to both healthy tissue and tumor genetics … Overall, combining the advantages of tumor-homing bacteria and CAR-T cells provides a new strategy for tumor recognition and, in turn, builds the foundation for engineered communities of living therapies.”

Immunotherapies using CAR-T cells have proven successful in treating some types of blood cancers, but their efficacy against solid tumors remains elusive. A key challenge facing tumor-antigen targeting immunotherapies like CAR-T is the identification of suitable targets that are specifically and uniformly expressed on solid tumors, the authors noted. “A key challenge of antigen-targeted cell therapies relates to the expression patterns of the antigen itself, which makes the identification of optimal targets for solid tumor cell therapies an obstacle for the development of new CARs.” Solid tumors express heterogeneous and nonspecific antigens and are poorly infiltrated by T cells. As a result, the approach carries a high risk of fatal on-target, off-tumor toxicity, wherein CAR-T cells attack the targeted antigen on healthy vital tissues with potentially fatal effects. “Few tumor-associated antigens (TAAs) identified on solid tumors are tumor restricted, and thus, they carry a high risk of fatal on-target, off-tumor toxicity because of cross-reactivity against proteins found in vital tissues,” the team continued.

Previous studies have shown that, unlike CAR-T cells, which require “considerable engineering to target and infiltrate solid tumors,” some species of bacteria can selectively colonize and preferentially grow within the hostile tumor microenvironments (TMEs) of immune-privileged tumor cores, and can be engineered as antigen-independent platforms for therapeutic delivery.

In this study, Vincent, Candice Gurbatri, and colleagues combine probiotic therapy with CAR-T cell therapy to create a two-stage probiotic-guided CAR-T cell (ProCAR) platform, whereby T cells are engineered to sense and respond to synthetic CAR targets that are delivered by solid tumor-colonizing probiotic bacteria. “This approach leverages the antigen independence of tumor-seeking microbes to create a combined cell therapy platform that broadens the scope of CAR-T cell therapy to include difficult-to-target tumors,” the investigators explained.

Using synthetic gene circuit engineering on a well-characterized non-pathogenic strain of E. coli, Vincent et al. created a probiotic that could infiltrate and cyclically release synthetic CAR targets directly to the tumor core, effectively “tagging” the tumor tissue. “With this system, bacterial growth reaches a critical population density exclusively within the niche of the solid TME and subsequently triggers lysis events that cyclically release genetically encoded payloads in situ,” they further explained.

Then, CAR-T cells that were programmed to recognize the probiotic-delivered synthetic antigen tags could be generated that homed in on the tagged solid tumors, killing the tumor cells in situ. The scientists also engineered probiotics that co-released chemokines in addition to synthetic targets to further enhance CAR-T cell recruitment to the tumor, further boosting therapeutic response.

Vincent et al. tested the resulting probiotic-guided CAR-T cell platform in humanized and immunocompetent mouse models of leukemia, colorectal cancer, and breast cancer and showed that it resulted in the safe reduction of tumor volume. “Collectively, these mouse model data demonstrate the use of engineered probiotics to selectively grow within the TME niche and safely release combinations of CAR-T cell enhancing payloads in situ,” they wrote. The team acknowledged that further development of the system will be needed before it can be considered for clinical application. Nevertheless, they stated, “We have demonstrated an approach to engineering interactions between living therapies, in which tumor-colonizing probiotics have been repurposed to guide the cytotoxicity of engineered T cells.”

In a related Perspective, Eric Bressler, PhD, and Wilson Wong, PhD, at Boston University Biomedical Engineering and Biological Design Center, also noted, “Translation of the ProCAR system to the clinic will depend on scalability to larger tumors and attenuation of bacterial strains for safety.” However, they concluded, “The study of Vincent et al. is an important proof-of-concept for a potential approach to treating heterogeneous, immunologically cold, and poorly infiltrated solid tumors.”

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A handful of targeted protein degrader drugs have already reached clinical application, with many additional therapies entering the pipeline as companies and academic researchers continue to develop new molecules and mechanisms. So far, the field has primarily focused on cancer therapeutics because aberrant proteins in cancer cells are obvious targets. However, targeted protein degradation technology could eventually be applied to myriad diseases and disorders, including those affecting the central nervous system.

Designing/optimizing degrader molecules

“We’re about to translate awesome new modalities that have really incredible pharmacology into drugs that we believe will make a huge difference to patients,” says Stewart L. Fisher, PhD, chief scientific officer, C4 Therapeutics.

C4 Therapeutics is one of many biotech companies developing and testing PROteolysis TArgeting Chimeras, known as PROTACs. These are small-molecule drugs that recruit specific E3 ubiquitin ligases to transfer polyubiquitin chains onto target proteins, thereby marking them for degradation by the cell’s native proteasome.

Unlike many existing drugs, such as inhibitors, targeted protein degraders need not bind to an active site on a protein. So long as the degrader forms a ternary complex with the E3 ligase, the protein can be ubiquinated, and degradation will occur. “This is opening up space where we can go after traditionally undruggable proteins,” notes Fisher. “We’re not limited to just that binding site.”

Most existing PROTACs rely on bifunctional degradation activation compounds (biDACs), which are heterobifunctional molecules featuring one site that binds a target protein and another that binds an E3 ligase. However, targeted protein degradation can also be achieved using monofunctional degradation activation compounds (monoDACs), also known as molecular glues or glue degraders. These bind to either the E3 ligase or the target protein and chemically modify the surface, prompting protein-protein interactions that ultimately cause the E3 ligase to bind to the target protein, resulting in degradation.

Fisher says that monoDACs tend to be smaller molecules than biDACs, which could simplify optimization with respect to bioavailability and compliance with drug development guidelines. However, monoDACs’ reliance on protein-protein interactions can weaken their ability to select the target of interest.

“One of the things that I think differentiates us at C4 Therapeutics is that we use both of these approaches as the targets present opportunities,” maintains Fisher. “I’m not aware of other biotech firms in this space that have the capability to focus on both monoDACs and biDACs.” C4 Therapeutics currently has four PROTACs in preclinical trials: an IKZF1-targeting monoDAC for treating hematologic malignancies, and a BRD9-targeting biDAC for treating sarcoma.

All of C4 Therapeutics’ degrader molecules recruit the same E3 ligase, known as cereblon. Along with the Von Hippel–Lindau (VHL) ligase, cereblon is currently one of the most commonly recruited E3 ligases in targeted protein degradation. Fisher says that his company decided to invest deeply in cereblon because it is involved in the molecular action of well-known drugs such as thalidomide. Clinical history indicates that the ligase’s action is well tolerated and unlikely to cause severe side effects.

Expanding the E3 ligase toolbox

Although PROTACs have been on the scene for a relatively short time, resistance mechanisms have already cropped up in preclinical trials. Most frequently, cancer cells evolve to downregulate the E3 ligases that the PROTACs depend on for polyubiquination of their target proteins. Furthermore, some proteins of interest are not effectively degraded using cereblon or VHL. Developing PROTACs that recruit E3 ligases other than VHL or cereblon could help bypass resistance mechanisms and expand the range of viable targets. In particular, some scientists have highlighted the potential advantages of focusing on E3 ligases that serve essential pathway roles. Taking this approach could make it harder for cells to downregulate them in response to PROTAC application.

“The creation of novel E3 ligands is the future of targeted protein degradation,” says Jing Liu, PhD, executive director of medical chemistry, Cullgen. The company has identified multiple ligands that bind E3 ligases not previously exploited for targeted protein degradation, and has confirmed that these ligands can be incorporated into bifunctional degrader molecules.

Liu says that roughly 50% of Cullgen’s research and development efforts are aimed at developing novel E3 ligands, whereas the remaining 50% of these efforts focus on developing the company’s existing internal pipeline of targeted protein degraders. Cullgen has built a library of linkers with different chemical and physical properties, allowing for efficient drug optimization.

For example, Cullgen previously reported on its creation of potent and selective degraders for tropomyosin receptor kinase A (TRKA), a key target for cancer treatments. However, the initial degrader molecules—CG416 and CG428—showed low oral bioavailability, so company scientists returned to the laboratory and created second-generation degraders—CG1037 and CG1054—for the same targets. These second-generation molecules showed higher oral bioavailability in mouse models without sacrificing efficacy or causing significant side effects.

Liu points to this process of developing TRKA degraders as a proof-of-concept example, saying, “We can utilize such pinpoint targeting capabilities to develop degraders with unique selectivity profiles to treat different diseases.”

Implementing location specificity

Suresh Kumar, PhD, senior director and head of discovery, Progenra, says that avoiding resistance isn’t the only reason to develop PROTACs that recruit E3 ligases other than cereblon and VHL.

“If your target is a membrane protein, and you want to degrade that protein with a PROTAC,” Kumar says, “that job is better done with a membrane-targeted E3 ligase than with a nuclear-located ligase.”

For example, K-Ras, a well-known yet famously elusive oncological therapeutic target, is a membrane protein. Kumar says that to his knowledge, Progenra is “currently the only company that has a membrane-targeted ligase.” At multiple conferences in September and October 2020, Kumar and his colleagues presented experimental results from the development of a potent, membrane-targeted PROTAC capable of degrading K-Ras with high specificity.

Over the past 15 years, Progenra has developed and utilized a proprietary platform that it calls UbiPro, which consists of a series of enzyme activity assays that “closely replicate physiological milieu.” Progenra uses this platform for drug discovery involving both E3 ligases and deubiquitinases, another group of enzymes in the UPS. The platform has the capacity for high-throughput screening, with a panel of over 30 purified E3 ligases that can be applied for profiling and selectivity.

Kumar expects that Progenra and the rest of the PROTAC field will eventually expand far beyond cancer therapeutics. “Our ligases have extremely high relevance to human biology, with implications in diseases ranging from cancer to Parkinson’s disease, Alzheimer’s disease, and inflammatory disorders,” he insists. “All these human diseases have an underlying problem at the fundamental cellular level in terms of degrading proteins—either lack of degradation or excessive degradation.” Progenra is currently evaluating novel PROTACs as anti-inflammatory agents but has not yet made further details public.

Bypassing E3 ligases entirely

Even as PROTAC technology continues to advance, there remain limitations. Recruiting specific E3 ligases means relying on a relatively narrow set of chemical structures that bind those ligases. Those molecules can present design challenges and limit target scope. Amphista Therapeutics decided to let other companies tackle PROTACs, and instead secured funding to pursue novel methods of targeted protein degradation.

“The ubiquitin proteasome system is, in many ways, one of the most rubbish enzyme systems there is, because it’s really very poorly selective,” says Ian Churcher, PhD, chief scientific officer, Amphista Therapeutics. “If you get substrates close enough for long enough to the ubiquitin proteasome system, they will be degraded. And that’s really what we set out to do.”

Amphista has designed multiple “magnet” ligands that are believed to recruit multiple UPS proteins and activate multiple parallel degradation pathways that depend on critical cellular components. This approach makes it more difficult for cancers to develop resistance. Researchers incorporate these magnet ligands into bifunctional molecules that bind to target proteins and bring them into proximity with the UPS.

“We don’t believe anyone has ever used these molecules before in this way,” remarks Churcher. “We often think of it as next-generation targeted protein degradation. It’s an amazing field with huge potential, but we want to expand that potential into more drug targets, better profiles, and better dosing routes for patients.”

Amphista is currently developing two types of small-molecule drugs that utilize these novel mechanisms. One recruits deubiquitinase enzymes, whereas the other recruits the proteasome directly. Amphista plans to bring its first molecule into the clinic in 2023.

Initiating protein self-sabotage

Jian Jin, PhD, director of the Mount Sinai Center for Therapeutics Discovery, is the lead author on a February 2020 paper in Nature Chemical Biology describing MS1943, a first-in-class selective degrader for histone methyltransferase EZH2. This degrader relies on a technique known as hydrophobic tagging, which Jin says has been understudied by the biomedical community thus far.

Hydrophobic tagging involves attaching a bulky hydrophobic chemical group to a small-molecule binder of the target protein—in this case, EZH2 inhibitor C24. Jin indicates that the mechanism of degrader action is not yet fully elucidated, but scientists believe that the presence of the large hydrophobic group causes the protein to misfold, which ultimately triggers its degradation via the natural action of the UPS.

MS1943 showed high potency in triple-negative breast cancer cells while sparing normal cells. Furthermore, the compound showed high oral bioavailability, which Jin hypothesizes may be due in part to the molecule’s smaller size compared to most PROTACs.

“Now that we have published this approach, I think more and more research groups will explore this technology,” predicts Jin. “We are actively optimizing these compounds and will hopefully progress into clinical studies.”

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Last month, Metabolon announced the close on $72 million in combined debt and equity financing. This round of financing included Perceptive Advisors as a new participant. EW Healthcare Partners and other existing investors also participated in the financing.

“The incremental funding will help accelerate our growth and expand our client base, in addition to helping further our research and development programs in machine learning to enable novel biomarker discovery and expand our precision medicine platform,” notes Rohan (Ro) Hastie, PhD, president and CEO, Metabolon.

According to William LeFew, PhD, Metabolon’s director of data science, the company uses machine learning to automate routine tasks and teachable processes so its scientists can focus their efforts on the challenges that most require their expertise.

“As part of our data science initiatives, we leverage machine learning to extract insight and recognize data patterns computationally,” he says. “Machine learning improves our data curation throughput, identifies biochemical signatures, and detects anomalies to accurately and rapidly ensure quality control. These capabilities improve coverage, quality, turnaround time, and, ultimately, our clients’ study success.”

LeFew provided three examples of machine learning at work at Metabolon.

Streamlined quality control

In the laboratory, time is always of the essence and attention to detail is paramount. Late-stage analysis revealing flaws in original data can invalidate days or weeks of work, derailing timelines and production deliverables.

“Machine learning,” LeFew explains, “enables us to provide clients with a compressed quality control cycle to detect failure modes much earlier than is possible with a purely manual process.”

For example, with several product lines, stringent requirements are tested via statistical analysis once curation is complete. A fully manual process would require several days of work. According to LeFew, the autocuration utility, combined with automated statistical analyses targeted at product requirements, makes it possible to detect failure modes instantly after initial raw data production, prompting immediate human review to classify samples that would likely fail quality control checks days later, reducing time and labor that would otherwise be spent on samples doomed to fail at a downstream quality control step.

“By detecting failure right after initial raw data review, we can run backup samples immediately,” he adds. “This saves a significant amount of time in our process, ultimately improving our total turnaround time for client projects while freeing up employee time to focus on shippable results rather than reruns.”

Faster data curation

In textbook machine learning, labeling problems are solved by learning a classification model from unbiased ground-truth data. In real applications, however, the matter may be significantly complicated by the practices and protocols used to produce the training data.

As an example, Metabolon generates liquid chromatography/mass spectrometry (LC/MS) data from which metabolites’ presence is inferred. Historically, expert curators examined these data with software assistance to confirm or deny the compounds’ presence. Each sample processed on Metabolon’s platforms, LeFew continues, is examined for the presence or absence of every one of the tier-one identified compounds in Metabolon’s metabolite knowledgebase. All samples processed on Metabolon’s Precision Metabolomics platform are curated against the company’s proprietary library of more than 5,200 unique metabolites.

“Machine learning allows us to achieve this same high-quality data, but much faster,” he asserts. “We can bring a data set directly to quality control through machine learning, saving time by automatically performing initial curation. Machine learning also allows us to quickly determine with certainty which compounds are present and the ones that were never present, significantly reducing or even eliminating the need for human experts to make these trivial decisions.”

Built on historical curations, machine learning feeds an autocuration utility that can curate many routine compounds. Consider cholesterol, says LeFew, which is readily found in human plasma and, therefore, is not an efficient use of staff expertise.

“With the support of machine learning tools, we can leverage our human expert curators’ skills in delving for the presence of ethylparaben sulfate, which often presents with interfering ions, or differentiating between compounds like isoleucylglycine and alanylvaline,” he details. “These compounds are not chromatographically separated but have distinct MS/MS fragmentation, usually containing a tremendous variety of information.”

Continued knowledge expansion

LeFew notes that there is still much to be discovered in terms of new metabolites and their impact on life sciences research and drug development.

“The body of literature, reports, and insights produced by Metabolon’s internal experts have served as the basis for a collaboration with data science to develop a shared vocabulary, a knowledgebase, and software to support the recording of continued knowledge expansion,” he maintains. “Future data science collaborations with these experts will automatically surface relevant historical knowledge to expert staff on each and every experiment run with Metabolon.”

William LeFew, PhD, is Metabolon’s director of data science. He leads a team that leverages machine learning to improve data curation throughput, identify biochemical signatures, and detect anomalies to accurately and rapidly ensure quality control.

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Broadening the base of metabolomics users are the suppliers of metabolomics systems. Besides elaborating instrumentation platforms around key technologies such as mass spectrometry, these suppliers are refining associated workflows and software.

This work isn’t being performed in a supplier bubble. Indeed, several suppliers are partnering with research institutions to help metabolomics technology become more user-friendly, even as it gains sophistication and power.

Grappling with a systemic disease

Bruker recently indicated that it has partnered with the Australian National Phenome Centre (ANPC) to support a new “frontline response to combat the COVID-19 threat.” Researchers at the ANPC are using instrumentation from Bruker to help them perform metabolic analyses on samples taken from COVID-19 patients and controls.

“The metabolic effects of SARS-CoV-2 infection on human blood plasma were characterized using multiplatform metabolic phenotyping with nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography–mass spectrometry (LC-MS),” ANPC researchers reported in a recent study.¹ “[We constructed] an exceptionally strong hybrid NMR-MS model that enabled detailed metabolic discrimination between the groups and their biochemical relationships.”

By probing the profound metabolic alterations that accompany COVID-19, the researchers identified markers “distinctive of a multisystem involvement” and “consistent with the reported extensive microvascular effects that would be expected to compromise multiple organ functions.”

Going forward, the researchers hope to build models that will predict variation in the severity of the disease, help clinicians understand differential responses to therapeutic interventions, and address other uncertainties. “One unanswered question about COVID-19,” noted Lucy Woods, PhD, business unit manager, Phenomics & Metabolomics, Bruker, “is why sufferers experience such a wide range of symptoms.”

Woods adds that metabolomics researchers must obtain reproducible measurements across assays and acquire an accurate picture of the presence of metabolites. Although extensive analyte libraries are available, a large proportion of analytes remain unidentified, necessitating the use of orthogonal techniques.

According to Bruker, ANPC researchers will use several of the company’s systems, including the Avance IVDr NMR platform and the Impact II, timsTOF Pro, and Solarix MS platforms, as well as various data modeling approaches, to perform metabolic analyses of the molecular, physical, and biochemical characteristics of blood plasma and urine samples to create informative translational models. These models, Bruker asserts, will predict variation in the severity of the disease and help clinicians understand differential responses to therapeutic interventions.

Coping with growing sample numbers

As more researchers explore the use of metabolomics and sample numbers increase, there is a need to enhance throughput without sacrificing coverage, identification, and accessibility. Many metabolites or lipids can exist in different isomeric forms, some biologically active and others not. They need to be properly identified if their biological significance is to be understood.

To facilitate discovery work, Waters offers high-resolution mass spectrometers, the Synapt XS high-resolution mass spectrometer and the Select Series Cyclic IMS system, both enabled with ion mobility separation technology that provides orthogonal structural information for identification. Ion mobility increases coverage, condenses chromatographic run times, and captures the sample complexity.²

In a metabolomics study that processes, for example, 1,000 samples, “ultraperformance liquid chromatography (UPLC) ion mobility MS can decrease analysis time from 1,300 to 200 hours,” says Suraj Dhungana, PhD, global biomedical research market segment manager, Waters. “For structural characterization of metabolites, our Cyclic IMS ion mobility mass spectrometer provides unique, on-demand variable ion mobility resolution. When isomeric metabolites cannot be separated using liquid chromatography, we can separate ions using ion mobility based on their rotationally averaged collision cross section (CCS).”

By exploiting its “racetrack” geometry, the Cyclic IMS system can improve ion mobility resolution as needed to separate small differences in CCS values by increasing the number of times the ions cycle around a mobility cell—the racetrack. This allows separation and very clean fragment ion spectrums for library matching.

To address the identification challenge for the natural products research community, Waters has partnered with a research group led by Roger G. Linington, PhD, at Simon Fraser University. This group maintains the Natural Products Atlas, a database of microbially derived natural product structures. The searchable version of the database contains 25,000 microbial compounds that can be directly queried with Waters discovery metabolomics software (Progenesis QI) and screening software (UNIFI).

“For added flexibility, we offer desorption electrospray ionization (DESI) and MALDI sources for discovery, targeted, and spatial studies,” adds Dhungana. “Metabolomics or lipidomics experiments and a DESI imaging experiment can be performed on the same instrument platform by simply swapping the source.”

Ensuring food quality and security

The food industry uses metabolomics to address the taste or smell of foods, develop new functional foods, and improve fermented food quality, says Eberhardt R. Kuhn, PhD, marketing manager, Food and Consumer Products, Shimadzu Scientific Instruments. Other applications include discovering patterns of abundance ratios of certain metabolites to confirm geographical origin of foods and beverages to verify food authenticity and prevent food fraud and identification of dietary biomarkers to screen for food allergens.

Shimadzu offers a variety of MS products for metabolomics research, such as triple-quadrupole LC-MS, quadrupole time-of-flight (Q-TOF) LC-MS, triple-quadrupole gas chromatography MS, and MALDI-TOF MS, as well as software platforms and databases that simplify workflows.

“Because it has a footprint the size of a sheet of paper, the MALDImini-1 Digital Ion Trap mass spectrometer can be placed on a workbench for a more convenient, efficient workflow,” asserts Kuhn. “The system’s digital ion trap uses rectangular wave RF to allow ion trapping up to 70,000 Da. Furthermore, the tandem MS (MS/MS) and three-stage MS (MS³) functionality allows comprehensive structural analysis of unknown metabolite biomolecules.”

When analyzing LC-MS/MS data, the procedure known as peak picking is a huge bottleneck requiring visual confirmation. Equipped with algorithms developed using artificial intelligence, Shimadzu’s Peakintelligence software automatically detects peaks appearing in chromatograms, substantially reducing the need for visual confirmation.

Shimadzu’s Metabolites Method Package Suite is a compilation of seven existing products including LC-MS/MS method packages, multiple reaction monitoring (MRM) libraries, and a GC-MS metabolite database. It enables the analysis of more than 1,900 metabolites without needing to investigate separation conditions, MRM optimization, or parameter settings. Metabolites span both hydrophilic and hydrophobic compounds.

The Metabolites Method Package Suite also incorporates the Multi-omics Analysis Package, which supports both regular and large-volume data analysis and interpretation, and contains metabolic pathways and other contour maps, making it easy to visualize fluctuations in the quantitative values of metabolites across metabolic pathways. Data filtering functions and statistical analysis can be applied to the network of compound relationships.

Accommodating different levels of expertise

Metabolomics provides a comprehensive snapshot of small molecules in a biological system. In medicine, metabolomics aids in understanding disease progression at the biochemical level revealing disease metabolic signatures; in an industrial setting, it can be used to optimize the production of monoclonal antibodies.

A metabolomics pilot study may total roughly 50 samples, while a study consisting of multiple experimental groups can reach several hundreds, and population-based biobank and repository studies can surpass thousands. As study size increases, high-quality quantitative data must be generated consistently over extended periods.

“Since metabolomics can be used to answer a variety of scientific questions, new scientists are entering the field with little to no knowledge on the use of LC-MS. We are also seeing traditional LC-MS users who are not familiar with the analytical requirements and nuances trying their hand at metabolomics,” says Amanda Souza, metabolomics program manager, Thermo Fisher Scientific.

This year, the company launched two new instruments as part of its Orbitrap Exploris mass spectrometer portfolio, the Orbitrap Exploris 240 Mass Spectrometer and the Orbitrap Exploris 120 Mass Spectrometer. These systems offer an easy-to-use and intuitive user interface for the mass spectrometry novice, and they come with application-specific method templates for metabolomics and lipidomics experiments providing recommended parameter settings. Instrument calibration is streamlined and accomplished by clicking on a check box.

To provide valuable biological insights, robust reproducible measurements and confident metabolite identifications are required. Collaborative efforts show the Orbitrap Exploris 240 MS robustly generates reproducible measurements over multiple days with sub-ppm mass accuracy, consistent signal response, and long-term stability. In addition, AcquireX acquisition allows for more unique unknown compounds with fragmentation spectra for increased annotation confidence.

Lastly, Compound Discoverer software can support large-scale metabolomics data analysis across multiple sample batches while providing statistical analysis, annotation tools, and pathway mapping.

Balancing perfection and practicality

“Researchers are recognizing that complete unbiased coverage of the metabolome is not practical,” says Steven M. Fischer, technical market director, Academia and Government, Agilent Technologies. “In the early days of metabolomics, a lot of effort was made trying to find the ‘perfect’ extraction protocol. That has now given way to more pragmatic approaches, whereby analysts focus on subsets of the metabolome.

“The result is less breadth, but more certainty about what was detected and how much was present. Coverage and identification issues will become more challenging as researchers try to extend metabolomics to single-cell analysis and tissue imaging.”

To help researchers’ address issues such as compound coverage, identification, and quantification, Agilent develops various technologies. Many of Agilent’s technologies—gas chromatography (GC), liquid chromatography (LC), capillary electrophoresis (CE), and supercritical fluid chromatography (SFC)—can be used in metabolomics analysis after they have been coupled to a mass spectrometer, such as a single-quadrupole, triple-quadrupole, TOF, or Q-TOF instrument. Each mass spectrometer has its advantages and disadvantages.

In addition to instrumentation, Agilent develops separation columns and solid-phase extraction (SPE) kits for metabolomics. For example, Agilent’s hydrophilic interaction liquid chromatography column (HILIC-Z) is designed to provide highly reproducible results in analyses of polar metabolites by high-performance liquid chromatography (HPLC). An SPE material (Captiva EMR-lipid) developed by Agilent allows for selective removal of lipids from biological samples, and it helps tremendously with the challenge of sample preparation. Specialized software enables metabolomics analysis.

The range of problems that can be studied with metabolomics will continue to expand. In the near term, metabolomics will focus on developing standardized methods, says Fischer. Such methods, he adds, reflect a growing interest in data sharing that is due, in part, to an expanding community of metabolomics researchers.

References
1. Kimhofer T, Lodge S, Whiley L, et al. Integrative Modeling of Quantitative Plasma Lipoprotein, Metabolic, and Amino Acid Data Reveals a Multiorgan Pathological Signature of SARS-CoV‑2 Infection. J. Proteome Res. 2020; August 17: online. DOI: 10.1021/acs.jproteome.0c00519.
2. King AM, Mullin LG, Wilson ID, et al. Development of a Rapid Profiling Method for the Analysis of Polar Analytes in Urine Using HILIC–MS and Ion Mobility Enabled HILIC–MS. Metabolomics 2019; 15: 17. DOI: 10.1007/s11306-019-1474-9.

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To bring the nervous system’s complexities into focus and discern openings for new treatments, new technologies are being developed. For example, optogenetics technology is refining our view of the brain’s structure and function, and new blood tests may allow us to glimpse Alzheimer’s disease in patients a decade before the first symptoms appear. Such tools may bring rapid progress against neurological disease.

Diagnosis: Alzheimer’s disease

Even simply diagnosing neurological disorders such as Alzheimer’s disease is a challenge. Because Alzheimer’s disease symptoms overlap significantly with other dementias, a decisive diagnosis depends on expensive and invasive procedures. Positron emission tomography (PET) imaging can reveal the characteristic amyloid protein deposits on the brain, and cerebrospinal fluid (CSF) testing can track biomarkers. But for large-scale screening, nothing would beat a blood test.

This year, a particularly promising blood biomarker candidate emerged: pT217, or tau protein phosphorylated at position 217. Blood measurements of pT217 correlate extremely well with its levels in CSF and with the PET imaging of tau pathology in the brain. Plus, it’s nearly undetectable in people who don’t have Alzheimer’s.

“I don’t think we’re going to get much better than this for Alzheimer’s disease,” says Nicholas Ashton, PhD, a neuroscientist at the University of Gothenburg, Sweden, who was not involved in the study. “The next step is that it needs to be on an automated platform.”

A pT217 blood test that uses mass spectrometry has been developed at the Washington University School of Medicine in St. Louis and licensed by C2N Diagnostics. The company is hard at work developing a commercial assay. “[We have] strong expertise in taking an academic-grade assay like that and industrializing it,” says Joel Braunstein, MD, co-founder and CEO of C2N Diagnostics. “We have a roadmap based on our experience of optimizing an existing assay that is about to reach the clinic.”

In addition to improving throughput and lowering costs, the team seeks to characterize how the biomarker behaves in different populations, including different racial and ethnic groups and people who may have additional health conditions.

Scientists at C2N have spent the last few years developing a mass spectrometry assay that reliably measures the ratio between two peptides in the blood, amyloid beta 42 and amyloid beta 40, an indicator that the amyloid plaques responsible for Alzheimer’s disease have begun to form in the brain.

“We now understand,” Braunstein asserts, “how those analytes behave, in the presence of different conditions and in different populations, and that test will soon be on the market.” He adds that this understanding will serve the company well as it works to optimize the pTau217 assay for widespread use.

Targeting the microbiome to protect the brain

In the early 1800s, when James Parkinson first described the “shaking palsy” that would later bear his name, he noted that in addition to tremors and muscle weakening, his patients also suffered with constipation. Curiously, digestive symptoms often precede the onset of motor symptoms, and over the last two decades, researchers have begun to suspect that Parkinson’s originates in the digestive system.

“Fast forward a couple hundred years, and now we have identified that diseases like Parkinson’s and others can emanate via the enteric nervous system,” says David H. Donabedian, PhD, co-founder and CEO of Axial Biotherapeutics. The enteric nervous system (ENS) encompasses the neurons that serve the digestive tract, and it is connected to the brain via the vagus nerve.

Sarkis Mazmanian, PhD, professor of microbiology at the California Institute of Technology and co-founder of Axial Biotherapeutics, believes the microbiome could be the key to Parkinson’s disease. The disease results when improperly folded alpha-synuclein (αSyn) protein forms aggregates that damage neurons in the brain. Mazmanian’s research has revealed that certain bacteria that produce amyloid proteins can accelerate αSyn aggregation and propagation to the brain.

“What’s really interesting,” Donabedian notes, “is that we’ve found higher levels of these amyloid-producing bacteria in Parkinson’s patients compared to their spouses and matched controls.” Rather than kill the amyloid-producing bacteria, which could throw the entire microbial ecosystem out of balance, Axial is developing small-molecule drugs that inhibit the bacteria from producing the problematic proteins. “The molecules we’ve developed,” Donabedian explains, “inhibit the aggregation and production of amyloids without killing the bugs.”

In July, Axial received a $440,000 research grant from the Michael J. Fox Foundation to create an in vitro model of the enteric nervous system. The model will allow a more detailed look at which cells are implicated in propagating the amyloids from the gut to the brain. Understanding more about the basic biology will help inform dosages and better predict the effect of the drugs.

Axial has demonstrated preclinical proof of concept in animal models and presented early clinical data showing the safety and tolerability of one of the company’s small-molecule candidates. According to Donabedian, efficacy data are expected later this year, and a second, more potent molecule is in the pipeline. “It’s all lining up as it relates to our mechanism,” Donabedian asserts. “We’re showing improvement.”

Optogenetics lights the way to improving deep brain stimulation

Parkinson’s patients currently take oral medication to relieve dyskinesia, but after years on the drug, the benefits eventually wane. Some patients find relief through deep brain stimulation (DBS), a procedure that implants a pacemaker-like device that sends electrical signals to electrodes placed inside the brain. Although DBS can dramatically reduce symptoms, the surgery to implant the device is technically challenging, and it helps only about 8–10% of Parkinson’s patients.

DBS uses electrical pulses to stimulate neurons in the subthalamic nucleus (STN), an almond-size region deep in the brain. It’s unclear exactly how that stimulation leads to a reduction of symptoms. Optogenetics lets researchers genetically program individual neurons to express a light-activated protein and to activate those neurons with pulses of light. In this way, researchers can observe the effects of stimulating very specific neurons, and no others.

At the Center for Interdisciplinary Research in Biology (CIRB), Collège de France, director of research Laurent Venance, PhD, set out to understand on a cellular level how DBS calms Parkinson’s symptoms. “Our hope,” he relates, “was to identify a more superficial target for which we could have less invasive access.”

Parkinson’s motor dysfunction has been associated with hyperactivity of a type of neuron called pyramidal cells, and DBS can settle them down again. “Pyramidal cells of the motor cortex are much more superficial than the STN,” Venance points out, “so it was a good target, and we started there.”

The researchers showed that DBS not only normalized the activity of pyramidal cells in the motor cortex, but also activated neurons called somatostatin-expressing GABAergic interneurons. Using optogenetics, the researchers selectively stimulated these neurons in a mouse model of Parkinson’s disease. The treatment improved the animal’s motor function considerably, suggesting that stimulating these somatostatin-expressing interneurons could be a less invasive target for relieving Parkinson’s dyskinesia.

“You have a unique opportunity with optogenetics to be highly specific in terms of a neuronal subpopulation,” stresses Venance. “You can remotely control a subpopulation of neurons and fine-tune the firing rate. Since it is the firing rate that is at the center of this Parkinsonian state, it was a good tool to specifically manipulate this subpopulation and choose the precise frequency of activation.”

Pooling tumor antigens to supercharge the immune system

Glioblastoma is the most commonly diagnosed brain tumor, and there is no cure. Targeted therapies have proved unsuccessful because the tumors are highly heterogeneous and genetically unstable.

“If you could make a vaccine from the patient’s own tumor, plus tumors of other patients that would have other antigenic profiles, you would have the entire tumor landscape covered,” says Joe Elliot, the managing director of ERC-USA, the American subsidiary of the Epitopoietic Research Corporation (ERC), which is headquartered in Belgium.

While developing this immunotherapeutic approach, ERC launched a Phase II trial in 2014 at the University of California, Irvine, for its cancer vaccine candidate, ERC-1671. This year, the company added a second site at Dana-Farber Cancer Institute. Located in Boston, Dana-Farber is the largest brain cancer referral center in the United States. To date, 84 patients have been treated, and with the second site, ERC-USA is, according to Elliott, “very optimistic that [it] will be able to significantly accelerate recruitment and complete this Phase II study within 12–18 months.”

ERC-1671 has also been approved for use under the FDA’s Right to Try Act, which allows its use by patients who have already tried all approved treatment options but who don’t qualify for a clinical trial.

Avastin (bevicuzimab) is currently approved to treat relapsed glioblastoma, and average survival is around four months, says Daniela Bota, MD, associate professor of neurology at UC Irvine and the principal investigator of the clinical trial. Patients get randomized to either avastin plus the vaccine or avastin plus placebo. “At the time of recurrence, patients who are getting placebo are allowed to cross over,” Bota details. “We need more work to confirm this, but our patients who have crossed over after progressing on Avastin live around 12 months.”

The personalized treatment contains tissue from the patient’s own tumor as well as tumors from three other patients. The allogeneic tumor tissue is carefully chosen for its major histocompatibility complex mismatch, that is, its potential to generate a strong rejection response. Irradiation prevents cancer cells from proliferating and forming new tumors. “They are still metabolizing, and they are still functioning cells,” explains Elliott. “But the DNA is destroyed, so they can’t reproduce.”

According to Bota, the side effects appear mild. “The patients feel well,” she declares. “They are very excited to participate in this treatment, and the number of patients who live one year or longer is going up.”

Untangling the mechanisms of multiple sclerosis

As powerful as the immune system is for fighting disease, it’s a scourge when turned on the wrong target. In multiple sclerosis (MS), immune cells attack the myelin sheath protecting the nerve cells, disrupting communication between the brain and the body. Symptoms range from muscle weakness and tremors to loss of vision, speech difficulties, and unsteady gait.

Recently, a type of immune cell called T helper 17 (Th17) emerged as a possible driver of autoimmune diseases, including MS. In mouse models of MS, researchers successfully staved off disease symptoms by inhibiting Th17 cell differentiation.

Th17 cells produce the cytokine IL-17, and drugs targeting IL-17 are currently used to treat certain autoimmune conditions, including psoriasis and some types of arthritis. However, clinical trials testing the anti-IL-17 drug secukinimab in autoimmune uveitis, an eye disease related to MS, failed to show efficacy.

Uveitis is being researched in mouse models by Rachel Caspi, PhD, head of the Immuoregulation Section and chief of the Laboratory of Immunology at the National Eye Institute. Working with mice that are prone to the disease, she was surprised to discover that knocking out the gene for IL-17A did not affect their disease symptoms.

“That started raising question marks,” Caspi says. “We hypothesized that in mice that are susceptible, there might be a circuit operating which compensates for lack of IL-17.” Caspi and colleagues thought that this circuit might, like many biological mechanisms, be part of a system that provides redundancy to protect against harmful loss of function. “Cytokines are not there to give us autoimmune disease, they are there to protect us against pathogens,” she explains. “In that context, compensation by other cytokines makes sense.”

The team found that IL-17A dampened the pathogenic activity of Th17 cells through a feedback system. IL-17A signals the cells to produce more IL-24, which slows down the release of other inflammatory cytokines by the Th17 cells.

Caspi says that at this point, it is a “leap of faith” that the findings in mice could translate to improved treatments for MS. In human cells, IL-17A exerts the same feedback control over Th17 cells’ release of cytokines, but that finding hasn’t been tested in patient samples yet. But if inhibiting IL-17 in humans does cause Th17 cells to pump out more cytokines, that might suggest better ways to silence the harmful inflammation wrought by the cells.

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In June 2014, Nick Loman, PhD, professor of microbiology at the University of Birmingham in the United Kingdom, took to Twitter to publish the first data that had been collected using a revolutionary new sequencing instrument. The data, presented in a graph affectionately known as a “wiggle plot,” depicted how an electrical current had changed over time as a DNA strand rapidly traversed a bacterial channel. Loman’s team had converted the graph’s peaks and valleys into the underlying DNA sequence of Pseudomonas aeruginosa.

Loman was an early devotee of the minION, a portable DNA sequencer, the first instrument produced by British biotech company Oxford Nanopore Technologies select ONT). His tweet came 25 years almost to the day after the concept of nanopore sequencing was hatched by David Deamer, PhD, research professor of biomolecular engineering at University of California, Santa Cruz.

In June 1989, “something clicked,” Deamer recalls, as he hit upon the central insight of nanopore sequencing after he considered what would happen if a strand of DNA were to pass through a channel in a membrane under a voltage.

At the time, genome scientist George Church, PhD, professor at Harvard Medical School, was working on similar ideas. A few years later, in 1995, Deamer, Church, Dan Branton, PhD, professor of biology, emeritus at Harvard University, and others developed their ideas into a patent application. According to Branton, the Harvard patent office thought, “This is such a wild idea. It’s never going to work.” Church’s advocacy, however, proved persuasive. Harvard relented and filed the application with the U.S. Patent Office, which granted the patent.

Realizing a vision

In 2005, ONT was founded as Oxford Nanolabs by Hagan Bayley, PhD, professor of chemical biology at the University of Oxford. Gordon Sanghera, PhD, was hired as founding CEO. Also participating in the company’s founding was Spike Willcocks, PhD, who was with the IP Group at the time and is now ONT’s chief business development officer. Sanghera secured the company’s first major investment—£500,000 from the IP Group—over drinks in an Oxford pub a few blocks from Bayley’s laboratory.

It wasn’t until February 2012, at the premier AGBT select Advances in Genome Biology and Technology) conference in Marco Island, FL, that Clive Brown, ONT’s chief technology officer, previewed the MinION, in a talk entitled, “Single Molecule ‘Strand’ Sequencing Using Protein Nanopores and Scalable Electronic Devices.” Brown had previously worked at Solexa, the British next-generation sequencing select NGS) company acquired by Illumina in 2007. His task was to somehow unseat the technology he had helped to build, a platform that had captured a huge portion of the NGS market.

ONT is competing not just with Illumina but also with Pacific Biosciences and MGI, as well as with startups just entering the sequencing game. Excitement is brewing over PacBio’s long, accurate HiFi reads, and MGI’s new sequencing platform with CoolMPS chemistry that the company claims can deliver a $100 genome select announced at AGBT this year, just before the pandemic).

ONT’s technology has evolved significantly since 2012. In addition to the MinION, ONT’s pocket-sized device with up to 512 nanopore channels, the ONT product lineup includes the larger GridION, a compact benchtop device designed to run and analyze up to five MinION Flow Cells while generating up to 150 Gb of data. For larger applications, ONT has developed the PromethION, a benchtop system that is ONT’s highest throughput sequencer with 48 flow cells that are capable of generating up to 8 Tb of data.

A newer product, the Flongle, is an adapter for the MinION or GridION that makes these instruments quicker and more accessible for smaller tests and experiments. Albert Vilella, PhD, a bioinformatics consultant, tells GEN that the ability to do a $100 experiment with the Flongle flowcell, something ONT has pointed at in their recent updates, would be a “game changer” and, he expects, would lead to increased adoption of ONT’s technology. None of the competition, Villela asserts, “are anywhere near” being able to deploy technology at that price.

The Plongle, essentially a 96-well-plate-compatible Flongle that can carry out larger numbers of small, quick tests in parallel, is due to be released soon.

Brown noted at the London Calling conference that every time he thinks of one of these “crazy names,” it “turns out to mean something rude in Australia.” And the SmidgION, also in development, will be ONT’s smallest device yet, designed for use with smartphones or other mobile, low-power devices.

Now, 15 years after its inception, ONT sees an opportunity to realize its full potential. Sanghera has long pointed to the ability of the ONT sensor to provide a rapid readout of DNA, like a pinprick test for diabetics. The COVID-19 pandemic offers just such an opportunity. And the stakes could scarcely be higher.

From generating sequences to diagnoses

With a high-profile announcement in early August, ONT signaled its determination to make an impact in the diagnostics of COVID-19 as the pandemic pushed academic scientists and companies to innovate at extraordinary speeds. ONT announced the rollout of LamPORE, a COVID-19 test, in an agreement with the United Kingdom’s Department of Health and Social Care.

LamPORE is designed to work on swabs and saliva samples. One MinION can hold up to 1,500 barcoded patient samples and complete a run in about 90 minutes.

“LamPORE has the potential to deliver a highly effective and, crucially, accessible global testing solution,” said Sanghera. “Not only for COVID-19 but for a range of other pathogens.”

LamPORE is the marriage of two processes, loop-mediated isothermal amplification select LAMP) and nanopore sequencing. LAMP is a relatively low-maintenance process to amplify DNA with high specificity and efficiency. It can be done cheaply in a single tube at a constant temperature.

Developed two decades ago by a group of Japanese researchers, LAMP has enjoyed a recent resurgence due to its applicability in COVID-19 diagnostics. Other groups developing COVID diagnostics, including Color, Sherlock Biosciences, and STOPCovid, also rely on LAMP for amplification.

After amplification, LamPORE uses nanopore sequencing to identify three genes of the SARS-CoV-2 virus. The method can differentiate between the presence of the virus and errors that can occur during amplification—a source of false-positive results. In addition, the test includes an internal control of human mRNA to identify errors in sample collecting select for example, poor swabbing procedure union which can be a source of false-negative results.

In addition to SARS-CoV-2, ONT is developing LamPORE to test for multiple pathogens within a single sample, including influenza A select H1N1 and H3N2 union influenza B, and respiratory syncytial virus. As Keith Robison, PhD, longtime genomics blogger, wrote, “More widespread deployment and use of respiratory virus tests could be one thin silver lining from the dark cloud of the pandemic.”

LamPORE is garnering excitement, in large part, due to its scalability, which could provide screening of frontline workforces and rapid screening in areas such as airports, nursing homes, and schools. Regulatory submissions for LamPORE are underway and awaiting approval.

Changing sequencing on the fly

Adaptive sequencing—a type of selective sequencing—builds a decision point into the sequencing process depending on whether a sequence of interest is present. To understand adaptive sequencing, it is important to first understand how nanopore sequencing works select see the sidebar entitled “Nanopore Sequencing’s Nuts and Bolts”).

If the region of interest is present, the sequencing continues. If not, the voltage is reversed, the DNA strand is ejected, and the nanopore is freed up for a new strand. This decision point is made through a process that matches the DNA sequence to a reference sequence.

With this technology, a researcher can have selective sequencing without upfront preparation or sample enrichment. And it allows for dynamic changes during the process, by controlling pore voltages in real time.

During a typical sequencing run, explains Michael Schatz, PhD, associate professor of computer science and biology at Johns Hopkins University, the data can be redundant or from irrelevant regions of the genome. “Adaptive sequencing changes all of this,” Schatz notes, because it can selectively target the reads that are relevant for a given project.

The “killer application,” he explains, is for targeted sequencing—when a researcher is interested in a particular set of genes. When researchers in Schatz’s lab targeted 148 genes associated with hereditary cancer using adaptive sequencing, they were able to sequence the genes with one flow cell instead of the standard five or six.

Schatz points to adaptive sequencing’s utility in metagenomics, selectively sequencing genomes of interest and enriching low-abundance material. Lastly, Schatz notes that they are currently working toward extending the approach to cDNA and direct RNA sequencing to allow for the selective sequencing of transcripts. He tells GEN that he can see a day when “all nanopore sequencing will use this approach for DNA and RNA sequencing projects.”

Redefining “long”

One of the great advantages of long-read sequencing—the forte of ONT and PacBio—is the ability to read through complex, highly repetitive regions of DNA. Despite the tremendous advancements in sequencing technology since the completion of the Human Genome Project, scientists had been unable to complete the contiguous sequence of an entire chromosome, from end to end, until the task was undertaken by the Telomere-to-Telomere select T2T) consortium.

The T2T, an open program to work on generating the first complete assembly of the human genome, is headed by Karen Miga, PhD, assistant research scientist at the UC Santa Cruz Genomics Institute, and by Sergey Koren, PhD, and Adam Phillippy, PhD, both from the Genome Informatics Section, Computational and Statistical Genomics Branch at the National Human Genome Research Institute. Last July, in Nature, the T2T reported the first gapless, telomere-to-telomere assembly of a human chromosome.

A particularly difficult step in assembling a complete chromosome is generating assemblies of repetitive DNA regions. Miga and colleagues were motivated to establish the T2T consortium by asking, “Can high-coverage ultra-long sequencing resolve complete assemblies of the human genome?”

Although nanopore sequencing was at the core of generating the high-coverage, ultra-long-read sequencing of the complete genome select from hydatidiform mole CHM13 union the team used a multiplatform approach, one that included PacBio and Illumina platforms, as well as complementary technologies for quality improvement and validation, such as polishing technology from 10x Genomics and optical map technology from BioNano Genomics.

This project was performed on a haploid genome, but Miga notes that the group has its sights set on diploid samples. Miga noted during her London Calling talk in 2019 that “[for too long] we’ve accepted an incomplete human reference genome with hundreds of gaps.” The aim at T2T is to shift the standard in genomics to completeness and quality. Miga asserts that we are entering a new era that “demands complete, high-quality chromosome assemblies.” If that is indeed the case, it will be interesting to watch the role ONT plays in the next generation of sequencing.

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Moreover, lysins can infiltrate a biofilm, a bacterial community that normally offers bacteria extra protection from antibiotics. When biofilms are treated with antibiotics, only the organisms on the surface of the matrix are killed. In contrast, lysins dissolve biofilms from the top down. The bacteria burst open, revealing the next layer of the biofilm, making it vulnerable to further lysin exposure.

Graham Hatfull, PhD, a professor at the University of Pittsburgh specializing in phage biology, says there are vast numbers of different lysins, each with specific cell wall targets, creating a huge space for discovery and development. The lysins are relatively cheap and easy to produce. For several Gram-positive bacteria—which have only a single membrane located interior to the cell wall, giving lysins direct access to the peptidoglycan targets—good antibacterial activity has been shown in vitro. In short, Fischetti says, “They work.”

One reason is that the research did not leave Rockefeller University until about 10 years ago. That’s when Yonkers, NY-based biotech Contrafect approached Fischetti about licensing the technology to develop lysins as a therapeutic. Raymond Schuch, PhD, a research assistant professor in Fischetti’s lab at the time, joined Contrafect, where he is now vice president of research.

Schuch tells GEN that he made the move to Contrafect because it opened a whole new area of translational research that “we usually don’t think about in research laboratories.” Schuch continues: “We spent years identifying these lysins, defining their characteristics, showing that they confer therapeutic benefits in animals.” He wanted “to continue along the pathway of the development.”

In addition to lysins, Contrafect develops amurins, phage-encoded lytic antimicrobial peptides. Still in the discovery phase, these peptides have been shown to have in vitro activity against Gram-negative bacteria. The electron micrograph shows lysis of the Gram-negative bacterium Pseudomonas aeruginosa. select Lysis occurred over 20 minutes.) Treatments to counter Pseudomonas pathogens are much sought after because the pathogens cause multidrug-resistant nosocomial infections.

Over the past decade, the small company of 25 employees has taken the Staphylococcus aureus lysin Exebacase into the clinic, completing Phase I and Phase II of a trial. Contrafect is currently enrolling patients in Phase III. This is the first and only lysin to enter human clinical trials in the United States. The data showed that Exebacase, given in combination with antibiotics, improved clinical outcomes in patients with Staphylococcus aureus bacteremia, including endocarditis, when compared to antibiotics alone.

Although Exebacase is further down the clinical trial pipeline than any phage therapy in the United States, phage therapy has attracted all the public attention. Few have heard of lysins. “I don’t really know why,” remarks Fischetti. “That’s the problem.” One reason may be that lysins have not yet been approved for use in the compassionate care cases that tend to garner attention. When asked if there are any known disadvantages to using lysin over phage, Fischetti is adamant: “There are none.”

Path of least resistance

One of the major problems with phage therapy is the ability of bacteria to develop resistance, similar to the resistance that is rampant with antibiotics. In the recent cases where phages were used for compassionate care, the medical teams opted to use a cocktail approach, believing that one or even two phages could lose efficacy at some point due to the onset of bacterial resistance.

For example, in the case last year of the 15-year-old cystic fibrosis patient Isabelle Carnell-Holdaway, who was treated for a disseminated Mycobacterium abscessus infection, three phages were used. Hatfull, who supplied the phages, says, “Three was a number that gave some confidence that we wouldn’t see resistance.” The more phages, he notes, “the more effectively you can counter that concern.”

Hatfull observes that lysins, unlike phages, are associated with “very low or even undetectable levels” of bacterial resistance. This, he maintains, is “a major advantage.”

Why is resistance not a problem for lysins? Fischetti says that “it’s keyed into the way that [the phages] have evolved.” Lysins are essential for the survival and release of the phage. Their target is a critical component of the bacterial cell wall that is essential and cannot be changed easily. For this reason, lysins tend to retain their potency against bacteria, which find it far more difficult to acquire resistance to phage lysins than to phages or antibiotics.

Lysin smoothies

Fischetti has recently teamed up with Lumen Bioscience, a Seattle-based company founded in 2017 with technologies for bioengineering spirulina, a photosynthetic microbe consumed worldwide as a nutritional supplement and food source. The blue-green algae can allow lysins to be produced for a fraction of the cost associated with conventional techniques. In fact, Fischetti asserts that algae can take the cost of lysins “down to pennies a dose.”

Producing lysins traditionally requires a biomanufacturing system and complicated purification processes. Algae are extremely easy to grow in enormous quantities select they grow in tap water union and they are the only microbes that can be commercially farmed at these scales.

Brian Finrow, the CEO of Lumen, tells GEN that the Fischetti laboratory will generate the lysins and that Lumen will carry out the protein engineering and other activities to create therapeutic strains of spirulina and carry those forward to FDA-supervised clinical trials.

Fischetti says that the spirulina could be used as a “lysin factory,” producing large quantities of lysin to be purified. But it is not implausible that someone could eat spirulina that is expressing lysin to have the enzymes coat their intestinal tract. He adds that this technology and collaboration opens interesting possibilities for lysin, including moving into the veterinary field. select As they are currently made, lysins are too expensive to be used for veterinary purposes.)

Time will tell

Hatfull is a proponent of phage therapy, but he says that “the proof-of-principle studies [using lysins] are encouraging.” As with every young field, there are many unresolved questions. Will lysins be able to access niches of bacterial infection in vivo, as these enzymes are larger than typical antibiotic molecules? And although the early data in Contrafect’s clinical trial regarding the host’s immune response look promising, might immune responses to the lysin limit their action in patients for extended or repeated periods?

In addition, the utility of lysins for Gram-negative pathogens remains unclear because of the need to get the lysins past the outer bacterial membrane. Gram-negative bacteria, in contrast to Gram-positive bacteria, have a second membrane located exterior to the cell wall, making access to the peptidoglycan layer more challenging. Fishcetti’s laboratory is working on this problem: the team has lysins that, when modified, can get past the outer membrane. But development of the Gram-negative lysins is behind the development of the Gram-positive lysins.

Phage and bacteria have been evolving together for a billion years, Fischetti explains. They have been battling back and forth, building systems so that nobody loses—and nobody wins—because “whoever wins, loses.”

Phage therapy is trying to take a billion-year-old, established system, where nobody is supposed to win and force the phages to win, notes Fischetti. He adds, “That is not going to happen easily.” One challenge is that we lack a full understanding of the mechanisms of bacterial resistance. “We know about CRISPR,” he says, “but we don’t know about all of the other bacterial defense systems against phage.”

Fortunately, resistance to lysins has not been seen in the 20 years of working with them. Bacteria have built up resistance to both phages and antibiotics, but “they don’t know how to handle a lysin,” notes Fischetti. The lysins, he says, “take the bacteria by surprise.” Fischetti notes that his laboratory tries to force resistance. Doing so is proving to be difficult. If resistance is difficult for scientists to achieve in the laboratory, it may also be difficult for bacteria to achieve in nature.

Lysins could buy us time select maybe decades) by killing antibiotic-resistant bacteria until new methods are discovered, Fischetti speculates. Using lysins, he adds, is simply taking advantage of something that phages have figured out—of something that has helped them survive—and using it to our own benefit.

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Because a patient’s microbiome consists of interacting elements—including elements that extend beyond the microbiome itself—these elements cannot be seen in isolation. Rather, they are dynamic parts of a systemic whole. Touch any one of them, and the effects of doing so may ripple outward in unpredictable ways—unless the elements and their interactions are thoroughly understood.

Fortunately, comprehensive knowledge of the microbiome has been and continues to be accumulated by researchers. Even better, this knowledge is being exploited in clinical applications. As this article shows, these applications are being developed by firms of various types—biotechnology companies, contract research organizations, and personal care companies.

Antibodies, not antibiotics

“We are covered by, and protected by, and interacted with by vast microbial ecosystems,” says Julius Goepp, MD, founder of Scaled Microbiomics. Everywhere the body comes into contact with the outside environment, you’ll find a thriving community of microbes. This includes places that are obviously “external”—like skin and hair (including the skin and hair of underarms and nostrils)—as well as places that we consider to be “internal,” like the gastrointestinal tract.

“The surface of our gut is continuous with the outside world,” Goepp points out. The miracle of our gut, he continues, is that it can transport “two pounds of very nasty material [while keeping] it one cell layer away from our precious, sterile, inside tissue.”

But one cell layer can prove precarious protection, especially if that microbial ecosystem gets out of balance. The gut’s microbial ecosystem, Goepp suggests, is like the Amazon rainforest ecosystem in that it is resilient, but only to a point. When subjected to stressors such as prolonged exposure to antibiotics, a diet high in certain additives and low in fiber, and environmental pollution, the microbial ecosystem can be tipped far enough out of balance that a new normal becomes established. This is called dysbiosis, and it’s increasingly linked with a number of noncommunicable diseases, such as diabetes, neurodegenerative diseases, and cancer.

For instance, hereditary colorectal cancer has been linked to an excess of two damaging types of bacteria. The first, a strain of Bacteroides fragilis, attacks the protective layer separating the microbiome from the colon cells. The second is a type of Escherichia coli that produces a DNA-damaging metabolite called colibactin. When B. fragilis breaks down the protective layer, E. coli may gain access to the intestinal epithelial cells, and colibactin may wreak havoc with the DNA.

In a retrospective longitudinal study of colorectal cancer patients, the telltale signature of colibactin-induced mutations was found in 20% of tumors. Evidence suggests that most of that damage was sustained during childhood. “Had there been an intervention to stop that genomic damage from happening, those people might not have had their cancer,” Goepp suggests.

Goepp has high hopes for the antibody approach. “We’re applying an old, established, mature technology to a new organ, a new series of cell types, and a new series of signaling molecules,” he says. “We believe that we can use our methodology to learn how disease cascades work and how they can be disrupted.”

Human-first discovery

“The microbiome is a new organ that we discovered just 12 to 15 years ago,” says Sonia Timberlake, PhD, vice president of research for Finch Therapeutics. “There’s a lot of complex and unknown biology.”

To tackle that complex biology, Finch is taking a human-first approach that draws on years of data from fecal microbiota transplants. The company is leveraging its long-standing association with OpenBiome, the first public stool bank, to move beyond fecal transplants and begin delivering full-spectrum microbiota in pill form. Two of OpenBiome’s founders, Mark Smith, PhD, and Zain Kassam, MD, are also founders of Finch, and now serve as Finch’s chief executive officer and chief medical officer, respectively.

OpenBiome was founded to expand access to fecal transplants for the treatment of Clostridioides difficile overgrowth, which can cause debilitating inflammation in the gastrointestinal tract. A naturally occurring component of the microbiome, C. difficile can get out of hand because it grows back faster than other species after a course of antibiotics. Once established, C. difficile can prove extremely hard to unseat, even with more antibiotics. A fecal transplant reintroduces a diverse population of naturally occurring gut bacteria, and in theory, once antibiotics are stopped, these bacteria can resume their normal abundances and keep C. difficile in check.

In addition to producing full-spectrum microbiota products, Finch is developing “rationally selected microbiota” products, a strategy the company hopes will enable it to scale up the technology. “Because each healthy donor’s community is slightly different, and each patient’s community before and after treatment is slightly different, you can start to ask which microbes are the active ingredients,” Timberlake explains. A full community transplant contains approximately 500 different microbial strains, but only 50 to 100 of them might take hold in the recipient. Using high-throughput DNA sequencing and metabolomics, scientists at Finch can identify which species correlate with the patients who get better.

Identifying the species most important for healing the gut is a key step in scaling up production. Instead of extracting a full community of microbes from an individual donor for every patient, a laborious process, investigators can grow and develop rationally selected strains into a more streamlined, off-the-shelf product. “Your product is much simpler, and you can really understand the safety at a gene-by-gene level,” Timberlake points out. She adds, however, that few established industrial protocols work well for culturing human gut bacteria, and growing them at scale remains a major technical challenge.

Based on the success of microbiome transplants for treating C. difficile, Finch hopes to develop treatments for inflammatory bowel disease (IBD), an autoimmune disease that causes inflammation of the gastrointestinal tract. IBD includes Crohn’s disease and ulcerative colitis, and it can be managed with medications or surgery. These treatments, which can involve powerful immune suppressants, are often expensive and unpleasant, and they work only in a fraction of patients.

“This field has so much promise to treat a lot of diseases increasing in our population,” Timberlake maintains. “I really think these therapies are going to be very safe, and they have a lot of potential.”

Skin deep

Like the gut, the skin hosts a teeming community of microbes, but studies of this microbiome began much more recently. Skin dysbiosis has been correlated with some skin disorders, such as atopic dermatitis (AD) and dandruff. Researchers are working to understand exactly which microbes might be involved, and whether treatments directed at those microbes can lessen the symptoms.

AD is a common, allergy-related skin inflammation that usually begins in childhood. Babies who are born by cesarean section are at increased risk for AD, explains Cécile Clavaud, PhD, project leader, Skin Microbiome Unit, Research and Innovation, L’Oréal. These babies have a very different community of microbes on their skin than babies born vaginally.

The typical treatment for AD involves corticosteroids to decrease the itching and redness associated with a flare-up. “This has a short-term effect,” says Clavaud. “The redness decreases but comes back a few weeks later.” She notes that watching changes in the microbiome over time reveals an overgrowth of Staphylococcus aureus bacteria in the days leading up to an outbreak of AD. “During the crisis, you have much lower diversity,” she continues. “You have increasing S. aureus, and other bacteria decrease. One type of bacteria is taking all the space, all the food, everything.”

L’Oréal researchers demonstrated that applying an emollient containing a probiotic extract could extend the time between AD outbreaks. “This extract will stimulate innate immunity and help the microbiome to recover its right diversity,” Clavaud asserts. The inactivated probiotic agent stimulates production of certain cytokines by immune cells in the skin. These not only help keep S. aureus in check, they also have anti-inflammatory activity that reduces the redness and itching characteristic of AD.

In their studies of the scalp, scientists at L’Oréal have shown that people with dandruff have a disruption in the ratio of two bacteria. A healthy scalp has more Propionibacterium acnes and less Staphylococcus epidermidis, while in dandruff sufferers, the ratio is reversed.

Dandruff is typically associated with an excess of yeast in the Malassezia genus, and over-the-counter shampoos that treat dandruff generally contain antifungal agents. L’Oréal is working on a formula specifically targeting S. epidermidis. “By targeting the bad bacteria, we limit its growth, and there’s an additional improvement in the symptoms,” Clavaud maintains.

Our understanding of the skin microbiome is only just beginning. Clavaud and her colleagues recently published the genome sequence of Malassezia restricta to better understand its role in the scalp community and how it may contribute to dandruff. They are also working to uncover the roles of environmental pollution and aging on the microbiome. “The story is continuing,” declares Clavaud.

Ultra-high-resolution genomic profiling

When Clinical Microbiomics set up shop in 2015, it began by offering 16S gene sequencing for microbiome samples. It wasn’t long, however, before the company’s clients began requesting more advanced genomic analysis.

“Most of our clients are not bioinformaticians,” says H. Bjørn Nielsen, PhD, chief scientific officer of Clinical Microbiomics. To serve those clients better, Clinical Microbiomics provides various individualized services at different stages of a project’s development. “We have built a large framework of different bioinformatics tools,” Nielsen points out, and that framework enables the company to provide a powerful analysis specific to the project at hand. “It’s a very custom, tailored analysis that we’re providing,” he asserts.

Some clients, for instance, request guidance even before beginning the study, to determine an appropriate number of samples to include for the best chance of seeing an effect. Once samples have been collected, Clinical Microbiomics extracts and sequences DNA and applies bioinformatics tools to extract meaning from the data.

In 2014, Nielsen and colleagues at the Technical University of Denmark pioneered a technique called shotgun metagenomics. The method allows identification of new microbial species without the need for reference sequences, based on identification of co-abundant genes. Using shotgun metagenomics, Clinical Microbiomics provides ultra-high-resolution microbiome analysis to accurately detect differences in microbes of the same species.

Ultra-high-resolution analysis reveals each person’s microbiome as a genetically unique entity. Two people can harbor the same bacterial species, but each one’s population of that species could be genetically distinct from the other. “Then you have n of 1,” Nielsen explains. “So, how do you do any statistics with that?” Clinical Microbiomics uses statistical methods to construct phylogenetic trees of all these genetically distinct bacterial populations. By doing this, it may be possible to associate one branch of the tree with a particular host characteristic. “We have found many cases of this already, where there are clades of bacteria that are associated with a human phenotype,” Nielsen notes.

Another approach is to look at functional differences, rather than taxonomic ones. By identifying species that encode particular biochemical pathways, Clinical Microbiomics can uncover clues to possible mechanisms driving certain phenotypes.

“One of the new things we have started offering also is absolute abundances,” Nielsen says. “This is a bit of a game changer for certain types of analysis.” Relative abundances can be misleading, he points out, because a species that appears to be rising in abundance may actually be staying the same, while other species are dying off. Absolute abundances can provide critical information for understanding the role of microbiome metabolites in certain conditions.

Clinical Microbiomics aims to help academic or pharmaceutical researchers make sense of their data in the context of clinical applications. “There are lots of very good companies that have a clinical focus,” Nielsen relates. “They just want to understand how the microbiome works, and they don’t have a team of bioinformaticians for that. We can help them with that, so they can have their focus on the medical side.”

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Antibody–drug conjugates (ADCs) are an emerging class of targeted therapeutics with the potential to improve the therapeutic index seen with traditional chemotherapy. An ADC consists of three components: a monoclonal antibody or antibody fragment (which targets a tumor-associated antigen); a highly cytotoxic entity; and a chemical linker to conjugate the first two components. Binding of the antibody to the cell surface triggers internalization, and processing within endosomes or lysosomes releases the potent cell-killing molecule. An ADC’s mechanism of action combines the targeting power of an antibody with a potent cytotoxic agent, making it possible to eradicate cancer cells more effectively and selectively, while reducing the side effects which undermine patient quality of life (Figure 1).

Realizing the potential of an ADC depends on optimizing each part of the three-component ADC jigsaw puzzle. There are several factors which affect the likely clinical success of an ADC, including antibody choice, levels of heterogeneity and of target expression, payload potency and mechanism of action, conjugation strategy, and the choice of cleavable versus non-cleavable linker. To ensure a wide enough therapeutic window, the conjugate should be stable in plasma to prevent premature release of toxin in systemic circulation, and make use of a cytotoxic payload that once released within the cell is of sufficient potency to eradicate the tumor.

Clincal status

Currently, seven ADCs have been approved for therapeutic use: brentuximab vedotin (Adcetris^®), trastuzumab emtansine (Kadcyla^®), inotuzumab ozogamicin (Besponsa^®), gemtuzumab ozogamicin (Mylotarg^®), polatuzumab vedotin (Polivy), enfortumab vedotin (Padcev), and trastuzumab deruxtecan (Enhertu^®). Adcetris, Polivy, and Padcev are conjugated to the tubulin inhibitor monomethyl auristatin E (MMAE), and Kadcyla to the tubulin inhibitor mertansine (DM1), via maleimide linkers. Besponsa and Mylotarg are conjugated to the DNA-damaging agent calicheamicin via an acid cleavable linker, and Enhertu is conjugated to the topoisomerase I inhibitor, deruxtecan, via a maleimide linkage. The clinical and commercial potential of ADC technology is reflected in the burgeoning pipeline. Nearly 90 ADCs entered clinical trials in 2019.²

Payload development

First-generation ADCs, which incorporated familiar chemotherapeutic agents such as methotrexate, vinblastine, and doxorubicin, lacked sufficient potency even though they had been conjugated to antibodies to increase their specificity. Although the cBR96-doxorubicin conjugate showed promise in preclinical studies and progressed to a Phase II trial, it was insufficiently efficacious. This was largely attributed to under-potency of the doxorubicin payload, as it is estimated that 4–12 million doxorubicin molecules are required to kill a cell. Antigen expression levels are generally under 1 million copies per cell, making it difficult to achieve a critical concentration of toxin.³

Until recently, the majority of ADCs in development employed toxins from three product classes⁴: calicheamycins (Wyeth/Pfizer), maytansines (ImmunoGen), and auristatins (Seattle Genetics). This gives an indication of how difficult it is to find suitable payloads for use in the ADC field.

To be effective, a toxin is required to possess sufficient potency to elicit efficacy. Cytotoxic molecules for use in ADC design would ideally have subnanomolar potency for optimal efficacy. Payload concentrations in target cells are often low due to poor tumor localization and insufficient internalization. Toxins should also possess a functional group for conjugation of a linker, or be capable of being chemically modified to generate an appropriate site to allow for release in tumor cells. Additionally, the cytotoxin should be cost effective to manufacture.

Tubulin inhibitors from the auristatin and maytansine classes have been widely used.⁵ Auristatins block tubulin assembly and cause G2/M phase cell cycle arrest. Maytansine, a naturally occuring isolate from the African shrub Maytenus ovatus, is also a potent inhibitor of microtubulin assembly. While free drug potency is generally in the range of 10⁻⁹ to 10⁻¹¹ M for tubulin inhibitors,⁶ and these conjugates have been approved in several indications, they are not as effective in indications with lower expression of target antigen or cells that are less sensitive to tubulin inhibition. There has therefore been a push toward design of payloads to address targets where tubulin ADCs are proving to be insufficiently active, such as colon cancer.

DNA-damaging agents are the other major toxin class used in the development of ADCs, providing a real shift in potency to the subnanomolar range, with some analogues exhibiting activity in the low picomolar range. These include the calicheamicin payload employed in both inotuzumab ozogamicin and gemtuzumab ozogamicin, as well as in duocarmycins, topoisomerase inhibitors, pyrrolobenzodiazepines (PBDs), and indolinobenzodiazepines (IGNs). The increase in potency allows for targeting of tumor-specific antigens that are expressed at lower density. As DNA-interacting payloads are active in nonproliferating cells, targets can be widened to include tumor-initiating cells (TICs).

Toxicity and stability

Recently, clinical trial data have suggested that PBD-containing ADCs cause significant toxicity issues in patients. A Phase III trial of the CD33-targeting vadastuximab talirine was discontinued due to an unfavorable safety profile, for example.⁷ PBDs are based on naturally occuring, antitumor antibiotics that bind to the DNA minor groove in a sequence-specific manner. PBD dimers can form interstrand and intrastrand DNA crosslinks by covalently binding to the nucleophilic C2-amino group of a guanine base. Clinical trial failures have led to a shift from crosslinkers toward DNA monoalkylators, such as the IGNs, which have similar potency to PBDs. Crosslinking IGNs also have unfavourable toxicity profiles, but DNA-alkylating IGNs have not shown prolonged or delayed toxicity.⁸

In first-generation ADCs, conjugate stability was a second factor undermining efficacy, and this factor becomes even more critical as more potent payloads are developed. The first ADCs conjugated the payload primarily via cysteine and lysine residues that are naturally present in the antibody structure. However, the acid-cleavable linker used in gemtuzumab ozogamicin has been associated with nonspecific release of the drug in circulation. Second-generation ADCs often use maleimide-based conjugation chemistry (but this has a propensity to deconjugate via a retro-Michael reaction) and cross-conjugate to thiol-containing proteins such as albumin.

Advances in the field have led to conjugation technologies with improved stability, such as ring-opened maleimides that are stabilized against deconjugation, and Iksuda Therapeutics’ vinyl pyridine-based PermaLink^® technology, which does not undergo the retro-Michael reaction.⁹ Third-generation ADCs make use of site-specific conjugation strategies such as engineered cysteines and nonnatural amino acid engineering.¹⁰

Emerging preclinical and clinical data will continue to guide the progress of ADCs, as this important therapeutic class increasingly offers benefits in efficacy and tolerabilitiy over current standard-of-care chemotherapies. These results will also help inform the ongoing innovation in payload and conjugation strategies that have the potential to improve efficacy in solid tumors, and broaden the range of indications that can be treated.

References

1. Cancer Survival Statistics. Cancer Research UK. Accessed April 2019.
2. Beacon ADC Landscape Infographic H1 2020. Beacon Targeted Therapies, Hanson Wade. Published January 2020. Accessed April 2019.
3. Khera E, Thurber GM. Pharmacokinetic and Immunological Considerations for Expanding the Therapeutic Window of Next-Generation Antibody-Drug Conjugates. 2018; BioDrugs 32(5): 465–480.
4. Lyon R. Drawing lessons from the clinical development of antibody-drug conjugates. Drug Discov. Today: Technol. 2018; 30: 105–109.
5. Beacon ADC Digest. March 2019. Preclinical ADC landscape. Beacon Targeted Therapies, Hanson Wade.
6. Diamantis N, Udai B. Antibody-drug conjugates—an emerging class of cancer treatment. Br. J. Cancer 2016; 114: 362–367.
7. Jackson PJM, Kay S, Pysz I, Thurston DE. Use of pyrrolobenzodiazepines and related covalent-binding DNA-interactive molecules as ADC payloads: Is mechanism related to systemic toxicity? Drug Discov. Today: Technol. 2018; 30: 71–83.
8. Miller ML, Shizuka M, Wilhelm A, et al. A DNA-Interacting Payload Designed to Eliminate Cross-Linking Improves the Therapeutic Index of ADCs. Mol. Cancer Ther. 2018; 17(3): 650–660.
9. Frigerio M, Kyle AF. The Chemical Design and Synthesis of Linkers Used in Antibody Drug Conjugates. Curr. Top. Med. Chem. 2017; 17(32): 3393–3424.
10. Beck A, Goetsch L, Dumontet C, Corvaïa N. Strategies and challenges for the next generation of antibody-drug conjugates. Nat. Rev. Drug Discov. 2017; 16(5): 315–337.

Jenny Thirlway, PhD, is Senior Director and Development Program Team Leader, Iksuda Therapeutics.

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If regenerative medicine is to succeed and deliver much-desired cures, it will need to create living tapestries as closely woven and richly detailed as those found in natural, intact tissues. Such tapestries, it happens, are emerging from regenerative medicine’s workshops, which are employing diverse materials and realizing varied designs.

The materials include bioinks, microscale scaffolds (collagen matrices and vascular structures), extracellular vesicles, immunomodulatory and other signaling factors, and—of course—stem cells. All these materials are being incorporated into dermal fillers, cartilage, bone tissue, and even more ambitious works.

Soon, regenerative medicine will attempt cures for a vast array of disorders, including cancer, diabetes, cardiovascular disease, autoimmune diseases, and bone and joint inflammations. Already, the discipline’s looms are working toward 3D printed organs. Such masterpieces could end donor organ shortages while avoiding rejection issues.

Photocurable regenerative fillers

Type I collagen is the basic building block in connective tissues. To mass produce recombinant human collagen (rhCollagen), CollPlant Biotechnologies uses tobacco plants, genetically engineered with the five genes responsible for the protein’s synthesis.

The tobacco plant was selected as the host for collagen synthesis for three main reasons: the well-known tobacco genome facilitates manipulation; tobacco is not part of the food and feed chain; and the large biomass leaves can be harvested in 6–8 weeks and the plants reharvested through three growing cycles.

CollPlant’s rhCollagen is the basis of the company’s BioInk products, which are used for 3D bioprinting. To produce a BioInk, CollPlant begins by chemically modifying rhCollagen so that it undergoes crosslinking when exposed to light. Next, polymers are added to mimic the target tissue’s physical properties. Finally, biological constituents make each BioInk biologically tissue specific.

“Our rhCollagen is ‘virgin’ with many cell-binding domains,” says Yehiel Tal, CollPlant’s CEO, “and it shows zero immune response compared to products that are based on tissue-extracted collagen.”

“BioInks are injected through very fine nozzles,” he continues. “We reduce the shear force significantly during the deposition then return it to the original viscosity to maintain high cell viability. After deposition, our BioInks can be photocured quickly with accurate positioning while enabling high-production throughput.”

“We jointly develop products with leading companies and are also building a portfolio of medical aesthetic products in which the common denominator is a collagen-based formulation,” Tal adds.

The market-dominant dermal filler, hyaluronic acid (HA), is hydrophilic, has good skin-lifting capacity, and is easily injected with a reasonable longevity. But HA does not regenerate the tissue; collagen adds the biological properties necessary for tissue rejuvenation.

CollPlant is developing dermal fillers that are comprised of collagen and HA and that are showing good tissue integration and skin-lifting capacity in ongoing preclinical studies. These fillers may leverage the features developed for photocuring BioInks. That is, upon illumination with a flashlight, they may enable post-injection skin sculpting and in vivo crosslinking. Additional products in the pipeline include 3D bioprinted regenerative breast implants and an injectable implant to address breast-shaping applications.

Hololithographic bioprinting

High fidelity and resolution are two of the biggest challenges in bioprinting. To overcome these challenges, Prellis Biologics has developed the Holograph X, a bioprinter that leverages a multiphoton laser system to perform holographic stereolithography. The Holograph X, which Prellis Biologics is developing and commercializing in partnership with Cellink, can print structures with micron-level resolution. These structures include hollow, permeable channels that can mimic capillaries and ultrafine vessels.

“Tissues and organs need a biomimetic microvascular network to survive,” comments Erin Stephens, PhD, director of tissue engineering at Prellis Biologics. “Resolution is key to allowing sufficient nutrient and oxygen exchange.

“Our hologram-based process starts by printing a high-resolution scaffold, followed by seeding the scaffold with cells. The types of microfluidic devices you can print with hololithography are very different than those printed with extrusion printers. We can build different shapes, different topographies, and different flow networks.”

“At Prellis,” she adds, “we continually push the limits of resolution and speed. Cells and living tissue need the detail; they need the capillary beds. We can print 240,000 voxels (3D pixels) per second. Printing a cubic centimeter with 1-µm resolution and a 1% fill factor takes us 11 hours compared to about 3 years for other similar technologies currently on the market.”

The required 3D scaffold architectures are organ dependent; that is, different organs have different flow requirements. Ideal organ-specific scaffolds have yet to be systematically investigated. To facilitate scaffold design, Prellis Biologics offers TissueWorkshop, a software platform that reduces the time required for scaffold design and iteration. TissueWorkshop accomplishes in seconds to minutes tasks that would keep conventional 3D modeling software busy for hours or days. The company also supplies Vascular Tissue Blanks if users need a starting point.

Stephens believes that the field will evolve with bursts of momentum that will drive new breakthroughs, and that interdisciplinary amalgamation will be critical to assemble all the information needed to get closer to true organ transplant. “Prellis has made huge strides in the field of regenerative medicine and that has already been incredibly impactful toward future healthcare,” he declares. “But there is still a lot to do.”

Prodding the body’s own stem cells

To replace or regenerate tissue, you could introduce new stem cells to a patient’s body. Alternatively, you could activate or stimulate the body’s own stem cells. The latter approach is being developed by Histogen. The company is focused on exposing a patient’s own stem cells to proteins and growth factors that have been produced by fibroblasts grown under simulated embryonic conditions, that is, in low oxygen and suspension cultures. The fibroblasts, which are induced to become multipotent stem cells, generate a liquid complex containing soluble biologicals with a diverse range of embryonic-like proteins. The biologicals can stimulate a patient’s own stem cells and allow regenerative applications to do animal-derived materials.

Histogen collects not only soluble proteins and growh factors, but also soluble and insoluble human extracellular matrix (hECM) materials. The hECM materials include components associated with stem cell niches in the body and scarless healing of fetal skin.

“Our in vitro studies demonstrated that the hECM supports the regulation and proliferation of human mesenchymal stem cells, as well as cell surface markers showing the maintenance of stemness, and induces the upregulation of aggrecan and Collagen II,” states Gail Naughton, PhD, Histogen’s founder and chief scientific officer. “These are important components in hyaline cartilage regeneration in damaged knees.”

In rat, rabbit, and goat models, hECM supported the regeneration of homogeneous hyaline cartilage and mature vascularized bone, whereas control knees showed only scar tissue formation. The tissue regeneration recapitulated the formation of cartilage, mineralization of the cartilage to form bone, and maturation of the cartilage to form a joint surface of a kind that is seen in embryogenesis.

The hyaline cartilage was fully attached to the adjacent cartilage, and a clear tide mark was seen. A 2020 U.S. clinical trial will study the hECM in regenerating hyaline cartilage in focal knee lesions.

The soluble hECM’s ability to reverse damage to degenerating spinal discs was also assessed. In a spinal disc study, a soluble extracellular matrix material reversed inflammation and protease activity and stimulated aggrecan secretion in a thrombin-induced ex vivo rabbit model. In vivo studies showed that within four weeks post treatment, disc height increased as compared to the control, and MRI analysis demonstrated regeneration of the disc tissue.

Histogen recently merged with Conatus Pharmaceuticals and is now listed on the NASDAQ exchange under HSTO.

Matrix-bound nanovesicles

In 2016, in the laboratory of Stephen F. Badylak, DVM, PhD, MD, small extracellular vesicles were seen to be physically embedded within the structural components of the extracellular matrix. This observation stimulated research that culminated in the characterization of entities now known as matrix-bound nanovesicles (MBVs).

Although MBVs are similar to fluid-phase extracellular vesicles, such as cellular-produced exosomes, the similarities between the two vesicle types end with size and shape. According to Raphael Crum, an MD-PhD candidate in Badylak’s laboratory, the characteristics that make these vesicles a unique population, and exciting for research, include their structure, function, and therapeutic potential.

Structurally, MBVs consist of a lipid membrane that transports cell signaling molecules, including microRNA (miRNA), (phospho)lipids, and proteins, that can influence processes such as macrophage activation, inflammation and fibrosis, tissue repair and remodeling, and wound healing. When MBVs are removed from their parent extracellular matrices, they recapitulate the same reconstructive remodeling and immunomodulatory properties seen with whole matrix biomaterials. MBV applications include more efficacious therapies for rheumatoid arthritis.

“Due to the broad immunomodulatory properties of MBVs and their cargoes, it is possible that MBVs might be used in the treatment of many other diseases and conditions stemming from a dysregulated immune response,” suggests Crum. The Badylak laboratory is currently investigating several other MBV applications in a variety of models of immune-mediated disease.

Crum believes as scientists develop a better understanding of the cellular and molecular biology involved in tissue regeneration in response to natural and synthetic biomaterials and cell-based therapies, the knowledge will facilitate the design of focused biomaterials that can operate in diverse patient and disease niches. Eventually, translational and personalized biomaterials will be developed.

Advancing manufacturing

Tissue-engineered medical products (TEMPs) represent a burgeoning industry, one that has drawn much attention to cell and gene therapies. Yet TEMPS, insists Tom Bollenbach, PhD, chief technology officer of the Advanced Regenerative Manufacturing Institute (ARMI), should also stimulate a reexamination of manufacturing issues.

At ARMI, a Manchester, NH-based nonprofit organization, the mission is to make consistent and cost-effective manufacturing practical by producing modular and scalable GMP-compliant processes and integrated technologies, and by developing standardized manufacturing practices. Programs are supported by awards, member-matching contributions, membership fees, and other grants and contracts.

In December 2016, the Department of Defense awarded ARMI $80M to operate BioFabUSA, which launched mid-2017. BioFabUSA is a 150+ member, public-private partnership comprising companies, academic institutions, and not-for-profit organizations in the TEMP ecosystem. Members are eligible for funding. To date, over $33.3M has been approved for TEMP manufacturing projects. BioFabConsulting, a premier regulatory consulting firm for complex TEMPs, supports members.

BioFabUSA has identified capability gaps preventing scalable, consistent, and cost-effective manufacturing. These include the need for a robust supply chain of more consistent raw materials, increased automation, more appropriately designed equipment, better measurement and data management tools, methods for the preservation and transport of cells and tissues, and scalable manufacturing processes and methodologies.

To close these capability gaps and help achieve BioFabUSA’s goals, membership teams are collaborating on the development of the modular Tissue Foundry manufacturing system. The Tissue Foundry, which integrates many developing platform technologies, can be reconfigured to produce any TEMP, and, ultimately, will be validated to allow GMP-compliant manufacturing.

“In the past, TEMP manufacturing seemed too complicated and too costly,” says Bollenbach. “Manufacturing improvements, new tools, measurement technologies, and manufacturing data will drive the cost reductions needed to attract investors and more widely advance the field.”

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