I Can See You Clearly Now: Use of Aptamer-Based Therapeutics to Treat Age-Related Macular Degeneration

What do you enjoy looking at the most? A grinning baby? A beautiful sunset? A cherished loved one? What if you learned that you would no longer be able look at those ever again? For the nearly two million people who are diagnosed with some form of age-related macular degeneration (AMD) each year in the U.S., this is a brutally cold reality (Brown et al., 2005).

Two forms of AMD exist (American Macular Degeneration Foundation, 2016). The first form, known as the “dry” version, constitutes the majority of AMD cases. The dry version is characterized by the thinning of the macula (the portion of the retina responsible for the fine details we can see) and by the buildup of drusen (yellow clumps). The second form, known as the “wet” version, only makes up 10% of AMD cases. The growth of new blood vessels that grow into the layer between the white of the eye and the retina causes the wet version. Being very weak, the blood vessels tend to leak behind the macula, which causes the macula to distort and impair vision. In both types, the peripheral vision remains intact, but the central vision becomes compromised and blurry.

Although the wet version is not as prevalent as the dry form, several treatments do exist. One subset of these vision-saving drugs are based on aptamers (Drolet, Green, Gold, & Janjic, 2016), similar to molecules that underlie SomaLogic’s unique protein measurement technology.

Several decades ago, vascular endothelial growth factor (VEGF) emerged as a key player for eye disorders, such as AMD. VEGF was responsible for not only the leakage of blood vessels, but also inducing the growth of new ones, which had been observed in the wet form of AMD. Treatment of AMD with aptamer-based drugs became attractive for several reasons. For administering a drug into the eye, only a small amount of the aptamer would be required.  The addition of the drug to the eye has a reduced chance of triggering an immune response. Also, the retention of the drug in the eye typically is much longer than elsewhere in the body. With an aging population with deteriorating eyesight, demand for an effective AMD drug definitely existed.

As evidence mounted for VEGF being an attractive target for AMD, concern also grew that inhibiting all the different forms of VEGF could lead to undesirable side effects. Therefore, a group of researchers at a biotech company called NeXagen (which later became NeXstar, and was subsequently acquired by Gilead) focused on a short and relatively abundant version of VEGF, so-called VEGF-165, which seemed to be a target for inhibition without the same range of side effects.

After a decade involving development, optimization, and clinical trials, the aptamer inhibitor for VEGF-165 (referred to now as Macugen) became the first aptamer-based therapeutic to receive FDA approval. This drug indeed improved the vision of many wet AMD sufferers. In its first year, the sales of Macugen were ~$185 million dollars (U.S). But, as often happened, Macugen soon had competition from compounds that targeted all versions of VEGF, and which outperformed Macugen.

During the time that Macugen was being developed, the scientists also began development of an inhibitor towards another protein of interest, platelet-derived growth factor (PDGF). They had learned that the new blood vessels in the eye become less reactive to VEGF inhibition as they mature. Thus, inhibiting both VEGF and PDGF may reduce the spread of slightly more mature blood vessels. In AMD animal models, combining Macugen and a PDGF inhibitor worked better than the use of either one alone.

The Ophthotech Corporation took this new PDGF inhibitor (now called Fovista) through several phases of clinical testing. In a Phase 1 study, the combined use of Fovista and Lucentis (a Macugen competitor drug aimed at VEGF) significantly increased the number of participants whose vision improved compared to individuals who received only Lucentis. This study also yielded a first for AMD treatment: The new problematic blood vessels began to disappear for all the participants who received the combination therapy. Further positive results in Phase 2 studies prompted Ophthotech  to move forward with Phase 3 clinical studies.

PDGF and VEGF are not the only targets that have had new aptamers inhibitors created. A potent aptamer-based inhibitor has been made to Complement component 5 (C5), another protein implicated in AMD. This aptamer (called Zimura by Ophthotech) is showing promise in early clinical trials.

Decades ago, AMD patients did not have a lot of options for how to preserve their sight. With more drugs coming onto the market and new technology being developed, the potential of maintaining clearer vision becomes greater. Also, the reality for AMD patients becomes rosier. They have a better chance of prolonging their ability to clearly witness the beauty around them and see the smiles of happy loved ones.

References

American Macular Degeneration Foundation. (2016). About Macular Degeneration. Retrieved on August 19, 2016 from https://www.macular.org/about-macular-degeneration.

Brown, G. C., Brown, M. M., Sharma, S., Stein, J. D., Roth, Z., Campanella, J., & Beauchamp, G. R. (2005). The burden of age-related macular degeneration: a value-based medicine analysis. Trans Am Ophthalmol Soc, 103, 173-184; discussion 184-176.

Drolet, D. W., Green, L. S., Gold, L., & Janjic, N. (2016). Fit for the Eye: Aptamers in Ocular Disorders. Nucleic Acid Ther, 26(3), 127-146. doi:10.1089/nat.2015.0573



Do You Know if Your Antibodies were Validated?

Increase productivity immediately! Get the results faster! Get more funding now! Publish first! These are just a few of the internal and external time pressures that many scientists face every day regardless of geography or lab setting. To meet these demands, a scientist may find he or she must order critical experimental laboratory reagents from vendors promising quick delivery. The reagent arrives quickly, as promised, and the scientist conducts the experiment, presuming that the vendor and/or manufacturer has spent the time and energy needed to test the quality of the reagent and its usefulness for the particular experiment type being done. As it turns out, this is an often wrong presumption to make, resulting in multiple bad outcomes.

The US spends close to $800 million dollars a year on conducting research that use protein-binding reagents. A whopping $350 million of it is wasted due to bad reagents, particularly antibodies that fail to perform as expected (Bradbury & Plückthun, 2015). These are significant wasted resources that could have been used to further the scientist’s research more productively. So, how can the “faulty antibodies” be avoided in the first place?

The simplest answer is to validate the antibodies. But who should validate, and to what degree?

It seems obvious to state that the manufacturer should invest the time and resources to fully validate their antibody products before making them available. Actually, this may not be feasible because the sheer volume of antibodies that would need to be tested in numerous different assay formats that have unlimited number of buffers/conditions. Nevertheless, several companies are taking up the torch to validate some of their antibody products (Baker, 2015a).

Efforts have been made to establish third party groups to help validate antibodies. Several websites and a few antibody companies are gathering/sharing reviews, data or articles using particular antibodies, such as the Antibodypedia, Antibody Validation Channel, Biocompare, St. John’s Laboratory, and CiteAb (Baker, 2015a; Freedman et al., 2016). Some companies, including ThermoFisher Scientific and Abgent, are even offering to validate an antibody for the wary scientist.

But ultimately, the burden of responsibility for validating an antibody for a particular use or uses falls on the end user. According to a survey put out by the Research Antibodies and Standards Task Force (set up by the Global Biological Standards Institute), the vast majority of seasoned researchers (~6 or more years post-training) realize that this is the case (Freedman et al., 2016). But the same survey also revealed that less than 45% of researchers who recently completed their training take the time to validate their antibodies (Freedman et al., 2016).

What would prevent a scientist from making sure the antibody is good and the results are trustworthy? The very same survey revealed that time, money, and delay in research were the primary reasons why a scientist may elect to not validate an antibody (Freedman et al., 2016). Here’s the head-scratcher: If the antibody yields a false-positive result or unreproducible result, the researcher would have already wasted money, time, and experienced a huge set back in their research. It would appear that more seasoned researchers have learned this lesson, but it will take time for those with less experience to come to this realization.

What if the researchers who recently completed their training do not fully know how to validate their antibodies, but want to? Yale University’s David Rimm (having falling victim to dubious antibody performance and now a champion for antibody validation) developed a flowchart of methods that can be used for validating an antibody, including the use of cell-lines that have the expression of the antigen knocked down (Baker, 2015b; Bordeaux et al., 2010). The scientists could also perform labor-intensive pull-downs followed by mass spectrometry to identify the protein(s) that bound to the antibody.

Why are antibodies so difficult? Like life, antibodies can be complicated. They are created in either living animals or in cell-lines, which can lead to variations in antibody composition, post-translational modifications (chemical changes made after the antibody or protein is created) or complete loss of the product if a cell-line dies/fails to grow. This can cause batch-to-batch antibody variability or cause a product to become unavailable.

Aside from variations due to the origination from animals or cells, other factors can affect antibody performance. For example, the purification of the antibodies from animal blood or from cell-lines can vary in quality and in the amount of contaminating proteins. Improper shipping, handling, or storage (wrong conditions or using past the expiration date) of the antibodies may cause them to unfold and lose activity. Another source of performance problems can be attributed to the antigen used in antibody development, which may possess a different set of post-translational modifications compared to its counterpart found in tissue or other biological sample, thus affecting antibody binding. If the experimental conditions are different from those used to create the antibody, the antigen’s antibody binding-site could become obscured by either the antigen adopting a different conformation or the antigen forming different complexes with other biologics. Yet, another source of performance issues could be attributed to the antibody’s specificity: The antibodies may bind to proteins that are similar to the intended target or to extremely abundant proteins. These are by no means the complete set of reasons for the problems seen with antibodies in research (for a more complete list, see a recent review by Michael Weller (Weller, 2016)).

Clearly, antibodies can be variable not only in their composition, but also in their performance. It would be ideal if a scientist could use affinity reagents that were less prone to variability, such as SomaLogic’s SOMAmer® reagents. SOMAmers, which are made of chemically synthesized modified single-stranded DNAs, can be used in most laboratory assays in place of antibodies. Their chemical origin greatly reduces batch-to-batch variability and the other issues that arise when using antibodies derived from animals or cell-lines. The methodology used to create SOMAmer reagents also includes measures to improve the specificity and enhance affinity. The methodology can be adjusted to generate SOMAmer reagents better suited for binding the desired target in the experimental conditions. SomaLogic researchers characterize what each SOMAmer reagent binds to by using mass spectrometry for high abundance targets, pull downs, and binding assays using very similar proteins to check for specificity. Although this level of characterization gives the user some confidence, it is still up to the researcher to confirm that the specific SOMAmer reagent will work for their specific need.

The old saying “slow and steady wins the race” can apply when it comes to research. Time and money should always be invested to validate binding reagents – or other critical assay components – that will be used for an intended experiment. The external/internal pressures may never go away, but at least fewer resources will have been wasted and more meaningful and reproducible research can happen.

References

Baker, M. (2015a). Antibody anarchy: A call to order. Nature, 527(7579), 545-551. doi:10.1038/527545a

Baker, M. (2015b). Reproducibility crisis: Blame it on the antibodies. Nature, 521(7552), 274-276. doi:10.1038/521274a

Bordeaux, J., Welsh, A., Agarwal, S., Killiam, E., Baquero, M., Hanna, J., . . . Rimm, D. (2010). Antibody validation. Biotechniques, 48(3), 197-209. doi:10.2144/000113382

Bradbury, A., & Plückthun, A. (2015). Reproducibility: Standardize antibodies used in research. Nature, 518(7537), 27-29. doi:10.1038/518027a

Freedman, L. P., Gibson, M. C., Bradbury, A. R., Buchberg, A. M., Davis, D., Dolled-Filhart, M. P., . . . Rimm, D. L. (2016). [Letter to the Editor] The need for improved education and training in research antibody usage and validation practices. Biotechniques, 61(1), 16-18. doi:10.2144/000114431

Weller, M. G. (2016). Quality Issues of Research Antibodies. Anal Chem Insights, 11, 21-27. doi:10.4137/ACI.S31614



SomaLogic announces that it has joined the iCarbonX Digital Life Alliance

SomaLogic announces that it has joined the iCarbonX Digital Life Alliance

Agreement includes development of a China-based joint venture and an equity investment by the iCarbonX ecosystem in SomaLogic

BOULDER, Colo. – January 5, 2017 – SomaLogic announced today that it has agreed to join the “Digital Life Alliance” established by iCarbonX, the China-based company founded in 2015 to build a “Global Digital Health Ecosystem that can define each person’s ‘digital life’ based on a combination of individual’s biological, behavioral and psychological data, the Internet and artificial intelligence.” Under the agreement between the companies, SomaLogic will provide proteomics data and applications expertise to the Alliance to accelerate the ecosystem’s development. Also under the agreement, iCarbonX and SomaLogic will establish a joint venture in China to provide the SOMAscan® proteomics assay for research and health applications in China. The iCarbonX ecosystem will also make an equity investment in SomaLogic to help accelerate these efforts.

“We founded iCarbonX with the goal of hastening the day when each person can receive truly useful digital health information, derived from many different aspects of their physical being, that they can use to live a healthier—and thus happier and fuller—life,” said Jun Wang, Founder and CEO of iCarbonX. “Our new SomaLogic colleagues share that vision deeply, and they provide the world-leading expertise in proteomics that we need to realize that vision sooner.”

SomaLogic’s proprietary technology, built over 15 years of intensive developmental work, is now empowering researchers across academia and business to measure human proteins as easily as nucleic acids. “Proteomics will provide us with essential digital knowledge for helping to understand each individual’s constantly changing health status,” said Wang, “and SomaLogic’s technology uniquely provides the breadth and depth of protein-based information we need to integrate into the ecosystem we are building.”

“Jun Wang and iCarbonX are at the forefront of the transformation of healthcare that we and many others are working hard to realize,” said Larry Gold, Chairman and Founder of SomaLogic. “All of us at SomaLogic are excited that Jun and his colleagues recognize the primary contribution of proteomic information in bringing about that transformation, and that they have selected our technology and scientists to partner with them in making it happen.”

Under this new agreement SomaLogic and iCarbonX will establish a joint venture in China, which will include installing SomaLogic’s SOMAscan proteomics assay in China to support the Digital Life Alliance as well as many other Chinese research and clinical concerns. “The joint venture with our iCarbonX colleagues significantly expands SomaLogic’s global reach,” said Byron Hewett, SomaLogic’s Chief Executive Officer. “iCarbonX is the best partner possible for helping us gain access to the enormous Chinese healthcare-related markets.”

Through the ecosystem it is building, iCarbonX has made a substantial equity investment in SomaLogic, but specific financial details were not released.

Earlier today at the 1st Digital Life Summit & Digital Life Alliance Global Recruitment Conference in Shenzhen, China, Wang announced that Somalogic and six other companies and organizations are founding members of iCarbonX’s Digital Life Alliance. SomaLogic, HealthTell, PatientsLikeMe, GALT, Inc. , Robustnique and AOBiome join Imagu Vision Technologies, which iCarbonX acquired in September 2016

About SomaLogic

SomaLogic is transforming healthcare by applying our proprietary protein-measurement technology to enable the precise monitoring of each individual’s health and wellness status in real time. We work with many different partners across research, health management, pharmaceutical development, and other health-related fields to build applications on our “Wellness Chip Platform,” a single cost-effective and reliable testing platform that provides actionable and timely information to patients and healthcare providers across a wide range of diseases and conditions. Our SOMAmer® and SOMAscan® technologies also have multiple applications across the life sciences, and are available to the entire biomedical scientific community for their own research needs. For more information, visit www.somalogic.com.

Contact information

Fintan R. Steele, Ph.D., VP, Corporate Communications

fsteele@somalogic.com

+1 720-214-3080 – T

+1 617-816-9834 – M

Time. Is it on My Side?

Time. Is it on My Side?

Time. There never seems to be enough of it. With our hectic lives, even the simplest of inconveniences, such as a car breaking down or a heart attack, can totally sour the afternoon and derail our well-laid plans. Wouldn’t it be nice to have advance warning for when we might expect to encounter an interruption to those plans? In part to gain us such a portent, several groups recently assessed a new technology for determining an individual’s cardiovascular disease risk, and the development of a warning test. Their combined efforts may indeed allow us to better plan our lives, or at least serve as a wake-up call that we need to change something in order to have more time to live.

The new technology that could play a crucial role in granting us more time is the SOMAscan® assay, which currently measures changes in over 1,130 proteins. To assess the practicality of SOMAscan in cardiovascular disease research, a research group led by Rob Gerszten at Beth Israel Deaconess Medical Center looked at “controlled heart attacks” to identify protein differences between pre- and post-heart attack in patients’ blood. Aside from identifying biomarkers that are well-established for heart injuries, the researchers also found several not previously seen. They also looked for biomarkers related to other traits that are known to elevate a person’s risk for cardiovascular disease (e.g. age, smoking, cholesterol levels, etc.). They noted the candidate biomarkers they discovered using SOMAscan may shed light into novel pathways that could in some way relate back to the development of cardiovascular disease. The researchers also noted that the SOMAscan assay was faster compared to mass spectrometry, an important consideration when assaying a large number of participants.

In addition to evaluating the SOMAscan assay for biomarker discovery, the research group also evaluated the accuracy of the individual SOMAmer® reagents in identifying their intended target proteins. Using mass spectrometry, the researchers found that all the SOMAmers tested did indeed hit the right targets.

In related work, a research group led by Peter Ganz at University of California San Francisco used the SOMAscan assay to identify a potential prognostic test for true cardiovascular risk in patients with stable coronary heart disease (CHD). The researchers initially analyzed plasma samples from CHD patients who took part in the Heart and Soul study (a study initially intended to assess how mental health affects heart disease patients) for biomarkers that could stratify risk, a measurement that has proved challenging when using traditional or even genetic methods. From the initial phase, the group identified nine proteins that passed the statistical rigors.

To further assess the accuracy of the nine-protein panel, Ganz and his group conducted another round of SOMAscan testing on samples from a completely different set of individuals (participants in the HUNT3 study whose medical data and samples were collected and could be used for further medical or social science research), they verified their findings from the Heart and Soul samples. The researches also evaluated paired samples from the Heart and Soul study participants to determine if the individuals’ risk change as a cardiovascular event approached. They found that indeed that the closer an individual came to a cardiovascular event, the greater the change for the nine-protein panel results when compared to baseline values. These changes were, in turn, shown to be a more reliable and accurate measure than the current clinical standards for assessing cardiovascular risk.

The related findings of these two research groups underline the ability of the SOMAscan assay to benefit cardiovascular disease research, and suggest that we are inching closer to being able to fine-tune our prediction of when a cardiovascular event may happen. Which in turn, may grant us the time we need to accomplish all our well-laid plans.

References:

Ngo, D., Sinha, S., Shen, D., Kuhn, E. W., Keyes, M. J., Shi, X., . . . Gerszten, R. E. (2016) Aptamer-Based Proteomic Profiling Reveals Novel Candidate Biomarkers and Pathways in Cardiovascular Disease. Circulation, 134(4), 270-285. doi:10.1161/CIRCULATIONAHA.116.021803

Ganz, P., Heidecker, B., Hveem, K., Jonasson, C., Kato, S., Segal, M. R., . . . Williams, S. A. (2016) Development and Validation of a Protein-Based Risk Score for Cardiovascular Outcomes Among Patients With Stable Coronary Heart Disease. JAMA, 315(23), 2532-2541. doi:10.1001/jama.2016.5951



The Toll of Aging on the Quest for Gold

Although the summer Olympics are over for another four years, the world is again amazed at the amount of training these Herculean athletes endure in order to capture gold. A young body usually recovers quite easily after an intense training session; thus, permitting young athletes to continue their quest. However, age inevitably starts to take its toll on the body. Repairing damaged muscles becomes increasingly more difficult with advancing age, though “why” the ability to regenerate muscle is lost is largely unknown. To try to figure it out, a research group led by Jerome Feige and C. Florian Bentzinger at the Nestlé Institute of Health Sciences decided to look the effects of age on muscle stem cells, in the context of their normal contribution to muscle regeneration.

The research group started with comparing gene expression in muscle stem cells from young and old mice. They found that the cells from the injured old mice showed lower expression of genes involved in the cell cycle regulation, higher expression of JAK-STAT and MAP kinase pathway-associated genes (major cell signaling pathways for a variety of functions), and multiple changes in the gene expression for proteins associated with the extracellular matrix (ECM) receptor pathway. The ECM findings were of particular interest, suggesting a role for ECM in muscle regeneration.

To confirm the ECM’s contribution to the muscle regeneration, the group used the SOMAscan® assay to directly measure and compare changes in protein levels in homogenized muscles from injured or uninjured, and young or old mice. They found that many ECM proteins showed higher levels in the old uninjured mice, an observation that is consistent with the usual increase in fibrosis seen in aging muscles. However, when looking at the injured mice, they saw that, in young mice, elevated levels of fibronectin occurred quickly after injury. However, in the old injured mice there was significantly less fibronectin present.

Upon further investigation, the overall importance of fibronectin in regenerating injured muscles solidified. Originating predominantly from lineage-positive cells (stem cells expressing markers seen in mature cells) near the sites of injury, fibronectin serves as an ideal substrate for muscle stem cells to adhere. The researchers found that loss of this ideal substrate led to alterations in several signaling cascades, in line with previous observations from other groups. These alterations may contribute to the aged muscle cells’ increased susceptibility to anoikis (cell-death induced by failure to anchor onto matrix).

Digging even deeper, the investigators looked into the role of a protein called focal adhesion kinase (FAK), which they noted is a known inhibitor of anoikis and dependent on fibronectin for activation. Like fibronectin, they observed the FAK levels to decrease with age in muscle stem cells, which may account for the increased susceptibility of the aged muscle stem cells to anoikis.

Is fibronectin the key to the fountain of youth for aged muscle stem cells? The researchers saw that aged muscle stem cells showed improved adhesion to matrices that included fibronectin, which reduced the cells’ susceptibility to anoikis and slightly improved their proliferation. The inclusion of fibronectin in the matrix also restored the FAK activity and subcellular localization in the aged muscle stem cells. When the researchers injected injured old mice with fibronectin, they saw more FAK in cells undergoing myogenesis, and the localization of the FAK within the cells was comparable to that of cells from young mice. In addition to the improved signaling, the researchers saw improved proliferation of the cells that would go on to become muscle cells. The resulting muscle fibers indeed showed fewer immature muscle fibers than seen in control mice, suggesting that the effects of age on muscle regeneration were mitigated by injection of fibronectin.

For the aging Olympian athletes, fibronectin injections would probably be seen as a new form of doping. For the rest of the aging populace, however, increasing fibronectin may be the winner as a way to maintain a more active lifestyle well into “old” age.

Link to paper: http://rdcu.be/i9ib (Lukjanenko et al., 2016)

Resources

Lukjanenko, L., Jung, M. J., Hegde, N., Perruisseau-Carrier, C., Migliavacca, E., Rozo, M., . . . Bentzinger, C. F. (2016) Loss of fibronectin from the aged stem cell niche affects the regenerative capacity of skeletal muscle in mice. Nat Med. doi:10.1038/nm.4126