Putting the Pain on Ice: IL-6 SOMAmer Reagent Inhibits Rheumatoid Arthritis

The seasons are changing. The days are getting shorter. The air is getting cool and crisp. Soon, the outdoors will be covered by a lovely blanket of fresh snow. As serene as this image may be, the fact is that the changing seasons maybe painful for those who suffer from rheumatoid arthritis (RA) (Savage et al., 2015).

There are several treatments for RA available today (briefly reviewed in Hirota et al., 2016), including non-steroidal anti-inflammatory drugs, small molecule disease-modifying anti-rheumatic drugs, and a growing number of biologics that specifically target inflammatory pathway components. None of these therapeutics can cure the disease, but they can make life a bit more comfortable for RA sufferers (though not without some significant side-effects).

One protein target of particular interest for new RA treatments is interleukin-6 (IL-6) (briefly reviewed in Hirota et al., 2016). As a cytokine (a protein involved in the communication between cells), IL-6 is involved in the immune response, inflammation, hematopoiesis (making of new blood cells) and bone metabolism. During an RA response, IL-6 and the IL-6 receptor-α together bind to signal transducing protein gp130 (CD130), which activates the cell signaling pathway known as “JAK-STAT3” and phosphorylation (the addition of phosphates) of STAT3.

A research team led by Dr. Masao Hirota recently developed a new approach to inhibit the IL-6 signaling pathway (Hirota et al., 2016). They developed SL1026, a SOMAmer reagent that has a strong binding affinity for both human and monkey IL-6.

Dr. Hirota and his team determined that SL1026 inhibits IL-6-induced STAT3 phosphorylation in human peripheral blood lymphocytes (a type of white blood cell). First, they incubated whole blood with IL-6 alone, or with IL-6 in combination with either SL1026 or tocilizumab (a current drug that specifically inhibits IL-6 signaling in RA). The researchers then isolated and analyzed the cells and, as expected, IL-6 treatment increased STAT3 phosphorylation. Treatment of the cells with either SL1026 or tocilizumab inhibited STAT3 phosphorylation.

The researchers tested whether or not SL1026 could delay the actual progression of RA in a primate collagen-induced arthritis model (an established model system for evaluating the therapeutic and preventative effects of existing RA drugs). Similar to human RA, the monkeys experienced an autoimmune-mediated polyarthritis, joint inflammation and erosion of bone and cartilage. While one group of monkeys went untreated, a subset of the monkeys received intravenous doses of SL1026. The animals were monitored and scored for behavior and movement. The researchers also scored the degree of swelling and rigidity in numerous joints.

SL1026 not only reduced arthritis symptoms in the monkey model, but also delayed arthritis onset in treated monkeys for 20 days compared to 13 days for the untreated monkeys. The monkeys treated with higher doses of SL1026 also had a reduced arthritis score at day 34 that was significantly different than the untreated monkeys’ score.

One concern with any potential new therapeutic is an allergic reaction on the part of the recipient. To see if this might happen, the team measured levels of anti-SL1026 antibodies. None were identified in the plasma of treated monkeys, indicating that the SL1026 did not trigger an immune/allergic response. In addition, the researchers noted that the SL1026 was well tolerated by the monkeys, and no adverse events occurred.

The efforts put forth by Dr. Hirota and his team established that SOMAmer reagents can be effective therapeutics as a result of their exquisite binding affinities for their target proteins. This particular work demonstrates that the SL1026 is a potent antagonist of the IL-6 signaling pathway. SL1026 may be a promising drug candidate for RA and potentially in other IL-6 mediated diseases. If it makes it to market, SL1026 may prove to be a better treatment option for RA suffers during these cooler and darker days.

References

Hirota, M., Murakami, I., Ishikawa, Y., Suzuki, T., Sumida, S., Ibaragi, S., . . . Schneider, D. J. (2016). Chemically Modified Interleukin-6 Aptamer Inhibits Development of Collagen-Induced Arthritis in Cynomolgus Monkeys. Nucleic Acid Ther, 26(1), 10-19. doi:10.1089/nat.2015.0567

Savage, E. M., McCormick, D., McDonald, S., Moore, O., Stevenson, M., & Cairns, A. P. (2015). Does rheumatoid arthritis disease activity correlate with weather conditions? Rheumatol Int, 35(5), 887-890. doi:10.1007/s00296-014-3161-5



Interleukin-16 is a Novel Protein Biomarker for Rheumatoid Arthritis Disease and Treatment Response

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by increasingly severe joint inflammation and degeneration. It is also not widely understood at the molecular level, which limits the efficacy of current treatment choices as well as new drug discovery and development. Robust biomarkers are particularly needed, as the current measurements used (C-reactive protein levels and erythrocyte sedimentation rate) are non-specific for RA.

The protein matrix metalloproteinase 3 (MMP-3), which plays a role in cartilage and bone degradation, is associated with RA and has been suggested as a potential biomarker for early disease onset. However, MMP-3 levels do not consistently correlate to clinical response in early disease.

To address the need for better biomarkers, Atsuko Murota and his team at the Keio University School of Medicine used the SOMAscan assay to find novel markers associated with both MMP-3 and RA. The Murota team also sought to identify new biomarkers associated with RA disease status, including response to treatment.

The study was done in two phases.

Phase 1: Screening for RA-associated proteins

Murota’s team used the SOMAscan assay to identify proteins in serum from subjects in three groups:

  • People with untreated RA (n = 28)
  • People with primary Sjögren’s syndrome (pSS), an autoimmune disease (n = 30)
  • Healthy controls with no history of autoimmune disease (n = 30)

The team measured protein levels in each subject’s serum, validating the SOMAscan results with a series of traditional immunoassays. They found 10 proteins in the serum of RA subjects were elevated more than 1.5-fold over healthy controls and more than 1.2-fold over pSS patients. On further examination, only the cytokine interleukin 16 (IL-16) was significantly increased in untreated patients with RA compared to both pSS patients and healthy controls. IL-16 is a proinflammatory cytokine associated with a number of immune-mediated disorders, and was shown in this study to be closely associated with MMP-3 and the most characteristic protein biomarker of RA.

Phase 2: Correlating IL-16 levels with clinical outcomes

The researchers then engaged a second set of subjects to determine whether or not IL-16 levels changed with treatment. They recruited people in three groups:

  • Subjects with untreated RA and osteoarthritis as controls
  • Methotrexate-naïve subjects treated with methotrexate during the study (n = 28)
  • Subjects treated with a combination of methotrexate and a biologic: abatacept (n = 11), tocilizumab (n = 7) or infliximab (n = 22)

Clinical and laboratory assessments for each subject were made at baseline and then after 12 weeks of treatment (with various therapeutic agents or placebo). The team confirmed that IL-16 expression was associated with clinical response during the early treatment phase of RA. IL-16 decreased in all treated subjects except those treated with infliximab.

The significance of IL-16 in the pathogenesis of RA is not yet understood, and more studies (with more subjects) are required to determine the role that IL-16 plays in this disease. However, the fact that this team has demonstrated its unambiguous association with RA is a promising start, and offers a potentially robust biomarker for monitoring disease progression and treatment.

 

References

  1. Murota A et al. (2016) Serum Proteomic Analysis Identifies Interleukin 16 as a Biomarker for Clinical Response during Early Treatment of Rheumatoid Arthritis. Cytokine 78:87–93. doi: 10.1016/j.cyto.2015.12.002



SOMAmer Reagents: Next-generation Aptamers

Aptamers are short single-stranded oligonucleotides that fold into diverse and intricate molecular structures that can bind with high affinity and specificity to a variety of proteins, peptides and small molecules. Since they were first described in 1990, more than 10,000 publications applying aptamers to many diverse areas of biomedical science have been published.

Traditional aptamers, which consist of unmodified DNA or RNA, can be selected against ~30% of human proteins. However, there are many other biologically and medically important proteins for which the standard RNA and DNA SELEX (Systematic Evolution of Ligands by EXponential enrichment) process does not yield adequate aptamer reagents. To overcome the limitations of traditional aptamers, SomaLogic scientists had developed a next generation aptamer platform (Slow Off-rate Modified Aptamers, or SOMAmers), and published the first example of the use of these reagents in a highly multiplexed proteomic assay in 2010.1

Affinity and Specificity

Like aptamers, SOMAmers are short, single-stranded deoxyoligonucleotides selected in vitro from large random libraries for their ability to bind to discrete molecular targets. To give them greater target diversity as well as high affinity, SomaLogic scientists endowed them with protein-like properties by adding functional groups that mimic amino acid side chains, thereby expanding their chemical diversity.1 Based on this work, SOMAmer reagents today are engineered with dU residues functionalized at the 5-position with different protein-like moieties (e.g., benzyl, 2-napthyl or 3-indolyl-carboxamide). These modifications can interact with more epitopes on a greater range of target molecules, largely as a result of the novel secondary and tertiary structures formed within the SOMAmer reagent itself. 2

SOMAmer reagents are selected not just for their affinity for their respective target proteins, but also for low dissociation rates (slow off-rates) with their targets. By introducing this second, kinetics-based element of specificity into the selection process (and thus into the reagent itself), SOMAmer reagents can be used in protein assays without requiring a second detection ligand. In addition, researchers using SOMAmer reagents can use a large amount of a non-specific polyanionic competitor1 to disrupt any non-specific binding interactions.

Stability

Nucleic acid–based reagents are susceptible to nuclease degradation, and unfortunately, most aptamers degrade quickly. The hydrophobic modifications made to the 5-position of deoxyuridine nucleotides in SOMAmer reagents results in a substantial increase in resistance to DNase-mediated degradation in comparison to aptamers, though the degree of improvement depends idiosyncratically on both the sequence and the nature of 5-position modification.

Sensitivity

Writing in a 2016 review of a 2010 article, Brody3 states that the best SOMAmer reagents allow limits of detection (LODs) at approximately 50 fM, and that approximately half the available analytes show measured LODs below 1 pM.

Conclusion

Compared with aptamers, SOMAmers can be selected that have higher affinity to and specificity for more diverse proteins, and are less vulnerable to nuclease degradation. These qualities, along with the other stability and production advantages aptamers have over antibodies, make SOMAmer reagents attractive for use in virtually every protein measurement application from the laboratory to the clinic.

References

  1. Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249(4968):505–510
  2. Ellington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346(6287):818–822
  3. Gold L et al. (2010) Aptamer-Based Multiplexed Proteomic Technology for Biomarker Discovery. PLoS ONE 5(12): e15004. doi:10.1371/journal.pone.0015004
  4. Rohlof JC et al. (2014) Nucleic Acid Ligands with Protein-like Side Chains: Modified Aptamers and Their Use as Diagnostic and Therapeutic Agents. Mol Ther Nucleic Acids 3(10): e201. doi: 10.1038/mtna.2014.49
  5. Brody EN et al. (2010) High-content Affinity-based Proteomics: Unlocking Protein Biomarker Discovery. Expert Review of Molecular Diagnostics 10(8): 1013–1022. doi: 10.1586/erm.10.89



Why Pharmaceutical Companies Are Ahead of Academic Scientists on the Topic of Antibody Validation

Recently, we’ve noticed a number of commentary articles appearing discussing the problems of validation and reproducibility of antibodies:

Antibody anarchy: A call to order

Reproducibility: Standardize antibodies used in research

Reproducibility crisis: Blame it on the antibodies

The authors and interviewees have primarily been associated with academic institutions or antibody suppliers. However, this is not a new a new issue and pharmaceutical scientists have been aware of it and taking action on it for a number of years.

In 2012, the Ligand Binding Assay Focus Group of the Biotechnology section of the American Association of Pharmaceutical Scientists published a white paper as part of their “Ligand Binding Assays in the 21st Century Laboratory” series of publications. The paper is “Recommendations for Characterization and Supply of Critical Reagents.” In this publication the authors summarize the key considerations for the generation, production, characterization, qualification, documentation, and management of critical reagents in ligand binding assays (LBAs). Ligand binding assays include any type of assay using affinity reagents.

LBAs are used during every phase of the drug development process from discovery to post-market monitoring. Pharmaceutical scientists jobs, and the products’ efficacy and safety, depend upon LBA reagent quality and consistency. Therefore, great importance is placed on the structural integrity, stability, and performance of LBA reagents.

Many of the critical reagents you might use in LBAs, such as monoclonal antibodies, polyclonal antibodies, fusion proteins, enzymes, or detection molecules, are produced via biological processes and are thus inherently prone to variability among different production lots.

Since the lifespan of a drug, from development to the end of its production, can span more than 20 years, establishing criteria and a stable supply for critical reagents, such as antibodies, is critical. This is something that most academic labs don’t have to concern themselves with, as their project timelines are much shorter and the impact on users is less severe than the safety and efficacy of a drug.

LBAs may be used anywhere from six weeks to six months in the preclinical stage, six months to five years during the clinical development stage, and possibly more than 20 years to support post-marketing activities and manufacturing. Even in the preclinical stage it’s critical that reagents be trustworthy, as the assays may get passed on from teams in the preclinical groups to teams in clinical research. Nobody wants to pass along a bad assay.

In September 2015, ThermoFisher announced it is sponsoring an International Working Group on Antibody Validation. This will be an independent group with some funding supplied by ThermoFisher. The interesting thing we noticed is that not one member of the working group is from pharma or biotech, instead they are all from universities or academic research centers. Pharmaceutical scientists seem to have grappled with this issue already and come up with some solutions. Unfortunately, it’s harder for pharmaceutical scientists to share their information as readily as those in universities and government research institutions. Perhaps these new teams working on the issue of antibody validation should engage with some pharmaceutical scientists and diagnostic company scientists, as both of these types of entities have had to solve these antibody issues already.

What are your thoughts on antibodies as critical reagents and how to validate them?



From Deep Genotype to Deep Phenotype

In the 60+ years since the description of the double helix, immense technical progress has been made in reading genomic variations and applying those findings to the prognosis and even diagnosis of disease states. But genomic studies are inherently limited: the genome does not exist in isolation from its multiple environments (nuclear, cellular, organismal, and beyond). Thus, there is usually no ascertainable direct path from genomic data to phenotype, except in cases where a single gene mutation results in obvious phenotypic alterations. Many of the so-called rare diseases such as Tay-Sachs, cystic fibrosis, and Duchenne muscular dystrophy are examples of a direct causal path, but even in these cases there can be wide, medically relevant phenotypic variations that are not easily explained – if at all — by genomic analyses. And it is unclear that simply sequencing more genomes will get us to the phenotypic knowledge needed to accurately diagnose, treat or even prevent disease onset.

Fortunately, there are ways to assess phenotype deeply in ways that are medically meaningful. Today, the tools are now at hand to “see” phenotype well beyond the resolution limits of the human eye, down to the levels of the individual proteins that can tell the presence, or even foretell the onset, of multiple diseases and conditions.

These new tools are only recently available: The ability to measure proteins widely and deeply as a way of more deeply visualizing phenotype has lagged well behind genomics, in large part because of the sheer complexity of the proteome when compared to the genome. While the genome is generally fairly static across multiple cell and tissue types, the proteome varies widely in identities and concentrations of particular proteins over the same spaces. Traditional technologies to assess protein differences have had to compromise: Either measure a few proteins across many concentrations (antibodies/ELISAS), or measure many proteins across a limited concentration space (mass spectrometry, 2-D gel electrophoresis).

SomaLogic was founded to discover and apply a new protein measurement technology that could visualize thousands of proteins across a wide range of concentrations. The technology we developed, SOMAmer® reagents and the SOMAscan® assay, opens up a whole new way of deeply reading the phenotype in a way that can provide critical new information that will guide personalized medical decision making.

Although our goal as a company is to apply our technology to the transformation how diseases are detected, diagnosed and treated, it became clear to us early that the ability to rapidly and reliably measure protein changes in a variety of research settings was a huge unmet need across the life sciences field. Thus, once we had satisfied ourselves that the technology far exceeded anything else available, we made it available to researchers worldwide to accelerate their own proteomics work.

Please contact us to learn more about how SomaLogic can help you accelerate your research by deepening your phenotypic knowledge.



Unbiased Biomarker Discovery in Duchenne Muscular Dystrophy

Although we have known the genetic cause of Duchenne muscular dystrophy since 1986, our knowledge of the actual biology of the disease and its progression is still incomplete. This lack of understanding seriously compromises our efforts to find effective new treatments, as well as new diagnostic tests that can help patients and their caregivers manage disease progression.

This paper, the result of a focused collaboration between industry, advocacy and Duchenne patient advocates, describes the first truly large-scale, unbiased biomarker discovery in Duchenne patients vs. controls, using the SOMAscan assay.

A total of 44 proteins were identified, 24 of which are up and 20 that are down in Duchenne patients as compared to controls. Some of these were expected (and confirmatory of previous studies), but others were not, and suggest new approaches for diagnosis, prognosis and novel therapeutic discovery for this devastating disease.

Hathout Y et al. (2015) Large–scale biomarker discovery in Duchenne muscular dystrophy. Proc Natl Acad Sci USA Early edition (online only) on May 26, 2015. http://www.pnas.org/cgi/doi/10.1073/pnas.1507719112