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Ablynx Develops Nano Antibody for Treatment of Rare Clotting Disorder

Last week, Ablynx announced substantial progress in the development of the nano antibody drug caplicizumab to treat acquired thrombotic thrombocytopenic purpura (aTTP), a rare, but life-threatening autoimmune disease. The Belgian biopharmaceutical company has submitted a Marketing Authorization Application (MAA) to the European Medicines Agency (EMA) for approval. If accepted, caplicizumab will not only be the first therapeutic specifically indicated for the treatment of aTTP, but also the first approved nano antibody drug on the market.

aTTP is characterized by the autoimmune impairment of ADAMTS13, an enzyme that normally cleaves multimeric von Willebrand factor (vWF) into its functional form. Without the function of ADAMTS13, multimeric vWF forms aggregates with platelets in the blood. Low free platelet count and excess clotting result in thrombotic complications and a significant risk of organ damage due to the blockages of blood flow to tissues.

The current standard of care for aTTP involves immunosuppression and daily plasma exchange transfusion, in which a patient’s plasma is replaced with donor plasma to remove platelet-vWF aggregates. Caplicizumab is an anti-vWF nano antibody that prevents the formation of aggregates by blocking the interaction of multimeric vWF complexes with platelets.

While dozens of monoclonal antibodies have been approved by the FDA for therapeutic use (with hundreds more undergoing clinical trials), caplicizumab is the first therapeutic nano antibody. Nano antibodies are single-domain antibody fragments that bear full antigen binding capacity like monoclonal antibodies, but have a smaller size and unique structure, giving them features of small-molecule drugs. Nano antibodies are more stable than conventional monoclonal antibodies, allowing for multiple administration routes, and can be humanized to lower toxicity and immunogenicity. Because they are encoded by single genes, nano antibodies are easier and more cost-effective than traditional antibodies to engineer and manufacture.

Currently, caplicizumab is undergoing Phase III clinical trials and a three-year follow-up study has been initiated to determine the long-term safety and efficacy of this drug. Ablynx aims to commercialize caplicizumab in North America and Europe upon the trial’s conclusion and approval of BLA filing in 2018.

With the obvious advantages of nano antibodies over conventional monoclonal antibodies as biological drugs, caplicizumab is likely only the first of many to come.

Allele Researchers Engineer Modified Nanoantibodies to Increase Sensitivity in Biochemical Assays

Researchers at Allele have published new work demonstrating a novel application for nanoantibodies (nAbs) in direct signal amplification. nAbs have distinguishable qualities that set them apart from their traditional IgG counterparts, including significantly smaller size, better stability, and excellent specificity. However, because of their small size, there are no suitable secondary antibodies for traditional assays like immunohistochemistry, immunofluorescence, and other biochemical assays that require an enhanced signal.

The researchers engineered a modified nAb, termed “nAb Plus,” to directly amplify nAb signal detection through the addition of a small scaffolding protein containing numerous reporter binding sites. nAb Plus bypasses the need for secondary antibodies or additional amplification steps, streamlining biochemical assays and decreasing costs of reagents. The authors demonstrate the use of nAb Plus using immunohistochemistry, an assay typically requiring one or more signal amplification steps. However, nAb Plus could also be incorporated in any biochemical assay needing signal enhancement.

Abstract: Revealing the spatial arrangement of molecules within a tissue through immunohistochemistry (IHC) is an invaluable tool in biomedical research and clinical diagnostics. Choosing both the appropriate antibody and amplification system is paramount to the pathologic interpretation of the tissue at hand. The use of single domain VHH nanoantibodies (nAbs) promise more robust and consistent results in IHC, but are rarely used as an alternative to conventional immunoglobulin G (IgG) antibodies. nAbs are originally obtained from llamas and are the smallest antigen-binding fragments available. To determine whether the unique biophysical properties of nAbs give them an advantage in IHC, we first compared a basic fibroblast growth factor nAb to polyclonal IgG antibodies using tissue isolated from pancreatic adenocarcinoma. The nAb was extremely effective in antigen signal detection and allowed for a more streamlined and reproducible protocol. Furthermore, because nAbs are expressed in Escherichia coli from a single gene, they are quite amenable to genetic engineering. As such, we then covalently bound a highly biotinylated amplifier protein to basic fibroblast growth factor and p16 nAbs (termed nAb Plus), resulting in improved IHC sensitivity. The use of a biotinylated nAb Plus not only achieved local, covalent signal amplification, but also eliminated the need for a secondary antibody and subsequent amplification steps. These results highlight nAbs as valuable alternatives to conventional IgG antibodies, decreasing overall processing time and costs of reagents while increasing sensitivity and reproducibility across individual IHC assays.

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Stem Cell Therapies: What’s Approved, What Isn’t, and Why Not?

With acceptance of stem cell therapies growing, so have controversies surrounding regulations.

Desperate to heal sports injuries, top professional athletes have been known to pay tens of thousands of dollars for experimental stem cell treatments that many used to find controversial. But now, stem cell therapies have become more mainstream and are no longer limited to professional athletes. Stem cell clinics offer both medical and non-medical treatments with claims of improving aesthetics and quality of life.

One recent study found over 400 websites – with the largest portion in the United States – advertising stem cell-based therapies (1); another found over 570 U.S. clinics offering stem cell interventions (2), giving more evidence that the market for stem cell therapies in the U.S. is growing at an accelerated rate. Yet these therapies are too often based on unfounded claims and lack proper clinical trials or authorized regulation. Despite what some clinics claim, very few stem cell treatments are currently available that are actually approved by the Food and Drug Administration (FDA). Hematopoietic stem cells harvested from bone marrow are routinely used in transplant procedures to treat patients with cancer or other blood or immune system disorders. Banking of umbilical cord blood is FDA-regulated and its use is approved for certain indications. Otherwise, consumers should be wary of claims by stem cell clinics implying FDA-approval.

So why aren’t more FDA-approved stem cell therapies available?

The FDA has strict regulations on using stem cell products in humans. In most cases, stem cell-based products are categorized the same way as pharmaceutical drugs. Therefore, each new therapy must go through a rigorous process including pre-clinical animal trials, phased clinical studies, and pre-market review by the FDA prior to offering the treatment in the clinic.

And with stringent regulatory requirements comes prohibitive costs. Research animals, Phase I-III clinical trials, and the regulatory demands for good manufacturing practice (GMP) labs result in an extraordinarily costly process that may hinder the progress of new therapies. The cost of developing a new drug has even been estimated to reach billions of dollars.

Nevertheless, a complete lack of regulation of stem cell therapies – as is seen in many of the stem cell clinics springing up worldwide – is clearly problematic. Alarmingly, many clinics advertise claims related to medical diseases for which there is no scientific consensus that supports their safety or efficacy. Premature commercialization of unproven therapies not only puts patients at risk, but also jeopardizes the credibility of still-developing stem cell products.

One of the most exciting outlooks for stem cell therapy is the prospect of using one’s own stem cells for personalized medicine. Should the development of an autologous stem cell product really be regulated the same way as a pharmaceutical drug, which is aimed at treating huge populations of people? If not, how should stem cell products be regulated?

In an effort to make the transition of novel stem cell products to the clinic more seamless, some countries have made significant changes in regulations. For instance, in 2014, Japan broke out a separate regulatory system for stem cell products that softened legislation dramatically to require only limited safety and efficacy data. Some argue that countries with softer regulations and less stringent safety and efficacy milestones, such as Japan, have poised themselves to become the likely pioneers in the field of regenerative medicine.

Regulatory frameworks for the clinical application of stem cell products are still evolving in most countries, including the U.S. In March, the Reliable and Effective Growth for Regenerative health Options that improve Wellness (REGROW) Act was introduced to congress. This change in legislation would remove some of the regulatory hurdles that hinder the progress of biologic therapies.

Regardless, the FDA needs to establish a more reasonable regulatory system that can evaluate the safety and efficacy of stem cell products in a more efficient manner.

1.  Berger, I., et al., Global Distribution of Businesses Marketing Stem Cell-Based Interventions. Cell Stem Cell, 2016. 19(2): p. 158-62.
2.  Turner, L. and P. Knoepfler, Selling Stem Cells in the USA: Assessing the Direct-to-Consumer Industry. Cell Stem Cell, 2016. 19(2): p. 154-7.


CRISPR: Growing in Popularity, But Problems Remain

The CRISPR gene-editing technology is taking the scientific world by storm, but researchers are still uncovering the platform’s potential and pitfalls.

The “Democratization” of Gene Editing
The idea of modifying human genomes using homology directed repair (HDR) has been around for decades. HDR is a widely-used repair mechanism to fix double-strand breaks in the cell’s DNA. By supplying an exogenous, homologous piece of DNA to the cell and increasing the probability of HDR occurring, changes in the DNA sequence can be introduced to the targeted area.

Some of the first gene editing platforms taking advantage of HDR used engineered endonucleases such as zinc finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs). Both ZFNs and TALENs required a custom protein to target a specific DNA sequence, making them pricey and very difficult to engineer. The more recently developed CRISPR/Cas9 platform works differently: instead of using a protein, the Cas enzyme uses a small guide RNA to locate the targeted DNA, which is then cut. CRISPR is more low tech and “user-friendly” than other platforms, as users are no longer required to do the onerous chore of protein engineering. Now, the ability to modify genomes can be done without extensive training or expensive equipment. CRISPR can be used for knocking out genes, creating reporter or selection genes, or modifying disease-specific mutations—by practically anyone with basic knowledge of molecular biology.

The CRISPR Revolution
Because of its relative simplicity and accessibility, it is no wonder that life scientists from all over the world are eager to incorporate CRISPR into their research. One of the most remarkable things about CRISPR technology is how quickly its popularity has allowed the platform to evolve. The discovery revealing that CRISPR can be used for RNA-programmable genome editing was first published in 2012 (1). Since then, about 2,000 manuscripts have been published including this technology and millions of dollars have been invested into CRISPR-related research and start-up companies. Scientists have developed a myriad of applications for CRISPR, which can be used in practically any organism under the sun. The popularity and progress of gene editing promises revolutionary advancements in virtually every scientific field: from eradicating disease-carrying mosquitoes to creating hypoallergenic eggs and even curing genetic diseases in humans.

There is no doubt that CRISPR has enormous potential – the widespread interest and rapid progress are evidence of that. The main reason CRISPR has been so widely adapted is because development and customization is way less labor-intensive and time-consuming than previous methods. But material preparation is a miniscule part of the CRISPR platform. Contrary to popular belief, just because CRISPR is easier to use than other methods of gene editing, it is not “easy.”

Hitting the Bull’s-Eye
To understand the limitations of this gene editing technology, it’s important to understand more about how CRISPR works. The CRISPR/Cas9 system functions by inducing double-strand breaks at a specific target and allowing the host DNA repair system to fix the site of interest. Cells have two major repair pathways to fix these types of breaks, Non-Homologous End Joining (NHEJ) and homology directed repair (HDR). NHEJ is faster and more active than HDR and does not require a repair template, so NHEJ is the principle means by which CRISPR/Cas9-induced breaks are repaired. If the editing goal is to induce insertions or deletions through NHEJ to cut out a part of a gene, then using the CRISPR platform is less complicated. But directing CRISPR to correct a gene with exogenous DNA does not work very well, as the rate of HDR occurring at the site of interest is often very low, sometimes less than 1%. For precise genome-editing through CRISPR, it is essential to have HDR while minimizing damaging NHEJ events.

Increasing the rate of HDR is not trivial. Recent research has shown that the conditions for the two repair pathways vary based on the cell type, location of the gene, and the nuclease used (2). Besides, optimizing CRISPR to better control HDR versus NHEJ events is only half the battle. One of the greatest challenges in using CRISPR is to be able to quickly and accurately detect different genome-editing events. Many scientists measure changes by sequencing. A more sophisticated method is to use droplet digital PCR (ddPCR) (3), but many labs have limited accessibility to ddPCR systems. After screening multiple clones and detecting the desired genetic edit comes what is usually the most laborious and time-consuming step: pure clonal isolation. Because each individual cell is affected by CRISPR independently, isogenic cell lines must be established.

Improving CRISPR
CRISPR has become the gold standard for many scientists now that the potential to perform gene editing has become so universal. Top journals expect isogenic lines for characterizing genes and mutations, and many researchers feel pressured to include such experiments to stay competitive in grant proposals. Moreover, a federal biosafety committee has recently approved the first study in patients using this genome-editing technology. To keep up with the pace of this rapidly moving field, significant improvements in CRISPR technology need to be made.

First, a better grasp of the basic principles behind CRISPR is likely to lead to improvements in targeting and efficiency. For example, understanding how the cell type, locus, and genomic landscape affect the targeting and cutting of Cas9 could lead to higher efficiencies of HDR. Similarly, engineering Cas9 and the guide RNA to maximize on-target activity could also accelerate the technology’s success. Screening the ability of the guide RNA to target the intended sequence can be performed in vitro by assessing the Cas9-mediated cuts on a PCR-amplified fragment of DNA; this method often gives a good indication of what to expect in cells. Another hindrance to CRISPR technology is that currently, all methods to detect genome changes require destruction of the cells of interest. Development of an assay to detect chromosomal changes in live cells – reminiscent of live-cell RNA detection – would immensely improve the processes of clonal screening and isolation.

Besides the technical challenges that come with maximizing on-target edits is that Cas9 frequently has off-target effects, producing insertions or deletions at unintended sites. Online algorithms can predict where some of these cuts are likely to occur, but currently, there is no efficient method to identify all possible off-target sites (4). To complicate things further, no two human genomes are identical. Due to genetic variation, predicting off-target effects based on reference genomes remains a challenge. Because so much of the hype around CRISPR surrounds the potential to treat human diseases, it is imperative to make sure that CRISPR does not introduce detrimental changes elsewhere in the genome before therapeutic use in humans. Standard screening methods and biological assays need to be established to robustly assess potential damage done to other sites in the genome and to measure its impact on cell function and mutagenesis.

Despite its shortcomings, the hub of activity surrounding CRISPR has been astounding. And with the force of thousands of scientists working on this technology, the possibilities for the future of CRISPR are boundless.




  1. Jinek, M., et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012. 337(6096): p. 816-21.
  2.  Miyaoka, Y., et al., Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing. Sci Rep, 2016. 6: p. 23549.
  3.  Hindson, B.J., et al., High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem, 2011. 83(22): p. 8604-10.
  4.  Stella, S. and G. Montoya, The genome editing revolution: A CRISPR-Cas TALE off-target story. Bioessays, 2016. 38 Suppl 1: p. S4-s13.

Researchers use GFP nano antibody to study organ growth

Single-domain nano antibodies have a broad range of applications in biochemistry due to their small size, high affinity, and high specificity. Now, a team of researchers from the University of Basel and the University of Zurich has demonstrated that nano antibodies can be used for research in complex living organisms such as Drosophila, uncovering another new and exciting application for nano antibodies.

The team used nano antibodies to develop an assay for studying morphogens, molecules that regulate the pattern of tissue growth and the positions of various cell types within tissue. Morphogens form long-range concentration gradients from a localized source, ultimately determining the fate and arrangement of cells that respond to that gradient. Drosophila is a classic model system for understanding how morphogens regulate organ development. One morphogen called Dpp controls uniform proliferation and growth of the wing imaginal disc. Yet because Dpp is an extracellular, diffusible protein, it is difficult to immobilize in situ. Therefore, despite over 20 years of studying the role of Dpp as a morphogen, the lack of a dynamic system for controlling Dpp gradients has prevented researchers from understanding precisely how Dpp governs development of the wing disc.

By developing a novel synthetic system using nano antibodies, the researchers were able to modulate the concentration gradient of Dpp at the protein level. Their system—coined “morphotrap”—uses a membrane-bound GFP nano antibody to “trap” GFP-tagged Dpp at different locations along the wing imaginal disc. By tethering Dpp in a controlled spatial manner, researchers were able to determine how Dpp gradients affect wing disc development. They discovered that the gradient of Dpp is required for the patterning of the wing disc but not for lateral growth, disproving one of the field’s popular theories that address the role of Dpp. In addition to resolving the controversy with respect to the role of Dpp as a morphogen, this study pioneers a new method for using nano antibodies in situ.

“Dpp spreading is required for medial but not for lateral wing disc growth.”
Harmansa S., Hamaratoglu F., Affolter M., Caussinus E.
Nature. 2015 Nov 19;527(7578):317-22. doi: 10.1038/nature15712. Epub 2015 Nov 9.

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