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.

Tags: Allele Biotech, anti-GFP, camelid antibodies, Fluorescent proteins, morphogens, nanobodies, VHH

A breakdown of your burning nAb questions

Allele Biotechnology just released its latest batch of nAbs (nano antibodies), the first wave on a long list of new antibodies to come! You might have a few questions about how these “antibodies of the future”, as we call them, can help your research:  What can I use them for?  How much should I use?  And how do they work compared to a traditional antibody? 
 
To answer these questions, we need to first discuss some antibody basics.  Conventional antibodies (your typical mouse or rabbit derived antibody) have a “Y” shape and tightly bind targeted antigens as a result of two factors.  The first is affinity between each monomer Fab fragment and the antigen.  The second is the fact that traditional antibodies are di-valent, i.e. they have two identical binding sites for each antigen, which is known as avidity. 
 
When developing a nano-antibody, we screen and select our clones to have extremely high affinity as a monomer.  This is because nAbs are mono-valent VHH fragments. The intrinsic high affinity VHHs possess for their antigens can make up for the lack of multivalency (avidity).  As a result, nAb binding is often superior to conventional antibody binding, which leads to superior performance in a variety of biological assays (immunoprecipitation, immune-staining, FACS staining, immunofluorescent imaging, etc.). 
 
Each nAb is roughly one tenth (1/10) the size of a traditional antibody.  The small size and stable conformation of nano-antibodies enable pinpointed localization of target antigens and allow access to antigen and cellular regions generally restrictive to larger antibodies. As a result of this smaller size, when measured by weight 1mg of a nAb is equivalent to 5 – 10mg of a traditional antibody (the lower end takes di-valency into account).  When substituting a nAb for a traditional antibody you can use as little as one tenth (1/10) the amount by weight. 
 
There are a couple of different ways to use nAbs.  The first is immobilizing the nano-antibody on a resin (i.e. magnetic-agarose resin) for immunoprecipitation.  The nano-antibody will not be released from the resin upon elution so you will not have contaminating bands.  The second method is direct labeling with a fluorescent dye or hapten.  nAb’s are compatible with standard NHS-ester amine chemistry binding.  This enables single or multiple fluorophore labeling per antibody.  Moving forward, additional platforms will be released that allow for a more flexible and adaptable labeling system, allowing you to harness nAbs for any biological assay you can imagine.  Have some suggestions? Don’t hesitate to let us know by emailing at nAb@allelebiotech.com. Or call 858-587-6645 and ask for a nAb expert.

Tags: nAb, nano-antibody, nanobody, VHH

Press Release: Allele Biotech Takes Major Step into Nano Antibody Leadership Position

SAN DIEGO–(BUSINESS WIRE)–Allele Biotechnology & Pharmaceuticals Inc., a San Diego based life sciences company with a focus on novel technology development, releases the first group of a brand new class of antibodies against crucial biological targets to the research market. This week, Allele launches nano-antibodies isolated from llamas against human bFGF, P16, VEGF, and TNFa, which are all important targets in the field of cancer biology.

Nano-Antibodies (also known as nAb™, Nanobodies®, Single Domain Antibodies, Camelid Antibodies and VHH antibodies) represent the future in antibody technology of Allele’s interest. “Camelid antibodies have been an area of intense research activities at Allele because they have desirable features that no other antibody has. These tiny antibodies outperform conventional antibodies in many ways and thrive in extreme conditions, eventually they will occupy a significant portion of the antibody reagent market,” said Dr. Jiwu Wang, CEO and founder of Allele Biotechnology. This first wave of novel reagents has been meticulously tested for immunohistochemistry (IHC) in human cancer tissues; some of these antibodies also performed well in cross-species reactivity in mouse and rat while others are highly suitable for advanced applications such as flow cytometry and antigen immunoprecipitation.

This is the first release in a long-term effort to generate and commercialize hundreds of nano-antibody derived capture tools. “Our nano-antibody project is based on years of internal technology development partially funded by the National Institute of Drug Abuse of the NIH,” according to Allele’s Marketing Director, Abbas Hussain. “The nAb product line will shortly encompass a wide range of high value targets that are applicable to both basic and clinically relevant research. It will also feature cutting edge conjugation technologies that enable fluorescent imaging and electron microscopy techniques being developed at Allele.”

Since the ability to generate monoclonal antibodies was discovered in 1975, antibodies have been used in virtually every branch of biomedical research and development. In the past decade there has been a shift toward harnessing antibody technology for therapy, as illustrated by large number of antibody-based drugs on the market today. Allele’s nAb development has been one of the targets of investment from Yifang Ventures and Yuan Capital.

“Nanobodies® is a trademark of Ablynx; nAb as nano antibodies is under copyright of Allele Biotechnology, all rights reserved.”
Contacts

Allele Biotechnology & Pharmaceuticals Inc.
Abbas Hussain, 858-587-6645
Director of Sales & Marketing
6404 Nancy Ridge Dr.
San Diego, CA 92121
abbashussain@allelebiotech.com

Tags: nAb, nano-antibody, nanobody, VHH

Press Release: Allele Biotechnology & Pharmaceuticals Closes Purchase of cGMP Facility for Production of Clinical-Grade Cells for Cell Therapy Applications

SAN DIEGO–(BUSINESS WIRE)–Allele Biotechnology & Pharmaceuticals, Inc. (“Allele”), a leader in the development of specialized cells for pharmaceutical drug discovery and regenerative medicine, today announced that it has closed the purchase of a new facility intended for its cGMP (current good manufacturing practices) production of clinical-grade cells for cell therapy applications.

The 18,000 square-foot facility, located near the main headquarters of Allele in San Diego, California, will be the center of production of human induced pluripotent stem cells (hiPSCs) using Allele’s proprietary synthetic mRNA platform, a technology that generates hiPSCs with neither the random integration of foreign DNA nor the use of whole virus or virus-based elements, drawbacks that are common to other technologies for making hiPSCs. Such “footprint-free” cells will be produced by Allele for industrial and academic partnerships, as well as Allele’s own efforts in the area of cellular therapeutics.

hiPSCs, as cells that have the potential to become any cell in the human body, hold great promise for therapies that can alleviate or cure human disease. Towards this end, Allele has recently made a number of advances regarding the differentiation of hiPSCs towards cells of specific lineages, such as neural progenitor cells, neurons, astrocytes, mesenchymal stem cells, cardiomyocytes, skeletal muscle cells, hepatocytes, and adipocytes, including brown fat cells. These cells would also be produced in the cGMP facility when intended for specific therapies.

“This dedicated facility will help us to realize a number of our visions in bringing the benefits of pluripotent stem cells to society,” said Jiwu Wang, Ph.D., President and CEO of Allele. “The first step in helping people in need with all the stem cell technologies developed in labs is to clear a path to move them from bench to bedside, which requires high-quality, controlled production that can be monitored by the FDA. Together with our licensees, drug development partners, investors, and individuals who would like to participate in banking hiPSCs for research and therapy, we anticipate even faster pace in our business development in this area.”

Contacts

Allele Biotechnology & Pharmaceuticals, Inc.
Jiwu Wang, 858-587-6645
info@allelebiotech.com

Tags: GMP iPSCs, GMP Stem Cells

What Does It Take to Bring New Nano Antibodies (nAbs) to the Hands of Researchers?

Judging from the hundreds of papers published using camelid VHH antibodies as reagents, there are probably thousands of researchers who have experience with this type of antibodies by now. We like to call the ~15kD camelid VHH antibody nano antibody or nAb^TM. Once someone experiences how well a nAb works for co-IP using a fluorescent protein as tag, they often wonder what it takes to bring nAbs to broader use.

The success of a nAb project starts with the antigen presentation. It is critical to build the capability to produce large quantities of recombinant antigen for immunization. At Allele, our scientists also established some unique presentation formats for traditionally difficult targets (e.g. large membrane proteins).

After llama immunization, the next step is screening. With the goal of creating large scale nano antibodies against diverse targets, we have developed multiple high throughput screening methods to cover very large, diverse libraries generated from immunized animals. The technologies will continue to evolve as the scale of nAb generation continues to expand. We have the ability to functionally screen for site-blocking antibodies and antibodies that only recognized natively folded targets, or targets in their naturally occurring presentations.

A nAb isolation project does not end with the obtaining of a cDNA clone. Or, if it does, the nAb is probably not as great as what Allele Biotech has been offering. In our hands, all nAbs go through an engineering step beginning with the generation of a 3D structural model of the isolated clone. We use structure-guided design to alter the protein, allowing us to improve its properties. This includes increasing affinity, solubility, or altering the protein to improve performance for specific applications. We also like to use known structures of traditional monoclonal antibodies to assist camelid VHH antibody engineering against specific targets.

With a finalized clone in hand, the next step is to establish protocols for commercial production. The Allele team spends a tremendous amount of effort aimed solely at high-yield, low-cost recombinant VHH antibody production in a variety of formats, so that the costs for other scientists to take advantage of these great reagents can be kept as low as possible.

Last but not the least, nAb labeling, including conjugating stable soluble VHH antibody to solid supports for immunoprecipitation or to fluorophores for detection, requires additional expertise and tight operation control. However, our vision is to have a modular system for antibody labeling that will enable the end user to select from a variety of fluorophores and other detection tags, which can be instantaneously and irreversibly coupled via simple mixing.

Note added: we work with commercial (diagnostic and clinical) partners from developing nAbs all the way to the market. We have expert scientists available to customers and licensees for consultation and troubleshooting antibody- and imaging-related questions and problems.

Tags: alpaca antibody, antibodies against membrane proteins, antibody engineering, camelid antibodies, GFP-nAb, GFP-Trap, llama antibody, mNeonGreen-nAb, nAb, nanoantibodies, nanoantibody, VHH