nAb

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.

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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

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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 nAbTM. 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.

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When Great is not Good Enough—VHH Antibodies Engineered for 10 Fold Affinity Increase

Single Domain antibodies (VHH fragments, nanobodies, or as we call them, nAbs) have been generated by injecting llamas with ligand-bound GPCR for the purpose of obtaining crystals of active-state structures. Such structural information could be critical in understanding drug functions and screening for new drugs. The unique ability of VHH fragments to fit into protein-protein complex crevices and hold proteins together was demonstrated by two Nature publications from Brian Kobilka’s group at Stanford ([1, 2], also see Allele Newsletter of Sep 4th, 2013). The nano antibody used in those studies, Nb80, showed affinity towards only the active state of the target GPCR.

However, even with an antibody as great as Nb80, the authors were only able to co-crystal GPCR beta2-adrenoceptor (b2AR) with high affinity agonists, not its natural agonists such as adrenaline. In yet another Nature paper published just now, the Kobilka lab showed that Nb80 could be further improved by 10 times in affinity, through in vitro evolution [3]. They presented Nb80 on the surface of yeast using an existing yeast display system, then applied standard limited mutagenesis and magnetic separation technologies for screening. After about 5 rounds of selection, a new version of VHH Nb6B9 was isolated that bound to ligand-loaded GPCR with a kD of 6.4 nM. For the first time, a co-crystal of b2AR-adrenoline was made.

Rasmussen et al. Nature, 2011 Structure of a nanobody-stabilized active state of the b2 adrenoceptor
Rasmussen et al. Nature, 2011 Crystal structure of the b2 adrenergic receptor–Gs protein complex
Ring et al. Nature, 2013 Adrenaline-activated structure of b2-adrenoceptor stabilized by an engineered nanobody

Update here http://www.allelebiotech.com/nab

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Visualizing Endogenous Synaptic Proteins in Living Neurons

The recently published method is based on the generation of disulfide-free “intrabodies”, a structure from the 10th fibronectin type III domain known as FingRs. These affinity molecules were fused to GFP for direct fluorescence miscroscopy. The FingRs do not need di-sulfite bonds and are therefore better folders in mammalian cells. Specifically, a library was screened with in vitro display to identify FingRs that bind two synaptic proteins, Gephyrin and PSD95. After the initial selection, the researchers from USC secondarily screened binders using a cellular localization assay to identify potential FingRs that bind at high affinity in an intracellular environment. As it turned out, only 10-20% of the original positive clones bind well inside the cells, suggesting this type of further screening was a critical step.

The expression of intrabody is transcriptionally regulated by the target protein through a ZFN-repressor fusion. This transcriptional control system matches the expression of the intrabody to that of the target protein regardless of the target’s expression level. This design virtually eliminates unbound FingR, resulting in very low background that allows unobstructed visualization of the target proteins. As result, the FingRs presented in this study enabled live cell visualization of excitatory and inhibitory synapses, and apparently without affecting neuronal function.

Technically, the reason to use in vitro mRNA display was required by the need to use a large library (>10exp12, beyond the limit of the more commonly used phase display) to find good binders. A similar visualization system can be established using more potent affinity domains such as the VHH single-domain antibodies that have only one, sometimes dispensable, di-sulfite bond. The VHH domain nanobodies can be more easily isolated from camelid animals. Another improvement to the visualization system can be made by using stronger, superresolution-ready FPs such as mNeonGreen or mMaple to enable single molecule imaging, which is particularly interesting for studying synapses and applied to the BRAIN initiative.

Gross et al. Neuron, June 2013, http://www.ncbi.nlm.nih.gov/pubmed/23791193

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