nAb: Camelid Antibodies, Nanobodies, 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.”

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

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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.”


Allele Biotechnology & Pharmaceuticals, Inc.
Jiwu Wang, 858-587-6645

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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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Lab Skills You Stopped Being Proud Of

Molecular biologists who were in graduate school in the 90’s learned how to isolate plasmid DNA from E. coli cultures by a method called “boil-prep” during their first lab rotation. This process involved mixing the bacterial cell pellet in a little bit of detergent, salt and sucrose, dabbed with some fresh lysozyme, and then you are ready to cook, literally! Bacterial cell membranes are disrupted by boiling this soup in a beaker of water over a Bunsen Burner for one minute, and the debris (containing the broken cell membrane and attached chromosomal DNA) is collected by centrifugation in a microfuge at top speed for 10 minutes. Then comes the step that differentiates a true master of lab skills versus a rotation student—if you knew just the right amount of bacterial culture to begin with and handled the E coli pellet by the right techniques, a skillful lab person could collect nearly all the liquid without disturbing the pellet. Pouring out the plasmid-containing supernatant without dislodging the goo on the side/bottom of the tube was such a desirable skill that would not only give you your plasmid but also give you admiration from fellow lab members. That is, of course, if you were doing it before the mid-90’s, because after the introduction of miniprep spin columns by Qiagen, nobody, even the true masters of boil-preps (or its contemporary alkali prep that also involves pelleting by centrifugation and careful removal of tiny volume of liquids surrounding small pellets) would be showing off those skills any longer.

It is actually never easy or fun to collect liquid surrounding small amount of beads or pellets as you always have to struggle to remove as much liquid as possible while trying not to lose any of the beads

Some of the old-timers used to also be very proud of being able to pour a “sequencing gel” (a very thin ~40 cm x 30 cm polyacrylamide gel). I still remember the first time I reported to the second rotation lab at USC. After describing the lab research, the PI showed me around the lab and complained how “Sarah destroyed all my sequencing gel plates”. But consider this, in order to avoid any greasy spot on either plate, you needed to wash both of them fanatically if not religiously. Why? You would have just about a minute’s time to pour non-polymerized acrylamide without leaking from the sides or bubbles forming anywhere in the DNA running lanes, and then inserting a pair of paper-thin combs, all at a speed quicker than TEMED/AP-catalyzed acrylamide polymerization. Good thing that after capillary sequencing was invented, we all happily retired our sequencing-gel pouring skills with a collective sigh of relief.

Technology will always move forward, so will the skills lab researchers will be required to perfect. Using a spin column is very much a “skill-less” technique in contrast to collecting pellets and washing beads after centrifugation, but when there is a choice, people will chose the method that requires “less skills”, such as the spin-column format as the preferred platform for the new FP-nAb™ products.

BTW, like to have information on the spin column kit? Here it is:

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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

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