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

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VHH Nanobodies in Superresolution Imaging and More

From the large number of recent publications using GFP-Trap beads, it appears that GFP-Trap is on the way to becoming one the most popular tags for co-IP thanks to its unparalleled “cleanness” of precipitated protein bands and its quantitative binding capabilities. As described previously, the antibody conjugated on the GFP-Trap beads is a single-domain antigen binding module from camelid single-chain antibodies. Termed VHH, this domain is only ~12 kD and can fit into structures that other types of antibodies cannot. We have successfully created VHH antibodies against a number of neural factors as a research project for the NIDA/NIH.

VHH antibodies are often called nanobodies as a result of their size (1.5 – 2.5nm) and binding affinity ( GFP-trap has a binding affinity of 0.59nM). In addition to their use for co-IP, VHH antibodies have proven themselves as a resilient tool for various other applications. Anti-GFP nanobodies, for example, are currently used to enhance the fluorescence of GFP (GFP-trap booster utilizes the same VHH binding antibody coupled to a fluorescent dye); others have used VHH antibodies that can insert into certain part of GFP to dim the fluorescence signal . More recently, Ries et al. published in Nature Methods that the anti-GFP nanobodies offered a simple and versatile method for super-resolution imaging (i.e. PALM)-previously super-resolution imaging requires photoconvertible fluorescent proteins (such as Eos, mClavGR2). With dye-conjugated nanobodies, generating fusions to these newer FPs is no longer needed, however, using the nanobody super-imaging method requires fixing and permeabilizing the cells.

When using anti-GFP VHH reagents you need to be aware that other fluorescent proteins can also be recognized, if they were derived from the avGFP (jellyfish GFP). Also, some GFPs are not recognized if they are from another species, or engineered such as our mWasabi. We are producing newer and brighter GFP/YFPs based on the lancelet YFP protein to offer alternative series that will not be cross-recognized by the GFP-Trap antibodies.

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Finding the Best Capture Reagents

As capture reagents, monoclonal antibodies are the most widely used reagents for specifically detecting and quantifying proteins due to their very high specificity. However, development of monoclonal antibodies is time-consuming and expensive. In addition, many antigens prove to be non-immunogenic or extremely toxic, and therefore cannot be used to generate antibodies in animals. Furthermore, the large size of monoclonal antibodies (150 kDa) may limit their use in cases where more than one binding reagent competes for space to recognize closely juxtaposed epitopes. These limitations could arguably be the biggest hurdles to using monoclonal antibodies as capture reagents for a systematic study of the complete human proteome or for clinical applications of advanced proteomics.

Therefore, alternative capture reagents with high specificity, high affinity, and flexible size and structure that can be easily and cost-effectively produced are urgently needed in order to accelerate proteomic research. Single-chain variable-fragment (scFv) antibodies have been commonly used as alternatives in this regard. scFv is comprised of only the light chain and heavy chain variable regions connected by a peptide linker and with a molecular weight of 27 kDa. Since scFv retains the antigen-binding site of the variable regions, it inherits the specificity of an intact antibody and affinity. In addition, scFv can be easily expressed in yeast or in E. coli with yields in milligrams per liter. scFv can be linked to Fc of desired species specificity and maintain binding properties. If necessary, there is also the option of converting scFv into other antibody formats such as Fab or full IgG by simple cloning steps. The converted antibodies can also be efficiently expressed and purified in yeast or E. coli.

More recently, single domain antibodies that exist in nature were discovered that can be as small as half the size of scFv, and judging from the available data, superior in binding capabilities to scFv or even traditional IgG antibodies. This type of affinity molecules, termed VHH isolated from camelid animals or nurse shark, can be highly expressed in E. coli, linked to a fluorescent protein marker, or chemically conjugated to HRP or other signal generating moieties through a one step reaction.

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    BioTechniques Publishes Article on Single Domain Antibodies

    Many blogs start by asking “Did you know…” to intrigue you to read along. So here it goes:

    Did you know that there are more than 300,000 antibodies that are commercially available? And yes, many antibody companies are still generating more antibodies at ever faster pace and in a more systematic way. There are companies that plan to make peptide or short protein fragments for making antibodies against all human proteins or subproteome, others develop antibodies particularly suitable for demanding assays such as ChIP-CHIP. Government activities such as the National Cancer Institute (NCI)’s Clinical Proteomic Technologies Initiative (CPTI) and the Road Map program under the NIH Director’s Office also set goals of producing comprehensive sets of widely usable, renewable, affinity reagents for clinical cancer samples or the human proteome. Apparently people do not think the 300,000 available antibodies are sufficient for what they do.

    Did you know that conventional antibodies commonly used as reagents are ~150kDa in molecular weight and can hardly be used inside live cells? Ulrich Rothbauer, professor in the department of biology at Ludwig Maximilians University, who is working with colleagues to develop tools to study cellular processes in living cells. “These antibodies have to assemble four different chains, two heavy and two light, and they’re assembled by disulfide bonds that cannot be correctly formed in the reducing environment of the cytoplasm. You cannot express such a huge complex molecule in living cells. You can [introduce] them by microinjection, for example, but it’s not applicable for high-throughput cell imaging.” [1] Antibody fragments such as scFv, Fab, and similar derivatives have been developed over the years to certain level of success, but not as widely accepted or practically amenable to replacing conventional antibodies.

    Did you know that camel, llama, and shark naturally produce single heavy chain antibodies that can function as 13-16kDa fragments (yes if you have read previous Allele Blogs They can easily be produced in bacteria, used directly inside live cells via transgene, fused to other proteins as a fusion tag, linked to DNA oligos as a detection module, or immobilized on beads for pull down or co-IP. Currently, these antibodies need to be selected by display after obtaining immunized antibody libraries. There is generally no commercial service for creating custom camelid antibodies at this time due to patent and other issues. Existing products are available for jelly fish GFP and DsRed derived RFP fusions. Publications using such a limited number of camelid antibodies have been amazing so far—dozens in top journals within the last few months and after only a short period of time since product launch.

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    Original BioTechniques Article

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