Nanobody Services

• Antigen purification by SEC (required prerequisite, if not perfomed by user)

• Basic or Full Selection Service:

  • antigen validation

  • 2 rounds of magnetic sorting

  • 2 rounds of fluorescence activated cell sorting

  • clonal isolation and Sanger sequencing of clones and NGS of the enriched library

  • Full service also includes cloning for bacterial expression

• Nanobody expression and purification

• Additional Services

  • Affinity Maturation

  • Counterscreening

  • Affinity Measurement and Epitope Binning

The CMI currently uses a synthetic yeast display library, developed in the Kruse lab, a based on a consensus framework derived from llama antibody genes with variable complementary determining region loops (CDRs) designed from known structures of nanobodies against protein antigens.

Original Reference:
McMahon, C. et al.Yeast surface display platform for rapid discovery of conformationally selective nanobodies. Nat Struct Mol Biol25,289–296 (2018).

The majority of antibodies used in the laboratory are of mammalian origin and have a conventional bivalent architecture consisting of two heavy chains and two light chains. The heavy and light chains have terminal variable domains responsible for antigen specificity, V_Hand V_L. While camelids also produce conventional antibodies, the majority of circulating antibodies have a different architecture, consisting only of two heavy chains, called heavy chain antibodies.¹ The smaller variable domains of camelid antibodies (V_HH domains), also called nanobodies, allow them to bind in clefts of folded proteins that are not accessible by larger heterodimeric Fab and single-chain variable domain (scFv) fragments. Nanobodies have been particularly useful as tools to facilitate structural studies and to modulate function of many proteins including integral membrane receptors.² Unlike conventional antibodies or antibody fragments which have multiple obligate interchain and intrachain disulfide bonds, the camelid V_HH domain framework (which can have up to two disulfides), generally retains function in the reducing environment of the cytoplasm. Combined with their small size, this makes nanobodies particularly useful tools for intracellular applications such as super-resolution live-cell imaging.^3,4 In addition to being useful laboratory tools, nanobodies are plausible scaffolds for drug development. Nanobody scaffolds have been humanized⁵ and the first nanobody drug (Caplacizumab) was approved by the FDA.⁶

While camelid heavy chain antibodies can be produced by immunization of camels, llamas or alpacas, the process requires a large animal husbandry facility and is time consuming and expensive. The Kruse lab has developed a synthetic yeast display library based on a consensus framework derived from llama antibody genes with variable complementary determining region loops (CDRs) designed from known nanobody structures.⁷ The CMI antibody discovery platform currently offers nanobody production services based on this unique HMS resource. 

Antibodies derived using surface display technologies, such as yeast display, offer several unique features rarely accessible by animal immunization.⁸ The most practical advantage of surface display technologies is that antibody fragments are sequence-verified and renewable, allowing for rapid subcloning into a variety of expression systems for production in many formats. Specificity can be expanded or restricted by including secondary or counter screens with additional antigens. In vitro selection methods have been used to achieve selective recognition of chemical modifications and proteins with single surface-exposed amino acid differences, where conventional immunization has failed. In vitro selection methods avoid immunological tolerance, which can restrict the production of antibodies produced in mammals against highly conserved antigenic targets. Affinity maturation to improve affinity or selectivity, by generation of secondary libraries of mutagenized variants, can be performed at any stage using the same selection process. Surface display methods can also be used to directly select human antibody frameworks, avoiding the need to humanize animal frameworks for conversion to biologics.

1. Hamers-Casterman, C. et al.Naturally occurring antibodies devoid of light chains. Nature363,446–448 (1993).
2. Manglik, A., Kobilka, B. K. & Steyaert, J. Nanobodies to Study G Protein-Coupled Receptor Structure and Function. Annu. Rev. Pharmacol. Toxicol.57,19–37 (2017).
3. Pleiner, T. et al.Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation. Elife4,e11349 (2015).
4. Schumacher, D., Helma, J., Schneider, A. F. L., Leonhardt, H. & Hackenberger, C. P. R. Nanobodies: Chemical Functionalization Strategies and Intracellular Applications. Angew. Chem. Int. Ed. Engl.57,2314–2333 (2018).
5. Vincke, C. et al.General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold. J Biol Chem284,3273–3284 (2009).
6. Kaplon, H. & Reichert, J. M. Antibodies to watch in 2019. MAbs11,219–238 (2019).
7. McMahon, C. et al.Yeast surface display platform for rapid discovery of conformationally selective nanobodies. Nat Struct Mol Biol25,289–296 (2018).
8. Bradbury, A. R. M., Sidhu, S., Dübel, S. & McCafferty, J. Beyond natural antibodies: the power of in vitro display technologies. Nat Biotechnol29,245–254 (2011).
9. Wörn, A. & Pluckthun, A. Stability engineering of antibody single-chain Fv fragments. J Mol Biol305,989–1010 (2001).