A cDNA Immunization Strategy to Generate Nanobodies against Membrane Proteins in Native Conformation.

Thomas Eden,Stephan Menzel,Gudrun Dubberke,Friedrich Koch-Nolte,Friedrich Haag,Fabienne Seyfried,Birte Albrecht,Philine Bergmann,Marion Nissen,Janusz Wesolowski

doi:10.3389/fimmu.2017.01989

Abstract

Nanobodies (Nbs) are soluble, versatile, single-domain binding modules derived from the VHH variable domain of heavy-chain antibodies naturally occurring in camelids. Nbs hold huge promise as novel therapeutic biologics. Membrane proteins are among the most interesting targets for therapeutic Nbs because they are accessible to systemically injected biologics. In order to be effective, therapeutic Nbs must recognize their target membrane protein in native conformation. However, raising Nbs against membrane proteins in native conformation can pose a formidable challenge since membrane proteins typically contain one or more hydrophobic transmembrane regions and, therefore, are difficult to purify in native conformation. Here, we describe a highly efficient genetic immunization strategy that circumvents these difficulties by driving expression of the target membrane protein in native conformation by cells of the immunized camelid. The strategy encompasses ballistic transfection of skin cells with cDNA expression plasmids encoding one or more orthologs of the membrane protein of interest and, optionally, other costimulatory proteins. The plasmid is coated onto 1 µm gold particles that are then injected into the shaved and depilated skin of the camelid. A gene gun delivers a helium pulse that accelerates the DNA-coated particles to a velocity sufficient to penetrate through multiple layers of cells in the skin. This results in the exposure of the extracellular domains of the membrane protein on the cell surface of transfected cells. Repeated immunization drives somatic hypermutation and affinity maturation of target-specific heavy-chain antibodies. The VHH/Nb coding region is PCR-amplified from B cells obtained from peripheral blood or a lymph node biopsy. Specific Nbs are selected by phage display or by screening of Nb-based heavy-chain antibodies expressed as secretory proteins in transfected HEK cells. Using this strategy, we have successfully generated agonistic and antagonistic Nbs against several cell surface ecto-enzymes and ligand-gated ion channels.

Highlights

Nanobodies (Nbs), single-domain antibodies derived from camelid heavy-chain antibodies, are 10-fold smaller than conventional antibodies (Figure 1) and exhibit a number of advantageous features [1,2,3,4]
Nbs can bind epitopes that are inaccessible to conventional antibodies, e.g., cryptic functional epitopes such as the active site of an enzyme, the ligand-binding site of an ion channel, or the virus-binding site of a cell surface receptor [5]
The first Nb expected to be licensed for clinical applications year is directed against a soluble protein

Summary

Introduction

Nanobodies (Nbs), single-domain antibodies derived from camelid heavy-chain antibodies, are 10-fold smaller than conventional antibodies (Figure 1) and exhibit a number of advantageous features [1,2,3,4]. We describe a cDNA immunization strategy that has allowed us to generate Nbs and Nb-based heavy-chain antibodies against membrane proteins from immunized llamas [8, 20,21,22]. T cell-mediated cytolysis of transfected cells can facilitate uptake of vesicles containing the membrane protein by antigen-specific B cells (see Mechanism of Antibody Induction by Genetic Immunization).

Results

Conclusion