Generation of High‐Specificity Antibodies against Membrane Proteins Using DNA‐Gold Micronanoplexes for Gene Gun Immunization

Abstract

Membrane proteins are the molecular interface of the cell and its environs; however, studies of membrane proteins are highly technically challenging, mainly due to instability of the isolated protein. Towards the production of antibodies that recognize properly folded and stabilized forms of membrane protein antigen, we describe a DNA-based immunization method for mice that expresses the antigen in the membranes of dendritic cells, thus allowing direct presentation to the immune system. This genetic immunization approach employs a highly efficient method of biolistic delivery based on DNA-gold micronanoplexes, which are complexes of micron-sized gold particles that allow dermal penetration and nanometer-sized gold particles that provide a higher surface area for DNA binding than micron gold alone. In contrast to antibodies derived from immunizations with detergent-solubilized protein or with protein fragments, antibodies from genetic immunization are expected to have a high capacity for binding conformational epitopes and for modulating membrane protein activity. © 2018 by John Wiley & Sons, Inc.

In vitro affinity maturation of antibody against membrane-bound GPCR molecules

“…Yeast display was previously used to mature the affinity of an scFv against the transmembrane protein transferrin receptor (Cho and Shusta 2010), in which detergent-solubilized transferrin receptor was used as a probe to enrich the yeast cells with high binding abilities to the probe. However, detergent solubilization often alters the functional conformation of a GPCR; thus, this method is not always possible to mature an antibody against a specific functional conformation (Geppetti et al 2015; Hansen et al 2018; Hotzel et al 2011). The average sizes of budding yeast and CHO cells are 4000 nm and 10,000 nm respectively, and the difference of their average surface areas is 5.76-fold.…”

“…For instance, yeast display has been used to mature an anti-transferrin receptor (TfR) scFv by using detergent-solubilized cell lysates as probes (Tillotson et al 2015). The problem is that detergent-solubilized protein probes may not guarantee to possess native conformations (Hansen et al 2018; Hotzel et al 2011; Wilkinson et al 2015). Phage display has been used in affinity maturation for antibodies against GPCR molecules (CCR4, CC chemokine receptor 4 and formyl-peptide receptor 1) on plasma membrane of intact human cells (Hagemann et al 2014; Krebs et al 2001); thus, these GPCR molecules are in native conformations.…”

Therapeutic Monoclonal Antibodies against Cancer: Present and Future

“…Animals can be immunized with known antigens using several strategies. The first would be to subclone the cDNA coding for the desired antigen into an eukaryotic expression vector, bind the plasmid to gold nanoparticles, and use a biolistic particle delivery (gene gun) for the particle-mediated DNA immunization [139,140]. This procedure allowed generating therapeutic antibodies against CCR9 [141,142].…”

Bacteriophages as Potential Tools for Use in Antimicrobial Therapy and Vaccine Development

“…Naked DNA molecules can be degraded, requiring various methods to extend the time of transgene expression and increase the efficiency of transfection by transporting DNA to target cells (e.g., electroporation, biolistic delivery based on DNA–gold micronanoplexes, lipid-based nanoparticles (LBNs)). In addition, optimization of the immunogenicity of such vaccines is also needed based on, e.g., codon optimization, genetic adjuvants, and sophisticated booster-priming schemes [ 56 , 59 , 60 , 61 ]. Nowadays, no commercial DNA vaccines are also known to be used for the prevention of infectious diseases in humans, but due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, intensive work is underway on potential vaccines (to limit the global spread of infection).…”