Site-Specific Antibody Conjugation Strategy to Functionalize Virus-Based Nanoparticles

Abstract

Amine/thiol-reactive chemistries are commonly used to conjugate antibodies to pharmaceuticals or nanoparticles. Yet, these conjugation strategies often result in unfavorable outcomes such as heterogeneous antibody display with hindered biological activity or aggregation due to multivalent interactions of the antibody and nanoparticles. Here, we report the application of a site-specific and enzymatically driven antibody conjugation strategy to functionalize virus-based nanoparticles (VNPs). Specifically, an azide-handle was introduced into the Fc region of a set of immunoglobulins using a two-step enzymatic reaction: (1) cleavage of N-linked glycan in the Fc region by a glycosidase and (2) conjugation of a chemically reactive linker (containing an azide functional handle) using a microbial transglutaminase. Conjugation of the azide-functional antibodies to several VNPs was achieved by making use of strain-promoted azide–alkyne cycloaddition. We report the conjugation of three immunoglobulin (IgG) isotypes (human IgG from sera, anti-CD47 Rat IgG2a, κ, and Trastuzumab recombinant humanized IgG1, κ) to the plant virus cowpea mosaic virus (CPMV) and the lysine mutant of tobacco mosaic virus (TMVlys) as well as bacteriophage Qβ. Site-specific conjugation resulted in stable and functional antibody-VNP conjugates. In stark contrast, the use of heterobifunctional linkers targeting thiols and amines on the antibodies and VNPs, respectively, led to aggregation due to nonspecific and multivalent coupling between the antibodies and VNPs. We demonstrate that antibody-VNP conjugates were functional, and Trastuzumab-displaying VNPs targeted HER2-positive SKOV-3 human ovarian cancer cells. This bioconjugation strategy adds to the portfolio of methods that can be used for designing functional antibody-VNP conjugates.

Materials promoting viral gene delivery

“…14C). 378 These examples highlight that natural proteins can efficiently enhance viral delivery. However, concerns of potential immunogenicity of xenogeneic materials exist.…”

Site-selective modification strategies in antibody–drug conjugates

“…Other reports have detailed the attachment of azide polymers, 271 photosensitiser motifs 272 or virus nanoparticles. 273 Recent research offers some reservations concerning the selectivity of mTG for the conserved PWEEQYNST sequence (containing Q295) in the Fc region. 274 Trastuzumab was deglycosylated with PNGase F, and then a small azidoamine was attached with mTG.…”

Field-Effect Sensors for Virus Detection: From Ebola to SARS-CoV-2 and Plant Viral Enhancers

“…Uses as templates for inorganic and synthetic compounds have led to biohybrid materials of convincing properties (Douglas and Young, 1998;Bittner et al, 2013;Vilona et al, 2015;Tiu et al, 2016;Wen and Steinmetz, 2016;Lee et al, 2017;Zhang et al, 2018;Eiben et al, 2019), such as high-capacity battery electrodes or spatially ordered dye ensembles for light-harvesting. If employed as immobilization scaffolds for biomolecules, from peptides and antibodies up to enzymes, plant VLPs exhibit special advantages (Sapsford et al, 2006;Werner et al, 2006;Comellas-Aragones et al, 2007;Minten et al, 2011;Aljabali et al, 2012;Pille et al, 2013;Uhde-Holzem et al, 2016;Roeder et al, 2017;Dickmeis et al, 2018;Koch et al, 2018b;Tian et al, 2018;Yuste-Calvo et al, 2019a;Aves et al, 2020;Park et al, 2020). This has laid the foundation for novel plant virus-supported biocatalytic nanomaterials (Carette et al, 2007;Cardinale et al, 2012;Koch et al, 2015;Besong-Ndika et al, 2016;Cuenca et al, 2016;Brasch et al, 2017;Schwarz et al, 2017;Aumiller et al, 2018;Chakraborti et al, 2019), and for biodetection formats that may serve as blueprints for novel SARS-CoV-2 sensor layouts, as outlined in the following.…”