Humanization and Expression of IgG and IgM Antibodies in Plants as Potential Diagnostic Reagents for Valley Fever

Abstract

Monoclonal antibodies (mAbs) are important proteins used in many life science applications, from diagnostics to therapeutics. High demand for mAbs for different applications urges the development of rapid and reliable recombinant production platforms. Plants provide a quick and inexpensive system for producing recombinant mAbs. Moreover, when paired with an established platform for mAb discovery, plants can easily be tailored to produce mAbs of different isotypes against the same target. Here, we demonstrate that a hybridoma-generated mouse mAb against chitinase 1 (CTS1), an antigen from Coccidioides spp., can be biologically engineered for use with serologic diagnostic test kits for coccidioidomycosis (Valley Fever) using plant expression. The original mouse IgG was modified and recombinantly produced in plants as IgG and IgM isotypes with human kappa, gamma, and mu constant regions. The two mAb isotypes produced in plants were shown to maintain target antigen recognition to CTS1 using similar reagents as the Food and Drug Administration (FDA)-approved Valley Fever diagnostic kits. As none of the currently approved kits provide antibody dilution controls, humanization of antibodies that bind to CTS1, a major component of the diagnostic antigen preparation, may provide a solution to the lack of consistently reactive antibody controls for Valley Fever diagnosis. Furthermore, our work provides a foundation for reproducible and consistent production of recombinant mAbs engineered to have a specific isotype for use in diagnostic assays.

Glyco-engineered pentameric SARS-CoV-2 IgMs show superior activities compared to IgG1 orthologues

“…Whereas, IgG1 carries one conserved, Fc-located GS, usually decorated with N-acetylglucosamine (GlcNAc) or galactose-terminating complex N-glycans (Stadlmann et al ., 2008). Glyco-engineered ΔXTFT line was used since it generates Abs with largely homogeneous and reproducible N-glycosylation profiles lacking plant specific residues (Jugler et al ., 2022; Strasser et al ., 2008). Moreover ΔXTFT-derived IgGs often exhibit increased functional activities compared to orthologues produced in CHO cells or wild type plants (Forthal et al ., 2010; Loos et al ., 2015; Zeitlin et al ., 2011).…”

“…While the production of IgG1 has been demonstrated in multiple cases using plant-based expression systems, only a few studies report the generation of more difficult to express Abs like IgG3 and dimeric IgA (Goritzer et al ., 2020; Kallolimath et al ., 2020; Kallolimath et al ., 2021; Sun et al ., 2021; Teh et al ., 2021; Vasilev et al ., 2016). Although an IgG1 to IgM switched mAb was produced recently (Jugler et al ., 2022), only one previous report describes the plant-based production of multimeric IgM (Loos et al ., 2014). The study presents a proof-of-concept for the multimeric assembly of an anti-cancer IgM in plants.…”

A novel plant‐made monoclonal antibody enhances the synergetic potency of an antibody cocktail against the SARS‐CoV‐2 Omicron variant

“…cDNA was generated from cells of the 11D7 hybridoma using a RNeasy kit for total RNA isolation (Qiagen), followed by first‐strand generation by the Reverse Transcription System (Promega). The coding sequences of mAb variable regions of were generated by PCR using degenerate primers (Wang et al ., 2000), cloned into the CloneJET PCR Cloning Kit (Thermo Scientific), and sequenced by Sanger sequencing as described previously (Jugler et al ., 2022).…”

“…The authors thank Dr. K. Kibler for technical assistance, Dr. Y. Li for sharing SARS‐CoV‐2 B.1.1.529 stock and H. Lai for sharing the recombinant RBD proteins of various SARS‐CoV‐2 variants. We also thank Dr. D. Lake and F. Grill for their advice in gene rescue and contributions described in Jugler et al ., 2022, and Clemens Gruenwald‐Gruber (Core Facility Mass Spectrometry, BOKU VIENNA) for MS analyses. This research was supported by a sponsored research agreement (FP00026198) funding to Q. Chen Lab via Arizona State University by the Halberd Corporation.…”