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 glycoengineered Nicotiana benthamiana plants via transient expression 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.

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.…”

Clinical Laboratory Utility of a Humanized Antibody in Commercially Available Enzyme Immunoassays for Coccidioidomycosis

“…The variable regions of 4H2, a murine monoclonal antibody (MAb) to CTS1, were cloned, sequenced, and genetically fused into human IgG and IgM Fc-containing plasmids to form a chimeric mouse V-region–human Fc region antibody construct, previously detailed ( 21 ). The chimeric plasmids were subjected to agroinfiltration in Nicotiana benthamiana leaves, followed by harvest and purification as described elsewhere ( 21 , 24 ).…”

“…The variable regions of 4H2, a murine monoclonal antibody (MAb) to CTS1, were cloned, sequenced, and genetically fused into human IgG and IgM Fc-containing plasmids to form a chimeric mouse V-region–human Fc region antibody construct, previously detailed ( 21 ). The chimeric plasmids were subjected to agroinfiltration in Nicotiana benthamiana leaves, followed by harvest and purification as described elsewhere ( 21 , 24 ). The resultant plant-produced humanized MAbs (P-4H2 IgG and P-4H2 IgM) were evaluated for their performance on two commercially available enzyme immunoassays (EIAs) that detect human IgG and IgM antibodies specific for Coccidioides spp., described below.…”

“…This finding further suggests that CTS1 is a component of the antigenic preparation used in each EIA, whether intentional or not. Therefore, in this report, we evaluate an anti-CTS1 humanized monoclonal antibody produced in plants as chimeric IgG and IgM isotypes ( 21 ) in both IMMY and Meridian FDA-approved Coccidioides EIA kit tests.…”