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.

Keywords: IgG; IgM; Valley Fever (VF); coccidioidomycosis; diagnostics; humanization; plant-made diagnostics.

Figure 1

Temporal expression of 4H2 Isotypes in Nicotiana benthamiana leaves. ΔXTFT N. benthamiana leaves were infiltrated with P-4H2 IgG or P-4H2 IgM gene constructs and leaf proteins were extracted 2–9 days post infiltration (PDI). The expression of P-4H2 IgG (A) and P-4H2 IgM (B) human chimeras was analyzed by a sandwich ELISA that detects only fully assembled IgG or IgM (containing both human kappa light chain and either human gamma or human mu heavy chain) from total soluble plant protein extracts. Mean ± SEM from at least two independent experiments is plotted.

Figure 2

Purification of P-4H2 IgG and P-4GH2 IgM from Nicotiana benthamiana leaves. Affinity-purified, humanized P-4H2 IgG (A) and P-4H2 IgM (B) were separated by SDS-PAGE on 4%–20% polyacrylamide gels under reducing (lanes 1 and 2) and non-reducing (lanes 3 and 4) conditions. One representative of multiple experiments is shown. Lanes 1 and 3, plant-made P-4H2 IgG (A) or P-4H2 IgM (B). Lanes 2 and 4, human IgG (A) or IgM (B) isotype control. M, molecular weight marker. 2.5 μg of protein was loaded per lane.

Figure 3

Western blot analysis of 4H2 IgG purified from Nicotiana benthamiana leaves. Purified recombinant P-4H2 IgG was separated under reducing (A,B) or non-reducing (C) conditions by SDS-PAGE and proteins was transferred to PVDF membranes. Proteins were then detected with antibodies specific for human kappa chain (A,C) or human gamma chain (B) to verify protein identity and assembly. Lane 1, Humanized P-4H2 IgG (1 μg per lane in A, 0.5 μg per lane in B,C), Lane 2, Human kappa IgG isotype reference standard (1 μg per lane in A, 0.5 μg per lane in B,C).

Figure 4

Western blot analysis of purified P-4H2 IgM. Recombinant 4H2 IgM purified from Nicotiana benthamiana leaves was separated under reducing (A,B) or non-reducing (C) conditions by SDS-PAGE and proteins were then transferred to PVDF membranes. The membranes were incubated with antibodies against human kappa chain (A,C) or human mu chain (B) to verify protein identity and IgM assembly. Lane 1, Humanized P-4H2 IgM (1 μg per lane in A, 0.5 μg per lane in B,C), Lane 2, Human kappa IgM isotype control (1 μg per lane in A, 0.5 μg per lane in B,C).

Figure 5

Plant-Made 4H2 Isotypes Specifically Bind to Target Antigen. Serial dilutions of P-4H2 IgG and P-4H2 IgM isotypes were incubated with CTS1 immobilized on ELISA plates. An HRP-conjugated anti-human IgG or anti-human IgM secondary antibody was used to detect the specific interaction between the plant-made IgG or IgM isotype (P-4H2 IgG, P-4H2 IgM) with CTS1, respectively. The parental mouse IgG (M-4H2 IgG) was used as a positive control, which was detected with an anti-mouse IgG secondary antibody conjugated to HRP. Mean ± SEM is plotted from three independent experiments.

Figure 6

Plant-Made 4H2 Isotypes Bind to Target Antigen on Western Blot. Recombinant CTS1 and Coccidioides posadasii mycelial culture supernatant were separated by SDS-PAGE (12%) under reducing conditions and either stained with Coomassie Blue (A) or transferred to PVDF membrane. Membranes were then probed with the parental M-4H2 IgG (B), the humanized, P-4H2 IgG (C), or the humanized, P-4H2 IgM (D), followed by the appropriate secondary HRP-conjugated antibody and development with a colorimetric substrate. Lane 1, Recombinant CTS1. Lane 2, Coccidioides posadasii mycelial culture supernatant. M, molecular weight marker.