An efficient approach for SARS-CoV-2 monoclonal antibody production via modified mRNA-LNP immunization

Abstract

Throughout the COVID-19 pandemic, many prophylactic and therapeutic drugs have been evaluated and introduced. Among these treatments, monoclonal antibodies (mAbs) that bind to and neutralize SARS-CoV-2 virus have been applied as complementary and alternative treatments to vaccines. Although different methodologies have been utilized to produce mAbs, traditional hybridoma fusion technology is still commonly used for this purpose due to its unmatched performance record. In this study, we coupled the hybridoma fusion strategy with mRNA-lipid nanoparticle (LNP) immunization. This time-saving approach can circumvent biological and technical hurdles, such as difficult-to-express membrane proteins, antigen instability, and the lack of posttranslational modifications on recombinant antigens. We used mRNA-LNP immunization and hybridoma fusion technology to generate mAbs against the receptor binding domain (RBD) of SARS-CoV-2 spike (S) protein. Compared with traditional protein-based immunization approaches, inoculation of mice with RBD mRNA-LNP induced higher titers of serum antibodies and markedly increased serum neutralizing activity. The mAbs we obtained can bind to SARS-CoV-2 RBDs from several variants. Notably, RBD-mAb-3 displayed particularly high binding affinities and neutralizing potencies against both Alpha and Delta variants. In addition to introducing specific mAbs against SARS-CoV-2, our data generally demonstrate that mRNA-LNP immunization may be useful to quickly generate highly functional mAbs against emerging infectious diseases.

Keywords: COVID-19; Lipid nanoparticle (LNP); Modified mRNA; Monoclonal antibodies; SARS-CoV-2.

Graphical abstract

Fig. 1

Design and physicochemical characterization of the modified RBD mRNA-LNP used for immunization. (A) Schematic illustration of SARS-CoV-2 RBD mRNA; comprises a 5′ cap, 5′ UTR, signal peptide, antigen (RBD of the spike protein), 3′ UTR, and 3′ poly(A) tail. (B) Schematic representation of the LNP-encapsulated RBD mRNA. (C) Cryo-TEM imaging was performed on RBD mRNA-loaded LNPs; the LNPs consisted of D-Lin-MC3-DMA:DSPC:Cholesterol:DMG-PEG 2000 at 50:10:38.5:1.5 molar ratios. Scale bar, 100 nm. (D) Agarose gel electrophoresis of RBD mRNA-LNP was performed with and without 1% Triton X-100 to assess mRNA integrity. Naked mRNA was used as a control. L = single-stranded RNA ladder, 1 = RBD mRNA alone, 2 = RBD mRNA-LNP (in 1X TE), 3 = disrupted RBD mRNA-LNP (in 1% Trition X-100). (E) Average size, zeta potential and PDI distribution were measured with dynamic light scattering (DLS); Encapsulation efficiency (EE%) was determined with a Ribogreen assay kit.

Fig. 2

In vitro characterization of immunogen expressed from RBD mRNA-LNP. Schematic of the RBD mRNA-LNP transduction protocol for HEK293T cells. (A, B) RBD protein expression level was measured in HEK293T cells transfected with RBD mRNA-LNP by Western blotting (A) and ELISA (B), using an anti-RBD antibody. Mock transfected HEK293T cells served as a negative control.

Fig. 3

Immunization with RBD mRNA-LNP elicits a robust antigen-specific immune response in mice. (A) Schematic of immunization regimens in mice. Female BALB/c mice (n = 3 per group) were immunized by three i.m. injections of RBD mRNA-LNP at 2-week intervals or by three i.p. injections of recombinant RBD protein at 3-week intervals. Red droplets indicate blood draws. After the final booster, the spleens were harvested and fused with NS1 cells to generate hybridomas, which were screened for RBD-mAbs. (B) ELISA was used to determine the titers of preimmune and hyperimmune sera against RBD mRNA-LNP or RBD protein after three immunizations. Mouse sera were collected before immunization and after the third immunization and incubated with RBD protein-coated plates. The absorbance values were read at O.D. 450 nm.

Fig. 4

Generation and characterization of mAbs from hybridomas. (A) Hybridomas were generated, and serial dilutions of the cell culture supernatants were screened for RBD WT-His binding ability by ELISA. (B) The binding affinities of hybridoma culture supernatants were also assessed for RBDs of different SARS-CoV-2 variants by ELISA. EpEX protein served as a negative control. (C) Neutralization assays were performed with hybridoma culture supernatants targeting four SARS-CoV-2 VOC pseudoviruses. Assays were performed in triplicate; each point represents the mean ± SEM. IC₅₀ values were calculated with GraphPad Prism software.

Fig. 5

RBD-mAb-3 exhibits potent binding and neutralization of SARS-CoV-2 Delta. (A) The binding of RBD-mAb-3 to different variants of SARS-CoV-2 RBDs was determined by ELISA analysis. EpEX served as a negative control. (B) Epitope mapping of RBD-mAb-3 using mutagenesis. RBD-mAb-3 binding to mutant RBDs with single or multiple alanine mutations was normalized to its binding to wild-type (WT) RBD; measurements were made with cellular ELISA. (C) Neutralization assays were performed on SARS-CoV-2 Alpha and Delta pseudoviruses with purified RBD-mAb-3. Assays were performed in triplicate; each point represents the mean ± SEM. IC₅₀ values were calculated with GraphPad Prism software. (D) RBD-mAb-3 neutralizes SARS-CoV-2 Delta according to PRNT. The PRNT₅₀ value was calculated with GraphPad Prism software. Assays were performed in triplicate, and points represent the mean ± SD.

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