key: cord-0811138-gmh0158g
authors: Hsu, Fu-Fei; Liang, Kang-Hao; Kumari, Monika; Chen, Wan-Yu; Lin, Hsiu-Ting; Cheng, Chao-Min; Tao, Mi-Hua; Wu, Han-Chung
title: An Efficient Approach for SARS-CoV-2 Monoclonal Antibody Production via Modified mRNA-LNP Immunization
date: 2022-04-22
journal: bioRxiv
DOI: 10.1101/2022.04.20.488878
sha: 5ccf7624ab79d781cde0623316f74692aaed03b3
doc_id: 811138
cord_uid: gmh0158g

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. In addition, 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.

As the world continues to grapple with the coronavirus COVID-19 pandemic (Zhu 51 N, et al., 2020; Sun P, et al., 2020) , monoclonal antibodies (mAbs) have become 52 increasingly used in applications such as basic research, diagnosis, therapeutics for 53 SARS-CoV-2 infection (Hwang YC, et al., 2022; Chapman AP, et al., 2021) . 54

Fortunately, the hybridoma technology developed by Georges Kohler and Cesar 55

Milstein in 1975 has made it possible to obtain large numbers of mAbs for these 56 purposes (Köhler G, et al., 1975) . Despite its widespread application, hybridoma 57 technology still suffers from some limitations. For example, the conventional 58 methodology used for hybridoma generation involves multiple injections of a protein 59 antigen with or without adjuvant (Chiarella P, et al., 2008) . It is sometimes a major 60 challenge to prepare high-quality protein antigen for immunization, yet this step is 61 necessary to generate high-precision mAbs that are able to recognize the native viral 62 antigen (Takeda H, et al., 2015) . Purification of protein antigens is also a time 63

consuming and labor-intensive process, as optimized protocols must be created for each 64 7 In our formulation, the particle size distribution and surface charge were suitable for 135 cellular uptake by endocytosis pathways. Next, we wanted to compare the strengths and durations of antibody responses between 150 mice immunized with RBD mRNA-LNP and those subjected to a traditional protein-151 based approach. BALB/c mice were immunized via intramuscular injection with RBD 152 mRNA-LNP at 2-week intervals or received intraperitoneal injection with recombinant 153 RBD protein in CFA-IFA adjuvant at 3-week intervals. A schematic representation of 154 the immunization schedule is shown in Figure 3A . Pre-immune and immune serum 155 were respectively collected before the priming injection (week 0) and one week after 156 the second booster. Subsequently, the RBD-binding antibody titers of the serum 157 samples were evaluated by ELISA. Sera from mice immunized with the RBD mRNA-158 LNP exhibited significantly higher titers against RBD protein, as compared to the mice 159 immunized with the protein and adjuvant ( Figure 3B ). This result suggests that RBD 160 8 mRNA LNP is highly immunogenic in mice and is suitable for subsequent production 161 of mAbs against RBD. As such, the RBD mRNA-LNP-immunized mice were used for 162 cell fusion. One week after the final injection, splenocytes were harvested from the 163 mice and fused with NS1/1-Ab4-1 (NS-1) mouse myeloma cells. The resulting fused 164 hybrid cells were cultured and selected in HAT tissue culture media. Nevertheless, like many other RNA viruses, SARS-CoV-2 has a high mutation rate, and 171 some mutations may cause new variants to be more transmissible and virulent than the 172 original SARS-CoV-2 wild-type strain, reported in late 2019 (Harvey WT, et al., 2021) . 173

Over the course of the pandemic, five SARS-CoV-2 lineages have been designated by 174 the World Health Organization as variants of concern (VOCs), including Alpha 175 RBD-mAb-6 bound well to wild-type RBD and most RBD variants, but their binding 184

to Omicron RBD was relatively weak. RBD-mAb-3 bound strongly to wild-type RBD, 185

Alpha RBD, and Delta RBD, but had no detectable binding to the Beta, Gamma and showed that Y453A and Q474A mutations significantly decreased the binding of RBD-207 mAb-3, suggesting that these residues contribute to the RBD-mAb-3 epitope. We then 208 further examined the neutralizing abilities of purified RBD-mAb-3 toward SARS-CoV-209 2 Alpha and Delta pseudoviruses. Notably, RBD-mAb-3 exhibited high neutralizing 210 capacities for Alpha and Delta variants, with respective IC50 values of 10.99 and 11.12 211 ng/ml ( Figure 5C ). Given the specificity and binding affinity of RBD-mAb-3 towards 212 the Delta RBD variant, we also tested its neutralization potential in a plaque reduction 213 neutralization test (PRNT) using authentic SARS-CoV-2 Delta variant ( Figure 5D ). 214 RBD-mAb-3 effectively neutralized viral infection with a PRNT50 value of 12.71 ng/ml. 215

Collectively, these data indicate that RBD-mAb-3 is a potent neutralizing mAb for 216 SARS-CoV-2 Delta variant. this study, we demonstrated this potential by using RBD mRNA-LNP as a novel 235 immunogen for hybridoma-based mAb production. 236

The main advantage of mRNA-LNP immunization is that the mRNA is easily 237 

The DNA template contained a T7 promoter followed by a codon optimized wild-type 256 

A novel coronavirus from patients 516 with pneumonia in china

Understanding of COVID-19 based on current 518 evidence

Monoclonal antibodies for COVID-19 therapy and SARS-CoV-2 detection

Rapid development of neutralizing and diagnostic SARS-526

COV-2 mouse monoclonal antibodies

Continuous cultures of fused cells secreting antibody of 528 predefined specificity

Mouse monoclonal antibodies in biological research: 530 strategies for high-throughput production

Production of 533 monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen 534 and biotinylated liposome-based interaction assay

Generation of murine monoclonal antibodies by 536 hybridoma technology

Expression and 539 purification of SARS coronavirus proteins using SUMO-fusions

Hybridoma technology a versatile method for isolation of monoclonal antibodies, 543 its applicability across species, limitations, advancement and future perspectives

DNA immunization as a technology platform for monoclonal 546 antibody induction

mRNA as a transformative technology 548 for vaccine development to control infectious diseases

Lipid nanoparticles for mRNA delivery

mRNA vaccine: a potential 553 therapeutic strategy

The promise of 555 mRNA vaccines: a biotech and industrial perspective

mRNA vaccines -a new era in 557 vaccinology

Lipid-based nanoparticle formulations for small 559 molecules and RNA drugs

mRNA, a revolution in 561 biomedicine

N 1 -Methylpseudouridine substitution enhances the 564 26 performance of synthetic mRNA switches in cells

SARS-CoV-2 variants, spike mutations 569 and immune escape

Structural basis for the recognition of 571

SARS-CoV-2 by full-length human ACE2

Neutralizing monoclonal antibodies for treatment of COVID-19

Development of 576 therapeutic antibodies for the treatment of diseases

The mRNA 578 vaccine development landscape for infectious diseases

Mice with megabase humanization of their 584 immunoglobulin genes generate antibodies as efficiently as normal mice

Omicron-specific mRNA vaccine induced potent neutralizing antibody

against Omicron but not other SARS-CoV-2 variants

Generation and 593 characterization of monoclonal antibodies against dengue virus type 1 for epitope 594 mapping and serological detection by epitope-based peptide antigens

CHC promotes 597 tumor growth and angiogenesis through regulation of HIF-1alpha and VEGF 598 signaling

6-well plates for 16 h. Then, the cells were seeded into 96-well plates at a density of 3 384 × 10 4 cells per well and incubated for one day. The cells were fixed in 4% 385 paraformaldehyde in PBS for 15 min at room temperature, and then the cell membranes 386were permeabilized using 0.1% Triton X-100 at room temperature for 10 min. The 387 plates were blocked using 5% milk, and 100 ng/ml RBD-mAb was added to each well 388 for 1 h at room temperature. Next, the horseradish peroxidase (HRP)-conjugated anti-389 mouse antibody (1:2000) was added for 1 h at room temperature. The binding capacities 390 of each RBD-mAb to the RBD mutants were examined by ELISA. 391 392

RBD-mAb-3 was serially diluted in PBS and pre-incubated with 100 plaque-forming 394 units (PFU) of SARS-CoV-2 for 1 h at 37°C. The mixtures were added to pre-seeded 395 Vero E6 cells for 1 h at 37°C. The virus-containing culture medium was then removed 396 and replaced with DMEM containing 2% FBS and 1% methyl-cellulose for an 397 additional 4-day incubation. The cells were fixed with 10% formaldehyde overnight 398 and stained with 0.5% crystal violet for 20 min. The plates were then washed with tap 399 water, and plaque numbers formed were counted for each dilution. Virus without RBD-400 mAb-3 served as a control. Each experiment was performed in triplicate. Plaque We are indebted to the Human Therapeutic Ab R&D Core facility of BioTReC, 505Academia Sinica, for their assistance with Ab production and analysis. We also thank 506 the National RNAi Core Facility at BioTReC for providing the SARS-CoV-2 variant 507 pseudoviruses. 508 509 510 511 512 24