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High-throughput saturation mutagenesis generates a high-affinity antibody against SARS-CoV-2 variants using protein surface display assay on a human cell [1]

['Ye Yang', 'State Key Laboratory Of Natural', 'Biomimetic Drugs', 'Department Of Biomedical Engineering', 'College Of Future Technology', 'Peking University', 'Beijing', 'Shuo Liu', 'Graduate School Of Chinese Academy Of Medical Sciences', 'Peking Union Medical College']

Date: 2023-03 

A New method for the rapid generation of a SARS-CoV-2 Antibody against Diverse RBD Variants with higher affinity

We integrated chip-based DNA oligo synthesis technology and a single cell-based assay to develop a method for the rapid generation of an antibody with higher affinity against diverse SARS-CoV-2 variants. The method comprised of four steps, including the development of a single cell-based assay, the construction of an antibody library, the screening of positive cells using fluorescence-activated cell sorting (FACS) technology and the decoding of antibody sequence using the DNA sequencing technology.

First, we developed a single cell-based assay, in which an antibody was displayed on the surface of a mammalian cell and bound to a targeted protein, such as S-RBD herein. To ensure the antibody to be assayed on the surface of mammalian cells, we adopted the structure of CAR-T, in which anti-CD19 single-chain fragment variable (scFv) was replaced with a SARS-CoV-2 neutralizing antibody, such as REGN10987 that has been authorized for emergency use (EUA) [11, 37]. In brief, the N-terminal 21-aa signal peptide and C-terminal 24-aa peptide of REGN10987 scFv were retained for the purpose of its correct transportation and transmembrane to plasma membrane. The whole scFv fragment was inserted into the 5’ end of the mCherry encoding region via a T2A tag, packaged into lentivirus, and then expressed in HEK 293T cells (Fig 1A). After 48h, the cells were treated with S-RBD conjugated with fluorescein isothiocyanate (RBD-FITC), and examined by confocal fluorescence microscopy. The positive cells expressing mCherry (red) were labeled green (Fig 1B).

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TIFF original image Download: Fig 1. Construction of an Antibody Surface Display System in HEK 293T. (A) Schematic diagram of antibody scFv surface display system on mammalian cells. The VL and VH domain of scFv receptor construct used in anti-CD19 CAR was replaced with a SARS-CoV-2 neutralizing antibody REGN10987. VH, variable region of Ig heavy-chain; VL, variable region of Ig light-chain. (B) Confocal microscopic images of REGN10987 scFv displayed on HEK 293T cells. HEK 293T cells were infected by lentivirus packaged with a plasmid directing surface-expression of REGN10987 scFv. Infected cells (red) were fixed with DAPI nuclear staining followed by detection with biotinylated RBD, and then with streptavidin-FITC (green). Merged staining patterns are shown. Scale bar: 20 μm. (C) RBD bound to REGN10987 scFv-expressing HEK 293T cells at different titers. Concentration of RBD was from 0 nM to 1,000 nM. RBD-FITC signal was measured by flow cytometry. (D) Confocal microscopic images of mutated REGN10987 scFv displaying on HEK 293T cells. Several key amino acids in CDRs were mutated into alanine. Mutated amino acid was showed in S1B Fig. Transfected cells (red) were fixed with DAPI nuclear staining followed by detection with biotinylated RBD, and then with streptavidin-FITC (green). Scale bar: 100 μm. (E) RBD-His added to RBD-FITC competitively bound to REGN10987 scFv-expressing HEK 293T cells. Concentration of RBD-FITC was 10 nM, while different titers of RBD-His was from 0 nM to 10,000 nM. RBD-FITC signal was measured by flow cytometry. https://doi.org/10.1371/journal.ppat.1011119.g001

Furthermore, to assess RBD binding specificity, a dose-binding affinity curve was observed with the increase of the RBD-FITC concentration, as quantified by FACS (Fig 1C). In contrast, when treated with 10 nM RBD-FITC, the cells were competitively treated with the different concentration of non-FITC RBD, and the fluorescence signal gradually weakened (Figs 1E and S1A). Interestingly, when a few of key amino acids located in complementarity-determining regions (CDRs) of REGN10987 scFv were mutated into alanine, the binding affinity of REGN10987 scFv to RBD-FITC was weakened or even disappeared (Figs 1D and S1B). Together, all these results demonstrate the successful establishment of a single-cell based assay that allows the rapid screening of antibody Fvs according to their binding affinity to a targeted antigen, such as a SARS-CoV-2 variant.

Second, we designed and synthesized a comprehensive library of full-length variable region of heavy chain (VH, 120 aa) and light chain (VL, 110 aa) of REGN10987 scFv protein, in which each amino acid in VH and VL domain of the protein was mutated into 19 other natural amino acids. To do so, we synthesized 4,370 mutation primers on an integrated chip (Fig 2A). Through tuning the concentration of those mutation primers to 100 ng/μL in a PCR, a library of mutated REGN10987 scFv with ~2 custom-designed point mutations was efficiently generated (Figs 2B and S2). The library was then inserted into the 5’ end of the mCherry encoding region in the vector as described in Fig 1A. The constructed plasmids were transformed into E. coli cells and a mutant library of over 107 mutants was obtained for the following screening.

Third, mutagenesis plasmid library was packaged into lentivirus and then transfected into HEK 293T cells in less than 0.3 multiplicity of infection (MOI) to guarantee no more than one copy of REGN10987 scFv variant per cell. This original cell library was defined as P0 (pre-screening) group and used for normalization later. After 48 hours, the mutant library of 107 HEK 293T cells was incubated with 2 nM wild type RBD-FITC. The top 1.1% FITC-positive cells (defined as P1 group) and the top 5% FITC-negative cells (defined as N group) were collected according to the fluorescence signal density, respectively. Then, P1 group cells were cultured for several days and divided into two parts, and one half was used for the second round of selection and another half for later sequencing (Fig 2C). The top 10.1% FITC-positive cells (defined as P2 group) were collected in the second-round screening (S3A Fig). If needed, more rounds of selection could be performed.

Finally, we used the next generation of DNA sequencing technology to decode the enriched REGN10987 scFv variants. In brief, total RNAs were extracted from P0, P1, P2 and N groups of cells, respectively and used for the next generation sequencing. Biological replicates (n = 4) were conducted to avoid deviation. All of the designed mutations (4,370) were observed in P0 group. The enrichment scores of nonsynonymous mutations were set as the log 2 -scaled ratio of the frequencies of transcripts between the sorted populations (P1/P0, P2/P0 and N/P0, respectively).

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TIFF original image Download: Fig 2. High-throughput Saturation Mutagenesis Screen. (A) A novel method for the generation of primers for high-throughput saturation mutagenesis. High-throughput chip-synthesized oligo was used to produce saturation mutagenesis primers of REGN10987 scFv. (B) Flow chart of obtaining REGN10987 scFv saturation mutagenesis library. High-throughput saturation mutagenesis primers were used to construct a full-length REGN10987 scFv lentiviral vector. (C) Timeline for establishment and screening of mutagenesis library. REGN10987 scFv vector-packaged lentivirus (MOI < 0.3) was transduced to HEK 293T cells and a mammalian cell mutant library (P0) was obtained after one round of FACS. After the incubation with SARS-CoV-2 RBD-FITC, a second round of FACS yielded RBD positive and negative groups (P1, N). https://doi.org/10.1371/journal.ppat.1011119.g002

The RBD variants initially screened also include the one of SARS-CoV-2 Beta variant (Lineage B.1.351), which have N501Y, K417N and E484K mutations. The top 1.41% FITC-positive cells (defined as BP1 group) were collected and followed by deep sequencing. The pre-screening cell library was also sequenced (defined as BP0 group) (S3 Fig). Delta S1 (Lineage B.1.617.2)-FITC probe, containing L452R and E484Q in RBD and D614G in S1, was used in the single-cell assay. Until the third round, the FITC-positive cells (defined as DP3 group) clustered distinctly (S4 Fig) due to the low binding ability of REGN10987 to Delta S1 protein. All of FITC-positive cells in three rounds and its pre-screening cell library, which were defined as DP1, DP2, DP3 and DP0 group, respectively, were deeply sequenced.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1011119

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