Fig. 1: Comparison of dual- and single-chain mRNA-encoded antibody systems.
a Schematic of antibody expression using the conventional dual-chain approach with separate heavy chain (HC) and light chain (LC) mRNAs. The IgG HC mRNA encodes the variable (VH) and constant (CH) domains of the heavy chain, while the IgG LC mRNA encodes the variable (VL) and constant (CL) domains of the light chain. Both mRNAs are delivered into the cytoplasm and translated into HC and LC proteins, respectively. The HC and LC are assembled in the endoplasmic reticulum, transported to the cell membrane, and secreted outside the cells. b Schematic of single-chain (SC) mRNA-based antibody expression. The LC and HC mRNAs are linked into a single mRNA strand using 12 repeats of Gly-Gly-Gly-Gly-Ser (G4S) linker ((G4S)12). The SC-IgG mRNA is translated into a fully assembled HC-LC complex. c Representative immunostaining images of aIgG expression in A549 cells. Glycosylphosphatidylinositol (GPI)-anchored PGT121s (PGT121 aIgG) expressed from dual- or single-chain mRNAs localize on the cell membrane (AF-488, green). The nuclei were stained with DAPI (blue). The Scale bar represents 20 µm. d Quantification of PGT121 aIgG expression in Expi293F cells from dual- and single-chain mRNAs. A total of 75 µg of each mRNA was used for transfection in 75 × 106 Expi293F cells. Data from n = 3 independent experiments are shown as individual values and their mean. e Capillary electrophoresis-based Western blot (JESS) analysis of PGT121 aIgG expression from dual- or single-chain mRNAs produced in Expi293F cells. The antibodies were detected using HRP-conjugated anti-human IgG antibody, with chemiluminescence signals normalized to the highest value. f JESS analysis of single-chain mRNA-expressed PGT121 aIgG under reducing conditions. g Combinational treatment of multiple antibodies. In the dual-chain mRNA system, partial or complete heavy-light mismatched antibodies (highlighted in red and purple) can form, whereas the SC mRNA system produces antibodies with correctly paired heavy and light chains. h TZM-bl neutralization assays against tier 2 SHIV strains from clades A, B, and C. The 50% inhibitory concentrations (IC50) are summarized in the table. All neutralization assays were performed in duplicates and repeated at least twice in independent assays (*: IC50 values from literature). i Correlation of IC50 values between mRNA-encoded and conventional bnAbs from in vitro neutralization assays. Statistical comparison by Pearson correlation: r(10) = 0.6921, p = 0.0126 (two-tailed), 95% CI [0.1960, 0.9061]. a, b, g Created in BioRender. Santangelo, P. (2025) https://BioRender.com/fowauum.
