Bacterial expression of a human recombinant monoclonal antibody Fab fragment against hepatitis B surface antigen

Abstract

The Fab fragment was cloned from the monoclonal cell line TAPC301-CL4, which was produced using the Epstein-Barr virus (EBV) transformation method. This cell line produces a human monoclonal antibody (CL4MAb) against the hepatitis B surface antigen (HBsAg). This MAb was shown to have hepatitis B virus (HBV) neutralizing activity in chimpanzees. The Fab fragment was produced by subjecting the heavy and light chain antibody genes of the TAPC301-CL4 cell line to reverse transcription-polymerase chain reaction, cloning the products in the plasmid vector pFab1-His2 and introducing the plasmid into bacteria. Sequence analyses of the CL4Fab fragment revealed that the light and heavy chains belong to the Vk3a and VH3 groups of the immunoglobulin (Ig) family, respectively. An enzyme-linked immunosorbent assay confirmed that specificity of the recombinant CL4Fab antibody against HBsAg was the same as that of the parental MAb. Flow cytometric analysis using PLC/PRF/5 (Alexander) cells, which express HBsAg, showed the reactivities of the CL4MAb and CL4Fab antibody were the same. These results suggest that the recombinant CL4Fab antibody produced by Escherichia coli using the new vector-primer system developed for human IgG Fab fragments has a very high affinity for the HBsAg and may be useful clinically. A source for generation of human MAb for human therapy with very stable and specific expression was thus produced by isolating antibodies from EBV-transformed cell lines.

Algae as Protein Factories: Expression of a Human Antibody and the Respective Antigen in the Diatom Phaeodactylum tricornutum

“…The human monoclonal IgG1 antibody CL4mAb from cell-line TAPC301-CL4 recognizes the Hepatitis B surface antigen (HBsAg) and was selected for heterologous expression in P. tricornutum as it was already successfully expressed in other systems like tobacco and bacteria [22], [23]. Gene sequences for light and heavy chain (LC/HC) were adapted to P. tricornutum specific codon-usage and expressed as GFP fusion proteins in P. tricornutum .…”

Characteristics Peripheral Blood IgG and IgM Heavy Chain Complementarity Determining Region 3 Repertoire before and after Immunization with Recombinant HBV Vaccine

“…Maeda et al studied the B cell receptor (BCR) sequence associated with the response to HBV vaccination and the synthesis of anti-HBsAg monoclonal antibodies, and developed HBsAb transgenic tomatoes [4–6]. Several other studies have utilized BCR sequencing and phage display technology to detect HBsAbs with high affinity to HBsAg [7–9].…”

Generation and characterization of a single‐gene mouse–human chimeric antibody against hepatitis B surface antigen

“…Although several recombinant antibodies against hepatitis B surface antigen have been reported in the literature, none is available for clinical uses 8–10 . Earlier we reported on an anti‐HBs mouse monoclonal 5S and a single‐chain variable fragment (scFv) derived from it 11 .…”

“…Although several recombinant antibodies against hepatitis B surface antigen have been reported in the literature, none is available for clinical uses. [8][9][10] Earlier we reported on an anti-HBs mouse monoclonal 5S and a single-chain variable fragment (scFv) derived from it. 11 This antibody binds to HBsAg with high affinity (equilibrium dissociation constant [K D ] = 0.889 nM) and the scFv generated from this hybridoma retained the high affinity and epitope specificity.…”