Characterization of molecular features, antigen-binding, and in vitro properties of IgG and IgM variants of 4E10, an anti-HIV type 1 neutralizing monoclonal antibody.
The human monoclonal antibody 4E10 has been generated previously by immortalization of peripheral blood cells from an HIV-1-infected individual. This antibody binds to the linear epitope NWFDIT on gp41 and exhibits exceptional neutralizing activity against a broad spectrum of primary HIV-1 isolates. In the present study, molecular features, immunoreactivity, and functional activity of 4E10 were studied. The original hybridoma-derived 4E10 was of subtype IgG(3). Analysis of the variable segment of the heavy chain (VH) demonstrated extensive somatic mutations compared to the closest homologous germline gene VH1-69. Most amino acid substitutions occurred in the complementarity-determining region (CDR) 2, characteristic for an antigen-driven somatic maturation. The heavy chain of the CDR3 (H3) is of unusual length and cannot be attributed with certainty to any specific D(H) locus. To enable mass production and to prolong the in vivo half-life, 4E10 was subsequently cloned as IgG(1) in Chinese hamster ovary (CHO) cells. In additional studies, 4E10 was class switched to the IgM isotype. Binding to the linear epitope NWFDIT was not significantly changed after the cloning procedures. However, in vitro studies revealed dramatic differences in the neutralizing potential. The antiviral activity could be greatly enhanced by change of IgG(3) to IgG(1). In contrast, the IgM isotype almost completely lost its neutralizing potential.
Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, A-1190 Vienna, Austria. R.Kunert@iam.boku.ac.at
Renate
R
Kunert
Susanne
S
Wolbank
Gabriela
G
Stiegler
Robert
R
Weik
Hermann
H
Katinger
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000936:Antigen-Antibody Complex / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000235:genetics; D018095:Germ-Line Mutation; D015497:HIV-1 / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics* / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D007075:Immunoglobulin M / Q000235:genetics* / Q000276:immunology; D008214:Lymphocytes / Q000276:immunology; D008969:Molecular Sequence Data; D009500:Neutralization Tests
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D022801:Complementarity Determining Regions; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007075:Immunoglobulin M