Introduction of germline residues improves the stability of anti-HIV mAb 2G12-IgM.
Immunoglobulins M (IgMs) are gaining increasing attention as biopharmaceuticals since their multivalent mode of binding can give rise to high avidity. Furthermore, IgMs are potent activators of the complement system. However, they are frequently difficult to express recombinantly and can suffer from low conformational stability. Here, the broadly neutralizing anti-HIV-1 antibody 2G12 was class-switched to IgM and then further engineered by introduction of 17 germline residues. The impact of these changes on the structure and conformational stability of the antibody was then assessed using a range of biophysical techniques. We also investigated the effects of the class switch and germline substitutions on the ligand-binding properties of 2G12 and its capacity for HIV-1 neutralization. Our results demonstrate that the introduced germline residues improve the conformational and thermal stability of 2G12-IgM without altering its overall shape and ligand-binding properties. Interestingly, the engineered protein was found to exhibit much lower neutralization potency than its wild-type counterpart, indicating that potent antigen recognition is not solely responsible for IgM-mediated HIV-1 inactivation.
Department of Biotechnology, Vienna Institute of BioTechnology at BOKU, University of Natural Resources and Life Sciences, Vienna, Austria.
Veronika
V
Chromikova
Department of Biotechnology, Vienna Institute of BioTechnology at BOKU, University of Natural Resources and Life Sciences, Vienna, Austria.
Alexander
A
Mader
Department of Chemistry, Division of Biochemistry, Vienna Institute of BioTechnology at BOKU, University of Natural Resources and Life Sciences, Vienna, Austria; Department for Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Austria.
Stefan
S
Hofbauer
Center for Integrated Protein Science Munich at Chair of Biomolecular NMR Spectroscopy, Department of Chemistry, Technical University Munich, Garching, Germany; Institute of Structural Biology, Helmholtz Center Munich, Neuherberg, Germany.
Christoph
C
Göbl
Center for Integrated Protein Science Munich at Chair of Biomolecular NMR Spectroscopy, Department of Chemistry, Technical University Munich, Garching, Germany; Institute of Structural Biology, Helmholtz Center Munich, Neuherberg, Germany; Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, Austria.
Tobias
T
Madl
Department of Medicine, Division of Infectious Diseases, University of CA, Irvine, USA.
Johannes S
JS
Gach
Department of Biotechnology, Vienna Institute of BioTechnology at BOKU, University of Natural Resources and Life Sciences, Vienna, Austria.
Stefan
S
Bauernfried
Department of Chemistry, Division of Biochemistry, Vienna Institute of BioTechnology at BOKU, University of Natural Resources and Life Sciences, Vienna, Austria.
Paul G
PG
Furtmüller
Department of Medicine, Division of Infectious Diseases, University of CA, Irvine, USA.
Donald N
DN
Forthal
Department of Applied Genetics and Cell Biology, Vienna Institute of BioTechnology at BOKU, University of Natural Resources and Life Sciences, Vienna, Austria.
Lukas
L
Mach
Department of Chemistry, Division of Biochemistry, Vienna Institute of BioTechnology at BOKU, University of Natural Resources and Life Sciences, Vienna, Austria. Electronic address: christian.obinger@boku.ac.at.
Christian
C
Obinger
Department of Biotechnology, Vienna Institute of BioTechnology at BOKU, University of Natural Resources and Life Sciences, Vienna, Austria. Electronic address: renate.kunert@boku.ac.at.
Renate
R
Kunert
D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology / Q000494:pharmacology; D001483:Base Sequence; D016466:CHO Cells; D003412:Cricetulus; D015870:Gene Expression; D057809:HEK293 Cells; D015483:HIV Antibodies / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology / Q000494:pharmacology; D015699:HIV Envelope Protein gp120 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000276:immunology; D015497:HIV-1 / Q000187:drug effects* / Q000254:growth & development; D006801:Humans; D017578:Immunoglobulin Class Switching / Q000235:genetics; D007075:Immunoglobulin M / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D009500:Neutralization Tests; D011487:Protein Conformation; D015202:Protein Engineering; D055550:Protein Stability; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; D007075:Immunoglobulin M; D011994:Recombinant Proteins
Antibody engineering; Germline; HIV; IgM; Protein stability