Extending mass spectrometry contribution to therapeutic monoclonal antibody lead optimization: characterization of immune complexes using noncovalent ESI-MS.
Monoclonal antibodies (mAbs) have taken on an increasing importance for the treatment of various diseases including cancers, immunological disorders, and other pathologies. These large biomolecules display specific structural features, which affect their efficiency and need, therefore, to be extensively characterized using sensitive and orthogonal analytical techniques. Among them, mass spectrometry (MS) has become the method of choice to study mAb amino acid sequences as well as their post-translational modifications. In the present work, recent noncovalent MS-technologies including automated chip-based nanoelectrospray MS and traveling wave ion mobility MS were used for the first time to characterize immune complexes involving both murine and humanized mAb 6F4 directed against human JAM-A, a newly identified antigenic protein (Ag) overexpressed in tumor cells. MS-based structural insights evidenced that heterogeneous disulfide bridge pairings of recombinant JAM-A alter neither its native structure nor mAbs 6F4 recognition properties. Investigations focused on mAb:Ag complexes revealed that, similarly to murine mAb, humanized mAb 6F4 binds selectively up to four antigen molecules with a similar affinity, confirming in this way the reliability of the humanization process. Noncovalent MS appears as an additional supporting technique for therapeutic mAbs lead characterization and development.
Atmanene
Cédric
C
Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), IPHC-DSA, University of Strasbourg UDS, CNRS, UMR7178, 25 rue Becquerel, 67087 Strasbourg, France.
Wagner-Rousset
Elsa
E
Malissard
Martine
M
Chol
Bertrand
B
Robert
Alain
A
Corvaïa
Nathalie
N
Van Dorsselaer
Alain
A
Beck
Alain
A
Sanglier-Cianférani
Sarah
S
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification; D000936:Antigen-Antibody Complex / Q000737:chemistry*; D015815:Cell Adhesion Molecules / Q000276:immunology; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry; D004364:Pharmaceutical Preparations / Q000737:chemistry*; D011485:Protein Binding; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D015815:Cell Adhesion Molecules; D004220:Disulfides; C407243:F11R protein, human; D007136:Immunoglobulins; D010446:Peptide Fragments; D004364:Pharmaceutical Preparations; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins