Hinge influences in murine IgG binding to Cryptococcus neoformans capsule

• Published in: Immunology Vol. 165; no. 1; pp. 110 - 121
• Main Authors: Oliveira, Diane Sthefany Lima de, Paredes, Verenice, Caixeta, Adrielle Veloso, Henriques, Nicole Moreira, Wear, Maggie P., Albuquerque, Patrícia, Felipe, Maria Sueli Soares, Casadevall, Arturo, Nicola, André Moraes
• Format: Journal Article
• Language: English
• Published: England Wiley Subscription Services, Inc 01.01.2022
• Subjects: Amino Acid Sequence; Animals; Antibodies; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - immunology; antibody; Antigens; Antigens, Bacterial - chemistry; Antigens, Bacterial - immunology; Bacterial Capsules - chemistry; Bacterial Capsules - immunology; Binding; Binding Sites, Antibody; Capsular polysaccharides; capsule; Cell Line; CHO Cells; Constant region; Cricetulus; Cryptococcosis; Cryptococcosis - immunology; Cryptococcus neoformans; Cryptococcus neoformans - immunology; Enzyme-linked immunosorbent assay; Epitopes - chemistry; Epitopes - immunology; Fungi; Immunofluorescence; Immunoglobulin G; Immunoglobulin G - chemistry; Immunoglobulin G - immunology; isotype; Isotypes; Mice; Polysaccharides; Recombinant Fusion Proteins; Structure-Activity Relationship; isotype; capsule; antibody; Cryptococcus neoformans; immunofluorescence
• ISSN: 0019-2805; 1365-2567
• DOI: 10.1111/imm.13411

Decades of studies on antibody structure led to the tenet that the V region binds antigens while the C region interacts with immune effectors. In some antibodies, however, the C region affects affinity and/or specificity for the antigen. One example is the 3E5 monoclonal murine IgG family, in which the mIgG3 isotype has different fine specificity to the Cryptococcus neoformans capsule polysaccharide than the other mIgG isotypes despite their identical variable sequences. Our group serendipitously found another pair of mIgG1/mIgG3 antibodies based on the 2H1 hybridoma to the C. neoformans capsule that recapitulated the differences observed with 3E5. In this work, we report the molecular basis of the constant domain effects on antigen binding using recombinant antibodies. As with 3E5, immunofluorescence experiments show a punctate pattern for 2H1‐mIgG3 and an annular pattern for 2H1‐mIgG1; these binding patterns have been associated with protective efficacy in murine cryptococcosis. Also as observed with 3E5, 2H1‐mIgG3 bound on ELISA to both acetylated and non‐acetylated capsular polysaccharide, whereas 2H1‐mIgG1 only bound well to the acetylated form, consistent with differences in fine specificity. In engineering hybrid mIgG1/mIgG3 antibodies, we found that switching the 2H1‐mIgG3 hinge for its mIgG1 counterpart changed the immunofluorescence pattern to annular, but a 2H1‐mIgG1 antibody with an mIgG3 hinge still had an annular pattern. The hinge is thus necessary but not sufficient for these changes in binding to the antigen. This important role for the constant region in antigen binding could affect antibody biology and engineering. In contrast with a prevailing Immunology tenet, the immunoglobulin constant region does affect how the variable region binds to the antigen. To understand the mechanism behind this, we engineered recombinant antibodies with identical variable regions but different heavy chain constant sequences, including hybrids between two different isotypes. We found differences in fine specificity between antibodies of different isotypes and determined that the murine IgG3 hinge is necessary, but not sufficient, for such changes in antigen recognition.