Very few substitutions in a germ line antibody are required to initiate significant domain exchange.
2G12 is a broadly neutralizing anti-HIV-1 monoclonal human IgG1 antibody reactive with a high-mannose glycan cluster on the surface of glycoprotein gp120. A key feature of this very highly mutated antibody is domain exchange of the heavy-chain variable region (V(H)) with the V(H) of the adjacent Fab of the same immunoglobulin, which assembles a multivalent binding interface composed of two primary binding sites in close proximity. A non-germ line-encoded proline in the elbow between V(H) and C(H)1 and an extensive network of hydrophobic interactions in the V(H)/V(H)' interface have been proposed to be crucial for domain exchange. To investigate the origins of domain exchange, a germ line version of 2G12 that behaves as a conventional antibody was engineered. Substitution of 5 to 7 residues for those of the wild type produced a significant fraction of domain-exchanged molecules, with no evidence of equilibrium between domain-exchanged and conventional forms. Two substitutions not previously implicated, A(H14) and E(H75), are the most crucial for domain exchange, together with I(H19) at the V(H)/V(H)' interface and P(H113) in the elbow region. Structural modeling gave clues as to why these residues are essential for domain exchange. The demonstration that domain exchange can be initiated by a small number of substitutions in a germ line antibody suggests that the evolution of a domain-exchanged antibody response in vivo may be more readily achieved than considered to date.
Huber
Michael
M
Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Le
Khoa M
KM
Doores
Katie J
KJ
Fulton
Zara
Z
Stanfield
Robyn L
RL
Wilson
Ian A
IA
Burton
Dennis R
DR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D057134:Antibodies, Neutralizing / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D000080908:Broadly Neutralizing Antibodies; D015483:HIV Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000276:immunology*; D015497:HIV-1 / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry / Q000276:immunology; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
C529157:2G12 monoclonal antibody; D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000080908:Broadly Neutralizing Antibodies; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; D007135:Immunoglobulin Variable Region; D011134:Polysaccharides; C514793:gp120 protein, Human immunodeficiency virus 1