Very Few Substitutions in a Germ Line Antibody Are Required To Initiate Significant Domain Exchange

Abstract

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 domainexchanged antibody response in vivo may be more readily achieved than considered to date.

2G12-Expressing B Cell Lines May Aid in HIV Carbohydrate Vaccine Design Strategies

“…3A). Neither immunogen was able to activate the 2G12 gl cells at concentrations of 50 g/ml (data not shown), supporting previous studies that failed to detect binding of soluble 2G12 gl IgG to glycans (21).…”

“…We have previously shown that only 5 amino acid substitutions in the germ line of 2G12 WT are required to induce approximately 27% domain exchange antibody (21). In addition, we have shown that a non-domain-exchanged variant of 2G12 that has an essentially unchanged binding site is unable to bind gp120 or neutralize HIV pseudovirus but can bind the yeast pathogen Candida albicans displaying an array of Man␣1,2Man epitopes (20).…”

“…The V(D)J sequences of 2G12 WT, 2G12 I19R, and 2G12 gl were previously described (10,20,21). About 10 g of both heavy-and lightchain-linearized (SalI and NheI, respectively) expression vectors was added to 5 ϫ 10 6 K46 cells in 500 l Dulbecco's modified Eagle's medium (DMEM) and incubated in 4-mm cuvettes on ice for 5 min.…”

“…In a second study, we showed that a domain-exchanged antibody could be generated from a germ line version of the antibody with a reasonably small number of mutations (21). For example, only 5 or 8 substitutions in germ line 2G12 (2G12 gl) were required to achieve 27% and 37% domain exchange, respectively (determined by size exclusion of the Fab fragment [1]).…”

PGV04, an HIV-1 gp120 CD4 Binding Site Antibody, Is Broad and Potent in Neutralization but Does Not Induce Conformational Changes Characteristic of CD4

“…Mock-treated or endo-␤-N-acetylglucosaminidase H (endo-H)-and endo-F-treated BaL gp120 was diluted in phosphate-buffered saline (PBS) and coated at 5 g/ml overnight at 4°C as previously described (9). Briefly, 5-fold serial dilutions of the MAbs, in 1% bovine serum albumin (BSA) in PBS, were added at a starting concentration of 10 g/ml.…”