Multiple binding capabilities utilized by specific protein-to-protein interactions in molecular recognition events are being documented increasingly but remain poorly understood at the molecular level. We identified five unrelated peptides that compete with each other for binding to the paratope region of the monoclonal anti-p24 (HIV-1) antibody CB4-1 by using a synthetic positional scanning combinatorial library XXXX[B1,B2,B3,X1,X2,X3]XXXX (14 mers; 68,590 peptide mixtures in total) prepared by spot synthesis. Complete sets of substitution analogs of the five peptides revealed key interacting residues, information that led to the construction of binding supertopes derived from each peptide. These supertope sequences were identified in hundreds of heterologous proteins, and those proteins that could be obtained were shown to bind CB4-1. Implications of these findings for immune escape mechanisms and autoimmunity are discussed.

All IgG-type antibodies are N-glycosylated in their Fc part at Asn-297. Typically, a fucose residue is attached to the first N-acetylglucosamine of these complex-type N-glycans. Antibodies lacking core fucosylation show a significantly enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and an increased efficacy of anti-tumor activity. In cases where the clinical efficacy of an antibody is to some extent mediated by its ADCC effector function, afucosylated N-glycans could help to reduce dose requirement and save manufacturing costs. Using Chinese hamster ovary (CHO) cells as a model, we demonstrate here that heterologous expression of the prokaryotic enzyme GDP-6-deoxy-d-lyxo-4-hexulose reductase within the cytosol can efficiently deflect the fucose de novo pathway. Antibody-producing CHO cells that were modified in this way secrete antibodies lacking core fucose as demonstrated by MALDI-TOF mass spectrometry and HPAEC-PAD monosaccharide analysis. Engineering of the fucose de novo pathway has led to the construction of IgGs with a strongly enhanced ADCC effector function. The method described here should have broad practical applicability for the development of next-generation therapeutic antibodies.

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The murine mAb CB4-1 raised against p24 (HIV-1) recognizes a linear epitope of the HIV-1 capsid protein. Additionally, CB4-1 exhibits cross-reactive binding to epitope-homologous peptides and polyspecific reactions to epitope nonhomologous peptides. Crystal structures demonstrate that the epitope peptide (e-pep) and the nonhomologous peptides adopt different conformations within the binding region of CB4-1. Site-directed mutagenesis of the fragment variable (Fv) region was performed using a single-chain (sc)Fv construct of CB4-1 to analyze binding contributions of single amino acid side chains toward the e-pep and toward one epitope nonhomologous peptide. The mutations of Ab amino acid side chains, which are in direct contact with the Ag, show opposite influences on the binding of the two peptides. Whereas the affinity of the e-pep to the CB4-1 scFv mutant heavy chain variable region Tyr32Ala is decreased 250-fold, the binding of the nonhomologous peptide remains unchanged. In contrast, the mutation light chain variable region Phe94Ala reduces the affinity of the nonhomologous peptide 10-fold more than it does for the e-pep. Thus, substantial changes in the specificity can be observed by single amino acid exchanges. Further characterization of the scFv mutants by substitutional analysis of the peptides demonstrates that the effect of a mutation is not restricted to contact residues. This method also reveals an inverse compensatory amino acid exchange for the nonhomologous peptide which increases the affinity to the scFv mutant light chain variable region Phe94Ala up to the level of the e-pep affinity to the wild-type scFv.