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J. Biol. Chem., Vol. 278, Issue 16, 14265-14273, April 18, 2003

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Mapping the Paratope of Anti-CD4 Recombinant Fab 13B8.2 by Combining Parallel Peptide Synthesis and Site-directed Mutagenesis^*

Cédric Bès^§, Laurence Briant-Longuet^¶, Martine Cerutti, Frédéric Heitz^**, Samuel Troadec, Martine Pugnière, Françoise Roquet, Franck Molina, Florence Casset, Damien Bresson, Sylvie Péraldi-Roux, Gérard Devauchelle, Christian Devaux^¶, Claude Granier, and Thierry Chardès^§§

From  CNRS UMR 5094, Institut de Biotechnologie et Pharmacologie, Faculté de Pharmacie, 15 Avenue Charles Flahault, 34093 Montpellier Cedex 5, France, ^¶ CNRS UMR 5121, Laboratoire Infections Rétrovirales et Signalisation Cellulaire, Institut de Biologie, 4 Boulevard Henri IV, 34060 Montpellier Cedex 2, France,  Institut National de la Recherche Agronomique-CNRS UMR 5087, Laboratoire de Pathologie Comparée, 30380 Saint-Christol-Lez-Alès, France, ^** CNRS UPR 1086, Centre de Recherche en Biochimie Macromoléculaire, 1919 Route de Mende, 34293 Montpellier Cedex 5, France, and  Synt:em, Parc Scientifique G. Besse, 30035 Nimes Cedex 1, France

We analyzed antigen-binding residues from the variable domains of anti-CD4 antibody 13B8.2 using the Spot method of parallel peptide synthesis. Sixteen amino acids, defined as Spot critical residues (SCR), were identified on the basis of a 50% decrease in CD4 binding to alanine analogs of reactive peptides. Recombinant Fab 13B8.2 mutants were constructed with alanine residues in place of each of the 16 SCR, expressed in the baculovirus cell system, and purified. CD measurements indicated that the mutated proteins were conformationally intact, with a -sheet secondary structure similar to that of wild-type Fab. Compared with the CD4-binding capacity of wild-type Fab 13B8.2, 11 light (Y32-L, W35-L, Y36-L, H91-L, and Y92-L) and heavy chain (H35-H, R38-H, W52-H, R53-H, F100K-H, and W103-H) Fab single mutants showed a decrease in CD4 recognition as demonstrated by enzyme-linked immunosorbent assay, BIAcore, and flow cytometry analyses. The five remaining Fab mutants showed antigen-binding properties similar to those of wild-type Fab. Recombinant Fab mutants that showed decreased CD4 binding also lost their capacity to inhibit human immunodeficiency virus promoter activation and the antigen-presenting ability that wild-type Fab displays. Molecular modeling of the 13B8.2 antibody paratope indicated that most of these critical residues are appropriately positioned inside the putative CD4-binding pocket, whereas the five SCR that were not confirmed by mutagenesis show an unfavorable positioning. Taken together, these results indicate that most of the residues defined by the Spot method as critical matched with important residues defined by mutagenesis in the whole protein context. The identification of critical residues for CD4 binding in the paratope of anti-CD4 recombinant Fab 13B8.2 provides the opportunity for the generation of improved anti-CD4 molecules with more efficient pharmacological properties.

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