Mapping the paratope of anti-CD4 recombinant Fab 13B8.2 by combining parallel peptide synthesis and site-directed mutagenesis.
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 beta-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.
Bès
Cédric
C
CNRS UMR 5094, Institut de Biotechnologie et Pharmacologie, Faculté de Pharmacie, 15 Avenue Charles Flahault, 34093 Montpellier Cedex 5, France.
Briant-Longuet
Laurence
L
Cerutti
Martine
M
Heitz
Frédéric
F
Troadec
Samuel
S
Pugnière
Martine
M
Roquet
Francoise
F
Molina
Franck
F
Casset
Florence
F
Bresson
Damien
D
Péraldi-Roux
Sylvie
S
Devauchelle
Gérard
G
Devaux
Christian
C
Granier
Claude
C
Chardes
Thierry
T
D000409:Alanine / Q000737:chemistry; D000906:Antibodies / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D017951:Antigen Presentation; D016367:Baculoviridae / Q000378:metabolism; D015704:CD4 Antigens / Q000378:metabolism*; D002942:Circular Dichroism; D004306:Dose-Response Relationship, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007376:Interleukin-2 / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism
C110023:13B8-2 monoclonal antibody; D000906:Antibodies; D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D007376:Interleukin-2; D010455:Peptides; D011993:Recombinant Fusion Proteins; D000409:Alanine