In vitro affinity maturation of an anti-PSA antibody for prostate cancer diagnostic assay.
Prostate-specific antigen (PSA) is a serum marker that is widely used for the diagnosis of prostatic diseases. Various subforms of free PSA, which are associated with prostate cancer differently, have been identified in sera. Thus, specific detection of certain subforms could permit discrimination between benign and malignant cases. Although the monoclonal antibody 5D3D11 displays the desired selectivity, its relative weak binding affinity prevents its development into an effective diagnostic tool. The directed-evolution strategy presented here succeeds in enhancing affinity and immunoassay sensitivity while maintaining selectivity. Starting without structural data, we constructed four independent phage-display single-chain variable fragment (scFv) libraries targeting hot spots from CDR-L1, H1, H2, and H3. Mutations derived from each library were combined, yielding further affinity gains. This constitutes the first demonstration of additivity for independently selected complementarity-determining region (CDR) hot-spot mutations. The X-ray structure of the Fab' 5D3D11-PSA complex (after it became available) inspired the design of two new libraries targeting CDR-L3 that resulted in other higher-affinity variants. Attempts at combining the new variants with previous ones did not result in further gains, suggesting that mutations from the two strategies provide alternative but noncomplementary solutions for affinity enhancement of 5D3D11. The results can be interpreted to provide a plausible explanation for the observed lack of additivity. Finally, with respect to the wild-type scFv, the best binders show an enhancement of sensitivity in sandwich immunoassay. Its ability to discriminate between prostate cancer sera and benign prostatic hyperplasia sera has now been confirmed through the dosage of 63 patients.
CEA, iBiTec-S, Service de Pharmacologie et d'Immunoanalyse, Laboratoire d'Ingénierie des Anticorps pour la Santé, Equipe mixte CEA/bioMérieux, Gif-sur-Yvette F-91191, France.
Bruno H
BH
Muller
Alexandra
A
Savatier
Guillaume
G
L'Hostis
Narciso
N
Costa
Marc
M
Bossus
Sandrine
S
Michel
Catherine
C
Ott
Laurence
L
Becquart
Alain
A
Ruffion
Enrico A
EA
Stura
Frédéric
F
Ducancel
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000915:Antibody Affinity; D001483:Base Sequence; D022801:Complementarity Determining Regions / Q000235:genetics / Q000276:immunology; D006801:Humans; D007118:Immunoassay / Q000379:methods; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019151:Peptide Library; D017430:Prostate-Specific Antigen / Q000097:blood / Q000737:chemistry / Q000276:immunology*; D011470:Prostatic Hyperplasia / Q000175:diagnosis / Q000276:immunology; D011471:Prostatic Neoplasms / Q000097:blood / Q000175:diagnosis* / Q000276:immunology*; D012680:Sensitivity and Specificity; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D019151:Peptide Library; D057127:Single-Chain Antibodies; D017430:Prostate-Specific Antigen