In vitro affinity maturation of an anti-PSA antibody for prostate cancer diagnostic assay.


Abstract

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.

Submission Details

ID: FVRfPNPe3

Submitter: Marie Ary

Submission Date: Oct. 24, 2018, 9:10 a.m.

Version: 1

Publication Details
Muller BH;Savatier A;L'Hostis G;Costa N;Bossus M;Michel S;Ott C;Becquart L;Ruffion A;Stura EA;Ducancel F,J Mol Biol (2011) In vitro affinity maturation of an anti-PSA antibody for prostate cancer diagnostic assay. PMID:22019475
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
5YD3 2018-06-06 1.35 Crystal structure of the scFv antibody 4B08 with epitope peptide
5YD4 2018-06-06 1.35 Crystal structure of the scFv antibody 4B08 with epitope peptide (mutation T6A)
5YD5 2018-06-06 1.96 Crystal structure of the scFv antibody 4B08 with epitope peptide (mutation N3A)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
91.1 5D3D11_scFv P01630 KV2A6_MOUSE