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.
Submitter: Marie Ary
Submission Date: Oct. 24, 2018, 9:10 a.m.
|Number of data points||68|
|Assays/Quantities/Protocols||Experimental Assay: Kd ; Experimental Assay: k_off ; Experimental Assay: k_on ; Experimental Assay: Relative increase in affinity (IC50_var/IC50_WT) ; Derived Quantity: Kd fold gain (over WT scFv)|
|Libraries||Binding analysis of variants from CDR hot-spot mutagenesis and structure-guided randomization (Table 2) ; Competition phage ELISA of 5D3D11_scFv variants from structure-guided libraries (Table 3) and combined mutations from CDR hot-spot and struc-guided random (Table 4: f-46, f-411) ; Competition phage ELISA of 5D3D11_scFv variants from CDR hot spot mutagenesis (Table 1)|
|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)|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|