Affinity Maturation of an Ovarian Cancer-Targeting Peptide.

Ovarian cancer is among the leading causes of cancer deaths in women. The low survival rate is in part due to inadequate means of detection, which emphasizes the need to develop effective screening techniques. Peptide J18 was previously discovered using phage display technology and was found to specifically target and image human ovarian tumors in a SKOV-3 xenografted mouse model. Here, affinity maturation of J18 was carried out to improve its binding affinity by conducting an alanine scan of the peptide. A cell-based modified ELISA using SKOV-3 cells was carried out and dose-response curves were modelled using non-linear regression. The results showed that substitution of serine-5 (S5) and aspartic acid-6 (D6) with alanine decreased the EC value of the original peptide J18 from 45.02 µM (39.15-52.94 95% confidence interval; CI) to 19.54 (9.70-38-51 95% CI) and 6.92 (5.2-9.80 95% CI), respectively. Next, a double alanine substitution of S5 and D6 (J18-S5A-D6A) was investigated to further increase the binding affinity. The EC value was determined to be 4.22 (2.16-16.14 95% CI), thus improving the binding affinity approximately 10-fold. To ensure that J18-S5A-D6A maintained specificity for ovarian cancer cells, the peptide was evaluated against ovarian cancer SKOV-3, pancreatic cancer Mia-Paca-2, prostate cancer LNCaP, and embryonic kidney HEK 293 cells using a modified ELISA and fluorescent microscopy. Both assays showed significantly increased binding of J18-S5A-D6A to SKOV-3 cells compared to the other cell lines. Based on these results, peptide J18-S5A-D6A may be used as an improved imaging agent of ovarian cancer.