Ovarian cancer is the fifth leading cause of cancer deaths in women, which is directly related to inadequate diagnostic methods. Phage display technology may be used to develop peptide‐displaying filamentous phage as cancer detection and imaging agents. Such modalities offer advantages in regard to the high specificity and affinity of peptides, as well as increased avidity by displaying multiple peptides per virion. Additionally, the large size of filamentous phage (0.9 μm) allows for signal amplification by labeling with multiple imaging moieties. Phage display technology was previously used to select phage clones with specific binding to human ovarian cancer cells (SKOV‐3). In short, a 15‐mer fUSE5 phage display library was injected into the tail vein of a nude mouse without tumors, and unbound phage were collected after 15 min of circulation. This pre‐cleared library was then subjected to four rounds of selection in nude mice carrying xenografted SKOV‐3 tumors. During each round, phage were allowed to circulate for 1 h, after which the mouse was sacrificed and the tumor was excised. Tumor‐bound phage were collected by 3‐[(3‐cholamideopropyl) dimethylammonio]‐1‐propanesulfonate (CHAPS) elution, and displayed peptides were identified by DNA sequencing. From the last round of selection, 31 phage clones were identified, and each was subjected to micropanning experiments, in which the binding ratio for SKOV‐3‐to‐normal human ovarian (HS‐832) cells were evaluated. These results showed that phage clones pJ18 and pJ24 exhibited specificity for ovarian cancer cells, exhibiting SKOV‐3‐to‐HS‐832 ratios of 6.57 and 3.55, respectively. To evaluate the tumor targeting capabilities, pJ18, pJ24, and WT phage were labeled with a near‐infrared fluorophore (AF680), and employed in biodistribution and optical imaging studies in mice bearing xenografted SKOV‐3 tumors. In brief, mice were injected with 1012 virions of phage, and the animals were sacrificed after 4 h. The tumor, organs and tissues were excised and fluorescent intensity was measured using a Xenogen IVIS 200 Imaging System. Tumor uptake of pJ24 was significantly higher compared to WT phage. For all phage (pJ18, pJ24, and WT), the liver showed increased fluorescent uptake compared to other organs, indicating that the phage were excreted through the reticuloendothelial system. Next, mice carrying xenografted SKOV‐3 tumors were injected with 1012 virions of AF680‐labeled phage. The animals were imaged pre‐injection (0 h), and after 2 h and 4 h of circulation. The results showed that the ovarian tumors were easily localized and exhibited ample tumor‐to‐background contrast. Further, the tumor fluorescent intensity of pJ18 and pJ24, was significantly higher (p<0.001, p<0.05, respectively) in comparison to WT after 2 h. The fluorescent intensity was lower for both clones after 4 h, however, the uptake of pJ18 remained significantly higher (p<0.01) compared to WT. Taken together, these results indicate that phage clones pJ18 and pJ24 target and image ovarian SKOV‐3 tumors in ...
Pancreatic cancer is characterized by a 5-year survival rate of 3%, in part due to inadequate detection methods. The small size of peptides offers advantages regarding molecular targeting. Thus, peptides may be used in detection of pancreatic cancer. Here, peptides that target pancreatic cancer cells were selected using phage display technology using a 15-mer fUSE5 library. Phage were pre-cleared against immortalized pancreatic cells (hTERT-HPNE), followed by selections against pancreatic cancer (Mia Paca-2) cells. Next-generation sequencing identified two peptides, MCA1 and MCA2, with a Log2 fold change (Mia Paca-2/ hTERT-HPNE) >1.5. Modified ELISA and fluorescent microscopy showed that both peptides bound significantly higher to Mia Paca-2 cells, and not to hTERT-HPNE, embryonic kidney (HEK 293), ovarian (SKOV-3) and prostate cancer (LNCaP) cell lines. Further characterization of MCA1 and MCA2 revealed EC50 values of 16.11 µM (95% CI [9.69, 26.31 µM]) and 97.01 µM (95% CI [58.64, 166.30 µM]), respectively. Based on these results, MCA1 was selected for further studies. A competitive dose response assay demonstrated specific binding and an IC50 value of 2.15 µM (95% CI [1.28, 3.62 µM]). Taken together, this study suggests that MCA1 may be used as a pancreatic cancer targeting ligand for detection of the disease.
Phage display has been widely used to develop high‐affinity peptide ligands against proteins and cell lines. Such peptides are typically selected using biopanning to exhibit significant binding affinity and specificity against a molecular target. Rarely are the same peptides identified from separate phage display selections, since the presence of these ligands in a selection typically signify specific binding to the target. However, here, we report the identification of the same peptide (TG1) from two different phage display selections against the hemagglutinin (HA) epitope and human pancreatic cancer (Mia Paca‐2) cells. Phage display selections were conducted to identify a peptide with high affinity and specificity for the HA epitope. In brief, a pre‐cleared 15‐mer fUSE5 phage library was subjected to four rounds of selections against 0.04–4 μg HA. Following the last round of selection, the phage DNA was amplified using PCR and submitted to next‐generation sequencing. These results identified peptide TG1 (RNVPPIFNDVYWIAF) as the most prevalent ligand in the selection. Modified enzyme‐linked immunosorbent assay (ELISA) results showed that 109 nM TG1 bound significantly higher (p<0.05) to 5 μg immobilized HA compared to a non‐relevant peptide J18 known to bind ovarian adenocarcinoma (SKOV‐3) cells. In a separate, previously reported phage display selection, the same 15‐mer fUSE5 phage library was subjected to selections against human pancreatic cancer (Mia‐Paca‐2) cells. Again, next‐generation sequencing results identified TG1 as the third most prevalent peptide. Modified ELISA results using 10 μM TG1, and a known pancreatic cancer‐targeting peptide MCA2, showed that both ligands bound significantly (p<0.01) to Mia‐Paca‐2 cells compared to dimethyl sulfoxide (DMSO; vehicle). A literature search revealed that TG1 was identified by two separate research groups conducting phage display selections against caveolin protein and mouse embryos. These results indicate that the peptide is an indiscriminate binder. Thus, we sought to elucidate the binding properties of peptide TG1 and phage TG1 (pTG1). Modified ELISA with 10 μM TG1, MCA2, J18, or DMSO were carried out against SKOV‐3 and human normal pancreatic (hTERT‐HPNE) cells. Results showed that TG1 bound significantly higher (p<0.01) to both SKOV‐3 and hTERT‐HPNE cells compared to DMSO. As expected MCA2 showed no binding to either cell line. Peptide J18 showed significant binding (p<0.05) to SKOV‐3 cells. Phage qPCR analysis was performed to further elucidate the binding properties of the pTG1. 1010 V/mL pTG1, pancreatic cancer‐targeting pMCA1, or WT phage was incubated with Mia‐Paca‐2, SKOV‐3, or hTERT‐HPNE cells. After washing with Tris‐buffered saline (TBS), bound phage were eluted with 2.5% 3‐cholamidopropyl dimethylammonio 1‐propanesulfonate (CHAPS). Collected phage virions (5 μL) were quantified by qPCR using Fast SYBR Green Master Mix on an Applied Biosystems StepOnePlus Real‐Time qPCR System (Applied Biosystems, CA). The results revealed that pTG1 demonstrated s...
Pancreatic cancer is the fourth leading cause of cancer deaths in the U.S., characterized by a 5‐year survival rate of 3%. The dire prognosis is due to inadequate detection methods. However, peptides offer advantages regarding in vivo molecular targeting due to their small size and ample binding affinity. Thus, peptides may be used in imaging of pancreatic cancer for the detection of the disease. Bacteriophage (phage) display technology using a 15‐mer fUSE5 peptide library (1013 virions) was previously performed by an initial negative selection round against 106 normal human pancreatic (hTERT‐HPNE) cells. Unbound phage were amplified in E. coli K91BK and isolated using PEG/NaCl precipitation. Phage (1013 virions) were then subjected to four subsequent rounds of positive selection against 106 human pancreatic cancer (Mia Paca‐2) cells. Following the last round, amplicons were produced by PCR and submitted to next‐generation sequencing. The results identified two clones, pMCA1 and pMCA2, which were comparatively prevalent in the fourth positive round. In order to evaluate the pancreatic cancer binding properties of phage‐displayed peptides (MCA1 and MCA2), the ligands were synthesized with an N‐terminal GSG spacer and a biotin group. A modified ELISA and fluorescent microscopy were performed, in which 10 μM MCA1, MCA2, J18 (negative control), or DMSO (vehicle) was incubated with Mia Paca‐2, hTERT‐HPNE, ovarian adenocarcinoma SKOV‐3, prostate cancer LNCaP, or immortalized human embryonic kidney HEK 293 cells for 1 h. For the modified ELISA, bound peptides were probed by HRP‐conjugated streptavidin, and detected spectrophotometrically at 405 nm after the addition of ABTS. For fluorescent microscopy, bound peptides were detected by FITC‐conjugated streptavidin. Results showed that MCA1 and MCA2 exhibited significantly (p<0.01) higher binding to Mia Paca‐2 cells, compared to J18 and DMSO, while no binding was detected to hTERT‐HPNE, SKOV‐3, LNCaP, or HEK 293 cells. Fluorescent microscopy validated these results by showing significantly (p<0.0001) increased fluorescent intensity of MCA1 and MCA2 to Mia Paca‐2. However, only minimal fluorescent intensity was observed to hTERT‐HPNE, SKOV‐3, LNCaP, or HEK 293 cells. This study indicates that peptides MCA1 and MCA2 specifically target pancreatic cancer cells. Hence, these ligands may be utilized in further studies to improve the detection of pancreatic cancer.
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 EC50 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 EC50 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.
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