Phage Display's Prospects for Early Diagnosis of Prostate Cancer

Abstract

Prostate cancer (PC) is the second most diagnosed cancer among men. It was observed that early diagnosis of disease is highly beneficial for the survival of cancer patients. Therefore, the extension and increasing quality of life of PC patients can be achieved by broadening the cancer screening programs that are aimed at the identification of cancer manifestation in patients at earlier stages, before they demonstrate well-understood signs of the disease. Therefore, there is an urgent need for standard, sensitive, robust, and commonly available screening and diagnosis tools for the identification of early signs of cancer pathologies. In this respect, the "Holy Grail" of cancer researchers and bioengineers for decades has been molecular sensing probes that would allow for the diagnosis, prognosis, and monitoring of cancer diseases via their interaction with cell-secreted and cell-associated PC biomarkers, e.g., PSA and PSMA, respectively. At present, most PSA tests are performed at centralized laboratories using high-throughput total PSA immune analyzers, which are suitable for dedicated laboratories and are not readily available for broad health screenings. Therefore, the current trend in the detection of PC is the development of portable biosensors for mobile laboratories and individual use. Phage display, since its conception by George Smith in 1985, has emerged as a premier tool in molecular biology with widespread application. This review describes the role of the molecular evolution and phage display paradigm in revolutionizing the methods for the early diagnosis and monitoring of PC.

Keywords: PC; affinity selection; electrochemical biosensor; electrochemical impedance spectroscopy; enzyme-linked immunosorbent assay (ELISA); free-prostate-specific antigen (F-PSA); label-free immunosensor; landscape phage; molecular evolution; phage ELISA; phage capture assay; phage display; prostate-specific antigen (PSA); prostate-specific matrix antigen (PSMA); recombinant antibodies; total-prostate-specific antigen (t-PSA).

Figure 1

(a): Sandwich PSA ELISA (A) vs. Phage PSA ELISA (B): The capture antibody (A), or phage (B) immobilized onto ELISA plates bind the analyte protein, and the detection antibodies linked to the enzyme are added to catalyze the appearance of a colored or fluorescent product. (b): PSA biosensors. A molecular interface linked to a transducer binds the analyte and generates a signal for changes in mass, capacitance, resistance, surface plasmon resonance, etc.

Figure 2

(Left) Electron microscopy image of the wild-type phage fd. Blue and red arrows depict the sharp and blunt ends of the phage capsid with the attached minor coat proteins p3/p6 and p7/p9, respectively (five copies each). Major coat protein p8 (~2700 copies) forms the tubular capsid around viral single-stranded DNA (scale bar: 100 nm, the length of the phage capsid ~1 µm). (Right) Peptide phage-displayed libraries. There are two essential types of phage display—display in the minor coat protein p3, and display in the major coat protein p8. (A) Phage vector fd-tet composed of 4000 copies of the p8 major coat protein (blue) and five copies of minor coat proteins p3 (pink), pVI (black), pIX (gray) and pVII (purple) each. (B,C) p3 and p8 phage display libraries. A random peptide (red) is fused to every copy of either p3 or p8 proteins. Adapted with modifications from [47]. The nomenclature of the types of phage display systems is provided in the phage display review [42].

Figure 3

Configuration of a 5-mer peptide displayed on bacteriophage (phage) M13. Computer rendering of a ~10 nm length for the surface of electron density maps of M13 (left), fusion phage with 5-mer peptide inserted in all copies of p8 proteins (center), and a rendering of the differences between images (right). A cylinder of 2.5 nm radius was added to images to mask the essentially identical interior features of the phages. About half of each coat protein is visible in phage surface images. Adapted from [46].

Figure 4

Vectors and libraries. In the nucleotide sequences corresponding to the part of recombinant gene gpVIII, encoding the N-terminal part of the major coat protein, randomized structures are designated as nnk, where n = A, T, G, or C, and k = G or T. Restriction sites for PstI and BamHI are underlined. N-terminal amino acid structures of mature recombinant pVIII proteins in libraries are indicated by capital single letters according to amino acid abbreviations. Randomized amino acids are designated by small letters (a–h in the f8/8 library and a–i in the f8/9 library). Amino acids are numbered as in vector phage f8-6 [41]. The nomenclature of the types of phage display vectors and libraries is provided in [38,42].

Figure 5

Selection of landscape phage interacting with PC-cell-associated antigens. The most common phage survey strategy is affinity selection, called ‘biopanning’ which enriches phage particles whose displayed peptides bind the target cells in culture or whole tissues in living animals. To use biopanning for selection of landscape phages against a variety of different PC, the researchers add the library to the immobilized target cells, wash away non-bound phage, elute bound phage particles, and amplify them. After 2–4 rounds of selection, they propagate individual clones, and analyze them. This procedure was named biopanning, because it is reminiscent of panning—the process of extraction of gold particles from sand.

Figure 6

Selectivity and specificity of phage probes. Phage probes selected from preliminary screening assays were incubated with target PC3 cells, control cells, or serum-treated wells of a 96-well cell culture plate. Phages associated with cells or serum were titered in bacteria and the ratio of phage output to phage input was expressed as recovery % to obtain the measure of the selectivity of a particular clone. The % recovery of the control phage bearing an unrelated peptide relative to the selected phage probe was indicative of the probe’s specificity. (A) f8/8 library, (B) f8/9 library. Adapted from [56,75] with permission from Elsevier.

Figure 7

Immunofluorescence microscopic demonstration of phage EPTHSWAT’s interaction with PC-3M cells at 15 min and 1 h in comparison with the control non-relevant phage VPEGAFSS. Adapted with modifications from [53].

Figure 8

Schematic illustration of biopanning for t-PSA (f-PSA and PSA-ACT). The f8/8 landscape phage library was added to the dishes with different immobilized forms of PSA. Unbound phages were washed away, and bound phages were eluted and used as a sub-library in the next round of biopanning. After three rounds, the individual phage clones were propagated, and their DNA segments corresponding to gpVIII were sequenced to determine the corresponding phage-displayed peptide sequences. Detailed procedures can be found in [25,37,100].

Figure 9

The phages can be conjugated to the gold electrode surface using carbodiimide chemistry (A), or immobilized to the gold sensor through physical adsorption [36] (B) and analyzed by atomic force microscope (A) or electron microscopy (B). As shown in panels (A,B), myriads of filamentous phage particles were attached to the gold surface to generate an intercrossing random network. Adapted with modifications from [36,49] with permission from Elsevier.

Figure 10

Standard curve for t-PSA. The schematic illustration of the phage-based ELISA for t-PSA detection is shown in the insert. The selected phage was loaded into wells of a 96-well plate. After coating the wells overnight, probes with different concentrations of t-PSA were added, and the plate was incubated at 37 °C. Then, mAb, IgG-HRP, and OPD were added successively. The absorbance values at 492 nm were linear with t-PSA concentration from 3.3 to 330 ng mL⁻¹. The limit of detection was calculated to be 1.6 ng mL⁻¹. Adapted from with permission of ELSEVIER [25,49].

Figure 11

The calibration curves of dual immunosensors after immunological recognition with different concentrations of f-PSA (left) and t-PSA (right). Adapted from [49] with permission of Elsevier.

Figure 12

The selectivity assay of P1- ERNSVSPS immunosensor (A) and P5- ATRSANGM immunosensor (B) for f-PSA and t-PSA (total of f-PSA, and PSA-ACT) in comparison with other common cancer biomarkers (AFP, CA125, CA15-3, CA19-9, and hK-2) present in the serum as controls. The samples were dropped onto the phage-covered immunosensors and incubated for same time. EIS assay was performed for the above immunosensors as described. Adapted from [49] with permission from Elsevier.