PSMA mimotope isolated from phage displayed peptide library can induce PSMA specific immune response

Cell Research (1999), 9, 271-280

ZHU Zhong Yu¹, Cui Ping ZHONG², Wei Feng XU¹, Guo Mei LIN¹, George QW YE³, Yong Yong JI¹, Bing SUN^1,*, Ming YEH^1,*

1. Shanghai Institute of Cell Biology, Chinese Academy of Sciences.
2. Department of Histology, Shanghai Medical University.
3. Yes Biotech Laboratories LTD, Canada

ABSTRACT

Prostate-specific membrane antigen (PSMA) is a cell surface glycoprotein expressed predominantly in prostate secretory acinar epithelium and prostate cancer cells as well as in several extraprostatic tissues. Mouse monoclonal antibody 4G5 specific to the extracellular domain of PSMA was used to screen two phage displayed peptide libraries (9aa linear and 9aa cys library). Three 4G5-reactive phagotopes were identified. Sequence analysis of isolated clones demonstrated that the interaction motif "VDPA/SK" has high homology to 719-725aa on PSMA. Immunohistochemical staining of the prostate cancer sample with the PSMA-mimic phagotope (mimotope) immunized serum antibodies demonstrate that the mimotope isolated from the phage displayed peptide libraries can induce PSMA specific immune response in vivo.

Key words: PSMA, mimotope, phage displayed peptide library, immunohistochemistry.

INTRODUCTION

Prostate carcinoma is one of the most common cancer in men[1],[2]. The death rate from prostate carcinoma is also increasing in China. To improve this situation, new markers are needed for early and accurate staging and treatment as well as better means to follow disease progress. One such potential marker is prostate specific membrane antigen (PSMA).

PSMA is a type II membrane glycoprotein of Mr ~ 100, 000 with folate hydrolase-type and neuropeptidase-type activity[3-5]. PSMA is highly expressed in benign prostate secretory acinar epithelium, prostatic intraepithelial neoplasia and prostatic adenocarcinoma, and evidence suggests that PSMA expression is greastest in high-grade and hormone insensitive cancers. Given its membrane bound character, PSMA has been exploited as a marker for tumor detection and treatment with radioactive isotope or toxin labeled PSMA-specific antibodies. Especially, recent studies demonstrate that the monoclonal antibodies specific for the extracellular domain of PSMA are able to induce the internalization of PSMA[6]. Moreover, PSMA is also consistently expressed in the neovasculature of a wide variety of malignant neoplasms and maybe it is an effective target for Ab-based antineovasculature therapy[7].

With the development of phage displayed peptide library technology, it has been successfully applied to the epitope study and other relevant fields[8-10]. In this report, we employed the phage display peptide library to study the epitope recognized by mAb 4G5 and its immunogenicity to induce PSMA specific antibodies.

MATERIALS AND METHODS

mAb

Prostate membrane specific antigen specific monoclonal antibody 4G5 was from YES Biotech Laboratories Ltd, Canada. The antibody was generated from immunization with crude membrane extract of prostate carcinoma and screened out with synthesized 8-peptide ("ESKVDPSK" derived from the 716-723aa of PSMA sequence).

Phage peptide libraries and bacteria strain

Random phage peptide libraries expressing linear (pVIII 9aa)[11] or circular (pVIII 9aa Cys)[12] nonapeptides fused to pVIII of filamentous bacteriophage fd, wild type phage f1 and bacterial strain DH5aF' were all kindly provided by Dr. Paolo Monaci of IRBM (Istituto di Ricerche di Biologia Molecolare P. Angeletti SPA, Rome, Italy).

Affinity selection

Two rounds of affinity selection were performed as following, 200 ml mAb 4G5 in coating buffer (50 mM NaHCO₃, pH 9.6) at 2mg/ml was incubated in 96-well plate (Immuno plate Maxisorp, Nunc) at 4^oC overnight, then blocked with blocking buffer (l × PBS, 3% BSA, 0.05% Tween) at 37^oC for 1.5 h. 10¹¹ wild type phage f1 in 200 ml blocking buffer were added to the well and incubated at room temperature for 1 h. The plate was washed extensively with PBST (1 × PBS, 0.1% Tween), then about 1 × 10¹⁰ phages from either 9aa linear or 9aa cys circular peptide library in 200 ml blocking buffer were added to the well and incubated at room temperature for 2 h. After washing, the absorbed phages were eluted and neutralized with Tris-HCL as described[13]. The eluted phages were amplified by infecting DH5aF' and purified with 20% PEG/2.5 M NaCl precipi tation. It can also be stored for immunoscreening. The purified phages were used for next round of affinity selection.

Immunoscreening

After two rounds of affinity selection, DH5aF' cells were infected with eluted phage at a multiplicity of infection (m.o.i.) of 10^-3, the mixture was incubated at 37^oC for 30 min, then helper phage M13 K07 was added at a m.o.i. of 20-50, and incubated at 37^oC for another 15 min. The infected bacteria were centrifuged for 5 min at 3000 g, the supernatant was discarded and bacteria pellet was washed three times in 1ml of LB medium to eliminate the non-absorbed phages. 100 ml of the resuspended pellet with series of dilution (10^-3, 10^-4, 10^-5) were plated on plates containing Ampicillin (100 mg/ml), Kanamycin(50mg/ml) and IPTG (30 mg/ml). After incubation at 37^oC overnight, the plates containing 200-400 separated clones (10 cm plate) were layered with nitrocellulose filters and marked with needle. The filter was took out immediately and blocked with blocking buffer (5% non-fat milk power, 1 × PBS, 0.05% Tween-20, 0.05% NaN₃) at room temperature for two h. 10 mg mAb preincubated with 25 ml bacterial extract (prepared as described in Ref 13) and 25 ml f1 Phage (2.3 × 10 ¹³/ml) in 5 ml blocking buffer at room temperature for 1 h, were then added to the blocked filters. Filter with mAb mixture was then incubated at room temperature for 1.5 h and then extensively washed with washing buffer (1 × PBS, 0.05% Tween-20). Then secondary antibody (alkaline phosphatase conjugated Goat anti-mouse Abs, Sigma) diluted to 1/5000 in blocking buffer was incubated with the filter for 1.5 h at room temperature. The filters were then washed as above and developed by incubation with developing solution (330 mg/ml nitro blue tetrazolium, 165 mg/ml 5-bromo-4-chloro-3-indolephosphate in l00 mM Tris HCl, l00 m M NaCl, 5mM MgCl₂, pH 9.6) at room temperature for 10 min. Reaction was stopped by washing with water.

ELISA

In brief, multi-well plates (Immuno plate Maxisorp, Nunc) were coated overnight at 4^oC with the mAb 4G5 at a concentration of 2 mg/ml in 50 mM NaHCO₃ pH 9.6. After washing several times with PBS/0.05% Tween-20 (PBST), plates were incubated at 37^oC for 60 min with ELISA blocking buffer (5% non-fat dry milk in PBST). 4 × 10⁹ sample phages were diluted in 100 ml blocking buffer and then added to each well and allowed to bind for 1 h at 37 ^oC. The equal amount of wild type phage was used as a negative control. Plates were then washed with PBST and 100 ml/well of goat anti-mouse IgG HRP conjugated antibodies (Sigma, 1/5000 dilution in ELISA blocking buffer) were added. After incubation for 1 h at room temperature, plates were then washed and developed by adding 100 ml substrate TMB buffer and incubated at 37 ^oC in dark for 15 min. Optical density was measured in an ELISA reader at 450 nm.

Competitive ELISA

96-well ELISA plates were coated with 1 × 10⁹ phage/well of f1 or three phage clones. Fixed amount of immune serum (1:2700 dilution) is incubated in each well with increasing amount of competitor f1 (from 0 to 1.5 × 10 ⁹). After extensively washing, the HRP conjugated secondary antibody (goat anti-mouse IgG) was added. Following development, the optical density value was read at 450 nm.

DNA sequencing

Single stranded DNA of the positive clones was extracted from the amplified phages using DNA purification kit according to the instructions (Promega). Sequencing was performed by the Sanger dideoxy method with T7 sequencing kit (Pharmacia) according to the instructions.

Immunization of mice with selected phage

The identified positive phage clones were amplified in DH5aF' and purified by PEG/NaCl precipitation. Then the phages were resuspended in 1 × PBS at a concentration of 5 × 10 ¹² phage particles/ml. Ten to twelve-week old male C57BL/6 mice were immunized by i. p. 250 ml of positive phage clones emulsion (1:1 with CFA for the primary injection, IFA for the boost injections), using wild type phage f1 as control. The immunization was performed at week 0, 4 and 7 and bled at days 10 after second and third injection.

Absorbing immunized serum with wild type phage f1

The immune serum was absorbed with wild type f1. 20 ml serum was diluted 1 ml and mixed with 1 ml PBS containing 1 × 10¹³ f1 phage particles. Then the mixture was centrifuged at 200,000 g for 2 h, The supernatant was collected and used in immunohistochemical staining at 1:200 dilution.

Immunohistochemical staining

All the slides of prostate cancer, lung cancer, stomach cancer and colon cancer were prepared routinely. Endogenous peroxidase activity was quenched by 30 min incubation in 3% hydrogen peroxide. After washing, the slides were blocked with 5% normal goat serum for 30 min. The f1-absorbed mimotope immunized serum diluted in 1:200 were added to the slides as first layer antibody and incubated at 4 ^oC overnight. Immunized serum with wild type phage f1 was used as negative control. After washing extensively with 1 × PBS 0.05% Tween-20, HRP conjugated goat anti-mouse IgG was added and incubated at 37 ^oC for 1 h. After washing with PBS tween, the slides were developed by adding the substrate DAB and H₂O₂ according to the manufacture's instructions. The reaction was stopped by washing with water. Slides were counterstained lightly with Harris hematoxylin, dehydrated through a graded series of ethanol to xylene and coverslipped with permount.

RESULTS

Affinity selection

Two rounds of affinity selection were performed before the immunoscreening. After each round of affinity selection, the percentage of the blue clones was calculated from every eluted pool of phage, and the titer of the phage was counted (Tab 1). From both libraries, the increase of the phage titer and percentage of blue clones were observed, indicating the enrichment of specific ligand during the affinity selection. The increase of phage titers was in parallel with an increase of phages binding to 4G5 in ELISA. And low binding was observed with equal amounts of wild-type phage f1 (Fig 1).

Tab 1. Eluted phage titer after affinity selection with MAb 4G5.

1. Percentage of blue clones can be used as a parameter indicating the enrichment of functional phage peptide[11].

2. Titer indicates the number of phages eluted after affinity selection. In each round of affinity selection, the number of input phages was 1 × 10¹⁰.

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