| Presently prostate cancer ranks the second place in all emerging malignant tumors in theworld, and the first place in the developed countries. In view of the diagnosis rate of prostatecancer rising quickly in China, more attention should be paid. Prostate stem cellantigen(PSCA) is a kind of protein associated with prostate cancer and GPI-anchored protein,including123amino acids residues, and expressed on epithelial of prostate cancer cells,which was firstly found by Reiter, etc. PSCA has high specificity of prostate tissue, andanchors on cell surface instead of secretion, so PSCA gets wide attention as the potentialtarget of diagnosis and immunotherapy of prostate cancer.This study aims at developing anti-PSCA monoclonal antibody based on the research athome and abroad as well as the author’s laboratory. The study contents include: developanti-PSCA monoclonal antibody by adopting hybridoma technique, then screen out antibodyable to bind PSCA on the cell surface, and evaluate their antitumor activity in the mice modelbearing tumor and establish immunohistochemical method for diagnosis of prostate cancer.Besides, the relevant primary research about single-chain fragment were undertaken aftercloning gene of variable region of anti-PSCA monoclonal antibody.In developing anti-PSCA monoclonal antibody, it is a key to select and prepare anantigen for screening out the active monoclonal antibody. Firstly, according to literature, weselected21-101aa of the full length of PSCA, a prokaryotic expression vector of PSCA fusionprotein with a GST tag and a His tag was constructed. and a GST tag is helpful to increase thesolubility of the fusion protein, and a His tag is convenient for purifying. PSCA fusion proteinwas expressed in escherichia coli, which proved to be soluble by the subcellular localizationexperiment. After ultrasonic treatment, purification was performed with the supernate,high-purity PSCA fusion protein was obtained by two steps (Ni column and GST column).The PSCA fusion protein was confirmed by Western blot with antibodies of anti-GST,anti-His and anti-PSCA.BALB/c mice were immunize with the PSCA fusion protein as antigen,6monoclonalantibodies were obtained by hybridoma technique. Titer and subtypes of every antibody wasdetermined, and the specificity of monoclonal antibody was verified by ELISA and Westernblot with standard PSCA Protein (Abnova). Then flow cytometry were applied to screen outthe monoclonal antibody has activity to bind PSCA on the surface with several cell lines,including P815-PSCA (mice’s mast cell line expressing as PSCA), LNCaP(hormone-dependent prostate cancer cell line), PC-3(hormone-independent prostate cancercell line) and DU-145(hormone-independent prostate cancer cell line). P815-PSCA cell linewas established early by our lab, and other3cell lines have also PSCA expression accordingto the literature. Finally,3monoclonal antibodies (19#,24#,31#) with the ability to bind PSCAon the cell surface were screened out, and they had similar binding characteristics on all4cell lines. Western blot were undertaken with PC-3cell and DU-145cell lysate respectively, as aresult, a strip as the same size (approximately18kDa) as commercial anti-PSCA rabbitpolyclonal antibody was obtained, and it provided a base for further study and application.To evaluate the antitumor activity of PSCA mAbs in mice, we selected thehormone-dependent prostate cancer cell line DU-145as the study object. The DU-145celllines was derived from an untreated white male with prostate cancer and nowadays is widelyused, and its characteristics have shown obviously: has tumorigenic feature in mice, it doesnot express PSA or androgen receptor, and may be similar to hormone-independent prostatecancer, and may be a model for the most advanced forms of prostate cancer.Firstly, it can be observed by the laser-scanning confocal experiment that PSCA mAbcan bind to surface of DU-145cell at4°C and internalized into the cells with incubation at37°C. The influence of PSCA mAb to DU-145growth was observed by means of the lightmicroscope and the cell activity were determined by MTT, it was concluded that PSCA mAbhas certain inhibiting effect.To evaluate the inhibitory effects in animal model, the athymic mice model wasestablished with DU-145. Human prostate cancer xenograft mice models were built byinjection of DU-145cell in the back of male mice. Results showed, the group (injection of1×106cell/mice) was observed that all member formed tumor, which was confirmed as cancerwith histopathologic diagnosis, indicating that DU-145human prostate cancer xenograft micewas established successfully.The the therapeutic efficacy of anti-PSCA mAbs was evaluated in human prostate cancerxenograft mouse models by using the DU-145. During the experiment, the tumor in the micein PBS group and antibody groups was growing larger and larger, but tumor forming delayed2weeks in antibody group, and on40days after injection of antibody, the average tumorvolume of31#mAb group had significant difference with t-test comparing to group of PBS,and group of PSCA mAb19and24did not. This result indicated that PSCA mAb31#had apotential application future.In order to establish the immunohistochemical method of PSCA mAb being used forpathological diagnosis of prostate cancer of clinical specimens, immunohistochemical methodwas adopted first for the tumor specimens of mice bearing tumor, PSCA mAb showed intensepositive reaction, staining deeply on the membrane and plasma of cancer cells. These resultshows PSCA mAb can mightily bind to prostate cancer cell.General, clinical specimens must be evaluated with Gleason score system in thepathological diagnosis. Gleason score of clinic specimens between1and4is rare, are mostbetween5and10, and score6,7are most common. Specimen of Gleason score6and7wereused normally in some research. In our study,4specimens evaluated as6,7,8and10respectively by Gleason score system has certain representativeness. PSCA mAb staining indifferent Gleason score specimens are from medium to high intensity, light for little portion.In positive area, staining located on cell membrane, plasma, with wide staining range.Comparing to normal prostate tissue, there was a clear difference on staining intensity and range in prostate cancer. Staining intensity of specimens of different Gleason score is different,and Gleason score of specimens is basically consonant with staining intensity. Result ofimmunohistochemistry of clinical specimens of prostate cancer showed that PSCA mAb couldbind to cancer cell specifically, and has good specificity and sensitivity, demonstrating thatimmunohistochemistry of PSCA mAb for diagnose of prostate cancer Preliminary established.For the purpose of further genetically engineered antibody modification for PSCA mAb,antibody variable region gene of3PSCA mAb(19#,24#,31#) was cloned by5’RACE, andwas analyzed inclusive of BLAST homology comparison, IMGT V region analysis andSignalP signal peptide analysis. As a result, the variable gene of antibodies was reliablyobtained, and the leading peptide, framework region and complementary determining regionwere divided.Based on these sequences, taking PSCA mAb31#as the example, the single-chainfragment (scFv) was constructed in the form of VL-linker-VH, and it showed that it is oftypical3-D structure of antibody after prediction in SWISS-MODEL. On the design principleto form disulfide bond, Amino acids at H44/L104were selected to be changed to Cysteine toshape disulfide-stabilized Fv (dsFv).3D structure prediction shows that dsFv has expectedintrachain disulfide bond, which is helpful to its stability. Then prokaryotic expression vectorof scFv and dsFv with a His tag was constructed, and scFv and dsFv were induced andexpressed in Escherichia coli, and the subcellular localization experiment shows it expressedas the form of inclusion body in Escherichia coli. The scFv and dsFv were purified withinclusion body through ultrasonic treatment, washing, denaturing and Ni column purification,and finally, the single-chain antibody was obtained by means of Gradient dialysis method andrenaturation. SDS-PAGE and Western blot result shows refolded dsFv has expected intrachaindisulfide bond. The binding activity of scFv and dsFv was verified by ELISA method underthe condition of adopting commercial PSCA protein (Abnova) as antigen, which laid theexperimental foundation for further Genetic engineering antibody modification.The above-mentioned experimental results show: active anti-PSCA mAbs have beenprepared, and have higher potential value in the field of pathological diagnosis and treatmentof prostate cancer. Acquisition of variable region gene of PSCA mAb and successful design ofdisulfide-stabilized Fv (dsFv) will be an experimental basis for follow up study on geneticallyengineered antibody. |
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