The Significance Of Prostate Specific Membrane Antigen (PSMA) In The Pathological Diagnosis And Immunotherapy Of Carcinoma Of Prostate

The carcinoma of prostate is the most common cancer in males in America, ranking as the second most common cause of cancer-related deaths, just after carcinoma of the lung. As going to an aged society, the incidence and mortality of carcinoma of prostate are increasing in China, and carcinoma of prostate is often discovered at advanced stage (exceeding the limits of the gland) or when already metastatic. At this stage, there is no curative therapy. To improve this situation, new biomarkers are needed to help the diagnosis and treatment of carcinoma of prostate, one such potential marker is prostate specific membrane antigne (PSMA).PSMA is a type II transmembrane glycoprotein expressed on the surface of prostate epithelial cells. Its expression is elevated in carcinoma of prostate, particularly in advanced cancer and hormone -refractory cancer. Given the physical properties of PSMA and its behavior in relation to disease progression, PSMA is a potential diagnostic and therapeutic molecule. For a long period, monoclonal antibodies against inner domain of PSMA have been widely utilized clinically, however, their applications are limited in such area as in vivo imaging of the carcinoma of prostate and immunotherapeutic application because they can not pass through the plasma membrane of viable cells to react with the epitope involved. Preparing monoclonal antibody specific for extracellular epitopes of PSMA provide the best way to conquer the above limitations.The differentiation of carcinoma of prostate is linked to the prognosis of patients and the efficiency to the treatment such as androgen deprivacation therapy. Gleason grade is a prevalent standard to evaluate the differentiation degree of carcinoma of prostate and used as a significant variable to predict the prognosis. PSMA is a well-established prostate-specific marker, but the correlation of its expression with Gleason grade remains unclear.Although treatments are available for organ-confined carcinoma of prostate, there is little effective treatment for metastatic disease, particularly for recurrent tumor after androgen deprivation therapy fails. Immunotherapy to carcinoma of prostate could be a safe, noninvasive approach. Several groups have recently reported DNA vaccine inthe treatment of carcinoma of prostate in preclinical trials. DNA vaccine can express protein with natural epitopes in vivo and stimulate the body to generate comprehensive immunity, such as antibodies, cytotoxic T lymphocytes (CTL) and immune memory. Furthermore, DNA vaccine is safe and cost-effective because DNA is relatively simple to purify in a large quantity. The level of PSMA expression is down-regulated by androgen, patients undergoing androgen deprivacation therapy exhibit increasing PSMA, so it may be a ideal target to construct DNA vaccine, especially for recurrent carcinoma of prostate after primary therapy fails.The purpose of this research is to investigate the significance of PSMA in the diagnosis and therapy in carcinoma of prostate. Briefly, we would develop monoclonal antibodies specific for extracellular epitopes of PSMA to provide a vehicle for immunotherapy to carcinoma of prostate. Then we would detect the expression of PSMA in carcinoma of prostate and find the correlation of PSMA expression with differentiation of carcinoma of prostate to evaluate whether PSMA could be used as a marker in Gleason grade. Furthermore, we would construct DNA vaccine expressing PSMA with which to immunize C57BL/6 mice, thus we would investigate the anti-tumor reaction of the DNA vaccine.Part 1 The Preparation of Monoclonal Antibodies Specific for the ExtracellularEpitopes of PSMAObjective To prepare monoclonal antibodies (mAb) specific for extracellular epitopes of prostate specific membrane antigen (PSMA) and provide a tool for the diagnosis and treatment for carcinoma of prostate.Methods A prokaryotic vector pET32b-psmac encoding extracellular fragment of PSMA was constructed and a fusion protein containing PSMA extracellular fragment was induced. Balb/c mice were immunized with purified protein. The splenocytes of the mice with the highest titer were fused with fusion partner myeloma cells SP2/0, the hybridoma cells were screened by immunofluorescence and immunohistochemical staining. Then the mAb was purified and labeled with fluorescein isothiacyanate (F1TC), the binding activity of the mAb to viable LNCaP cells was investigated.Results Eight hybridoma cell lines secreting monoclonal antibodies were obtained,the monoclonal antibodies exhibited high prostate-specificity when used in immunohistochemical staining. One antibody was found to be able to bind viable LNCaP cells.Conclusion Monoclonal antibodies specific for extracellular epitopes of PSMA were obtained and laid foundation for our further research. Moreover, it provided vehicle to the diagnosis and treatment of carcinoma of prostate.Part II The Correlation of PSMA Expression With Differentiation ofCarcinoma of ProstateObjective To detect PSMA expression in carcinoma of prostate and investigate the significance of PSMA in the pathological grade of carcinoma of prostate.Methods The sections of carcinoma of prostate tissue fixed in 4% formalin and embedded in paraffin were immunohistochemically stained with self-prepared mAb against PSMA, the .intensity of PSMA was obtained through digital image processing, and the data were statistically analyzed, PSA intensity was also obtained for comparison. - -Results The PSMA intensity was reversely correlated with the differentiation of carcinoma of prostate, the lower the differentiation of the carcinoma of prostate is, the higher the expression of PSMA will be. On the contrary, PSA shared no correlation. Interestingly, PSMA was detected both in cytoplasm and on membrane of cells in the well and moderately differentiated carcinoma of prostate, while in poorly differentiated carcinoma of prostate, it was predominantly located on cell membrane.Conclusion The intensity of PSMA was reversely correlated with differentiation of carcinoma of prostate, so PSMA may be a marker in pathological grade of carcinoma of prostate. PSMA expression is elevated in poorly differentiated carcinoma of prostate and mainly located on the membrane, which was more accessible by antibodies, so PSMA could be an ideal molecule for immunotherapy of poorly differentiated carcinoma of prostate.Part III The Anti-tumor Reaction of DNA vaccine Expressing PSMAObjective To construct DNA vaccine expressing PSMA and analyze its resultant protection to the challenge of tumor cells so as to find a new means for the treatment to carcinoma of prostate.Methods A eukaryotic vector pCDNA3.1-PSMA encoding full-length PSMA was constructed. The C57BL/6 mice in PSMA group were immunized with endotoxin-free pCDNA3.1-PSMA by intramuscular injection; the mice in PSMA+ODN group were injected with pCDNA3.1-PSMA along with adjuvant CpG oligodeoxynucleotides 1826 ( CpG ODN 1826); the mice in pCDNA group were injected with pCDNA3.1 as control. After 4 times of immunization, humoral immunity was measured through indirect ELISA, cellular immunity was measured by lactate dehydrogenase (LDH) release method. B16 melanoma cells with a C57BL/6 mice species background were transfected with pCDNA3.1-PSMA and selected by G418. All immunized C57BL/6 mice were challenged by implanting PSMA-expressing B16 cells subcutaneously, the tumor free survival time and growth of tumor were observed to verify the anti-tumor response of the vaccine.Results DNA vaccine, alone or with CpG ODN 1826, elicited PSMA-specific antibody with an average titer of 1:160. More importantly, DNA vaccine expressing PSMA induced CTL reaction, and CpG ODN 1826 could enhance the reaction, the difference was of statistical significance (p<0.05).The tumor-forming time of mice immunized with DNA vaccine was delayed compared with pCDNA group, tumor free survival time of pCDNA group, PSMA groups PSMA+ODN group were 13.17+ 1.01, 19.67 + 2.24, 22.33 + 1.61 days respectively, the difference had statistical significance (p<0.05). On a follow-up of 26 days, all mice in pCDNA group developed tumor, but there were 2 mice (2/6), 3 mice(3/6) that had not developed tumor in PSMA group, PSMA+ODN group . All mice were sacrificed and the tumor mass were removed , the average weight of tumor mass in pCDNA group, PSMA group, PSMA+ODN group were 4.75 + 0.66g, 2.28 + 0.5lg> 1.10±0.70g respectively, and the difference had statistical significance (p<0.01) .Conclusion DNA vaccine expressing PSMA could generate anti-tumor reaction, and...