Anti-Tumor Efficacy of Gene Vaccine Expressing PSMA

6 Clin Oncol Cancer Res () 7: 6-5 DOI.7/s85--5-7 Anti-Tumor Efficacy of Gene Vaccine Expressing PSMA Xiao-ling YANG Jing LV Yue-hong ZHANG Bo NIU, Department of Laboratory Medicine, Shanxi Bo ai Hospital, Taiyuan, Shanxi Province, China. Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan, Shanxi Province, China. Biotechnology Research Laboratory, Capital Institute of Pediatrics, Beijing, China. Correspondence to: Bo NIU Tel: 86--8569 55 E-mail: Niub@6.com OBJECTIVE To observe anti-tumor effects of PVAX-PSMA gene vaccine. METHODS The PSMA gene was inserted into a mammalian expression vector, PVAX-, to construct the DNA vaccine candidate, and was then used to vaccinate C57BL/6 mice. Animals vaccinated with PVAX- and NaCl were used as controls. Anti-PSMA antibody was detected in sera of the animals. The proliferation and cytotoxicity of the spleen cells were observed. The immunized mice were inoculated with RM- cells. The mice were inoculated with RM- cells, and then the mice were immunized. The anti-tumor efficacy of the gene vaccine was evaluated by the ratio of tumor formation, tumor volume, tumor mass before and a er gene vaccination and evaluated by survival rate of the immunized mice. RESULTS High level of anti-psma antibody was induced in the PVAX-PSMA group. The splenocytes from PVAX-PSMA group were stimulated to produce strong proliferation responses and significant cytotoxic T-cells (CTL) activity. After the mice were immunized with PVAX-PSMA gene, tumor occurrence was decreased, and the growth velocity of tumor was markedly reduced, resulting in prolonged tumor-free time (P <.5). CONCLUSION PVAX-PSMA gene vaccine has significant antitumor effects and provides an experimental basis for primary prevention and immunotherapy of prostate cancer. KEY WORDS: prostate cancer, prostate-specific membrane antigen, gene vaccine. Copyright by Tianjin Medical University Cancer Institute & Hospital and Springer Introduction Received August, ; accepted November 6,. E-mail: 8cocr@gmail.com Tel (Fax): 86--5 99 Prostate cancer has the highest incidence rate in men and accounts for 9% of all new cancer diagnosis. In 7, approximately 8,89 new cases of prostate cancer and 7,5 deaths due to prostate cancer were estimated in the US alone []. Early screening to detect prostate has reduced the mortality rate of prostate cancer. The expression level of the prostate specific membrane antigen (PSMA) has been proposed as a useful marker, indicating how late the prostate cancer has been [-5]. PSMA, a type II transmembrane glycoprotein with a Mr of kd, is found in metastatic prostate cancer, hormone refractory tumors, the vascular endothelium of some carcinomas and sarcomas, and limited its expression in normal cells and in benign prostatic

Clin Oncol Cancer Res () 7: 6-5 7 secretory-acinar epithelium [6,7]. Gene vaccine is a new approach to immunization and immunotherapy. In the years following the publication of the initial demonstration of the ability of plasmid DNA in vivo to generate protective immune responses, DNA vaccines have entered into a variety of clinical trials for vaccines against various infectious diseases and for therapies against cancer. In this study, we introduced the gene encoding the specific antigen PSMA into a mammalian expression vector PVAX- in C57BL/6 mice bearing RM- cells obtained from injection of the tumor cells, for the purpose of observing the anti-tumor effect of the recombinant vaccine. Materials and Methods Materials Forty-five C57BL/6 mice were purchased from the Experimental Animal Center of Shanxi Medical University; recombinant plasmid PVAX-PSMA and RM- cell were produced in our laboratory; eukaryotic expression vector PVAX- was obtained from Invitrogen Co; LDH assay kit was purchased from Promega Co; a large number of ultrapure plasmid kits for fast isolation were purchased from Boda Co; Triton X- was produced by Amresco Co; MTT assay kit was obtained from Promega Co; Mitomycin C was purchased from Sigma. Methods Preparation and purification of recombinant plasmid The plasmid PVAX- and recombinant plasmid PVAX- PSMA were extracted with a large number of ultrapure plasmid kits for fast isolation. Endotoxin in plasmid solution was removed by Triton X- phase separation. Recombinant plasmid was dissolved by sterile saline. The concentration of recombinant plasmid was diluted into g/l with sterile saline. Injection of the plasmid Fifteen C57BL/6 mice were randomly divided into PVAX-PSMA group, PVAX- group and NaCl group (5 in each). Five mg/l of procaine, μl for each were injected into the quadriceps of the mice days before they were immunized. The method of immunization: μl of the recombinants (the concentration of recombinants was g/l) were injected into the quadriceps of each C57BL/6 mouse in the groups at the st, th, and st day. All the experimental procedures and animal maintenance met the strict guidelines of institutional animal ethics committee. Identification of anti-psma antibody in immunized mice Serum was extracted from each mouse in the groups days after each time the mice were immunized, and the extracted serum was preserved at - C. Anti-PSMA antibody was detected through indirect immunofluorescence assay. Proliferation of T lymphocyte in the spleen The spleen cells were obtained from the mice and then prepared into single cell suspension. The stimulated cells were prepared as follows. RM- cells with mitomycin C was cultured at 7 C for h. The ratio of the spleen cells to the stimulated cells was 5:, and they were cultured in a 96-well plate as controls. And then we operated manually the MTT assay kit. Formulation of proliferation index of the Lymphocytes: (A57 value in experiment group A57 value in stimulated cell group)/a57 value in spleen control group. Detection of CTL in immunized mice Killing effect of RM- on spleen cells was detected using LDH assay kit. RM- cells, as target cells were inoculated into a 96-well plate with the density of cells/well, with different amounts of effector cells added in order to reach the ratios of effector cells to target cells being 5:, 5: and :, respectively, and then cultured at 7 C for h. And then we operated manually the LDH assay kit. Formulation of killing rate of CTL: (experiment release-natural release of effector cells natural release of target cells)/(target cell maximum natural release of target cells) %. Establishment of tumor xenografts and assessment of growth There were 5 immunized mice in each group. At the st day, the immunized mice were inoculated. ml of tumor cell, RM- ( /ml) through subcutaneous injection. The process of tumor growth was recorded every week. The length (a) and width (b) of the tumor were measured by caliper regularly. The tumor volume was then calculated by the formula: volume = (a b )/. Preventive effects of gene vaccine Fifteen new mice were divided randomly into the groups, PVAX-PSMA group, PVAX- group and NaCl group. The mice were inoculated. ml tumor cell, RM- ( /ml) through subcutaneous injection. When the tumors grew tactile, μl recombinants (the concentration of the recombinants was g/l) were injected into the quadriceps of each C57BL/6 mouse in the groups at the st, th, and st day. The process of tumor growth was recorded every 8 day. The length (a) and width (b) of the tumor were measured using caliper regularly. The tumor volume was then calculated by the formula: volume = (a b )/. Statistical analysis Results were expressed as Mean ± SD, and one-way analysis of variance (ANOVA) was used for evaluating statistical significance. A value of P <.5 was considered statistically significant.

8 Clin Oncol Cancer Res () 7: 6-5 Results Detection of anti-psma antibody There was no anti-psma antibody in the sera of the mice in PVAX- group and NaCl group, but high level of anti-psma antibody was induced in the PVAX-PS- MA group. After the first immunization, antibody titer was :; after the second immunization, antibody titer was :; after the third immunization, antibody titer was :6 (Table ). Moreover, during a period of time, the antibody concentration appeared to go up as the time went by. Detection of proliferation effects on spleen T lymphocytes When the RM- cells with mitomycin C was cultured together, PVAX-PSMA group could induce obvious proliferation effects on spleen lymphocyte. The differences in proliferation rate between the PVAX-PSMA group and each of the control groups were significant (P <.5, Fig.). Detection of CTL When the ratio of effector cells to target cells was 5:, PVAX-PSMA group could induce a effective killing activity of CTL, causing stronger cellular immune response, and its killing rate was 7.%. When contrasted with the killing rate of the CTL in the PVAX-PSMA group, the killing rate in the PVAX- group and in NaCl group was.5% and.8%, respectively. The differences in killing rate between the PVAX-PSMA group and each of the control groups were significant (P <.5, Fig.). Preventive effects of gene vaccine The tumor forming rate was % in both NaCl group and PVAX- group, and was % in PVAX-PSMA group. The tumor in NaCl group and PVAX- group grew fast, and the differences in tumor volume and tumor mass between the PVAX-PSMA group and each of the control groups were significant (P <.5, Table ). In PVAX-PSMA group, tumor-free time was longer and survival rate increased after the mice were inoculated RM-cells compared with the other groups. Therefore, the lifetime of the mice in the PVAX-PSMA group was significantly different than that of the mice in the other control groups (P <.5, Fig.). Anti-tumor effects of the vaccine C57BL/6 mice (5 per group) were first challenged by giving. ml RM- tumor cells ( /ml) subcutaneously. Fifteen days after tumor challenged, the tumor forming rate in each of the groups was %, and the tumor volume was approximate mm. The mice in each of the groups were vaccinated with PVAX- PSMA, PVAX- and NaCl, respectively for times with a -day interval in between. In the NaCl group and PVAX- group, the tumor grew fast. The changes in the tumor volume indicated that PVAX-PSMA could significantly inhibit the growth of RM- cells in vivo. At day 6, the tumor volume in the PVAX-PSMA group was only (.7 ±.8) cm, significantly less than the tumor volume of (.78 ±.) cm in the NaCl group and also less than that of (.6 ±.9) cm in the PVAX- group. The differences in growth velocity of tumor between the PVAX-PSMA group and each of the control groups were significant (P <.5, Fig.). Table. The antibody level in the sera of the mice. Antibody titer After the first immunization After the second immunization After the third immunization NaCl PVAX- Vaccination NaCl PVAX- Vaccination NaCl PVAX- Vaccination : - - + - - + - - + : - - + - - + - - + : - - - - - + - - + :6 - - - - - - - - + Proliferation index.5.5.5.5.5 I II III Times of immunization Fig.. The proliferation of spleen T lymphocytes in the C57BL/6 mice. P <.5 compared with NaCl group and PVAX- group. I, NaCl group stimulated by RM- cells; II, PVAX- group stimulated by RM- cells; III, PVAX-PSMA group stimulated by RM- cells. Specificlysis (%) 5 5 5 5 5 5 5 Effector/Target PVAX-PSMA PVAX- N Fig.. Cytotoxicity T-lymphocyte (CTL) assay.

Clin Oncol Cancer Res () 7: 6-5 9 Survival rate NaCl PVAX- PVAX-PSMA..8.6.. 8 5 9 56 6 7 Times after tumor challenge (d) Table. Tumor volume, tumor mass and tumor forming rate in mice after immunotherapy (n = 5, Mean ± SD). Group Tumor volume (cm ) Tumor mass (g) Tumor forming rate (%) NaCl.96 ±. 6. ±.57 PVAX-.88 ±. 6.55 ±. PVAX-PSMA.5 ±.57.87 ±.8 P <.5 compared with NaCl group and PVAX- group. Fig.. The comparison of mouse survival rate. Tumor volume (cm ) NaCl.5.5.5.5 8 6 8 56 6 Tumor volume (cm ).5.5.5.5 PVAX- 8 6 8 56 6 Tumor volume (cm )...8.6.. PVAX-PSMA 8 6 8 56 6 Fig.. Inhibitory effects of the groups on tumor growth in mouse in vivo (n = 5). Discussion Prostate cancer is the most common neoplasia diagnosed in men in developed countries. In this study, we showed for the first time that the tumor biomarker PSMA, a multi-functional cell surface ectopeptidase [], was endowed with an efficient signaling activity in prostate cancer cells. In prostate cancer cells, PSMA is expressed at -fold higher level than in normal prostate epithelium. Expression levels are increased with disease progression, with the highest level in metastatic diseases, hormone refractory cancers, and higher-grade lesions [7]. Endothelial-expression of PSMA in the neovasculature of a variety of non-prostatic solid malignancies has also been detected [8]. Gene vaccine could provide a good therapeutic possibility against tumors. DNA vaccines are simple rings of DNA containing a gene encoding an antigen, and a promoter/terminator to make the gene express in mammalian cells. They are a promising new approach for generating all types of desired immunity: cytolytic T lymphocytes (CTL), T helper cells and antibodies. This study further explored gene therapy of prostate cancer using recombinant plasmid PVAX-PSMA. The key point of gene therapy is to establish an effective gene delivering system. In this study, a highly safe vector PVAX- was selected to construct a recombinant vector. Recombinant plasmid PVAX-PSMA was then used to vaccinate C57BL/6 mice. Animals vaccinated with PVAX- or NaCl were used as controls. The immunized mice were inoculated RM- cells for the purpose of observing the anti-tumor effects of the recombinant vaccine. The mechanisms may be: i) It has been clear for many years that CD + T cells have a key role in helping B cells produce antibody [9]. ii) The majorities of candidate tumor antigens are intracellular and expressed only as peptides in the groove of the MHC class I molecule. Only CD8 + T cells can recognize these peptides and, after recognition, develop into effector CTLs capable of killing the target cells []. In this study, the splenocytes from PVAX-PSMA group were stimulated to produce strong proliferation responses and significant cytotoxic T-cells (CTL) activity. After the mice were immunized with PVAX-PSMA gene, tumor occurrence was decreased, and the growth velocity of tumor was markedly reduced, resulting in prolonged tumor- free time (P <.5). In a word, gene vaccine PVAX-PSMA may provide a feasible and effective way in prostate cancer treatment. Conflict of interest statement No potential conflicts of interest were disclosed.

5 Clin Oncol Cancer Res () 7: 6-5 References Jemal A, Siegel R, Ward E, et al. Cancer statistics, 7. CA Cancer J Clin 7; 57:. Rajasekaran AK, Anilkumar G, Christiansen JJ. Is prostate-specific membrane antigen a multifunctional protein? Am J Physiol Cell Physiol 5; 88: C975-C98. Chandran SS, Banerjee SR, Mease RC, et al. Characterization of a targeted nanoparticle functionalized with a urea-based inhibitor of prostate-specific membrane antigen (PSMA). Cancer Biol Ther 8; 7: 97-98. Chu TC, Shieh F, Lavery LA, et al. labeling tumor cells with fluorescent nanocrystal-aptamer bioconjugates. Biosens Bioelectron 6; : 859-866. 5 Milowsky MI, Nanus DM, Kostakoglu L, et al. Vascular targeted therapy with anti-prostate-specific membrane antigen monoclonal antibody J59 in advanced solid tumors. J Clin Oncol 7; 5: 5-57. 6 Sokoloff RL, Norton KC, Gasior CL, et al. A dualmonoclonal sandwich assay for prostate-specific membrane antigen: Levels in tissues, seminal fluid and urine. Prostate ; : 5. 7 Slovin SF. Targeting novel antigens for prostate cancer treatment: focus on prostate-specific membrane antigen. Expert Opin Ther Targets 5; 9: 56-57. 8 Chang SS, Reuter VE, Heston WD, et al. Department of Urologic Surgery VUMCNTUSA. Comparison of antiprostate-specific membrane antigen antibodies and other immunomarkers in metastatic prostate carcinoma. Urology ; 57: 79-8. 9 Stevenson FK, Ottensmeier CH, Johnson P, et al. DNA vaccines to attack cancer. Proc Natl Acad Sci U S A. ; : 66-5. Oxford JS. The end of the beginning: Vaccines for the next 5 years. Vaccine 8; 6: 679-68.