Prostate stem cell antigen (PSCA) is a highly glycosylated cell surface protein which is overexpressed in several malignancies including prostate, pancreas, and urinary bladder cancers. Tumor suppression has been reported by anti-PSCA antibody. Small and high affinity single chain antibodies (scFv) have been introduced as effective agents for cancer immunotargeting approaches. In the present study, we used a phage antibody display library of scFv and selected two antibodies against two immunodominant epitopes of PSCA by panning process. The reactivity of the scFvs for the corresponding epitopes was determined by phage ELISA. The binding specificity of antibodies to PSCA-expressing prostate cancer cell line, DU-145, was analyzed by flow cytometry. The antiproliferative and apoptotic induction effects were evaluated by MTT and Annexin-V assays, respectively. Results represented functional scFv C5-II which could bind specifically to DU-145 cells and significantly inhibited the proliferation of these cells (61%) with no effect on PSCA-negative cells. The antibody also induced apoptosis in the PSCA expressing cells. The percentage of the apoptotic cells after 24？hrs of exposure to 500？scFv/cell was 33.80%. These results demonstrate that the functional anti-PSCA scFv C5-II has the potential to be considered as a new agent for targeted therapy of prostate cancer. 1. Introduction Prostate stem cell antigen is a cell surface antigen belonging to the Thy-1/Ly-6 family of glycosylphosphatidylinositol (GPI) anchored proteins [1]. PSCA expression in normal tissues has shown to be predominantly prostate specific. However, less expression of PSCA has also been detected in other normal tissues including placenta, stomach, and kidney [2]. Elevated levels of PSCA have been reported in over 80% of prostate cancer specimens and in all cases of bone metastasis from prostate cancer patients [3]. The overexpression of PSCA has also been reported in most bladder and pancreatic cancers [4–6]. In the cases of prostate cancer, high levels of PSCA expression have widely been correlated with high Gleason score, advanced tumor stage, seminal vesicle involvement, progression to androgen-independent disease, and bone metastasis [7–10]. Although the role of PSCA in intercellular signaling has been shown, little is known about the regulatory mechanism or biological functions of PSCA [11, 12]. It has been suggested that PSCA could act as both tumor suppressor and tumor promoting antigen based on tumor type, the microenvironment of the tumor, and the crosstalk between PSCA and other
References
[1]
R. E. Reiter, Z. Gu, T. Watabe et al., “Prostate stem cell antigen: a cell surface marker overexpressed in prostate cancer,” Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 4, pp. 1735–1740, 1998.
[2]
J. Dannull, P.-A. Diener, L. Prikler et al., “Prostate stem cell antigen is a promising candidate for immunotherapy of advanced prostate cancer,” Cancer Research, vol. 60, no. 19, pp. 5522–5528, 2000.
[3]
Z. Gu, G. Thomas, J. Yamashiro et al., “Prostate stem cell antigen (PSCA) expression increases with high gleason score, advanced stage and bone metastasis in prostate cancer,” Oncogene, vol. 19, no. 10, pp. 1288–1296, 2000.
[4]
N. Amara, G. S. Palapattu, M. Schrage et al., “Prostate stem cell antigen is overexpressed in human transitional cell carcinoma,” Cancer Research, vol. 61, no. 12, pp. 4660–4665, 2001.
[5]
P. Argani, C. Rosty, R. E. Reiter et al., “Discovery of new markers of cancer through serial analysis of gene expression: prostate stem cell antigen is overexpressed in pancreatic adenocarcinoma,” Cancer Research, vol. 61, no. 11, pp. 4320–4324, 2001.
[6]
E. G. Grubbs, Z. Abdel-Wahab, D. S. Tyler, and S. K. Pruitt, “Utilizing quantitative polymerase chain reaction to evaluate prostate stem cell antigen as a tumor marker in pancreatic cancer,” Annals of Surgical Oncology, vol. 13, no. 12, pp. 1645–1654, 2006.
[7]
K.-R. Han, D. B. Seligson, X. Liu et al., “Prostate stem cell antigen expression is associated with gleason score, seminal vesicle invasion and capsular invasion in prostate cancer,” Journal of Urology, vol. 171, no. 3, pp. 1117–1121, 2004.
[8]
Z. Zhigang and S. Wenlu, “External beam radiotherapy (EBRT) suppressed prostate stem cell antigen (PSCA) mRNA expression in clinically localized prostate cancer,” Prostate, vol. 67, no. 6, pp. 653–660, 2007.
[9]
Z. Zhigang and S. Wenlu, “Complete androgen ablation suppresses prostate stem cell antigen (PSCA) mRNA expression in human prostate carcinoma,” Prostate, vol. 65, no. 4, pp. 299–305, 2005.
[10]
J. Y. Joung, S. O. Yang, I. G. Jeong et al., “Reverse transcriptase-polymerase chain reaction and immunohistochemical studies for detection of prostate stem cell antigen expression in prostate cancer: potential value in molecular staging of prostate cancer,” International Journal of Urology, vol. 14, no. 7, pp. 635–643, 2007.
[11]
M. L. Moore, M. A. Teitell, Y. Kim et al., “Deletion of PSCA increases metastasis of TRAMP-induced prostate tumors without altering primary tumor formation,” Prostate, vol. 68, no. 2, pp. 139–151, 2008.
[12]
N. Saeki, J. Gu, T. Yoshida, and X. Wu, “Prostate stem cell antigen: a Jekyll and Hyde molecule?” Clinical Cancer Research, vol. 16, no. 14, pp. 3533–3538, 2010.
[13]
M. P. Deonarain, C. A. Kousparou, and A. A. Epenetos, “Antibodies targeting cancer stem cells: a new paradigm in immunotherapy?” MAbs, vol. 1, pp. 12–25, 2009.
[14]
M. N. Wente, A. Jain, E. Kono et al., “Prostate stem cell antigen is a putative target for immunotherapy in pancreatic cancer,” Pancreas, vol. 31, no. 2, pp. 119–125, 2005.
[15]
S. Ross, S. D. Spencer, I. Holcomb et al., “Prostate stem cell antigen as therapy target: tissue expression and in vivo efficacy of an immunoconjugate,” Cancer Research, vol. 62, no. 9, pp. 2546–2553, 2002.
[16]
P. Holliger and P. J. Hudson, “Engineered antibody fragments and the rise of single domains,” Nature Biotechnology, vol. 23, no. 9, pp. 1126–1136, 2005.
[17]
K. Chester, B. Pedley, B. Tolner et al., “Engineering antibodies for clinical applications in cancer,” Tumor Biology, vol. 25, no. 1-2, pp. 91–98, 2004.
[18]
P. A. Emanuel, J. Dang, J. S. Gebhardt et al., “Recombinant antibodies: a new reagent for biological agent detection,” Biosensors and Bioelectronics, vol. 14, no. 10-11, pp. 751–759, 2000.
[19]
S. K. Batra, M. Jain, U. A. Wittel, S. C. Chauhan, and D. Colcher, “Pharmacokinetics and biodistribution of genetically engineered antibodies,” Current Opinion in Biotechnology, vol. 13, no. 6, pp. 603–608, 2002.
[20]
N. E. Weisser and J. C. Hall, “Applications of single-chain variable fragment antibodies in therapeutics and diagnostics,” Biotechnology Advances, vol. 27, no. 4, pp. 502–520, 2009.
[21]
T. Yokota, D. E. Milenic, M. Whitlow, and J. Schlom, “Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin forms,” Cancer Research, vol. 52, no. 12, pp. 3402–3408, 1992.
[22]
J. S. Huston, J. McCartney, M. S. Tai et al., “Medical applications of single-chain antibodies,” International Reviews of Immunology, vol. 10, no. 2-3, pp. 195–217, 1993.
[23]
S. S. J. Leong and W. N. Chen, “Preparing recombinant single chain antibodies,” Chemical Engineering Science, vol. 63, no. 6, pp. 1401–1414, 2008.
[24]
C. E. Hagemeyer, C. von Zur Muhlen, D. von Elverfeldt, and K. Peter, “Single-chain antibodies as diagnostic tools and therapeutic agents,” Thrombosis and Haemostasis, vol. 101, no. 6, pp. 1012–1019, 2009.
[25]
F. Nejatollahi, S. J. Hodgetts, P. J. Vallely, and J. P. Burnie, “Neutralising human recombinant antibodies to human cytomegalovirus glycoproteins gB and gH,” FEMS Immunology and Medical Microbiology, vol. 34, no. 3, pp. 237–244, 2002.
[26]
F. Nejatollahi, Z. Malek-Hosseini, and D. Mehrabani, “Development of single chain antibodies to P185 tumor antigen,” Iranian Red Crescent Medical Journal, vol. 10, no. 4, pp. 298–302, 2008.
[27]
S. Ahmad, P. Sweeney, G. C. Sullivan, and M. Tangney, “DNA vaccination for prostate cancer from preclinical to clinical trials-where we stand?” Genetic Vaccines and Therapy, vol. 10, article 9, 2012.
[28]
D. Karan, S. Dubey, P. van Veldhuizen, J. M. Holzbeierlein, O. Tawfik, and J. B. Thrasher, “Dual antigen target-based immunotherapy for prostate cancer eliminates the growth of established tumors in mice,” Immunotherapy, vol. 3, no. 6, pp. 735–746, 2011.
[29]
T. Olafsen, Z. Gu, M. A. Sherman et al., “Targeting, imaging, and therapy using a humanized antiprostate stem cell antigen (PSCA) antibody,” Journal of Immunotherapy, vol. 30, no. 4, pp. 396–405, 2007.
[30]
J. V. Leyton, T. Olafsen, M. A. Sherman et al., “Engineered humanized diabodies for microPET imaging of prostate stem cell antigen-expressing tumors,” Protein Engineering, Design and Selection, vol. 22, no. 3, pp. 209–216, 2009.
[31]
J. V. Leyton, T. Olafsen, E. J. Lepin et al., “Humanized radioiodinated minibody for imaging of prostate stem cell antigen-expressing tumors,” Clinical Cancer Research, vol. 14, no. 22, pp. 7488–7496, 2008.
[32]
Z. Gu, J. Yamashiro, E. Kono, and R. E. Reiter, “Anti-prostate stem cell antigen monoclonal antibody 1G8 induces cell death in vitro and inhibits tumor growth in vivo via a Fc-independent mechanism,” Cancer Research, vol. 65, no. 20, pp. 9495–9500, 2005.
[33]
D. C. Saffran, A. B. Raitano, R. S. Hubert, O. N. Witte, R. E. Reiter, and A. Jakobovits, “Anti-PSCA mAbs inhibit tumor growth and metastasis formation and prolong the survival of mice bearing human prostate cancer xenografts,” Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 5, pp. 2658–2663, 2001.
[34]
R. Reiter and O. N. Witte, “Antibody against prostate stem cell antigen (PSCA),” Patent no: EP 1514876 A2, 2005.
[35]
R. Reiter, O. N. Witte, D. C. Saffran, and A. Jakobovits, “Anti-PSCA antibodies,” Patent no: US 6790939 B2, 2004.
[36]
S. Matsueda, A. Yao, Y. Ishihara et al., “A prostate stem cell antigen-derived peptide immunogenic in HLA-A24—prostate cancer patients,” Prostate, vol. 60, no. 3, pp. 205–213, 2004.
[37]
E. S. Antonarakis and M. A. Carducci, “Targeting angiogenesis for the treatment of prostate cancer,” Expert Opinion on Therapeutic Targets, vol. 16, no. 4, pp. 365–376, 2012.
[38]
J. Lian, X. Wu, F. He et al., “A natural BH3 mimetic induces autophagy in apoptosis-resistant prostate cancer via modulating Bcl-2-Beclin1 interaction at endoplasmic reticulum,” Cell Death and Differentiation, vol. 18, no. 1, pp. 60–71, 2011.
[39]
H. Albrecht, G. L. Denardo, and S. J. Denardo, “Development of anti-MUC1 di-scFvs for molecular targeting of epithelial cancers, such as breast and prostate cancers,” Quarterly Journal of Nuclear Medicine and Molecular Imaging, vol. 51, no. 4, pp. 304–313, 2007.
[40]
S. A. Parker, I. L. Diaz, K. A. Anderson, and C. A. Batt, “Design, production, and characterization of a single-chain variable fragment (ScFv) derived from the prostate specific membrane antigen (PSMA) monoclonal antibody J591,” Protein Expression and Purification, vol. 89, no. 2, pp. 136–145, 2013.
[41]
A. Morgenroth, M. Cartellieri, M. Schmitz et al., “Targeting of tumor cells expressing the prostate stem cell antigen (PSCA) using genetically engineered T-cells,” Prostate, vol. 67, no. 10, pp. 1121–1131, 2007.
