Abstract
Purpose
Despite great progress in the diagnosis and treatment of localized prostate cancer (PCa), there remains a need for new diagnostic markers that can accurately distinguish indolent and aggressive variants. One promising approach is the antibody-based targeting of prostate stem cell antigen (PSCA), which is frequently overexpressed in PCa. Here, we show the construction of a molecular imaging probe comprising a humanized scFv fragment recognizing PSCA genetically fused to an engineered version of the human DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT), the SNAP-tag, enabling specific covalent coupling to various fluorophores for diagnosis of PCa. Furthermore, the recombinant immunotoxin (IT) PSCA(scFv)-ETA′ comprising the PSCA(scFv) and a truncated version of Pseudomonas exotoxin A (PE, ETA′) was generated.
Methods
We analyzed the specific binding and internalization behavior of the molecular imaging probe PSCA(scFv)-SNAP in vitro by flow cytometry and live cell imaging, compared to the corresponding IT PSCA(scFv)-ETA′. The cytotoxic activity of PSCA(scFv)-ETA′ was tested using cell viability assays. Specific binding was confirmed on formalin-fixed paraffin-embedded tissue specimen of early and advanced PCa.
Results
Alexa Fluor® 647 labeling of PSCA(scFv)-SNAP confirmed selective binding to PSCA, leading to rapid internalization into the target cells. The recombinant IT PSCA(scFv)-ETA′ showed selective binding leading to internalization and efficient elimination of target cells.
Conclusions
Our data demonstrate, for the first time, the specific binding, internalization, and cytotoxicity of a scFv-based fusion protein targeting PSCA. Immunohistochemical staining confirmed the specific ex vivo binding to primary PCa material.
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References
Afshar-Oromieh A et al (2013) PET imaging with a [68Ga]gallium-labelled PSMA ligand for the diagnosis of prostate cancer: biodistribution in humans and first evaluation of tumour lesions. Eur J Nucl Med Mol Imaging 40:486–495. doi:10.1007/s00259-012-2298-2
Afshar-Oromieh A et al (2015) The diagnostic value of PET/CT imaging with the (68)Ga-labelled PSMA ligand HBED-CC in the diagnosis of recurrent prostate cancer. Eur J Nucl Med Mol Imaging 42:197–209. doi:10.1007/s00259-014-2949-6
Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NB, Hamid M (2012) scFv antibody: principles and clinical application. Clin Dev Immunol 2012:980250. doi:10.1155/2012/980250
Akhtar NH, Pail O, Saran A, Tyrell L, Tagawa ST (2012) Prostate-specific membrane antigen-based therapeutics. Adv Urol 2012:973820. doi:10.1155/2012/973820
Amoury M et al (2013) SNAP-tag based agents for preclinical in vitro imaging in malignant diseases. Curr Pharm Des 19:5429–5436
Antignani A, Fitzgerald D (2013) Immunotoxins: the role of the toxin. Toxins (Basel) 5:1486–1502. doi:10.3390/toxins5081486
Bang S, Nagata S, Onda M, Kreitman RJ, Pastan I (2005) HA22 (R490A) is a recombinant immunotoxin with increased antitumor activity without an increase in animal toxicity. Clin Cancer Res 11:1545–1550. doi:10.1158/1078-0432.CCR-04-1939
Barth S (2002) Technology evaluation: BL22. NCI Curr Opin Mol Ther 4:72–75
Barth S, Huhn M, Wels W, Diehl V, Engert A (1998) Construction and in vitro evaluation of RFT5(scFv)-ETA′, a new recombinant single-chain immunotoxin with specific cytotoxicity toward CD25+ Hodgkin-derived cell lines. Int J Mol Med 1:249–256
Barth S et al (2000a) Recombinant anti-CD25 immunotoxin RFT5(SCFV)-ETA′ demonstrates successful elimination of disseminated human Hodgkin lymphoma in SCID mice. Int J Cancer 86:718–724
Barth S et al (2000b) Ki-4(scFv)-ETA′, a new recombinant anti-CD30 immunotoxin with highly specific cytotoxic activity against disseminated Hodgkin tumors in SCID mice. Blood 95:3909–3914
Becker N, Benhar I (2012) Antibody-based immunotoxins for the treatment of cancer. Antibodies 1:39–69
Bokhorst LP et al (2016) Correlation between stage shift and differences in mortality in the European Randomized study of Screening for Prostate Cancer (ERSPC). BJU Int. doi:10.1111/bju.13505
Bruell D et al (2003) The recombinant anti-EGF receptor immunotoxin 425(scFv)-ETA′ suppresses growth of a highly metastatic pancreatic carcinoma cell line. Int J Oncol 23:1179–1186
Cao Y, Mohamedali KA, Marks JW, Cheung LH, Hittelman WN, Rosenblum MG (2013) Construction and characterization of novel, completely human serine protease therapeutics targeting Her2/neu. Mol Cancer Ther 12:979–991. doi:10.1158/1535-7163.MCT-13-0002
Carey LA et al (2012) TBCRC 001: randomized phase II study of cetuximab in combination with carboplatin in stage IV triple-negative breast cancer. J Clin Oncol 30:2615–2623. doi:10.1200/JCO.2010.34.5579
Cathomas R et al (2012) Efficacy of cetuximab in metastatic castration-resistant prostate cancer might depend on EGFR and PTEN expression: results from a phase II trial (SAKK 08/07). Clin Cancer Res 18:6049–6057. doi:10.1158/1078-0432.CCR-12-2219
Chen L, Qiu X, Wang R, Xie X (2014) The efficacy and safety of docetaxel plus thalidomide vs docetaxel alone in patients with androgen-independent prostate cancer: a systematic review. Sci Rep 4:4818. doi:10.1038/srep04818
Cizeau J, Grenkow DM, Brown JG, Entwistle J, MacDonald GC (2009) Engineering and biological characterization of VB6-845, an anti-EpCAM immunotoxin containing a T-cell epitope-depleted variant of the plant toxin bouganin. J Immunother 32:574–584. doi:10.1097/CJI.0b013e3181a6981c
Colcher D, Pavlinkova G, Beresford G, Booth BJ, Choudhury A, Batra SK (1998) Pharmacokinetics and biodistribution of genetically-engineered antibodies. Q J Nucl Med 42:225–241
Cremer C et al (2015) Novel angiogenin mutants with increased cytotoxicity enhance the depletion of pro-inflammatory macrophages and leukemia cells ex vivo. Cancer Immunol Immunother 64:1575–1586. doi:10.1007/s00262-015-1763-8
Cunha GR, Hayward SW, Wang YZ, Ricke WA (2003) Role of the stromal microenvironment in carcinogenesis of the prostate. Int J Cancer 107:1–10. doi:10.1002/ijc.11335
DeVita VT Jr, Chu E (2008) A history of cancer chemotherapy. Cancer Res 68:8643–8653. doi:10.1158/0008-5472.CAN-07-6611
Elsasser-Beile U, Wolf P, Gierschner D, Buhler P, Schultze-Seemann W, Wetterauer U (2006) A new generation of monoclonal and recombinant antibodies against cell-adherent prostate specific membrane antigen for diagnostic and therapeutic targeting of prostate cancer. Prostate 66:1359–1370. doi:10.1002/pros.20367
Fang YQ, Wu JY, Li TC, Liu W, Gao L, Luo Y (2015) Nanoparticle mediated chemotherapy of hormone refractory prostate cancer with a novel combi-molecule. Am J Transl Res 7:1440–1449
Farwell WR, Linder JA, Jha AK (2007) Trends in prostate-specific antigen testing from 1995 through 2004. Arch Int Med 167:2497–2502
Feld J, Barta SK, Schinke C, Braunschweig I, Zhou Y, Verma AK (2013) Linked-in: design and efficacy of antibody drug conjugates in oncology. Oncotarget 4:397–412. doi:10.18632/oncotarget.924
Gioux S, Choi HS, Frangioni JV (2010) Image-guided surgery using invisible near-infrared light: fundamentals of clinical translation. Mol Imaging 9:237–255
Gu Z et al (2000) Prostate stem cell antigen (PSCA) expression increases with high gleason score, advanced stage and bone metastasis in prostate cancer. Oncogene 19:1288–1296. doi:10.1038/sj.onc.1203426
Han KR et al (2004) Prostate stem cell antigen expression is associated with gleason score, seminal vesicle invasion and capsular invasion in prostate cancer. J Urol 171:1117–1121. doi:10.1097/01.ju.0000109982.60619.93
Hassan R et al (2014) Phase 1 study of the antimesothelin immunotoxin SS1P in combination with pemetrexed and cisplatin for front-line therapy of pleural mesothelioma and correlation of tumor response with serum mesothelin, megakaryocyte potentiating factor, and cancer antigen 125. Cancer 120:3311–3319. doi:10.1002/cncr.28875
Hehmann-Titt G, Schiffer S, Berges N, Melmer G, Barth S (2013) Improving the therapeutic potential of human Granzyme B for targeted cancer therapy. Antibodies (Basel) 2:19–49
Heidenreich A et al (2014a) EAU guidelines on prostate cancer. part 1: screening, diagnosis, and local treatment with curative intent-update 2013. Eur Urol 65:124–137. doi:10.1016/j.eururo.2013.09.046
Heidenreich A et al (2014b) EAU guidelines on prostate cancer. Part II: Treatment of advanced, relapsing, and castration-resistant prostate cancer. Eur Urol 65:467–479. doi:10.1016/j.eururo.2013.11.002
Hillerdal V, Ramachandran M, Leja J, Essand M (2014) Systemic treatment with CAR-engineered T cells against PSCA delays subcutaneous tumor growth and prolongs survival of mice. BMC Cancer 14:30. doi:10.1186/1471-2407-14-30
Hristodorov D, Nordlohne J, Mladenov R, Huhn M, Fischer R, Thepen T, Barth S (2014) Human microtubule-associated protein tau mediates targeted killing of CD30(+) lymphoma cells in vitro and inhibits tumour growth in vivo. Br J Haematol 164:251–257. doi:10.1111/bjh.12626
Hussain AF, Kampmeier F, von Felbert V, Merk HF, Tur MK, Barth S (2011) SNAP-tag technology mediates site specific conjugation of antibody fragments with a photosensitizer and improves target specific phototoxicity in tumor cells. Bioconjug Chem 22:2487–2495. doi:10.1021/bc200304k
Hussain AF, Amoury M, Barth S (2013) SNAP-tag technology: a powerful tool for site specific conjugation of therapeutic and imaging agents. Curr Pharm Des 19:5437–5442
Jorissen H et al (2009) Production and characterisation of monoclonal antibodies against RAI3 and its expression in human breast cancer. BMC Cancer 9:200. doi:10.1186/1471-2407-9-200
Kampmeier F, Ribbert M, Nachreiner T, Dembski S, Beaufils F, Brecht A, Barth S (2009) Site-specific, covalent labeling of recombinant antibody fragments via fusion to an engineered version of 6-O-alkylguanine DNA alkyltransferase. Bioconjug Chem 20:1010–1015. doi:10.1021/bc9000257
Kampmeier F et al (2010) Rapid optical imaging of EGF receptor expression with a single-chain antibody SNAP-tag fusion protein. Eur J Nucl Med Mol Imaging 37:1926–1934. doi:10.1007/s00259-010-1482-5
Kaplan G, Lee F, Onda M, Kolyvas E, Bhardwaj G, Baker D, Pastan I (2016) Protection of the furin cleavage site in low-toxicity immunotoxins based on pseudomonas exotoxin A. Toxins (Basel). doi:10.3390/toxins8080217
Knowles SM et al (2014) Quantitative immunoPET of prostate cancer xenografts with 89Zr- and 124I-labeled anti-PSCA A11 minibody. J Nucl Med 55:452–459. doi:10.2967/jnumed.113.120873
Kolberg K, Puettmann C, Pardo A, Fitting J, Barth S (2013) SNAP-tag technology: a general introduction. Curr Pharm Des 19:5406–5413
Kreitman RJ et al (2012) Phase I trial of anti-CD22 recombinant immunotoxin moxetumomab pasudotox (CAT-8015 or HA22) in patients with hairy cell leukemia. J Clin Oncol 30:1822–1828. doi:10.1200/JCO.2011.38.1756
Kuroda K, Liu H, Kim S, Guo M, Navarro V, Bander NH (2010) Saporin toxin-conjugated monoclonal antibody targeting prostate-specific membrane antigen has potent anticancer activity. Prostate 70:1286–1294. doi:10.1002/pros.21164
Lam JS et al (2005) Prostate stem cell antigen is overexpressed in prostate cancer metastases. Clin Cancer Res 11:2591–2596. doi:10.1158/1078-0432.CCR-04-1842
Lepin EJ et al (2010) An affinity matured minibody for PET imaging of prostate stem cell antigen (PSCA)-expressing tumors. Eur J Nucl Med Mol Imaging 37:1529–1538. doi:10.1007/s00259-010-1433-1
Leyton JV, Olafsen T, Lepin EJ, Hahm S, Bauer KB, Reiter RE, Wu AM (2008) Humanized radioiodinated minibody for imaging of prostate stem cell antigen-expressing tumors. Clin Cancer Res 14:7488–7496. doi:10.1158/1078-0432.CCR-07-5093
Long RM, Morrissey C, Fitzpatrick JM, Watson RW (2005) Prostate epithelial cell differentiation and its relevance to the understanding of prostate cancer therapies. Clin Sci (Lond) 108:1–11. doi:10.1042/CS20040241
Madu CO, Lu Y (2010) Novel diagnostic biomarkers for prostate cancer J. Cancer 1:150–177
Matthey B, Engert A, Klimka A, Diehl V, Barth S (1999) A new series of pET-derived vectors for high efficiency expression of Pseudomonas exotoxin-based fusion proteins. Gene 229:145–153
Michalk I et al (2014) Characterization of a novel single-chain bispecific antibody for retargeting of T cells to tumor cells via the TCR co-receptor CD8. PLoS One 9:e95517. doi:10.1371/journal.pone.0095517
Morgenroth A et al (2007) Targeting of tumor cells expressing the prostate stem cell antigen (PSCA) using genetically engineered T-cells. Prostate 67:1121–1131. doi:10.1002/pros.20608
Niesen J et al (2015a) In vitro effects and ex vivo binding of an EGFR-specific immunotoxin on rhabdomyosarcoma cells. J Cancer Res Clin Oncol 141:1049–1061. doi:10.1007/s00432-014-1884-z
Niesen J et al (2015b) Novel EGFR-specific immunotoxins based on panitumumab and cetuximab show in vitro and ex vivo activity against different tumor entities. J Cancer Res Clin Oncol 141:2079–2095. doi:10.1007/s00432-015-1975-5
Olafsen T et al (2007) Targeting, imaging, and therapy using a humanized antiprostate stem cell antigen (PSCA) antibody. J Immunother 30:396–405. doi:10.1097/CJI.0b013e318031b53b
Panowksi S, Bhakta S, Raab H, Polakis P, Junutula JR (2014) Site-specific antibody drug conjugates for cancer therapy MAbs 6:34–45. doi:10.4161/mabs.27022
Pastan I, Hassan R, FitzGerald DJ, Kreitman RJ (2007) Immunotoxin treatment of cancer. Annu Rev Med 58:221–237. doi:10.1146/annurev.med.58.070605.115320
Pavlinkova G, Beresford GW, Booth BJ, Batra SK, Colcher D (1999) Pharmacokinetics and biodistribution of engineered single-chain antibody constructs of MAb CC49 in colon carcinoma xenografts. J Nucl Med 40:1536–1546
Pieczonka CM, Telonis D, Mouraviev V, Albala D (2015) Sipuleucel-T for the treatment of patients with metastatic castrate-resistant prostate cancer: considerations for clinical practice. Rev Urol 17:203–210
Reichert JM (2014) Antibodies to watch in 2014: mid-year update. MAbs 6:799–802. doi:10.4161/mabs.29282
Reiter RE et al (1998) Prostate stem cell antigen: a cell surface marker overexpressed in prostate cancer. Proc Natl Acad Sci USA 95:1735–1740
Robinson MK et al (2005) Quantitative immuno-positron emission tomography imaging of HER2-positive tumor xenografts with an iodine-124 labeled anti-HER2 diabody. Cancer Res 65:1471–1478. doi:10.1158/0008-5472.CAN-04-2008
Ross S et al (2002) Prostate stem cell antigen as therapy target: tissue expression and in vivo efficacy of an immunoconjugate. Cancer Res 62:2546–2553
Saeki N, Gu J, Yoshida T, Wu X (2010) Prostate stem cell antigen: a Jekyll and Hyde molecule? Clin Cancer Res 16:3533–3538. doi:10.1158/1078-0432.CCR-09-3169
Schrama D, Reisfeld RA, Becker JC (2006) Antibody targeted drugs as cancer therapeutics nature reviews drug discovery. Nat Rev Drug Discov 5:147–159. doi:10.1038/nrd1957
Schwemmlein M et al (2006) A CD33-specific single-chain immunotoxin mediates potent apoptosis of cultured human myeloid leukaemia cells. Br J Haematol 133:141–151. doi:10.1111/j.1365-2141.2005.05869.x
Scott AM, Allison JP, Wolchok JD (2012) Monoclonal antibodies in cancer therapy. Cancer immunity 12:14
Sheikh NA et al (2013) Sipuleucel-T immune parameters correlate with survival: an analysis of the randomized phase 3 clinical trials in men with castration-resistant prostate cancer. Cancer Immunol Immunother 62:137–147. doi:10.1007/s00262-012-1317-2
Sheng J et al (2015) The efficacy of combining EGFR monoclonal antibody with chemotherapy for patients with advanced nonsmall cell lung cancer: a meta-analysis from 9 randomized controlled trials. Medicine (Baltimore) 94:e1400. doi:10.1097/MD.0000000000001400
Singh R, Samant U, Hyland S, Chaudhari PR, Wels WS, Bandyopadhyay D (2007) Target-specific cytotoxic activity of recombinant immunotoxin scFv(MUC1)-ETA on breast carcinoma cells and primary breast tumors. Mol Cancer Ther 6:562–569. doi:10.1158/1535-7163.MCT-06-0604
Singh CK, Ojha A, Bhatanagar RK, Kachru DN (2008) Detection and characterization of recombinant DNA expressing vip3A-type insecticidal gene in GMOs-standard single, multiplex and construct-specific PCR assays. Anal Bioanal Chem 390:377–387. doi:10.1007/s00216-007-1714-0
Small AC, Oh WK (2012) Bevacizumab treatment of prostate cancer. Expert Opin Biol Ther 12:1241–1249. doi:10.1517/14712598.2012.704015
Sonn GA et al (2016) Fluorescent image-guided surgery with an anti-prostate stem cell antigen (PSCA) diabody enables targeted resection of mouse prostate cancer xenografts in real time. Clin Cancer Res 22:1403–1412. doi:10.1158/1078-0432.CCR-15-0503
Stahnke B et al (2008) Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes. Mol Cancer Ther 7:2924–2932. doi:10.1158/1535-7163.MCT-08-0554
Stocker M, Tur MK, Sasse S, Krussmann A, Barth S, Engert A (2003) Secretion of functional anti-CD30-angiogenin immunotoxins into the supernatant of transfected 293T-cells. Protein Expr Purif 28:211–219
Sundaresan G et al (2003) 124I-labeled engineered anti-CEA minibodies and diabodies allow high-contrast, antigen-specific small-animal PET imaging of xenografts in athymic mice. J Nucl Med 44:1962–1969
Tagawa ST et al (2013) Phase II study of Lutetium-177-labeled anti-prostate-specific membrane antigen monoclonal antibody J591 for metastatic castration-resistant prostate cancer. Clin Cancer Res 19:5182–5191. doi:10.1158/1078-0432.CCR-13-0231
Tannock IF et al (2004) Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer. N Engl J Med 351:1502–1512. doi:10.1056/NEJMoa040720
Taylor RM, Severns V, Brown DC, Bisoffi M, Sillerud LO (2012) Prostate cancer targeting motifs: expression of alphanu beta3, neurotensin receptor 1, prostate specific membrane antigen, and prostate stem cell antigen in human prostate cancer cell lines and xenografts. Prostate 72:523–532. doi:10.1002/pros.21454
Tse BW, Jovanovic L, Nelson CC, de Souza P, Power CA, Russell PJ (2014) From bench to bedside: immunotherapy for prostate cancer. Biomed Res Int 2014:981434. doi:10.1155/2014/981434
Tur MK et al (2003) Recombinant CD64-specific single chain immunotoxin exhibits specific cytotoxicity against acute myeloid leukemia cells. Cancer Res 63:8414–8419
Vacchelli E et al (2015) Trial watch: Tumor-targeting monoclonal antibodies for oncological indications. Oncoimmunology 4:e985940. doi:10.4161/2162402X.2014.985940
Valerio M et al (2014) The role of focal therapy in the management of localised prostate cancer: a systematic review. Eur Urol 66:732–751. doi:10.1016/j.eururo.2013.05.048
Vallabhajosula S et al (2016) Radioimmunotherapy of metastatic prostate cancer with (1)(7)(7)Lu-DOTAhuJ591 anti prostate specific membrane antigen specific monoclonal antibody. Curr Radiopharm 9:44–53
von Felbert V et al (2016) A specific photoimmunotheranostics agent to detect and eliminate skin cancer cells expressing EGFR. J Cancer Res Clin Oncol 142:1003–1011. doi:10.1007/s00432-016-2122-7
Weidle UH, Georges G, Brinkmann U (2012) Fully human targeted cytotoxic fusion proteins: new anticancer agents on the horizon. Cancer Genom Proteom 9:119–133
Weidle UH, Tiefenthaler G, Schiller C, Weiss EH, Georges G, Brinkmann U (2014) Prospects of bacterial and plant protein-based immunotoxins for treatment of cancer. Cancer Genom Proteom 11:25–38
Wente MN et al (2005) Prostate stem cell antigen is a putative target for immunotherapy in pancreatic cancer. Pancreas 31:119–125
Woitok M et al (2016) The efficient elimination of solid tumor cells by EGFR-specific and HER2-specific scFv-SNAP fusion proteins conjugated to benzylguanine-modified auristatin F. Cancer Lett 381:323–330. doi:10.1016/j.canlet.2016.08.003
Wolf P et al (2010) Preclinical evaluation of a recombinant anti-prostate specific membrane antigen single-chain immunotoxin against prostate cancer. J Immunother 33:262–271. doi:10.1097/CJI.0b013e3181c5495c
Wu AM, Reiter RE, Lepin EJ, Marks JD, Zhou Y (2010) High affinity anti-prostate stem cell antigen (PSCA) antibodies for cancer targeting and detection. Google Patents
Wu H et al (2014) Prostate stem cell antigen antibody-conjugated multiwalled carbon nanotubes for targeted ultrasound imaging and drug delivery. Biomaterials 35:5369–5380. doi:10.1016/j.biomaterials.2014.03.038
Yokota T, Milenic DE, Whitlow M, Schlom J (1992) Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin forms. Cancer Res 52:3402–3408
Zhigang Z, Wenlu S (2007) Prostate stem cell antigen (PSCA) mRNA expression in prostatic intraepithelial neoplasia: implications for the development of prostate cancer. Prostate 67:1143–1151. doi:10.1002/pros.20610
Ziada A et al (2004) The use of trastuzumab in the treatment of hormone refractory prostate cancer; phase II trial. Prostate 60:332–337. doi:10.1002/pros.20065
Acknowledgements
This work was funded by the ForSaTum project, sponsored within the NRW-EU Ziel 2-Programm “Regionale Wettbewerbsfähigkeit und Beschäftigung 2007–2013” (ERFE). We would like to thank Radoslav Mladenov and Nina Berges (Department of Experimental Medicine and Immunotherapy, Institute of Applied Medical Engineering, RWTH Aachen University Clinic, Aachen, Germany) for their help with immunohistochemistry and confocal microscopy. We also thank Dr. Richard M. Twyman for support in preparation of the manuscript draft.
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In accordance with the Helsinki Declaration of 1975, primary tissue samples were obtained during routine clinical practice at the University Hospital Giessen approved by the appropriate ethics committee.
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Kessler, C., Pardo, A., Tur, M.K. et al. Novel PSCA targeting scFv-fusion proteins for diagnosis and immunotherapy of prostate cancer. J Cancer Res Clin Oncol 143, 2025–2038 (2017). https://doi.org/10.1007/s00432-017-2472-9
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DOI: https://doi.org/10.1007/s00432-017-2472-9