BACKGROUND: Prostate-specific membrane antigen (PSMA) is a validated target for the imaging and therapy of prostate cancer. Here, we report the detailed characterization of four novel murine monoclonal antibodies (mAbs) recognizing human PSMA as well as PSMA orthologs from different species. METHODS: Performance of purified mAbs was assayed using a comprehensive panel of in vitro experimental setups including Western blotting, immunofluorescence, immunohistochemistry, ELISA, flow cytometry, and surface-plasmon resonance. Furthermore, a mouse xenograft model of prostate cancer was used to compare the suitability of the mAbs for in vivo applications. RESULTS: All mAbs demonstrate high specificity for PSMA as documented by the lack of cross-reactivity to unrelated human proteins. The 3F11 and 1A11 mAbs bind linear epitopes spanning residues 226-243 and 271-288 of human PSMA, respectively. 3F11 is also suitable for the detection of PSMA orthologs from mouse, pig, dog, and rat in experimental setups where the denatured form of PSMA is used. 5D3 and 5B1 mAbs recognize distinct surface-exposed conformational epitopes and are useful for targeting PSMA in its native conformation. Most importantly, using a mouse xenograft model of prostate cancer we show that both the intact 5D3 and its Fab fragment are suitable for in vivo imaging. CONCLUSIONS: With apparent affinities of 0.14 and 1.2 nM as determined by ELISA and flow cytometry, respectively, 5D3 has approximately 10-fold higher affinity for PSMA than the clinically validated mAb J591 and, therefore, is a prime candidate for the development of next-generation theranostics to target PSMA. Prostate 77:749-764, 2017. © 2017 Wiley Periodicals, Inc.

Laboratory of Structural Biology Institute of Biotechnology Czech Academy of Sciences Vestec Czech Republic

Munich Center for Integrated Protein Science and Lehrstuhl für Biologische Chemie Technische Universität München Freising Weihenstephan Germany

The Russell H Morgan Department of Radiology and Radiological Science Johns Hopkins Medical Institutions Baltimore Maryland

Zobrazit více v PubMed

Siegel RL, Miller KD, Jemal A. Cancer statistics. CA Cancer J Clin 2015;65(1):5–29. PubMed

Huber F, Montani M, Sulser T, Jaggi R, Wild P, Moch H, Gevensleben H, Schmid M, Wyder S, Kristiansen G. Comprehensive validation of published immunohistochemical prognostic biomarkers of prostate cancer—What has gone wrong? A blueprint for the way forward in biomarker studies. Br J Cancer 2015;112(1):140–148. PubMed PMC

Bostwick DG, Pacelli A, Blute M, Roche P, Murphy GP. Prostate specific membrane antigen expression in prostatic intraepithelial neoplasia and adenocarcinoma: A study of 184 cases. Cancer 1998;82(11):2256–2261. PubMed

Chang SS, O’Keefe DS, Bacich DJ, Reuter VE, Heston WD, Gaudin PB. Prostate-specific membrane antigen is produced in tumor-associated neovasculature. Clin Cancer Res 1999;5(10): 2674–2681. PubMed

Wernicke AG, Kim S, Liu H, Bander NH, Pirog EC. Prostate-specific membrane antigen (PSMA) expression in the neovasculature of gynecologic malignancies: Implications for PSMA-targeted therapy. Appl Immunohistochem Mol Morphol 2016, doi: 10.1097/PAI.0000000000000297 PubMed DOI

Gordon IO, Tretiakova MS, Noffsinger AE, Hart J, Reuter VE, Al-Ahmadie HA. Prostate-specific membrane antigen expression in regeneration and repair. Mod Pathol 2008;21(12): 1421–1427. PubMed

Barinka C, Rojas C, Slusher B, Pomper M. Glutamate carboxypeptidase II in diagnosis and treatment of neurologic disorders and prostate cancer. Curr Med Chem 2012;19(6):856–870. PubMed PMC

Foss CA, Mease RC, Cho SY, Kim HJ, Pomper MG. GCPII imaging and cancer. Curr Med Chem 2012;19(9):1346–1359. PubMed PMC

Kiess AP, Banerjee SR, Mease RC, Rowe SP, Rao A, Foss CA, Chen Y, Yang X, Cho SY, Nimmagadda S, Pomper MG. Prostate-specific membrane antigen as a target for cancer imaging and therapy. Q J Nucl Med Mol Imaging 2015;59(3): 241–268. PubMed PMC

Sacha P, Knedlik T, Schimer J, Tykvart J, Parolek J, Navratil V, Dvorakova P, Sedlak F, Ulbrich K, Strohalm J, Majer P, Subr V, Konvalinka J. iBodies: Modular synthetic antibody mimetics based on hydrophilic polymers decorated with functional moieties. Angew Chem Int Ed Engl 2016;55(7):2356–2360. PubMed PMC

Haberkorn U, Eder M, Kopka K, Babich JW, Eisenhut M. New strategies in prostate cancer: Prostate-specific membrane antigen (PSMA) ligands for diagnosis and therapy. Clin Cancer Res 2016;22(1):9–15. PubMed

Kratochwil C, Giesel FL, Stefanova M, Benesova M, Bronzel M, Afshar-Oromieh A, Mier W, Eder M, Kopka K, Haberkorn U. PSMA-targeted radionuclide therapy of metastatic castration-resistant prostate cancer with 177Lu-labeled PSMA-617. J Nucl Med 2016;57(8):1170–1176. PubMed

Rowe SP, Macura KJ, Mena E, Blackford AL, Nadal R, Antonarakis ES, Eisenberger M, Carducci M, Fan H, Dannals RF, Chen Y, Mease RC, Szabo Z, Pomper MG, Cho SY. PSMA-based [(18)F]DCFPyL PET/CT is superior to conventional imaging for lesion detection in patients with metastatic prostate cancer. Mol Imaging Biol 2016;18(3):411–419. PubMed PMC

Hlouchova K, Barinka C, Konvalinka J, Lubkowski J. Structural insight into the evolutionary and pharmacologic homology of glutamate carboxypeptidases II and III. FEBS J 2009;276(16): 4448–4462. PubMed

Hohberg M, Eschner W, Schmidt M, Dietlein M, Kobe C, Fischer T, Drzezga A, Wild M. Lacrimal glands may represent organs at risk for radionuclide therapy of prostate cancer with [(177)Lu] DKFZ-PSMA-617. Mol Imaging Biol 2016;18(3):437–445. PubMed

Barinka C, Ptacek J, Richter A, Novakova Z, Morath V, Skerra A. Selection and characterization of Anticalins targeting human prostate-specific membrane antigen (PSMA). Protein Eng Des Sel 2016;29(3):105–115. PubMed

Dassie JP, Hernandez LI, Thomas GS, Long ME, Rockey WM, Howell CA, Chen Y, Hernandez FJ, Liu XY, Wilson ME, Allen LA, Vaena DA, Meyerholz DK, Giangrande PH. Targeted inhibition of prostate cancer metastases with an RNA aptamer to prostate-specific membrane antigen. Mol Ther 2014;22(11): 1910–1922. PubMed PMC

Zhu C, Bandekar A, Sempkowski M, Banerjee SR, Pomper MG, Bruchertseifer F, Morgenstern A, Sofou S. Nanoconjugation of PSMA-targeting ligands enhances perinuclear localization and improves efficacy of delivered alpha-particle emitters against tumor endothelial analogues. Mol Cancer Ther 2016; 15(1):106–113. PubMed PMC

Wiehr S, Buhler P, Gierschner D, Wolf P, Rolle AM, Kesenheimer C, Pichler BJ, Elsasser-Beile U. Pharmacokinetics and PET imaging properties of two recombinant anti-PSMA antibody fragments in comparison to their parental antibody. Prostate 2014;74(7):743–755. PubMed

Ellis RJ, Kaminsky DA, Zhou EH, Fu P, Chen WD, Brelin A, Faulhaber PF, Bodner D. Ten-year outcomes: The clinical utility of single photon emission computed tomography/computed tomography capromab pendetide (Prostascint) in a cohort diagnosed with localized prostate cancer. Int J Radiat Oncol Biol Phys 2011;81(1):29–34. PubMed

Liu H, Moy P, Kim S, Xia Y, Rajasekaran A, Navarro V, Knudsen B, Bander NH. Monoclonal antibodies to the extracellular domain of prostate-specific membrane antigen also react with tumor vascular endothelium. Cancer Res 1997;57(17): 3629–3634. PubMed

Holland JP, Divilov V, Bander NH, Smith-Jones PM, Larson SM, Lewis JS. 89Zr-DFO-J591 for immunoPET of prostate-specific membrane antigen expression in vivo. J Nucl Med 2010;51(8): 1293–1300. PubMed PMC

Kampmeier F, Williams JD, Maher J, Mullen GE, Blower PJ. Design and preclinical evaluation of a 99mTc-labelled diabody of mAb J591 for SPECT imaging of prostate-specific membrane antigen (PSMA). EJNMMI Res 2014;4(1):13. PubMed PMC

Tagawa ST, Akhtar NH, Nikolopoulou A, Kaur G, Robinson B, Kahn R, Vallabhajosula S, Goldsmith SJ, Nanus DM, Bander NH. Bone marrow recovery and subsequent chemotherapy following radiolabeled anti-prostate-specific membrane antigen monoclonal antibody j591 in men with metastatic castration-resistant prostate cancer. Front Oncol 2013;3:214. PubMed PMC

Tagawa ST, Milowsky MI, Morris M, Vallabhajosula S, Christos P, Akhtar NH, Osborne J, Goldsmith SJ, Larson S, Taskar NP, Scher HI, Bander NH, Nanus DM. Phase II study of Lutetium-177-labeled anti-prostate-specific membrane antigen monoclonal antibody J591 for metastatic castration-resistant prostate cancer. Clin Cancer Res 2013;19(18):5182–5191. PubMed PMC

Barinka C, Mlcochova P, Sacha P, Hilgert I, Majer P, Slusher BS, Horejsi V, Konvalinka J. Amino acids at the N- and C-termini of human glutamate carboxypeptidase II are required for enzymatic activity and proper folding. Eur J Biochem 2004;271(13): 2782–2790. PubMed

Tykvart J, Sacha P, Barinka C, Knedlik T, Starkova J, Lubkowski J, Konvalinka J. Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II. Protein Expr Purif 2012;82(1):106–115. PubMed PMC

Rovenska M, Hlouchova K, Sacha P, Mlcochova P, Horak V, Zamecnik J, Barinka C, Konvalinka J. Tissue expression and enzymologic characterization of human prostate specific membrane antigen and its rat and pig orthologs. Prostate 2008;68(2): 171–182. PubMed

Tykvart J, Navratil V, Sedlak F, Corey E, Colombatti M, Fracasso G, Koukolik F, Barinka C, Sacha P, Konvalinka J. Comparative analysis of monoclonal antibodies against prostate-specific membrane antigen (PSMA). Prostate 2014;74(16):1674–1690. PubMed

Peknicova J, Capkova J, Cechova D, Sulcova B. Preparation and characterization of a monoclonal antibody against boar acrosin. Folia Biol (Praha) 1986;32(4):282–285. PubMed

Smith-Jones PM, Vallabahajosula S, Goldsmith SJ, Navarro V, Hunter CJ, Bastidas D, Bander NH. In vitro characterization of radiolabeled monoclonal antibodies specific for the extracellular domain of prostate-specific membrane antigen. Cancer Res 2000;60(18):5237–5243. PubMed

Myszka DG. Improving biosensor analysis. J Mol Recognit 1999;12(5):279–284. PubMed

Banerjee SR, Ngen EJ, Rotz MW, Kakkad S, Lisok A, Pracitto R, Pullambhatla M, Chen Z, Shah T, Artemov D, Meade TJ, Bhujwalla ZM, Pomper MG. Synthesis and evaluation of Gd(III)—Based magnetic resonance contrast agents for molecular imaging of prostate-specific membrane antigen. Angew Chem Int Ed Engl 2015;54(37):10778–10782. PubMed PMC

Yang X, Mease RC, Pullambhatla M, Lisok A, Chen Y, Foss CA, Wang Y, Shallal H, Edelman H, Hoye AT, Attardo G, Nimmagadda S, Pomper MG. [(18)F]Fluorobenzoyllysinepentanedioic acid carbamates: New scaffolds for positron emission tomography (PET) imaging of prostate-specific membrane antigen (PSMA). J Med Chem 2016;59(1):206–218. PubMed PMC

O’Keefe DS, Bacich DJ, Heston WD. Comparative analysis of prostate-specific membrane antigen (PSMA) versus a prostate-specific membrane antigen-like gene. Prostate 2004;58(2):200–210. PubMed

Kiess AP, Minn I, Chen Y, Hobbs R, Sgouros G, Mease RC, Pullambhatla M, Shen CJ, Foss CA, Pomper MG. Auger radiopharmaceutical therapy targeting prostate-specific membrane antigen. J Nucl Med 2015;56(9):1401–1407. PubMed PMC

Bouchelouche K, Capala J, Oehr P. Positron emission tomography/computed tomography and radioimmunotherapy of prostate cancer. Curr Opin Oncol 2009;21(5):469–474. PubMed PMC

Milowsky MI, Nanus DM, Kostakoglu L, Sheehan CE, Vallabhajosula S, Goldsmith SJ, Ross JS, Bander NH. Vascular targeted therapy with anti-prostate-specific membrane antigen monoclonal antibody J591 in advanced solid tumors. J Clin Oncol 2007;25(5):540–547. PubMed

Nakajima T, Mitsunaga M, Bander NH, Heston WD, Choyke PL, Kobayashi H. Targeted, activatable, in vivo fluorescence imaging of prostate-specific membrane antigen (PSMA) positive tumors using the quenched humanized J591 antibody-indocyanine green (ICG) conjugate. Bioconjug Chem 2011;22(8): 1700–1705. PubMed PMC

Osborne JR, Akhtar NH, Vallabhajosula S, Anand A, Deh K, Tagawa ST. Prostate-specific membrane antigen-based imaging. Urol Oncol 2013;31(2):144–154. PubMed PMC

Ng QS, Goh V, Milner J, Padhani AR, Saunders MI, Hoskin PJ. Acute tumor vascular effects following fractionated radiotherapy in human lung cancer: In vivo whole tumor assessment using volumetric perfusion computed tomography. Int J Radiat Oncol Biol Phys 2007;67(2):417–424. PubMed

Larson SM, Carrasquillo JA, Cheung NK, Press OW. Radioimmunotherapy of human tumours. Nat Rev Cancer 2015;15(6): 347–360. PubMed PMC

Sofou S Radionuclide carriers for targeting of cancer. Int J Nanomedicine 2008;3(2):181–199. PubMed PMC

Wittrup KD, Thurber GM, Schmidt MM, Rhoden JJ. Practical theoretic guidance for the design of tumor-targeting agents. Methods Enzymol 2012;503:255–268. PubMed PMC

Troyer JK, Beckett ML, Wright GL Jr. Location of prostate-specific membrane antigen in the LNCaP prostate carcinoma cell line. Prostate 1997;30(4):232–242. PubMed

Development and therapeutic evaluation of 5D3(CC-MLN8237)3.2 antibody-theranostic conjugates for PSMA-positive prostate cancer therapy

Targeting Prostate Cancer Using Bispecific T-Cell Engagers against Prostate-Specific Membrane Antigen

A gut-restricted glutamate carboxypeptidase II inhibitor reduces monocytic inflammation and improves preclinical colitis

Glycoforms of human prostate-specific membrane antigen (PSMA) in human cells and prostate tissue

Structural basis of prostate-specific membrane antigen recognition by the A9g RNA aptamer

Engineered Fragments of the PSMA-Specific 5D3 Antibody and Their Functional Characterization

Development of 5D3-DM1: A Novel Anti-Prostate-Specific Membrane Antigen Antibody-Drug Conjugate for PSMA-Positive Prostate Cancer Therapy

Cellular Delivery of Bioorthogonal Pretargeting Therapeutics in PSMA-Positive Prostate Cancer

Evaluation of 111In-DOTA-5D3, a Surrogate SPECT Imaging Agent for Radioimmunotherapy of Prostate-Specific Membrane Antigen