title: A Novel ¹¹¹In-Labeled Anti-Prostate-Specific Membrane Antigen Nanobody for Targeted SPECT/CT Imaging of Prostate Cancer.
abstract: Prostate-specific membrane antigen (PSMA) is overexpressed in prostate cancer (PCa) and a promising target for molecular imaging and therapy. Nanobodies (single-domain antibodies, VHH) are the smallest antibody-based fragments possessing ideal molecular imaging properties, such as high target specificity and rapid background clearance. We developed a novel anti-PSMA Nanobody (JVZ-007) for targeted imaging and therapy of PCa. Here, we report on the application of the (111)In-radiolabeled Nanobody for SPECT/CT imaging of PCa.
  
  
  METHODS
  A Nanobody library was generated by immunization of a llama with 4 human PCa cell lines. Anti-PSMA Nanobodies were captured by biopanning on PSMA-overexpressing cells. JVZ-007 was selected for evaluation as an imaging probe. JVZ-007 was initially produced with a c-myc-hexahistidine (his) tag allowing purification and detection. The c-myc-his tag was subsequently replaced by a single cysteine at the C terminus, allowing site-specific conjugation of chelates for radiolabeling. JVZ-007-c-myc-his was conjugated to 2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (p-SCN-DTPA) via the lysines, whereas JVZ-007-cys was conjugated to maleimide-DTPA via the C-terminal cysteine. PSMA targeting was analyzed in vitro by cell-binding experiments using flow cytometry, autoradiography, and internalization assays with various PCa cell lines and patient-derived xenografts (PDXs). The targeting properties of radiolabeled Nanobodies were evaluated in vivo in biodistribution and SPECT/CT imaging experiments, using nude mice bearing PSMA-positive PC-310 and PSMA-negative PC-3 tumors.
  
  
  RESULTS
  JVZ-007 was successfully conjugated to DTPA for radiolabeling with (111)In at room temperature. (111)In-JVZ007-c-myc-his and (111)In-JVZ007-cys internalized in LNCaP cells and bound to PSMA-expressing PDXs and, importantly, not to PSMA-negative PDXs and human kidneys. Good tumor targeting and fast blood clearance were observed for (111)In-JVZ-007-c-myc-his and (111)In-JVZ-007-cys. Renal uptake of (111)In-JVZ-007-c-myc-his was initially high but was efficiently reduced by coinjection of gelofusine and lysine. The replacement of the c-myc-his tag by the cysteine contributed to a further reduction of renal uptake without loss of targeting. PC-310 tumors were clearly visualized by SPECT/CT with both tracers, with low renal uptake (<4 percentage injected dose per gram) for (111)In-JVZ-007-cys already at 3 h after injection.
  
  
  CONCLUSIONS
  We developed an (111)In-radiolabeled anti-PSMA Nanobody, showing good tumor targeting, low uptake in nontarget tissues, and low renal retention, allowing excellent SPECT/CT imaging of PCa within a few hours after injection.
authors.affiliation: Department of Urology, Erasmus MC, Rotterdam, The Netherlands Departments of Nuclear Medicine and Radiology, Erasmus MC, Rotterdam, The Netherlands k.chatalic@erasmusmc.nl.
authors.forename: Kristell L S
authors.initials: KL
authors.lastname: Chatalic
authors.affiliation: Department of Urology, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Joke
authors.initials: J
authors.lastname: Veldhoven-Zweistra
authors.affiliation: Laboratory Experimental Surgical Oncology, Department of Surgery, Erasmus MC, Rotterdam, The Netherlands; and.
authors.forename: Michiel
authors.initials: M
authors.lastname: Bolkestein
authors.affiliation: Department of Urology, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Sander
authors.initials: S
authors.lastname: Hoeben
authors.affiliation: Laboratory Experimental Surgical Oncology, Department of Surgery, Erasmus MC, Rotterdam, The Netherlands; and.
authors.forename: Gerben A
authors.initials: GA
authors.lastname: Koning
authors.affiliation: Department of Radiology and Nuclear Medicine, Radboud University Medical Center, Nijmegen, The Netherlands.
authors.forename: Otto C
authors.initials: OC
authors.lastname: Boerman
authors.affiliation: Departments of Nuclear Medicine and Radiology, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Marion
authors.initials: M
authors.lastname: de Jong
authors.affiliation: Department of Urology, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Wytske M
authors.initials: WM
authors.lastname: van Weerden
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry*; D001345:Autoradiography; D045744:Cell Line, Tumor; D003545:Cysteine / Q000737:chemistry; D005434:Flow Cytometry; D043425:Glutamate Carboxypeptidase II / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007668:Kidney / Q000000981:diagnostic imaging; D008297:Male; D051379:Mice; D064847:Multimodal Imaging; D050997:Nanomedicine; D009368:Neoplasm Transplantation; D009842:Oligopeptides / Q000737:chemistry; D019151:Peptide Library; D011471:Prostatic Neoplasms / Q000175:diagnosis / Q000000981:diagnostic imaging*; D017434:Protein Structure, Tertiary; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry; D061905:Single-Domain Antibodies / Q000737:chemistry*; D015899:Tomography, Emission-Computed, Single-Photon; D014057:Tomography, X-Ray Computed
chemical_list: D000954:Antigens, Surface; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D019151:Peptide Library; D016271:Proto-Oncogene Proteins c-myc; D061905:Single-Domain Antibodies; D006639:Histidine; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II; D003545:Cysteine
keywords: 111In; Nanobody; PSMA; SPECT; prostate cancer