title: In vitro characterization of radiolabeled monoclonal antibodies specific for the extracellular domain of prostate-specific membrane antigen.
abstract: Prostate-specific membrane antigen (PSMA) is a well-characterized cell surface antigen expressed by virtually all prostate cancers (PCas). PSMA has been successfully targeted in vivo with (111)In-labeled 7E11 monoclonal antibody (mAb; ProstaScint; Cytogen, Princeton, NJ), which binds to an intracellular epitope of PSMA. This work reports the in vitro characterization of three recently developed mAbs that bind the extracellular domain of PSMA (PSMAext). Murine mAbs J415, J533, J591, and 7E11 were radiolabeled with 131I and evaluated in competitive and saturation binding studies with substrates derived from LNCaP cells. J415 and J591 were conjugated to 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid labeled with (111)In. The uptake and cellular processing of these antibodies were evaluated in viable LNCaP cells. All four mAbs could be labeled with 131I up to a specific activity of 350 MBq/mg with no or little apparent loss of immunoreactivity. Competition assays revealed that J415 and J591 compete for binding to PSMAext antigen. J533 bound to a region close to the J591 binding epitope, but J533 did not interfere with J415 binding to PSMA. mAb 7E11 did not inhibit the binding of J415, J533, or J591 (or vice versa), consistent with earlier work that these latter mAbs bind PSMAext whereas 7E11 binds the intracellular domain of PSMA. Saturation binding studies demonstrated that J415 and J591 bound with a similar affinity (Kds 1.76 and 1.83 nM), whereas J533 had a lower affinity (Kd, 18 nM). In parallel studies, all four mAbs bound to a similar number of PSMA sites expressed by permeabilized cells (1,000,000-1,300,000 sites/cell). In parallel studies performed with viable LNCaP cells, J415, J533, and J591 bound to a similar number of PSMA sites (i.e., 600,000-800,000 sites/cell), whereas 7E11 bound only to a subpopulation of the available PSMA sites (95,000 sites/cell). This apparent binding of 7E11 to viable cells can be accounted for by a 5-7% subpopulation of permeabilized cells produced when the cells were trypsinized and suspended. Up to five DOTA chelates could be bound to either J415 or J591 without compromising immunoreactivity. A comparison of the cellular uptake and metabolic processing of the 131I- and (111)In-labeled antibodies showed a rapid elimination of 131I from the cell and a high retention of (111)In. All four mAbs recognized and bound to similar numbers of PSMAs expressed by ruptured LNCaP cells (i.e., the exposed intracellular and extracellular domains of PSMA). By comparison to J415 and J591, J533 had a lower binding affinity. Both J415 and J591 recognized and bound to the same high number of PSMAs expressed by intact LNCaP. By contrast, 7E11 bound to fewer sites expressed by intact LNCaP cells (i.e., the exposed extracellular domain of PSMA). Both J415 and J591 are promising mAbs for the targeting of viable PSMA-expressing tissue with diagnostic and therapeutic metallic radionuclides.
authors.affiliation: Department of Radiology, New York Presbyterian Hospital-Weill Medical College of Cornell University, New York 10021, USA. psj2001@med.cornell.edu
authors.forename: P M
authors.initials: PM
authors.lastname: Smith-Jones
authors.forename: S
authors.initials: S
authors.lastname: Vallabahajosula
authors.forename: S J
authors.initials: SJ
authors.lastname: Goldsmith
authors.forename: V
authors.initials: V
authors.lastname: Navarro
authors.forename: C J
authors.initials: CJ
authors.lastname: Hunter
authors.forename: D
authors.initials: D
authors.lastname: Bastidas
authors.forename: N H
authors.initials: NH
authors.lastname: Bander
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism / Q000493:pharmacokinetics; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000276:immunology* / Q000378:metabolism; D000954:Antigens, Surface / Q000276:immunology* / Q000378:metabolism; D001667:Binding, Competitive; D002268:Carboxypeptidases / Q000276:immunology* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002614:Chelating Agents / Q000493:pharmacokinetics; D004355:Drug Stability; D043425:Glutamate Carboxypeptidase II; D006801:Humans; D018796:Immunoconjugates / Q000276:immunology* / Q000378:metabolism / Q000493:pharmacokinetics; D007205:Indium Radioisotopes; D007457:Iodine Radioisotopes / Q000627:therapeutic use; D007553:Isotope Labeling; D007700:Kinetics; D008297:Male; D011471:Prostatic Neoplasms / Q000276:immunology / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011786:Quality Control; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D002614:Chelating Agents; D018796:Immunoconjugates; D007205:Indium Radioisotopes; D007457:Iodine Radioisotopes; D002268:Carboxypeptidases; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II