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Immunoreactive Evaluation of the Extracellular Epitope of Prostate-specific Membrane Antigen: in Vitro Diagnostic and Therapeutic Potential


Affiliations
1 Regent University College of Science and Technology, School of Informatics, Health and Allied Sciences, Accra, Ghana University of Texas MD Anderson Cancer Center, Department of Genitourinary Medical Oncology, Laboratory of Experimental Therapeutics, Houston, TX 77030, United States
     

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Prostate-specific membrane antigen (PSMA) has been successfully targeted in vivo with 111In-labeled 7E11 monoclonal antibody, which binds to an intracellular epitope of PSMA. This work reports the in vitro characterization and evaluation of three other commonly used mAbs (J415, J533, and J591) that bind the extracellular epitope of PSMA (PSMAext). Briefly, 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 111In. The uptake and cellular processing of these antibodies were evaluated in viable LNCaP cells. 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. 7E11 mAb did not inhibit the binding of J415, J533, or J591 (or vice versa). 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 performed with viable LNCaP cells, J415, J533, and J591 bound to a similar number of PSMA sites, whereas 7E11 bound only to a subpopulation of the available PSMA sites. All four mAbs recognized and bound to similar numbers of PSMAs expressed by ruptured LNCaP cells (i.e., the exposed intracellular and extracellular epitopes of PSMA). 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 epitope of PSMA). These results indicate that both J415 and J591 are promising mAbs for the targeting of viable PSMA-expressing tissue with diagnostic and therapeutic metallic radionuclides

Keywords

Pca, Prostate Cancer, Mab, Monoclonal Antibody, Psma, Prostate-specific Membrane Antigen, Dota, 1, 4, 7, 10- Tetraazacyclododecane-n, N', N?, N??-tetraacetic Acid, Hplc, Highperformance Liquid Chromatography, Dtpa, Diethylenetriaminepentaacetic Acid.
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  • Immunoreactive Evaluation of the Extracellular Epitope of Prostate-specific Membrane Antigen: in Vitro Diagnostic and Therapeutic Potential

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Authors

Stanley S. Moffatt
Regent University College of Science and Technology, School of Informatics, Health and Allied Sciences, Accra, Ghana University of Texas MD Anderson Cancer Center, Department of Genitourinary Medical Oncology, Laboratory of Experimental Therapeutics, Houston, TX 77030, United States
Derrick E. Ansie
Regent University College of Science and Technology, School of Informatics, Health and Allied Sciences, Accra, Ghana University of Texas MD Anderson Cancer Center, Department of Genitourinary Medical Oncology, Laboratory of Experimental Therapeutics, Houston, TX 77030, United States

Abstract


Prostate-specific membrane antigen (PSMA) has been successfully targeted in vivo with 111In-labeled 7E11 monoclonal antibody, which binds to an intracellular epitope of PSMA. This work reports the in vitro characterization and evaluation of three other commonly used mAbs (J415, J533, and J591) that bind the extracellular epitope of PSMA (PSMAext). Briefly, 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 111In. The uptake and cellular processing of these antibodies were evaluated in viable LNCaP cells. 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. 7E11 mAb did not inhibit the binding of J415, J533, or J591 (or vice versa). 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 performed with viable LNCaP cells, J415, J533, and J591 bound to a similar number of PSMA sites, whereas 7E11 bound only to a subpopulation of the available PSMA sites. All four mAbs recognized and bound to similar numbers of PSMAs expressed by ruptured LNCaP cells (i.e., the exposed intracellular and extracellular epitopes of PSMA). 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 epitope of PSMA). These results indicate that both J415 and J591 are promising mAbs for the targeting of viable PSMA-expressing tissue with diagnostic and therapeutic metallic radionuclides

Keywords


Pca, Prostate Cancer, Mab, Monoclonal Antibody, Psma, Prostate-specific Membrane Antigen, Dota, 1, 4, 7, 10- Tetraazacyclododecane-n, N', N?, N??-tetraacetic Acid, Hplc, Highperformance Liquid Chromatography, Dtpa, Diethylenetriaminepentaacetic Acid.

References