Prostate-specific Membrane Antigen Targeting Research Articles

Assessment of an 18F-Labeled Phosphoramidate Peptidomimetic as a New Prostate-Specific Membrane Antigen–Targeted Imaging Agent for Prostate Cancer

Prostate-specific membrane antigen (PSMA) is a transmembrane protein commonly found on the surface of late-stage and metastatic prostate cancer and a well-known imaging biomarker for staging and monitoring therapy. Although (111)In-labeled capropmab pendetide is the only approved agent available for PSMA imaging, its clinical use is limited because of its slow distribution and clearance that leads to challenging image interpretation. A small-molecule approach using radiolabeled urea-based PSMA inhibitors as imaging agents has shown promise for prostate cancer imaging. The motivation of this work is to explore phosphoramidates as a new class of potent PSMA inhibitors to develop more effective prostate cancer imaging agents with improved specificity and clearance properties. N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB) was conjugated to S-2-((2-(S-4-amino-4-carboxybutanamido)-S-2-carboxyethoxy)hydroxyphosphorylamino)-pentanedioic acid (Phosphoramidate (1)), yielding S-2-((2-(S-4-(4-(18)F-fluorobenzamido)-4-carboxybutanamido)-S-2-carboxyethoxy)hydroxyphosphorylamino)-pentanedioic acid (3). In vivo studies were conducted in mice bearing either LNCaP (PSMA-positive) or PC-3 (PSMA-negative) tumors. PET images were acquired at 1 and 2 h with or without a preinjection of a nonradioactive version of the fluorophosphoramidate. Tissue distribution studies were performed at the end of the 2 h imaging sessions. Phosphoramidate (1) and its fluorobenzamido conjugate (2) were potent inhibitors of PSMA (inhibitory concentration of 50% [IC(50)], 14 and 0.68 nM, respectively). PSMA-mediated tumor accumulation was noted in the LNCaP versus the PC-3 tumor xenografts. The LNCaP tumor uptake was also blocked by the administration of nonradioactive (2) prior to imaging studies. With the exception of the kidneys, tumor-to-tissue and tumor-to-blood ratios were greater than 5:1 at 2 h. The strong kidney uptake may be due to the known PSMA expression in the mouse kidney, because significant reduction (>6-fold) in kidney activity was seen in mice injected with (2). (18)F-labeled phosphoramidate (3) is a representative of a new class of PSMA targeting peptidomimetic molecules that shows great promise as imaging agents for detecting PSMA+ prostate tumors.

Preclinical Evaluation of Novel Glutamate-Urea-Lysine Analogues That Target Prostate-Specific Membrane Antigen as Molecular Imaging Pharmaceuticals for Prostate Cancer

Prostate-specific membrane antigen (PSMA) is expressed in normal human prostate epithelium and is highly up-regulated in prostate cancer. We previously reported a series of novel small molecule inhibitors targeting PSMA. Two compounds, MIP-1072, (S)-2-(3-((S)-1-carboxy-5-(4-iodobenzylamino)pentyl)ureido)pentanedioic acid, and MIP-1095, (S)-2-(3-((S)-1carboxy-5-(3-(4-iodophenyl)ureido)pentyl)ureido)pentanedioic acid, were selected for further evaluation. MIP-1072 and MIP-1095 potently inhibited the glutamate carboxypeptidase activity of PSMA (K(i) = 4.6 +/- 1.6 nmol/L and 0.24 +/- 0.14 nmol/L, respectively) and, when radiolabeled with (123)I, exhibited high affinity for PSMA on human prostate cancer LNCaP cells (K(d) = 3.8 +/- 1.3 nmol/L and 0.81 +/- 0.39 nmol/L, respectively). The association of [(123)I]MIP-1072 and [(123)I]MIP-1095 with PSMA was specific; there was no binding to human prostate cancer PC3 cells, which lack PSMA, and binding was abolished by coincubation with a structurally unrelated NAALADase inhibitor, 2-(phosphonomethyl)pentanedioic acid (PMPA). [(123)I]MIP-1072 and [(123)I]MIP-1095 internalized into LNCaP cells at 37 degrees C. Tissue distribution studies in mice showed 17.3 +/- 6.3% (at 1 hour) and 34.3 +/- 12.7% (at 4 hours) injected dose per gram of LNCaP xenograft tissue, for [(123)I]MIP-1072 and [(123)I]MIP-1095, respectively. [(123)I]MIP-1095 exhibited greater tumor uptake but slower washout from blood and nontarget tissues compared with [(123)I]MIP-1072. Specific binding to PSMA in vivo was shown by competition with PMPA in LNCaP xenografts, and the absence of uptake in PC3 xenografts. The uptake of [(123)I]MIP-1072 and [(123)I]MIP-1095 in tumor-bearing mice was corroborated by single-photon emission computed tomography/computed tomography (SPECT/CT) imaging. PSMA-specific radiopharmaceuticals should provide a novel molecular targeting option for the detection and staging of prostate cancer.

In vivo targeted killing of prostate tumor cells by a synthetic amoebapore helix 3 peptide modified with two γ-linked glutamate residues at the COOH terminus

We previously designed a pro-cytolytic peptide to target prostate-specific membrane antigen (PSMA)-positive prostate tumor cells. The backbone of the peptide was derived from the cell lytic amoebapore H-3 domain, which becomes completely inactive upon modification by two glutamate residues linked to the ε-amide group of the COOH-terminal lysine through γ-linkages (H-3Glu2). This modified H-3 domain regains its lytic activity against PSMA-positive cells (LNCaP) after the γ-linked glutamate residues are cleaved by PSMA. Our previous in vitro results demonstrate that the modified amoebapore peptide has strong cytolytic activity towards PSMA-positive cells and very little activity towards PSMA-negative cells. In the present study, the in vivo efficacy of this modified peptide was examined in human LNCaP prostate tumor xenografts in nude mice. The results showed significantly decreased tumor size and PSA levels in treated mice as compared to control mice. As well, 5/12 of the treated mice were tumor-free. Peptide distribution studies showed that peptide levels in the prostate tumors maintained a steady concentration for approximately 6 hours. Single-dose toxicity studies showed no toxic effects of the peptide when administered intraperitoneally or intravenously at a dose of 30 mg/kg.

In vitro targeted photodynamic therapy with a pyropheophorbide‐a conjugated inhibitor of prostate‐specific membrane antigen

The lack of specific delivery of photosensitizers (PSs), represents a significant limitation of photodynamic therapy (PDT) of cancer. The biomarker prostate-specific membrane antigen (PSMA) has attracted considerable attention as a target for imaging and therapeutic applications for prostate cancer. Although recent efforts have been made to conjugate inhibitors of PSMA with imaging agents, there have been no reports on PS-conjugated PSMA inhibitors for targeted PDT of prostate cancer. The present study focuses on the use of a PSMA inhibitor-conjugate of pyropheophorbide-a (Ppa-conjugate 2) for targeted PDT to achieve apoptosis in PSMA+ LNCaP cells. Confocal laser scanning microscopy with a combination of nuclear staining and immunofluorescence methods were employed to monitor the specific imaging and PDT-mediated apoptotic effects on PSMA-positive LNCaP and PSMA-negative (PC-3) cells. Our results demonstrated that PDT-mediated effects by Ppa-conjugate 2 were specific to LNCaP cells, but not PC-3 cells. Cell permeability was detected as early as 2 hr by HOE33342/PI double staining, becoming more intense by 4 hr. Evidence for the apoptotic caspase cascade being activated was based on the appearance of poly-ADP-ribose polymerase (PARP) p85 fragment. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay detected DNA fragmentation 16 hr post-PDT, confirming apoptotic events. Cell permeability by HOE33342/PI double staining as well as PARP p85 fragment and TUNEL assays confirm cellular apoptosis in PSMA+ cells when treated with PS-inhibitor conjugate 2 and subsequently irradiated. It is expected that the PSMA targeting small-molecule of this conjugate can serve as a delivery vehicle for PDT and other therapeutic applications for prostate cancer.

Targeted delivery of cisplatin to prostate cancer cells by aptamer functionalized Pt(IV) prodrug-PLGA–PEG nanoparticles

Cisplatin is used to treat a variety of tumors, but dose limiting toxicities or intrinsic and acquired resistance limit its application in many types of cancer including prostate. We report a unique strategy to deliver cisplatin to prostate cancer cells by constructing Pt(IV)-encapsulated prostate-specific membrane antigen (PSMA) targeted nanoparticles (NPs) of poly(D,L-lactic-co-glycolic acid) (PLGA)-poly(ethylene glycol) (PEG)-functionalized controlled release polymers. By using PLGA-b-PEG nanoparticles with PSMA targeting aptamers (Apt) on the surface as a vehicle for the platinum(IV) compound c,t,c-[Pt(NH(3))(2)(O(2)CCH(2)CH(2)CH(2)CH(2)CH(3))(2)Cl(2)] (1), a lethal dose of cisplatin was delivered specifically to prostate cancer cells. PSMA aptamer targeted delivery of Pt(IV) cargos to PSMA(+) LNCaP prostate cancer cells by endocytosis of the nanoparticle vehicles was demonstrated using fluorescence microscopy by colocalization of green fluorescent labeled cholesterol-encapsulated NPs and early endosome marker EEA-1. The choice of linear hexyl chains in 1 was the result of a systematic study to optimize encapsulation and controlled release from the polymer without compromising either feature. Release of cisplatin from the polymeric nanoparticles after reduction of 1 and formation of cisplatin 1,2-intrastrand d(GpG) cross-links on nuclear DNA was confirmed by using a monoclonal antibody for the adduct. A comparison between the cytotoxic activities of Pt(IV)-encapsulated PLGA-b-PEG NPs with the PSMA aptamer on the surface (Pt-NP-Apt), cisplatin, and the nontargeted Pt(IV)-encapsulated NPs (Pt-NP) against human prostate PSMA-overexpressing LNCaP and PSMA(-) PC3 cancer cells revealed significant differences. The effectiveness of PSMA targeted Pt-NP-Apt nanoparticles against the PSMA(+) LNCaP cells is approximately an order of magnitude greater than that of free cisplatin.

Clinical Trials of Cancer Therapies Targeting Prostate-Specific Membrane Antigen

Prostate cancer is the most common non-cutaneous cancer of men in the United States and represents their second-leading cause of cancer-related death. Metastatic disease is largely resistant to conventional chemotherapies, and targeted therapies are urgently needed. Prostate-specific membrane antigen (PSMA) is a prototypical cell-surface marker of prostate cancer. PSMA is an integral, non-shed, type 2 membrane protein with abundant and nearly universal expression in prostate carcinoma, but has limited extra-prostatic expression. In addition, PSMA is expressed in the neovasculature of other solid tumors. These findings have spurred development of PSMA-targeted therapies for cancer, and first-generation products have entered clinical testing. Vaccine approaches have included recombinant protein, nucleic acid and cell-based strategies, and anti-PSMA immune responses have been demonstrated in the absence of significant toxicity. Therapy with drug-conjugated and radiolabeled antibodies has yielded objective clinical responses as measured by reductions in serum prostate-specific antigen and/or imageable tumor volume. However, responses were observed in a minor fraction of patients and at doses near the maximum tolerated dose. Overall, these initial studies have provided measured proof of concept for PSMA-based therapies, and second-generation antibody and vaccine products may hold the key to exploit PSMA for molecularly targeted therapy of prostate and other cancers.

Significance of micrometastases in pelvic lymph nodes detected by real‐time reverse transcriptase polymerase chain reaction in patients with clinically localized prostate cancer undergoing radical prostatectomy after neoadjuvant hormonal therapy

To clarify the significance of micrometastases in pelvic lymph nodes in patients treated by radical prostatectomy (RP) for prostate cancer after neoadjuvant hormonal therapy (NHT). The study included 52 patients with clinically localized prostate cancer who received NHT followed by RP. The expression of prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) in 989 lymph nodes isolated from the 52 patients were assessed by a fully quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR). We regarded specimens in which either PSA or PSMA mRNA were positive as showing the 'presence of micrometastasis'. Lymph node specimens were also stained immunohistochemically with an antibody against PSA. Pathological examinations detected tumour cells in 11 lymph nodes from four patients, and real-time RT-PCR further identified micrometastasis in 40 lymph nodes from 19 patients with no pathological evidence of nodal involvement. The presence of micrometastatic cancer cells was confirmed by immunohistochemical staining in 19 lymph nodes from 11 patients with pathologically negative nodes. The presence of micrometastases was significantly associated with other conventional prognostic variables, including the pretreatment serum PSA level, biopsy Gleason score and surgical margin status. The biochemical recurrence-free survival rate in patients with no micrometastasis was significantly higher than that in those with micrometastasis. Furthermore, multivariate analysis identified the presence of micrometastasis as an independent factor predicting biochemical recurrence. Although residual foci of atrophic prostate cancer cells in resected lymph nodes after NHT can be difficult to diagnose by routine pathological examination, the present results show the usefulness of quantitative real-time RT-PCR targeting PSA and PSMA genes for detecting micrometastatic tumour foci in pelvic lymph nodes from patients with localized prostate cancer treated by NHT followed by RP. Furthermore, the present findings suggest that micrometastases in pelvic lymph nodes might be, at least partly, important in the development of biochemical recurrence in some patients undergoing RP after NHT.

Differing effects of microtubule depolymerizing and stabilizing chemotherapeutic agents on t-SNARE–mediated apical targeting of prostate-specific membrane antigen

Prostate-specific membrane antigen (PSMA) is a protein up-regulated in the vast majority of prostate cancers. Antibodies to PSMA have proved highly specific for prostate cancer cells, and the therapeutic potential of such antibodies is currently being assessed in clinical trials. We have previously shown that PSMA at the cell surface of polarized epithelial cells is predominantly expressed at the apical plasma membrane and that microtubule depolymerization abolishes apical PSMA targeting. In the current report, we implicate a functional role for a target membrane soluble N-ethylmaleimide-sensitive factor adaptor protein receptor, syntaxin 3, in the microtubule-dependent apical targeting of PSMA. PSMA and syntaxin 3 are similarly localized to the apical plasma membrane of the prostatic epithelium and Madin-Darby canine kidney cells. Introduction of a point mutation into syntaxin 3 abolishes its polarized distribution and causes PSMA to be targeted in a nonpolarized fashion. Additionally, treatment of polarized Madin-Darby canine kidney cells with vinblastine, a microtubule depolymerizing chemotherapeutic agent, causes both syntaxin 3 and PSMA to redistribute in a nonpolarized fashion. However, following treatment with the microtubule stabilizing chemotherapeutic agent Taxotere, both syntaxin 3 and PSMA continue to localize in a polarized manner at the apical plasma membrane. Thus, microtubule depolymerizing and stabilizing chemotherapeutic drugs might exact similar cytotoxic effects but have disparate effects on protein targeting. This phenomenon might have important clinical implication, especially related to antibody-mediated immunotherapy, and could potentially be exploited for therapeutic benefit.

PEGylated J591 mAb loaded in PLGA-PEG-PLGA tri-block copolymer for targeted delivery: In vitro evaluation in human prostate cancer cells

J591 monoclonal antibody (mAb) has high affinity for prostate specific membrane antigen (PSMA) on prostate cancer (PCA) cells. We coupled polyethylene glycol-J591 (PEGylated J591) to a salicyl hydroxamic acid (SHA)–derivatized polyethylenimine (PEI)/DNA-βgal vector to investigate the specificity and efficiency of targeting PSMA in PCA cells through encapsulation. Coupling was facilitated via the high affinity interaction between phenyl(di)boronic acid (PDBA) and SHA molecules yielding J591/PEG/PEI/DNA-βgal polyplex. After encapsulation with poly( d, l-lactic- co-glycolic acid)- b-polyethylene glycol- b-poly( d, l-lactic- co-glycolic acid) (PLGA-PEG-PLGA) tri-block copolymer, 8–10-fold increment of gene transfection levels were attained at the optimum concentration of 0.25% (w/v) using Pluronic F68 tri-block copolymer as a control. The enhanced transfection efficiency was attributed to increased internalization and uptake of the radiolabeled plasmid in the presence of PLGA-PEG-PLGA tri-block copolymer. The release of plasmid DNA (pDNA) from microparticles containing SHA–PEI-complexed pDNA showed little initial burst release followed by a 5% release over 48 h. The release accelerated thereafter and approximately 60% was released after 28 days. Deconvolution confocal microscopy showed polyplex/microparticle formulation localized in the cell nucleus as opposed to the polyplex without PLGA-PEG-PLGA indicating that an optimal concentration of PLGA-PEG-PLGA tri-block copolymer can be utilized to enhance endocytic process of J591-mediated targeting of PCA cells.

Successful in vivo tumor targeting of prostate-specific membrane antigen with a highly efficient J591/PEI/DNA molecular conjugate

We have utilized a novel polyethylenimine (PEI)/DNA-betagal vector to investigate the specificity and efficiency of immuno-targeting prostate-specific membrane antigen (PSMA). Coupling of the PSMA-specific monoclonal antibody, J591, to the vector was facilitated via the high-affinity interaction between phenyl(di)boronic acid and salicylhydroxamic acid molecules. Highly efficient gene delivery by this prostate cancer (PCA)-targeted J591/polyethylene glycol (PEG)/PEI/DNA-betagal vector was demonstrated in PSMA-positive cells relative to controls, resulting in significant growth inhibition in vitro when the J591/PEG/PEI/DNA-p53 was used. Competition with free antibody resulted in about 90% reduction in both J591 internalization and betagal gene delivery, indicating specificity for PSMA-positive cells. More importantly, testing the efficiency of the J591/PEG/PEI/DNA-betagal targeting vector in an orthotopic PCA model in nude mice resulted in up to a 20-fold increase in gene delivery over the untargeted vector controls. The in vivo organ distribution profile also revealed betagal expression predominantly in the tumor, which was more than 1 log higher than the next highest level of expression in the lung. Furthermore, with the targeted vector containing the gene for yellow fluorescent protein or biotinylated J591, we further demonstrate in vivo that vector-mediated gene delivery is specific for both tumor cells and tumor-associated neovasculature in PSMA-positive tumors. These results suggest the potential for further optimization of this novel vector in the context of therapeutic gene delivery.

N-glycosylation and microtubule integrity are involved in apical targeting of prostate-specific membrane antigen: implications for immunotherapy

Prostate-specific membrane antigen (PSMA) is an important biomarker expressed in prostate cancer cells with levels proportional to tumor grade. The membrane association and correlation with disease stage portend a promising role for PSMA as an antigenic target for antibody-based therapies. Successful application of such modalities necessitates a detailed knowledge of the subcellular localization and trafficking of target antigen. In this study, we show that PSMA is expressed predominantly in the apical plasma membrane in epithelial cells of the prostate gland and in well-differentiated Madin-Darby canine kidney cells. We show that PSMA is targeted directly to the apical surface and that sorting into appropriate post-Golgi vesicles is dependent upon N-glycosylation of the protein. Integrity of the microtubule cytoskeleton is also essential for delivery and retention of PSMA at the apical plasma membrane domain, as destabilization of microtubules with nocodazole or commonly used chemotherapeutic Vinca alkaloids resulted in the basolateral expression of PSMA and increased the uptake of anti-PSMA antibody from the basolateral domain. These results may have important relevance to PSMA-based immunotherapy and imaging strategies, as prostate cancer cells can maintain a well-differentiated morphology even after metastasis to distal sites. In contrast to antigens on the basolateral surface, apical antigens are separated from the circulation by tight junctions that restrict transport of molecules across the epithelium. Thus, antigens expressed on the apical plasma membrane are not exposed to intravenously administered agents. The ability to reverse the polarity of PSMA from apical to basolateral could have significant implications for the use of PSMA as a therapeutic target.

Update on Fused Capromab Pendetide Imaging of Prostate Cancer

The primary objective of this overview is to apprise clinical urologists and oncologists of the current state of fused multimodality imaging of prostate cancer, which can be applied to optimize treatment by ensuring that a patient's disease is characterized as well as current imaging technology permits. The focus of this study is the monoclonal antibody capromab pendetide, which targets prostate specific membrane antigen (PSMA), a type II membrane glycoprotein strongly associated with prostate cancer. Identifying where capromab pendetide uptake occurs can be done accurately if this functional imaging modality is combined with a modality that provides anatomic detail, such as computed tomography (CT) or magnetic resonance imaging (MRI). Image fusion, or coregistration, which is overlaying the functional images of capromab pendetide uptake on the anatomic CT or MRI images, provides a detailed map of cancer localization inside and outside the prostate gland. This same principle of fusing functional images on anatomic images is the basis for enormous growth of positron emission tomography with CT during the past 2 years. Positron emission tomography imaging has a different functionality base than does capromab pendetide, and thus the 2 modalities should be complementary. However, the key to both functional imaging modalities is accurate fusion with anatomic images, which is illustrated in our case reports. The cases cited demonstrate the need to optimize every phase of imaging from patient preparation to reading and reporting increased PSMA concentration seen on the fused images. Reference is also made to applying capromab pendetide/CT fused imaging to radiation therapy planning.

Clinical Use of Monoclonal Antibody HuJ591 Therapy: Targeting Prostate Specific Membrane Antigen

Prostate specific membrane antigen (PSMA) is a cell surface peptidase highly expressed by malignant prostate epithelial cells and vascular endothelial cells of numerous solid tumor malignancies but not normal vascular endothelium in benign tissues or neoplastic epithelial cells of nonprostate malignancies. Monoclonal antibody (mAb) J591 recognizes the extracellular domain of PSMA. The current status of clinical trials using mAb J591 is reviewed. The mouse antibody was deimmunized by replacing murine immunoglobulin sequences with human immunoglobulin sequences, resulting in a nonimmunogenic antibody HuJ591. Results of completed and ongoing phase 1 and 2 clinical trials using mAb J591 are presented. A phase I clinical trial with murine J591 indicated that it possesses high affinity for prostate cancer metastases in bone and soft tissue. A phase I clinical trial showed that HuJ591 was well tolerated, demonstrated excellent targeting to disseminated prostate cancer sites and did not result in a host immune response to the antibody. A phase II clinical trial was initiated to study the efficacy of combining HuJ591 with low dose interleukin-2. Two phase I studies in patients with prostate cancer are in progress using the beta-emitting radiometals 90yttrium and 177lutetium linked via a DOTA chelate to HuJ591. Preliminary results from an ongoing phase I trial of 111indium labeled mAb HuJ591 in patients with advanced solid tumors showed that HuJ591 specifically targets nonprostate cancers. Early results of clinical studies indicate that mAb HuJ591 represents targeted therapy to PSMA and has therapeutic potential in prostate cancer and other urological and solid tumor malignancies.

Tumor target prostate specific membrane antigen (PSMA) and its regulation in prostate cancer

Prostate specific membrane antigen (PSMA), is a unique membrane bound glycoprotein, which is overexpressed manifold on prostate cancer as well as neovasculature of most of the solid tumors, but not in the vasculature of the normal tissues. This unique expression of PSMA makes it an important marker as well as a large extracellular target of imaging agents. PSMA can serve as target for delivery of therapeutic agents such as cytotoxins or radionuclides. PSMA has two unique enzymatic functions, folate hydrolase and NAALADase and found to be recycled like other membrane bound receptors through clathrin coated pits. The internalization property of PSMA leads one to consider the potential existence of a natural ligand for PSMA. In this review we have discussed the regulation of PSMA expression within the cells, and significance of its expression in prostate cancer and metastasis.

964. Prostate Tumor Specific Gene Delivery Mediated by a Novel Antibody/Polyethylenimine/DNA Polyplex Targeting Prostate Specific Membrane Antigen

964. Prostate Tumor Specific Gene Delivery Mediated by a Novel Antibody/Polyethylenimine/DNA Polyplex Targeting Prostate Specific Membrane Antigen

Biological and therapeutic properties of a novel, fully human monoclonal antibody targeting prostate specific membrane antigen

Biological and therapeutic properties of a novel, fully human monoclonal antibody targeting prostate specific membrane antigen