Prostate-specific Membrane Antigen Antibody Research Articles

Preclinical Efficacy of a PSMA-Targeted Actinium-225 Conjugate (225Ac-Macropa-Pelgifatamab): A Targeted Alpha Therapy for Prostate Cancer.

Initially, prostate cancer responds to hormone therapy, but eventually resistance develops. Beta emitter-based prostate-specific membrane antigen (PSMA)-targeted radionuclide therapy is approved for the treatment of metastatic castration-resistant prostate cancer. Here we introduce a targeted alpha therapy (TAT) consisting of the PSMA antibody pelgifatamab covalently linked to a macropa chelator and labeled with actinium-225 and compare its efficacy and tolerability with other TATs. The in vitro characteristics and in vivo biodistribution, antitumor efficacy, and tolerability of 225Ac-macropa-pelgifatamab (225Ac-pelgi) and other TATs were investigated in cell line- and patient-derived prostate cancer xenograft models. The antitumor efficacy of 225Ac-pelgi was also investigated in combination with the androgen receptor inhibitor darolutamide. Actinium-225-labeling of 225Ac-pelgi was efficient already at room temperature. Potent in vitro cytotoxicity was seen in PSMA-expressing (LNCaP, MDA-PCa-2b, and C4-2) but not in PSMA-negative (PC-3 and DU-145) cell lines. High tumor accumulation was seen for both 225Ac-pelgi and 225Ac-DOTA-pelgi in the MDA-PCa-2b xenograft model. In the C4-2 xenograft model, 225Ac-pelgi showed enhanced antitumor efficacy with a T/Cvolume (treatment/control) ratio of 0.10 compared with 225Ac-DOTA-pelgi, 225Ac-DOTA-J591, and 227Th-HOPO-pelgifatamab (227Th-pelgi; all at 300 kBq/kg) with T/Cvolume ratios of 0.37, 0.39, and 0.33, respectively. 225Ac-pelgi was less myelosuppressive than 227Th-pelgi. 225Ac-pelgi showed dose-dependent treatment efficacy in the patient-derived KuCaP-1 model and strong combination potential with darolutamide in both cell line- (22Rv1) and patient-derived (ST1273) xenograft models. These results provide a strong rationale to investigate 225Ac-pelgi in patients with prostate cancer. A clinical phase I study has been initiated (NCT06052306).

Abstract 3386: Development of next generation extracellular vesicle based urine tests for prostate cancer

Abstract Introduction and Objective: Non-invasive urine and blood biomarkers are commercially available to assess the risk of clinically significant prostate cancer (csPCa) and the need for a biopsy. However, these tests are limited in their specificity resulting in a significant number of men still undergoing biopsy to avoid missing a csPCa. Exosomes and other extracellular vesicles (EVs) provide a platform for blood and urine biomarkers since they are released by all types of cells and preserve molecular constituents from their cells of origin within lipid membranes. The EPI urine test from Exosome Diagnostics (ExoDx) is currently the only commercially available EV based cancer diagnostic test; it uses three well established EV RNA markers to estimate the risk of csPCa. Studies by us and others have shown that an expanded urine EV PCa marker panel would improve disease stratification if these markers could be incorporated into a clinical grade assay. Our objective in this current study is to identify and characterize the EV RNA transcripts that are enriched by using antibodies for prostate specific membrane antigen (PSMA) to capture urinary EVs that originated from PCa cells. This is the first step in the development of a next generation urinary EV test with increased specificity for csPCa within a rigorous and reproducible assay format. Methods: Urine EVs from patients with csPCa and from healthy controls were prepared using the ExoDx clinical platform. These total EV samples were then compared with EVs enriched by immunocapture either with a PSMA specific antibody or with an isotype IgG control reagent. RNA was prepared from each of these three types of urinary EV samples and analyzed using RNAseq. Results: RNAseq analysis of urinary total EVs, PSMA-antibody captured EVs and isotype IgG control captured EVs showed excellent mapping to transcriptome regions. We detected over 600 genes differentially enriched by PSMA antibody vs. isotype IgG EV capture and identified 18 genes from a candidate list of prostate biomarkers that have been assembled based on previous studies. A principal component analysis of 196 differentially expressed transcripts obtained from EVs enriched by PSMA immunocapture vs IgG controls showed excellent discrimination between prostate cancer patients and healthy controls. By using a feature selection approach to optimize for discrimination between prostate cancer patients and healthy controls we identified a novel 6-gene signature that, when derived from a PSMA capture EV dataset, shows a superior AUC for the accuracy of cancer detection (greater than 0.95) than that obtained from applying the same 6-gene signature on total EVs or on isotype IgG controls. Conclusions: EV PSMA immuno-capture provides a robust approach to expanding the panel of PCa biomarkers that can be incorporated into clinical grade PCa urine EV assays, supporting the translation of pre-clinical discovery into clinical practice. Citation Format: Sandra M. Gaston, Douglas Roberts, Sudipto Chakrabortty, Emily Mitsock, Kailey Babcock, Rikky Xing, Benjamin Spieler, Radka Stoyanova, Mark L. Gonzalgo, Chad Ryan Ritch, Bruno Nahar, Alan Pollack, Dipen J. Parekh, Seth Yu, Johan Skog, Sanoj Punnen. Development of next generation extracellular vesicle based urine tests for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3386.

Bispecific PSMA antibodies and CAR-T in metastatic castration-resistant prostate cancer.

Prostate cancer is the most common cancer among men and the second leading cause of cancer-related deaths in men in the United States. The treatment paradigm for prostate cancer has evolved with the emergence of a variety of novel therapies which have improved survival; however, treatment-related toxicities are abundant and durable responses remain rare. Immune checkpoint inhibitors have shown modest activity in a small subset of patients with prostate cancer and have not had an impact on most men with advanced disease. The discovery of prostate-specific membrane antigen (PSMA) and the understanding of its specificity to prostate cancer has identified it as an ideal tumor-associated antigen and has revived the enthusiasm for immunotherapeutics in prostate cancer. T-cell immunotherapy in the form of bispecific T-cell engagers (BiTEs) and chimeric antigen receptor (CAR) T-cell therapy have shown exceptional success in treating various hematologic malignancies, and are now being tested in patients with prostate cancer with drug design centered on various target ligands including not just PSMA, but others as well including six-transmembrane epithelial antigen of the prostate 1 (STEAP1) and prostate stem cell antigen (PSCA). This summative review will focus on the data surrounding PSMA-targeting T-cell therapies. Early clinical studies with both classes of T-cell redirecting therapies have demonstrated antitumor activity; however, there are multiple challenges with this class of agents, including dose-limiting toxicity, 'on-target, off-tumor' immune-related toxicity, and difficulty in maintaining sustained immune responses within a complex and overtly immunosuppressive tumor microenvironment. Reflecting on experiences from recent trials has been key toward understanding mechanisms of immune escape and limitations in developing these drugs in prostate cancer. Newer generation BiTE and CAR T-cell constructs, either alone or as part of combination therapy, are currently under investigation with modifications in drug design to overcome these barriers. Ongoing innovation in drug development will likely foster successful implementation of T-cell immunotherapy bringing transformational change to the treatment of prostate cancer.

The Importance of Prostate-Specific Membrane Antigen Expression in Salivary Gland Tumors.

Prostate-specific membrane antigen (PSMA) is a cell membrane protein expressed by prostate tissues. It is not prostate specific and is also expressed by some non-prostatic solid neoplasms. Our study aimed to investigate the potential role of PSMA in salivary gland tumors. The present study was designed to retrospectively analyze our cases that presented with salivary gland tumors. The files of 105 patients were reviewed and their paraffin embedded blocks were retrieved from the pathology department. Immunohistochemical examination and staining were done using PSMA antibody. Tumor tissue PSMA immunohistochemical staining was scored semi-quantitatively with the modified quartile approach. Negative staining was scored 0, >0% and ≤25% tissue expression was considered weak (score 1), >25% and ≤50% tissue expression was considered mild (score 2), >50% and ≤75% tissue expression was considered moderate (score 3), and >75% tissue expression was considered strong (score 4). Eighty-eight patients (55 males, 33 females) were included in the study. Forty-eight patients had pleomorphic adenoma (PA), 35 had Warthin's tumor (WT), two had mucoepidermoid carcinoma, two had adenoid cystic carcinoma, and one had squamous cell carcinoma. There was statistically significant difference in terms of PSMA expression between PA and WT (p=0.003). PSMA expression was high in PA and absent in WT. PSMA is a potential source of inspiration for future studies on the development of novel diagnostic and theranostic investigations of salivary gland tumors. Prospective studies targeting intratumoral PSMA in salivary gland tumors should be planned.

Tumor-Targeting Ability of Novel Anti-Prostate-Specific Membrane Antigen Antibodies.

Patients with prostate-specific membrane antigen (PSMA)-positive tumors can benefit from PSMA-targeted therapy; thus, we have constructed a phage-displayed synthetic antibody library for the production of novel PSMA antibodies with superior PSMA-targeting ability, favoring clinical management. The binding affinities of anti-PSMA antibodies were verified by an enzyme-linked immunosorbent assay (ELISA). Several in vitro and in vivo experiments, including cellular uptake, internalization, and cytotoxicity studies, micro single photon emission computed tomography (microSPECT)/CT, and biodistribution studies, were performed to select the most promising antibody among six different antibodies. The results showed the target affinities of our antibodies in the ELISA assays (7A, 8C, 8E, and 11A) were comparable to the existing antibodies (J591). The half-maximal effective concentrations of 7A, 8C, 8E, 11A, and J591 were 2.95, 6.64, 5.50, 2.08, and 4.79, respectively. The radiochemical yield of 111In-labeled antibodies ranged from 30% to 50% with high radiochemical purity (>90%). In the cellular uptake studies, the accumulated radioactivity of 111In-J591, 111In-7A, and 111In-11A increased over time. The internalized percentage of 111In-11A was the highest (32.14% ± 2.06%) at 48 h after incubation, whereas that of 111In-J591 peaked at 22.43% ± 4.38% at 24 h and dropped to 13.52% ± 3.03% at 48 h postincubation. Twenty-four hours after injection, radioactivity accumulation appeared in the LNCaP xenografts of the mice injected with 111In-11A, 111In-8E, 111In-7A, and 111In-J591 but not in the xenografts of the 111In-8C-injected group. Marked liver uptake was noticed in all groups except the 111In-11A-injected group. Moreover, the killing effect of 177Lu-11A was superior to that of 177Lu-J591 at low concentrations. In conclusion, we successfully demonstrated that 11A IgG owned the most optimal biological characteristics among several new anti-PSMA antibodies and it can be an excellent PSMA-targeting component for the clinical use.

Telomerase positive CTCs with PSMA high expression associated with prostate cancer metastasis.

BackgroundWhether circulating tumor cells (CTCs) with prostate-specific membrane antigen (PSMA) high expression was related to the metastatic progress in prostate cancer (PCa) remains explored. This study aimed to provide evidence to elucidate this relationship via the telomerase reverse transcriptase (TERT)-based CTC detection method.MethodsA total of 71 patients were enrolled and divided into the local PCa group (n=44) and metastatic PCa group (n=27). TERT-based CTC detection (TBCD) was used to detect CTCs. CTCs single-cell sequencing data were analyzed using gene ontology (GO) functional classification and enrichment.ResultsThe mean ‘TERT+ CTCs’ number was 6.11±9.63 in the metastatic group and 4.09±3.41 in the local group. GO enrichment analysis for 77 prostate CTCs single-cell sequencing confirmed that proliferation-related terms were enriched in the PSMA-high expression group, and 27 metastasis-related gene panels also had high expression in this group. Then, PSMA antibody was applied to mark the ‘TERT+ CTCs’. The proportion of patients with ‘TERT+ PSMA+ CTCs’ was positively associated with the Gleason score. Furthermore, the proportion of ‘TERT+ PSMA+ CTCs’ patients was 48.15% in the metastatic group, significantly higher than 22.72% in the local group.ConclusionsThis study suggested that TERT positive CTCs with high PSMA expression were associated with the PCa metastatic progress.

An IgG-based bispecific antibody for improved dual targeting in PSMA-positive cancer.

The prostate‐specific membrane antigen (PSMA) has been demonstrated in numerous studies to be expressed specifically on prostate carcinoma cells and on the neovasculature of several other cancer entities. However, the simultaneous expression of PSMA on both, tumor cells as well as tumor vessels remains unclear, even if such “dual” expression would constitute an important asset to facilitate sufficient influx of effector cells to a given tumor site. We report here on the generation of a PSMA antibody, termed 10B3, which exerts superior dual reactivity on sections of prostate carcinoma and squamous cell carcinoma of the lung. 10B3 was used for the construction of T‐cell recruiting bispecific PSMAxCD3 antibodies in Fab‐ and IgG‐based formats, designated Fabsc and IgGsc, respectively. In vitro, both molecules exhibited comparable activity. In contrast, only the larger IgGsc molecule induced complete and durable elimination of established tumors in humanized mice due to favorable pharmacokinetic properties. Upon treatment of three patients with metastasized prostate carcinoma with the IgGsc reagent, marked activation of T cells and rapid reduction of elevated PSA levels were observed.

The prostate-specific membrane antigen (PSMA)-targeted radiotracer 18F-DCFPyL detects tumor neovasculature in metastatic, advanced, radioiodine-refractory, differentiated thyroid cancer.

Prostate-specific membrane antigen (PSMA; also termed glutamate carboxypeptidase II (GCP II)) is abundantly expressed in prostate cancer. It has been shown recently that PSMA is expressed in neovasculature of differentiated thyroid cancer. In this study, we show that 18F-DCFPyl might detect neovasculature in advanced, metastatic differentiated thyroid cancer (DTC). We first stained the preserved lymph node samples of three patients with DTC who had undergone total thyroidectomy and neck dissection for cervical lymph node metastatic disease to identify PSMA expression, with the PSMA antibody (DAKO Monoclonal). Then, we performed 18F-DCFPyl imaging in two other advanced DTC patients with elevated serum thyroglobulin (Tg), indicative of residual disease. We compared the findings with contemporaneous FDG PET/CT scan, conventional Imaging (CT,MRI) and whole-body scan performed with I123/I131. All the three lymph node samples stained positive for PSMA expression in the neovasculature. In the first imaged patient, 18F-DCFPyl detected activity within the retropharyngeal CT contrast-enhancing lymph node. Compared to FDG PET/CT, the 18F-DCFPyl scan showed a greater SUV (3.1 vs 1.8). In the second imaged patient, 18F-DCFPyl showed intense uptake in the L3 vertebra (not seen on the post treatment 131I scan or the 18F-FDG PET/CT). MRI of the lumbar spine confirmed the presence of sclerotic-lytic lesion at the location, consistent with metastatic disease. Our exploratory study is proof of principle, that the prostate cancer imaging agent 18F-DCFPyl may prove useful for the localization of metastases, in patients with metastatic RAI-refractory DTC by detecting neoangiogenesis within the tumor.

Abstract 5663: Affinity tuned XmAb®2+1 PSMA x CD3 bispecific antibodies demonstrate selective activity in prostate cancer models

Abstract Bispecific antibodies enable therapeutic modalities inaccessible to traditional mAbs, such as T cell engagers that bind both CD3 on T cells and a target antigen on tumor cells. These agents provide synthetic immunity by expanding, activating, and redirecting T cells against a target of interest. While targeting lineage-restricted antigens such as CD19 or CD20 have found clinical success in hematopoietic cancers, targeting solid tumors requires high expressing tumor associated antigens (TAAs) with minimal normal tissue expression. Prostate cancer (PC) is one of the most prevalent cancers in men, and end stage (castration-resistant prostate cancer) has no curative treatment option. Prostate specific membrane antigen (PSMA), a type II transmembrane protein with a large extracellular domain, has long generated interest as a therapeutic target. It is highly overexpressed in PC compared to normal tissue, and its expression has been shown to correlate with malignancy. Previous attempts to target PSMA include antibody-based radiotherapy and antibody drug conjugates, which have shown some success but can be hampered by the inherent toxicity of the modality. Building upon the XmAb® heterodimeric Fc platform, we generated bispecific antibodies in an XmAb 2+1 Fab2-scFv-Fc format that are bivalent for PSMA and monovalent for CD3. Reducing the affinity of both specificities encouraged avid binding and strong activity on high PSMA expressing cell lines while minimizing reactivity on low expressing cell lines, matching PSMA's differential expression pattern. To ensure biologically valid antigen densities were being considered, IHC was conducted on paraffin embedded arrays of tumor tissue, normal tissue, and cell lines. Upon matching the staining intensity between the three sample types, we identified cell lines with corresponding PSMA antigen density that could serve as proxies of tumor and normal tissue. In vitro T cell-dependent cellular cytotoxicity (TDCC) assays on the relevant cell lines confirmed selective potency on high versus low expressing cells. Citation Format: Alex Nisthal, Matthew Dragovich, Erik W. Pong, Veronica Zeng, Michael Hedvat, Christine Bonzon, Kendra Avery, Rumana Rashid, Connie Ardila, Umesh S. Muchhal, Gregory L. Moore, Seung Y. Chu, John R. Desjarlais. Affinity tuned XmAb®2+1 PSMA x CD3 bispecific antibodies demonstrate selective activity in prostate cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5663.

PSA targeted dual-modality manganese oxide–mesoporous silica nanoparticles for prostate cancer imaging

Studies have shown the potential of nanomaterials for the accurate and early detection of cancer. The aim of the present study was to design and evaluate the value of prostate-specific membrane antigen (PSA)-targeted manganese oxide–mesoporous silica nanoparticles (Mn–Msns) for the detection of prostate cancer. Mn–Msns were prepared, and then conjugated with the PSA antibody and Cy7 to create the multimodality PSA-Mn-Msn-Cy7. Their particle size, zeta potential, stability and magnetic resonance imaging (MRI) features of the nanoparticles were characterized. Optical and MR imaging were evaluated in cell and tumor-bearing mouse models. The Mn in tissues was measured by inductively coupled plasma mass spectrometry. The fabricated nanoparticles were stable and showed good T1relaxivity. The targeted nanoparticles accumulated to a great extent in prostate cancer cells in vitro but not in noncancerous cells. In vivo studies further demonstrated a targeted distribution of PSA-Mn-Msn-Cy7 to cancer tissues as shown by high optical and T1 signals. The targeted distribution was also confirmed by determining the Mn content in the cancer tissues. Our data demonstrate that PSA targeted fluorescence and MR dual-functional nanoparticle can visualize prostate cancer and can be used as NIRF/MR contrast agents.

Therapeutic effects of human monoclonal PSMA antibody-mediated TRIM24 siRNA delivery in PSMA-positive castration-resistant prostate cancer.

Background and Aims: Prostate specific membrane antigen (PSMA) is specifically expressed on prostate epithelial cells and markedly overexpressed in almost all prostate cancers. TRIM24 is also up-regulated from localized prostate cancer to metastatic castration-resistant prostate cancer (CRPC). Because of the high relevance of TRIM24 for cancer development and the universal expression of PSMA in CPRC, we investigated the efficacy of human monoclonal PSMA antibody (PSMAb)-based platform for the targeted TRIM24 siRNA delivery and its therapeutic efficacy in CRPC in vivo and in vitro.Methods: The therapeutic complexes were constructed by conjugating PSMAb and sulfo-SMCC-protamine, and encapsulating TRIM24 siRNA. Flow cytometry, immunofluorescence, and fluorescence imaging were performed to detect the receptor-binding, internalization, and targeted delivery of PSMAb-sulfo-SMCC-protamine (PSP)-FAM-siRNA complex (PSPS) in vitro and in vivo. CCK-8, plate-colony formation, apoptosis, cell cycle, and Transwell assays were performed to evaluate the therapeutic potential of the PSP-TRIM24 siRNA complex in vitro, whereas the in vivo therapeutic efficacy was monitored by small animal imaging, radiography, and micro CT.Results: We confirmed that PSP could efficiently protect siRNA from enzymatic digestion, enable targeted delivery of siRNA, and internalize and release siRNA into PSMA-positive (PSMA+) prostate cancer cells in vitro and in vivo. Silencing TRIM24 expression by the PSP-TRIM24 siRNA complex could dramatically suppress proliferation, colony-formation, and invasion of PSMA+ CRPC cells in vitro, and inhibit tumor growth of PSMA+ CRPC xenografts and bone loss in PSMA+ CRPC bone metastasis model without obvious toxicity at therapeutic doses in vivo.Conclusion: PSMAb mediated TRIM24 siRNA delivery platform could significantly inhibit cell proliferation, colony-formation, and invasion in PSMA+ CRPC in vitro and suppressed tumor growth and bone loss in PSMA+ CRPC xenograft and bone metastasis model.

Preparation of QDs@SiO2/Polystyrene Composite Particles for Cancer Cells Detection

We report a sandwich immunoassay strategy for specific isolation and detection of cancer cells. Prostate cancer LNCaP cells overexpressing PSMA (Prostate-specific membrane antigen) were used as the model target to evaluate the performance of immunoassay. Inorganic/polymer nanocomposites with Janus-like structure and enhanced fluorescence properties were synthesized and applied for immobilization of QDs and the antibodies. PSMA antibodies were covalently conjugated to the QDs@SiO2/PS Janus-like composite nanoparticles to construct the fluorescent probes. Magnetic probes conjugated with polyclonal antibodies were used to capture and isolate the cancer cells. Results indicated high potential of the unique nanoprobes in specific isolation and detection of cancer cells.

PSMA Antibody-Conjugated Pentablock Copolymer Nanomicellar Formulation for Targeted Delivery to Prostate Cancer.

The main purpose of this study was to develop a prostate-specific membrane antigen (PSMA) antibody-conjugated drug-loaded nanomicelles using MPEG--PLA-PCL-PLA-PEG-NH2 pentablock copolymer for targeted delivery of hydrophobic anticancer drugs to prostate cancer cells. During this experiment, monomers of L-lactide, ε-caprolactone, poly(ethylene glycol)-methyl ether, and poly(ethylene glycol)-NH2 were used to prepare pentablock copolymer using the ring opening technique. The pentablock nanomicellar (PBNM) formulation was prepared by the evaporation-rehydration method. The resultant pentablock nanomicelles were then conjugated with PSMA antibody resulting in PSMA-Ab-PTX-PBNM. Both the block copolymers and the nanomicelles were analyzed by hydrogen nuclear magnetic resonance (H-NMR), Fourier-transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD). The obtained nanomicelles (NM) were then analyzed for size and zeta potential using dynamic light scattering-dynamic laser scattering (DLS) and then further submitted to H-NMR and TEM analyses. The XRD, FTIR, and the H-NMR analyses confirmed the structure of the pentablock copolymers. The average size for conjugated nanomicellar was 45nm ± 2.5nm. The average (ζ-potential) was around - 28mV. H-NMR and FTIR analysis done on PSMA-coupled paclitaxel-loaded PBNM showed peaks characteristic of the drug (paclitaxel) and the polymer, confirming the successful encapsulation. TEM analysis showed well-defined spherical morphology and confirmed the size range obtained by the DLS. In vitro release studies revealed sustained slow of PTX in phosphate buffer solution (PBS). Confocal scanning microscopy (TEM) of coumarin6-loaded in PBNM indicated that pentablock nanomicelles were internalized into the prostate cancer (PC-3) cells. Cell proliferation assay showed that nanomicelles ferried paclitaxel into the PC-3 cells and subsequently reduced the cell proliferation. The results depict PTX-PBNM-Ab as a suitable carrier for targeted delivery of drugs to prostate cancer cells.

Potential use of prostate specific membrane antigen (PSMA) for detecting the tumor neovasculature of brain tumors by PET imaging with 89Zr-Df-IAB2M anti-PSMA minibody.

Tumor angiogenesis has attracted increasing attention because of its potential as a valuable marker in the differential diagnosis of brain tumors as well as a novel therapeutic target. Prostate-specific membrane antigen (PSMA) is expressed by the neovasculature endothelium of some tumors, with little to no expression by the tumor cells or normal vasculature endothelium. The aim of this study was to investigate the potential of PSMA for the evaluation of the tumor neovasculature of various brain tumors and the possibility of detecting PSMA expression in brain tumors using PET imaging with 89Zr-Df-IAB2M (anti-PSMA minibody). Eighty-three tissue specimens including gliomas, metastatic brain tumors, primary central nervous system lymphomas (PCNSL), or radiation necroses were analyzed by immunohistochemical staining with PSMA antibody. 89Zr-Df-IAB2M PET scans were performed in three patients with recurrent high-grade gliomas or metastatic brain tumor. PSMA was highly expressed in the vascular endothelium of high-grade glioma and metastatic brain tumor, whereas PSMA was poorly expressed in the vascular endothelium of PCNSL and radiation necrosis. PSMA expression in high-grade gliomas and a metastatic brain tumor was clearly visualized by PET imaging with 89Zr-Df-IAB2M. Furthermore, a trend toward a positive correlation between the degree of 89Zr-Df-IAB2M uptake and PSMA expression levels in tumor specimens was observed. PET imaging of PSMA using 89Zr-Df-IAB2M may have potential value in the differential diagnosis of high-grade glioma from PCNSL or radiation necrosis as well as in the prediction of treatment efficacy and assessment of treatment response to bevacizumab therapy for high-grade glioma.

Abstract 5697: Proteomic analysis of prostate cancer-related exosomes isolated by anti-PSMA antibody beads

Abstract Purposes of the study: Exosome has been demonstrated to be a useful non-invasive biomarker for several cancers including prostate cancer. For better understanding cancer-derived exosome, their proteomic analysis is necessary. The aim of this study is to establish the method for isolation and analysis of prostate cancer-related exosomes. Experimental procedures: Total exosomes were isolated from the conditioned media of LNCaP cells by ultracentrifugation. Then, exosomes positive for CD9 or prostate specific membrane antigen (PSMA) were isolated by magnetic beads conjugated with anti-CD9 or -PSMA antibody, respectively. Isolated exosomes were subjected to proteomic analysis. The PSMA positive fraction was also collected by anti-PSMA beads from diluted serum of 3 prostate cancer patients and analyzed by proteomics. Results: A total of 126, 139 and 13 proteins were detected in the LNCaP exosome fractions that were isolated by ultracentrifugation, anti-CD9 beads and anti-PSMA beads, respectively. Out of 126 proteins identified in ultracentrifuge isolated exosome, 56 and 6 protein were found in exosomes that were isolated by anti-CD9 beads and anti-PSMA beads, respectively. Seven proteins were commonly detected in exosomes that were isolated by both beads. Fifty, 54 and 48 proteins were identified in the PSMA positive fraction from serum of 3 prostate cancer patients (P1: localized prostate cancer, P2: advanced prostate cancer and P3: castration resistant prostate cancer). Thirty-three proteins were detected in one patient, 22 in two patients and 25 in three patients. Most proteins detected in all patients were serum- or immunoglobulin-related proteins. The numbers of proteins that were detected only in P2 and P3 were 16 and 8, respectively. Six proteins were found in both P2 and P3 patients. Conclusion: In the present study, we analyzed prostate cancer-related exosomes that were isolated by immunoaffinity-based method. Although most proteins were serum- or immunoglobulin-related proteins, proteins that had been reported as a cancer marker were also detected, which could be candidates for exosomal marker of prostate cancer. Citation Format: Kosuke Mizutani, Kyojiro Kawakami, Yasunori Fujita, Kengo Horie, Koji Kameyama, Masafumi Ito, Takashi Deguchi. Proteomic analysis of prostate cancer-related exosomes isolated by anti-PSMA antibody beads [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5697. doi:10.1158/1538-7445.AM2017-5697

Co-Delivery of Docetaxel and p44/42 MAPK siRNA Using PSMA Antibody-Conjugated BSA-PEI Layer-by-Layer Nanoparticles for Prostate Cancer Target Therapy.

How to overcome the low accumulation of chemotherapeutic agent in tumor tissue and exhibit multitherapeutics remains an ongoing challenge for cancer treatment. Here, a simple method is demonstrated that used to prepare prostate-specific membrane antigen antibody (PSMAab )-conjugated fluorescent bovine serum albumin (BSA)-branched polyethylenimine layer-by-layer nanoparticles (BSA-PEILBL NPs) for co-delivery of docetaxel (DTX) and p44/42 mitogen-activated protein kinase (MAPK) small interfering RNA (p44/42 MAPK siRNA) as synergistic and selective inhibition of cancer cell proliferation platform. The results show the levels of α-tubulin and p44/42 MAPK in CWR22R cells are significantly reduced after treatment with PSMAab -conjugated DTX/BSA-PEILBL /siRNA NPs. Consequently, the 50% cellular growth inhibition (IC50 ) values of the NPs loaded with both DTX and p44/42 MAPK siRNA are ≈2.1-fold less than those for the NPs only loaded with DTX. The median survival significantly prolongs from 18 d to upward 45 d compared to mice that receive same dose (12 mg kg-1 ) of free DTX. The results suggest this synergistic delivery system may be a promising clinical treatment in prostate cancer.

Prostate-Specific Membrane Antigen-Directed Therapy for Metastatic Castration-Resistant Prostate Cancer.

Prostate-specific membrane antigen (PSMA) is highly expressed on both benign and malignant prostatic tissue. Prostate-specific membrane antigen-directed therapy is conceptually promising, with a potential to additionally serve as a theranostic model in management of advanced prostate cancer. To date, various approaches have been devised and tested, including radiolabeled PSMA antibodies and inhibitor and antibody-drug conjugates. However, development and progress have faced challenges in determining the optimal combination of payload, PSMA-binding moiety, and linker technology. We review the available clinical data to date in PSMA-directed therapies and discuss the challenges faced.

Abstract 1312: PSMA antibody functionalized docetaxel-loaded magnetic nanoparticles for prostate cancer therapy

Abstract Objectives: Prostate cancer (PrCa) is the second most leading cause of cancer-related death in men in the United States. Chemotherapy (Docetaxel, Dox) is currently the most common first-line therapeutic option. However, adverse side effects and chemo-resistance of docetaxel limit its clinical use. Improving docetaxel targeted delivery and its activity at the tumor site using a targeted nanoparticle system could be an attractive strategy for PrCa therapy. Prostate Specific Membrane Antigen (PSMA) is highly overexpressed in PrCa cells, thus is a highly attractive molecular target for PrCa therapy. In this study, we developed and determined anti-cancer efficacy of a novel docetaxel loaded, PSMA targeted magnetic nanoparticle (PSMA-MNP-Dox) formulation for PrCa therapy. Methods: Docetaxel loaded magnetic nanoparticle (MNP-Dox) formulation is composed of an iron oxide core coated with cyclodextrin (for drug loading) and F127 polymer (for particle stability and chemosensitization). Therapeutic efficacy of this unique nanoparticle formulation was evaluated using clinically relevant cell line models (C4-2, PC-3, and DU-145) through cell proliferation and colony formation assays. Molecular effects of this formulation on apoptosis, anti-apotosis, and drug resistance associated proteins were evaluated using immunoblotting assays. Contrast imaging property of MNP-Dox formulation was examined using Phantom Gel MR imaging model. For active targeting, PSMA antibody conjugation to this formulation was achieved through N-hydroxysuccinimide group containing PEG polymer. Active targeting potential of this formulation was evaluated in PSMA+ (C4-2) and PSMA- (PC-3) cell lines, C4-2 generated tumor xenografts. Results: MNP-Dox formulation showed optimal particle size and zeta potential which can efficiently internalized in PrCa cells. Our formulation showed anti-cancer efficacy in prostate cancer cell lines. Additionally, it induces the expression of apoptosis associated proteins, Bax and Bad, cleaved PARP, and caspase 3, and downregulated the expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL. Moreover, it also inhibited the expression of chemoresistance associated proteins (PSMA and MDR1). Our PSMA antibody targeted MNPs-Dox formulation exhibited a profound uptake pattern in PSMA+ cells (C4-2) compared to PSMA null (PC-3)- cells, suggesting its targeting potential. A similar targeting potential was also observed in ex-vivo studies while using C4-2 tumor xenografts, however, no intense targeting was observed in normal tissues due to lack of PSMA expression. Conclusion: PSMA antibody functionalized MNP-Dox formulation can efficiently target PSMA + PrCa cells and deliver docetaxel into prostate tumors. This targeted drug delivery system could reduce the dose of docetaxel required to kill cancer cells, thus minimizing long-term docetaxel associated systemic toxicity and drug-resistance. Citation Format: Prashanth Kumar Bhusetty Nagesh, Nia Johnson, Vijaya K.N. Boya, Pallabita Chowdhury, Aditya Ganju, Bilal Hafeez, Sheema Khan, Meena Jaggi, Subhash C. Chauhan, Murali M. Yallapu. PSMA antibody functionalized docetaxel-loaded magnetic nanoparticles for prostate cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1312.

PSMA targeted docetaxel-loaded superparamagnetic iron oxide nanoparticles for prostate cancer

Docetaxel (Dtxl) is currently the most common therapeutic option for prostate cancer (PC). However, adverse side effects and problems associated with chemo-resistance limit its therapeutic outcome in clinical settings. A targeted nanoparticle system to improve its delivery to and activity at the tumor site could be an attractive strategy for PC therapy. Therefore, the objective of this study was to develop and determine the anti-cancer efficacy of a novel docetaxel loaded, prostate specific membrane antigen (PSMA) targeted superparamagnetic iron oxide nanoparticle (SPION) (J591-SPION-Dtxl) formulation for PC therapy. Our results showed the SPION-Dtxl formulation exhibits an optimal particle size and zeta potential, which can efficiently be internalized in PC cells. SPION-Dtxl exhibited potent anti-cancer efficacy via induction of the expression of apoptosis associated proteins, downregulation of anti-apoptotic proteins, and inhibition of chemo-resistance associated protein in PC cell lines. J591-SPION-Dtxl exhibited a profound uptake in C4-2 (PSMA+) cells compared to PC-3 (PSMA−) cells. A similar targeting potential was observed in ex-vivo studies in C4-2 tumors but not in PC-3 tumors, suggesting its tumor specific targeting. Overall, this study suggests that a PSMA antibody functionalized SPION-Dtxl formulation can be highly useful for targeted PC therapy.

Nanoconjugation of PSMA-Targeting Ligands Enhances Perinuclear Localization and Improves Efficacy of Delivered Alpha-Particle Emitters against Tumor Endothelial Analogues.

This study aims to evaluate the effect on killing efficacy of the intracellular trafficking patterns of α-particle emitters by using different radionuclide carriers in the setting of targeted antivascular α-radiotherapy. Nanocarriers (lipid vesicles) targeted to the prostate-specific membrane antigen (PSMA), which is unique to human neovasculature for a variety of solid tumors, were loaded with the α-particle generator actinium-225 and were compared with a PSMA-targeted radiolabeled antibody. Actinium-225 emits a total of four α-particles per decay, providing highly lethal and localized irradiation of targeted cells with minimal exposure to surrounding healthy tissues. Lipid vesicles were derivatized with two types of PSMA-targeting ligands: a fully human PSMA antibody (mAb) and a urea-based, low-molecular-weight agent. Target selectivity and extent of internalization were evaluated on monolayers of human endothelial cells (HUVEC) induced to express PSMA in static incubation conditions and in a flow field. Both types of radiolabeled PSMA-targeted vesicles exhibit similar killing efficacy, which is greater than the efficacy of the radiolabeled control mAb when compared on the basis of delivered radioactivity per cell. Fluorescence confocal microscopy demonstrates that targeted vesicles localize closer to the nucleus, unlike antibodies which localize near the plasma membrane. In addition, targeted vesicles cause larger numbers of dsDNAs per nucleus of treated cells compared with the radiolabeled mAb. These findings demonstrate that radionuclide carriers, such as PSMA-targeted lipid-nanocarriers, which localize close to the nucleus, increase the probability of α-particle trajectories crossing the nuclei, and, therefore, enhance the killing efficacy of α-particle emitters.