PSMA-positive Prostate Cancer Cells Research Articles

Evaluation of a bimodal, matched pair theranostic agent targeting prostate-specific membrane antigen

BackgroundProstate cancer affects 1 in 6 men, and it is the second‑leading cause of cancer-related death in American men. Surgery is one of the main treatment modalities for prostate cancer, but it often results in incomplete resection margins or complete resection that leads to nerve damage and undesirable side effects. In the present work, we have developed a new bimodal tracer, NODAGA-sCy7.5 PSMAi (prostate-specific membrane antigen inhibitor), labeled with the true matched theranostic pair 64Cu/67Cu and a near-infrared fluorescent dye. This agent could potentially be used for concomitant PET imaging, optical surgical navigation, and targeted radiopharmaceutical therapy. MethodsA prostate-specific membrane antigen (PSMA)-targeting urea derivative was conjugated to NODAGA for copper radiolabeling and to the near-infrared fluorophore sulfo-Cy7.5 (sCy7.5). Binding studies were performed in PSMA-positive PC-3 PIP cells, as well as uptake and internalization assays in PC-3 PIP cells and PSMA-negative PC-3 wild type cells. Biodistribution studies of the 64Cu-labeled compound were performed in PC-3 PIP- and PC-3 tumor-bearing mice, and 67Cu biodistributions of the agent were obtained in PC-3 PIP tumor-carrying mice. PET imaging and fluorescence imaging were also performed, using the same molar doses, in the two mouse models. ResultsThe PSMA conjugate bound with high affinity to PSMA-positive prostate cancer cells, as opposed to cells that were PSMA-negative. Uptake and internalization were rapid and PSMA-mediated in PC-3 PIP cells, while only minimal non-specific uptake was observed in PC-3 cells. Biodistribution studies showed specific uptake in PC-3 PIP tumors, while accumulation in PC-3 tumor-bearing mice was low. Furthermore, tumor uptake of the 67Cu-labeled agent in the PC-3 PIP model was statistically equivalent to that of 64Cu. PET and fluorescence imaging at 0.5 nmol per mouse also demonstrated that PC-3 PIP tumors could be clearly detected, while PC-3 tumors showed no tumor accumulation. ConclusionsNODAGA-sCy7.5-PSMAi was specific and selective in detecting PSMA-positive, as opposed to PSMA-negative, tumors in mouse models of prostate cancer. This bioconjugate could potentially be used for PET staging with 64Cu, targeted radiopharmaceutical therapy with 67Cu, and/or image-guided surgery with sCy7.5.

PSMA-targeted small-molecule drug-conjugates with valine-citrulline and phosphoramidate cleavable linkers

In this study, we present a modular synthesis and evaluation of two prostate-specific membrane antigen (PSMA) targeted small molecule drug conjugates (SMDCs) incorporating the potent chemotherapeutic agent monomethyl auristatin E (MMAE).These SMDCs are distinguished by their cleavable linker modules: one utilizing the widely known valine-citrulline linker, susceptible to cleavage by cathepsin B, and the other featuring a novel acid-labile phosphoramidate-based (PhosAm) linker. Both SMDCs maintained nanomolar affinity to PSMA. Furthermore, we confirmed the selective release of the payload and observed chemotherapeutic efficacy specifically within PSMA-positive prostate cancer cells, while maintaining cell viability in PSMA-negative cells. These findings not only validate the efficacy of our approach but also highlight the potential of the innovative pH-responsive PhosAm linker. This study contributes significantly to the field and also paves the way for future advancements in targeted cancer therapy.

Preclinical evaluation of [58mCo]Co-DOTA-PSMA-617 for Auger electron therapy of prostate cancer

Prostate-specific membrane antigen (PSMA), highly expressed in prostate cancer, is a promising target for radionuclide therapy. Auger electron-emitting radionuclides are well suited for targeted radionuclide therapy if they can be delivered close to the DNA of the targeted cells. This preclinical study evaluated the theranostic pair [55/58mCo]Co-DOTA-PSMA-617 for PET imaging and Auger electron therapy of prostate cancer. [58mCo]Co-DOTA-PSMA-617 was successfully prepared with > 99% radiochemical yield and purity. In vitro, uptake and subcellular distribution assays in PSMA-positive prostate cancer cells showed PSMA-specific uptake with high cell-associated activity in the nucleus. Incubation with [58mCo]Co-DOTA-PSMA-617 reduced cell viability and clonogenic survival in a significant dose-dependent manner (p < 0.05). Biodistribution of xenografted mice showed high specific tumor uptake of the cobalt-labeled PSMA ligand for all time points with rapid clearance from normal tissues, which PET imaging confirmed. In vivo, therapy with [58mCo]Co-DOTA-PSMA-617 in tumor-bearing mice demonstrated significantly increased median survival for treated mice compared to control animals (p = 0.0014). In conclusion, [55/58mCo]Co-DOTA-PSMA-617 displayed excellent in vitro and in vivo properties, offering significant survival benefits in mice with no observed toxicities.

Dominant negative TGFβ receptor II and truncated TIM3 enhance the antitumor efficacy of CAR-T-cell therapy in prostate cancer

BackgroundThe immune checkpoint molecules, Transforming growth factor beta receptor II (TGFβRII) and T cell immunoglobulin and mucin domain 3 (TIM3), have been identified as contributors to T cell immune suppression in prostate cancer. The objective of this investigation was to improve the tumor killing capability of prostate-specific membrane antigen (PSMA)-chimeric antigen receptor T (CAR-T) cells by targeting TIM3 and TGFβRII simultaneously. MethodsTo generate dnTGFβRII-trTIM3-PSMA-CAR-T (DT-PSMA-CAR-T) cells, the surface of PSMA-CAR-T cells was overexpressed with dominant negative TGFβRII (dnTGFβRII) and truncated extracellular TIM3 (trTIM3). The efficacy of DT-PSMA-CAR-T cells was assessed through in vitro killing experiments and animal experiments. ResultsThe DT-PSMA-CAR-T cells demonstrated the ability to eradicate PSMA-positive prostate cancer cells, even in the presence of exogenous TGF-β and/or TIM3 activating antibodies. In addition, the cells demonstrated the ability to eliminate tumor tissue in an immunodeficient mouse model transplanted with GAL9-PSMA-PC3 cells in vitro, prolonging survival without significant toxic side effects. ConclusionsThis study emphasizes that upregulating dnTGFβRII and trTIM3 on the surface of T cells can potentially diminish the inhibitory effects of TGFβRII and TIM3.

Abstract 1787: Design of bispecific adapters for universal CAR T cell therapies of solid tumors

Abstract Recent success in the use of chimeric antigen receptor (CAR) T cell therapies to treat hematologic cancers has encouraged the exploration of CAR T cell technologies to treat solid tumors. For establishment of a productive immunologic synapse between the CAR T cell and cancer cell, multiple CARs on the T cell surface must engage multiple tumor antigens on the cancer cell surface. Whereas these binding interactions are mediated in classical CAR T cells by direct recognition of the tumor antigen by the scFv on the CAR, in the case of universal CAR T cells this recognition must be created by a bispecific adapter that can bridge between the tumor antigen and the CAR. While a diversity of bispecific adapters have been explored in the literature, the bispecific adapter used here is comprised of a low molecular weight ligand that binds avidly to the desired tumor antigen, the yellow dye fluorescein (FL) that binds tightly to an anti-fluorescein scFv on the CAR, and a polyethylene glycol (PEG) spacer that spans between the two. The length of this PEG spacer appears to be critical to ensure that 1) the bispecific adapter is long enough to mediate engagement of the universal CAR T cell with the cancer cell, and 2) the resulting intercellular distance is optimal for activating the necessary co-receptor interactions.In our study, we have utilized protein structure data and molecular modeling to estimate the optimal adapter spacer lengths for bridging between our CAR T cell and two important target cells, namely PSMA positive prostate cancer cells and fibroblast activation protein (FAP) expressing cancer associated fibroblasts. We then synthesized the desired bispecific adapters with a variety lengths of PEG spacer and evaluated their effects in mediating the cytotoxicity of our universal anti-FL CAR T cells both in vitro and in vivo. Our results reveal that the optimal spacer lengths in the bispecific adapters for targeting PSMA (DUPA-FL) and FAP (FAPL-FL) are different and depend on the locations of the adapter binding pockets on their respective protein targets, as predicted by molecular modeling. Specifically, a shorter PEG spacer is required for maximal cytoxicity when employing the PSMA-targeted adapter, whereas a much longer PEG spacer is needed to achieve maximal cell killing with the FAP-targeted adapter. In addition, spacer length can also be adjusted to avoid overactivation-induced T cell exhaustion. Taken together, our studies demonstrate that instead of optimizing the hinge length of a CAR for each antigen epitope, our bispecific adapter/universal CAR platform provides a highly flexible strategy for optimizing the intermembrane distance between the universal CAR T cell and cancer cell to achieve maximal target cell killing with minimal CAR T cell exhaustion. Citation Format: Suilan Zheng, Bo Huang, Yashapal Singh, Haiyan Chu, Suresh K. Bowroju, Md Sazzadul Bari, Madduri Srinivasarao, Sudarsan R. Kasireddy, Ramesh Mukkamala, Laurie Beitz, Byoung Ryu, Andrew M. Scharenberg, Michael Jensen, Philip S. Low. Design of bispecific adapters for universal CAR T cell therapies of solid tumors [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 1787.

Abstract 2638: Preclinical development of ADCT-212, a PSMA-targeted antibody-drug conjugate employing the pyrrolobenzodiazepine dimer SG2000 for PSMA-expressing cancers

Abstract Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase that is highly expressed in nearly all prostate cancers with the highest expression in metastatic castration-resistant prostate cancer. Moreover, PSMA is expressed in the neovasculature that supplies most non-prostatic solid tumors, including carcinomas of the lung, colon, breast, kidney, liver, and pancreas. ADCT-212 is an antibody-drug conjugate composed of the human IgG1 antibody 2A10 directed against human PSMA, site-specifically conjugated using GlycoConnectTM technology to PL1801, which contains HydraspaceTM, a valine-alanine cleavable linker and the pyrrolobenzodiazepine (PBD) dimer warhead SG2000 (drug-antibody ratio of ~1.8). The purpose of this study was to characterize the in vitro and in vivo anti-tumor activity of ADCT-212 in human cancer cell lines and xenograft models and to determine its tolerability and pharmacokinetic (PK) in the rat. In vitro, ADCT-212 demonstrated potent cytotoxicity in a panel of PSMA-positive prostate cancer cell lines, whereas its activity was greatly reduced in PSMA-negative cell lines. ADCT-212 was efficiently internalized by PSMA-expressing LNCaP cells. Trafficking to the lysosomes started as early as 30 minutes and peaked at 1-2 hours post treatment. In line with the mechanism of action of the PBD dimer, ADCT-212 produced DNA interstrand cross-links (ICLs) that peaked by 12 hours and persisted for up to 36 hours post-treatment. In contrast, the peak of DNA ICLs formation for SG2000, the PBD dimer warhead alone, was observed immediately after 2-hour incubation, while a non-targeted PBD-ADC did not yield any appreciable DNA ICLs. Moreover, ADCT-212 showed indirect bystander killing activity in PSMA-negative PC3 cells incubated with conditioned medium from ADCT-212-treated LNCaP cells. In vivo, ADCT-212 showed strong antitumor activity against CWR22Rv1 and LNCaP prostate cancer xenograft models. In the CWR22Rv1 model, a tumor with heterogeneous PSMA expression, ADCT-212 achieved dose-dependent antitumor activity when administered as single dose at 2, 4 or 6 mg/kg, which resulted in increased survival compared to the control animals. In the LNCaP model, a single dose of ADCT-212 at 5 or 10 mg/kg resulted in strong and specific antitumor activity. Conversely, in the PSMA-negative PC3 xenograft model, ADCT-212 did not show anti-tumor activity compared to the controls, highlighting its target-mediated antitumor activity. ADCT-212 was tolerated as a single 20 mg/kg dose in male rats, with exposure data being indicative of a linear PK profile with a half-life of approximately 12 days. In conclusion, ADCT-212 demonstrated potent and specific in vitro and in vivo anti-tumor activity while it was stable and well tolerated in the rat, warranting further development of ADCT-212 into the clinic. Citation Format: Francesca Zammarchi, Karin Havenith, Lolke de Haan, Ian Kirby, Narinder Janghra, Veronica Gil, Pedro Alves, Kristina Zaitseva, Meghann Kerr, Ben Leatherdale, Afroze Patel, Marie Thoelke, Shiran Huang, John A Hartley, Patrick H van Berkel. Preclinical development of ADCT-212, a PSMA-targeted antibody-drug conjugate employing the pyrrolobenzodiazepine dimer SG2000 for PSMA-expressing cancers [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 2638.

SiAKR1C3@PPA complex nucleic acid nanoparticles inhibit castration-resistant prostate cancer in vitro.

AKR1C3, as a crucial androgenic enzyme, implicates the androgen biosynthesis and promoting prostate cancer cell growth in vitro. This study provides a new gene therapy strategy for targeting AKR1C3 to treat castration-resistant prostate cancer. siAKR1C3@PPA is assembled from PEG3500, PAMAM, Aptamer-PSMA, and siRNA for AKR1C3. We analyzed the relationship between AKR1C3 expression and the survival rate of prostate cancer patients based on the GEPIA online database to perform disease-free survival, and found that AKR1C3 may be an important factor leading to poor prognosis in prostate cancer. Considering AKR1C3 as a therapeutic target for castration-resistant prostate cancer, we constructed a complex nucleic acid nanoparticle, siAKR1C3@PPA to investigate the inhibitory effect on castration-resistant prostate cancer. Aptamer-PSMA acts as a target to guide siAKR1C3@PPA into PSMA-positive prostate cancer cells and specifically down regulate AKR1C3. Cyclin D1 was decreased as a result of siAKR1C3@PPA treatment. Changes in Cyclin D1 were consistent with decreased expression of AKR1C3 in LNCaP-AKR1C3 cells and 22RV1 cells. Furthermore, in the LNCaP-AKR1C3 group, 1070 proteins were upregulated and 1015 proteins were downregulated compared to the LNCaP group according to quantitative 4D label-free proteomics. We found 42 proteins involved in cell cycle regulation. In a validated experiment, we demonstrated that PCNP and CINP were up-regulated, and TERF2 and TP53 were down-regulated by western blotting. We concluded that siAKR1C3@PPA may arrest the cell cycle and affect cell proliferation.

Abstract 6401: PSMA-positive membranes secreted from prostate cancer cells have potency to transform vascular endothelial cells into an angiogenic state

Abstract Introduction &amp; objectives: Prostate-specific membrane antigen (PSMA) is highly expressed in poorly differentiated, metastatic, and castration-resistant prostate cancers. Recently, 68Ga-PSMA Positron Emission Tomography/Computed Tomography (PET/CT) has been successfully developed as an effective diagnostic tool for prostate cancer. However, the pathophysiological functions of PSMA in prostate tumors remain unclear. Materials &amp; methods: We examined the protein expression of PSMA in tumor endothelial cells in human prostate tumors by immunohistochemistry. Prostate cancer tissues were resected by robotic surgery in 2019 at Ehime University from patients with prostate cancer. In vitro, we prepared conditioned medium derived from a PSMA-positive human prostate cancer cell line, LNCaP, cultured on collagen I gels. We then examined PSMA expression in human umbilical vascular endothelial cells (HUVECs) cultured with the conditioned medium. We assessed angiogenic activities by treatment of HUVECs with LNCaP-derived conditioned medium using a tube formation assay that mimics angiogenesis. Results: Immunohistochemical positivity of PSMA and CD31, a marker of endothelial cells, and PSMA-expressing tumor endothelial cells were observed in four of 33 prostate cancer patients (12.1%). We also found that the 10,000 × g pellet fraction of the LNCaP-derived conditioned medium containing PSMA-positive membranes, including microvesicles, transformed HUVECs from “PSMA-negative” to “PSMA-positive.” Mass spectrometry revealed that the fraction contained an important growth factor. Furthermore, treating HUVECs with the 10,000 × g pellet fraction of the LNCaP-derived conditioned medium significantly promoted tube formation, mimicking angiogenesis in a PSMA-dependent manner. Conclusions: Our findings revealed the existence of PSMA-positive tumor endothelial cells in human prostate tumors, which enhances tumor angiogenesis in prostate cancer tissues. The endocytic process of the PSMA-positive microvesicles in normal endothelial cells may be an attractive target to develop novel anti-angiogenic drugs, which could inhibit transformation of normal endothelial cells to tumor endothelial cells (Watanabe et al., The Prostate. 2021. in press). Citation Format: RYUTA WATANABE, MASASHI MAEKAWA, TAKASHI KIYOI, MIE KURATA, NORIYOSHI MIURA, TADAHIKO KIKUGAWA, SHIGEKI HIGASHIYAMA, TAKASHI SAIKA. PSMA-positive membranes secreted from prostate cancer cells have potency to transform vascular endothelial cells into an angiogenic state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6401.

Glutamate-urea-based PSMA-targeted PLGA nanoparticles for prostate cancer delivery of docetaxel

Targeted drug delivery is a tool to make treatment more specific, selective, and effective and to prevent unwanted complications. Prostate specific membrane antigen (PSMA) is a useful biomarker in order to monitor and control prostate cancer. Glutamate-Urea-R (Glu-Urea-R) is a PSMA enzyme inhibitor capable of binding to this surface marker of prostate cancer cell in an efficient and special manner. The aim of this project was to develop a docetaxel-loaded nanoparticle of poly (lactic-co-glycolic acid) polyethylene glycol which is cojugated to a urea-based anti-PSMA ligand named glutamate-urea-lysine (glu-urea-lys) for targeted delivery of docetaxel in prostate cancer. The obtained nanoparticles, prepared by nanoprecipitation method, were spheres with a particle size of around 150 nm and zeta potential of −7.08 mV. Uptake studies on the PC3 (as PSMA negative) and LNCaP (as PSMA positive) cells demonstrated that drug uptake was efficient by the PSMA positive cells. IC50 of targeted NPs on LNCaP cell line compared to non-targeted ones was reduced by more than 70% in three different incubation times of 24, 48, and 72 h. In conclusion, the nanoparticles are expected to specifically transport docetaxel to PSMA-positive prostate cancer cells and consequently, enhance the antitumor efficacy of docetaxel on these cells.

Synthesis and in vitro preliminary evaluation of prostate-specific membrane antigen targeted upconversion nanoparticles as a first step towards radio/fluorescence-guided surgery of prostate cancer.

Over the last decade, upconversion nanoparticles (UCNP) have been widely investigated in nanomedicine due to their high potential as imaging agents in the near-infrared (NIR) optical window of biological tissues. Here, we successfully develop active targeted UCNP as potential probes for dual NIR-NIR fluorescence and radioactive-guided surgery of prostate-specific membrane antigen (PSMA)(+) prostate cancers. We designed a one-pot thermolysis synthesis method to obtain oleic acid-coated spherical NaYF4:Yb,Tm@NaYF4 core/shell UCNP with narrow particle size distribution (30.0 ± 0.1 nm, as estimated by SAXS analysis) and efficient upconversion luminescence. Polyethylene glycol (PEG) ligands bearing different anchoring groups (phosphate, bis- and tetra-phosphonate-based) were synthesized and used to hydrophilize the UCNP. DLS studies led to the selection of a tetra-phosphonate PEG(2000) ligand affording water-dispersible UCNP with sustained colloidal stability in several aqueous media. PSMA-targeting ligands (i.e., glutamate-urea-lysine derivatives called KuEs) and fluorescent or radiolabelled prosthetic groups were grafted onto the UCNP surface by strain-promoted azide-alkyne cycloaddition (SPAAC). These UCNP, coated with 10 or 100% surface density of KuE ligands, did not induce cytotoxicity over 24 h incubation in LNCaP-Luc or PC3-Luc prostate cancer cell lines or in human fibroblasts for any of the concentrations evaluated. Competitive binding assays and flow cytometry demonstrated the excellent affinity of UCNP@KuE for PSMA-positive LNCaP-Luc cells compared with non-targeted UCNP@CO2H. Furthermore, the binding of UCNP@KuE to prostate tumour cells was positively correlated with the surface density of PSMA-targeting ligands and maintained after 125I-radiolabelling. Finally, a preliminary biodistribution study in LNCaP-Luc-bearing mice demonstrated the radiochemical stability of non-targeted [125I]UCNP paving the way for future in vivo assessments.

Targeted Nanobubbles Carrying Indocyanine Green for Ultrasound, Photoacoustic and Fluorescence Imaging of Prostate Cancer.

ObjectiveTo construct prostate-specific membrane antigen (PSMA)-targeting, indocyanine green (ICG)-loaded nanobubbles (NBs) for multimodal (ultrasound, photoacoustic and fluorescence) imaging of prostate cancer.MethodsThe mechanical oscillation method was used to prepare ICG-loaded photoacoustic NBs (ICG NBs). Then, PSMA-binding peptides were connected to the surface of ICG NBs using the biotin–avidin method to make targeted photoacoustic NBs, namely, PSMAP/ICG NBs. Their particle sizes, zeta potentials, and in vitro ultrasound, photoacoustic and fluorescence imaging were examined. Confocal laser scanning microscopy and flow cytometry were used to detect the binding ability of the PSMAP/ICG NBs to PSMA-positive LNCaP cells, C4-2 cells, and PSMA-negative PC-3 cells. The multimodal imaging effects of PSMAP/ICG NBs and ICG NBs were compared in nude mouse tumor xenografts.ResultsThe particle size of the PSMAP/ICG NBs was approximately 457.7 nm, and the zeta potential was approximately −23.5 mV. Both confocal laser scanning microscopy and flow cytometry confirmed that the PSMAP/ICG NBs could specifically bind to both LNCaP and C4-2 cells, but they rarely bound to PC-3 cells. The ultrasound, photoacoustic and fluorescence imaging intensities of the PSMAP/ICG NBs in vitro positively correlated with their concentrations. The ultrasound and photoacoustic imaging effects of the PSMAP/ICG NBs in LNCaP and C4-2 tumor xenografts were significantly enhanced compared with those in PC-3 tumor xenografts, which were characterized by a significantly increased duration of ultrasound enhancement and heightened photoacoustic signal intensity (P < 0.05). Fluorescence imaging showed that PSMAP/ICG NBs could accumulate in LNCaP and C4-2 tumor xenografts for a relatively long period.ConclusionThe targeted photoacoustic nanobubbles prepared in this study can specifically bind to PSMA-positive prostate cancer cells and have the ability to enhance ultrasound, photoacoustic and fluorescence imaging of PSMA-positive tumor xenografts. Photoacoustic imaging could visually display the intensity of the red photoacoustic signal in the tumor region, providing a more intuitive imaging modality for targeted molecular imaging. This study presents a potential multimodal contrast agent for the accurate diagnosis and assessment of prostate cancer.

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.

Antitumor Activity of MEDI3726 (ADCT-401), a Pyrrolobenzodiazepine Antibody-Drug Conjugate Targeting PSMA, in Preclinical Models of Prostate Cancer.

Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase that is highly expressed in nearly all prostate cancers with the highest expression in metastatic castration-resistant prostate cancer (mCRPC). The prevalence of increased surface expression and constitutive internalization of PSMA make it an attractive target for an antibody-drug conjugate (ADC) approach to treating patients with mCRPC. MEDI3726 (previously known as ADCT-401) is an ADC consisting of an engineered version of the anti-PSMA antibody J591 site specifically conjugated to the pyrrolobenzodiazepine (PBD) dimer tesirine. MEDI3726 specifically binds the extracellular domain of PSMA and, once internalized, releases the PBD dimer to crosslink DNA and trigger cell death. In vitro, MEDI3726 demonstrated potent and specific cytotoxicity in a panel of PSMA-positive prostate cancer cell lines, consistent with internalization and DNA interstrand crosslinking. In vivo, MEDI3726 showed robust antitumor activity against the LNCaP and the castration-resistant CWR22Rv1 prostate cancer cell line xenografts. MEDI3726 also demonstrated durable antitumor activity in the PSMA-positive human prostate cancer patient-derived xenograft (PDX) LuCaP models. This activity correlated with increased phosphorylated Histone H2AX in tumor xenografts treated with MEDI3726. MEDI3726 is being evaluated in a phase I clinical trial as a treatment for patients with metastatic castrate-resistant prostate cancer (NCT02991911). Mol Cancer Ther; 17(10); 2176-86. ©2018 AACR.

Development of a Ga-68 labeled PET tracer with short linker for prostate-specific membrane antigen (PSMA) targeting

Glu-Urea-Lys (GUL) derivatives have been reported as prostate-specific membrane antigen (PSMA) agent. We developed derivatives of GUL conjugated with NOTA or DOTA via a thiourea linker and tested their feasibility as PSMA imaging agents after labeling with 68Ga. NOTA-GUL and DOTA-GUL were synthesized and labeled with 68Ga using generator-eluted 68GaCl3 in 0.1 M HCl in the presence of 1 M NaOAc at pH 5.5. The stabilities of 68Ga-labeled compounds in human serum were tested at 37.5 °C. A competitive binding assay was performed using the PSMA-positive prostate cancer cell line 22Rv1 and [125I]MIP-1072 (PSMA-specific binding agent) as a tracer. Biodistribution and micro-PET studies were performed using 22Rv1-xenograft BALB/c nude mice. The radiolabeling efficiency of NOTA-GUL (>99%) was higher than that of DOTA-GUL (92%). The IC50 of Ga-NOTA-GUL was 18.3 nM. In the biodistribution study, tumor uptake of 68Ga-NOTA-GUL (5.40% ID/g) was higher than that of 68Ga-DOTA-GUL (4.66% ID/g) at 1 h. Tumor/muscle and tumor/blood uptake ratios of 68Ga-NOTA-GUL (31.8 and 135, respectively) were significantly higher than those of 68Ga-DOTA-GUL (16.1 and 31.1, respectively). The tumor/kidney uptake ratio of 68Ga-NOTA-GUL was 3.4-fold higher than that of 68Ga-DOTA-GUL. 68Ga-NOTA-GUL showed specific uptake to PSMA positive tumor xenograft and was blocked by co-injection of the cold ligand. In conclusion, we successfully synthesized 68Ga-NOTA-GUL and 68Ga-DOTA-GUL for prostate cancer imaging. 68Ga-NOTA-GUL showed better radiochemical and biodistribution results. 68Ga-NOTA-GUL may be a promising PSMA targeting radiopharmaceutical.

Synthesis and evaluation of a novel near-infrared fluorescent probe based on succinimidyl-Cys-C(O)-Glu that targets prostate-specific membrane antigen for optical imaging

Prostate-specific membrane antigen (PSMA), which is highly expressed in both localized and metastatic prostate cancer (PCa), is an ideal target for imaging and therapy of PCa. We previously reported radiolabeled asymmetric urea derivatives asa PSMA-targeting radiotracer for single-photon emission computed tomography (SPECT) and positron emission tomography (PET) imaging. Here, based on these radiopharmaceutical probes, we designed a novel near-infrared (NIR) fluorescent imaging probe (800CW-SCE) by chemical conjugation between IRDye 800CW-Maleimide and an asymmetric urea compound, known as PSMA inhibitor, for optical imaging. In the in vitro cellular uptake study, 800CW-SCE was internalized into PSMA-positive PCa cells (LNCaP cells) but not into PSMA-negative PCa cells (PC-3 cells). Moreover, in the in vivo imaging study, the probe was highly accumulated in LNCaP tumors but not in PC-3 tumors, and remained in LNCaP tumors until 24h after intravenous administration. These results suggest that the potent NIR conjugate may contribute to clinical intraoperative optical imaging.

Enhanced growth inhibition of prostate cancer in vitro and in vivo by a recombinant adenovirus-mediated dual-aptamer modified drug delivery system

The peptide aptamer DUP-1 targets prostate-specific membrane antigen (PSMA)-negative cells, while the RNA aptamer A10-3.2 targets PSMA-positive prostate cancer cells. Moreover, the tumor-suppressor gene phosphatase and tensin homolog (PTEN) and the chemotherapeutic agent doxorubicin (DOX) effectively inhibit prostate cancer, and a recombinant adenovirus (Ad5) mediates high gene transfer efficiency. Here, we design a dual-aptamer modified tumor targeting gene and DOX delivery system mediated by recombinant adenovirus (A10-3.2(DOX)/DUP-1-PEG-Ad5, ADDP-Ad5). DUP-1 and A10-3.2 are connected to the adenovirus through polyethylene glycol (PEG), PTEN is integrated into Ad5, and DOX is embedded into the double chain of aptamer A10-3.2. The PEG-modification rate of Ad5 is 98.70 ± 2.43%. The DUP-1 and A10-3.2 modified products yield 80.40 ± 1.36% and 82.20 ± 2.14%, respectively. The uptake of ADDP-Ad5 and the expression of the reporter gene are enhanced by the system in PSMA-positive LNCaP and PSMA-negative PC3 human prostate cancer cells. ADDP-Ad5 significantly inhibits the cell growth of both LNCaP and PC3 cells. More importantly, ADDP-Ad5 is active in vivo against LNCaP and PC3 tumor xenografts and exhibits no significant toxicity to the mice. Therefore, ADDP-Ad5 may have clinical potential in prostate cancer therapy.

Discovery of PSMA-specific peptide ligands for targeted drug delivery

Prostate-specific membrane antigen (PSMA) has been widely used as a biomarker and targeting receptor for prostate cancer therapy because of its overexpression in most prostate cancer cells. In this study, a novel combinatorial phage biopanning procedure was developed to discover PSMA-specific peptides that can be potentially used as ligands for targeted drug delivery to prostate cancer cells. Five rounds of biopanning against recombinant human PSMA extracellular domain (ECD), PSMA-positive LNCaP cells, and LNCaP xenografts in nude mice were conducted. Various affinity assays were conducted to identify high-affinity peptides for PSMA ECD and PSMA-positive prostate cancer cells. Among them, the GTI peptide shows the highest affinity as well as specificity to PSMA in prostate cancer cells. The apparent Kd values of the GTI peptide to PSMA-positive LNCaP and C4-2 cells are 8.22μM and 8.91μM, respectively. The GTI peptide can specifically deliver the proapoptotic peptide D(KLAKLAK)2 to LNCaP cells to induce cell death. In the biodistribution study, the GTI peptide shows the highest uptake in C4-2 xenografts, while its uptake in other major organs, such as the liver and spleen, are either low or negligible. Compared to its scrambled control (random permutation of the GTI peptide), the GTI peptide exhibits higher and more specific uptake in C4-2 xenografts. All the results suggest that the GTI peptide is a potentially promising ligand for PSMA-targeted drug delivery for prostate cancer therapy.

Systemic Administration and Targeted Radiosensitization via Chemically Synthetic Aptamer-siRNA Chimeras in Human Tumor Xenografts.

Radiation therapy is a highly effective tool for treating all stages of prostate cancer, from curative approaches in localized disease to palliative care and enhanced survival for patients with distant bone metastases. The therapeutic index of these approaches may be enhanced with targeted radiation-sensitizing agents. Aptamers are promising nucleic acid delivery agents for short interfering RNAs (siRNA) and short hairpin RNAs (shRNA). We have previously developed a radiation-sensitizing RNA aptamer-shRNA chimera that selectively delivers DNA-PK targeting shRNAs to prostate-specific membrane antigen (PSMA) positive cells in the absence of transfection reagents. Although these chimera are effective, their synthesis requires in vitro transcription and their evaluation was limited to intratumoral administration. Here, we have developed a second-generation aptamer-siRNA chimera that can be assembled through the annealing of three separate chemically synthesized components. The resulting chimera knocked down DNA-PK in PSMA-positive prostate cancer cells, without the need of additional transfection reagents, and enhanced the efficacy of radiation-mediated cell death. Following intravenous injection, the chimera effectively knocked down DNA-PK in established subcutaneous PSMA-positive tumors. Systemic treatment with these radiation-sensitizing agents selectively enhanced the potency of external beam radiation therapy for established PSMA-positive tumors.

A Theranostic “SMART” Aptamer for Targeted Therapy of Prostate Cancer

A Theranostic “SMART” Aptamer for Targeted Therapy of Prostate Cancer

Abstract 664: Amanitin-based antibody-drug conjugates targeting the prostate-specific membrane antigen

Abstract Antitumoral activity of monoclonal antibodies can be dramatically enhanced by conjugation to toxic small molecules. Beside the recent approval of Kadcyla (T-DM1) and Adcetris (SGN-35) more than 30 antibody-drug conjugates (ADC) have entered clinical trials, promising to strengthen the therapeutic capabilities for cancer treatment in the next decade. Surprisingly most ADCs are based on one of few toxic compounds only and on an even smaller number of toxicity mechanisms: Most antibodies are coupled to the microtubuli-targeting auristatins and maytansines. Toxins that operate through such a mechanism could suffer from limited activity in different cancer indications and in cells expressing resistance mechanisms. Accordingly the use of new drugs that function via alternative toxicity mechanisms could enhance the therapeutic potential of ADCs. In the present study we evaluated the antitumoral potency of a monoclonal antibody targeting the prostate-specific membrane antigen (PSMA) conjugated to small molecules from the amatoxin family. PSMA is a membrane antigen overexpressed in prostate cancer and an attractive target for an ADC approach, as it shows low expression by most normal tissues and sufficient internalization after antibody binding. Amanitin, the most well-known toxin of the amatoxin family, binds to the eukaryotic RNA pol II and thereby inhibits the cellular transcription at very low concentrations. In our experiments, we tested several random- and site-specific strategies to covalently conjugate amanitin to the antibody and generated conjugates with low aggregation and high affinity for the target antigen. Using a series of PSMA-expressing cells we compared the cytotoxic activity of stable and cleavable linker ADCs and the stability of such constructs in plasma. Overall we observed picomolar activity of ADCs after incubation for three to five days with PSMA-positive prostate cancer cells independent of the hormone-sensitivity status. Moreover we demonstrated high activity of amanitin-based anti-PSMA ADCs in prostate cancer xenograft models. The data encourage the evaluation of these agents in a clinical advanced prostate cancer study. Note: This abstract was not presented at the meeting. Citation Format: Torsten Hechler, Michael Kulke, Christoph Mueller, Andreas Pahl, Jan Anderl. Amanitin-based antibody-drug conjugates targeting the prostate-specific membrane antigen. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 664. doi:10.1158/1538-7445.AM2014-664