Prostate-specific Membrane Antigen Positive Cells Research Articles

45Ti targeted tracers for PET imaging of PSMA

PurposePositron Emission Tomography is an important molecular imaging technique for detection and diagnoses of various disease states. This work aims to develop novel titanium-45 (t½ = 3.08 h) PET tracers using Prostate Specific Membrane Antigen (PSMA) targeting vectors for imaging of prostate cancer as proof of concept for this relatively unexplored isotope. ProceduresTitanium-45 was produced on the University of Alabama at Birmingham (UAB) TR24 cyclotron using proton bombardments on natural scandium foils and separated using procedures described previously [1]. After purification, Titanium-45 was used to radiolabel two PSMA-targeting molecules; DFO-DUPA and LDFC-DUPA. Radiochemical yields were determined via radio-high purity liquid chromatography (radioHPLC). The radiolabeled compounds were tested both in vitro and in vivo using PSMA+ cell lines (LNCaP and 22Rv1) and PSMA− cell lines (PC3). ResultsTitanium-45 was produced and purified in yields suitable for research studies. Radiochemical yields of up to 98 ± 1% were achieved with DFO-DUPA and 92 ± 7% with LDFC-DUPA. PSMA specific targeting was observed in vitro in PSMA positive cells (LNCaP (0.6% ± 0.05%) and confirmed by blocking (0.15% ± 0.04%) (P < 0.0001)), compared to uptake in the PSMA negative cells (PC3 (0.07% ± 0.008%)) and confirmed by blocking (0.07% ± 0.01%) (P = 0.5253). In vivo studies demonstrated statistically significant uptake in LNCaP tumors (2.3% ± 0.3% ID/g) compared to PC3 tumor uptake (0.1% ± 0.07%). ConclusionsThis work shows that titanium-45 can be used to radiolabel PSMA targeting compounds with high radiochemical yields. These radiolabeled compounds remain intact in serum for at least two half-lives of titanium-45, showing that these compounds would be appropriate for implementation in the clinical setting. This study shows the feasibility of using titanium-45 as positron emitting radiometal for use in imaging PSMA+ prostate cancer, and illustrates that further research is in this area is warranted.

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.

Investigating PSMA-Targeted Radioligand Therapy Efficacy as a Function of Cellular PSMA Levels and Intratumoral PSMA Heterogeneity.

Prostate-specific membrane antigen (PSMA) targeting radioligands deliver radiation to PSMA-expressing cells. However, the relationship between PSMA levels and intralesion heterogeneity of PSMA expression, and cytotoxic radiation by radioligand therapy (RLT) is unknown. Here we investigate RLT efficacy as function of PSMA levels/cell, and the fraction of PSMA+ cells in a tumor. RM1 cells expressing different levels of PSMA (PSMA-, PSMA+, PSMA++, PSMA+++; study 1) or a mix of PSMA+ and PSMA- RM1 (study 2, 4) or PC-3/PC-3-PIP (study 3) cells at various ratios were injected into mice. Mice received 177Lu- (studies 1-3) or 225Ac- (study 4) PSMA617. Tumor growth was monitored. Two days post-RLT, tumors were resected in a subset of mice. Radioligand uptake and DNA damage were quantified. 177Lu-PSMA617 efficacy increased with increasing PSMA levels (study 1) and fractions of PSMA positive cells (studies 2, 3) in both, the RM1 and PC-3-PIP models. In tumors resected 2 days post-RLT, PSMA expression correlated with 177Lu-PSMA617 uptake and the degree of DNA damage. Compared with 177Lu-PSMA617, 225Ac-PSMA617 improved overall antitumor effectiveness and tended to enhance the differences in therapeutic efficacy between experimental groups. In the current models, both the degree of PSMA expression and the fraction of PSMA+ cells correlate with 177Lu-/225Ac-PSMA617 tumor uptake and DNA damage, and thus, RLT efficacy. Low or heterogeneous PSMA expression represents a resistance mechanism to RLT.See related commentary by Ravi Kumar and Hofman, p. 2774.

Anti-PSMA 124I-scFvD2B as a new immuno-PET tool for prostate cancer: preclinical proof of principle

BackgroundProstate cancer (PCa) is the second leading cause of cancer-related death in the Western population. The use in oncology of positron emission tomography/computed tomography (PET/CT) with emerging radiopharmaceuticals promises accurate staging of primary disease, restaging of recurrent disease and detection of metastatic lesions. Prostate-specific membrane antigen (PSMA) expression, directly related to androgen-independence, metastasis and progression, renders this tumour associate antigen a good target for the development of new radiopharmaceuticals for PET. Aim of this study was to demonstrate in a preclinical in vivo model (PSMA-positive versus PSMA-negative tumours) the targeting specificity and sensitivity of the anti-PSMA single-chain variable fragment (scFv) labelled with 124I.MethodsThe 124I-labeling conditions of the antibody fragment scFvD2B were optimized and assessed for purity and immunoreactivity. The specificity of 124I-scFvD2B was tested in mice bearing PSMA-positive and PSMA-negative tumours to assess both ex-vivo biodistribution and immune-PET.ResultsThe uptake fraction of 124I-scFvD2B was very high on PSMA positive cells (range 75–91%) and highly specific and immuno-PET at the optimal time point, defined between 15 h and 24 h, provides a specific localization of lesions bearing the target antigen of interest (PSMA positive vs PSMA negative tumors %ID/g: p = 0.0198 and p = 0.0176 respectively) yielding a median target/background ratio around 30–40.ConclusionsPreclinical in vivo results of our immuno-PET reagent are highly promising. The target to background ratio is improved notably using PET compared to SPECT previously performed.These data suggest that, upon clinical confirmation of sensitivity and specificity, our anti-PSMA 124I-scFvD2B may be superior to other diagnostic modalities for PCa. The possibility to combine in patients our 124I-scFvD2B in multi-modal systems, such as PET/CT, PET/MR and PET/SPECT/CT, will provide quantitative 3D tomographic images improving the knowledge of cancer biology and treatment.

Abstract 565: A novel T-cell costimulatory Humabody® VH therapeutic for PSMA-positive tumors

Abstract Agonistic monoclonal antibodies targeting CD137/4-1BB have shown much preclinical promise but their clinical development has been slowed due to a poor therapeutic index, in particular liver toxicity. CB307 is a novel half-life extended bispecific Humabody VH targeting CD137 (4-1BB) and prostate specific membrane antigen (PSMA). The design of CB307 enables agonism of CD137 selectively in the presence of PSMA positive tumour cells and in this way enable tumour selective T cell activation whilst minimising systemic activation. The molecular weight of CB307 is less than 50 kDa (around a third of the size of a standard IgG) and it does not contain an Fc domain, thereby avoiding interaction with Fc receptors. Half life extension is achieved through the inclusion of a VH domain with specificity for human serum albumin (HSA). The identification of CB307 using the Crescendo Mouse™ which develops fully human VH domains in a background devoid of light chains will be described along with characterisation of the key properties of the molecule in in vitro and in vivo models. In dual target binding assays CB307 shows potent co-binding to both PSMA and CD137 targets and mediates CD137 signalling in an NFKB cell reporter assay in the presence of PSMA positive cells but not PSMA negative cells. Co-incubation of primary human T-cells from healthy individuals or cancer patients together with PSMA positive tumour cells and CD3 stimulation induces T-cell activation and cytokine release. In an in vivo model using NSG mice engrafted with human PBMCs the growth of PSMA positive DU145 prostate tumour cells is inhibited by a surrogate bispecific. Together these data support progression of CB307 into clinical development. Citation Format: James W. Legg, Brian McGuinness, Sophie Archer, Phil Bland-Ward, Verena Brucklacher, Jenny Craigen, Carolyn Edwards, Emma Hames, Jay Majithiya, pavel Pisa, Bhindu Revi, Nikki Royale, Angelica Sette, Yumin Teng, Lorraine Thompson, Wembin Wang, Chris Wilson, Chris Wyre, Chris Rossant. A novel T-cell costimulatory Humabody® VH therapeutic for PSMA-positive tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 565.

Development of prostate specific membrane antigen targeted ultrasound microbubbles using bioorthogonal chemistry

Prostate specific membrane antigen (PSMA) targeted microbubbles (MBs) were developed using bioorthogonal chemistry. Streptavidin-labeled MBs were treated with a biotinylated tetrazine (MBTz) and targeted to PSMA expressing cells using trans-cyclooctene (TCO)-functionalized anti-PSMA antibodies (TCO-anti-PSMA). The extent of MB binding to PSMA positive cells for two different targeting strategies was determined using an in vitro flow chamber. The initial approach involved pretargeting, where TCO-anti-PSMA was first incubated with PSMA expressing cells and followed by MBTz, which subsequently showed a 2.8 fold increase in the number of bound MBs compared to experiments performed in the absence of TCO-anti-PSMA. Using direct targeting, where TCO-anti-PSMA was linked to MBTz prior to initiation of the assay, a 5-fold increase in binding compared to controls was observed. The direct targeting approach was subsequently evaluated in vivo using a human xenograft tumor model and two different PSMA-targeting antibodies. The US signal enhancements observed were 1.6- and 5.9-fold greater than that for non-targeted MBs. The lead construct was also evaluated in a head-to-head study using mice bearing both PSMA positive or negative tumors in separate limbs. The human PSMA expressing tumors exhibited a 2-fold higher US signal compared to those tumors deficient in human PSMA. The results demonstrate both the feasibility of preparing PSMA-targeted MBs and the benefits of using bioorthogonal chemistry to create targeted US probes.

Abstract 5263: Prostate-specific membrane antigen (PSMA)-targeting nanobioconjugate-encapsulated green tea polyphenol EGCG for prostate cancer prevention and therapy

Abstract Initially we introduced the idea of employing nanotechnology to improve the outcome of natural agents in chemoprevention and coined the term ‘Nanochemoprevention’. Recently we reported the efficacy of chitosan nanoparticles encapsulating (-)-epigallocatechin-3-gallate (EGCG) for prostate cancer (PCa) prevention and therapy. Here we employed the concept of targeted uptake of therapeutic nanoparticles, a potentially powerful technology to enhance the efficacy of EGCG for PCa management. We developed chitosan nanobioconjugates encapsulating EGCG and surface functionalized with the A10 2′-fluoropyrimidine RNA aptamers that recognize the extracellular domain of the PSMA, henceforth called chit-EGCG-Apt. Cellular binding and uptake of the fluorescent nanosystem was detected suggesting PSMA specific internalization and accumulation. Further, we observed that chit-EGCG-Apt lead to a PSMA specific increase of anti-proliferative activity in PCa cells, with a better efficacy in PSMA positive cells (LNCaP and C4-2) than in PSMA negative (PC3 and DU145) cells and a decrease in IC50 by at least half the dose of the native agent. Further, we also observed a significant inhibition in the colony formation potency of PCa cells when treated with chit-EGCG-Apt as compared to the native EGCG with the effects more distinct in PSMA positive cells. Next, we established that EGCG in chit-EGCG-Apt retains its mechanistic identity and observed that the treatment resulted in significant (i) shift in Bax/Bcl-2 ratio in favor of apoptosis, (ii) increase in PARP cleavage, (iii) inhibition in caspase 3, 7, 8 and 9, and other key regulators of apoptosis, (iv) modulation of death receptor pathway proteins, and (v) cyclin dependent kinase inhibitors in addition to arrest of the cells in S phase of the cell cycle. Importantly, these effects were only visible at higher doses of the native EGCG. The nanobioconjugate also demonstrated a significant inhibition of invasive potential of several PCa cells and 3-dimensional spheroid BME cell invasion assay. Further, chit-EGCG-Apt treated mice implanted with androgen sensitive CWR22R(nu)1 cells showed significant inhibition in tumor growth and decreased serum PSA levels reciprocating our in vitro findings. In tumor tissues of mice treated with Chit-EGCG-Apt there was significant (i) induction of poly (ADP-ribose) polymerases cleavage, (ii) increase in the protein expression of Bax with concomitant decrease in Bcl-2, (iii) activation of caspases, and (iv) reduction in Ki-67 and proliferating cell nuclear antigen, as compared to native EGCG. We further observed inhibition of angiogenesis, invasion and metastasis markers in these tissues. Translation of these data to appropriate animal model systems could pave way for prostate cancer therapy in humans. Citation Format: Imtiaz A. Siddiqui, Dhruba J. Bharali, Rahime Jashari, Vaqar M. Adhami, Shaker A. Mousa, Hasan Mukhtar. Prostate-specific membrane antigen (PSMA)-targeting nanobioconjugate-encapsulated green tea polyphenol EGCG for prostate cancer prevention and 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 5263.

Abstract 251: Aptamer conjugated prostate specific membrane antigen (PSMA) targeting EGCG nanobioconjugate for prostate cancer prevention and treatment

Abstract Earlier we introduced the idea of employing nanotechnology to improve the outcome of natural agents in chemoprevention and coined the term ‘Nanochemoprevention’. Recently we reported the efficacy of chitosan nanoparticles encapsulating green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG) for prostate cancer (PCa) therapy. Here we employed the idea of targeted uptake of therapeutic nanoparticles, a potentially powerful technology. Using PCa as a model, we report the efficacy of EGCG encapsulated in chitosan nanoparticles surface functionalized with the A10 2′-fluoropyrimidine RNA aptamers that recognize the extracellular domain of the prostate-specific membrane antigen (PSMA), henceforth called chit-EGCG-Apt. Prepared bioconjugates were spherical in shape with a unimodal size distribution, with diameter around 200 nm and a zeta potential of -30 mV. Cellular binding and uptake of the fluorescent nanosystem was detected suggesting PSMA specific internalization and accumulation. Next, we determined the EGCG release kinetics and observed a sustained and constant release of EGCG from the nanobioconjugates. To test the biological efficacy, we first determined the efficacy of chit-EGCG-Apt for its cytotoxic response in several human prostate carcinoma cells. We observed that chit-EGCG-Apt lead to an increased anti-proliferative activity in PCa cells, with a better efficacy in PSMA positive cells (LNCaP and C4-2) than in PSMA negative PC3 cells and a decrease in IC50 by at least half the dose of the native agent. Further, we also observed a significant inhibition in the colony formation potency of PCa cells when treated with chit-EGCG-Apt as compared to the native EGCG with the effects more distinct in PSMA positive cells. Our next goal was to ascertain if EGCG in chit-EGCG-Apt retains its mechanistic identity. Chit-EGCG-Apt treatment (10 and 20 μM) to LNCaP cells resulted in significant (i) shift in Bax/Bcl-2 ratio in favor of apoptosis, (ii) increase in PARP cleavage, (iii) inhibition in caspase 3, 7, 8 and 9, and other key regulators of apoptosis, (iv) modulation of DR4, DR5, TNFR1, FADD, TRADD death receptor pathway proteins, (v) increase in the levels of annexin V positive cells, (vi) increase in TdT positive cells (additional information to suggest induction of apoptosis), and (vii) modulation of p21, p27, CDK 4 and 6 cyclin dependent kinase inhibitors in addition to arrest of the cells in S phase of the cell cycle. Importantly, these effects were only visible at 40 μM of the native EGCG. The nanobioconjugate also demonstrated a significant inhibition of invasive potential of several PCa cells in scratch wound assay and 3-dimensional spheroid BME cell invasion assay. Translation of these data to appropriate animal model systems could pave way for prostate cancer therapy in humans. Citation Format: Imtiaz A. Siddiqui, Dhruba J. Bharali, Minakshi Nihal, Vaqar M. Adhami, Rahime Jashari, Shaker A. Mousa, Hasan Mukhtar. Aptamer conjugated prostate specific membrane antigen (PSMA) targeting EGCG nanobioconjugate for prostate cancer prevention and treatment. [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 251. doi:10.1158/1538-7445.AM2014-251

Abstract 3919: A preclinical model of laparoscopy demonstrating the feasibility of detecting PSMA positive cells with a NIR fluorophore (YC-27): implications for strategies to decrease positive margins during prostatectomy.

Abstract Introduction: Depending on the stage there is approximately a 4-54% positive surgical margin (+SM) following prostatectomy. Patients with +SM face the associated expense and impaired functional outcomes of salvage radiotherapy. To circumvent this, a low-molecular weight urea-based near infrared (NIR) fluorophore (YC-27) was developed to target the prostate specific membrane antigen (PSMA) for use in intraoperative imaging. PSMA is a transmembrane protein expressed in normal prostate and upregulated in cancer. PSMA expression correlates with Gleason score, advanced stage and PSA. We optimized dosing and timing in a small animal model for laparoscopic resection of PSMA positive tumors. Secondly, we demonstrate the feasibility of applying such a system in a porcine model. Methods: PSMA positive and negative tumors were established contralaterally on the flank of athymic male nude mice. YC-27 was administered intravenously at varying doses (9.5, 19.1, 39.7 [ug/Kg]) and sequentially imaged using a custom built laparoscopic system to obtain probe kinetics (1, 2, 3, 4, 6, 8, 10, 24 hrs post IV injection). Arbitrary pixel count was used to evaluate dosing and timing for signal to noise ratio. NIR guided laparoscopic resection was performed on mice with established PSMA positive and negative tumors 6 hrs post injection of 39.7 ug/Kg YC-27. In a porcine model, prestained human xenografts generated in a murine host were implanted behind the peritoneum of the abdominal cavity, and exploratory NIR laparoscopy was performed. In a porcine model, the kidney was observed with our laparoscopic NIR system after intravenous YC-27 administration. Results: In a murine model, the highest signal to noise ratio was determined to occur 6 hrs after injecting 39.7 ug/Kg YC-27. Using these parameters, NIR guided laparoscopic surgery facilitated full resection of the xenograft. In a porcine model, exploratory laparoscopy with our NIR system allowed detection of a prestained tumor implanted behind the peritoneum. In another experiment, strong fluorescent signal was observed from the porcine kidney immediately after IV injection of YC-27 which may be attributed to specific binding to PSMA, renal clearance, or a combination of both. Conclusions: YC-27 allows intraoperative localization of PSMA in real time using a prototype NIR guided laparoscopic surgical system. Pilot studies in large animals demonstrated excellent potential for clinical translation. Citation Format: Brian P. Neuman, John B. Eifler, Mark Castanares, Wasim H. Chowdhury, Ying Chen, Martin G. Pomper, Ronald Rodriguez. A preclinical model of laparoscopy demonstrating the feasibility of detecting PSMA positive cells with a NIR fluorophore (YC-27): implications for strategies to decrease positive margins during prostatectomy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3919. doi:10.1158/1538-7445.AM2013-3919

Abstract C30: Use of targeted magnetic nanoparticles for imaging in prostate cancer

Abstract Objective: Imaging techniques better than those conventionally used are needed to improve prostate cancer (PC) staging and read out of therapeutic effects in real time in treated patients. We aimed to perform preclinical evaluation of newly developed well-characterized, biocompatible, PC-targeted magnetic nanoparticles (MNPs) targeted to prostate cancer by conjugation with J591 (from N. Bander, Cornell, USA), an antibody which binds specifically to prostate specific membrane antigen (PSMA) which is expressed on the surface of ∼90% of PCs including castrate resistant prostate cancers; this binding results in internalization. The use of targeted MNPs should enhance the specificity and sensitivity of magnetic resonance imaging (MRI) to enable better staging of patients with PC and future targeted delivery of therapy. Methods: MNPs were prepared, engineered to the appropriate size and conjugated with J591. There was no compromise in J591 cell binding or specificity for PSMA positive cells due to the conjugation, and Inductively Coupled Plasma Optical Emission Spectrometry and Prussian blue staining for iron indicated increased uptake of MNPs that were conjugated with the antibody. In vivo studies were performed in immunodepressed nude mice with subcutaneous LNCaP-LN3 (PSMA-positive) xenografts (post euthanasia using an 11.7T NMR system) or on live mice with orthotopic LNCaP xenografts, using a 16.4T MRI imaging system following intravenous injection of MNPs. Results: Similar enhancement of MRI was obtained by NMR after injection of MNP or J591-MNP conjugates. Live imaging of mice given systemic J591-MNPs showed uptake into the prostate tumors. Conclusions: The data indicate the promise of this technique in enabling imaging of small clusters of human prostate cancer cells. This should enable the effects of therapy to be determined by imaging in real time, improving patient management. Citation Format: Pamela J. Russell, C Soekmadji, B Thierry, G Cowin, T Strait-Gardner, N Verma, Wang Xiaochun, A Khatri. Use of targeted magnetic nanoparticles for imaging in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C30.

Selective Gene Therapy for Prostate Cancer Cells Using Liposomes Conjugated with IgM Type Monoclonal Antibody against Prostate-Specific Membrane Antigen

Prostate cancer cells express prostate-specific membrane antigen (PSMA). We developed an IgM type monoclonal antibody against PSMA. The antibody was coupled to poly-L-lysine and thereafter this conjugate was mixed with cationic liposomes containing plasmid DNA. The antibody-liposome complex was tested whether it could deliver the gene of interest selectively to the PSMA positive cells. As assessed by beta-galactosidase reporter gene, the transfection efficiency was 13.2% with anti-PSMA-liposome complex as compared to 4% with control IgM liposome complex. In contrast, no such differences were observed in PSMA negative PC-3, DU145 and T24 cells. Furthermore, in the suicide gene therapy in vitro with thymidine kinase gene plus ganciclovir system, anti-PSMA liposome complex demonstrated a selective growth inhibitory effect on PSMA positive LNCaP cells but not on PSMA negative cell lines.