Human Prostate-specific Membrane Antigen Research Articles

Novel Monoclonal Antibodies Recognizing Human Prostate-Specific Membrane Antigen (PSMA) as Research and Theranostic Tools.

Prostate-specific membrane antigen (PSMA) is a validated target for the imaging and therapy of prostate cancer. Here, we report the detailed characterization of four novel murine monoclonal antibodies (mAbs) recognizing human PSMA as well as PSMA orthologs from different species. Performance of purified mAbs was assayed using a comprehensive panel of in vitro experimental setups including Western blotting, immunofluorescence, immunohistochemistry, ELISA, flow cytometry, and surface-plasmon resonance. Furthermore, a mouse xenograft model of prostate cancer was used to compare the suitability of the mAbs for in vivo applications. All mAbs demonstrate high specificity for PSMA as documented by the lack of cross-reactivity to unrelated human proteins. The 3F11 and 1A11 mAbs bind linear epitopes spanning residues 226-243 and 271-288 of human PSMA, respectively. 3F11 is also suitable for the detection of PSMA orthologs from mouse, pig, dog, and rat in experimental setups where the denatured form of PSMA is used. 5D3 and 5B1 mAbs recognize distinct surface-exposed conformational epitopes and are useful for targeting PSMA in its native conformation. Most importantly, using a mouse xenograft model of prostate cancer we show that both the intact 5D3 and its Fab fragment are suitable for in vivo imaging. With apparent affinities of 0.14 and 1.2 nM as determined by ELISA and flow cytometry, respectively, 5D3 has approximately 10-fold higher affinity for PSMA than the clinically validated mAb J591 and, therefore, is a prime candidate for the development of next-generation theranostics to target PSMA. Prostate 77:749-764, 2017. © 2017 Wiley Periodicals, Inc.

Enhancement of PSMA-Directed CAR Adoptive Immunotherapy by PD-1/PD-L1 Blockade

Chimeric antigen receptor (CAR) T cell therapy in hematologic malignancies has shown remarkable responses, but the same level of success has not been observed in solid tumors. A new prostate cancer model (Myc-CaP:PSMA(+)) and a second-generation anti-hPSMA human CAR T cells expressing a Click Beetle Red luciferase reporter) were used to study hPSMA targeting and assess CAR T cell trafficking and persistence by bioluminescence imaging (BLI). We investigated the antitumor efficacy of human CAR T cells targeting human prostate-specific membrane antigen (hPSMA), in the presence and absence of the target antigen; first alone and then combined with a monoclonal antibody targeting the human programmed death receptor 1 (anti-hPD1 mAb). PDL-1 expression was detected in Myc-CaP murine prostate tumors growing in immune competent FVB/N and immune-deficient SCID mice. Endogenous CD3+ T cells were restricted from the centers of Myc-CaP tumor nodules growing in FVB/N mice. Following anti-programmed cell death protein 1 (PD-1) treatment, the restriction of CD3+ T cells was reversed, and a tumor-treatment response was observed. Adoptive hPSMA-CAR T cell immunotherapy was enhanced when combined with PD-1 blockade, but the treatment response was of comparatively short duration, suggesting other immune modulation mechanisms exist and restrict CAR T cell targeting, function, and persistence in hPSMA expressing Myc-CaP tumors. Interestingly, an “inverse pattern” of CAR T cell BLI intensity was observed in control and test tumors, which suggests CAR T cells undergo changes leading to a loss of signal and/or number following hPSMA-specific activation. The lower BLI signal intensity in the hPSMA test tumors (compared with controls) is due in part to a decrease in T cell mitochondrial function following T cell activation, which may limit the intensity of the ATP-dependent Luciferin-luciferase bioluminescence signal.

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.

68Ga-PSMA PET/CT Detects the Location and Extent of Primary Prostate Cancer.

Twenty-one patients with biopsy-proven prostate cancer underwent 68Ga-PSMA-HBED-CC (68Ga-PSMA) PET/CT at a median of 4 d (range, 0-47 d) before radical prostatectomy. Based on a 6-segment model, the Gleason score and proportion of tumor tissue within each segment (segmental tumor burden, or STB) as determined by histopathology (STBHP) were correlated with SUVmax and STB as determined by different SUV cutoffs for 68Ga-PSMA PET (STBPET1-6). Furthermore, the involvement of seminal vesicles and other extracapsular extension were assessed by histopathology and PET/CT. Histopathology-positive segments (n = 100 of 126; 79%) demonstrated a significantly higher mean ± SD SUVmax (11.8 ± 7.6) than histopathology-negative segments (4.9 ± 2.9; P < 0.001). Receiver-operating-characteristic analysis revealed an optimal SUVmax cutoff of 6.5 for discrimination of histopathology-positive segments from histopathology-negative segments (area under the curve, 0.84; P < 0.001), which gave 67% sensitivity, 92% specificity, a 97% positive predictive value, a 42% negative predictive value, and 72% accuracy. STBPET3 as determined by (2 × blood SUV) + (2 × SD) correlated best with STBHP (Pearson ρ = 0.68; P < 0.001; mean difference ± SD, 19% ± 15%). PET/CT correctly detected invasion of seminal vesicles (n = 11 of 21 patients; 52%) with 86% accuracy and tumor spread through the capsule (n = 12; 57%) with 71% accuracy. 68Ga-PSMA PET/CT accurately detected the location and extent of primary prostate cancer. Our preliminary findings warrant further investigation of 68Ga-PSMA PET/CT in conjunction with needle biopsy.

A Novel Prostate-Specific Membrane-Antigen (PSMA) Targeted Micelle-Encapsulating Wogonin Inhibits Prostate Cancer Cell Proliferation via Inducing Intrinsic Apoptotic Pathway

Prostate cancer (PCa) is a malignant tumor for which there are no effective treatment strategies. In this study, we developed a targeted strategy for prostate-specific membrane-antigen (PSMA)-positive PCa in vitro based on 2-(3-((S)-5-amino-1-carboxypentyl)ureido) pentanedioic acid (ACUPA) modified polyethylene glycol (PEG)-Cholesterol micelles containing wogonin (WOG), which was named ACUPA-M-WOG. ACUPA-M-WOG was conventionally prepared using a self-assembling method, which produced stable particle size and ζ potential. Moreover, ACUPA-M-WOG showed good drug encapsulating capacity and drug release profiles. Fluorescence activated cell sorting (FACS) results suggested that ACUPA modified PEG-Cholesterol micelles could effectively enhance the drug uptake on PSMA(+) PCa cells, and the cytotoxicity of ACUPA-M-WOG was stronger than other controls according to in vitro cellular proliferation and apoptosis assays, separately through methyl thiazolyl tetrazolium (MTT) and Annexin V/Propidium Iodide (PI) staining. Finally, the molecular mechanisms of ACUPA-M-WOG’s effects on human PSMA(+) PCa were investigated, and were mainly the intrinsic or extrinsic apoptosis signaling pathways. The Western blot results suggested that ACUPA-M-WOG could enhance the WOG-induced apoptosis, which was mainly via the intrinsic signaling pathway rather than the extrinsic signaling pathway. In conclusion, ACUPA-M-WOG was successfully developed for WOG-selective delivery to PSMA(+) PCa cells and had stronger inhibition than free drugs, which might make it an effective strategy for PSMA(+) PCa.

Selection and characterization of Anticalins targeting human prostate-specific membrane antigen (PSMA).

Although prostate carcinoma (PCa) is by far the most commonly diagnosed neoplasia in men, corresponding diagnostic and therapeutic modalities have limited efficacy at present. Anticalins comprise a novel class of binding proteins based on a non-immunoglobulin scaffold that can be engineered to specifically address molecular targets of interest. Here we report the selection and characterization of Anticalins that recognize human prostate-specific membrane antigen (PSMA), a membrane-tethered metallopeptidase constituting a disease-related target for imaging and therapy of PCa as well as solid malignancies in general. We used a randomized lipocalin library based on the human lipocalin 2 (Lcn2) scaffold together with phage display and ELISA screening to select PSMA-specific variants. Five Anticalin candidates from the original panning were expressed in Escherichia coli as soluble monomeric proteins, revealing affinities toward PSMA down to the low nanomolar range. Binding characteristics of the most promising candidate were further improved via affinity maturation by applying error-prone PCR followed by selection via phage display as well as bacterial surface display under more stringent conditions. In BIAcore measurements, the dissociation constant of the best Anticalin was determined as ∼500 pM, with a substantially improved dissociation rate compared with the first-generation candidate. Finally, immunofluorescence microscopy revealed specific staining of PSMA-positive tumor cell lines while flow cytometric analysis confirmed the ability of the selected Anticalins to detect PSMA on live cells. Taken together, Anticalins resulting from this study offer a viable alternative to antibody-based PSMA binders for biomedical applications, including in vivo imaging of PCa or neovasculature of solid tumors.

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.

Abstract A193: Fully human bispecific antibodies induce potent anti-tumor effects against prostate tumors in mice

Abstract Despite multiple new treatment options for patients with castrate-resistant prostate cancer (CRPC), this is still an area of un-met need. Prostate specific membrane antigen (PSMA) is highly expressed in prostate cancer and expression is maintained in CRPC, making it an attractive target for late stage prostate cancer. We have developed a novel full-length human IgG bispecific antibody that binds to human and monkey PSMA and CD3 and results in T cell activation via the CD3 complex in the presence of PSMA-expressing tumors. In cell-based assays, our PSMAxCD3 bispecific resulted in target cell-dependent activation and cytokine release by human T cells, and efficient redirected T cell lysis of prostate tumor cells. Of note, in the absence of PSMA-expressing tumor cells, no T cell activation was seen. Our bispecific antibody also resulted in redirected killing by cynomolgus monkey T cells, enabling pre-clinical toxicity studies. To examine bispecific anti-tumor efficacy in vivo, we developed xenogenic and syngeneic tumor models. In studies with human tumor cells, NOD SCID IL2R gamma deficient (NSG) mice were co-implanted with human peripheral blood mononuclear (PBMC) cells plus human prostate tumor cells (22Rv1 or C4-2), and the mice treated immediately with PSMAxCD3 i.p. Significant inhibition of tumor growth was observed against both human prostate tumors with dose levels as low as 0.004mg/kg. Importantly, PSMAxCD3 bispecific antibodies could also suppress growth of established C4-2 tumors when PBMCs were transferred i.p.. Our PSMAxCD3 bispecific was also tested in mice engrafted with human hematopoietic CD34+ stem cells. Newborn SIRPα BALB/c-Rag2null IL2rγnull (BRG) pups were engrafted with hCD34+ fetal liver cells as hematopoietic progenitor cells, which gave rise to human T, B, and NK cells, as well as granulocytes, monocytes, and DCs. Once engraftment was achieved (3 months later), mice were given C4-2 tumor cells subcutaneously. Eight days after tumor implantation, mice were treated with PSMAxCD3 (0.4mg/kg) followed by twice weekly doses. The PSMAxCD3 bispecific reduced tumor growth in this model. To examine the effects of our lead-candidate bispecifics in an immune-competent model, we engineered mice to express both human CD3 and PSMA in the corresponding murine loci. T cells from these mice were efficiently activated by antibodies to human CD3, and the expression of human PSMA allowed assessment of effects on normal tissues that may express PSMA. T cell activation, proliferation and cytokine production in response to our bispecific antibodies were examined in the absence of tumor as well as the absence of target. To assess anti-tumor efficacy in this model, a murine prostate cancer line expressing human PSMA was generated and tumors cells were implanted s.c. into these mice. Our bispecific antibodies significantly suppressed tumor growth when mice were treated at the time of implantation and also when treatment was delayed until tumors were established. Moreover, T cells were still present at normal levels in the spleen of mice treated with our bispecific at the end of the experiment and the treatment was well-tolerated. Taken together, these studies show potent anti-tumor activity of our PSMAxCD3 bispecific in several different tumor models and also demonstrate that treatment was well-tolerated in mice expressing human PSMA and human CD3. Citation Format: Alison Crawford, Kristin Vazzana, Jeffrey VanValkenburgh, Lauric Haber, Jennifer Principio, Cagan Gurer, Kara Olson, Eric Smith, Gavin Thurston, Jessica R. Kirshner. Fully human bispecific antibodies induce potent anti-tumor effects against prostate tumors in mice. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A193.

T cells bearing a chimeric antigen receptor against prostate-specific membrane antigen mediate vascular disruption and result in tumor regression.

Aberrant blood vessels enable tumor growth, provide a barrier to immune infiltration, and serve as a source of protumorigenic signals. Targeting tumor blood vessels for destruction, or tumor vascular disruption therapy, can therefore provide significant therapeutic benefit. Here, we describe the ability of chimeric antigen receptor (CAR)-bearing T cells to recognize human prostate-specific membrane antigen (hPSMA) on endothelial targets in vitro as well as in vivo. CAR T cells were generated using the anti-PSMA scFv, J591, and the intracellular signaling domains: CD3ζ, CD28, and/or CD137/4-1BB. We found that all anti-hPSMA CAR T cells recognized and eliminated PSMA(+) endothelial targets in vitro, regardless of the signaling domain. T cells bearing the third-generation anti-hPSMA CAR, P28BBζ, were able to recognize and kill primary human endothelial cells isolated from gynecologic cancers. In addition, the P28BBζ CAR T cells mediated regression of hPSMA-expressing vascular neoplasms in mice. Finally, in murine models of ovarian cancers populated by murine vessels expressing hPSMA, the P28BBζ CAR T cells were able to ablate PSMA(+) vessels, cause secondary depletion of tumor cells, and reduce tumor burden. Taken together, these results provide a strong rationale for the use of CAR T cells as agents of tumor vascular disruption, specifically those targeting PSMA. Cancer Immunol Res; 3(1); 68-84. ©2014 AACR.

PSMA-specific CAR-engineered T cells eradicate disseminated prostate cancer in preclinical models.

Immunology-based interventions have been proposed as a promising curative chance to effectively attack postoperative minimal residual disease and distant metastatic localizations of prostate tumors. We developed a chimeric antigen receptor (CAR) construct targeting the human prostate-specific membrane antigen (hPSMA), based on a novel and high affinity specific mAb. As a transfer method, we employed last-generation lentiviral vectors (LV) carrying a synthetic bidirectional promoter capable of robust and coordinated expression of the CAR molecule, and a bioluminescent reporter gene to allow the tracking of transgenic T cells after in vivo adoptive transfer. Overall, we demonstrated that CAR-expressing LV efficiently transduced short-term activated PBMC, which in turn were readily stimulated to produce cytokines and to exert a relevant cytotoxic activity by engagement with PSMA+ prostate tumor cells. Upon in vivo transfer in tumor-bearing mice, CAR-transduced T cells were capable to completely eradicate a disseminated neoplasia in the majority of treated animals, thus supporting the translation of such approach in the clinical setting.

Abstract 3238: The transmembrane domain of prostate-specific membrane antigen: Its role in oligomerization and activation in prostate cancer and nonprostatic neovasculature

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related deaths among American men. The high expression of the membrane protein called prostate specific membrane antigen (PSMA) is implicated in PCa invasiveness as well as neovasculature of nonprostatic solid tumors. Little is known about the exact role of PSMA in PCa progression albeit it is one of the most validated biomarker for the diagnosis and detection of PCa. The functional form of PSMA occurs as a non-covalent homodimer, which is a common motif among bitopic membrane proteins. Further, it is widely recognized that many membrane proteins participate in intracellular signal transduction through the lateral association of their transmembrane domains (TMDs), but this has not been directly demonstrated in PSMA. We hypothesize that PSMA TMD participates in oligomerization and subsequent activation of PSMA. Thus, to gain insight into the non-covalent assembly of PSMA, we aim to determine if the isolated PSMA TMD peptide forms a dimer and if the treatment of full-length PSMA with exogenous PSMA TMD peptide will perturb its properties. Studies using isolated TMDs have been widely used to probe protein-protein interactions and provide useful information on protein assembly under membrane mimetic systems. We identified the amino acid sequence and designed the isolated PSMA TMD peptide based on a known human PSMA sequence (UniProt accession code: Q04609). Two Lysine residues ware appended at both the N- and C-termini to increase the solubility of this highly hydrophobic peptide in polar solvents. Our initial studies revealed that the TMD has twenty-four residues and contains two repeat units of the Small-XXX-Small motif. This sequence is known to be crucial in inducing a strong self-assembly as observed in the bitopic protein Glycophorin A. We prepared the PSMA TMD peptide by microwave-assisted solid phase synthesis and purified by high performance liquid chromatography. The peptide was analyzed by MALDI-TOF mass spectrometry to establish homogeneity and by circular dichroism to examine its secondary structure in membrane mimetic environment. We investigated the oligomerization of the peptide using SDS-PAGE gel shift and fluorescence self-quenching assays. We tested the effect of the peptide against PSMA using an established assay. Herein we report on the oligomerization and PSMA binding properties of PSMA TMD. Our findings may shed light on the role of PSMA TMD in PSMA oligomerization and its potential as a model peptide that target transmembrane helices. It may also aid in the rational design and development of peptide probes for protein-protein interactions as well as therapeutics that target cancer- and clinically-relevant membrane proteins. Citation Format: Brianna S. Berg, Brandan M. Cook, Nathan Beattie, Cliff E. Berkman, Jonel P. Saludes. The transmembrane domain of prostate-specific membrane antigen: Its role in oligomerization and activation in prostate cancer and nonprostatic neovasculature. [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 3238. doi:10.1158/1538-7445.AM2014-3238

Abstract 798: Androgen receptor, PI3K/mTOR and PSMA: Exploring and exploiting the interplay between therapeutic targets in prostate cancer

Abstract Introduction: Androgen receptor (AR), the PI3K/mTOR signaling pathway and prostate-specific membrane antigen (PSMA) represent significant potential targets for prostate cancer therapy. The present study examined the cross-regulation and co-targeting of these molecular pathways. Methods: Cytotoxicity and the kinetics of antigen expression were evaluated in prostate cancer cell lines (LNCaP and C4-2) that vary according to androgen dependence. Expression of PSMA, prostate-specific antigen (PSA), and AR was evaluated in cells cultured in rapamycin and AR inhibitors. Cells were tested for susceptibility to PI3K/mTOR inhibitors used alone and in combination with PSMA ADC, a fully human PSMA monoclonal antibody conjugated to the microtubule disrupting agent monomethyl auristatin E (MMAE). Potential drug synergy or antagonism was evaluated using the Bliss independence method. Results: In androgen-dependent LNCaP cells, rapamycin exerted antiproliferative effects that were accompanied by an increase in AR expression and signaling in the absence of any significant effect on PSMA expression. In androgen-independent C4-2 cells, rapamycin increased AR expression/signaling and PSMA expression in the absence of any significant anti-proliferative effect. AR inhibitors synergized with PSMA ADC in LNCaP and C4-2 cells, but PI3K/mTOR pathway inhibitors synergized with PSMA ADC in C4-2 cells only. More modest synergy was observed by combining non-targeted microtubule inhibitors with inhibitors of AR or mTOR. Compared with rapamycin, GDC0941 (PI3K inhibitor) and MK-2206 (Akt inhibitor), both upstream of mTOR, synergized less strongly with PSMA ADC in C4-2 cells. Conclusions: In this study, AR and PI3K/mTOR pathways exhibited cross-regulation impacting PSMA expression. PSMA ADC synergized with AR and mTOR inhibitors via a multimodal mechanism involving increased PSMA expression and disruption of microtubule function. The findings support clinical exploration of regimens combining PSMA-targeted therapies with inhibitors of the AR and/or PI3K/mTOR signaling pathways. Citation Format: Jose D. Murga, Wells W. Magargal, Sameer M. Moorji, Vincent A. DiPippo, William C. Olson. Androgen receptor, PI3K/mTOR and PSMA: Exploring and exploiting the interplay between therapeutic targets in prostate cancer. [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 798. doi:10.1158/1538-7445.AM2014-798

Biochemical characterization of prostate-specific membrane antigen from canine prostate carcinoma cells

Prostate-specific membrane antigen (PSMA) remains an important target for diagnostic and therapeutic application for human prostate cancer. Model cell lines have been recently developed to study canine prostate cancer but their PSMA expression and enzymatic activity have not been elucidated. The present study was focused on determining PSMA expression in these model canine cell lines and the use of fluorescent small-molecule enzyme inhibitors to detect canine PSMA expression by flow cytometry. Western blot and RT-PCR were used to determine the transcriptional and translational expression of PSMA on the canine cell lines Leo and Ace-1. An endpoint HPLC-based assay was used to monitor the enzymatic activity of canine PSMA and the potency of enzyme inhibitors. Flow cytometry was used to detect the PSMA expressed on Leo and Ace-1 cells using a fluorescently tagged PSMA enzyme inhibitor. Canine PSMA expression on the Leo cell line was confirmed by Western blot and RT-PCR, the enzyme activity, and flow cytometry. Kinetic parameters Km and Vmax of PSMA enzymatic activity for the synthetic substrate (PABGγG) were determined to be 393 nM and 220 pmol min(-1) mg protein(-1) , respectively. The inhibitor core 1 and fluorescent inhibitor 2 were found to be potent reversible inhibitors (IC50 = 13.2 and 1.6 nM, respectively) of PSMA expressed on the Leo cell line. Fluorescent labeling of Leo cells demonstrated that the fluorescent PSMA inhibitor 2 can be used for the detection of PSMA-positive canine prostate tumor cells. Expression of PSMA on Ace-1 was low and not detectable by flow cytometry. The results described herein have demonstrated that PSMA is expressed on canine prostate tumor cells and exhibits similar enzymatic characteristics as human PSMA. The findings show that the small molecule enzyme inhibitors currently being studied for use in diagnosis and therapy of human prostate cancer can also be extended to include canine prostate cancer. Importantly, the findings demonstrate that the potential of the inhibitors for use in diagnosis and therapy can be evaluated in an immunocompetent animal model that naturally develops prostate cancer before use in humans.

Abstract A262: Antiandrogen modulation of prostate-specific membrane antigen (PSMA): Dynamics and synergy with PSMA-targeted therapy.

Abstract Introduction: Recent approvals of new prostate cancer (PCa) drugs and a growing number of pipeline agents have created opportunities for designing rational drug combinations. Potent antiandrogens such as enzalutamide and abiraterone affect expression of numerous androgen-regulated molecules, including PSMA, a well-characterized cell-surface target for PCa therapy. Methods: Cytotoxicity and the kinetics of antigen expression were evaluated in PCa cell lines (LNCaP, C4-2 and 22Rv1) that vary according to androgen dependence and level of PSMA expression. Expression of PSMA and prostate-specific antigen (PSA) was evaluated over time in cells cultured in enzalutamide or abiraterone. Reversibility was examined following drug washout. Cells were tested for susceptibility to antiandrogens used alone and in combination with PSMA ADC, which is a fully human PSMA monoclonal antibody conjugated to the microtubule disrupting agent monomethyl auristatin E (MMAE). Potential drug synergy or antagonism was evaluated using the Combination Index and Bliss independence methods. Results: In androgen-dependent LNCaP cells, enzalutamide and abiraterone exerted antiproliferative effects that were accompanied by a 2- to 3-fold increase in PSMA expression and a decrease in PSA expression. PSMA expression reached maximum levels after four weeks’ culture in enzalutamide and returned to baseline levels within days following enzalutamide removal. In androgen-independent C4-2 cells, enzalutamide and abiraterone increased PSMA expression in the absence of any significant anti-proliferative effect. PSMA ADC exhibited synergistic antitumor activity with both enzalutamide and abiraterone in LNCaP and C4-2 cells. Lesser effects were observed using 22Rv1 cells, an androgen-independent cell line with low basal expression of PSMA. Conclusions: Enzalutamide and abiraterone significantly and reversibly augmented PSMA expression and potentiated the activity of PSMA ADC in androgen-dependent and -independent PCa cell lines in vitro. In androgen-independent cells, the effects on PSMA expression and PSMA ADC activity were uncoupled from any anti-proliferative effect of the antiandrogens. The findings support clinical exploration of regimens that combine potent antiandrogens and PSMA-targeted therapies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A262. Citation Format: Jose D. Murga, Wells W. Magargal, Sameer M. Moorji, Vincent A. DiPippo, William C. Olson. Antiandrogen modulation of prostate-specific membrane antigen (PSMA): Dynamics and synergy with PSMA-targeted therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A262.

Evaluation of Phage Display Discovered Peptides as Ligands for Prostate-Specific Membrane Antigen (PSMA)

The aim of this study was to identify potential ligands of PSMA suitable for further development as novel PSMA-targeted peptides using phage display technology. The human PSMA protein was immobilized as a target followed by incubation with a 15-mer phage display random peptide library. After one round of prescreening and two rounds of screening, high-stringency screening at the third round of panning was performed to identify the highest affinity binders. Phages which had a specific binding activity to PSMA in human prostate cancer cells were isolated and the DNA corresponding to the 15-mers were sequenced to provide three consensus sequences: GDHSPFT, SHFSVGS and EVPRLSLLAVFL as well as other sequences that did not display consensus. Two of the peptide sequences deduced from DNA sequencing of binding phages, SHSFSVGSGDHSPFT and GRFLTGGTGRLLRIS were labeled with 5-carboxyfluorescein and shown to bind and co-internalize with PSMA on human prostate cancer cells by fluorescence microscopy. The high stringency requirements yielded peptides with affinities KD∼1 µM or greater which are suitable starting points for affinity maturation. While these values were less than anticipated, the high stringency did yield peptide sequences that apparently bound to different surfaces on PSMA. These peptide sequences could be the basis for further development of peptides for prostate cancer tumor imaging and therapy.

Cloning and characterization of canine prostate‐specific membrane antigen

Prostate-specific membrane antigen (PSMA) is a promising biomarker in the diagnosis of prostate cancer and a potential target for antibody-based therapeutic strategies. We isolated the canine PSMA cDNA and investigated the cellular and biochemical characteristics of the recombinant protein as a potential target for animal preclinical studies of antibody based-therapies. Canine PSMA cDNA was isolated by PCR, cloned into expression vectors and transfected into COS-1 and MDCK cells. The biosynthesis and glycosylation of the recombinant protein were investigated in pulse-chase experiments, the cellular localization by confocal laser microscopy, the mode of association of PSMA with the membrane with solubilization in different detergents and its quaternary structure in sucrose-density gradients. Canine PSMA shows 91% amino acid homology to human PSMA, whereby the major difference is a longer cytoplasmic tail of canine PSMA compared to its human counterpart. Canine PSMA is trafficked efficiently along the secretory pathway, undergoes homodimerization when it acquires complex glycosylated mature form. It associates with detergent-resistant membranes, which act as platforms along its intracellular trafficking. Confocal analysis revealed canine PSMA at the cell surface, Golgi, and the endoplasmic reticulum. A similar distribution is revealed for human PSMA, yet with reduced cell surface levels. The cloning, expression, biosynthesis, processing and localization of canine PSMA in mammalian cells is described. We demonstrate that canine PSMA reveals similar characteristics to human PSMA rendering this protein useful as a translational model for investigations of prostate cancer as well as a suitable antigen for targeted therapy studies in dogs.

Immune response against prostate cancer cells induced by recombinant adenovirus encoding truncated prostate specific membrane antigen gene and 4-1BBL gene modified dendritic cells in vitro

Objective To study the cytotoxic T lymphocyte response induced by dendritic cells (DCs) transduced with recombinant adenovirus vector bearing truncated human prostate specific membrane antigen (tPSMA) gene and mouse 4-1BB ligand (m4-1BBL) gene against prostate cancer cells in vitro.Methods tPSMA cDNAs and m4-1BBL cDNAs were cloned into pDC316 vector,which was then transfected into HEK 293 cells with adenovirus backbone vector pBHGlox_E1,3Cre to obtain adenovirus expression vector Ad-tPSMA-IRES-m4-1BBL.tPSMA and m4-1BBL proteins were detected by using Western blotting.DCs generated from C57BL/6 mice were transduced with Ad-tPSMA-IRES-m4-1BBL,and the phenotype of DCs was analysed by flow cytometry; Proliferation of T cells stimulated by Ad-tPSMA-IRES-m4-1BBL-transduced DCs in allogeneic mixed lymphocyte reactions and cytotoxic cytotoxic T lymphocyte (CTL) activity induced by Ad-tPSMA-IRES-m4-1BBL-transduced DCs were detected by cell counting kit-8 (CCK-8) assay.Results Recombinant adenovirus Ad-tPSMA-IRES-m4-1BBL was constructed,and tPSMA gene and m4-1BBL gene were expressed correctly.pDC316-tPSMA-IRES-m4-1BBL-transduced DCs upregulated the expression of major histocompatibility complex (MHC) Ⅱ [(87.1 ± 4.7) ％],CD80 [(1.6 ± 5.4) ％] and CD86 [(80.1 ± 2.8) ％].Proliferation rate of T cells stimulated by pDC316-tPSMA-IRES-m4-1BBL-transduced DCs (A450 =0.61 ± 0.02) was significantly higher than that in DCs control group (0.38 ±0.13) and Ad-eGFP-transfected DCs group (0.42 ±0.18) (P ＜0.05).pDC316-tPSMA-IRES-m4-1BBL-transduced DCs induced obvious CTL specific cytotoxicity [(53.5 ± 0.1)％] against tPSMA-expressing prostate cancer cells in vitro.Conclusion DCs vaccines modified by recombinant adenovirus pDC316-tPSMA-IRES-m4-1BBL can induce effective CTL specific cytotoxicity against tPSMA-expressing prostate cancer cells. Key words: Prostate cancer; Dendritic cells; Recombinant adenovirus

Regression of Human Prostate Cancer Xenografts in Mice by AMG 212/BAY2010112, a Novel PSMA/CD3-Bispecific BiTE Antibody Cross-Reactive with Non-Human Primate Antigens

For treatment of patients with prostate cancer (PCa), we developed a novel T cell-engaging (BiTE) antibody designated AMG 212 or BAY2010112 that is bispecific for prostate-specific membrane antigen (PSMA) and the CD3 epsilon subunit of the T cell receptor complex. AMG 212/BAY2010112 induced target cell-dependent activation and cytokine release of T cells, and efficiently redirected T cells for lysis of target cells. In addition to Chinese hamster ovary cells stably expressing human or cynomolgus monkey PSMA, T cells redirected by AMG 212/BAY2010112 also lysed human PCa cell lines VCaP, 22Rv1, MDA PCa 2b, C4-2, PC-3-huPSMA, and LnCaP at half maximal BiTE concentrations between 0.1 and 4 ng/mL (1.8-72 pmol/L). No lysis of PSMA-negative human PCa cell lines PC-3 and DU145 was observed. The subcutaneous (s.c.) formation of tumors from PC-3-huPSMA cells in NOD/SCID mice was significantly prevented by once daily intravenous (i.v.) injection of AMG 212/BAY2010112 at a dose level as low as 0.005 mg/kg/d. Rapid tumor shrinkage with complete remissions were observed in NOD/SCID mice bearing established s.c. 22Rv1 xenografts after repeated daily treatment with AMG 212/BAY2010112 by either the i.v. or s.c. route. Of note, 22Rv1 tumors were grown in the absence of human T cells followed by intraperitoneal injection of T cells 3 days before BiTE treatment. No effects on tumor growth were observed in the absence of human T cells or AMG 212/BAY2010112. On the basis of these preclinical results, AMG 212/BAY2010112 appears as a promising new BiTE antibody for the treatment of patients with PSMA-expressing PCa.

Dendritic Cell Based PSMA Immunotherapy for Prostate Cancer Using a CD40-Targeted Adenovirus Vector

Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs) with prostate specific membrane antigen (PSMA) have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells). To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ). Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy.

CpG-ODN promotes specific anti-prostate cancer induced by dendritic cells vaccine

Objective To observe cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG-ODN) for the anti-prostate cancer effect induced by dendritic cells (DCs) transduced with recombinant adenovirus vector bearing truncated human prostate specific membrane antigen (tPSMA) gene. Methods Replication deficient adenovirus AdEasy-1 system was used to construct recombination adenovirus Ad-tPSMA and Ad-eGFP. Mouse derived DCs were transduced with Ad-tPSMA and Ad-eGFP, and cultured for 6 days in the presence of 10 μg/L GM-CSF and IL4 in RPMI 1640 containing 10% FCS. After culture with 1 mg/L CpG-ODN for 1 day, DCs were further matured with lipopolysaccharide (LPS) at a concentration of 1μg/L for 1 day. The phenotype of DCs was analyzed by using flow cytometry. T cells proliferation stimulated by DCs in allogeneic mixed lymphocyte reactions and the level of interleukin (IL)-2, interferon (IFN)-γ were detected by ELISA kit, and cytotoxic CTL activity induced by DCs was tested by CCK-8 assay. Results CpG-ODN up-regulated the expression of MHCⅡ (83. 8 ±3. 7)% , CD80 (79. 8 ±5. 6)% and CD86 (78. 3 ±2. 8)% in Ad-tPSMA-tranduced DCs (CpG/DCs-Ad-tPSMA). T cells proliferation stimulated by CpG/DCs-Ad-tPSMA was significantly higher than that in DCs control group, DCs-Ad-tPSMA group and DCs-Ad-eGFP group (P ＜0.05). CpG/DCs-Ad-tPSMA induced increased IL-2 (179. 64 ±2. 72) ng/L, and INF-7 (1581.75 ±28. 61) ng/L expression levels as compared to DCs control group, DCs-Ad-tPSMA group, and DCs-Ad-eGFP group (P＜0. 05), and induced more outstanding RM-1-tPSMA cell specific cytotoxic rate (40.7 ±1.4)%than DCs control group (10. 8 ± 1.7) % , DCs-Ad-tPSMA group (40.7 ± 1.4)% , and DCs-Ad-eGFP group (12.7 ± 1. 2)% (P＜0.05). Conclusion CpG-ODN can promote specific anti-prostate cancer induced by DCs modified with recombinant adenovirus vector bearing tPSMA gene. Key words: Prostate carcinoma; Dendritic cells; CpG-ODN