Surface Of Prostate Cancer Cells Research Articles

Abstract 3668: Single-dose activity of PSMA ADC against prostate cancer xenograft and explant tumors

Abstract Introduction: Prostate-specific membrane antigen (PSMA) is an attractive target for antibody-targeted therapy of prostate cancer due to its abundant and selective expression on the surface of prostate cancer cells. We have generated a novel antibody-drug conjugate (ADC) by linking a fully human PSMA monoclonal antibody to monomethylauristatin-E (MMAE), a potent tubulin inhibitor. Here, we describe the use of PSMA ADC to treat SCID mice bearing LNCaP xenografts or LuCaP explants derived from surgical resections of prostate cancer patients. Methods: PSMA expression in LNCaP and LuCaP tumor cells was evaluated by flow cytometry. Activity of PSMA ADC, assessed by decrease in tumor volume, was evaluated in LNCaP xenograft and LuCaP explant models. SCID mice were implanted subcutaneously with cultured LNCaP tumor cells or disaggregated human LuCaP 70 or LuCaP 77 prostate tumor tissue obtained from tumor-bearing mice. When average tumor size reached ∼300mm3, animals were randomized into groups of 9-10 mice and each animal was treated with a single intravenous dose of vehicle control, a control ADC of irrelevant specificity, or PSMA ADC. Treatment effects were assessed by measuring tumor volume and overall survival for up to 41 days post-treatment. Results: Surface expression of PSMA was assessed by flow cytometry, with a rank order of expression as follows: LNCaP > LuCaP 70, LuCaP 77, LuCaP 105 > LuCaP 96.1 > LuCaP 35V, LuCaP 141. Single-dose PSMA ADC demonstrated significant activity against LNCaP xenografts at a dose of 5 mg/kg (p<0.0001 relative to either vehicle or control ADC) and against LuCaP 77 explants at a dose of 10 mg/kg (p=0.0003 relative to either vehicle or control ADC). A trend toward delayed tumor growth was observed in the LuCaP 70 tumor model. Conclusion: Single-dose PSMA ADC showed activity against LNCaP xenografts and LuCaP explants derived from surgical resections of prostate cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3668. doi:10.1158/1538-7445.AM2011-3668

Abstract 425: Alteration of ADAM9 and integrin interaction regulated cancer invasion and metastasis potential

Abstract Alteration of integrin composition is highly correlated with cancer malignancy and metastasis. Recent evidence demonstrated the metastasis activities involved the interaction of integrin and ADAM gene family, such as ADAM8, ADAM9, ADAM10 and ADAM17. These may either stabilized or cleavage of integrin-matrice interaction at the lamellipodia of cancer cell. Mahimakar et al., recently demonstrated the interaction of ADAM9 with integrins alpha3, alpha6 and beta1 by pull down assay, suggests ADAM9 might work together with β1 class integrins. To further analyze their roles and possible downstream regulatory proteins, we perform knockdown of ADAM9 using shRNA and determine its role in cancer metastasis both in vitro and in vivo. Western blotting confirmed the knockdown of ADAM9 in prostate cancer, and oral cell carcinoma. Cell migration assay was performed using micro-slide Chemotaxis chambers either with or without collagen coating. Time-Lapse microscope analysis of cell migration was plotted and analyzed. Interestingly, we observed the opposite cell migration behavior in which faster and longer cell migration activities was observed in cancer cell with knockdown of ADAM9 expression in the chamber coated with collagen, whereas less migration activities was observed in chamber without collagen coating. This may reflect the possible role of ADAM9 in alteration of integrin composition or function. Flow Cytometry analyses of integrin composition on the surface of prostate cancer cells revealed no integrin composition change in knockdown strain compared to parental. We also notice highly expression of integrin alpha2beta1 and alpha6beta4 in C4-2 and PC3 cells. Analyses of signaling pathways demonstrated no significant changes in p-FAK, p-Erk and p-Akt, suggesting not through beta1 downstream signal regulation. Immunoprecipitation of ADAM9 demonstrated the highly associated with integrin beta4 but not beta1. Intra-cardiac injection of prostate cancer cell line, PC3-pSM2C, PC3-siADAM9 demonstrated 100% metastasis activities in PC3-pSM2C, and no any metastasis in mice with PC3-siADAM9. In summary, we notice ADAM9 may associated with the activation/stabilization of integrin complex. Knockdown of ADAM9 demonstrated decreased of cell migration on dish and metastasis when inoculated in mice, suggests hemidesmosome-ADAM9 interaction may involved in cancer migration and metastasis potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 425. doi:10.1158/1538-7445.AM2011-425

Is 37LRP cell surface receptor for PSP94?

Dear Editor, This has reference to the commentary ‘Growth inhibition properties of the putative prostate cancer biomarkers PSP94 and CRISP-3' by van Eynde et al. published online in your journal in 2010.1 PSP94 and CRISP-3 are both secretory proteins. Identifying cell surface-binding sites for both these molecules and understanding the downstream signaling would unravel their role(s) in prostate cancer. Though existence of cell surface binding sites for PSP94 has been indicated,2, 3 till date the identity of the ‘cell surface' receptor for PSP94 is not reported in literature. van Eynde et al. in their commentary state that PSP94 attaches to cell membranes and one of its binding partners is 37LRP (laminin receptor precursor). The reference which they have cited has reviewed the original study by Annabi et al.4 which proposes the involvement of 37LRP in binding to a 15-mer peptide representing amino acids 31–45 of PSP94 (PCK3145) to cell surface and not the PSP94 protein per se. These experiments were conducted on HT1080 cell line which is a human fibrosacoma cell line and similar studies in prostate cell lines have not yet been reported. Annabi et al.5 in another study have also reported that transfection of HT1080 with 67LR plasmid increases cell adhesion to PCK3145 peptide. 67LR is mature form of the laminin receptor present on the cell surface. 37LRP is a 37-kDa precursor protein which is predominantly cytoplasmic. Studies distinguishing between the expression of 37LRP and 67LR on prostate cancer cell surface are also not available. More importantly, the native PSP94 protein binding to prostate cell surface via 37LRP is yet to be demonstrated. van Eynde et al. further quote that binding of PSP94 to cell surface receptors inhibits MMP9 secretion and VEGF signaling. These activities again are reported only for the PCK3145 peptide and not the native protein as per the cited study by Lamy et al.6 Extrapolating the studies carried out on a peptide and assigning a function to native protein without experimental evidence can be misleading. In our opinion, these facts should be taken into account while commenting on PSP94 and its receptor interactions.

Ligation of Prostate Cancer Cell Surface GRP78 Activates a Proproliferative and Antiapoptotic Feedback Loop: A ROLE FOR SECRETED PROSTATE-SPECIFIC ANTIGEN

GRP78, a well characterized chaperone in the endoplasmic reticulum, is critical to the unfolded protein response. More recently, it has been identified on the cell surface, where it has many roles. On cancer cells, it functions as a signaling receptor coupled to proproliferative/antiapoptotic and promigratory mechanisms. In the current study, we demonstrate that ligation of prostate cancer cell surface GRP78 by its natural ligand, activated α(2)-macroglobulin (α(2)M*), results in a 2-3-fold up-regulation in the synthesis of prostate-specific antigen (PSA). The PSA is secreted into the medium as an active proteinase, where it binds to native α(2)M. The resultant α(2)M·PSA complexes bind to GRP78, causing a 1.5-2-fold increase in the activation of MEK1/2, ERK1/2, S6K, and Akt, which is coupled with a 2-3-fold increase in DNA and protein synthesis. PSA is a marker for the progression of prostate cancer, but its mechanistic role in the disease is unclear. The present studies suggest that PSA may be involved in a signal transduction-dependent feedback loop, whereby it promotes a more aggressive behavior by human prostate cancer cells.

Abstract 3377: CXCL13-directed therapy against bone metastasis in hormone-refractory prostate cancer

Abstract Metastasis is responsible for most prostate cancer (PCa) related deaths; therefore, therapies designed to prevent or abrogate the spread of cancer cells and prognostic tests to better predict outcomes are greatly needed. Chemokines and their receptors are thought to play key roles in this multi-factorial process. We have recently reported, chemokine receptor CXCR5 and its ligand CXCL13 expression positively correlates with prostate tumor staging. We have also noted differential expression of CXCR5 by PCa cell lines (LNCaP, C4-2B, DU145, and PC3). By virtue of the presence of elevated CXCL13 levels in patient serum, CXCL13 secretion by bone marrow endothelium, and expression of CXCR5 on the surface of PCa cells, we hypothesized that gradient-dependent CXCR5-CXCL13 interactions are responsible in part for PCa cell bone metastases and inhibition of this axis may prove beneficial for prevention of bone metastasis and tumor progression. Luciferase-expressing PC3 cells were injected into the right carotid artery or directly into the tibia of nude mice. Before and after the development of bone metastases, groups were treated with control or anti-CXCL13 antibodies every third day and monitored for tumor progression or regression by non-invasive bioluminescent and microCT in vivo imaging. We demonstrate CXCL13 is present in bone marrow of xenograft tumor-bearing mice following intra-cardiac /intra-tibial injection of PC3 cells. This hormone-refractory PCa cell line established skeletal (osteolytic) metastases and tumor growth, but CXCL13 blockade significantly delayed prostate tumor formation, osteolysis, and spread to bones as well as growth, than compared to mice treated with control antibody. In conclusion, our study shows that the CXCL13-CXCR5 axis supports PCa bone metastasis and growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3377.

Abstract 3182: Decreased expression of PTEN enhances CXCR4-mediated proliferation and tumorigenesis in prostate cancer cells

Abstract The progression of human prostate cancer is a result of metastasis from the primary tumor to vital organs. Metastasis is a complex process that involves invasion, intravasation, extravasation, and metastatic colonization. To date, the biology underlying the various mechanisms of metastasis has not been fully elucidated. Chemokines are pro-inflammatory molecules that bind to chemokine receptors, which are members of the G-protein coupled receptor (GPCR) family. The interaction between chemokines and their receptors results in a diverse array of biological and biochemical functions, such as chemotaxis, hematopoiesis and angiogenesis. Likewise, neoplastic cells employ chemokines and their receptors to promote metastasis, and encourage cell survival. It has been observed that the CXCR4 chemokine receptor is overexpressed on the cell surface of prostate cancer cells, which directed metastatic cells to tissues where its CXCL12 ligand is overexpressed, such as the bones and lungs. The Phosphate and Tensin homolog deleted on chromosome 10 (PTEN) is the second most mutated tumor suppressor in human cancer, and has been shown to be mutated in metastatic prostate cancer cells. Gao et al demonstrated that reconstituted PTEN in lymphocytes down-regulated CXCR4-mediated chemotaxis towards its ligand, CXCL12. Additionally, progenitor PTEN-deficient smooth muscle cells (SMC) increased migration towards CXCL12-producing SMC, suggesting that an alteration in PTEN expression negatively regulated CXCR4-mediated events. We analyzed prostate cancer cells for the expression of CXCR4 by flow cytometry, and observed that CXCR4 was expressed in human metastatic prostate cancer cell lines, PC3 and LnCaP. We analyzed for the expression of PTEN, and observed no expression of PTEN in PC3 and LnCaP cells at the protein level by western blot, nor at the mRNA level in PC3 cells by PCR. Furthermore, when reconstituted with PTEN, PC3 cells demonstrated a mesenchymal to epithelial-like phenotypic change in morphology. Additionally, we observed that reconstituted PTEN inhibited CXCR4-meditated proliferation via an MTT assay. Therefore, we hypothesize that the absence of PTEN expression correlates with an up-regulation of CXCR4-mediated proliferation and tumorigenesis in prostate cancer cells. In summary, our results showed that PTEN induced an epithelial-like morphological change, and inhibited CXCR4-mediated proliferation in metastatic prostate cancer cell lines. In future studies, we will investigate if the reconstituted expression of functional PTEN regulates CXCR4-mediated metastatic events in prostate cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3182.

CXCL13 inhibition prevents bone metastasis in hormone-refractory prostate cancer (133.8)

Abstract We have recently reported, chemokine receptor CXCR5 and its ligand CXCL13 expression positively correlates with prostate tumor staging. We have also noted differential expression of CXCR5 by prostate cancer (PCa) cell lines. By virtue of the presence of elevated CXCL13 levels in patient serum, CXCL13 secretion by bone marrow endothelium, and expression of CXCR5 on the surface of PCa cells, we hypothesized that gradient-dependent CXCR5-CXCL13 interactions are responsible in part for PCa cell bone metastases and inhibition of this axis may prove beneficial for prevention of bone metastasis and tumor progression. Luciferase-expressing PC3 cells were injected into the right carotid artery or directly into the tibia of nude mice. Before and after the development of bone metastases, groups were treated with control or anti-CXCL13 antibodies every third day and monitored for tumor progression or regression by non-invasive bioluminescent and microCT in vivo imaging. We demonstrate CXCL13 is present in bone marrow of xenograft tumor-bearing mice following intra-cardiac /intra-tibial injection of PC3 cells. This hormone-refractory PCa cell line established skeletal (osteolytic) metastases and tumor growth, but CXCL13 blockade significantly delayed prostate tumor formation, osteolysis, and spread to bones as well as growth, than compared to mice treated with control antibody. In conclusion, our study shows that the CXCL13-CXCR5 axis supports PCa bone metastasis and growth.

Targeting Prostate Cancer Cells In Vivo Using a Rapidly Internalizing Novel Human Single-Chain Antibody Fragment

Human antibodies targeting prostate cancer cell surface epitopes may be useful for imaging and therapy. The objective of this study was to evaluate the tumor targeting of an internalizing human antibody fragment, a small-size platform, to provide high contrast in a mouse model of human prostate carcinoma. A prostate tumor-targeting single-chain antibody fragment (scFv), UA20, along with a nonbinding control scFv, N3M2, were labeled with (99m)Tc and evaluated for binding and rapid internalization into human prostate tumor cells in vitro and tumor homing in vivo using xenograft models. For the in vitro studies, the labeled UA20 scFv was incubated at 37 degrees C for 1 h with metastatic prostate cancer cells (DU145) to assess the total cellular uptake versus intracellular uptake. For the animal studies, labeled UA20 and N3M2 scFvs were administered to athymic mice implanted subcutaneously with DU145 cells. Mice were imaged with small-animal SPECT/CT with concomitant biodistribution at 1 and 3 h after injection. The UA20 scFv was labeled in 55%-65% yield and remained stable in phosphate buffer within 24 h. The labeled UA20 scFv was taken up specifically by prostate tumor cells. Internalization was rapid, because incubation at 37 degrees C for less than 1 h resulted in 93% internalization of total cell-associated scFvs. In animal studies, SPECT/CT showed significant tumor uptake as early as 1 h after injection. At 3 h after injection, tumor uptake was 4.4 percentage injected dose per gram (%ID/g), significantly greater than all organs or tissues studied (liver, 2.7 %ID/g; other organs or tissues, <1 %ID/g), except the kidneys (81.4 %ID/g), giving tumor-to-blood and tumor-to-muscle ratios of 12:1 and 70:1, respectively. In contrast, the control antibody exhibited a tumor uptake of only 0.26 %ID/g, similar to that of muscle and fat. Tumor-specific targeting was evidenced by reduced tumor uptake of nearly 70% on administration of a 10-fold excess of unlabeled UA20 scFv. Kidney uptake was nonspecific, consistent with the route of excretion by scFvs. The UA20 scFv showed rapid and specific internalization in prostate tumor cells in vitro and accumulation in prostate tumor xenografts in vivo, demonstrating the potential for future development for prostate cancer imaging and targeted therapy.

Antitumor activity of PSMA ADC after progression on docetaxel in a mouse xenograft model of human prostate cancer

3030 Background: Currently, there are no approved therapies for castration-resistant metastatic prostate cancer that has progressed following docetaxel therapy. Prostate-specific membrane antigen (PSMA) is an attractive target for antibody-targeted therapy of prostate cancer due to its abundant and restricted expression on the surface of prostate cancer cells. We have developed a novel antibody-drug conjugate (ADC) by linking a fully human PSMA monoclonal antibody to monomethylauristatin E (MMAE), a potent tubulin inhibitor. Here, we describe the use of PSMA ADC in a mouse model to treat xenografted human prostate tumors that have progressed following docetaxel therapy. Methods: Nude mice were implanted subcutaneously with 5 x 106 C4–2 human prostate cancer cells. Animals were first randomized to receive weekly intravenous (IV) doses of either 2 mg/kg docetaxel (n = 50) or vehicle (n = 10). Docetaxel significantly reduced tumor growth (p = 0.025) during the initial phase of the study; however, most of the tumors later progressed. When the tumor volume of an animal in the docetaxel group exceeded 400 mm3, the animal was rerandomized to receive continued docetaxel therapy (n = 18) or weekly IV doses of 6 mg/kg PSMA ADC (n = 18). Treatment effects were assessed by measuring tumor volume and overall survival. When tumor volume was assessed to be ≥2,000 mm3, animals would be sacrificed. Results: At 134 days following tumor implantation, the survival rate was 100% for animals in the PSMA ADC treatment group; 94% of these mice had tumor sizes &lt;100 mm3. In the continued docetaxel treatment group, 14 of 18 animals that were sacrificed when their tumors exceeded 2,000 mm3; the survival rate was 22%. Therefore, PSMA ADC treatment significantly shrank tumors and increased overall survival of animals compared to continued docetaxel treatment (p &lt; 0.0001). Conclusions: PSMA ADC had antitumor activity in mice to xenografted human prostate tumors that had progressed following docetaxel treatment. Treatment with PSMA ADC significantly extended survival in this setting. [Table: see text]

PR_b-targeted delivery of tumor necrosis factor-α by polymersomes for the treatment of prostate cancer

Targeted delivery of therapeutics is an active area of research with cancer being an important target because of the necessity for new and better cancer therapies. In this study we design and assess peptide functionalized polymer vesicles, or polymersomes, self assembled from poly(ethylene oxide)-b-poly(butadiene) (PEO-PBD) diblock copolymers for the treatment of prostate cancer. PR_b, a highly effective α5β1 targeting peptide that mimics the cell adhesion binding site in fibronectin, was recently developed in our group, and is here conjugated onto the surface of our polymer vesicles with controlled orientation, ensuring proper and uniform orientation of the targeting peptide at the interface. Polymer vesicles, although often proposed as highly advantageous drug delivery agents, have so far seen limited use as targeted delivery carriers with this study focusing on targeting the polymer vesicles to prostate cancer cells and delivering a therapeutic payload. PR_b-functionalized polymer vesicles are found to effectively internalize within prostate cancer cells after adhering specifically to α5β1 integrins expressed on the surface of prostate cancer cells. Internalization of polymer vesicles was found to be dependent on the surface concentration of PR_b. In addition polymer vesicles encapsulating a model protein therapeutic, tumor necrosis factor-α (TNFα), were delivered to LNCaP human prostate cancer cellsin vitro. Dramatic improvements in the cytotoxic potential of the delivered TNFα were achieved for PR_b-functionalized polymer vesicles, while polymer vesicles functionalized with the peptide GRGDSP were found to perform no better than non-functionalized polymer vesicles. The potency and specificity of these PR_b-targeted polymersomes show great promise for the targeted delivery of therapeutics.

Targeting CUB Domain-Containing Protein 1 with a Monoclonal Antibody Inhibits Metastasis in a Prostate Cancer Model

Through a whole-cell panning approach, we previously identified a panel of antibodies that bound to prostate cancer cell surface antigens. One such antigen, CUB domain-containing protein 1 (CDCP1), was recognized by monoclonal antibody 25A11 and is a single transmembrane molecule highly expressed in several metastatic cancers as well as on CD34(+)CD133(+) myeloid leukemic blast cells. We show CDCP1 expression on prostate cancer cell lines by real-time quantitative PCR (RT-qPCR), flow cytometry, and immunohistochemistry and on prostate cancer patient samples by RT-qPCR and immunohistochemical staining. In cell-based assays, antibody 25A11 inhibited prostate cancer cell migration and invasion in vitro. Further characterization showed that CDCP1 is internalized on antibody binding. When 25A11 was coupled to the cytotoxin saporin either directly or via a secondary antibody, both resulted in prostate cancer cell killing in vitro. In vivo targeting studies with an anti-CDCP1 immunotoxin showed significant inhibition of primary tumor growth as well as metastasis in a mouse xenograft model. These data provide support for continued evaluation of anti-CDCP1 therapy for potential use in cancer in primary and metastatic disease.

Targeting Gene Therapy for Prostate Cancer Cells by Liposomes Complexed with Anti-Prostate-Specific Membrane Antigen Monoclonal Antibody

Prostate-specific membrane antigen (PSMA) is a membrane-bound antigen expressed on the surface of prostate cancer cells, and this paper describes the use of an antibody against PSMA for targeting gene therapy. We coupled anti-PSMA monoclonal antibody with poly-L-lysine and then incubated it with plasmids. These complexes were then transfected with cationic liposomes into cells. The transfection efficiency of anti-PSMA- liposome complex was higher than that of normal IgG-liposome complex in PSMA-positive LNCaP cells. Furthermore, anti-PSMA-liposome complex containing a suicide gene, thymidine kinase, demonstrated a selective growth-inhibitory effect on LNCaP cells in vitro, but did not exert a significant effect on PSMA-negative cells. In an in vivo xenograft model of LNCaP cells in nu/nu mice, we administered the complexes via the tail vein. Judging on the basis of both 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining and luciferase assay findings, a significant enrichment of plasmid DNA was observed in LNCaP xenografts with anti-PSMA-liposome complex in comparison with normal IgG-liposome complex. However, the distribution of plasmid DNA did not change substantially in any other organs including the liver, kidney, lung, and spleen. Moreover, in suicide gene therapy, anti-PSMA-liposome complex exerted a significant inhibitory effect on the growth of LNCaP xenograft, in contrast to normal IgG-liposome complex.

Targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo

Targeted uptake of therapeutic nanoparticles in a cell-, tissue-, or disease-specific manner represents a potentially powerful technology. Using prostate cancer as a model, we report docetaxel (Dtxl)-encapsulated nanoparticles formulated with biocompatible and biodegradable poly(D,L-lactic-co-glycolic acid)-block-poly(ethylene glycol) (PLGA-b-PEG) copolymer and surface functionalized with the A10 2'-fluoropyrimidine RNA aptamers that recognize the extracellular domain of the prostate-specific membrane antigen (PSMA), a well characterized antigen expressed on the surface of prostate cancer cells. These Dtxl-encapsulated nanoparticle-aptamer bioconjugates (Dtxl-NP-Apt) bind to the PSMA protein expressed on the surface of LNCaP prostate epithelial cells and get taken up by these cells resulting in significantly enhanced in vitro cellular toxicity as compared with nontargeted nanoparticles that lack the PSMA aptamer (Dtxl-NP) (P < 0.0004). The Dtxl-NP-Apt bioconjugates also exhibit remarkable efficacy and reduced toxicity as measured by mean body weight loss (BWL) in vivo [body weight loss of 7.7 +/- 4% vs. 18 +/- 5% for Dtxl-NP-Apt vs. Dtxl-NP at nadir, respectively (mean +/- SD); n = 7]. After a single intratumoral injection of Dtxl-NP-Apt bioconjugates, complete tumor reduction was observed in five of seven LNCaP xenograft nude mice (initial tumor volume of approximately 300 mm3), and 100% of these animals survived our 109-day study. In contrast, two of seven mice in the Dtxl-NP group had complete tumor reduction with 109-day survivability of only 57%. Dtxl alone had a survivability of only 14%. Saline and nanoparticles without drug were similarly nonefficacious. This report demonstrates the potential utility of nanoparticle-aptamer bioconjugates for a therapeutic application.

Prostate-Specific Membrane Antigen Vaccines: Naked DNA and Protein Approaches

Prostate-specific membrane antigen (PSMA) is a relatively omnipresent molecule with a multiplicity of functions and has been shown to be a reasonable target for immunologic approaches such as vaccines or more directed therapy with radioactively labeled monoclonal antibodies against PSMA. Given the abundance of various glycoprotein and carbohydrate antigens expressed on the surface of prostate cancer cells and cell lines, PSMA stands out as another self-antigen that is not only expressed on cancer cells but also on neovasculature. Although vaccines are varied in their design and target goal, recent technology has afforded researchers the opportunity to induce recruitment of multiple effector cell populations, cytokines, and factors that can elicit cellular and humoral responses. This review serves to present unique approaches in vaccine development that can induce immunologic responsiveness to PSMA with potential impact on disease progression.

Development of optimal Lu-177 labeled monoclonal antibody (7E11) constructs for radioimmunotherapy of prostate cancer

4765 Background: Prostate-specific membrane antigen (PSMA) is highly over expressed on the surface of prostate cancer cells. Radioimmunoimaging using an anti-PSMA monoclonal antibody (7E11) coupled to Indium-111 via the bifunctional chelating agent (BFC) GYK-DTPA is currently FDA approved. In prior clinical studies evaluating therapeutic constructs of this antibody and BFC labeled with Y-90, the dose that could be safely administered was limited by toxicity secondary to loss of radionuclide from the BFC. We have investigated the use of kinetically inert cyclic BFCs (of the DOTA family) conjugated to this antibody and labeled with Lutetium-177 to produce immunoconjugates that reduce radiation doses to bone marrow thereby allowing administration of higher and potentially more efficacious doses. Methods: A number of DOTA based 7E11 immunoconjugates were generated using a variety of pH, ionic and temperature conditions. Characterization of the resultant immunoconjugates was performed using SDS-PAGE and HPSEC to assess purity and apparent molecular weight and MALDI-MS to determine ratios of BFC to antibody. Specificity and biological activity of the resultant conjugates were evaluated using an immunoaffinity binding ELISA that targets the N-terminal epitope on PSMA recognized by 7E11. Optimal radiolabeling conditions using Lu-177 have also been determined. Results: Conjugation of DOTA based BFC to 7E11 has been achieved using conditions that preserve the integrity of 7E11 and its ability to specifically bind PSMA. Molar ratios of linker to antibody ranged from 0.2 to 5.3. Purity of the immunoconjugates ranged from 96% to 98%. These conjugates have also been shown to be more stable than previous constructs resulting in lower levels of loss of radionuclide in both in vitro and in vivo studies. Conclusions: Based on these studies, a Lutetium-177 labeled radioimmunoconjugate that should allow administration of higher doses than previous constructs has been developed and is being scaled up for human clinical testing. No significant financial relationships to disclose.

Novel prime-boost combinations of PSMA-based vaccines for prostate cancer

2572 Background and Methods The development of vaccination strategies targeted to tumor antigens holds great promise for cancer therapy. Prostate-specific membrane antigen (PSMA) is an attractive candidate self-antigen for vaccination based on its expression signature. PSMA is abundantly expressed on the surface of prostate cancer cells and on the neovasculature of a wide array of other tumors, but PSMA has limited expression in normal extraprostatic tissues. In prostate cancer, PSMA expression increases with disease progression, becoming highest in high-grade, metastatic, hormone-refractory disease. Our main goals are to overcome tolerance to PSMA by active immunization and to induce recognition and rejection of tumor cells expressing PSMA. We are investigating non-replicating vaccine replicon particles (VRP) based on the alphavirus, Venezuelan equine encephalitis virus. VRP have demonstrated potency in animal models, and infect cells in the lymph nodes where the replicon drives production of large quantities of the heterologous antigen. Robust immune responses can be further amplified by VRP-induced cell apoptosis leading to cross-priming. We are also evaluating a complementary purified protein vaccine based on a novel recombinant soluble PSMA (rsPMSA) immunogen both alone and with VRP in prime-boost regimens. Results When used as single immunogens, VRP encoding full-length human PSMA elicited vigorous, durable Th1-biased cellular and humoral responses in normal BALB/c mice, whereas rsPSMA vaccines induced high levels of Th2-biased antibodies. We have now extended this analysis to human HLA-A2 transgenic mice, a more pertinent model for quantification of human Class I-restricted cellular responses, and to prime-boost regimens. Mice were immunized with VRP alone or in combination with rsPSMA boosts. PSMA-specific immune responses were measured with a panel of cellular and humoral immunoassays, including ELISPOT and intracellular cytokine staining for IFN-γ and IL-4 in T cell subsets. Conclusions Based on these translational findings, the rsPSMA protein vaccine has been advanced into a dose-escalating Phase I clinical trial in advanced prostate cancer, while the VRP-PSMA vaccine is in late-stage preclinical testing. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration AlphaVax, PSMA Development Company, LLC

Targeting novel antigens for prostate cancer treatment: focus on prostate-specific membrane antigen

Prostate-specific membrane antigen (PSMA) is a relatively omnipresent, but unique Type II dimeric transmembrane protein with a multiplicity of functions and has been shown to be a reasonable target for immunological approaches such as vaccines or more directed therapy with radioactively labelled monoclonal antibodies against PSMA. Given the abundance of various glycoprotein and carbohydrate antigens expressed on the surface of prostate cancer cells and cell lines, PSMA stands out as another ‘self’ antigen which is not only expressed on cancer cells, but on neovasculature. Although vaccines are varied in their design and target goal, recent technology has afforded researchers the opportunity to induce recruitment of multiple effector cell populations, cytokines and factors which can elicit both cellular and humoral responses. This review serves to present unique approaches in vaccine development which can induce immunological responsiveness with potential impact on disease progression and to introduce PSMA as a poten-tial target for multimodality therapies.

Mass Spectral Analyses of Labile DOTA-NHS and Heterogeneity Determination of DOTA or DM1 Conjugated Anti-PSMA Antibody for Prostate Cancer Therapy

Prostate specific membrane antigen (PSMA) is a well-characterized glycoprotein overexpressed on the surface of prostate cancer cells. The novel radiopharmaceutical 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) radiolabeled with Yttrium (90Y) or Indium (111In) conjugated with anti-PSMA genetically engineered humanized monoclonal antibody (huJ591) has been investigated to target prostate cancer cells. The immunoconjugate of huJ591 with the analog of the cytotoxic drug maytansine, DM1 (N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine) has also been developed at Millennium Pharmaceuticals. Activation of the DOTA molecule, resulting in 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid mono-(N-hydroxysuccinimidyl) ester (DOTA-NHS), allows conjugation with the anti-PSMA antibody through lysine residues in the antibody. The objectives of the study were to characterize the unstable chemical properties of DOTA-NHS before bioconjugation with huJ591, evaluate the binding profiles of DOTA to huJ591, and calculate trace metal elements (which may disturb 90Y or 111In labeling efficacy to the DOTA-huJ591 conjugate). A novel LC/MS/MS (Liquid Chromatography/Mass Spectrometry/Mass Spectrometry) quantitation method was developed to monitor the stability of DOTA-NHS in solid form and its bioconjugation chemistry reactions. Meanwhile, metal analysis was quantified by Inductively Coupled Plasma Mass Spectrometry (ICP/MS) to estimate the amounts of trace metals in DOTA-NHS and ensure radiolabeling efficiency of the conjugate at the radiopharmacy. MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry) was used to identify levels of DOTA or DM1conjugation in DOTA-huJ591 and DM1-huJ591 conjugates, respectively.

Fully human monoclonal antibodies to PSMA selectively target cytotoxins, radiotoxins and host immunity to prostate cancer

2546 Background: Prostate-specific membrane antigen (PSMA) is a type-2 membrane protein that is expressed abundantly on the surface of prostate cancer cells but not on normal human tissues. Furthermore, levels of PSMA expression increase with disease progression and are highest in hormone refractory and metastatic disease. This expression profile makes PSMA an attractive target for targeted antibody therapies for prostate cancer. We have evaluated fully human monoclonal antibodies (mAbs) against PSMA in preclinical settings in anticipation of human clinical trials. Methods: Using novel recombinant forms of PSMA and XenoMouse™ technology (Abgenix, Fremont, CA), we generated a panel of fully human IgG1 mAbs against PSMA. Characterization of these antibodies was conducted using a battery of Biacore, ELISA, flow cytometry, anti- PSMA Western blot and other functional assays. We have evaluated the mAbs' intrinsic cytotoxic effects as well as their ability to specifically deliver cytotoxic agents to PSMA-expressing tumor cells in vitro and in vivo. Results: We demonstrated that these mAbs specifically recognize conformational epitopes on PSMA with sub-nanomolar affinity. Naked mAbs reproducibly induced antibody-dependent cell-mediated cytotoxicity (ADCC) of human prostate cancer cells at nanomolar concentrations in vitro. Toxin-conjugated mAbs rapidly internalized and killed PSMA-expressing tumor cells with picomolar potency and >1000-fold selectivity, and long-term eradication of tumor was obtained without toxicity in mice xenografted with C4.2 cells. Similarly, mAbs labeled with a therapeutic beta-emitting isotope, 177Lu, selectively targeted PSMA-expressing xenografts, with a tumor uptake of 30% injected dose per gram of tumor. A single 400 μCi dose of 177Lu-labeled antibody was well tolerated and increased median survival times in the animals by 3.5 fold compared to untreated control animals (p<0.005). Conclusions: Based on these studies, a lead fully human antibody has been selected for human clinical testing in naked, radiolabeled and toxin-conjugated forms. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration PSMA Development Co. LLC

Fully human monoclonal antibodies to PSMA selectively target cytotoxins, radiotoxins and host immunity to prostate cancer

2546 Background: Prostate-specific membrane antigen (PSMA) is a type-2 membrane protein that is expressed abundantly on the surface of prostate cancer cells but not on normal human tissues. Furthermore, levels of PSMA expression increase with disease progression and are highest in hormone refractory and metastatic disease. This expression profile makes PSMA an attractive target for targeted antibody therapies for prostate cancer. We have evaluated fully human monoclonal antibodies (mAbs) against PSMA in preclinical settings in anticipation of human clinical trials. Methods: Using novel recombinant forms of PSMA and XenoMouse™ technology (Abgenix, Fremont, CA), we generated a panel of fully human IgG1 mAbs against PSMA. Characterization of these antibodies was conducted using a battery of Biacore, ELISA, flow cytometry, anti- PSMA Western blot and other functional assays. We have evaluated the mAbs' intrinsic cytotoxic effects as well as their ability to specifically deliver cytotoxic agents to PSMA-expres...