Generation and characterization of nanobodies targeting PSMA for molecular imaging of prostate cancer

Nanobodies show attractive characteristics for tumor targeting in cancer diagnosis and therapy. A radiolabeled nanobody binding the prostate‐specific membrane antigen (PSMA) could offer a noninvasive strategy to select prostate cancer patients eligible for PSMA‐targeted therapies. We here describe the generation, production and in vivo evaluation of anti‐PSMA nanobodies. Nanobodies were derived from heavy‐chain‐only antibodies, raised in immunized dromedaries. Binding characteristics were evaluated through ELISA and flow cytometry. Selected nanobodies were radiolabeled with ^99mTc at their hexahistidine tail, after which cell binding capacity and internalization were evaluated on PSMA^pos LNCaP and PSMA^neg PC3 cell lines. In vivo tumor targeting was analyzed in both LNCaP and PC3 xenografted mice through SPECT/microCT and tissue sampling. A panel of 72 generated clones scored positive on ELISA, all contributing to three nanobody groups, of which group 3 dominated with 70 clones. ELISA and FACS analysis led to the selection of two dominant nanobodies. ^99mTc‐labeled PSMA6 and PSMA30 both showed specific binding on LNCAP cells, but not on PC3 cells. ^99mTc‐PSMA30 internalized significantly more in LNCaP cells compared to ^99mTc‐PSMA6. Higher absolute tumor uptake and tumor‐to‐normal organ ratios were observed for ^99mTc‐PSMA30 compared with ^99mTc‐PSMA6 and a ^99mTc‐control nanobody in LNCaP but not in PC3 tumor‐bearing mice. PSMA30 nanobody has improved targeting characteristics both in vitro as well as in vivo compared with PSMA6 and the control nanobody, and was therefore selected as our in‐house‐developed lead compound for PSMA targeting. Copyright © 2014 John Wiley & Sons, Ltd.     

Prostate-specific membrane antigen associates with anaphase-promoting complex and induces chromosomal instability

Prostate-specific membrane antigen (PSMA) is a transmembrane protein highly expressed in advanced and metastatic prostate cancers. The pathologic consequence of elevated PSMA expression in not known. Here, we report that PSMA is localized to a membrane compartment in the vicinity of mitotic spindle poles and associates with the anaphase-promoting complex (APC). PSMA-expressing cells prematurely degrade cyclin B and exit mitosis due to increased APC activity and incomplete inactivation of APC by the spindle assembly checkpoint. Further, expression of PSMA in a karyotypically stable cell line induces aneuploidy. Thus, these findings provide the first evidence that PSMA has a causal role in the induction of aneuploidy and might play an etiologic role in the progression of prostate cancer.     

Inhibition of G9a promoted 5-fluorouracil (5-FU) induced gastric cancer cell apoptosis via ROS/JNK signaling pathway in vitro and in vivo

A histone methyltransferase G9a, encoded by euchromatic histone-lysine N-methyltransferase 2 (EHMT2), is up-regulated in various cancers, and is involved in their poor prognosis. In the study reported here, the abnormal expression of G9a in gastric cancer it was investigated in vitro and in vivo. Furthermore, the expression of G9a was revealed to have a negative correlation with chemotherapy response in gastric cancer patients. Next, the effect of G9a knockdown on fluorouracil (5-FU) induced cell apoptosis in gastric cancer cells was focused on. The results demonstrated that G9a knockdown significantly activated the expression level of phospho c-Jun N-terminal kinase (p-JNK) and increased the intracellular reactive oxygen species (ROS) levels in the gastric cancer cells. Inhibition of the ROS/JNK signaling partial reversed the effect of G9a knockdown on 5-FU treated gastric cancer cells. Down-regulation of G9a enhanced the sensitivity of 5-FU to the gastric cancer cells in vitro and in vivo, which was involved in the activation of the ROS/JNK signaling pathway. These results demonstrated that G9a could play a critical role in the sensitivity of chemotherapy for gastric cancer and might be a novel method for treating gastric cancer in the clinic.

A histone methyltransferase G9a, encoded by euchromatic histone-lysine N-methyltransferase 2 (EHMT2), is up-regulated in various cancers, and is involved in their poor prognosis.     

Targeted chemoimmunotherapy using drug-loaded aptamer-dendrimer bioconjugates

We reported an innovative, targeted chemoimmuno drug-delivery system. Although chemoimmunotherapy, as an alternative to or in combination with conventional therapeutic systems, has been in the forefront of recent oncological research, as presently configured, such systems face several major obstacles for efficient clinical application. Here, we establish a novel nano-platform for effective chemoimmunotherapy designed to overcome the drawbacks of conventional cancer therapies, describing a delivery system based on a dendrimer and a single-strand DNA-A9 PSMA (prostate-specific membrane antigen) RNA aptamer hybrid. Employing these vehicles, we demonstrate the promising possibility of this chemoimmuno therapeutic system against prostate cancer in in vivo and in vitro models.     

Enhanced Intrapulmonary Delivery of Anticancer siRNA for Lung Cancer Therapy Using Cationic Ethylphosphocholine-based Nanolipoplexes

Here, we report a cationic nanolipoplex as a pulmonary cellular delivery system for small-interfering RNA (siRNA). Six nanoliposomes differing in cationic lipids were formulated and screened in vitro and in vivo for cellular delivery functions in lung cells/tissues. Although the six nanoliposomes showed similar siRNA delivery efficiency in vitro, they exhibited significant differences in pulmonary cellular delivery functions in vivo. Among the various nanoliposomes, cationic dioleoyl-sn-glycero-3-ethylphosphocholine and cholesterol (ECL)-based nanoliposomes showed the highest pulmonary cellular delivery in vivo and the lowest cytotoxicity in vitro. The delivery efficiency of fluorescent siRNA in ECL nanoliposomes was 26.2-fold higher than that of naked siRNA in vivo. Treatment with Mcl1 (myeloid cell leukemia sequence 1)-specific siRNA (siMcl1) using ECL nanolipoplexes reduced target expression in B16F10 cell lines, whereas control, luciferase-specific siGL2 in ECL nanolipoplexes did not. In metastatic lung cancer mouse models induced by B16F10 or Lewis lung carcinoma (LLC) cells, intratracheal administration of siMcl1 in ECL nanolipoplexes significantly silenced Mcl1 mRNA and protein levels in lung tissue. Reduced formation of melanoma tumor nodules was observed in the lung. These results demonstrate the utility of ECL nanoliposomes for pulmonary delivery of therapeutic siRNA for the treatment of lung cancers and potentially for other respiratory diseases.     

荧光定量PCR检测前列腺特异膜抗原及其变异体DNA的方法学

目的拟建立以荧光定量逆转录聚合酶链反应（FQ-RT-PCR）检测前列腺特异性膜抗原（PSMA）及其变异体5（PSMA5）DNA的定量方法。方法自行设计三对引物,自制PC3.0-T-PSMA及PC3.0-T-PSMA5质粒作为标准品,采用FQ-RT-PCR检测前列腺癌LNCap细胞中PSMA及PSMA5 DNA的含量。结果标准曲线的斜率为-0.29,相关系数为0.995,成功地扩增出前列腺癌LNCap细胞中PSMA及PSMA5基因的目标DNA片段。结论应用自行设计的引物及标准品的FQ-RT-PCR能对前列腺癌细胞株中PSMA及变异体PSMA5的DNA进行定量PCR检测。     

Sensitizing hormone-refractory prostate cancer cells to drug treatment by targeting 14-3-3sigma

Advanced and hormone-refractory prostate cancer has long been considered as a chemoresistant disease. Recently, it was found that 14-3-3sigma expression increases as prostate tumor progresses, and that 14-3-3sigma contributes significantly to drug resistance in breast cancers. We, thus, hypothesized that advanced and hormone-refractory prostate cancers may have an increased level of 14-3-3sigma, which in turn may contribute to drug resistance in advanced and hormone-refractory prostate cancers. In this study, we tested this hypothesis and found that, indeed, the expression level of 14-3-3sigma in androgen-independent prostate cancer cell lines DU145, PC3, and CWR22RV are much higher than that in the androgen-dependent cell line LNCaP, and that the androgen-independent cells are more resistant to mitoxantrone and Adriamycin than the androgen-dependent cells. Depleting 14-3-3sigma expression in DU145 and CWR22RV by RNA interference significantly sensitized these cells to mitoxantrone and Adriamycin by abrogating G2-M checkpoint and increasing apoptosis, whereas restoring 14-3-3sigma expression in LNCaP cells enhanced drug resistance. We also showed that 14-3-3sigma deficiency caused nuclear localization of Cdc2 and dephosphorylation of the Tyr15 residue upon DNA damage. Based on these studies, we propose that therapeutic intervention targeting 14-3-3sigma may be useful for sensitizing hormone-refractory prostate cancers to chemotherapy by both G2-M checkpoint abrogation and apoptosis enhancement.     

Blocking the Glycolytic Pathway Sensitizes Breast Cancer to Sonodynamic Therapy

Inhibition of the increased aerobic glycolysis in cancer cells is a promising methodology for various malignant tumor therapies but is limited by systemic toxicity, at least in part. Recent studies suggest that dual restriction of glycolysis and mitochondrial function may overcome this issue. Sonodynamic therapy (SDT), a prospective therapeutic modality for cancers, has been reported to induce mitochondria-dependent cell damage. Here, we investigated the combined effect of SDT and 2-deoxyglucose (2DG), an anti-glycolytic agent, on breast cancer both in vitro and in vivo. In vitro, we found that, compared with a single treatment, SDT + 2DG co-treatment significantly decreased cell viability and increased cell apoptosis. Moreover, the generation of reactive oxygen species was enhanced and mitochondrial membrane potential (MMP) was reduced after SDT + 2DG co-treatment. Furthermore, the oxidative phosphorylation was also restrained after SDT + 2DG co-treatment, further to cause the blockage of ATP provision. In vivo, SDT + 2DG markedly reduced tumor volume and weight, consistent with the in vitro findings. Furthermore, toxicology tests concurrently indicated that the dosages of sinoporphyrin sodium and 2DG were comparatively tolerable. Generally, these results indicated that SDT + 2DG combination therapy may be an available, promising therapy for highly metastatic breast cancer.     

Tobacco mosaic virus for the targeted delivery of drugs to cells expressing prostate-specific membrane antigen

Prostate-specific membrane antigen (PSMA) is a membrane-bound protein that is preferentially expressed in the prostate gland and induced in many prostate cancers, making it an important target for new diagnostics and therapeutics. To improve the efficacy of nanoparticle formulations for the imaging and/or eradication of prostate cancer, we synthesized the PSMA-binding glutamic acid derivative DUPA and conjugated it to the external surface of tobacco mosaic virus (TMV) particles. DUPA-targeted TMV was subsequently loaded with the antineoplastic agent mitoxantrone (MTO) or conjugated internally with the fluorescent dye cyanine 5 (Cy5). We found that TMV particles could be efficiently decorated with DUPA and loaded with MTO or Cy5 while maintaining structural integrity. DUPA-targeted TMV particles were able to bind more efficiently to the surface of PSMA⁺ LNCaP cells compared to non-targeted TMV; but there was little difference in binding efficiency between targeted and untargeted TMV when we tested PSMA⁻ PC3 cells (both cell lines are prostate cancer cell lines). DUPA-targeted TMV particles were internalized by LNCaP cells enabling drug delivery. Finally, we loaded the DUPA-targeted TMV particles and untargeted control particles with MTO to test their cytotoxicity against LNCaP cells in vitro. The cytotoxicity of the TMV-MTO particles (IC₅₀ = 10.2 nM) did not differ significantly from that of soluble MTO at an equivalent dose (IC₅₀ = 12.5 nM) but the targeted particles (TMV-DUPA-MTO) were much more potent (IC₅₀ = 2.80 nM). The threefold increase in cytotoxicity conferred by the DUPA ligand suggests that MTO-loaded, DUPA-coated TMV particles are promising as a therapeutic strategy for PSMA⁺ prostate cancer and should be advanced to preclinical testing in mouse models of prostate cancer.

Prostate-specific membrane antigen (PSMA) is a membrane-bound protein that is preferentially expressed in the prostate gland and induced in many prostate cancers, making it an important target for new diagnostics and therapeutics.     

Retracted Article: SNHG3 promotes proliferation and invasion by regulating the miR-101/ZEB1 axis in breast cancer

Background: Dysregulated lncRNA expression contributes to the pathogenesis of human tumors via the lncRNAs functioning as oncogenes or tumor suppressors. Small nucleolar RNA host gene 3 (SNHG3) was demonstrated to be upregulated in breast cancer cells. However, the detailed roles and molecular mechanism of SNHG3 in breast cancer are largely unknown. Methods: The expression of SNHG3, miR-101, and zinc finger E-box-binding protein 1 (ZEB1) in breast cancer tissues and cells was detected using qRT-PCR. The effects of SNHG3 on cell proliferation and invasion were evaluated using MTT, EdU, and cell invasion assays. The protein levels of Ki-67, proliferating cell nuclear antigen (PCNA), matrix metalloproteinase MMP-2, and MMP-9 were analyzed using western blot analysis. A luciferase reporter assay and RNA immunoprecipitation (RIP) were performed to explore the interaction between SNHG3, ZEB1 and miR-101. A subcellular fractionation assay was used to detect the subcellular location of SNHG3. Xenograft tumor experiments were conducted to verify the role and mechanism of SNHG3 in breast cancer in vivo. Results: SNHG3 expression was upregulated in breast cancer tissues and correlated with poor prognosis. SNHG3 knockdown suppressed breast cancer cell proliferation and invasion, which was further demonstrated by high levels of proliferation marker proteins Ki-67/PCNA and metastasis-related proteins MMP-2/MMP-9. Additionally, SNHG3 was located in the cytoplasm of breast cancer cells. SNHG3 functioned as a molecular sponge for miR-101 in breast cancer cells. miR-101 was downregulated in breast cancer tissues and negatively correlated with SNHG3 expression. Moreover, ZEB1, a target of miR-101, was positively regulated by SNHG3 in breast cancer cells. ZEB1 mRNA expression was upregulated in breast cancer tissues and positively correlated with SNHG3 expression. Mechanistically, SNHG3 knockdown suppressed cell proliferation and invasion by upregulation of miR-101 and downregulation of ZEB1 expression in breast cancer cells in vitro and in vivo. Conclusion: SNHG3 promoted proliferation and invasion by regulating the miR-101/ZEB1 axis in breast cancer.

In the present study, we investigated the expression and functional roles of SNHG3 in breast cancer cells, as well as the underlying mechanism of SNHG3 involved in the progression of breast cancer in vitro and in vivo.     

Bufalin suppresses colorectal cancer cell growth through promoting autophagy in vivo and in vitro

Specific groups in Asia, including the Chinese, are more susceptible to colorectal cancer (CRC). The best strategy for anticancer drug action is to induce cancer cell apoptosis and autophagy. Bufalin is a potent inducer of apoptosis in some human cancer cell lines, but bufalin has barely been evaluated in colorectal cancer cells as a potent autophagy inducing agent. The aim of this study was to investigate the roles and interactions of bufalin in autophagy and the effects of the drug on human colorectal cancer. We applied bufalin and autophagy inhibitors (CQ and 3-MA) in LoVo cells to investigate their potential anticancer bioactivity under certain concentrations of bufalin to monitor autophagy and cell proliferation in vivo and in vitro. Bufalin induced autophagy of LoVo and inhibited proliferation of LoVo cells. Bufalin inhibited the expression of autophagy-related (ATG) proteins and tumor growth in vivo. Our studies identified that bufalin could potentially be a small molecule inhibitor for cancer therapy.

Specific groups in Asia, including the Chinese, are more susceptible to colorectal cancer (CRC).     

Hesperetin inhibits Eca-109 cell proliferation and invasion by suppressing the PI3K/AKT signaling pathway and synergistically enhances the anti-tumor effect of 5-fluorouracil on esophageal cancer in vitro and in vivo

Previously, we reported that hesperetin exhibited pro-apoptotic activity against esophageal cancer in vitro and in vivo. Here, we examined whether hesperetin inhibits cell proliferation and invasion and synergistically enhances the anti-tumor effect of 5-fluorouracil (5-FU) in esophageal cancer. Flow cytometry, EdU staining, and transwell invasion assays using Eca-109 cells showed that hesperetin induced cell cycle arrest at the G0/G1 phase and inhibited cell proliferation and invasion significantly. Moreover, hesperetin suppressed the expression of phosphorylated PI3K/AKT, cyclin D1, MMP-2, and MMP-9 and increased phosphorylated PTEN and p21 in Eca-109 cells. Hesperetin combined with 5-FU inhibited cell growth more effectively than did either drug alone in Eca-109 cells and in a xenograft mouse model. Hoechst 33258, Annexin V-PE/7-ADD double staining and TUNEL assay showed more apoptotic cells in the combination treatment group than in either individual treatment group. The combination down-regulated protein levels of Bcl-2 and up-regulated those of Bax, cleaved caspase-3, and cleaved caspase-9 more effectively than did either drug alone. These data suggest that hesperetin inhibited esophageal cancer cell proliferation and invasion by suppressing the PI3K/AKT signaling pathway. In conclusion, 5-FU and hesperetin exerted synergistic antitumor effects in vivo and in vitro and could constitute a novel therapeutic approach for esophageal cancer.

Effects of hesperetin on the proliferation and invasion of esophageal cancer cells and its synergistic anti-cancer effect with 5-FU.     

In silico and in vitro design of cordycepin encapsulation in liposomes for colon cancer treatment

Cordycepin or 3′-deoxyadenosine is an interesting anti-cancer drug candidate that is found in abundance in the fungus Cordyceps militaris. It inhibits cellular growth of many cancers including lung carcinoma, melanoma, bladder cancer, and colon cancer by inducing apoptosis, anti-proliferation, anti-metastasis and by arresting the cell cycle. Cordycepin has, however, poor stability and low solubility in water, resulting in loss of its bioactivity. Liposomes can be used to overcome these obstacles. Our aim is to improve cordycepin''s anti-colon cancer activity by liposome encapsulation. Cordycepin-encapsulated liposomes were designed and fabricated based on a combination of theoretical and experimental studies. Molecular dynamics (MD) simulations and free energy calculations suggest that phosphatidylcholine (PC) lipid environment is favorable for cordycepin adsorption. Cordycepin passively permeates into PC lipid bilayers without membrane damage and strongly binds to the lipids'' polar groups by flipping its deoxyribose sugar toward the bilayer center. Our fabricated liposomes containing 10 : 1 molar ratio of egg yolk PC : cholesterol showed encapsulation efficiency (%EE) of 99% using microfluidic hydrodynamic focusing (MHF) methods. In our in vitro study using the HT-29 colon cancer cell line, cordycepin was able to inhibit growth by induction of apoptosis. Cell viability was significantly decreased below 50% at 125 μg mL⁻¹ dosage after 48 h treatment with non-encapsulated and encapsulated cordycepin. Importantly, encapsulation provided (1) a 2-fold improvement in the inhibition of cancer cell growth at 125 μg mL⁻¹ dosage and (2) 4-fold increase in release time. These in silico and in vitro studies indicate that cordycepin-encapsulated liposomes could be a potent drug candidate for colon cancer therapy.

Cordycepin-encapsulated liposomes could be a potent drug candidate for cancer therapy.     

Bone morphogenetic protein-9 promotes the proliferation of non-small cell lung cancer cells by activating PI3K/Akt and Smad1/5 pathways

Non-small-cell lung carcinoma (NSCLC) is any type of epithelial lung cancer other than small cell lung carcinoma (SCLC), which accounts for about 85% of all lung cancers. Bone morphogenetic protein (BMP)-9 in humans is encoded by the growth differentiation factor 2 gene, which belongs to the transforming growth factor-beta superfamily. In the present study, we explored the role of BMP-9 in A549 and NCI-H1650 cell proliferation and its possible molecular mechanisms. 25 NSCLC patients were recruited to evaluate mRNA expression of BMP-9 to determine its clinicopathologic significance. We found that recombinant protein BMP-9 and overexpression of BMP-9 promoted A549 and NCI-H1650 cell proliferation in vitro, which was abolished by phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002). Western blot results revealed that BMP-9 significantly activated the PI3K/Akt and Smad1/5 pathway signaling. In vivo, BMP-9 promoted tumor growth and PI3K/Akt and Smad1/5 signaling pathways in an A549 or NCI-H1650 cell line-derived xenograft model. Knockdown BMP-9 or BMP-9 receptor ALK1 inhibited A549 cell growth in vitro and in vivo, which was associated with regulating the PI3K/Akt and Smad1/5 signaling pathways. These results demonstrated that BMP-9 promoted A549 and NCI-H1650 cell proliferation via PI3K/Akt and Smad1/5 signaling pathways.

Non-small-cell lung carcinoma (NSCLC) is any type of epithelial lung cancer other than small cell lung carcinoma (SCLC), which accounts for about 85% of all lung cancers.     

Nanomedicine-based combination of gambogic acid and retinoic acid chlorochalcone for enhanced anticancer efficacy in osteosarcoma

In this study, gambogic acid (GA) and retinoic acid chlorochalcone (RACC) co-loaded glycol chitosan nanoparticle was successfully developed and studied for its therapeutic efficacy against osteosarcoma cancer cells. The GA/RACC loaded glycol chitosan nanoparticles (RGNP) was nanosized and exhibited a controlled release of drug in either pH 7.4 and pH 5.0. Owing to the strong positive charge on the RGNP surface, efficiency cellular uptake was observed in cancer cells. Moreover, a synergistic combination of GA and RACC were effectively suppressed the tumor growth progression. The half maximal inhibitory concentration (IC50) values in MG63 cells were 0.89 μg/ml and 0.35 μg/ml for GA and RGNP after 24 h. The results clearly suggest the synergist effect of GA and RACC in effectively inhibiting the cancer cell proliferation. The RGNP as expected induced a remarkably higher apoptosis of cancer cells with ∼28%. Overall, combination of GA and RACC encapsulated in a nanocarrier could be an effective strategy to treat osteosarcoma. Future studies will focus on the in vivo evaluation of GA/RACC-loaded polymeric nanoparticles.     

Sonodynamic Therapy Based on Combined Use of Low Dose Administration of Epirubicin-Incorporating Drug Delivery System and Focused Ultrasound

Sonodynamic therapy (SDT) is currently considered as one of the promising minimally invasive treatment options for solid cancers. SDT is based on the combined use of a sonosensitizer drug and high-intensity focused ultrasound (HIFU) to produce cytotoxic reactive oxygen species (ROS) in and around neoplastic cells. Anthracycline drugs, including epirubicin (EPI), have been well known as effective sonosensitizers after interaction with focused ultrasound. Recently a new anticancer drug delivery system (DDS), NC-6300, has been developed that comprises EPI through an acid–labile hydrazone bond. In previous in vivo studies, NC-6300 showed basic drug safety and an excellent concentration property of EPI, and recently has been tested in clinical trials. For realizing minimally invasive cancer treatment, the present study demonstrated the effectiveness and feasibility of DDS-based SDT, which combined a small dose of NC-6300 and low energy of HIFU in mouse models of colon cancer and pancreatic cancer.     

KITENIN-targeting MicroRNA-124 Suppresses Colorectal Cancer Cell Motility and Tumorigenesis

MicroRNAs are increasingly implicated in the modulation of the progression of various cancers. We previously observed that KAI1 C-terminal interacting tetraspanin (KITENIN) is highly expressed in sporadic human colorectal cancer (CRC) tissues and hence the functional KITENIN complex acts to promote progression of CRC. However, it remains unknown that microRNAs target KITENIN and whether KITENIN-targeting microRNAs modulate CRC cell motility and colorectal tumorigenesis. Here, through bioinformatic analyses and functional studies, we showed that miR-124, miR-27a, and miR-30b negatively regulate KITENIN expression and suppress the migration and invasion of several CRC cell lines via modulation of KITENIN expression. Through in vitro and in vivo induction of mature microRNAs using a tetracycline-inducible system, miR-124 was found to effectively inhibit the invasion of CT-26 colon adenocarcinoma cells and tumor growth in a syngeneic mouse xenograft model. Constitutive overexpression of precursor miR-124 in CT-26 cells suppressed in vivo tumorigenicity and resulted in decreased expression of KITENIN as well as that of MYH9 and SOX9, which are targets of miR-124. Thus, our findings identify that KITENIN-targeting miR-124, miR-27a, and miR-30b function as endogenous inhibitors of CRC cell motility and demonstrate that miR-124 among KITENIN-targeting microRNAs plays a suppressor role in colorectal tumorigenesis.     

Expression and purification of prostate-specific membrane antigen in the baculovirus expression system and recognition by prostate-specific membrane antigen-specific T cells.

Antigen-specific immunotherapy of cancer depends on a consistent source of well-defined protein antigen. Production of recombinant protein offers the obvious solution to this problem but few comparisons of recombinant and native proteins in cellular immune assays have been reported. We report expression of a putative immunotherapy antigen, prostate-specific membrane antigen (PSMA), in insect cells using a baculovirus vector. T cells stimulated with recombinant PSMA or native PSMA derived from the LNCaP cell line recognized both native PSMA and recombinant, baculoviral PSMA. These data indicate that PSMA produced in Sf9 cells is immunologically cross-reactive with native PSMA and therefore suitable for immunotherapy as it is recognized by both cellular and humoral immune responses.     

In vitro and in vivo responses of advanced prostate tumors to PSMA ADC, an auristatin-conjugated antibody to prostate-specific membrane antigen

Prostate-specific membrane antigen (PSMA) is a membrane protein that is overexpressed manifold in prostate cancer and provides an attractive target for therapy. PSMA ADC is an antibody-drug conjugate (ADC) that consists of a fully human anti-PSMA monoclonal antibody conjugated to monomethylauristatin E through a valine-citrulline linker. In this study, the antitumor activity of PSMA ADC was evaluated against a panel of prostate cancer cell lines in vitro and in a novel in vivo model of taxane-refractory human prostate cancer. In vitro cell killing was efficient for cells with abundant PSMA expression (>10(5) molecules/cell; IC(50) ≤ 0.022 nmol/L) and 1,000-fold less efficient for cells with undetectable PSMA (IC(50) > 30 nmol/L). Intermediate potency (IC(50) = 0.80 nmol/L) was observed for cells with approximately 10(4) molecules of PSMA per cell, indicating a threshold PSMA level for selective cell killing. Similar in vitro activity was observed against androgen-dependent and -independent cells that had abundant PSMA expression. In vitro activity of PSMA ADC was also dependent on internalization and proper N-glycosylation/folding of PSMA. In contrast, less potent and nonselective cytotoxic activity was observed for a control ADC, free monomethylauristatin E, and other microtubule inhibitors. PSMA ADC showed high in vivo activity in treating xenograft tumors that had progressed following an initial course of docetaxel therapy, including tumors that were large (>700 mm(3)) before treatment with PSMA ADC. This study defines determinants of antitumor activity of a novel ADC. The findings here support the clinical evaluation of this agent in advanced prostate cancer.