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.     

Disulfide-constrained peptides that bind to the extracellular portion of the prostate-specific membrane antigen

The prostate-specific membrane antigen (PSMA) is a well-characterized surface antigen, overexpressed in the most advanced, androgen-resistant human prostate cancer cells. We sought to exploit PSMA cell surface properties as a target for short peptides that will potentially guide protein-based therapeutics, such as viral vectors, to prostate cancer cells. Two separate phage display peptide strategies were applied, in parallel, to purified PSMA protein bound to two separate substrates. We reasoned that peptide sequences common to both substrate selections would be specific binders of PSMA. Additionally, the design allowed for stringent cross-selections, where phage populations from one selection condition could be applied to the alternative substrate. These strategies resulted in a series of phage displayed peptides able to bind to PSMA by ELISA and direct binding assays, both with purified protein and in prostate cancer cells. Cell binding is competitively inhibited by purified PSMA. The synthesized peptides are capable of enhancing PSMA carboxypeptidase enzymatic activity, suggesting protein folding stabilization. The discovery of these peptides provides the foundation for subsequent development of peptide targeted therapeutics against prostate cancer.     

Dendritic cells transduced with HSV-1 amplicons expressing prostate-specific antigen generate antitumor immunity in mice

There is currently much interest in generating cytotoxic T lymphocyte (CTL) responses against tumor antigens as a therapy for cancer. This work describes a novel gene transfer technique utilizing dendritic cells (DCs), an extremely potent form of antigen-presenting cell (APC), and herpes simplex virus-1 (HSV-1) amplicons. HSV-1 amplicons are plasmid-based viral vectors that are packaged into HSV-1 capsids, but lack viral coding sequences. Amplicon vectors have been constructed that encode the model tumor antigen ovalbumin (HSV-OVA) and human prostate-specific antigen (HSV-PSA), a protein that is expressed specifically in prostate epithelium and prostate carcinoma cells. These amplicons were packaged using a helper virus-free system that produces vector stocks that are devoid of contaminating cytotoxic helper virus. Transduction of DCs with HSV-OVA or HSV-PSA and co-culture with CTL hybridomas results in specific activation, indicating that transduced DCs express these transgenes and process the tumor antigens for class I MHC presentation to CTL. Mice immunized with HSV-PSA-transduced DCs generate a specific CTL response that can be detected in vitro by a (51)Cr-release assay and are protected from challenge with tumors that express PSA. These results indicate that DCs transduced with HSV-1 amplicon vectors may provide a tool for investigation of the biology of CTL activation by DCs and a new modality for immunotherapy of cancer.     

Conjugation of an antibody to cross-linked fibrin for targeted delivery of anti-restenotic drugs

There is an urgent need to treat restenosis, a major complication of the treatment of arteries blocked by atherosclerotic plaque, using local delivery techniques. We observed that cross-linked fibrin (XLF) is deposited at the site of surgical injury of arteries. An antibody to XLF, conjugated to anti-restenotic agents, should deliver the drugs directly and only to the site of injury. An anti-XLF antibody (H93.7C.1D2/48; 1D2) was conjugated to heparin (using N-succinimidyl 3-(2-pyridyldithio)propionate), low molecular weight heparin (LMWH) (adipic acid dihydrazide) and rapamycin (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide), and the conjugates purified and tested for activity before use in vivo. Rabbits had their right carotid arteries de-endothelialised and then given a bolus of 1D2–heparin, 1D2–LMWH or 1D2–rapamycin conjugate or controls of saline, heparin, LMWH, rapamycin or 1D2 (±heparin bolus) and sacrificed after 2 or 4 weeks (12 groups, n=6/group). Rabbits given any of the conjugates had minimal neointimal development in injured arteries, with up to 59% fewer neointimal cells than those given control drugs. Rabbits given 1D2–heparin or 1D2–LMWH had an increased or insignificant reduction in luminal area, with positive remodelling, while the medial and total arterial areas of rabbits given 1D2–rapamycin were not affected by injury. Arteries exposed to 1D2–heparin or 1D2–rapamycin had more endothelial cells than rabbits given control drugs. Thus, XLF-antibodies can site-deliver anti-restenotic agents to injured areas of the artery wall, where the conjugates can influence remodelling, re-endothelialisation and neointimal cell density, with reduced neointimal formation.     

Engineering adeno-associated virus 2 vectors for targeted gene delivery to atherosclerotic lesions

Targeted delivery of biological agents to atherosclerotic plaques may provide a novel treatment and/or useful tool for imaging of atherosclerosis in vivo. However, there are no known viral vectors that possess the desired tropism. Two plaque-targeting peptides, CAPGPSKSC (CAP) and CNHRYMQMC (CNH) were inserted into the capsid of adeno-associated virus 2 (AAV2) to assess vector retargeting. AAV2-CNH produced significantly higher levels of transduction than unmodified AAV2 in human, murine and rat endothelial cells, whereas transduction of nontarget HeLa cells was unaltered. Transduction studies and surface plasmon resonance suggest that AAV2-CNH uses membrane type 1 matrix metalloproteinase as a surface receptor. AAV2-CAP only produced higher levels of transduction in rat endothelial cells, possibly because the virus was found to be affected by proteasomal degradation. In vivo substantially higher levels of both peptide-modified AAV2 vectors was detected in the brachiocephalic artery (site of advanced atherosclerotic plaques) and aorta, whereas reduced levels were detected in all other organs examined. These results suggest that in the AAV2 platform the peptides are exposed on the capsid surface in a way that enables efficient receptor binding and so creates effective atherosclerotic plaque targeted vectors.     

Artificial polymeric cells for targeted drug delivery.

Selectins are optimal biological molecules for targeted delivery of therapeutic agents because of their localized and carefully regulated expression in several human diseases, and their highly specific interactions with their counter receptors. In this study, we describe a targeted delivery system that can potentially deliver anti-inflammatory drug to sites of chronic inflammation using Poly(lactic-co-glycolic acid) (PLGA) and selectin-ligand chemistry. Biotinylated-sialyl Lewis(x) (sLe(x)), a carbohydrate that serves as a ligand to selectins, was attached to the surface of avidin-linked PLGA microspheres. These carbohydrate-coated microspheres mimic the adhesive behavior of leukocytes on selectins in flow chambers, displaying slow rolling under flow. The rolling velocity of these artificial leukocytes is similar to that displayed by leukocytes rolling on P- or E-selectin coated surfaces. We can tune rolling velocity, and hence residence time of capsules on surfaces, by changing the density of sialyl Lewis(x) on the microsphere surfaces. Therefore, we have made a targeted drug delivery vehicle that mimics the adhesive properties of leukocytes and is biodegradable.     

A nanoprobe for fluorescent monitoring of microRNA and targeted delivery of drugs

Multifunctional nano-materials that can be used to monitor the expression of specific biomarkers and serve as vehicles for controlled drug delivery are highly desirable. Herein, we report a new DNA-hybrid-gated core–shell upconversion nanoprobe (UCNP@MOF/DOX) for fluorescence analysis of microRNA-21 (miR-21), which also triggers the release of drug loaded in the probes for on-demand anti-cancer treatment. The nanoprobe is built on the merits of ultraviolet-visible light of upconversion nanoparticles (UCNPs) excited by near-infrared (NIR) and extraordinary loading capability of metal–organic frameworks (MOFs) for drug delivery. Controlled release of doxorubicin (DOX) from the nanoprobe by miR-21 underwent the following two-stage kinetics: a fast release stage specifically triggered by miR-21 and proportional to miR-21 concentration and a slow stage observed in both gated and ungated nanoprobes due to collapse of the UIO-66-NH₂ coatings via ligand exchange with phosphates. In addition, the nanoprobe showed good selectivity, a linear response towards miR-21 ranging from 4 nM to 500 nM, and a limit of detection in 4 nM, which precluded unintended payload leakage due to low-abundance endogenous miR-21 expression in normal cells. Moreover, based on a dual-targeted delivery system constituted by AS1411-mediated recognition and responsive release of DOX, a specific cytotoxic efficacy was observed in MCF-7 cells. The present work provides a smart and robust nanoprobe for real-time detection of miRNA and dual-responsive drug delivery in tumor cells.

A DNA-hybrid-gated core–shell upconversion nanoprobe is prepared for both fluorescent monitoring of miR-21 and on-demand delivery of DOX. It showed good selectivity towards miR-21 and demonstrated specific cytotoxic efficacy towards MCF-7 cells.     

Trastuzumab-cisplatin conjugates for targeted delivery of cisplatin to HER2-overexpressing cancer cells

Cisplatin is widely used for the treatment of numerous types of cancer, while its application is limited by the adverse side effects for its poor selectivity. Trastuzumab is a highly targeting protein to HER2 protein, and it is usually combined with paclitaxel or cisplatin for the treatment of HER2-overexpressing breast cancer. In the present work, we used trastuzumab as a targeting carrier for platinum drug delivery. In ELISA assays and immunofluorescence study, Tmab-1 exhibited high and specific binding affinity to HER2 protein and HER2-overexpressing SK-BR-3 cells. In cytotoxicity test, Tmab-1 showed promising antiproliferative activity to SK-BR-3 cells, while it hardly inhibited the growth of MCF-7 cells and MDA-MB-231 cells. The cell cycle arrest study showed Tmab-1 induced the cell cycle arrest mainly at G2/M phase. This work indicates that trastuzumab is an effective and potential targeting carrier for drug delivery.     

Internalizing antibody-based targeted gene delivery for human cancer cells

Gene therapy, a potential solution to hereditary and nonhereditary diseases, faces the challenges of safe and specific gene delivery. Cationic carrier molecules (e.g., liposome and polymers) that form noncovalent complexes with negatively charged DNA have been in use as nonviral gene delivery vectors. Although they tend to be relatively less efficient than viral systems, they have inherent advantages of flexibility and safety. Their derivatives, in conjugation with functional molecules such as peptides, proteins, growth factors, and antibodies, have been focused on to generate nanocarriers with low toxicity, high stability, high efficiency, and cell-specific targeting features. Here we describe internalizing polyclonal and monoclonal antibodies against a stress chaperone, mortalin/mtHsp70. We demonstrate that these internalizing anti-mortalin antibodies (i-mot Ab) could be employed for (1) internalization of nanoparticles (quantum dots, Qdots) and the generation of illuminating cells and (2) gene delivery. By using cancer and normal human cells in parallel, we further demonstrate that gene delivery can be specifically enhanced in human cancer cells if cationic polymer polyethylenimine (PEI) and i-mot Ab complex are used and may provide a novel cancer-targeting nanocarrier.     

Characterization of monoclonal antibodies for prostate-specific antigen and development of highly sensitive free prostate-specific antigen assays

BACKGROUND: The recent elucidation of the importance of serological free prostate-specific antigen (PSA) in the diagnosis of prostate cancer has created a demand for immunoassays specific for free PSA. METHODS: We developed and characterized 11 monoclonal antibodies with high affinities for PSA (Ka values from 1.1 x 10(8) to 1.8 x 10(10)L/mol), only 3 of which cross-react with human glandular kallikrein (hK2). Using these antibodies and PSA antibodies developed by others, in conjunction with time-resolved fluorometry, we developed ultrasensitive sandwich immunoassays specific for the free form of PSA. RESULTS: The analytical detection limit of these immunoassays is 0.001 microg/L. To our knowledge, this is the most sensitive free PSA assay reported to date. The free PSA immunoassays exhibit <1% cross-reactivity with PSA-alpha1-antichymotrypsin, show no cross-reactivity with hK2, and correlate well with established free PSA kits. The 11 antibodies developed by our group, in conjunction with 4 commercially available antibodies, were used to generate a putative epitope map of the PSA molecule. CONCLUSION: The highly sensitive free PSA immunoassays may be used for measuring PSA subfractions in female serum, an application currently impossible with other reported free PSA immunoassays.     

Sialyl LewisX mimic-decorated liposomes for anti-angiogenic everolimus delivery to E-selectin expressing endothelial cells

In this study, we developed novel E-selectin-targeting liposomes, i.e., 3′-(1-carboxy)ethyl sialyl LewisX (3′-CE sLeX) mimic liposomes, for targeted delivery of everolimus (EVE) in anti-angiogenic therapy. We investigated the uptake and efficacy of these E-selectin targeting liposomes in inflammatory cytokine-treated human umbilical vein endothelial cells (HUVECs). The uptake of EVE in 3′-CE sLeX mimic liposomes increased steadily and almost caught up with the uptake of plain EVE at 3 h, which was higher than that in PEGylated liposomes (PEG-liposomes). Inhibition of uptake by anti-E-selectin antibody suggested involvement of E-selectin-mediated endocytotic processes. Migration in cells treated with EVE/3′-CE sLeX mimic liposomes was suppressed by more than half when compared to the control. This treatment was also seen to significantly inhibit the formation of capillary tubes and networks. In addition, Thr389 phosphorylation of pS6 kinase, as a marker of mTOR activity, was remarkably suppressed to less than endogenous levels by EVE/3′-CE sLeX mimic liposomes. In conclusion, the present study demonstrated that EVE/3′-CE sLeX mimic liposomes were intracellularly taken up by E-selectin and prompted anti-angiogenic effects of EVE involved in the mTOR signaling pathway. However, moderate retention of EVE in the liposomes might limit the targeting ability of 3′-CE sLeX mimic liposomes.

Novel E-selectin-targeting liposomes deliver everolimus to E-selectin expressing endothelial cells and accelerate its anti-angiogenic effect.     

Anti-GAD antibody targeted non-viral gene delivery to islet beta cells.

An islet cell targeting polymeric gene carrier was synthesized by conjugating anti-GAD Fab' fragment to PEI via PEG linker (PEI-PEG-Fab'). The Fab' fragment was prepared from a murine monoclonal antibody against glutamic acid decarboxylase (GAD), which has been identified as one of the major auto-antigens expressed in islet cells, and used as a targeting moiety for islet cell targeting. The electrophoretic migration of plasmid DNA (pCMVLuc)/PEI-PEG-Fab' complexes in agarose gel was completely retarded above the N/P ratio of 2. The complexes demonstrated a size of 100-275 nm with an almost neutral surface charge. Confocal microscopy revealed that the PEI-PEG-Fab' complexes showed much higher cellular binding and uptake efficiency compared to PEI-PEG complexes. The PEI-PEG-Fab' showed about 10-fold higher transfection efficiency (relative luciferase activity) than PEI-PEG in GAD-expressing mouse insulinoma cells (MIN6), however the transfection efficiency of PEI-PEG-Fab' reduced to that of PEI-PEG in GAD negative cells (293) and in the presence of competitive free Fab'. Considering the neutral surface charge of its complexes with DNA, and selectivity toward the islet cells expressing a specific antigen, the PEI-PEG-Fab' conjugate could be thought as a potential candidate of the systemic gene therapy for the treatment of type I diabetes.     

Stable cationic microparticles for enhanced model antigen delivery to dendritic cells.

The objectives of this work were (i) to prepare physically stable cationic microparticles and (ii) to study the impact of the surface properties on microparticle phagocytosis and the phenotype of dendritic cells (DC). Protein loaded biodegradable microparticles from poly(lactic-co-glycolic acid) [PLGA] were produced in a micromixer-based w/o/w solvent evaporation procedure. Anionic particles were obtained by using polyvinyl alcohol (PVA) as stabilizing agent; for cationic surfaces cetyltrimethylammonium bromide (CTAB) and chitosan/PVA or DEAE-dextran/PVA blends were evaluated. In phagocytosis studies human monocytes and monocyte-derived DC were incubated with microparticles and analysed by flow cytometry. While CTAB modified microparticles lost their positive charge and aggregated due to CTAB desorption from the particle surface, the modification with chitosan and DEAE-dextran resulted in stable microparticles without cell toxicity. Due to a very low endotoxin content, phagocytosis of anionic and cationic microparticles did not induce an upregulation of maturation-associated surface markers on DC. DEAE-dextran modified microparticles showed an enhanced model protein delivery into phagocytic cells. Overall, PLGA microparticles are suitable vehicles for protein delivery to DC, which might be used for DC-based cell therapies.     

Local delivery of vaccinia virus expressing multiple costimulatory molecules for the treatment of established tumors

Successful immunotherapy of established tumors depends on overcoming the suppressive influence of the local tumor microenvironment. The direct injection of vaccinia virus expressing the B7.1 (CD80) costimulatory molecule into melanoma lesions resulted in local and systemic immunity with associated clinical responses. Therefore, we sought to evaluate the effects of a vaccinia virus expressing three costimulatory molecules, B7.1, ICAM-1, and LFA-3 (rV-TRICOM), in patients with metastatic melanoma. A standard dose escalation phase I clinical trial was performed. Thirteen patients were enrolled and 12 were available for follow-up. Local vaccination was feasible, with only low-grade injection site reactions associated with mild fatigue and myalgia reported. There was one occurrence of grade 1 vitiligo. Overall there was a 30.7% objective clinical response, with one patient achieving a complete response for more than 22 months. An inverse association was detected between anti-vaccinia antibody and anti-vaccinia T cell responses. Patients who failed to respond to vaccination but received high-dose interleukin-2 had a trend toward improved survival. Collectively, these results confirm the safety profile and feasibility of direct injection of vaccinia virus expressing multiple costimulatory molecules in patients with established tumors. Further clinical investigation is needed to better define the role of antigen, adjuvant cytokines, costimulation, and cross-presentation in the host immune response to local vaccination with vaccinia viruses expressing immunomodulatory molecules.     

前列腺特异抗原在前列腺癌诊断中的价值探讨

目的探讨游离前列腺特异抗原（ｆＰＳＡ）／总前列腺特异抗原（ｔＰＳＡ）比值在前列腺癌（ＰＣａ）鉴别诊断中的价值。方法检测６３名正常人、７５例前列腺增生和４０例前列腺癌患者的血清ｔＰＳＡ和ｆＰＳＡ，并求得ｆＰＳＡ／ｔＰＳＡ比值。ｔＰＳＡ测定采用放射免疫双抗体法，ｆＰＳＡ测定采用免疫放射双抗体夹心法。计量资料以ｘ±ｓ表示，两组均数比较采用ｔ检验，计数资料采用χ２检验。结果单独以ｔＰＳＡ＞２．７μｇ／Ｌ或ｆＰＳＡ＞０．６０μｇ／Ｌ作为对ＰＣａ的诊断指标，其特异性和阳性预测值分别为５３．３％、５０．７％和５６．０％、５０．７％，而以ｆＰＳＡ／ｔＰＳＡ比值＜０．０９３作为对ＰＣａ的诊断指标，其特异性与阳性预测值分别高达８０％和７９．５％，显著高于ｔＰＳＡ和ｆＰＳＡ（Ｐ＜０．００１）。结论ｆＰＳＡ／ｔＰＳＡ比值结合临床情况和影像学检查，可大大提高ＰＳＡ对ＰＣａ诊断的特异性，为ＰＣａ的早期诊断提供了新的依据。     

复合前列腺特异性抗原、总前列腺特异性抗原及其比值在前列腺癌诊断中的应用

目的:研究复合前列腺特异性抗原、总前列腺特异性抗原及其比值在鉴别前列腺癌和良性前列腺增生中的应用价值。方法:采用化学发光法检测30例前列腺癌患者、40例良性前列腺增生患者和30例对照组患者复合前列腺特异性抗原、总前列腺特异性抗原,计算并比较其比值。结果:前列腺癌患者的复合前列腺特异性抗原、总前列腺特异性抗原均高于对照组和良性前列腺增生患者,差异均具有统计学意义(P<0.05);其比值前列腺癌患者高于良性前列腺增生患者,差异有统计学意义(P<0.05);其相关性和共线性均不如前列腺癌组病例。结论:前列腺癌患者总前列腺特异性抗原的升高以复合前列腺特异性抗原升高为主,测定复合前列腺特异性抗原更有意义,结合其比值进行综合分析可以有效区分良性前列腺增生和前列腺癌。     

N-glycosylation and microtubule integrity are involved in apical targeting of prostate-specific membrane antigen: implications for immunotherapy

Prostate-specific membrane antigen (PSMA) is an important biomarker expressed in prostate cancer cells with levels proportional to tumor grade. The membrane association and correlation with disease stage portend a promising role for PSMA as an antigenic target for antibody-based therapies. Successful application of such modalities necessitates a detailed knowledge of the subcellular localization and trafficking of target antigen. In this study, we show that PSMA is expressed predominantly in the apical plasma membrane in epithelial cells of the prostate gland and in well-differentiated Madin-Darby canine kidney cells. We show that PSMA is targeted directly to the apical surface and that sorting into appropriate post-Golgi vesicles is dependent upon N-glycosylation of the protein. Integrity of the microtubule cytoskeleton is also essential for delivery and retention of PSMA at the apical plasma membrane domain, as destabilization of microtubules with nocodazole or commonly used chemotherapeutic Vinca alkaloids resulted in the basolateral expression of PSMA and increased the uptake of anti-PSMA antibody from the basolateral domain. These results may have important relevance to PSMA-based immunotherapy and imaging strategies, as prostate cancer cells can maintain a well-differentiated morphology even after metastasis to distal sites. In contrast to antigens on the basolateral surface, apical antigens are separated from the circulation by tight junctions that restrict transport of molecules across the epithelium. Thus, antigens expressed on the apical plasma membrane are not exposed to intravenously administered agents. The ability to reverse the polarity of PSMA from apical to basolateral could have significant implications for the use of PSMA as a therapeutic target.