Construction and characterization of an anti-asialoglycoprotein receptor single-chain variable-fragment-targeted melittin

Antibody-therapeutic agent conjugation to be delivered specifically to tumor cells is required for many target-based therapeutic strategies. In the present study, a recombinant immunotoxin was constructed by which melittin was fused to an anti-asialoglycoprotein receptor (ASGPR) single-chain variable fragment antibody (C1), and targeting ability and cytolytic efficacy of the fusion protein were studied. Our results suggested that the recombinant 29.4 kDa protein C1M was expressed in Escherichia coli as a soluble style. Binding of C1M to the surface of hepatocellular carcinoma (HCC) cells was confirmed by both immunohistochemistry and flow cytometry assays. C1M kept the hemolytic activity of melittin and exhibited cytolytic capacity to HepG2 cells at a concentration of 1.5 μg/mL, under which erythrocytes would not be lysed. The effects were greatly inhibited by coadministration with asialoorosomucoid, a natural ligand for ASGPR. These results suggested that C1M conferred targeting and ASGPR-specific cytotoxicity to HCC cells. This work makes it possible to further investigate its antihepatoma efficacy in vivo.     

肝脏去唾液酸糖蛋白受体特异性RNA适配子的SELEX筛选与鉴定

目的获得能够特异性高亲和力结合肝脏特异性去唾液酸糖蛋白受体（asialoglycoprotein receptor,ASGPR）的RNA适配子,为开发诊断和治疗肝脏疾病的靶向性试剂和药物奠定基础。方法合成一个长度为115nt含有25个随机序列的单链DNA随机文库,通过体外转录构建出单链RNA适配子随机文库,以肝脏ASGPR大亚基为靶蛋白,采用SELEX（systematic evolution of ligands by exponential enrichment）技术筛选具有高亲和力的AsGPR特异性RNA适配子;通过膜结合测定实验、凝胶阻滞实验鉴定筛选适配子对靶蛋白的特异性和亲和力。结果经过12轮筛选获得了具有高亲和力的肝脏ASGPR特异性RNA适配子。结论成功地筛选出了具有离亲和力的肝脏ASGPR特异性RNA适配子库。     

Hepatocyte-targeting gene delivery using a lipoplex composed of galactose-modified aromatic lipid synthesized with click chemistry

Highly efficient drug carriers targeting hepatocyte is needed for treatment for liver diseases such as liver cirrhosis and virus infections. Galactose or N-acetylgalactosamine is known to be recognized and incorporated into the cells through asialoglycoprotein receptor (ASGPR) that is exclusively expressed on hepatocyte and hepatoma. In this study, we synthesized a galactose-modified lipid with aromatic ring with click chemistry. To make a complex with DNA, termed ‘lipoplex’, we prepared a binary micelle composed of cationic lipid; dioleoyltrimethylammoniumpropane (DOTAP) and galactose-modified lipid (D/Gal). We prepared lipoplex from plasmid DNA (pDNA) and D/Gal and examined the cell specificity and transfection efficiency. The lipoplex was able to interact with ASGPR immobilized on gold substrate in the quartz-crystal microbalance (QCM) sensor cell. The lipoplex induced high gene expression to HepG2 cells, a human hepatocellular carcinoma cell line, but not to A549 cells, a human alveolar adenocarcinoma cell line. The treatment with asialofetuin, which is a ligand for ASGPR and would work as a competitive inhibitor, before addition of the lipoplexes decreased the expression to HepG2 cells. These results indicate that D/Gal lipoplex was incorporated into HepG2 cells preferentially through ASGPR and the uptake was caused by galactose specific receptor. This delivery system to hepatocytes may overcome the problems for gene therapy and be used for treatment of hepatitis and hepatic cirrhosis.     

Detection of Circulating Tumor Cells in Hepatocellular Carcinoma Using Antibodies against Asialoglycoprotein Receptor,Carbamoyl Phosphate Synthetase 1 and Pan-Cytokeratin

Background

Asialoglycoprotein receptor (ASGPR)-ligand-based separation combined with identification with Hep Par 1 or pan-cytokeratin (P-CK) antibody have been demonstrated to detect circulating tumor cells (CTCs) in hepatocellular carcinoma (HCC). The aim of this study was to develop an improved enrichment and identification system that allows the detection of all types of HCC CTCs.

Methods

The specificity of the prepared anti-ASGPR monoclonal antibody was characterized. HCC cells were bound by ASGPR antibody and subsequently magnetically isolated by second antibody-coated magnetic beads. Isolated HCC cells were identified by immunofluorescence staining using a combination of anti-P-CK and anti-carbamoyl phosphate synthetase 1 (CPS1) antibodies. Blood samples spiked with HepG2 cells were used to determine recovery and sensitivity. CTCs were detected in blood samples from HCC patients and other patients.

Results

ASGPR was exclusively expressed in human hepatoma cell line, normal hepatocytes and HCC cells in tissue specimens detected by the ASGPR antibody staining. More HCC cells could be identified by the antibody cocktail for CPS1 and P-CK compared with a single antibody. The current approach obtained a higher recovery rate of HepG2 cells and more CTC detection from HCC patients than the previous method. Using the current method CTCs were detected in 89% of HCC patients and no CTCs were found in the other test subjects.

Conclusions

Our anti-ASGPR antibody could be used for specific and efficient HCC CTC enrichment, and anti-P-CK combined with anti-CPS1 antibodies is superior to identification with one antibody alone in the sensitivity for HCC CTC detection.     

Autophagy potentiates the anti-cancer effects of the histone deacetylase inhibitors in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer death worldwide. Drug treatments for HCC have been largely unsuccessful. Histone deacetylase inhibitors can reactivate tumor suppressor genes in cancer cells and serve as potential anti-cancer drugs. Two potent HDAC inhibitors OSU-HDAC42 and SAHA induced autophagy in HCC cells as revealed by transmission electron microscopy, immunofluorescence and LC3-II accumulation. We found that SAHA and OSU-HDAC42 induced autophagy through downregulation of Akt/mTOR signaling and induction of ER stress response. Inhibition of autophagy by 3-MA or Atg5 knockout reduced SAHA-induced cytotoxicity, indicating that SAHA-induced autophagy led to cell death. Our results show that the combination of autophagy inducers with SAHA might be attractive for the treatment of HCC and pharmacological targeting of autophagy provides promise for the management of cancer therapy.     

Autophagy potentiates the anti-cancer effects of the histone deacetylase inhibitors in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer death worldwide. Drug treatments for HCC have been largely unsuccessful. Histone deacetylase inhibitors can reactivate tumor suppressor genes in cancer cells and serve as potential anti-cancer drugs. Two potent HDAC inhibitors OSU-HDAC42 and SAHA induced autophagy in HCC cells as revealed by transmission electron microscopy, immunofluorescence and LC3-II accumulation. We found that SAHA and OSU-HDAC42 induced autophagy through downregulation of Akt/mTOR signaling and induction of ER stress response. Inhibition of autophagy by 3-MA or Atg5 knockout reduced SAHA-induced cytotoxicity, indicating that SAHA-induced autophagy led to cell death. Our results show that the combination of autophagy inducers with SAHA might be attractive for the treatment of HCC and pharmacological targeting of autophagy provides promise for the management of cancer therapy.     

甲胎蛋白携带药物靶向治疗肿瘤

甲胎蛋白(alpha fetoprotein,AFP)是一种在胎儿发育时期高表达的蛋白质,它又是一种穿梭蛋白质,能够将营养物质输送给胚胎细胞.相似的是,在肝癌等恶性肿瘤发展时期,肿瘤细胞也高表达AFP及其受体,它们通过AFP受体摄取AFP及其运载的物质.因此,可以将AFP与抗癌药物结合,选择性攻击肿瘤细胞.AFP与药物...     

肝靶向配体半乳糖基白蛋白和多聚谷氨酸

化学合成两类去唾液酸糖蛋白受体(ASGPR)的人工配体——半乳糖基白蛋白(Gal_nHSA)和半乳糖基多聚-L-谷氨酸(Gal_nPLGA), 并以 ¹²⁵I标记的去唾液酸胎球蛋白(ASF)为标准配体,测定了合成配体抑制 ¹²⁵I-ASF与大鼠肝细胞膜ASGPR结合的IC₅₀值. 结果表明,Gal₁₂HSA、Gal₁₅HSA、Gal₂₆HSA、Gal₃₀HSA和Gal₃₄PLGA均能够有效地抑制 ¹²⁵I-ASF与ASGPR的结合,且前者与ASGPR的亲和力随半乳糖基化程度的增加而增加. 这些合成配体来源丰富、制备简单,适合于作为药物或基因肝靶向运送的导向配体.     

A novel vascular-targeting peptide for gastric cancer delivers low-dose TNFα to normalize the blood vessels and improve the anti-cancer efficiency of 5-fluorouracil

Various vascular-targeted agents fused with tumor necrosis factor α (TNFα) have been shown to improve drug absorption into tumor tissues and enhance tumor vascular function. TCP-1 is a peptide selected through in vivo phage library biopanning against a mouse orthotopic colorectal cancer model and is a promising agent for drug delivery. This study further investigated the targeting ability of TCP-1 phage and peptide to blood vessels in an orthotopic gastric cancer model in mice and assessed the synergistic anti-cancer effect of 5-fluorouracil (5-FU) with subnanogram TNFα targeted delivered by TCP-1 peptide. In vivo phage targeting assay and in vivo colocalization analysis were carried out to test the targeting ability of TCP-1 phage/peptide. A targeted therapy for improvement of the therapeutic efficacy of 5-FU and vascular function was performed through administration of TCP-1/TNFα fusion protein in this model. TCP-1 phage exhibited strong homing ability to the orthotopic gastric cancer after phage injection. Immunohistochemical staining suggested that and TCP-1 phage/TCP-1 peptide could colocalize with tumor vascular endothelial cells. TCP-1/TNFα combined with 5-FU was found to synergistically inhibit tumor growth, induce apoptosis and reduce cell proliferation without evident toxicity. Simultaneously, subnanogram TCP-1/TNFα treatment normalized tumor blood vessels. Targeted delivery of low-dose TNFα by TCP-1 peptide can potentially modulate the vascular function of gastric cancer and increase the drug delivery of chemotherapeutic drugs.     

Capacity limits of asialoglycoprotein receptor-mediated liver targeting

The abundant cell surface asialoglycoprotein receptor (ASGPR) is a highly selective receptor found on hepatocytes that potentially can be exploited as a selective shuttle for delivery. Various nucleic acid therapeutics that bind ASGPR are already in clinical development, but this receptor-mediated delivery mechanism can be saturated, which will likely result in reduced selectivity for the liver and therefore increase the likelihood for systemic adverse effects. Therefore, when aiming to utilize this mechanism, it is important to optimize both the administration protocol and the molecular properties. We here present a study using a novel ASGPR-targeted antibody to estimate ASGPR expression, turnover and internalization rates in vivo in mice. Using pharmacokinetic data (intravenous and subcutaneous dosing) and an in-silico target-mediated drug disposition (TMDD) model, we estimate an ASGPR expression level of 1.8 million molecules per hepatocyte. The half-life of the degradation of the receptor was found to be equal to 15 hours and the formed ligand-receptor complex is internalized with a half-life of 5 days. A biodistribution study was performed and confirmed the accuracy of the TMDD model predictions. The kinetics of the ASGPR shows that saturation of the shuttle at therapeutic concentrations is possible; however, simulation allows the dosing schedule to be optimized. The developed TMDD model can be used to support the development of therapies that use the ASGPR as a shuttle into hepatocytes.     

Combined therapeutic potential of nuclear receptors with receptor tyrosine kinase inhibitors in lung cancer

Cancer heterogeneity is a big hurdle in achieving complete cancer treatment, which has led to the emergence of combinational therapy. In this study, we investigated the potential use of nuclear receptor (NR) ligands for combinational therapy with other anti-cancer drugs. We first profiled all 48 NRs and 48 biological anti-cancer targets in four pairs of lung cell lines, where each pair was obtained from the same patient. Two sets of cell lines were normal and the corresponding tumor cell lines while the other two sets consisted of primary versus metastatic tumor cell lines. Analysis of the expression profile revealed 11 NRs and 15 cancer targets from the two pairs of normal versus tumor cell lines, and 9 NRs and 9 cancer targets from the primary versus metastatic tumor cell lines had distinct expression patterns in each category. Finally, the evaluation of nuclear receptor ligand T0901317 for liver X receptor (LXR) demonstrated its combined therapeutic potential with tyrosine kinase inhibitors. The combined treatment of cMET inhibitor PHA665752 or EGFR inhibitor gefitinib with T0901317 showed additive growth inhibition in both H2073 and H1993 cells. Mechanistically, the combined treatment suppressed cell cycle progression by inhibiting cyclinD1 and cyclinB expression. Taken together, this study provides insight into the potential use of NR ligands in combined therapeutics with other biological anti-cancer drugs.     

Liver-Targeting of Interferon-Alpha with Tissue-Specific Domain Antibodies

Interferon alpha (IFNα) is used for the treatment of hepatitis C infection and whilst efficacious it is associated with multiple adverse events including reduced leukocyte, erythrocyte, and platelet counts, fatigue, and depression. These events are most likely caused by systemic exposure to interferon. We therefore hypothesise that targeting the therapeutic directly to the intended site of action in the liver would reduce exposure in blood and peripheral tissue and hence improve the safety and tolerability of IFNα therapy. We genetically fused IFN to a domain antibody (dAb) specific to a hepatocyte restricted antigen, asialoglycoprotein receptor (ASGPR). Our results show that the murine IFNα2 homolog (mIFNα2) fused to an ASGPR specific dAb, termed DOM26h-196-61, could be expressed in mammalian tissue culture systems and retains the desirable biophysical properties and activity of both fusion partners when measured in vitro. Furthermore a clear increase in in vivo targeting of the liver by mIFNα2-ASGPR dAb fusion protein, compared to that observed with either unfused mIFNα2 or mIFNα2 fused to an isotype control dAb V_HD2 (which does not bind ASGPR) was demonstrated using microSPECT imaging. We suggest that these findings may be applicable in the development of a liver-targeted human IFN molecule with improved safety and patient compliance in comparison to the current standard of care, which could ultimately be used as a treatment for human hepatitis virus infections.     

VCAM-1 directed immunoliposomes selectively target tumor vasculature in vivo

Targeting the tumor vasculature and selectively modifying endothelial functions is an attractive anti-tumor strategy. We prepared polyethyleneglycol modified immunoliposomes (IL) directed against vascular cell adhesion molecule 1 (VCAM-1), a surface receptor over-expressed on tumor vessels, and investigated the liposomal targetability in vitro and in vivo. In vitro, anti-VCAM-1 liposomes displayed specific binding to activated endothelial cells under static conditions, as well as under simulated blood flow conditions. The in vivo targeting of IL was analysed in mice bearing human Colo 677 tumor xenografts 30 min and 24 h post i.v. injection. Whereas biodistribution studies using [3H]-labelled liposomes displayed only marginal higher tumor accumulation of VCAM-1 targeted versus unspecific ILs, fluorescence microscopy evaluation revealed that their localisations within tumors differed strongly. VCAM-1 targeted ILs accumulated in tumor vessels with increasing intensities from 30 min to 24 h, while control ILs accumulated in the tumor tissue by passive diffusion. ILs that accumulated in non-affected organs, mainly liver and spleen, primarily co-localised with macrophages. This is the first morphological evidence for selective in vivo targeting of tumor vessels using ILs. VCAM-directed ILs are candidate drug delivery systems for therapeutic anti-cancer approaches designed to alter endothelial function.     

CA215 and GnRH receptor as targets for cancer therapy

Two monoclonal antibodies (Mabs), RP215 and GHR106, were selected for the preclinical evaluations of anti-cancer drugs targeting various human cancers including those of the ovary, cervix, lung, and liver. Both Mabs were shown to react with pan cancer markers, which are over-expressed on the surface of almost all human cancers. RP215 Mab was shown to react with the carbohydrate-associated epitope(s) of cancer cell-expressed glycoproteins, mainly consisting of immunoglobulin superfamily (IgSF) proteins and mucins, generally known as CA215. GHR106 Mab was generated against the extracellular domain of human GnRH receptor, which is also highly expressed on the cancer cell surface. Preclinical studies were performed to evaluate the efficacy of these two Mabs as anti-cancer drugs for treating human cancers. High tumor specificity of RP215 Mab was demonstrated with immunohistochemical staining studies of various cancer cell lines, as well as normal and cancerous tissue sections. These two Mabs were shown to induce apoptosis as well as complement-dependent cytotoxicity upon treatment to many cultured cancer cells. Significant dose-dependent growth inhibition of tumor cells from several different tissue origins were demonstrated by nude mouse experiments. It was further demonstrated that GHR106 Mab can function as long-acting GnRH analogs in its biological actions. Efforts were made to generate human/mouse chimeric forms of the GHR106 Mab. Based on the results of these preclinical studies, we believe that these two Mabs, in chimeric or humanized forms, can be developed into suitable therapeutic agents for treatment of human cancers as anti-cancer drugs.     

CF102 an A3 adenosine receptor agonist mediates anti-tumor and anti-inflammatory effects in the liver

The Gi protein-associated A(3) adenosine receptor (A(3) AR) is a member of the adenosine receptor family. Selective agonists at the A(3) AR, such as CF101 and CF102 were found to induce anti-inflammatory and anti-cancer effects. In this study, we examined the differential effect of CF102 in pathological conditions of the liver. The anti-inflammatory protective effect of CF101 was tested in a model of liver inflammation induced by Concanavalin A (Con. A) and the anti-cancer effect of CF102 was examined in vitro and in a xenograft animal model utilizing Hep-3B hepatocellular carcinoma (HCC) cells. The mechanism of action was explored by following the expression levels of key signaling proteins in the inflamed and tumor liver tissues, utilizing Western blot (WB) analysis. In the liver inflammation model, CF102 (100 μg/kg) markedly reduced the secretion of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase in comparison to the vehicle-treated group. Mechanistically, CF102 treatment decreased the expression level of phosphorylated glycogen synthase kinase-3β, NF-κB, and TNF-α and prevented apoptosis in the liver. This was demonstrated by decreased expression levels of Fas receptor (FasR) and of the pro-apoptotic proteins Bax and Bad in liver tissues. In addition, CF102-induced apoptosis of Hep-3B cells both in vitro and in vivo via de-regulation of the PI3K-NF-κB signaling pathway, resulting in up-regulation of pro-apoptotic proteins. Taken together, CF102 acts as a protective agent in liver inflammation and inhibits HCC tumor growth. These results suggest that CF102 through its differential effect is a potential drug candidate to treat various pathological liver conditions.     

Optimization of lipase-catalyzed synthesis of novel galactosyl ligands for selective targeting of liposomes to the asialoglycoprotein receptor

The asialoglycoprotein receptor (ASGPR) is a potential target in the search for hepatic cancer drugs. However, application of ASGPR targeting in the clinic is limited by inefficient synthetic methods for the ligand. In this study, we designed and synthesized a novel galactosylated lipid with a mono-galactoside moiety using a lipase. Then we investigated the optimal reaction conditions and analyzed the targeting ability of liposomes modified with the galactosylated lipid. In an organic phase system, different lipases were used as catalysts to synthesize (5-cholesten-3b-yl) [(4-O-β-D-galactopyranosyl)D-glucitol-6] sebacate (CHS-SE-LA). Variables in enzymatic esterification, such as the type of enzyme and solvent, were explored by single-factor experiments. Optimal reaction conditions were determined through response surface methodology. The (CHS-SE-LA)-incorporated galactosylated liposome containing fluorescent dye was then prepared by thin-film hydration and a HepG2 cell transfection test used to confirm the targeting efficiency of galactosylated liposomes to hepatic cancer cells. The structure of CHS-SE-LA was identified by electrospray ionization or ESI and nuclear magnetic resonance or NMR. Under optimal conditions, the predicted yield of CHS-SE-LA was 94.3%, and the actual experimental value was 95.6 ± 1.35%, n = 3. For HepG2 cells, the cellular fluorescence intensities of liposomes modified with CHS-SE-LA (galactosylated liposomes [GAL-FL]) were as much as 2.6-fold (P < 0.01) the control liposomes (FL). Moreover, the presence of excess galactose significantly inhibited the uptake of GAL-FL suggesting ASGPR mediated uptake. The novel galactosylated ligand was synthesized enzymatically with high efficiency under mild conditions. Liposomes containing CHS-SE-LA have great potential as drug delivery carriers for hepatocyte-selective targeting.     

Molecular targeting of angiogenesis

The majority of pharmacological approaches for the treatment of solid tumors suffer from poor selectivity, thus limiting dose escalation (i.e., the doses of drug which are required to kill tumor cells cause unacceptable toxicities to normal tissues). The situation is made more dramatic by the fact that the majority of anticancer drugs accumulate preferentially in normal tissues rather than in neoplastic sites, due to the irregular vasculature and to the high interstitial pressure of solid tumors. One avenue towards the development of more efficacious and better tolerated anti-cancer drugs relies on the targeted delivery of therapeutic agents to the tumor environment, thus sparing normal tissues. Molecular markers which are selectively expressed in the stroma and in neo-vascular sites of aggressive solid tumors appear to be particularly suited for ligand-based tumor targeting strategies. Tumor blood vessels are accessible to agents coming from the bloodstream, and their occlusion may result in an avalanche of tumor cell death. Furthermore, endothelial cells and stromal cells are genetically more stable than tumor cells and can produce abundant markers, which are ideally suited for tumor targeting strategies. This review focuses on recent advances in the development of ligands for the selective targeting of tumor blood vessels and new blood vessels in other angiogenesis-related diseases.     

Detection of surface asialoglycoprotein receptor expression in hepatic and extra-hepatic cells using a novel monoclonal antibody

The asialoglycoprotein receptor (ASGPR) is a heterodimeric membrane protein which is involved in the internalization of desialylated glycoproteins and also in the binding and uptake of various pathogenic viruses. To facilitate the analysis of ASGPR expression, we generated a monoclonal antibody, termed ASSA-1, that is specific to the ASGPR H1 subunit based on ELISA and Western blots analysis. ASSA-1 also reacted to surface-displayed ASGPR in live cells thus enabling analysis of ASGPR expression by immunofluorescence flow cytometry, which we used to analyze established human liver cell lines previously confirmed to be positive for ASGPR mRNA expression. In agreement with previous reports, surface ASGPR was also detected in extra-hepatic cells and, surprisingly, even in human T cell lines, which was then further confirmed in activated, but not in resting, primary human peripheral blood lymphocytes. These observations suggest that ASGPR has a broad pattern of expression that even extends into cells from the immune system, which biological meanings still have to be analyzed. We expect that monoclonal antibody ASSA-1 will serve as a new powerful tool in analyzing the biological role of ASGPR in hepatic and extra-hepatic cells.     

Design and Development of a Robotized System Coupled to μCT Imaging for Intratumoral Drug Evaluation in a HCC Mouse Model

Hepatocellular carcinoma (HCC) is one of the most common cancer related deaths worldwide. One of the main challenges in cancer treatment is drug delivery to target cancer cells specifically. Preclinical evaluation of intratumoral drugs in orthotopic liver cancer mouse models is difficult, as percutaneous injection hardly can be precisely performed manually. In the present study we have characterized a hepatoma model developing a single tumor nodule by implantation of Hep55.1C cells in the liver of syngeneic C57BL/6J mice. Tumor evolution was followed up by µCT imaging, and at the histological and molecular levels. This orthotopic, poorly differentiated mouse HCC model expressing fibrosis, inflammation and cancer markers was used to assess the efficacy of drugs. We took advantage of the high precision of a previously developed robotized system for automated, image-guided intratumoral needle insertion, to administer every week in the tumor of the Hep55.1C mouse model. A significant tumor growth inhibition was observed using our robotized system, whereas manual intraperitoneal administration had no effect, by comparison to untreated control mice.