Zonghai Li

Development of T Cells Redirected to Glypican-3 for the Treatment of Hepatocellular Carcinoma

Purpose: The aim of our study is to elucidate whether T cells expressing GPC3-targeted chimeric antigen receptor (CAR) can efficiently eliminate GPC3-positive HCC cells and their potential in the treatment of HCC. Experimental Design: T cells expressing a first-generation and third-generation GPC3-targeted CAR were prepared using lentiviral vector transduction. The in vitro and in vivo cytotoxic activities of the genetically engineered CAR T cells were evaluated against various HCC cell lines. Results: GPC3-targeted CAR T cells could efficiently kill GPC3-positive HCC cells but not GPC3-negative cells in vitro. These cytotoxic activities seemed to be positively correlated with GPC3 expression levels in the target cells. In addition, T cells expressing the third-generation GPC3-targeted CAR could eradicate HCC xenografts with high level of GPC3 expression and efficiently suppress the growth of HCC xenografts with low GPC3 expression level in vivo. The survival of the mice bearing established orthotopic Huh-7 xenografts was significantly prolonged by the treatment with the third-generation GPC3-targeted CAR T cells. Conclusions: GPC3-targeted CAR T cells could potently eliminate GPC3-positive HCC cells, thereby providing a promising therapeutic intervention for GPC3-positive HCC. Clin Cancer Res; 20(24); 6418–28. ©2014 AACR.

Cationic microRNA-delivering nanovectors with bifunctional peptides for efficient treatment of PANC-1 xenograft model

Therapeutic strategies based on modulation of microRNA activity possess much promise in cancer therapy, but the in vivo delivery of microRNA to target sites and its penetration into tumor tissues remain great challenge. In this work, miR-34a-delivering therapeutic nanocomplexes with a tumor-targeting and -penetrating bifunctional CC9 peptide were proposed for efficient treatment of pancreatic cancers. In vitro study indicated that the nanoparticle-based miR-34a delivery systems could effectively facilitate cellular uptake and greatly up-regulate the mRNA level of miR-34a in PANC-1 cell lines. The up-regulation of miR-34a remarkably induced cell cycle arrest and apoptosis, suppressed the tumor cell migration and inhibited the target gene expressions such as E2F3, Bcl-2, c-myc and cyclin D1. More importantly, the in vivo systemic administration of the developed targeting miR-34a delivery systems in a pancreatic cancer model significantly inhibited tumor growth and induced cancer cell apoptosis. Such bifunctional peptide-conjugated miRNA-delivering nanocomplexes should have great potential applications in cancer therapy.

Epidermal growth factor receptor vIII enhances tumorigenicity and resistance to 5-fluorouracil in human hepatocellular carcinoma.

We investigated whether EGFRvIII contributes to tumorigenicity and resistance to 5-FU in HCC cell lines. Our results show that several HCC cell lines have EGFRvIII expression. EGFRvIII-positive HCC cells grew more rapidly and had a lower sensitivity to 5-FU than EGFRvIII-negative HCC cells. For further analysis of the biological characteristics of EGFRvIII, an EGFRvIII or EGFR expression cassette was introduced into the HCC cell line, Huh-7. Compared with Huh-7 cells and Huh7-EGFR cells, Huh7-EGFRvIII not only exhibit significantly increase of cell growth in vitro and in vivo but also show enhanced migration in vitro. Furthermore, 5-FU has significantly lower inhibition effect on Huh7-EGFRvIII cells then on both Huh-7 and Huh7-EGFR cells in vitro and in vivo. Collectively, these results demonstrate that EGFRvIII plays a pivotal role in tumorigenicity and enhanced 5-FU resistance of HCC.

An oligopeptide ligand-mediated therapeutic gene nanocomplex for liver cancer-targeted therapy

The epidermal growth factor receptor (EGFR) is over-expressed in a wide variety of epithelial-derived cancer cells. In this study, EGFR-targeted gene carriers were designed to complex the therapeutic acetylcholinesterase gene (AChE gene), which suppresses cell proliferation via inactivating mitogen-activated protein kinase and PI3K/Akt pathways in cells, for treatment of EGFR-positive liver cancers. Different amounts of target ligand YC21 (an oligopeptide composed of 21 amino acid units) were coupled with the PEI(600)-CD (PC) vectors composed of β-cyclodextrin (β-CD) and low-molecular-weight polyethylenimine (PEI, Mw 600) to form the EGFR-targeted gene vectors (termed as YPCs). The YPC vectors possessed the highly efficient gene delivery ability to the EGFR-positive liver cancer cells. YPCs could effectively promote AChE gene expression. The YPC/AChE complexes produced excellent gene transfection abilities in EGFR-positive liver cancer cells in vitro and in vivo.

Loss of miR-638 in vitro promotes cell invasion and a mesenchymal-like transition by influencing SOX2 expression in colorectal carcinoma cells

BackgroundColorectal carcinoma (CRC) is a major cause of cancer mortality. The aberrant expression of several microRNAs is associated with CRC progression; however, the molecular mechanisms underlying this phenomenon are unclear.MethodsmiR-638 and SRY-box 2 (SOX2) expression levels were detected in 36 tumor samples and their adjacent, non-tumor tissues from patients with CRC, as well as in 4 CRC cell lines, using real-time quantitative RT-PCR (qRT-PCR). SOX2 expression levels were detected in 90 tumor samples and their adjacent tissue using immunohistochemistry. Luciferase reporter and Western blot assays were used to validate SOX2 as a target gene of miR-638. The regulation of SOX2 expression by miR-638 was assessed using qRT-PCR and Western blot assays, and the effects of exogenous miR-638 and SOX2 on cell invasion and migration were evaluated in vitro using the HCT-116 and SW1116 CRC cell lines.ResultsWe found that miR-638 expression was differentially impaired in CRC specimens and dependent on tumor grade. The inhibition of miR-638 by an antagomiR promoted cell invasion and a mesenchymal-like transition (lamellipodium stretching increased and cell-cell contacts decreased, which was accompanied by the suppression of the epithelial cell marker ZO-1/E-cadherin and the upregulation of the mesenchymal cell marker vimentin). A reporter assay revealed that miR-638 repressed the luciferase activity of a reporter gene coupled to the 3′-untranslated region of SOX2. miR-638 overexpression downregulated SOX2 expression, and miR-638 inhibition upregulated SOX2 expression. Moreover, miR-638 expression levels were correlated inversely with SOX2 mRNA levels in human CRC tissues. The RNAi-mediated knockdown of SOX2 phenocopied the invasion-inhibiting effect of miR-638; furthermore, SOX2 overexpression blocked the miR-638-induced CRC cell transition to epithelial-like cells.ConclusionsThese results demonstrate that the loss of miR-638 promotes invasion and a mesenchymal-like transition by directly targeting SOX2 in vitro. These findings define miR-638 as a new, invasion-associated tumor suppressor of CRC.

Growth suppression of human hepatocellular carcinoma xenografts by a monoclonal antibody CH12 directed to epidermal growth factor receptor variant III

Human hepatocellular carcinoma (HCC) is considered difficult to cure because it is resistant to radio- and chemotherapy and has a high recurrence rate after curative liver resection. Epidermal growth factor receptor variant III (EGFRvIII) has been reported to express in HCC tissues and cell lines. This article describes the efficacy of an anti-EGFRvIII monoclonal antibody (mAb CH12) in the treatment of HCC xenografts with EGFRvIII expression and the underlying mechanism of EGFRvIII as an oncogene in HCC. The results demonstrated that CH12 bound preferentially to EGFRvIII with a dissociation constant (Kd) of 1.346 nm/liter. In addition, CH12 induces strong antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in Huh7-EGFRvIII (with exogenous expression of EGFRvIII) and SMMC-7721 (with endogenous expression of EGFRvIII) cells. Notably, CH12 significantly inhibited the growth of Huh7-EGFRvIII and SMMC-7721 xenografts in vivo with a growth inhibition ratio much higher than C225, a U. S. Food and Drug Administration-approved anti-EGFR antibody. Treatment of the two HCC xenografts with CH12 significantly suppressed tumor proliferation and angiogenesis. Mechanistically, in vivo treatment with CH12 reduced the phosphorylation of constitutively active EGFRvIII, Akt, and ERK. Down-regulation of the apoptotic protectors Bcl-xL, Bcl-2, and the cell cycle regulator cyclin D1, as well as up-regulation of the cell-cycle inhibitor p27, were also observed after in vivo CH12 treatment. Collectively, these results indicate that the monoclonal antibody CH12 is a promising therapeutic agent for HCC with EGFRvIII expression.

A gene nanocomplex conjugated with monoclonal antibodies for targeted therapy of hepatocellular carcinoma.

To enhance tumor-targeting abilities and therapeutic efficiency, a monoclonal antibody-conjugated gene nanocomplex was herein designed. The biodegradable cationic polyethylenimine-grafted-α,β-poly(N-3-hydroxypropyl)-DL-aspartamide (PHPA-PEI) was used for complexing pDNA to form the PHPA-PEI/pDNA nanoparticle, and then 9B9 mAb, an anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody, was conjugated to produce the PHPA-PEI/pDNA/9B9 mAb (PP9mN) complex. The PP9mN complex with the diameter of around 300 nm at its optimal weight ratio could be uptaken effectively by SMMC-7721 cells. The cytotoxicity of the PP9mN complex was much lower than that of PEI 25 kD in SMMC-7721, HepG2, Bel-7404 and COS-7 cell lines. The PP9mN complex possessed the highly efficient in vitro gene delivery ability to the hepatocellular carcinoma cells. The in vivo gene expression indicated that PP9mN could target to the tumor tissues effectively. By using the therapeutic AChE gene, it was found that the PP9mN complexes significantly enhanced the anti-tumor effect on tumor-bearing nude mice. Such monoclonal antibody-conjugated gene complex should have great potential applications in liver cancer therapy.

The synergistic effect of EGFR tyrosine kinase inhibitor gefitinib in combination with aromatase inhibitor anastrozole in non-small cell lung cancer cell lines.

BACKGROUND Several studies implicated that lung cancer progression was governed by the interaction between estrogen receptor (ER) and epidermal growth factor receptor (EGFR) signaling pathways. Combined targeting of EGFR and ER may have the synergistic effect in lung cancer treatment. The aim of this study was to explore the potential utility of inhibiting these two pathways with combination of anastrozole and gefitinib in non-small cell lung cancer (NSCLC) cell lines. MATERIALS AND METHODS The expression levels of ER (ER-α and ER-β) in lung cancer cell lines (A549, H460, SPC-A-1, H1299) and normal bronchus epithelial cell BEAS-2B were detected using real-time PCR and Western blot. Immunocytochemistry was used to locate ER-α and ER-β in cell line with highest ER expression levels. The cells were treated with anastrozole or gefitinib alone or in combination. The cell proliferation inhibition was detected by the CCK8 assay, cell cycle and apoptosis effects were detected by flow cytometry; the expression levels of phosphorylated-EGFR (p-EGFR), ERK, phosphorylated-ERK (p-ERK), AKT and phosphorylated-AKT (p-AKT) were detected by Western blot. RESULTS Among these cell lines the expression levels of ER in A549 cells were highest. In A549 cell line, ER-α was mainly localized in the cytoplasm, whereas ER-β was mainly localized in the cytoplasm and to a lesser degree in the nucleus. The combination of two drugs increased the proliferation inhibition rates for 24h, 48 h, 72 h to 37.66 ± 1.02%, 63.41 ± 2.02%, 70.50 ± 0.86%, respectively, which was closely associated with elevation of the G0/G1 phase fraction (P<0.05). Apoptosis rates of A549 cells treated with anastrozole, gefitinib alone or in combination were 10.72 ± 1.12%, 17.40±1.28%, 23.02 ± 2.32%, respectively (P<0.05). The synergistic effects of the combination therapy were accompanied by reduction of p-EGFR, p-ERK and p-AKT expression compared with individual treatment. CONCLUSIONS The results of this study suggest that the combination of anastrozole and gefitinib compared with either drug alone can maximally inhibit cell proliferation, induce apoptosis, and affect downstream signaling pathways. Our study supports functional interaction between the ER and the EGFR pathways in lung cancer and provides a clinically exploitable strategy for non-small cell lung cancer patients.

An EpCAM/CD3 bispecific antibody efficiently eliminates hepatocellular carcinoma cells with limited galectin-1 expression

There have been several studies suggesting that cancer stem cells (CSCs) contribute to the high rates of recurrence and resistance to therapies observed in hepatocellular carcinoma (HCC). Epithelial cell adhesion molecule (EpCAM) has been demonstrated to be a biomarker of CSCs and a potential therapeutic target in HCC. Here, we prepared two anti-EpCAM monoclonal antibodies (1H8 and 2F2) and an anti-EpCAM bispecific T cell engager (BiTE) 1H8/CD3, which was derived from 1H8, and used them to treat HCC in vitro and in vivo. The results demonstrated that all of the developed anti-EpCAM antibodies specifically bound to EpCAM. Neither anti-EpCAM monoclonal antibody had obvious anti-HCC activities in vitro or in vivo. However, anti-EpCAM BiTE 1H8/CD3 induced strong peripheral blood mononuclear cell-dependent cellular cytotoxicity in Huh-7 and Hep3B cells but not EpCAM-negative SK-Hep-1 cells. Notably, 1H8/CD3 completely inhibited the growth of Huh-7 and Hep3B xenografts in vivo. Treatment of the Huh-7 HCC xenografts with 1H8/CD3 significantly suppressed tumor proliferation and reduced the expression of most CSC biomarkers. Intriguingly, galectin-1 (Gal-1) overexpression inhibited 1H8/CD3-induced lymphocytotoxicity in HCCs while knockdown of Gal-1 increased the lymphocytotoxicity. Collectively, these results indicate that anti-EpCAM BiTE 1H8/CD3 is a promising therapeutic agent for HCC treatment. Gal-1 may contribute to the resistance of HCC cells to 1H8/CD3-induced lysis.

Identification and characterization of Ch806 mimotopes

The chimeric antibody 806 (Ch806) is a promising antitumor agent that recognizes both the epidermal growth factor receptor variant III (EGFRvIII) and the overexpressed epidermal growth factor receptor (EGFR) in cancer tissues but does not recognize the wild type EGFR in normal tissues. However, passive antibody immunization could not produce effective antitumor titers unless the immunization was administered repeatedly over long periods. To overcome this limitation, we generated epitope mimics that bind to Ch806 and tested whether the peptide mimics could induce the production of similar antibodies when actively immunizing mice with the peptides. We used the PH.D-12 phage display peptide library to identify peptides that bind to the monoclonal antibody (mAb) 12H23, which also recognizes similar epitopes of Ch806. Two mimotopes (WHTEILKSYPHE and LPAFFVTNQTQD) were shown to mimic the mAb 12H23 and Ch806 epitope using immunoassays. The mimotopes were conjugated to immunogenic carrier proteins and used to intraperitoneally immunize BALB/c mice. Interestingly, sera from the mice immunized with the isolated mimotopes not only recognize the recombinant or synthetic 806 eptitope, but can also recognize EGFR that is overexpressed in A431 cells and EGFRvIII expressed in Huh7-EGFRvIII cells, whereas sera from mice immunized with the control peptide-KLH (keyhole limpet hemocyanin) and carrier KLH alone failed to show a similar reactivity. Furthermore, in an antibody-dependent cellular cytotoxicity assay (ADCC), the mimotope-induced antibodies specifically lysed human Huh-7-EGFRvIII cells. Our data indicate that the isolated mimotopes reported here may potentially be used as new alternative agents for treating cancer with EGFRvIII expression or EGFR overexpression.