Energized Fusion Protein Research Articles

An arginine-rich cell penetrating peptide contained anti-gelatinase scFv-LDM fusion protein shows potent antitumor efficacy in pancreatic cancer.

Pancreatic cancer (PC) is one of the most dangerous cancers with less than 5% survival rate in 5 years. This study was to evaluate the antitumor activities of dFv-LDP-AE and dFv-R-LDP-AE, two energized fusion protein targeting gelatinases, on pancreatic cancer. The fusion protein dFv-LDP-AE consists of two tandem anti-gelatianses scFv and an enediyne antibiotic lidamycin (LDM) for receptor binding and cell killing. To improve the penetration capability, the fusion protein dFv-LDP-AE was integrated with an arginine-rich cell penetrating peptide (Arg)9 and then generated the fusion protein dFv-R-LDP-AE. The current study demonstrated that dFv-LDP and dFv-R-LDP had high affinity with the antigen gelatinases and PC cells, the integration of (Arg)9 could increase the penetration rate of fusion protein in SW-1990 and PANC-1 cells. After enediyne-energized with chromophore of lidamycin, the energized fusion protein dFv-LDP-AE and dFv-R-LDP-AE showed potent cytotoxicity to PC cells and could induced the robust cell apoptosis and necrosis in vitro. Western blot showed that dFv-R-LDP-AE could increase PARP cleavage, and inhibited the expression of VEGF, Cyclin D1, Cox-2 and Bcl-2 in SW-1990 and PANC-1 cells. In vivo, at a tolerated dosage, dFv-LDP, dFv-LDP-AE and dFv-R-LDP-AE inhibited tumor growth by 20.42%, 56.31% (P < 0.01, compared to that of control) and 74.2% (P < 0.05, compared to that of dFv-LDP-AE) in pancreatic cancer SW-1990 xenografted mice, respectively. Moreover, the results of in vivo optical imaging showed that fusion protein dFv-R-LDP displayed prominent accumulation in the tumor in SW-1990 xenografted mice and Capan-2 orthotopic transplanted mice. These results showed that dFv-R-LDP-AE possessed potent antitumor efficacy on PC, which indicating it could be a promising candidate for targeting therapy of PC.

A bispecific fusion protein and a bifunctional enediyne-energized fusion protein consisting of TRAIL, EGFR peptide ligand, and apoprotein of lidamycin against EGFR and DR4/5 show potent antitumor activity.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mainly induces apoptosis through the extrinsic death receptor-induced pathway by ligation with death receptor 4 (DR4) and death receptor 5 (DR5). On the basis of the antitumor activity to cancer cells and no cytotoxity to normal cells of TRAIL and the target of the epidermal growth factor receptor (EGFR) ligand peptide, the study constructed a new bispecific fusion protein and a new bifunctional enediyne-energized fusion protein and investigated their antitumor efficacy. Bispecific fusion protein Ec-LDP-TRAIL showed potent binding activity to cancer cell lines with a high expression of EGFR or DR4/DR5 such as A431 and H460 cells, whereas poor binding activity to NIH/3T3 cells with low expressing EGFR and DR4/DR5. Ec-LDP-TRAIL also showed more potent cytotoxicity to A431 and H460 cells than Ec-LDP, which could result from the TRAIL-inducing apoptosis. Results of an in-vivo efficacy study showed that Ec-LDP-TRAIL at a dose of 10 mg/kg decreased the growth of epidermoid carcinoma A431 xenografts by 80.19% (P < 0.01) on day 26. Immunohistochemical detection of nuclear antigen factor Ki-67 suggested that Ec-LDP-TRAIL effectively induced cell necrosis and inhibited cell proliferation of tumor. From IC50 values, bispecific and bifunctional energized fusion protein Ec-LDP-TRAIL-AE was more potent and selective in its cytotoxicity against different carcinoma cell lines than corresponding lidamycin in vitro and induction of the cleavage of poly(ADP-ribose)polymerase was observed in A431 cells treated with Ec-LDP-TRAIL-AE and lidamycin, respectively. Ec-LDP-TRAIL-AE also significantly inhibited the growth of A431 xenografts in a nude mouse model. These properties suggested that Ec-LDP-TRAIL and Ec-LDP-TRAIL-AE may be promising candidates for targeted cancer therapy.

Tissue factor-targeted lidamycin inhibits growth and metastasis of colon carcinoma

Colon cancer is the third most common cancer in the world. The overexpression of tissue factor (TF) in colon cancer cells makes it an ideal target for colon cancer therapy. The purpose of the present study was to develop a TF-targeting energized fusion protein, mlFVII-LDP-AE, which is composed of a mouse Factor VII light chain (mlFVII) as the targeting domain conjugated to the highly cytotoxic antibiotic lidamycin (LDM, LDP-AE) as the effector domain. The potential efficacy of mlFVII-LDP-AE for mouse colon cancer therapy was tested in a mouse colon cancer subcutaneous xenograft model and a live metastasis model in BALB/c mice. mlFVII-LDP-AE showed a tumor growth inhibition rate of 91.2% (at a dose of 0.8 mg/kg) and a tumor metastasis inhibition rate of 84.7% (at a dose of 0.6 mg/kg). The results showed that mlFVII-LDP-AE was able to effectively inhibit the growth and metastasis of mouse colon cancer. As human TF and FVII have features similar to those of mice, human FVII light chain (hlFVII)-targeted LDM (hlFVII-LDP-AE) may be expected to have therapeutic potential for human colon cancer.

Abstract 5478: A bispecific enediyne-energized fusion protein Ec-LDP-Hr-AE enhances anti-cancer efficacy on esophageal cancer cells by targeting EGFR and HER2.

Abstract Esophageal cancer is one of the most common cancers, and the 5-year survival rate is less than 10% due to lack of effective therapeutic agents. This study was to evaluate antitumor activity of Ec-LDP-Hr-AE, a recently developed bispecific enediyne-energized fusion protein, on esophageal carcinoma, targeting both EGFR (epidermal growth factor receptor) and HER2 (human epidermal growth factor receptor 2), because both EGFR and HER2 have been shown overexpression in over 30% of esophageal cancers. The fusion protein Ec-LDP-Hr-AE was encoded by a gene that was genetically constructed by fusing the oligopeptides ligand Ec (22 amino acids of EGF COOH-terminal) and Hr (VH CDR3 region of anti-HER2 C6.5 antibody) with lidamycin (LDM, an enediyne antibiotic with potent cytotoxicity) via two (G4S)3-linker. The encoded 18.4 kDa fusion protein was expressed in Escherichia coli and characterized its binding affinity with esophageal squamous cell carcinoma (ESCC) cells by flow-cytometry-based immunofluorescence assay. The results showed that Ec-LDP-Hr-AE protein bound to ESCC cells EC9706 and KYSE150 with high affinity (the Kd values were 5.283μmol/L and 3.562μmol/L, respectively). The potency of Ec-LDP-Hr-AE was determined on four human ESCC cell lines with differential expression of EGFR and HER2 using MTT assay. The bispecific Ec-LDP-Hr-AE showed potent cytotoxicity to EC1, EC109, EC9706 and KYSE150 cells with IC50 values in the picomolar level. Moreover, the bispecific Ec-LDP-Hr-AE was more potent than the monospecific counterparts (Ec-LDP-AE and LDP-Hr-AE) and LDM. Cell cycle distribution assay revealed that Ec-LDP-Hr-AE caused G2-M arrest in EC9706 and KYSE150 cells, in which the G2-M phase cells reached the peak with 0.1nmol/L of Ec-LDP-Hr-AE treatment. Ec-LDP-Hr-AE also induced cancer cell apoptosis in EC9706 and KYSE150 cells and the induction of apoptosis was in a dosage-dependent manner. The apoptotic index in the EC9706 cells exposed to 0.1, 0.5, or 1 nmol/L of Ec-LDP-Hr-AE were 15.0 ± 0.3%, 38.1 ± 0.6%, and 50.0 ± 0.4%, respectively. Western blotting showed that Ec-LDP-Hr-AE promoted caspase-3 and caspase-7 activities as well as PARP cleavage, indicating that Ec-LDP-Hr-AE-induced apoptosis was associated with mitochondria-caspase cascade. Moreover, Ec-LDP-Hr-AE inhibited KYSE150 cancer cell proliferation via decreasing phosphorylation of EGFR and HER2, and further exerted inhibition of the activation of their downstream signaling molecules (e.g. AKT and ERK). In summary, the bispecific energized fusion protein Ec-LDP-Hr-AE exhibited potent cytotoxicity to ESCC cells in vitro, leading to cell cycle arrest and apoptosis via targeting EGFR and HER2. These results suggest that Ec-LDP-Hr-AE could be a promising candidate for esophageal cancer targeting therapy. Citation Format: Guo Xiaofang, Xiaofei Zhu, Yue Shang, Shenghua Zhang, Yongsu Zhen, Wancai Yang. A bispecific enediyne-energized fusion protein Ec-LDP-Hr-AE enhances anti-cancer efficacy on esophageal cancer cells by targeting EGFR and HER2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5478. doi:10.1158/1538-7445.AM2013-5478

A cell penetrating peptide-integrated and enediyne-energized fusion protein shows potent antitumor activity

Arginine-rich peptides belong to a subclass of cell penetrating peptides that are taken up by living cells and can be detected freely diffusing inside the cytoplasm and nucleoplasm. This phenomenon has been attributed to either an endocytotic mode of uptake and a subsequent release from vesicles or a direct membrane penetration. Lidamycin is an antitumor antibiotic, which consists of an active enediyne chromophore (AE) and a noncovalently bound apoprotein (LDP). In the present study, a fusion protein (Arg)9–LDP composed of cell penetrating peptide (Arg)9 and LDP was prepared by DNA recombination, and the enediyne-energized fusion protein (Arg)9–LDP–AE was prepared by molecular reconstitution. The data in fixed cells demonstrated that (Arg)9–LDP could rapidly enter cells, and the results based on fluorescence activated cell sorting indicated that the major route for (Arg)9-mediated cellular uptake of protein molecules was endocytosis. (Arg)9–LDP–AE demonstrated more potent cytotoxicity against different carcinoma cell lines than lidamycin in vitro. In the mouse hepatoma 22 model, (Arg)9–LDP–AE (0.3mg/kg) suppressed the tumor growth by 89.2%, whereas lidamycin (0.05mg/kg) by 74.6%. Furthermore, in the glioma U87 xenograft model in nude mice, (Arg)9–LDP–AE at 0.2mg/kg suppressed tumor growth by 88.8%, compared with that of lidamycin by 62.9% at 0.05mg/kg. No obvious toxic effects were observed in all groups during treatments. The results showed that energized fusion protein (Arg)9–LDP–AE was more effective than lidamycin and would be a promising candidate for glioma therapy. In addition, this approach to manufacturing fusion proteins might serve as a technology platform for the development of new cell penetrating peptides-based drugs.

Factor VII Light Chain-Targeted Lidamycin Shows Intensified Therapeutic Efficacy for Liver Cancer

The overexpression of tissue factor (TF) observed in numerous cancer cells and clinical samples of human cancers makes TF an ideal target for cancer therapy. The purpose of this study is to develop a TF-targeting energized fusion protein hlFVII-LDP-AE, which is composed of a human Factor VII light chain (hlFVII) as the targeting domain conjugated to the cytotoxic antibiotic lidamycin (LDM, LDP-AE) as the effector domain. The potential efficacy of hlFVII-LDP-AE for cancer therapy was tested in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assays and in vivo with a BALB/c nude mouse xenograft model of human liver cancer line HepG2. The inhibitory concentration (IC(50)) value of hlFVII-LDP-AE varied from 0.15 to 0.64 nM for the various human tumor lines. hlFVII-LDP-AE showed a tumor growth inhibition rate of 90.6% at the dose of 0.6 mg/kg in in vivo animal experiments. The mechanism through which hlFVII-LDP-AE inhibits tumor growth also was determined by Hoechst 33342 staining and Tdt-mediated dUTP nick-end labeling (TUNEL) assay. hlFVII-LDP-AE causes tumor cell death through inducing chromatin condensation and cleavage of genomic DNA. These findings suggest that the hlFVII-LDP-AE protocol is efficacious and tolerated in the mouse model of human liver cancer HepG2 and has clinical applicability for treating cancer patients.

Factor VII light chain-targeted lidamycin targets tissue factor-overexpressing tumor cells for cancer therapy

The overexpression of tissue factor (TF) observed in numerous cancer cells and clinical samples of human cancers make TF an ideal target for cancer therapy. Here, we report an energized fusion protein, hlFVII-LDP-AE, which can be used for cancer therapy and is composed of a human Factor VII light chain (hlFVII) conjugated to the cytotoxic antibiotic lidamycin (LDM, LDP-AE). hlFVII-LDP-AE binds with specificity to TF expressed on tumor cells, resulting in internalization of the fusion protein and cytotoxicity induced by the LDM domain. The potential efficacy of hlFVII-LDP-AE for cancer therapy was examined in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and in vivo with a BALB/c nude mouse xenograft model of the human lung cancer line NCI-H292. hlFVII-LDP-AE caused chromatin condensation and cleavage of genomic DNA in NCI-H292 cells. In the MTT assays, the IC50 value of hlFVII- LDP-AE was 0.19 nM. In the in vivo tests, after two intravenous injections of hlFVII-LDP-AE at a dose of 0.6 mg/kg, the growth rate of the lung tumor xenograft was reduced to 15% of the control rate, and there was no excessive loss of body weight and inflammatory response in the mice. These findings suggest that hlFVII-LDP-AE is efficacious and tolerated in the mouse model of NCI-H292 human lung cancer examined and could have broad clinical applicability for treating cancer patients.

Antitumor effects of an engineered and energized fusion protein consisting of an anti-CD20 scFv fragment and lidamycin

Antibody-based fusion proteins are the next generation of antibody therapies for cancer and other diseases. CD20 antigen, which is overexpressed on cell membranes in nearly 95% of cases of B-cell Non-Hodgkin's Lymphoma, is an attractive target for the therapy of B-lymphoid malignancies. Lidamycin (LDM) is a potent enediyne-containing antitumor antibiotic that now has entered phase II clinical trials. In this study, we prepared an engineered fusion protein, scFv-LDP, consisting of an anti-CD20 scFv fragment and the apoprotein LDP of LDM using DNA recombination. After purification and refolding, scFv-LDP was found to bind specifically to CD20-positive lymphoma cells using ELISA and indirect immunofluorescent cytochemical staining assays. The energized fusion protein scFv-LDP-AE was obtained using molecular reconstitution of the active chromophore AE of LDM and scFv-LDP. MTT assay revealed potent cytotoxicity of scFv-LDP-AE to CD20-positive Raji and Daudi cells, with IC(50) values of 1.21×10(-11) and 6.24×10(-11) mol L(-1), respectively. An in vivo subcutaneous xenograft model of CD20-positive B cell lymphoma in BALB/c (nu/nu) mice was also utilized. Drugs were given intravenously on day 14 and 21 after tumor transplantation. In terms of maximal tolerated doses, scFv-LDP-AE at 0.3 mg kg(-1) suppressed tumor growth by 79.3%, and LDM at 0.05 mg kg(-1) by 68.6% (P<0.05). Results suggested scFv-LDP-AE could be a potential candidate for tumor-targeting therapy.

A Bispecific Enediyne-Energized Fusion Protein Containing Ligand-Based and Antibody-Based Oligopeptides against Epidermal Growth Factor Receptor and Human Epidermal Growth Factor Receptor 2 Shows Potent Antitumor Activity

The cooverexpression of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) observed in many human tumors and their synergistic interaction in the transformation of cells make these receptors important targets for the development of new targeted therapeutics. Targeting of EGFR and HER2 simultaneously has been pursued as a strategy with which to potentially increase efficiency and selectivity in therapy of certain cancers. This study was set to construct a bispecific energized fusion protein (Ec-LDP-Hr-AE) consisting of two oligopeptides against EGFR and HER2, and lidamycin, and investigate its antitumor efficacy. In vitro experiments measured the binding and internalization of bispecific Ec-LDP-Hr fusion protein. The potency of energized fusion proteins was also done in which the bispecific Ec-LDP-Hr-AE was compared with lidamycin (LDM) and its monospecific counterparts, Ec-LDP-AE and LDP-Hr-AE. In vivo, Ec-LDP-Hr-AE was given i.v. to nude mice bearing human ovarian carcinoma SK-OV-3 xenografts. Binding and internalization studies showed that bispecific fusion protein Ec-LDP-Hr bound to carcinoma cells specifically and then were internalized into the cytoplasm. Bispecific Ec-LDP-Hr-AE was more potent and selective in its cytotoxicity against different carcinoma cell lines than corresponding momospecific agents and LDM in vitro. In addition, Ec-LDP-Hr-AE significantly inhibited the growth of SK-OV-3 xenografts in nude mouse model. In vivo imaging study showed that FITC-labeled Ec-LDP-Hr was targeted and accumulated in the tumors. A ligand-based and an antibody-based oligopeptide fused to the enediyne antibiotic LDM created a new bispecific fusion protein with low molecular weight and more potent in vitro and in vivo antitumor activity (than momospecific fusion proteins).

Binding capability of the enediyne-associated apoprotein to human tumors and constitution of a ligand oligopeptide-integrated protein

The molecule of lidamycin that belongs to the chromoprotein family of antitumor antibiotics is composed of an apoprotein (LDP) and an enediyne chromophore. The enediyne moiety of the molecule is responsible for the potent cytotoxicity; however, the biological function of the apoprotein moiety, particularly its interaction with cancer cells, remains unclear. In present study, the binding capability of LDP to human tumors was detected for the first time by tissue microarray. LDP bound to various human tumors with significant difference from the corresponding normal tissues. Positive correlation between binding activity and the overexpression of VEGF and EGFR was confirmed by lung carcinoma tissue microarray. A fusion protein LG–LDP that consists of LDP and a ligand oligopeptide to EGFR was constructed by DNA recombination. LG–LDP showed augmented binding to EGFR-overexpressing cancer cells. Furthermore, an energized fusion protein LG–LDP–AE was prepared by integrating the active enediyne (AE) into LG–LDP molecule. By MTT assay, LG–LDP–AE displayed extremely potent cytotoxicity to cancer cells with IC50 approximate to 0.01 nM. The results indicate that LDP binds to various human tumors and it might serve as a delivery carrier by integration of ligand oligopeptide to manufacture motif-based, targeted fusion proteins for cancer.

Generation and antitumor effects of an engineered and energized fusion protein VL-LDP-AE composed of single-domain antibody and lidamycin

Type IV collagenase plays a pivotal role in invasion, metastasis and angiogenesis of tumor. Single domain antibodies are attractive as tumor-targeting vehicle because of their much smaller size compared with antibody molecules produced by conventional methods. Lidamycin (LDM) is a potent enediyne-containing antitumor antibiotic. In this study an engineered and energized fusion protein VL-LDP-AE composed of lidamycin and VL domain of mAb 3G11 directed against type IV collagenase was prepared using a novel two-step method. First a VL-LDP fusion protein was constructed by DNA recombination. Secondly VL-LDP-AE was obtained by molecular reconstitution. In MTT assay, VL-LDP-AE showed potent cytotoxicity to HT-1080 cells and KB cells with IC(50) values of 8.55 x 10(-12) and 1.70 x 10(-11) mol/L, respectively. VL-LDP-AE showed antiangiogenic activity in chick chrorioallantoic membrane (CAM) assay and tube formation assay. In in vivo experiments, VL-LDP-AE was proved to be more effective than free LDM against the growth of subcutaneously transplanted hepatoma 22 in mice. Drugs were given intravenously on day 3 and 10 after tumor transplantation. Compared in terms of maximal tolerated doses, VL-LDP-AE at 0.25 mg/kg suppressed the tumor growth by 89.5%, LDM at 0.05 mg/kg by 69.9%, and mitomycin at 1 mg/kg by 35%. Having a molecular weight of 25.2 kDa, VL-LDP-AE was much smaller than other reported antibody-based drugs. The results suggested that VL-LDP-AE would be a promising candidate for tumor targeting therapy. And the 2-step approach could serve as a new technology platform for making a series of highly potent engineered antibody-based drugs for a variety of cancers.

An enediyne-energized single-domain antibody-containing fusion protein shows potent antitumor activity

Single-domain antibodies are attractive as tumor-targeting vehicles because of their much smaller size than intact antibody molecules. Lidamycin is a macromolecular antitumor antibiotic, which consists of a labile enediyne chromophore (AE) and a noncovalently bound apoprotein (LDP). An enediyne-energized fusion protein VH-LDP-AE composed of single-domain antibody directed against type IV collagenase and lidamycin was prepared by a novel two-step method including DNA recombination and molecular reconstitution. VH-LDP-AE demonstrated extremely potent cytotoxicity to cancer cells and marked antiangiogenic activity in vitro. In the mouse hepatoma 22 model, drugs were administered intravenously as a single dose on day 1 with maximal tolerated doses. VH-LDP-AE (0.25 mg/kg) suppressed the tumor growth by 95.9%, whereas lidamycin (0.05 mg/kg) and mitomycin (1 mg/kg) by 79.6 and 51.1%, respectively. In the HT-1080 xenograft model in nude mice, drugs were given intravenously as a single dose on day 4 after tumor implantation. VH-LDP-AE at 0.25 mg/kg suppressed tumor growth by 76% (P<0.05) compared with that of lidamycin at 0.05 mg/kg (53%) on day 18. No obvious toxic effects were observed in all groups during treatments. The results showed that energized fusion protein VH-LDP-AE was more effective than lidamycin and mitomycin. These properties, together with its much smaller size than conventional antibody-based agents, suggested that VH-LDP-AE would be a promising candidate for cancer-targeting therapy. In addition, the two-step approach could serve as a new technology platform for making a series of highly potent engineered antibody-based drugs for a variety of cancers.