HER2-targeting recombinant protein with truncated pseudomonas exotoxin A translocation domain efficiently kills breast cancer cells.

The second domain of Pseudomonas exotoxin A (PEAII, residues 253-364) has been shown to facilitate translocation of extracellular and vesicular contents into the cytoplasm, and can transport heterologous molecules into target cells. Because full length PEAII may elicit a host immune response, we tried to identify the minimal PEAII translocation motif and use this fragment in combination with an antibody and constitutively active granzyme B (ImmunoGrB) to kill HER2-positive tumor cells. We constructed four ImmunoGrB fusion proteins containing different PEAII deletions and tested their abilities to kill HER2-positive cells. Our data showed that while a complete deletion of PEAII in ImmunoGrB resulted in an inability to kill cancer cells, ImmunoGrBs containing either PEAII (253-358aa) or PEAII (275-358aa) could efficiently kill HER2-positive SK-BR-3 cells. Most interestingly, the construct which contains only a furin cleavage site, named PEAII (275-280aa), could still induce SK-BR-3 apoptosis, although less efficiently. Moreover, delivery of the recombinant proteins by intramuscular plasmid injection led to an apparent tumor regression and prolonged animal survival in a nude mouse xenograft SK-BR-3 tumor model, indicating in vivo antitumor activity of the different PEAII containing ImmunoGrBs. Our results may help in understanding PEAII translocation and may lead to the development of useful tools or alternative therapy.

[Pro-apoptotic efficiencies of three reconstructed human caspase-8 on cervical cancer cell line HeLa].

BACKGROUND & OBJECTIVE: Apoptosis is a kind of evolutional high conservative cell death. Transferring high active pro-apoptotic molecules into cancer cells to induce apoptosis is a potential strategy for cancer gene therapy. Based on our previous generation of reconstructed human caspase-8, which can continuously induce apoptosis of cervical cancer cell line HeLa, by reversing its large and small subunits, this study was designed to investigate the pro-apoptotic efficiencies of 3 reconstructed human caspase-8 (Casp8CD, Rev8, and Rev8L) on HeLa cells, and to explore the feasibility of reconstructed human caspase-8 as potential apoptosis-inducing candidates. METHODS: The eukaryotic expression vectors pIRES2-EGFP carrying Casp8CD, Rev8, and Rev8L genes were transfected into HeLa cells, and breast cancer MCF-7 cells. Expressions and pro-apoptotic effects of Casp8CD, Rev8, and Rev8L genes were observed under fluorescent microscope, and their pro-apoptotic efficiencies were assessed by MTT assay and cells counting. The flexibilities of linking-peptides between subunits of Rev8 and Rev8L were analyzed by bioinformatics. RESULTS: Expressions of the 3 reconstructed caspase-8 genes were observed under fluorescent microscope, and the HeLa and MCF-7 cells expressing Rev8 or Rev8L genes displayed typical apoptotic volume decrease (AVD). MTT assay showed that compared with control cells, A(570) values of Rev8- and Rev8L-transfected cells began to decrease 20 h after transfection. Cell counting results indicated that cell death ratio of Casp8CD-, Rev8-, and Rev8L-transfected cells were 16.9%, 52.3%, and 47.7%, respectively, 24 h after transfection; and 12.9%, 51.6%,and 61.2%,respectively,48 h after transfection. Bioinformatics analysis showed that the linking-peptides between subunits of Rev8 and Rev8L were flexible. CONCLUSION: Rev8 and Rev8L molecules have similar pro-apoptotic effects and efficiencies, but over-expressed Casp8CD had no significant pro-apoptotic effects.

[Establishment of Hela cell line inducibly expressing human active granzyme B].

AIM: To construct inducible expression vector for human granzyme B gene and express it in Hela cell line. METHODS: Human active granzyme B gene was obtained by PCR and cloned into the inducible expression vector pIND. The resulting expression vector, together with a helper plasmid pVgRXR, was stably transfected into Hela cells using Lipofectamine 2000. The transfected cells were selected in medium containing G418 and zeocin. The resistant cells were induced with ponasterone A, and the optimal concentration of ponasterone A and time of induction were determined by immunocytochemical staining. Then the effects of the expressed granzyme protein on Hela cells were detected by MTT colorimetry and cytoskeletal staining. RESULTS: The Hela cells that inducibly expressed human active granzyme B were obtained. Induction with 30 micromol/L ponasterone A for 5 days caused the strongest expression of granzyme B. The induced cells appeared as either multinucleate giant cells or pyknotic small cells, and their growth was inhibited. Further analysis demonstrated cytoskeletal abnormality of multinucleate giant cells. CONCLUSION: The establishment of inducible expression system for active granzyme B lays the foundation for further research on biological function of granzyme B.

A caspase-6 and anti-human epidermal growth factor receptor-2 (HER2) antibody chimeric molecule suppresses the growth of HER2-overexpressing tumors.

Clinical studies have suggested that human epidermal growth factor receptor-2 (HER2) provide a useful target for antitumor therapy. We previously described the generation of a chimeric HER2-targeted immunocasp-3 protein. In this study, we extend the repertoire of chimeric proapoptotic proteins with immunocasp-6, a construct that comprises a HER2-specific single-chain Ab, a single-chain Pseudomonas exotoxin A, and an active caspase-6, which can directly cleave lamin A leading to nucleus damage and inducing programmed cell death. We demonstrate that the secreted immunocasp-6 molecule selectively recognizes and induces apoptosis in HER2-overexpressing tumor cells in vitro, but not in cells with undetectable HER2. The immunocasp-6 gene was next transferred into BALB/c athymic mice bearing human breast SK-BR-3 tumors by i.m. injection of liposome-encapsulated vectors, by intratumor injection of adenoviral vectors, or by i.v. injection of PBMC modified by retroviral infection. Regardless of the method used, expression of immunocasp-6 suppressed tumor growth and prolonged animal survival significantly. Our data show that the chimeric immunocasp-6 molecule can recognize HER2-positive tumor cells, promptly attack their nucleus, and induce their apoptotic death, suggesting the potential of this strategy for the treatment of human cancers that overexpress HER2.

[The cloning and expression of the truncated human bid gene and its pro-apoptotic effect on Hela cells].

AIM: To explore the expression of the truncated bid gene and its pro-apoptotic effect on Hela cells. METHODS: A full-length human bid gene was cloned by RT-PCR and confirmed by sequence analysis. By deleting the 60 amino acids of N-terminal the truncated bid (tbid) gene was obtained and then inserted into the eukaryotic expression vector pIRES2-EGFP. After the tbid gene was transfected into Hela cells under lipofectamine mediation, the effect of target gene expression on morphology and growth of Hela cells were observed under fluorescence and electron microscopes and analysed by TUNEL staining. RESULTS: pIRES2-EGFP containing tbid gene was constructed successfully. After Hela cells were transfected with GFP expression vector of tbid gene, tbid was expressed which was followed by decreased cell fluorescence intensity, poor cell growth, and cell death. Cell shrinkage and nuclear condensation, typical apoptotic characteristics, were observed by electron microscope observation and Tunel staining. CONCLUSION: The expression of tbid can effectively induce apoptosis of Hela cells.

Secreted antibody/granzyme B fusion protein stimulates selective killing of HER2-overexpressing tumor cells.

Targeted cell killing is required for effective treatment of cancers. We previously described the generation of a chimeric immunocasp-3 protein and its potent selective antitumor activity (Jia, L. T., Zhang, L. H., Yu, C. J., Zhao, J., Xu, Y. M., Gui, J. H., Jin, M., Ji, Z. L., Wen, W. H., Wang, C. J., Chen, S. Y., and Yang, A. G. (2003) Cancer Res. 63, 3257-3262). Here we extend the repertoire of another chimeric pro-apoptotic protein immunoGrB, which comprises an anti-HER2 single-chain antibody, a Pseudomonas exotoxin A translocation domain and active granzyme B. Human lymphoma Jurkat cells transfected with the immunoGrB gene expression vector were able to produce and secrete the chimeric protein. The immunoGrB molecule selectively recognized and destroyed HER2-overexpressing tumor cells both in vitro and in nude mouse after intramuscular injection of the immunoGrB expression plasmid. Further in vivo study showed that intravenous administration of immunoGrB gene-modified lymphocytes led to suppression of HER2-overexpressing tumor growth and prolonged animal survival because of continuous secretion of immunoGrB molecules into blood and lymph fluid. These results demonstrate that the chimeric immunoGrB molecule, which is capable of antibody-directed targeting and granzyme B-mediated killing, has therapeutic potential against HER2 tumors, especially in cases in which caspase-dependent apoptosis is inhibited.

[Growth inhibitory effects of recombinant granzyme B containing different N-terminal translocating peptides].

Translocating protein and translocating peptides have therapeutic potential against tumors by exposing the cytotoxic domains of toxic proteins to the cell cytosol. The aim of this study is to investigate the effect of N-terminally fused PE translocating peptides on granzyme B (GrBa) activity. PE II-GrBa fusion protein genes were constructed by replacing N-terminal signal and acidic dipeptide sequence of human granzyme B gene with two truncated translocating sequences of Pseudomonas exotoxin A (PE II aa 280-364/358) by recombinant PCR, and then cloned into pIND inducible expression vector. The resulting pIND-PE II-GrBa expression vectors were co-transfected with assistant plasmid pVgRXR into HeLa cells through lipofectamine, followed by selection on G418 and zeocin. The resistant cells were collected and induced with ponasterone A. Western blot analysis demonstrated that ponasterone A induction caused the expression of PE II-GrBa fusion proteins, and indirect immunofluorescence detected giant sized multinucleated cells, suggesting cytoskeletal and mitotic abnormalities as reported in our previous studies. Western blot, enzymatic activity assay and cell counting analysis indicated that two types of PE II-GrBa fusion proteins were capable of cleaving both endogenous and exogenous substrates of granzyme B, and inhibiting the growth of cells. The PE II (aa 280-358)-GrBa was shown to have higher serine protease activity and stronger growth inhibitory effect. Such inhibition was presumably associated with G2 arrest as determined by cell cycle analysis. These data prove that PE II-GrBa fusion proteins have cell inhibitory effect similar to GrBa, and that the shorter PE-derived peptide exerts less influence on GrBa activity. This study helps to optimize the construction of recombinant protein comprising translocating peptides and cytotoxic molecules for tumor cell killing.

[Targeted tumor suppression by a secreted fusion protein consisting of anti- erbB2 antibody and reversed caspase-3 to SKBr3 cells].

OBJECTIVE: To investigate the targeted killing effect to SKBr3 cells due to the expression of a secreted fusion protein consisting of anti-erbB2 antibody and reversed caspase-3. METHODS: A recombinant plasmid pCMV-e23scFv-PEII-revcasp 3 was constructed by subcloning reversed caspase-3 gene to the downstream of anti-erbB2 antibody and transfected into Jurkat cells. The cell lines which secreted expressing fusion protein stably were selected. The fusion protein in media was detected by ELISA and the media was used to culture human breast cancer SKBr3 cells. The recombinant plasmids with liposomes was administrated to BALB/C nude mouses bearing SKBr3 tumor by intramuscular injection. The targetting effect of the recombinant fusion protein caspase-3 was detected by indirect immunofluorescence staining. RESULTS: Fusion protein can be expressed and secreted by Jurkat cells stably and kill SKBr3 cells. Significant prolonged survival time (prolonged by 72%) and inhibition of tumor growth in vivo (within inhibition ratio of 77%) were seen in the group administered with recombinant plasmids. Indirect immunofluorescence staining showed that the recombinant fusion protein caspase-3 has targetting effect. CONCLUSION: Secreted expression of the fusion protein consisting of anti-erbB2 antibody and reversed caspase-3 can targetedly induce SKBr3 cells to death.

Specific tumoricidal activity of a secreted proapoptotic protein consisting of HER2 antibody and constitutively active caspase-3.

In this study, a novel approach to antitumor therapy was devised by generating a chimeric tumor-targeted killer protein, referred to as immunocasp-3, that comprises a single-chain anti-erbB2/HER2 antibody with a NH(2)-terminal signal sequence, a Pseudomonas exotoxin A translocation domain, and a constitutively active caspase-3 molecule. In principle, cells transfected with the immunocasp-3 gene would express and secrete the chimeric protein, which then binds to HER2-overexpressing tumor cells. Subsequent cleavage of the constitutively active capase-3 domain from the immunocasp-3 molecule and its release from internalized vesicles would lead to apoptotic tumor cell death. To test this strategy, we transduced human lymphoma Jurkat cells with a chimeric immunocasp-3 gene expression vector and showed that they not only expressed and secreted the fusion protein but also selectively killed tumor cells overexpressing HER2 in vitro. i.v. injection of the transduced Jurkat cells led to tumor regression in a mouse xenograft model because of continuous secretion of immunocasp-3 by the transduced cells. The growth of HER2-positive tumor cells in this model was inhibited by i.m. as well as intratumor injection of immunocasp-3 expression plasmid DNA, indicating that the immunocasp-3 molecules secreted by transfected cells have systematic antitumor activity. We conclude that the immunocasp-3 molecule, combining the properties of a tumor-specific antibody with the proapoptotic activity of a caspase, has potent and selective antitumor activity, either as cell-based therapy or as a DNA vaccine. These findings provide a compelling rationale for therapeutic protocols designed for erbB2/HER2-positive tumors.

[Effect of the truncated human apoptosis-inducing factor expression on apoptosis of HeLa cells].

AIM: To observe the expression of the truncated human apoptosis-inducing factor (AIF) gene and its apoptosis-inducing effect on HeLa cells. METHODS: Full-length human AIF gene was cloned by RT-PCR, then the truncated AIF gene was constructed by deleting the N-terminal mitochondrial location sequence (MLS), and inserted into the EGFP co-expression vector pIRES2-EGFP. After being transfected into HeLa cells with Lipofectamin, the expression of the truncated AIF gene and its effect on HeLa cells morphology and growth condition were detected by fluorescence microscope, immunohistochemical staining, indirect fluoroimmunoassay and electron microscope analysis. RESULTS: The eukaryotic expression vector pIRES2-EGFP containing of truncated human AIF gene was constructed successfully. The AIF protein could be detected in the transfected HeLa cells. After transfection, typical apoptotic feature of the transfected HeLa cells and a mass of cell death were observed under electron microscope. CONCLUSION: The expression of the truncated human AIF gene can induce apoptosis of the transfected human HeLa cells.

[Expression of a secreted fusion protein consisting of anti-HER2 antibody and reversed caspase-3 and its suppression on growth of SKOV3 cells].

AIM: To investigate the killing effect on SKOV3 cells by fusion protein HER2-specific antibody-reversed caspase-3 in the secreted form. METHODS: The reversed human caspase-3 gene was subcloned into pCMV-e23scFv-PEII-PEIII to construct recombinant eukaryotic expression vector pCMV-e23scFv-PEII-revcasp-3 and transfect Jurkat cells. The secreted expression of the in culture supernatant of transfected Jurkat cells was detected by ELISA. The suppression of the fusion protein on growth of SKOV3 cells was examined by co-culture with supernatant of transfected Jurkat cells. RESULTS: There was the secreted expression of the fusion protein in Jurkat cells. Expressed product had the activity of inducing SKOV3 cells to death. CONCLUSION: The fusion protein expressed in the secreted form can targeted towards SKOV3 cells and kill them.

[Construction of single-chain Fv Gene of Anti-HBsAg and its expression in COS-7 cells].

AIM: To subclone the V(L) gene and V(H) genes of anti-HBsAg and construct the single-chain Fv gene and to analyse the expression of the constructed gene in COS-7 cells. METHODS: A set of oligonucleotide primers were designed and used to amplify the V(H) and V(L) genes from Fab antibodies screened from phage antibody library. The products were cloned into pUC19 vector and their sequences were analysed. The V(H) and V(L) gene fragments were linked up by a peptide linker and a leader sequence added at 5' terminus of each gene (L-V(H)-Linker-V(L)) and designated as L-ScFv. The L-ScFv genes were inserted into the eukaryotic fusion protein expression vector pEGFP-N3 and transfected into COS-7 cells to express respectively. RESULTS: The coding sequences of V(H),V(L), linker and leader in all constructs were all correct. The expression of ScFv fusion protein was detectable by fluorescence microscope after transient expression in COS-7. The secretive expression of L-ScFv was confirmed by SDS-PAGE and Western blot analysis. And the binding specificity of the ScFvs with HBsAg were proved by indirect ELISA. CONCLUSIONS: Anti-HBsAg ScFv genes have been successfully constructed and expressed in COS-7 cells.