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.

[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.

[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.

[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.

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.

[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.

[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.

[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.

[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.

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.

[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.

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.