Gene Construction, Expression, Purification And Targeted Killing Of Immunoproapoptotic Molecule E23sFv-TD-tBID Against HER2 Positive Carcinoma

Recombinant immunotoxin is an important drug of targeted cancer therapy and is composed of a very potent protein toxin and a targeting moiety such as a recombinant antibody fragment or growth factor. Protein toxins commonly used include bacterial toxins such as PE and DT, and plant toxins such as ricin, PAP and saporin. The clinical efficacy of recombinant immunotoxins has been demonstrated in patients with malignant tumors. But the antitumor activity of recombinant immunotoxins in vivo is interfered by many factors, e.g, strong immunogenicity and poor penetration into tumors. One of methods is to develop new type of micromolecule recombinant immunotoxins to reduce their immunogenicity and improve their penetration into tumor cells.Bid (BH3-interacting death agonist), one of the BH3 domain-only pro-apoptotic Bcl-2 family member, is recognized as an intracellular linker connecting the death receptors pathway and mitochondrial death machinery through caspase-8 activation. Full-length Bid,normally present in cytosol, is cleaved by activated caspase-8 or other proteases to form tBid (carboxyl-terminal region of Bid) which translocates to the mitochondria causing cytochrome c (Cyt c) and AIF release. Because Bid can link the extrinsic and intrinsic cell death pathways and amplify death receptor signaling, we believe tBid could be a potential candidate for cancer therapy.HER2/neu (c-erbB2) proto-oncogene is located on chromosome 17q21 and encodes a Mr 185,000 transmembrane glycoprotein (p185HER2) that belongs to a subfamily of growth factor receptors having intrinsic tyrosine kinase activity. Amplification and overexpression of ERBB-2 is found in 25-30% of primary human breast cancers and is associated with a poor clinical outcome. Increased expression of ErbB2 is also observed in colon, prostate, lung, and gastric cancers, making it a popular therapeutic target for carcinomas. HER2/neu is an attractive target for immunotherapy.In this study we constructed a noval human antitumor immunotBID molecule e23sFv-TD-tBID made up of anti-HER2 scFv (e23sFv) and active truncated tBID. A 10-residue spacer, Ala-Gly-Asn-Arg-Val-Arg-Arg-Ser-Val-Gly, containging the recognition site for protease furin from diphtheria toxin translocation domain was incorporated between the antibody fragment and the active truncated BID.The recombinant gene was inserted into prokaryotic expression vector pQE30 and transformed into E.coli M15. The recombinant plasmid pQE30-e23sFv-TD-tBID expressed in inclsion body by the induction of IPTG. After purification by Ni-NTA chromatography and renaturation through dialysis, we get the fusion protein.In vitro target-specific binding and cytotoxic activity of e23sFv-TD-tBID were show on a variety of HER2-positive cell lines and primary breast tumor cells. On binding to HER2, it was rapidly internalized into the target cells, after proteolytic cleavage, accessed directly to the cytosol. To determine the therapeutic effects of e23sFv-TD-tBID, it was given i.p. to immunodeficient mice bearing s.c. human breast carcinomas. The immunoproapoptotic protein either a single dose or combinations with chemotherapy significantly inhibited tumor growth with no toxic effect on mice. Our results suggest that e23sFv-TD-tBID has therapeutic potential for HER2-positive tumors and deserves further development.Conclusion: We produced the fusion protein e23sFv-TD-tBID in E.coli. and described the characterization and antitumor activity in vitro and in vivo. These results show that this molecule may prove to be a valuble agent for immunotherapy of HER2-positive carcinomas.