Construction Of Energized Fusion Protein Consisting Of Anti-HER2 ScFv And Lidamycin And Its Antitumor Activity

Breast cancer is the most common malignancy in women, it now represents thesecond leading cause of death from cancer in women, and the number of new breastcancer cases has been increasing year by year. To increase the patients' survival ratesand their life quality, we need new measures of therapy. Molecular targeted cancertherapy has been developing rapidly. In present, many molecular agents for targetedtherapy have been used for breast cancer therapy in of clinical practice, such asHerceptin, Iressa and inhibitors of cell cycle regulator, COX-2 or PKC.The Her-2/neu (c-erbB2) proto-oncogene is a component of the four-memberfamily of closely related growth factor receptors that includes the epidermal growthfactor receptor Her-1 (EGFR), Her-3 (c-erbB3) and Her-4 (c-erbB4). The human geneis located on chomosome 17q21 and encodes a 185 kD protein with tyrosine kinaseactivity, also known by the designation P185. Structurelly, the protein hasextracellular, transmembrane, and cytoplasmic domains, the latter of which containsthe tyrosine kinase domain and shares significant homology, although is distinct,from EGFR. Her-2/neu is thought to function as a growth factor receptor and plays arole in cell differentiation, adhesion, and motility. Overexpression of HER-2, usuallyas a result of her-2 gene amplification, can result in malignant transformation of cellsand is seen in tumor tissue in up to 30% of patients with metastatic breast cancer(MBC) and part of patients with ovarian, lung, gastric, and oral cancers. HER-2overexpression is usually associated with a more aggressive tumor phenotype, andwomen with HER-2 positive tumor have a poor overall prognosis and faster relapse.So the overexpression of HER-2 has been an independent prognostic index forevaluating the malignancy of above cancers. Evidently, HER2 become an explicittarget for therapy of breast cancer and ovarian cancer.Antibody can carry agent to tumor region, so enhances the therapeutic effect anddecreases the toxicity. It has been shown that scFv (single chain variable Fragment)can penetrate into the solid tumor deeply. Compared to the whole antibody, scFvshows low immunogenicity and so induce less human anti-mouse antibody (HAMA)response. On the other hand, because of the absence of Fc, scFv can not execute theeffector function to kill target tumor cells. So a "warhead" molecule is neededincluding drugs, toxins, and cytokines.Molecular targeted therapy is currently having a tremendous impact on the daily practice of clinical oncology. The advent of Herceptin (trastuzumab), a humanizedmonoclonal antibody against extracellular domain of HER-2, represented a majorbreakthough in the treatment of women with HER-2-positive MBC. Pivotal trials ofHerceptin alone or in combination with taxanes have result in significant clinicalbenefit, and Herceptin plus taxanes as first-line therapy is now the standard of care ofHER-2-positive disease. Already, the use of Herceptin in the metastatic setting haschanged HER-2-positive status from a marker of poor prognosis to one of betteroverall outcome, and ongoing studies should expand further treatment options forpatients with HER-2-positive MBC. This agent was shown to significantly prolongthe progression-free survival as a single agent or in combination with standardchemotherapeutic agents.Lidamycin (LDM), an antibiotic produced by Streptomyces globisporus strainwhich was isolated in our institute, displayed extremely potent cytotoxicity againsttumor cells. LDM consists of a chomophore and an apoprotein, and the former has theability to attack DNA, whereas the latter plays the role as a protecting protein. Thetwo parts can be separated and reconstructed without affecting its activity.The first surface expression system was developed by Smith in 1985, whodisplayed the filamentous phage on the surface of bacteriophge. In recent years, thefilamentous bacteriophages have been used extensively for the display of largerepertoires of antibodies on their surface. Phage display enables rapid selection ofantibodies from a large single chain fragment variable (scFv) library by virtue of thebinding to certain target antigen. After three or more rounds of selection, the resultingphage population is markedly enriched for the scFvs that bind to the antigen.Antibodies against hundreds of target antigens have so far been obtained from phagedisplay antibody libraries, including cell-surface markers, peptide hormones, humanproteins, and carbohydrates. One of the prominent advantages of the phage-displaytechnology is that, once a library has been created, it can be used to select antibodiesthat bind to any target antigens of interest. A single phage-antibody library can bedistributed to thousands of users and serve as the source of cloned antibodies againstan unlimited array of antigens. This technology is also efficient, and the process ofselection and primary screening is very rapid and can be completed with 2-3 weeks.The selected phage clones are then subjected to immunodetection to confirm theirbinding activities. ELISA and Western blot are extensively used in immunodetection.Conventionally, the procedure usually involves subcloning and expression of the target fragments encoding the scFv to obtain significant quantities of antibodies,which proves to be time-consuming and laborious. To resolve this problem,researchers have recently foucused on the use of selected phage clones inimmunodetection or function studies. In this study, a phage-display scFv antibodylibrary constructed on the N terminus of pâ…¢protein of M13 filamentous phage.In this study, phage display has been used to select high affinity scFv to HER2,and molecule reconstitution was performed to construct energized fusion proteinwith extremely potent cytotoxicity to HER2 over-expressing tumor cells.1. Construction and selection of HER2 phage scFv libraryIn this study, mRNA was isolated from splenocytes of mice immunized withpurified ECD protein of HER2. Immunoglobulin variable fragments VH and VL wereamplified by RT-PCR and were connected by linker (Gly₄Ser)₃ to form the singlechain Fv (scFv) through a peptide by SOE-PCR. Then the scFv gene was cloned inphagemid pCANTAB5E, and transformed into E.coli TG1. The transformed cellswere infected by M13KO7 helper phage to get the primary phage display scFv library.Panning against breast cancer SKBR3 cells line was performed four times. At last, arecombinant phage display scFv library with a titre of 7.5×10⁷ was established.Random 20 clones were selected from primary and selected phage antibody library,through PCR identified, scFv fragment was inserted into 15 clones of primary phagelibrary and 20 clones in selected phage library. Random 18 clones' plasmids fromprimary library digested with BstNâ… showed different pattern, and clones fromselected library showed specially pattern enriched. The affinity of clones to HER2was identified by ELISA.Through 4 rounds of panning and screening, a phage-scFvnamed scFv6 binding to HER2 specifically was selected from the library. Bysequence analysis, the length of this gene is 732 bp, and 243 amino acid is encoded.This murine antibody is different from any other antibodies discovered before bysequence blast analysis.2. Construction, expression, purification and characterization analysis of energizedfusion protein of HER2(Fv-LDM)ScFv6 which has high affinity with HER2 antigen was cloned intoreconstruction plasmid pET-LDP, and plasmid pET-Fv-LDP was transformed intoE.coli BL21 (DE3) star^TM, fusion protein HER2(Fv-LDP) with His-tag wasexpressed when induced by IPTG. Through SDS-PAGE analysis, 7% of soluble fusion protein existed in periplasm of E.coli. Subsequently purified by IMAC(immobilized metal affinity chromatography). About 2 mg of protein was obtainedfrom 1 liter fermentation broth. The purity of this protein exceeded 90%. For thepreparation of energized fusion protein HER2 (Fv-LDM), we added AE to the PBSsolution of the HER2 (Fv-LDP) fusion protein with the molecular ratio of 5:1 for 12h at room temperature. After the reconstitution was completed, separation andpurification were preformed using PD-10 colomn to remove the free AE (SephadexG-25 column). Western-blot assay showed that fusion protein have affinity withpurified protein ECD of HER2. Cell ELISA and cell fluorescence showed thatfusion protein HER2 (Fv-LDP) had high affinity with SKBR3 and SKOV3 cells,but it was lower than that of commercial anti-HER2 mAb L87. By contrast, HER2(Fv-LDP) had little affinity with MCF7 cells. The level of HER2 mRNA andprotein in SKBR3 cells decreaced by the treatment with HER2 (Fv-LDM). Flowcytometry (FCM) assay of cell cycle showed that cells were arrested at G1 phasewith fusion protein HER2 (Fv-LDP) treatment, instead of G2/M phase withenergized fusion protein HER2 (Fv-LDM) treatment. Through Hoechest and HEdying, the appearance of cells and nuclei showed that energized fusion proteinHER2 (Fv-LDM) can induce cell apoptosis of SKBR3,SKOV3 and MCF7. ByMTT assay, HER2 (Fv-LDM) showed potent cytotoxicity to SKBR3, SKOV3,Moreover, the in vivo therapeutic efficacy of the energized fusion protein HER2(Fv-LDM) was investigated in human breast carcinoma SKBR3 xenograft in nudemice. In experiment, the SKBR3 tumor bearing mice were treated by single i.v doseon day 10 and day17 after subcutaneous tumor transplantation, inhibitory effect ofHER (Fv-LDM), LDM, HER2 (Fv-LDP) was detected through measuring thevolume of tumor. At dose 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, HER2 (Fv-LDP)exhibited highly effected potency in retarding growth of SKBR3 with inhibitionrate of 60%, 70%, 89.7%, while the inhibition rate of LDM (0.05mg/kg) is 56.8%.So the energized fusion protein can increase the tolerance dose of LDM, improvethe therapeutic effects, and become an antibody targeted medicine of goodperspective.