The Recombinant Defensin-based And Macropinocytosis-mediated Fusion Protein As Well As The Recombinant Bispecific ScFv Fusion Protein-- Preparation And Study Of Antitumor Activity

Part Ⅰ. Preparation of the defensin HBD2-based and macropinocytosis-mediated fusion protein and study of antitumor activity and mechanismPurpose:Recent studies have shown that oncogenic K-Ras mutant pancreatic cancer cells display intensive macropinocytosis to support their unique metabolic needs and point to the possible exploitation of this process in the design of anticancer targeted therapies. Therefore, it is of interest to employ albumin as a carrier for macropinocytosis-mediated targeting therapy. We designed and prepared a recombinant albumin-tethered defensin (DF-HSA) which consists of the human beta-defensin-2 (DF) as an "effector" molecule and the human serum albumin (HSA) as a macropinocytosis mediator, then investigated its antitumor efficacy against the K-Ras mutant pancreatic cancer.Experimental Design:The macropinocytosis intensity and cytotoxicity of DF-HSA were investigated in K-Ras mutant MIA PaCa-2 and wild-type BxPC-3 cells. The cytotoxicity and related mechanistic effects of DF-HSA were compared with HSA and the free defensin HBD2. Tumor-specific accumulation and therapeutic effect of DF-HSA were evaluated with human pancreatic carcinoma MIA PaCa-2 xenografts in athymic mice. The Human Gene Expression Microarrays were applied to evaluate the gene expression profile of MIA PaCa-2 cells after treated with HBD2 and DF-HSA.Results:Macropinocytosis-mediated internalization of DF-HSA was found in the K-Ras mutant MIA PaCa-2 cells. The intensity of macropinocytosis in MIA PaCa-2 cells was much higher than that of wild-type K-Ras-expressing BxPC-3 cells. Similarly, DF-HSA showed more potent cytotoxicity to MIA PaCa-2 cells than to BxPC-3 cells. Compared in terms of cytotoxicity, DF-HSA was stronger than free beta-defensin HBD2 and HSA. DF-HSA induced mitochondrial damage and mitochondrial pathway apoptosis. Notably, DF-HSA significantly inhibited the growth of human pancreatic carcinoma MIA PaCa-2 xenografts in athymic mice at well tolerated dose. By in vivo imaging, DF-HSA displayed specific biodistribution and continuously accumulated in the tumor. The changes of the expression profile induced by DF-HSA were obvious. These differently expressed genes involved in a variety of functions including gene expression and transcription regulation, cellular metabolic process, apoptotic process and so on.Conclusion:The albumin-tethered beta-defensin DF-HSA can enter into the K-Ras mutant pancreatic cancer cells through an intensive macropinocytosis-mediated process and exert potent therapeutic efficacy against the pancreatic carcinoma xenograft in athymic mice. Evidently. DF-HSA could be a promising agent in macropinocytosis-mediated targeting therapy.Part Ⅱ. Preparation of defensin HBD1-based dual scFv fusion protein β1-dFv-LDPAs one of the most important components in the innate immunity, beta-defensin 1 (HBD1) exhibits cytolytic activity against a range of tumor cells:moreover, it is more difficult for cancer cells to develop resistance. Studies have shown that the gene hbd-1 may serve as a potential tumor-suppressor. In addition, HBD1 may play a certain role in tumor immunity. Therefore, HBD1 might be used as an active agent in cancer treatment. On the other hand, over-expression of matrix metalloproteinases (MMPs) which play an important role in cancer progression and spread is considered to be a potential drug target for cancer therapy. In the MMP family, gelatinase (MMP2/9) is critical in cancer cell invasion and metastasis associated with extracellular matrix (ECM) degradation. Some biomedical evidence has shown that MMP-2/9 could be useful as therapeutic targets. This study was set to construct a HBD1-based anti-gelatinase dual scFv fusion protein β1-dFv-LDP which consists of an anti-gelatinase dual Fv fragment, a lidamycin-derived apoprotein LDP and the "warhead" agent HBD1 and investigate its antitumor efficacy. After constructed and sequenced, the expression vector PET-30a/β1-dFv-LDP was transformed into E.coli transetta (DE3). The expressed proteins mainly existed as insoluble inclusion bodies. After purification under denatured conditions, the β1-dFv-LDP fusion protein was refolded in a way of step-wise dialysis and approximately 20 mg soluble functional β1-dFv-LDP could be obtained from one liter fermentation broth. The fusion protein β1-dFv-LDP exhibited same affinity as dFv-LDP to the antigen gelatinase.Part Ⅲ. Preparation of a bispecific scFv fusion protein Fv(E)-LDP-Fv(M) that targets EGFR and gelatinaseBispecific antibody (bsAb), capable of binding two targets simultaneously, is one of the currently most attractive topics in the development of targeted anticancer drugs. Overexpression of EGFR that belongs to receptor tyrosine kinase family has been observed in a variety of human tumors, making it promising target for antibody therapeutics. In addition, matrix metalloproteinases (MMPs) play an important role in cancer progression and spread. It has been reported that gelatinases (MMP-2 and -9) are highly expressed in many cancers as compared to their adjacent normal tissues. Therefore, MMP-2/9 could be potential targets for cancer therapy. Lidamycin (LDM) is a macromolecular peptide antitumor antibiotic with extremely potent cytotoxicity. This study was set to construct a bispecific scFv fusion protein consisting of an anti-EGFR scFv, an anti-gelatinase scFv, and the LDM-derived apoprotein LDP which serves as the "scaffold" and the fitted pocket for the insertion of the extremely potent cytotoxic enediyne molecule. After being verified by sequencing, the recombinant plasmid pET30a(+)/Fv(E)-LDP-Fv(M) was transformed into E.coli Transetta(DE3). The expressed proteins mainly existed as insoluble inclusion bodies. After purification under denatured conditions, the Fv(E)-LDP-Fv(M) fusion protein was refolded in a way of step-wise dialysis and approximately 0.5 mg active Fv(E)-LDP-Fv(M) fusion proteins could be obtained from one liter fermentation broth. The fusion protein Fv(E)-LDP-Fv(M) exhibited good affinity to the antigens both of EGFR and gelatinase.