The Construction Of Engineered And Energized Fusion Proteins Consisting Of Lidamycin And Molecularly Small-sized Anti-gelatinase Antibody And Their Antitumor Activity

The study of antibody-drug immunoconjugate is a promising field in current anticancer drug development; however, the successful development of which is dependent, in part, on the effective targeting capability of antibody and on the biological activity of the drug conjugated. Gelatinase is abundantly expressed in a variety of malignancy, and palys an important role in cancer invasion, metastasis, angiogenesis and the homeostasis of tumor microenviroment. Murine mAb 3G11 developed in our lab can specifically bind to gelatinases and lidamycin (LDM, also called C-1027) is a potent antitumor antibiotic. Some targeted drugs based on 3G11 and LDM had been created and shown striking antitumor effects in vitro or in vivo. To further augment the antitumor efficacy of fusion proteins based on anti-gelatinases antibody fragments and LDM, the following investigations have been performed.1. Preparation of fusion protein dFv-LDP consisting of a tandem anti-gelatinase scFv and the apoprotein of lidamycin and its tumor targeting capability in vivoThe bivalent scFv was considered to possess the optimal tumor targeting capability in vivo. To further augment the antitumor efficacy of the fusion protein Fv-LDP-AE which consisting of an anti-gelatinase scFv and LDM, we constructed a fusion protein named dFv-LDP, which was comprised of a di-Fv in tandem and the apoprotein of LDM. The recombinant plasmid pET30a(+)/dFv-LDP, which was verified by enzyme-digestion and sequencing, was transformed into E.coli BL21 (DE3) and E.coli Rosetta(DE3)pLysS. The expression of fusion protein dFv-LDP was induced by 0.2 mM IPTG. It was found that the expression level of dFv-LDP in E.coli Rosetta(DE3)pLysS was higher as compared with that of in E.coli BL21(DE3). The expressed proteins was mainly existed as insoluble inclusion bodies. After purification by IMAC under denatured conditions, the dFv-LDP fusion protein was refolded in a way of step-wise dialysis and approximately 50 mg soluble functional dFv-LDP could be obtained from one liter fermentation broth and the purity of which was>95%. The refolded dFv-LDP exhibited significant binding specificity with antigen gelatinases as determined by ELISA, the affinity constant with gelatinase was 0.043μM, about 4-fold increase compared to that of Fv-LDP, which just contained a single scFv. The dFv-LDP fusion protein could specifically bind to different kinds of tumor cells such as Bel-7402, PG-BE1, HT-1080, HepG 2 and MCF-7 cells. The results of flow cytometry (FCM) showed that the fusion protein dFv-LDP could be internalized by tumor cells in a certain degree, and the internalization efficacy is a little higher in comparison to that of Fv-LDP. Howerver, the internalization efficacy of dFv-LDP and Fv-LDP was both not very apparent. As in vivo tumor targeting experiments, the fusion protein dFv-LDP showed strong tumor targeting capability in human hepatoma Bel-7402, lung carcinoma PG-BE1 and colon carcinoma HCT-116 xenografts, which was better in contrast to that of Fv-LDP. The dFv-LDP could locate and concentrate at the tumor site in less than 1 h. As shown, the fusion protein dFv-LDP had the feature of fast tumor targeting, and could carry more molecules of lidamycin to the tumor foci under the same condition.2. Optimization of the assembly efficiency for lidamycin chromophore AE bound to the apoprotein in dFv-LDP fusion proteinThe molecule of lidamycin (LDM), an antitumor antibiotic with extremely potent cytotoxicity to cultured cancer cells, can be dissociated to an apoprotein (LDP) and an active enediyne chromophore (AE). The detached AE can be reassembled with its apoprotein-containing fusion protein to endow the latter obtain the potent antitumor activity. However, the assembly of AE with LDP is influenced by several factors. To optimize the assembly ratio of the AE with the LDP-containing fusion protein dFv-LDP and investigate the complete information of several factors that influence the assembly efficiency, a method based on RP-HPLC was developed to analyze the assembly ratio and an orthogonal experimental design L9(34) was used to determine the effects of temperature, assembly time, pH and molecular ratio of AE to the fusion protein dFv-LDP. A calibration curve based on the LDM micromolar concentration against the peak-area of AE by HPLC was deduced. The order in which individual factors in the orthogonal experiment affected the assembly ratio were temperature>time>pH> molecular ratio of AE to protein and all the four factors were statistically significant (P<0.01). The optimal assembly conditions were temperature at 10℃, time of 12 h, pH7.0, and the molar ratio of AE:dFv-LDP at 5:1. The assembly ratio of AE with the fusion protein dFv-LDP was increased by 20% after condition optimization as compared to that obtained under traditional condition and the cytotoxicity in vitro was improved. In conclusion, the assembly efficiency of the chromophore AE with fusion protein dFv-LDP was improved after condition optimization and it would be useful for the development of this kind of LDP-containing fusion protein.3. The antitumor efficacy of enediyne-energized fusion protein dFv-LDP-AE in vivoThe results of optical imaging showed that the fusion protein dFv-LDP possessed well tumor targeting capability in vivo, then the antitumor efficacy was evaluated after it was assembled with the chromophore of lidamycin. MTT assay showed that the enediyne-energized fusion protein dFv-LDP-AE had potent cytotoxicity to kinds of cancer cell, with IC50 values varied from 1×10-10to 10-12 M, which was not beyond that of the intact lidamycin. Under the equimolar dosage level, the energized fusion protein dFv-LDP-AE showed higher tumor growth inhibition of murine hepatoma 22 (H22) in KM mice as compared to that of Fv-LDP-AE (dFv-LDP-AE 89.5%vs Fv-LDP-AE 84.2%). The antitumor efficacy was increased compared to lidamycin (73.6% inhibition rate), which indicated the therapeutic efficacy was inproved by antibody targeting delivery of lidamycin. dFv-LDP-AE also showed potent antitumor efficacy in Bel-7402 xenograft model, doses of 0.4 mg/kg,0.6 mg/k and 0.8 mg/kg suppressed tumor growth by 64.2%,73.1%, and 87.3%, respectively, while free LDM at 0.05 mg/kg showed the inhibition rate of 63.4%. By contrast, the tumor inhibition rate of energized fusion protein Fv-LDP-AE at 0.48 mg/kg (the equimolar dosage of dFv-LDP-AE at 0.8 mg/kg) was 73.9%, there was significant difference on inhibition rate (P<0.05) between dFv-LDP-AE and Fv-LDP-AE under equimolar dosage level, which suggested that the targeted delivery of lidamycin was improved by dFv-LDP.The shedding of tumor antigen from tumor site is a problem which is needed to consider. In order to further augment the antitumor efficacy of energized fusion protein dFv-LDP-AE, we attempt a new regimen of drug administration by the combination of dFv-LDP in a larger dosage and energized dFv-LDP-AE in a smaller dosage. As in lung carcinoma PG-BE1 xenograft, the naked fusion dFv-LDP at 10 mg/kg showed an inhibition rate of 77.5%, the energized fusion protein dFv-LDP-AE improved the tumor inhibition efficacy, with an inhibition rate of 91.3% and 94.2% at dosage 0.4 mg/kg and 0.6 mg/kg, respectively. Moreover, the tumor inhibition rate was further augmented in the groups of dFv-LDP (10 mg/kg) combined with dFv-LDP-AE (0.4 and 0.6 mg/kg), producing the initial tumor shrinkage in 5 of 6 mice, and the shrinkage ratio were 22.0% and 49.6% respectively. The antitumor efficacy was stepwise intensified in groups of dFv-LDP, dFv-LDP-AE and the combination, which was also verified by the immunohistochemical analysis of microvessel density CD31 and nuclear proliferation index Ki-67.It is known that sarcoma is a tumor with highly expressed. Using the human fibrosarcoma HT-1080 which was stably transformed luciferase-containing plasmid, we established an experimental metastasis model in athymic mice. The FITC-labeled fusion protein dFv-LDP showed apparent targeting characteristic toward experimental metastasis foci. In vivo animal experiment, fusion protein dFv-LDP at 10 mg/kg diaplayed a moderate antitumor efficacy, the number of tumor foci were 55.8% of the control group (P<0.01), the energized fusion protein dFv-LDP-AE at 0.4 and 0.6 mg/kg showed an increase of antitumor efficacy, the numbers of tumor foci were 41.4% and 25.1% of the control respectively (P<0.05 compared to that of dFv-LDP group). Moreover, the metastasis foci in lung of nude mice were further decreased in groups of dFv-LDP (10 mg/kg) plus dFv-LDP-AE (0.4 and 0.6 mg/kg), the number of which just 20.3%(P<0.05, compared to dFv-LDP at 0.4 mg/kg) and 13.1%(P<0.05, compared to dFv-LDP at 0.6 mg/kg) of the control group, indicating the augmented anti-metastasis efficacy by the combination of dFv-LDP and dFv-LDP-AE. The combination of fusion protein dFv-LDP and dFv-LDP-AE might has potential application in clinical therapy of fibrosarcoma.4. The construction of engineered and energized fusion protein consisting of lidamycin and heavy-chain CDR3 domain of anti-gelatinases scFv and its antitumor activityIt was demonstrated that the antitumor efficacy could be further augmented by the combination of unenergized fusion protein in a larger dosage and energized fusion protein in a smaller dosge, however, the increase of immunogenicity might be inevitable, the downsizing of molecule was a feasible approach to obviate this issue. In this study, we constructed, expressed and purified two recombinat fusion proteins, CDR3-LDP and CDR3-LDP-CDR3, which comprise of one or two heavy-chain CDR3 domain of antigelatinase scFv and the apoprotein of lidamycin. The results of ELISA showed that there were no significant difference between the affinity of CDR3-LDP and CDR3-LDP-CDR3 fusion protein, the affinity constants of which were 5.78×10-6 and 6.563×10-6 M, respectively. The results of immunofluorescence showed that fusion protein CDR3-LDP could bind well with M21 and HepG2 cancer cells. The energized fusion protein CDR3-LDP-AE displayed potent cytotoxicity to Bel-7402 cancer cell with IC50 value of 1.05×10-11 mol/L. In murine hepatoma 22 animal experiment, fusion protein CDR3-LDP (10 mg/kg) showed an inhibition rate of 56%, the energized fusion protein CDR3-LDP-AE at dosage 0.25 and 0.5 mg/kg displayed an inhibition rate of 78.8% and 87.1%(P<0.05 compared to CDR3-LDP fusion protein), respectively. After combination, the tumor growth inhibition rate was increased to 85.2%(P<0.05, compared to CDR3-LDP-AE at 0.25 mg/kg) and 92.7%(P<0.05, compared to CDR3-LDP-AE at 0.5 mg/kg), respectively.In summary, the bivalent fusion protein dFv-LDP increased the binding with gelatinases in vitro and tumor targeting capability in vivo as compared to that of monovalent fusion Fv-LDP. The energized dFv-LDP-AE also displayed better antitumor efficacy in contrast to that of Fv-LDP-AE. The combination of dFv-LDP in a larger dosage and dFv-LDP-AE in a smaller dosage had potent antitumor and antimetastasis efficacy in vivo, indicating that this pair of fusion proteins were promising for development of novel targeted therapy and might have potential application in future. The combination of antibody and its antibody-armed analogue may be of interest for other antibody-drug conjugates as a new approach to further augment the antitumor efficacy.