Nanoliposome-encapsulated Tumor Specific Antigen Protein Vaccine Preparation And Its Pharmacokines

Neoplasm is a life-threatening disease and the leading cause of death. The traditional therapies such as surgery, radiotherapy and chemotherapy are far from satisfaction. With the progress in immunology, cellular biology and molecular biology, tumor vaccines are regarded as a promising method and play an important role in the prevention and treatment of tumor. However, there are still many difficulties before the tumor vaccines could be used clinically. The lack of tumor specific antigens, the shortage of effective antigen-presenting tools, the limitation of MHC, the polymorph of tumor cells and the low bioavailability are the major factors. The Melanoma Antigen-associated Gene (MAGE) belongs to Cancer/Testis antigen, which was the first reported example of tumor specific antigens expressed in most malignant tumors but not in normal tissue except for testis and placenta, and has been used as the ideal target in tumor immunotherapy. Heat shock protein (HSP) participates in processing and presentation of tumor antigen and plays an important role in promoting anti-tumor immunity. Our recent researches have demonstrated that the fusion protein of MAGE1 and MAGE3 to M. tuberculosis HSP70 can lead to the enhancement of tumor vaccines potency in tumor immunotherapy, and we have prepared nanoemulsion encapsulated tumor antigen protein vaccine. To improve its antitumor immunoactivity and bioavailability and meet the requirement of pre-clinical experiments, based on the MAGE1, MAGE3 and HSP70 fusion protein, a short cycle, high expression and stabilized fermentation process of recombinant fusion protein has been used, which would be the basis for further purification and large-scale production of recombinant fusion protein. To prepare nanoliposome encapsulated tumor specific antigen derivative vaccine, the purified recombinant fusion protein was esterified and then was wrapped up with long circulating nano-liposome. Its immunological mechanisms and the dynamic rules of ADME in mice were studied.1. The structure and stability of biological characteristics of recombinant bacterial strain pET28a-MAGE1/HSP70/MAGE3(MHM)/BL21(DE3) were studied. The recombinant fusion protein expressed in inclusion bodies induced with IPTG. The expressed protein, binding with anti-MAGE1 mAb as well as anti- MAGE3 polyclonal Ab specifically, is over 30% of total bacteria protein. Then the engineering bacteria were cultured in shake flask. The inclusion bodies were washed, denatured, renatured and purified by affinity chromatography. The purity of the product was over 80%. The recombinant strain was subcultured for 50 passages, and the expression level of MHM,property of plasmid,as well as morphology,cultivation characters and biochemical reactions of various passages were studied.pET28a-MHM/BL21(DE3) showed a typical morphology of E.coli, and the characteristics of various passages were not significantly different from that of primary strain.The strain is suitable for large-scale production. 2. To match the requirement of pre-clinical experiments, the production and purification procedure of recombinant fusion protein were scaled up to pilot-scale. The bacteria were cultured in 20L DO feed-back fed-batch culture system. The effects of the composition of the fermentation medium, activation time, induction time, the range of pH and fed-batch carbon sources on the expression level of MHM and cell output were analyzed.We performed a triplicate fermentation. The expression level of recombinant fusion protein MHM and M3H was higher than 35 % of the total protein in E.coli. Three batches M3H protein were purified to homogeneity and the products amounted to 600-700mg/batch. Quality control was established by the tests of characteristics, purity, bioactivity and impurity residue of the fusion protein.3. To improve the efficiency of envelopment of protein by liposomes and the bioavailability of tumor antigen, the recombinant fusion protein was cross linked with stearic acid by chemical methods. The purified recombinant fusion protein was esterified and then was wrapped up with nano-liposome. Nanoliposome-encapsulated tumor specific antigen derivative vaccine (L-SA-MH) was prepared and its shape and size were detected by electron microscope and particle sizing system. The mean size of nano-liposomes was 87.9nm(CV=0.371), the encapsulation rate was 86%, and L-SA-MH had the required stability after stored in 4℃for 6 months. The anti-tumor immunity was tested by Enzyme-linked immunospot assay (ELISPOT) and Cytotoxicity assays. ELISPOT and Cytotoxicity assays showed the immunization using L-SA-MH vaccine enhanced the frequency of splenocytes secretingγ-IFN significantly compared with immunization using free protein vaccine alone. Cytotoxicity assays showed the MAGE3 B16 lysis of CTLs from mice immunized with L-SA-MH vaccine was greater than that from mice immunized with free protein vaccine. The fusion protein MH, SA-MH, L-MH and L-SA-MH vaccine could induce higher titer of anti-MAGE3 antibody than control groups. These results showed that nanoliposome had novel characters, and nanoliposome-encapsulated tumor specific antigen derivative vaccine could greatly enhance the potency of MAGE3 protein vaccines, and generate specific anti-tumor immunity against MAGE3 expression tumors.4. To clarify the process and properties of ADME and supply the pharmacokinetic parameters for safe use of nano-liposome L-SA-MH in clinics, ¹²⁵I-SA-MH and ¹²⁵I-L-SA-MH were prepared. The radioactivity in plasma or tissues or urine or feces was determined following trichloroacetic (TCA) precipitation of after a single intravenous injection. The concentration-time curves of ¹²⁵I-SA-MH and ¹²⁵I-L-SA-MH were proved to comply with two-compartment model. At the same amount of dosage, the distribution half life T1/2α, the elimination half life T1/2βand the AUC for plasma were increased after a single i.v. with ¹²⁵I-L-SA-MH compared i.v. with ¹²⁵I-SA-MH, and the total body clearance rate of ¹²⁵I-SA-MH was higher than that of ¹²⁵I-L-SA-MH. So liposome can protect drug and prolong the half time of SA-MH in serum and tissues, which enhanced drug bioavailability. ¹²⁵I-L-SA-MH was aggregated in most tissues especially in spleen while 25I-SA-MH in liver. Urinary system excretion test proved it to be the major pathway of ¹²⁵I-SA-MH and ¹²⁵I-L-SA-MH elimination.In summary, we constructed nanoliposome-encapsulated tumor specific antigen derivative vaccine, and it can induce tumor cell specific cellular and humoral immune reaction effectively. The enhanced immunity resulted in potential therapeutic effects against MAGE3-expressing tumors. The pharmacokinetic data indicate that it is tumor-targeting, delayed released and has higher bioavailability. Moreover, we established a stable production procedure of recombinant fusion protein and set up a standard of quality control. The data from this study grounds the large scale production and pre-clinical experiments of vaccine.