The Development Of Tumor Neovasculature Endothelial Cells Targeted Interferon-alpha2a

Interferons-alpha2a (IFN-a2a), a multifunctional regulatory cytokines, has many biological functions, including antiviral, antiproliferative, and immunomodulatory effects. It has approved worldwide for the treatment of variety of malignancies and virologic disease. Unfortunately, significant toxicities of IFN-α2a therapy, divided into constitutional, neurophsychiatric, hematologic and hepatic effects, impact on its spreading of clinical application.Cyclic peptide NGR (CNGRCVSGCAGRC), homing specifically to tumor vascular was screened by in vivo phage display. The receptor of NGR has demonstrated to be aminopeptidase N(CD13) which homes the tumor blood vessel endothelium cells specifically. The CD13/APN has been approved to mediate migration and invision of endothelial cells in the process of angiogenesis. A peptide containing the NGR motif, the main domain of CNGRCVSGCAGRC, has been coupled to different antitumor drugs, such as doxorubicin, cytokines and proapoptotic peptides, and yielded compounds with increased efficacy against tumors and lowered toxicity to normal tissues in mice.To enhance the antitumor effects of IFN-α2a while decrease the toxicity, wecoupled the cyclic peptide NGR to the C terminus of IFN-a2a (IFN-a2a-NGR), and hope that the yielding fusion protein could either bind with the CD13/APN homing specifically on tumor endothelial cells or inhibit angiogenesis that mediated with CD 13/APN. The results of our studies are as follow:1. We prepared periplasmic expression vector pET-22b(+)- IFN-a2a-NGR by recombinant DNA technology and transfected it into BL21(DE3) cells. The fusion protein expressed in inclusion bodies induced with IPTG. The expressed protein, binding with anti-IFN-a mAb as well as anti-NGR mAb specifically, is more than 10% of total bacteria protein. Then the engineering bacteria were cultured by shake flask. The inclusion bodies were washed, denatured, renatured and purified by affinity chromatography. The product had a purity of >95% and a specific activity of 4.2-5.5 X 108U/mg.2. To match the requirement of pre-clinical experiments and clinical trial, the production and purification procedure of IFN-a2a-NGR were scaled up to pilot-scale. The bacteria were cultured in 5L automatic fermentor and yielded about 200g wet bacteria per batch. Three batches IFN-a2a-NGR protein were purified to homogeneity and the products amounted to 250-300mg/batch. Quality control requirement was established based on the detections of characteristics, purity, bioactivity and impurity residue. In addition, the purified IFN-a2a-NGR was characterized to be a mixture of monomer, dimer and trimer by SDS-PAGE, Western blot, HPLC, peptide map and 15 amino acid sequence of N terminus.3. We observed coupling IFN-a2a to NGR can enhance the therapeutic index, markedly superior to that of untargeted IFN-a2a, in A549, SPC-A-1 and Lewis nude mice models. We also investigate that IFN-a2a-NGR can bind to vessels...