Preparation And Targeted Cancer Therapy Of Micromolecule Recombinant Immuotoxin GnRH-luffinS

Recombinant immunotoxin is an important drug of targeted cancer therapy and is composed of a very potent protein toxin and a targeting moiety such as a recombinant antibody fragment or growth factor. Protein toxins commonly used include bacterial toxins such as PE and DT, and plant toxins such as ricin, PAP and saporin. The clinical efficacy of recombinant immunotoxins has been demonstrated in patients with malignant tumors. For example, recombinant immunotoxin ONTAK, which consists of truncated DT and IL2, has been approved by the FDA for CTCL therapy. But the antitumor activity of recombinant immunotoxins in vivo is interfered by many factors, e.g, good immunogenicity and poor penetration into tumors. One of methods is to develop new type of micromolecule recombinant immunotoxins to reduce their immunogenicity and improve their penetration into tumor cells.LuffinP1 and luffinS2, seperated from the seeds of Luffa cylindrical, are two novel ribosome inactivating proteins (RIPs).The two small toxin molecules are RNA N-glycosidases with molecular weights of 5 kDa and 8 kDa, respectively, and are much smaller than any other RIPs so far investigated. Their IC50 values are 10-9M and 10-10M respectively and can be used as cytotoxic moiety of recombinant immunotoxins. TypeⅠof the gonadotropin releasing-hormone (GnRH), also called luteinizing hormone-releasing hormone (LHRH), is a neuro-decapeptide. After binding to its receptor on pituitary cells, it triggers the secretion of the luteinizing hormone (LH) and follicle stimulating hormone (FSH). These gonadotropin hormones control the activity of the gonads. GnRH receptors have been identified on tumor cells from mammary, endometrial, prostatic, hepatic and pancreatic tissues as well as on tumor cell lines. A growing interest has developed for the application of GnRH and related peptides in the fields of gynaecology and oncology. GnRH and its analogues have been used in treatment of hypogonadism and infertility. It is also used for the treatment of gonadotropin dependent cancers. Furthermore, this peptide binds to its receptor with high affinity (kd about 10-9M) and is not immunogenic, so GnRH is a very good candidate for the targeted moiety of recombinant immunotoxins. For example, LHRH-PE40 showed antitumor efficacy in preclinical study and has been produced for clinical phase testing. Therefore, recombinant immunotoxins, composed of GnRH used as targeted moiety and luffinP1 or luffinS2 used as cytotoxic moiety, are potential antitumor drugs which have low molecular weights, poor immunogenicity and strong penetration.We firstly cloned and expressed luffinP1 and luffinS2 in E. coli, and analyzed their protein biosynthesis inhibitory activity in cell-free systems. The protein with higher activity was used to construct recombinant immunotoxins along with GnRH. Then, the two recombinant immunotoxins of GnRH-luffinS2 and luffinS2-GnRH were designed and constructed. After the fusion proteins were expressed and purified, the in vitro cytotoxicity of the fusion proteins was determined by XTT method. Finally, because PEⅡfragment has the function of translocation and a protease recognition site, which can enhance efficacy of transmembrane and make targeted moiety and toxic moiety separate in cytoplasm, we constructed a novel recombinant immunotoxin GnRH-PEⅡ-luffinS, in which PEⅡw as inserted into GnRH-luffinS2, and analyzed the antitumor activity of the fusion protein in vitro and in vivo.In this study, the experiments were conducted as follow.1. Cloning, expression and activity assay of luffinP1 and luffinS2(1) The PCR primers were designed by Condon software, after PCR conditions were optimized, the genes of luffinP1 and luffinS2 were amplified. (2) LuffinP1 and luffinS2 were ligated with expression vectors pET-His, pET32a, pQE30 and pBV220, and recombinant vectors pET-His/luffinP1, pET-His/luffinS2, pET32a/luffinS2, pET32a/luffinP1, pQE30/luffinP1, pQE30/luffinS2, pBV220/luffinP1 and pBV220/luffinS2 were constructed. The results of expression showed that only recombinant vectors pET32a/luffinS2 and pET32a/luffinP1 of them can express target fusion proteins in soluble form. The soluble fusion proteins were purified by Ni-NTA chromatography method. After cleaved by rEK, the single rluffinP1 and rluffinS2 were produced. (3) Then, the protein bioinhibitory activitives were determined by rabbit reticulocyte lysate. The results showed that IC50 of rluffinP1 and rluffinS2 were 9.51μg/ml and 1.58μg/ml, respectively. So luffinS2 was selected as the cytotoxic moiety of recombinant immunotoxins in following experiment.2. Design, cloning, expression and activity assay of GnRH-luffinS2 and luffinS2-GnRH(1) Because the order of GnRH and luffinS2 may influence their activitives, the GnRH-luffinS2, which GnRH was fused to the N terminus of luffinS2, and the luffinS2-GnRH, which GnRH was fused to the C terminus of luffinS2, were designed, and their secondary structures were anticipated. (2) The PCR primers were designed by Condon software, the gene fragments of GnRH-luffinS2 and luffinS2-GnRH were amplified by optimized PCR reaction conditions. (3) the two gene fragments of GnRH-luffinS2 and luffinS2-GnRH were respectively ligated with expression vectors pET-His, pET32a, pQE30 and pBV220, and recombinant vectors pET-His/GnRH-luffinS2, pET-His/luffinS2-GnRH, pET32a/GnRH-luffinS2, pET32a/luffinS2-GnRH, pQE30/GnRH-luffinS2, pQE30/luffinS2-GnRH, pBV220/GnRH-luffinS2 and pBV220/luffinS2-GnRH were all constructed. The results of expression showed that only recombinant vectors of pET32a/GnRH-luffinS2 and pET32a/luffinS2-GnRH can express target fusion proteins in soluble form. The soluble fusion proteins were purified by Ni-NTA chromatography method. After cleaved by rEK, the rGnRH-luffinS2 and rluffinS2-GnRH were produced. (4) Then their cytotoxicity to Hela, A549, HepG-2, SP2/0 and CEF cells were analyzed by XTT method. The results showed that IC50 values of rGnRH-luffinS2 were 67.19μg/ml for Hela cells, 70.42μg/ml for A549 cells, 84.44μg/ml for HepG-2 cells and 106.25μg/ml for SP2/0 cells, respectively. The IC50 values of rluffinS2-GnRH were 79.5μg/ml for Hela cells, 76.7μg/ml for A549 cells, 96.2μg/ml for HepG-2 cells and 137μg/ml for SP2/0 cells, respectively. But the two fusion proteins showed no toxic effect to CEF cells. The results revealed that GnRH-luffinS2 and luffinS2-GnRH had low cytotoxicity to cancer cell lines3. Design, cloning, expression and activity assay of GnRH-PEⅡ-luffinS(1) Because PEⅡhas the function of translocation and includes a protease recognition site, recombinant immunotoxin GnRH-PEⅡ-luffinS was designed, in which PEⅡwas inserted into GnRH-luffinS2, and its secondary structure was anticipated. (2) The PCR primers were designed by Condon software, the gene fragment of GnRH-PEⅡ-luffinS was amplified by optimized PCR reaction conditions. (3) GnRH-PEⅡ-luffinS gene was inserted into expression vector pET32a and transformed into E.coli BL21. The recombinant plasmid pET32a/GnRH-PEⅡ-luffinS expressed insoluble protein. The inclusion bodies of fusion protein were extracted and purified by Ni-NTA chromatography method. After renaturation through dialysis and concentration through ultrafiltration, the fusion protein was cleaved by rEK in order to produce rGnRH-PEⅡ-luffinS. (4) The cytotoxicity to Hela, A549, HepG-2, SP2/0 and CEF cells was analyzed by XTT method. The result showed that IC50 values of rGnRH-PEⅡ-luffinS were 13.50μg/ml for Hela cells, 13.74μg/ml for A549 cells, 16.79μg/ml for HepG-2 cells and 26.07μg/ml for SP2/0 cells, respectively and had no toxic effect to CEF. To evaluate antitumor activity in vivo of rGnRH-PEⅡ-luffinS fusion protein, animal models with solid tumor were established through inoculated SP2/0 subcutaneously. The tumor weight inhibition rate of rGnRH-PEⅡ-luffinS against SP2/0 was 50.3%, when administrated continuously for ten days with 100μg per mice. The result showed that the GnRH-PEⅡ-luffinS had antitumor activitiy both in vitro and in vivo.In conclusion, luffinP1 and luffinS2 were expressed in prokaryotic expression system and the protein biosynthesis inhibition activity of rluffinS2 was higher than rluffinP1 in rabbit reticulocyte lysate. The recombinant toxins GnRH-luffinS2 and luffinS2-GnRH were constructed, in which GnRH was as targeted moiety and luffinS2 was as toxic moiety. The cytotoxicity of these fusion proteins in vitro was low. Finally, PEⅡ, which has the function of translocation and proteolytic cleavage site, was inserted into GnRH-luffinS2, and GnRH-PEⅡ-luffinS was designed and expressed in prokaryotic expression system, the cytotoxicity in vitro and in vivo showed that the novel recombinant toxin GnRH-PEⅡ-luffinS had a potential to be used for targeted cancer therapy.