The Pegylation Of Recombinant Human Plasminogen Kringle 5 And Preliminary Research On Its Antitumor Activity

Angiogenesis is the process that the tissues produce new vessel by ultilizingexisting ones.Tumor angiogenesis means the angiogenesis induced by tumor tissueduring its growth. In 1970s, Folkman gave a hypothesis that angiogenesis of tumorcontributes to its growth and metastasis and correspondingly the corruption andinhibition of tumor angiogenesis can delay the its growth and metastasis.Human plasminogen kringle 5(hk5), a kringle domain connecting to angiostatinin plasminogen, can inhibit the angiogeneis of tumor by inhibiting the proliferationand migration of endothelial cell and by inducing the hypopotosis of them. Hk5 is anewly discovered tumor angiogenesis inhibitor with high activity.In our lab,we usedrecombinant human plasminogen kringle 5 (rk5) expressed in Pichia yeast. Rhk5,consisting of 98 amino acid residues and having 3-paired disulfide bonds and 5 freeamines, is approximate 17.5kDa.Recently, recombinant human endostatin (rhEndo),with the similar antitumor mechanism like rhk5,has been approved to market.Likerhk5,rhEndo has low MW and short half-life and needs frequent dosing which willreduce the clinical compatibility, so we paid attention to the long-lasting preparation ofrk5 and the long-lasting is also an upgrading trend for recombinant protein andpolyipeptide drugs.The long-lasting modification methods of recombinant protein and polypeptidedrugs mainly include:recombinant DNA modification,PEGylation,sustained releasepreparations.Among the three measurements,PEGylation,deriving from 1970s andbelonging to protein in vitro chemical modification, is maturer and relatively easier tocarry out.PEGylation improves the pharmacokinetic properties of the protein andpolypeptide by changing the physico-chemical properties (increasing MW, decreasingPI,strengthening stability) and immunological characteristics(reducing immunogenicityand antigenicity), and acquire similar and even improved pharmacodynamicproperties at lower dosing frequence Firstly, three well-used species of active PEG were used to modify rhK5 andthree kinds of monopegylated rhk5 were prepared from modified mixtures by weakpositive ion exchange chromatography; secondly, a monopegylated rhk5(mPEG-SPA_20kD-rhK5) was selected by antitumor test in vivo; finally, its antitumoreffect was analysed furtherly.In the first part, designed experiment to find a relatively rational reactionconditions combination to gain a better monopegylation ratio from pH,molarratio(PEG/rhK5),tempreture, reaction period and protein concentration for threekinds of PEG by SDS-PAGE.Gained monopegylated rhk5 by weak positive ionexchange chromatography, and made sure the monopegylated rhk5 is indeed thePEG-rhK5 with the combination test of iodine staining, coomassie blue staining andWestern blotting and its purity is qualified with SDS-PAGE,which makes goodfoundation for afterword test.In the second part,at first,we authenticated two kinds of protein concentrationdetermination(BCA and Lowry)which were not interfered by pegylation, and gave astrong support to make dose comparable in serum concentration test and in vivoantitumor test;then, we test the serum concentration of three kinds of PEG-rhk5 bytwo kinds of path(intramuscular and intraperitoneal) in two kinds of mice,the resultsindicated three kinds of PEG-rhk5 could last longer periods with higher concentrationthan native rhk5,so we could decide the injection interval of PEG-rhk5;at last,wechose two kinds of tumor model (Lewis lung cancer and H22 liver cancer) and twoinjection paths (intramuscular and intraperitoneal) to select a PEG-rhk5 withrelatively higher antitumor activity. The dosage regimen is that the dose of PEG-rhk5is the same as rhk5,but lower dosage frequence.In Lewis lung cancer model withintramuscular path,the inhibiting ratio of tumor is 9ï¼…(rhK5),27ï¼…(mPEG-SPA_20kD-rhK5),8ï¼…(mPEG₂-NHS_20kD-rhK5),13ï¼…(mPEG₂-NHS_40kD-rhK5);InH22 liver cancer model with intraperitoneal path, the inhibiting ratio of tumor is14.2ï¼…,57.5ï¼…,17.2ï¼…,19.2ï¼…, correspondingly.So,the antitumor activity ofmPEG-SPA_20kD-rhK5 is better than other two kinds. In the third part,we chose mPEG-SPA_20kD-rhK5 for further study. Firstly, study thequality control key points,such as MW, PI, purity, residue PEG, residue rhK5,Ultraviolespectrum, CD, the results indicated pegylation could increase the MW and decrease thePI of the rhK5,but did not produce significant effect on the Ultraviole spectrum andCD of rhK5, the purity, residue PEG and residue rhK5 were qualified;then,we usedChicken Chorioallantotic Membrane(CAM)angiogenesis inhititing test to demonstratethat the mPEG-SPA_20kD-rhK5 had in vitro activity;next,utilized H22 liver cancer withintravenous dosage and three dose group (high,medium,low) to test the antitumoractivity of PEG-SPA_20kD-rhK5 and rhK5. PEG-SPA_20kD-rhK5 have the same dose asrhK5, but low dosage frequence.For PEG-SPA_20kD-rhK5,the inhibiting ratio of tumorin three dose group (high,medium,low) is 55.0ï¼…,63.5ï¼…,40.6ï¼…respectively;ForrhK5,it is 60.0ï¼…,63.5ï¼…,65.3ï¼…respectively. The statistic results indicated the samedosage groups of PEG-SPA20kD-rhK5 and rhK5 have no significant difference,andwhich demonstrated pegylation can achieve equal antitumor effect with low dosagefrquence, at the same time,we approached the distribution of PEG-SPA_20kD-rhK5 andrhK5 in tumor tissue by immunohistochemistry and ELISA,and we preliminaryapproached the antitumor mechanism of rhK5 by detecting the microvessel denisityand apoptosis in tumor tissue. The results suggested that pegylation profit the widerdistribution of rhK5 to tumor tissue,but had not effect on the antitumor mechanism ofrhK5.