Using Transgenic Mice To Generate Fully Human Anti-PCSK9 Monoclonal Antibodies

Cardiovascular diseases(CVD)is one of the highest mortality diseases at present in worldwide.Studies found that LDL-C level is one of the independent factors affecting cardiovascular disease.Therefore,the lower levels of LDL-C can alleviate hypercholesterolemia,atherosclerosis and coronary heart disease associated with lipid.In 2003,PCSK9 mutation was found firstly,similar to LDLR and ApoB which found previous associated with autosomal dominant familial hypercholesterolemia.Since then,the mutant and its biological function of PCSK9 had been gradually elucidated.PCSK9 is a kind of serine proteinase,mature PCSK9 is secreted into the blood and bind to LDLR,LDLR will be transferred to lysosome and degraded directly,then reduce the number of recycled PCSK9.LDLR is mainly responsible for transshipment of plasma LDL,so the LDL-C level is positively related to the level of PCSK9.In vitro experiment and PCSK9(-/-)mice in vivo test showed PCSK9 can adjust the expression levels of LDLR.In a word,PCSK9 inhibitors may be effective in the therapy of hyperlipidemia,and by adjusting the expression levels of LDL-C,finally prevention and treatment of cardiovascular diseases.Monoclonal antibody play an important role in the biological drugs research and development,especially fully human monoclonal antibody.Harbour transgenes mice introduced with human V regions,rat constant regions and mouse LCR and enhancer.Harbour mice immunized with hPCSK9 can get anti PCSK9 monoclonal antibodies.The monoclonal antibody from Harbour mice had normal assembly and mature process,so it shows higher affinity than the chimeric antibody and murine antibody.Human PCSK9 protein and plasmid expression of human PCSK9 from chempartner were as immunogen,Harbour transgenic mice immunized with three kinds of immunization programs,after four or five times immunizations,antiserum titer of some mice had good binding activity at dilution of 1:10000.Lymph nodes and splenocytes from Harbour mice were fused with SP2/0 by PEG or electric fusion.Protein-based binding assay,receptor binding blocking assay and cell based functional assay including PCSK9 mediated LDLR downregulation and PCSK9 mediated LDL uptake inhibition within HepG2 were conducted,and finally two positive clones in accordance with the requirementswere screened out,named 139G1C5 and 96F8C10.Using the isotyping determination kit,isotying of two clones were determined as IgG1 and IgG2 b respectively.Using protein G affinity chromatography purified antibodies were obtained,and named mAb016(139G1C5)and mAb023(96F8C10).The purity of two mAbs were > 90% by SEC-HPLC and SDS-PAGE analyzation.mAbs showed good binding activity within both protein-based binding assay and cell-based functional assay.This study successfully generated two monoclonal antibodies with high affinity,good binding activity and biological function,they provide solid basement for the development of fully human anti-PCSK9 monoclonal antibody.