生物技术药物 3,antibody

Lightvariableregion–VLLightconstantregion–CLHeavyvariableregion–VHHeavyconstantregion1-CH1Heavyconstantregion2-CH2Heavyconstantregion3-CH3Antigenbindingfragment–FabHingeConstantfragment-FcChapterIII.TherapeuticAntibodiesMadebyGeneEngineeringChapterIII.TherapeuticAntibodiesMadebyGeneEngineeringChapterIII.TherapeuticAntibodiesMadebyGeneEngineeringAntibodiesareimmunesystem-relatedproteinscalledimmunoglobulins.Eachantibodyconsistsoffourpolypeptides–twoheavychainsandtwolightchainsjoinedtoforma"Y"shapedmolecule.ChapterIII.TherapeuticAntibodies

MadebyGeneEngineeringTheimmunesystemcreatesbillionsofdifferentantibodieswithalimitednumberofgenesbyrearrangingDNAsegmentsduringBcelldevelopment,priortoantigenexposure.ChapterIII.TherapeuticAntibodies

MadebyGeneEngineeringOver15,000,000combinationsofVariable,DiversityandJoininggenesegmentsarepossible.Impreciserecombinationandmutationincreasethevariabilityintobillionsofpossiblecombinations.ChapterIII.TherapeuticAntibodies

MadebyGeneEngineeringWhenastemcellchangestobecomeaBcell,DNAsegmentsforbothheavy(VDJ)andlight(VJ)chainsarerandomlycombined.EachBcellendsupwithfunctionalgenesformakingonelightandoneheavychaincodingforanantibodyasamembrane-boundreceptor.Antibodyspecificitydependsonthegenefragmentsused.Antibodiesareproducedthatcanreactwithalmostanychemicalstructureinnature,includingourownproteins.ChapterIII.TherapeuticAntibodies

MadebyGeneEngineeringBcellsarespecializedwhitebloodcellsproducedinthebonemarrow.EachBcellcontainsmultiplecopiesofonekindofantibodyasasurfacereceptorforantigen.TheentirepopulationofBcellshastheabilitytospecificallybindtomillionsofdifferentantigens.WhentheantibodyonthesurfaceofaBcellbindstoanantigen,thecellcanbestimulatedtoundergoproliferationanddifferentiation.Thisprocessiscalledclonalselection.Thecellsproducedmakethesameantibody,butbecomememorycellsandplasmacells.ChapterIII.TherapeuticAntibodies

MadebyGeneEngineeringMiceareimmunizedwithanantigen,andBcellsisolatedfromtheblood.Thesecellsarefusedwithcancercellswhichgrowcontinuously.HybridcellsproducetheantibodyoftheBcell,butcannowgrowinculture.Thecellscanbeclonedorgrownfromsinglecoloniesandscreenedforspecificantibody.ChapterIII.TherapeuticAntibodies

MadebyGeneEngineeringChapterIII.TherapeuticAntibodies

MadebyGeneEngineeringApplicationofantibodiesintheclinics.Preventandtreatinfectiousdiseases.Invivodiseaselocalization.Immuneregulation.Cancertherapy.Limitationsofthetherapeuticantibodies.PK:T1/2isshort(20hrformousemAb),only1%reachthetarget.Humananti-mouseantibody:8to10ds,willleadtoneutralizetheantibody,andincreasetheclearanceoftheantibody.ChapterIII.TherapeuticAntibodiesMadebyGeneEngineering

III.TechnologiesformakingabetterantibodyIsolatethegenesencodingthevariableregionsofAb.CDR1,2,3arecomplementarydeterminingregions.DeterminetheAbspecificity.FR1,2,3,,4areframeworkregions.TheyareregionsbetweenCDRs,andfairlyconstant.III.Technologiesfor

makingabetterantibodyAntibodystructuresrevealedbyproteinbiochemicalmethods.

RT-PCRcloninggenesencodingVcandVH.Primers:5’primeraccordingtoFR1orSignalpeptidesequence.3’primeraccordingtotheFR4region.TemplateDNA:Spleencellsofimmunizedmouse;orhybridomacellline.III.Technologiesfor

makingabetterantibody：

MakingscFvFR1FR4FR2FR3Constructionofsinglechainvariableregionfragmentofantibody(scFv).

a.SchematicrepresentationofscFvgeneconstructionusingoverlappingPCR.Thevariableregionsofheavychain(VH)andlightchain(VL)wereamplifiedseparatelybythe1stPCR.TheVHandVLsegmentswereassembledbythesecondoverlapPCRtogeneratethescFvfragment.b.

AgarosegelanalysisofPCRproductofscFv.LaneM,DNAmarker(100bpladder);lane1,thescFvPCRproducts;lane2,negativecontrol(withouttemplates).III.Technologiesfor

makingabetterantibody：

MakingscFvNucleotideanddeducedaminoacidsequencesoftheconstructedHep27scFvcontainingVH,linkerpeptide,andVL.TheCDRregionsareindicatedbyboldcharacters.Thelinkerpeptideismarkedinbox.UnderliningindicatestherestrictionenzymesitesusedforcloningtheHep27scFvgene.III.Technologiesfor

makingabetterantibody：

MakingscFvProteinfractionationanalysisofscFv.A.12%SDSofthegelwasstainedwith0.25%CoomassieBrilliantBlueR-250.B.Westernblot.Afusionproteinwasprobedwithmouseanti-HisAb,followedbygoatanti-mousealkalinephosphataseconjugatedAb.ColorwasdevelopedbyusingNBT/BCIPalkalinephosphatasesubstrate.LaneM,proteinmolecularweightmarker;lane1,totalcellssample(noIPTGinduction);lane2,totalcellssample(plusIPTGinduction);lane3,culturesupernatant;lane4,insolublefraction;lane5,solublefraction.ThescFv(27kD)isdenotedbyanarrow.III.Technologiesformakingabetterantibody：

MakingscFvSDSandimmunoblottinganalysisofpurifiedscFv.

SDSstainedwithCBB.LaneM,proteinmolecularweightmarker;lane1,solubleproteinsafterrefolding;lane2,Flow-throughfraction;lane3,washedfraction;lane4,elutedfraction.B. ImmunoblottingofpurifiedHep27scFvstainedwithmouseanti-Hisantibody(IgG)andalkalinephosphatase-conjugatedanti-mouseIgG.Relativemolecularmass(kDa)ofstandardproteinsisshownontheleft.III.Technologiesformakingabetterantibody

MakingscFvTheeffectofHep27scFvonHCC-S102cellgrowthinvitro.HCC-S102cellswereincubatedwithvariousconcentrationsofHep27scFvfor72h.CellproliferationratewasassayedbytheMTSmethodandcalculatedasdescribedinthemethodssection.CellproliferationratereducedaftertreatmentwithvariousconcentrationsofscFvinadose-dependentmanner.Theratereducedto68%with20μg/mlofHep27scFvincubationbutdidnotchangeeitherinnegativecontrol(0μg