抗体噬菌体展示技术课件.ppt

1、Antibody Phage DisplayMeiling Xiong20180629Contents Introduction of Ab phage Display Technology Ab Formats for Phage Display Ab Libraries Construction Phage Ab Selection Methods&Strategies Phage Ab Screening Applications In vitro Affinity Maturation Expression&Purification of Phage Ab FragmentsIntro

2、duction of Phage Display TechnologyThe Ff bacteriophage structure Introduction of Phage Display TechnologyThe scheme of phagemid vectorIG region:intergenic region,usually contains the packing sequence and replication origin of minus and plus strandsMolecular tag:to facilitate library screening and f

3、or protein analysisRestriction enzyme recognition sites:useful for DNA recombination and gene manipulation;multiple cloning sites(MCS)Coat protein:PIII(larger protein,less than 5 copies,)PVIII(more than 5 copies,decreased length)Amber codon TAG:supE strains(glutamic acid codon),non-suppressor strain

4、s(stop codon)Protease cleavage sitePromoterSignal peptides:phage protein translocation,crucial for display levelSelective marker:for selection of infected host cellsIntroduction of Phage Display TechnologyNonlytic filamentous phage is the most often used for phage display,primarily the M13 and Fd st

5、rains.Proteins to be selected are infused to all five coat proteins,with pIII and pVIII most commonly used.pIII protein is essential for infection of bacteriaHelper phage:wild-type pIII helper phage and special helper phageAntigen immobilized on magnetic beads,polystryrene surfaces,or on columns,or

6、is used in solution as biotinylated antigen and later captured by immobilized streptavidin Advantages of Phage Display for Recombinant Antibody SelectionMore efficiently than through conventional hybridoma system.Cheaper to produce recombinant antibodies using bacteria,rather than mammalian cell lin

7、e.Easier to maintain and grow bacterial cultures for recombinant antibody production.Bypass immunization in antibody selection.Bypass the use of animal cells for production of antibodies.Producing the combinatorial library(ideally with 108 to 109 members)of functional antibodies to generate a larger

8、 repertoire of antibodies than those available through conventional hybridoma technology.Easy isolation and expression of the cloned gene in a bacterial host.Excellent potential to further improve binding properties of the selected antibody by protein engineering techniques.Capable of generating ant

9、ibodies against almost any desired antigen,including highly conserved or self-antigens,conformational variants,low immunogenic antigens,and also toxic components,which is not possible by in vivo immunization of animals.A number of starting material:proteins,peptides,haptens,cell lines,tissue slides,

10、or virus particlesAntibody FormatsThe most commonly used format:single-chain variable fragment(scFv)Simplicity of cloning process Fast and easy library generation A high display rate(small protein size 25 kDa)Less stable than Fab fragments Tend to form dimers(can be reduced with linker more than 20

11、amino acids)Antibody Formats Fab The light chain(VL-CL)and the Fd-domain(VH-CH1)of the heavy chain of an antibody.During bacterial expression,these two chains are synthesized separately,and secreted into the periplasm where they fold to form heterodimers.Fab exhibit higher stability than scFvs Posse

12、ss better PK and PD qualities than scFvs Easier to convert into full-length antibodies Clinical applications:abciximab,lucentis,cimzia.Antibody Formats Single domain antibody VHH:VH domain of camelid antibody,heavy chains only,IgNAR(new antigen receptor):shark antibody,heavy chains only,Unique CDRsA

13、ffibodiesAnticalinsDARPinsAvimersAffimersMonobodiesvNARAntibody Formats Multivalent fragmentsMiniantibodies are scFvs or Fabs connected via a flexible linker to self-associating structures such as helix bundles or leucine zippers.Diabodies are noncovalent dimers of scFvs,which spontaneously form dep

14、ending on the linker length between VH and VL.Another form of diabodies is two scFvs connected with a short linker.Fab-A is created by genetic fusion of the Fab Fd gene with the alkaline phosphatase(PhoA)gene and coexpressing the light chain gene.scFv-Fc are scFvs dimerized by the Fc domain.Immune l

15、ibraries:first,immunize an animal with an antigen and isolate the mRNA from B lymphocytes(for immunized animals)or peripheral blood B cells(for immunized donors).The mRNA is then reverse transcribed into cDNA,and the variable regions of expressed antibodies are amplified via PCR and cloned into a ph

16、age display vector.Advantages:Matured in vivoImmune libraries can be generated from any animal and even humans:mouse,human,chicken,rabbit,camelAny species that have been immunized,infected,or exposed to an antigen.Useful in analyzing natural humoral responses,for example,in patients with autoimmune

17、disease,viral infection,neoplastic diseases,etc.Antibody LibrariesNave natural libraries:universal antibody libraries generated from B-cells of nonimmunized donors and eliminate the need to construct new libraries for each antigen.lower affinities than those generated during in vivo affinity maturat

18、ion.to find good antibodies against diverse antigens,these libraries need to be very large.Advantages:Absolute freedom in antigen choice,including self,nonimmunogenic,and toxic Ags Several antibodies selected by phage display from human nave libraries have already been approved as drugs,such as raxi

19、bacumab,ramucirumab,necitumumab,or belimumab.Antibody LibrariesNave Semisynthetic libraries:Nave semisynthetic libraries are usually libraries that have been isolated from nonimmune hosts and where one or several CDRs were exchanged with synthetic peptides or were randomly mutated.This approach is a

20、 way to achieve high diversity without requiring a large number of donors and can generate specificities not normally included in natural repertoires.Advantages:Low immunogenicity in hosts since only a few of the CDRs are artificial These libraries can cover the entire repertoire of germ linesAntibo

21、dy LibrariesNave Synthetic librariesAdvantages:The principle advantage of nave synthetic libraries over semisynthetic libraries is that the biophysical parameters and codon usage of the framework region can be optimized for expressibility and stability.Advanced DNA synthesis methods such as TRIM,slo

22、nomics,or chip-based DNA photolithography offer the ability to precisely define the frequency of each amino acid at each position with optimized codons.CDRs can be of higher diversity,different in composition than biologically occurring CDRs,hence offering a potentially larger paratope space.Have be

23、en used to generate therapeutic antibodies,as well as antibodies for research and diagnostic applications.Antibody LibrariesStandard Fab Library ConstructionConstruction of Large Nave Fab LibraryAn efficient cloning method,in which restriction fragments instead of PCR products were used.VH fragments

24、 are isolated by digestion of plamid DNA purified from the primary repertoires,and cloned into the acceptor phagemid vector containing the light-chain(LC)repertoires.This innovation increases the size of the libraries dramatically.IgM-derived antibody repertoire were used.scFv Library ConstructionTo

25、 ensure that all five Ab classes are likely to be represented and increase the overall size of the final library,random hexamers are employed in the primary first-strand cDNA synthesis from PBL mRNA.Component VH and VL gene segments are amplified in separate PCR reactions,and initially cloned into t

26、wo different vectors,pCANTAB6 and pCANTAB3his6(see Fig.1).The latter is used for cloning the VL repertoire because it has the appropriate polylinker cloning sites for the digested VL fragments;the VH repertoire is cloned into pCANTAB6.A short linker from an existing scFv is cloned(together with an i

27、rrelevant or“dummy”VH)into the VL repertoire,upstream of the VL fragments.The VH and linker-VL repertoires are then amplified from their vectors,and the scFv construct is prepared using a simple two-fragment PCR assembly procedure.This construct is then cloned into pCANTAB6 to create the large nave

28、scFv libraryPolyclonal antibody library constructionPolyclonal antibody libraries(PCALs)are standardized mixtures of antibodies specific for an antigen or multi-Ag targets.They target multiple epitopes on poly-Ags,resulting in high-avidity binding and efficient triggering of effector functions.PCAL

29、generation usually involves the recovery of VL and VH repertoires,and their random pairing as Fabs into a phage-display vector.The library is positively and negatively selected.Selected VLVH gene pairs are then transferred in mass to a mammalian expression vector.The constructs are then transfected

30、into a mammalian cell line for expression.Phage Ab Selection Procedures and applicationsDiversity in Selection methods Immobilized Ag:solid supports,columns,BIAcore sensor chips Biotinylated Ag in solution to avoid conformational changes Prokaryotic or mammalian cells,fluorescenceactivated cell sort

31、ing,tissue sections,in vivo selection,etc.Elution Acid solutions(HCl).Glycine buffers;Basic solutions,triethylaming;Chaotropic agents;Dithiothreitol;Enzymatic cleavage;Competition methodsSelection of Abs for affinity or binding kineticsSelection on complex AgsSelection on cellsFinding new Ags with p

32、hage Ab librariesSelection for Ab stability and foldingIn vitro selection of antibodies for specific applicationsTissue panning for immunohistochemistry antibodies:antibody selection with formalin-fixed paraffin embedded(FFPE)tissue.Sandwich pair selection,complex-specific antibodies,and drug monito

33、ring:Drug monitoring:various forms(free antibody drug,antibody-target complex,or both)of antibody therapeutics can be easily tracked and quantified in PK assays,using anti-idiotype antibodies Complex-specific antibodies:guided selection method Sandwich pair selectionSite-specific antibody conjugatio

34、n using methods such as genetic fusion(enzyme,or fluorescent protein).Hapten-specific antibody selectionIsolation of anti-hapten specific antibody fragments from combinatorial libraries Hapten targets with molecular weight below 1000 Dalton They should be conjugated to a suitable immunogenic carrier

35、 protein for presentation To avoid the selection of antibodies specific for the carrier protein or the linker,we can use a method that utilizes two different hapten conjugates for alternative rounds of selection.The library can be immunized or nave.The nave library should be large but immunized libr

36、ary should be construct separately.Competitive DeselectionAntigens from a particular pathogen can be of variable immunogenicity,with the antigen that stimulates the strongest response being the immunodominant one.To obtain antibodies against the epitope of interest,a preadsorption panning is used.Th

37、is facilitates the molecular cloning of Mab fragments against non-immunodominant Ag determinants.The phage library is first preabsorbed on the Ag of interest to remove phage that react with the immunodominant epitope.The unbound phage are then incubated a second time with Ag and eluted and amplified

38、 according to normal protocols.Epitope-masking StrategyCapture-lift Screening procedureCapture-sandwich ELISAStrongly effective to select Abs against Ags from crude preparations.Abs against conformation-sensitive Ags can be selected.MAbs against a variety of Ag epitopes can be isolated from a single

39、 library.Both pAb and mAb can be used as capture Abs.Proximity-Guided SelectionIt involves the use of catalyzed reporter enzyme deposition(CARD),which is a method of signal amplification.CARD uses HRP-conjugated secondary antibody,biotin tyramine to biotinylate phage particles that bind around the s

40、ite of the HRP activity.These phage can be recovered on streptavidin-coated magnetic beads.This selection strategy can be sued to isolate phage Ab against cell surface markers,and other antigens,such as purified Ags,cell extracts,membrane preparations.Magnetic sorting for selection of antibodies to

41、cell-surface antigensFor selection of antibodies targeting cell-surface antigensA competitive cell-panning approach is used,in which target cells(positive cells)are precoated with magnetic beads,and mixed with an excess of unmodified Ag-negative cells.This method is more efficient than just several

42、rounds of negative selection on Ag-negative cells.Phage Ab screening applicationsScreening for affinity or kinetics of bindingScreening for bioactivity/function:receptor blocking or triggering(dimerization),virus or cytokine neutralizationSelection for a particular function:Ab with agonist or antago

43、nist activity for a given receptor,for drug discovery;Ab that dimerizes receptors;Ab internalization for gene transfer;Ab selection for cell survival or killing;Combining phage display with other procedures such as selection using a mammalian host cell or other cell systems.High-throughput selection

44、 and screeningScreening for affinity or kinetics of bindingDepending on the intended application,the binding of a molecule to its target is desired to be long-lived or short-lived.BIAcore technologyIn vitro affinity maturationMethods to generate mutations:Error-prone PCRDegenerate oligonucleotidesMu

45、tagenic strains of bacteria:mutD5-FITChain/CDR shufflingSite-directed mutagenesis,at restricted positions in the CDR regionRandom mutagenesis,mutations are introduced into the entire V regionTargeting random mutations to hotspots in antibody variable domains for affinity improvementExpression and pu

46、rification of Abs in different cell linesExpression and purification of scFvs and recombinant Fab in E.coliCytoplasmic inclusion bodies or expressed as correctly folded AbSeveral ways to enhance the proportion of correctly folded Abs and reduce aggregationPurification:His-tagged protein,IMAC,affinit

47、y chromatographyExpression of Antibody fragments in Pichia pastorisExpression of VHH Antibody Fragments in Saccharomyces cerevisiaeIntrabodiesExpression of scFvs and scFv Fusion Proteins in Eukaryotic CellsExpression of Antibody Fab Fragments and Whole Immunoglobulin in Mammalian CellsGuided Selecti

48、on StrategiesBlocking strategy to prevent selections of corss-reactive antibodies.In this strategy,closely related antigens,which should not be detected by the antibody,are added in excess to the antibody phage solution.Selection of complex-specific antibodies,an isotype-matched antibody is used for blocking capture antibody-specific phages.It is used to select antibodies against therapeutic antibody-target complexes.