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1、Antibody Phage Display,Meiling Xiong20180629,Contents,Introduction of Ab phage Display TechnologyAb Formats for Phage DisplayAb Libraries ConstructionPhage Ab Selection Methods&StrategiesPhage Ab Screening ApplicationsIn vitro Affinity MaturationExpression&Purification of Phage Ab Fragments,Introduc
2、tion of Phage Display Technology,The Ff bacteriophage structure,Introduction of Phage Display Technology,The scheme of phagemid vector,IG 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 cells,Introduction of Phage Display Technology,Nonlytic filamentous phage is the most often used for phage display,primarily the M13 and Fd
5、strains.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,o
6、r is used in solution as biotinylated antigen and later captured by immobilized streptavidin,Advantages of Phage Display for Recombinant Antibody Selection,More efficiently than through conventional hybridoma system.Cheaper to produce recombinant antibodies using bacteria,rather than mammalian cell
7、line.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 lar
8、ger 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
9、antibodies 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 slid
10、es,or virus particles,Antibody Formats,The most commonly used format:single-chain variable fragment(scFv)Simplicity of cloning processFast and easy library generationA high display rate(small protein size 25 kDa)Less stable than Fab fragmentsTend to form dimers(can be reduced with linker more than 2
11、0 amino acids),Antibody Formats,FabThe 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 scFvsPoss
12、ess better PK and PD qualities than scFvsEasier to convert into full-length antibodiesClinical applications:abciximab,lucentis,cimzia.,Antibody Formats,Single domain antibodyVHH:VH domain of camelid antibody,heavy chains only,IgNAR(new antigen receptor):shark antibody,heavy chains only,Unique CDRsAf
13、fibodiesAnticalinsDARPinsAvimersAffimersMonobodies,Antibody 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 dependi
14、ng 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 libr
15、aries: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 phage
16、 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 dis
17、ease,viral infection,neoplastic diseases,etc.,Antibody Libraries,Nave 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 maturati
18、on.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 raxib
19、acumab,ramucirumab,necitumumab,or belimumab.,Antibody Libraries,Nave 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
20、a 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 artificialThese libraries can cover the entire repertoire of germ lines,Antib
21、ody Libraries,Nave 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,s
22、lonomics,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
23、been used to generate therapeutic antibodies,as well as antibodies for research and diagnostic applications.,Antibody Libraries,Standard Fab Library Construction,Construction of Large Nave Fab Library,An efficient cloning method,in which restriction fragments instead of PCR products were used.VH fra
24、gments 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 Constru
25、ction,To 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 clone
26、d into two 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 w
27、ith an irrelevant 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 lar
28、ge nave scFv library,Polyclonal antibody library construction,Polyclonal 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 funct
29、ions.PCAL 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 tr
30、ansfected into a mammalian cell line for expression.,Phage Ab Selection Procedures and applications,Diversity in Selection methodsImmobilized Ag:solid supports,columns,BIAcore sensor chipsBiotinylated Ag in solution to avoid conformational changesProkaryotic or mammalian cells,fluorescenceactivated
31、cell sorting,tissue sections,in vivo selection,etc.ElutionAcid 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 Ag
32、s with phage Ab librariesSelection for Ab stability and folding,In vitro selection of antibodies for specific applications,Tissue panning for immunohistochemistry antibodies:antibody selection with formalin-fixed paraffin embedded(FFPE)tissue.Sandwich pair selection,complex-specific antibodies,and d
33、rug monitoring: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 antibodiesComplex-specific antibodies:guided selection methodSandwich pair selectionSite-specific antibody co
34、njugation using methods such as genetic fusion(enzyme,or fluorescent protein).,Hapten-specific antibody selection,Isolation of anti-hapten specific antibody fragments from combinatorial librariesHapten targets with molecular weight below 1000 DaltonThey should be conjugated to a suitable immunogenic
35、 carrier protein for presentationTo 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 immuniz
36、ed library should be construct separately.,Competitive Deselection,Antigens 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 i
37、s used.This 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
38、amplified according to normal protocols.,Epitope-masking Strategy,Capture-lift Screening procedure,Capture-sandwich ELISA,Strongly 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
39、from a single library.Both pAb and mAb can be used as capture Abs.,Proximity-Guided Selection,It 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 bi
40、nd around the site 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 o
41、f antibodies to cell-surface antigens,For 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 t
42、han just several rounds of negative selection on Ag-negative cells.,Phage Ab screening applications,Screening for affinity or kinetics of bindingScreening for bioactivity/function:receptor blocking or triggering(dimerization),virus or cytokine neutralizationSelection for a particular function:Ab wit
43、h agonist or antagonist 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-t
44、hroughput selection and screening,Screening for affinity or kinetics of binding,Depending on the intended application,the binding of a molecule to its target is desired to be long-lived or short-lived.BIAcore technology,In vitro affinity maturation,Methods to generate mutations:Error-prone PCRDegene
45、rate oligonucleotidesMutagenic 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 impro
46、vement,Expression and purification of Abs in different cell lines,Expression 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-ta
47、gged protein,IMAC,affinity 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 Mamm
48、alian Cells,Thank you!,Guided Selection Strategies,Blocking 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.,
