抗体噬菌体展示技术.pptx

单击此处编辑母版标题样式,Edit Master text styles,Second level,Third level,Fourth level,Fifth level,6,7,8,9,2019/2/15,#,Antibody Phage Display,Meiling Xiong,20230629,第1页,Contents,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 Fragments,第2页,Introduction of Phage Display Technology,The Ff bacteriophage structure,第3页,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 strands,Molecular tag,:to facilitate library screening and for protein analysis,Restriction 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 strains(stop codon),Protease cleavage site,Promoter,Signal peptides,:phage protein translocation,crucial for display,level,Selective marker,:for selection of infected host cells,第4页,Introduction of Phage Display Technology,Nonlytic filamentous phage is the most often used for phage display,primarily the M13 and Fd 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 bacteria,Helper phage:wild-type pIII helper phage and special helper phage,Antigen immobilized on magnetic beads,polystryrene surfaces,or on columns,or is used in solution as biotinylated antigen and later captured by immobilized streptavidin,第5页,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 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 10,8,to 10,9,members)of functional,antibodies,to generate a larger 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 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,cel,l,lines,tissue slides,or virus particles,第6页,Antibody,Formats,The 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 amino acids),第7页,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,Possess better PK and PD qualities than scFvs,Easier to convert into full-length antibodies,Clinical applications:abciximab,lucentis,cimzia.,第8页,Antibody,Formats,Single domain antibody,V,HH:VH domain of camelid antibody,heavy chains only,IgNAR(new antigen receptor):shark antibody,heavy chains only,Unique CDRs,Affibodies,Anticalins,DARPins,Avimers,Affimers,Monobodies,vNAR,第9页,Antibody,Formats,Multivalent fragments,Miniantibodies,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 depending 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,.,第10页,Immune libraries,: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 display vector.,Advantages:,Matured in vivo,Immune libraries can be generated from any animal and even humans:mouse,human,chicken,rabbit,camel,Any species that have been immunized,infected,or exposed to an antigen.,Useful in analyzing natural humoral responses,for example,in patients with autoimmune disease,viral infection,neoplastic diseases,etc.,Antibody,Libraries,第11页,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 maturation.,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 raxibacumab,ramucirumab,necitumumab,or belimumab.,Antibody,Libraries,第12页,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 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 artificial,These libraries can cover the entire repertoire of germ lines,Antibody,Libraries,第13页,Nave Synthetic libraries,Advantages:,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,slonomics,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 been used to generate therapeutic antibodies,as well as antibodies for research and diagnostic applications.,Antibody,Libraries,第14页,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 fragments 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.,第15页,scFv,Library,Construction,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 cloned 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 with 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,large nave scFv library第16页,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 functions.,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 transfected into a,mammalian cell line for expression,.,第17页,Phage Ab Selection Procedures and applications,Diversity 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 sorting,tissue sections,in vivo selection,etc.,Elution,Acid solutions(HCl).Glycine,buffers;Basic,solutions,triethylaming;Chaotropic agents;Dithiothreitol;Enzymatic cleavage;Competition methods,Selection of Abs for affinity or binding kinetics,Selection on complex Ags,Selection on cells,Finding new Ags with phage Ab libraries,Selection for Ab stability and folding,第18页,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 drug 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 antibodies,Complex-specific antibodies,:guided selection method,Sandwich pair selection,Site-specific antibody,conjugation,using methods such as genetic fusion(enzyme,or fluorescent protein).,第19页,Hapten-specific antibody selection,Isolation 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 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 library should be construct separately,.,第20页,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,is 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 amplified according to normal protocols.,第21页,Epitope-masking Strategy,第22页,Capture-lift Screening procedure,第23页,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 from a single,library.,Both pAb and mAb can be used as capture Abs.,第24页,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 bind 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 Agscell,extracts,membrane preparations.,第25页,Magnetic sorting for selection of antibodies to cell-surface antigens,For selection of antibodies targeting cell-surface antigens,A 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 rounds of negative selection on Ag-negative cells.,第26页,Phage Ab screening applications,Screening for affinity or kinetics of binding,Screening for bioactivity/function,:receptor blocking or triggering(dimerization),virus or cytokine neutralization,Selection for a particular function,:Ab with 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-throughput selection and screening,第27页,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,第28页,In,vitro affinity,maturation,Methods to generate mutations:,Error-prone PCR,Degenerate oligonucleotides,Mutagenic strains of bacteria,:mutD5-FIT,Chain/CDR shuffling,Site-directed mutagenesis,at restricted positions in the CDR region,Random mutagenesis,mutations are introduced into the entire V region,Targeting random mutations to,hotspots,in antibody variable domains for affinity,improvement,第29页,Expression and purification of Abs in different cell lines,Expression and purification of scFvs and recombinant Fab in E.coli,Cytoplasmic inclusion bodies or expressed as correctly folded Ab,Several ways to enhance the proportion of correctly folded Abs and reduce,aggregation,Purification:,His-tagged,protein,IMAC,affinity chromatography,Expression of Antibody fragments in,Pichia pastoris,Expression of VHH Antibody,Fragments in,Saccharomyces,cerevisiae,Intrabodies,Expression of scFvs and scFv Fusion,Proteins in,Eukaryotic,Cells,Expression of Antibody Fab,Fragments and,Whole Immunoglobulin in Mammalian Cells,第30页,Thank you!,第31页,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.,第32页,