抗體噬菌體展示技術(shù)ppt課件

1、Antibody Phage DisplayMeiling Xiong201806291；.ContentsContentsIntroduction of Ab phage Display TechnologyAb Formats for Phage DisplayAb Libraries ConstructionPhage Ab Selection Methods & StrategiesPhage Ab Screening ApplicationsIn vitro Affinity MaturationExpression & Purification of Phage A

2、b Fragments2；.Introduction of Phage Display TechnologyThe Ff bacteriophage structure 3；.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 facilitat

3、e library screening and for 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 (glutam

4、ic acid codon), non-suppressor strains (stop codon)Protease cleavage sitePromoterSignal peptides: phage protein translocation, crucial for display levelSelective marker: for selection of infected host cells4；.Introduction of Phage Display TechnologyNonlytic filamentous phage is the most often used f

5、or 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 bacteriaHelper phage: wild-type pIII helper phage and special helper phageAntigen immobilized on magneti

6、c beads, polystryrene surfaces, or on columns, or is used in solution as biotinylated antigen and later captured by immobilized streptavidin 5；.Advantages of Phage Display for Recombinant Antibody SelectionMore efficiently than through conventional hybridoma system. Cheaper to produce recombinant an

7、tibodies 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 108

8、to 109 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 antibod

9、y 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 st

10、arting material: proteins, peptides, haptens, cell lines, tissue slides, or virus particles6；.Antibody FormatsThe 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 th

11、an Fab fragmentsTend to form dimers (can be reduced with linker more than 20 amino acids)7；.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 peripla

12、sm where they fold to form heterodimers.Fab exhibit higher stability than scFvsPossess better PK and PD qualities than scFvsEasier to convert into full-length antibodiesClinical applications: abciximab, lucentis, cimzia. 8；.Antibody Formats Single domain antibodyVHH: VH domain of camelid antibody, h

13、eavy chains only,IgNAR (new antigen receptor): shark antibody, heavy chains only, Unique CDRsAffibodiesAnticalinsDARPinsAvimersAffimersMonobodiesvNAR9；.Antibody Formats Multivalent fragmentsMiniantibodies are scFvs or Fabs connected via a flexible linker to self-associating structures such as helix

14、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 (

15、PhoA) gene and coexpressing the light chain gene. scFv-Fc are scFvs dimerized by the Fc domain. 10；.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 revers

16、e transcribed into cDNA, and the variable regions of expressed antibodies are amplified via PCR and cloned into a phage 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

17、, 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 Libraries11；.Nave natural libraries: universal antibody libraries generated from B-cells of nonimmunized donors and

18、 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, nonimmunog

19、enic, 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 Libraries12；.Nave Semisynthetic libraries: Nave semisynthetic libraries are usually libraries that have

20、 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.Ad

21、vantages:Low immunogenicity in hosts since only a few of the CDRs are artificialThese libraries can cover the entire repertoire of germ linesAntibody Libraries13；.Nave Synthetic librariesAdvantages:The principle advantage of nave synthetic libraries over semisynthetic libraries is that the biophysic

22、al 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 c

23、odons.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 Libraries14；.Standard Fab Li

24、brary ConstructionConstruction of Large Nave Fab LibraryAn 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 th

25、e light-chain (LC) repertoires.This innovation increases the size of the libraries dramatically.IgM-derived antibody repertoire were used.15；.scFv Library ConstructionTo ensure that all five Ab classes are likely to be represented and increase the overall size of the final library, random hexamers a

26、re 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 th

27、e 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 a

28、mplified 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 library16；.Polyclonal antibody library constructionPolyclonal antibody libraries (PCALs) are standardized mixt

29、ures 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 p

30、hage-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.17；.Phage Ab Selection Procedures and applicationsDiversity in

31、Selection methodsImmobilized Ag: solid supports, columns, BIAcore sensor chipsBiotinylated Ag in solution to avoid conformational changesProkaryotic or mammalian cells, fluorescenceactivated cell sorting, tissue sections, in vivo selection, etc.ElutionAcid solutions (HCl). Glycine buffers; Basic sol

32、utions, triethylaming; Chaotropic agents; Dithiothreitol; Enzymatic cleavage; Competition methodsSelection of Abs for affinity or binding kineticsSelection on complex AgsSelection on cellsFinding new Ags with phage Ab librariesSelection for Ab stability and folding18；.In vitro selection of antibodie

33、s for specific applicationsTissue 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,

34、 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 conjugation using methods such as genetic fusion (enzyme, or fluorescent protein).19；

35、.Hapten-specific antibody selectionIsolation of anti-hapten specific antibody fragments from combinatorial librariesHapten targets with molecular weight below 1000 DaltonThey should be conjugated to a suitable immunogenic carrier protein for presentationTo avoid the selection of antibodies specific

36、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.20；.Competitive DeselectionAntigens

37、 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 n

38、on-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.21；.Epitope-masking Strategy22；.

39、Capture-lift Screening procedure23；.Capture-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 library.Both pAb and mAb can be used as capture Ab

40、s.24；.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 site of the HRP activity.These phage can be r

41、ecovered 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.25；.Magnetic sorting for selection of antibodies to cell-surface antigensFor selection o

42、f 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 rounds of negative selection on

43、Ag-negative cells.26；.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 antagonist activity for a give

44、n 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 screening27；.Screening 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 sh