1、.,Antibody Phage Display,Meiling Xiong 20180629,.,Contents,Introduction of Ab phage Display Technology Ab Formats for Phage Display Ab Libraries Construction Phage Ab Selection Methods multiple cloning sites (MCS) Coat protein: PIII (larger protein, less than 5 copies,) PVIII (more than 5 copies, de

2、creased 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,.,Introduction of Phage Display Tech

3、nology,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

4、 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,.,Advantages of Phage Display for Recombinant Antibody Selection,More efficiently than through co

5、nventional 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 anti

6、bodies. Producing the combinatorial library (ideally with 108 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 potenti

7、al 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 i

8、s not possible by in vivo immunization of animals. A number of starting material: proteins, peptides, haptens, cell lines, tissue slides, or virus particles,.,Antibody Formats,The most commonly used format: single-chain variable fragment (scFv) Simplicity of cloning process Fast and easy library gen

9、eration 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),.,Antibody Formats,Fab The light chain (VL-CL) and the Fd-domain (VH-CH1) of the heavy chain of an antibody. During bacterial expression, t

10、hese 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.,.,

11、Antibody Formats,Single domain antibody VHH: 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,.,Antibody Formats,Multivalent fragments Miniantibodies are scFvs or F

12、abs 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

13、-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 libraries: first, immunize an animal with an antigen and isolate the mRNA from B lymphocytes ( for immunized anim

14、als) 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 ev

15、en 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,.,Nave natural libr

16、aries: 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

17、 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,.,Nav

18、e 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

19、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,.,Nave Synthetic libraries Advantages: The princ

20、iple 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 a

21、bility 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 we

22、ll 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 fragments are isolated by digestion of plamid DNA

23、 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 Construction,To ensure that all five Ab classe

24、s 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, pCANTA

25、B6 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”

26、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,.

27、,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 gener

28、ation 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 i

29、nto a mammalian cell line for expression.,.,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 ce

30、ll 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

31、 Finding new Ags with phage Ab libraries Selection 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-sp

32、ecific 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

33、 selection Site-specific antibody conjugation using methods such as genetic fusion (enzyme, or fluorescent protein).,.,Hapten-specific antibody selection,Isolation of anti-hapten specific antibody fragments from combinatorial libraries Hapten targets with molecular weight below 1000 Dalton They shou

34、ld 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

35、. The nave library should be large but immunized 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

36、 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 ar

37、e then incubated a second time with Ag and eluted and 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 se

38、lected. MAbs against a variety of Ag epitopes can be isolated 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 secon

39、dary 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

40、Ags, cell extracts, membrane preparations.,.,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 mi

41、xed with an excess of unmodified Ag-negative cells. This method is more efficient than just several rounds of negative selection on Ag-negative cells.,.,Phage Ab screening applications,Screening for affinity or kinetics of binding Screening for bioactivity/function: receptor blocking or triggering (

42、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

43、with other procedures such as selection using a mammalian host cell or other cell systems. High-throughput 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,Method