抗体噬菌体展示技术

• 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 repertoire of antibodies than those available through conventional hybridoma technology.
• 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
AntiboΒιβλιοθήκη Baiduy Formats
• Fab ✓ The light chain (VL-CL) and the Fddomain (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.
• Advantages: ✓ Absolute freedom in antigen choice, including self, nonimmunogenic, and
Antibody Libraries
✓ 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.
▪ 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)
▪ Protease cleavage site
▪ Promoter
▪ Signal peptides: phage protein
Introduction of Phage Display Technology
▪ Nonlytic filamentous phage is the
most often used for phage display, primarily the M13 and Fd strains.
Antibody Formats
vNAR
• Single domain antibody
✓ VHH: VH domain of camelid antibody, heavy chains only,
✓ IgNAR (new antigen receptor): shark antibody, heavy chains only,
✓ 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.
▪ 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
Antibody Libraries
• Naïve natural libraries: universal
antibody libraries generated from B-cells of nonimmunized donors and eliminate the need to construct new libraries for each antigen.
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 strands
▪ 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
Antibody Phage Display
Meiling Xiong 20180629
Contents
• Introduction of Ab phage Display Technology
• Ab Formats for Phage Display
• Ab Libraries Construction
Antibody Libraries
• 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.
• 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 of Phage• DMiosrepelfaficyienftolyrthRanethcrooumgh binant
Antibody Selection •
conventional hybridoma system. Cheaper to produce recombinant
• Phage Ab Selection Methods & Strategies
• Phage Ab Screening Applications
• In vitro Affinity Maturation
• Expression & Purification
Introduction of Phage Display 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.
▪ 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)
• Capable of generating antibodies against
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)
antibodies using bacteria, rather than
mammalian cell line.
• Easier to maintain and grow bacterial cultures for recombinant antibody production.
• Bypass immunization in antibody selection.
✓ Unique CDRs • Affibodies • Anticalins • DARPins • Avimers • Affimers • Monobodies
Antibody Formats
• Multivalent fragments
✓ Miniantibodies are scFvs or Fabs connected via a flexible linker to selfassociating structures such as helix bundles or leucine zippers.