Download - Antibody engineering
Chapter-5: Antibody engineering (Part-2)
Presented By
Md. Abu Taher
Department Of Microbiology
Noakhali Science And technology University, Bangladesh
Sub Topic Pertaining to Antibody Engineering
DNA Sequencing Modeling the Combining Site Changing the Structure How to alter affinity and specificity or
both? Limitations of Hybridoma technology Recent Technologies Recombinant antibody method
Antibody Engineering
What? Why? Supplementary !
Why supplementary?
DNA Sequencing
???? When? After the desired antibody genes have been
cloned and the DNA sequence is determined using chain termination sequencing methods (Sanger method).– Chain termination sequencing methods/Sanger
method??– Requirements?– Process ?
Sequencing Process through Sanger method
DNA Sequencing-
Photographic ViewComparison between template (original strand/sequence of AB) and newly synthesized strand
Modeling the Combining Site Structure of a developed
Antibody! How? Store! Where?
Who builds Antibody model?
Uses? Justification? Construction a
computational model of a new antibody , purpose?
Changing the Structure
How? Increasing copy number? Which Ab is selected or produced? How to assesse the mutation?
How to alter affinity and specificity or both?? Changing the relative orientations of VH and VL domains at their
interface, Lengthening or shortening particular CDRs to enlarge or shrink
the binding pocket respectively, Increasing the flexibility of CDRs in the combining site, Removing or re-spacing some of the side chains that form the
combining site, Altering residues that do not contact antigen but help to form the
combining site through CDR-CDR and CDR-framework interactions
Purposes: The antibody-antigen interactions can be changed. The antibody can also be fused with other antibody molecules,
toxins, or enzymes.
Hybridoma TechnologyHybridoma technology is a technology of forming hybrid cell lines (called hybridomas ) by fusing an antibody - producing B cell with a myeloma (B cell cancer) cell that is selected for its ability to grow in tissue culture and for an absence of antibody chain synthesis. The antibodies produced by the hybridoma are all of a single specificity and are therefore monoclonal antibodies (in contrast to polyclonal antibodies ).
1) Immunization of a mouse2) Isolation of B cells from the spleen3) Cultivation of myeloma cells4) Fusion of myeloma and B cells5) Separation of cell lines6) Screening of suitable cell lines7) in vitro (a) or in vivo (b) multiplication8) Harvesting
Limitations of Hybridoma
1) More importantly, there is no practical way to alter the properties of Abs produced by hybridomas.
1) Requires specialized cell culture facilities(HAT medium)2) The main problem is the generation of immune response
against murine mAbs in human and the Abs are rapidly cleared from the body.
3) Time consuming and expensive
Recent Technologies
The recent technologies are based on-
1) Desired conformation of Ab and their functions2) DNA manipulation and mutagenesis 3) Criteria of bacteriophage replication
The techniques are –
1) Recombinant antibody method
2) Antibody engineering
Recombinant Antibody MethodRecombinant Antibody can be done
by making new combinations of H and L chains by mutating individual CDRs
By using this technology it is possible to generate new Abs with combination of heavy & light chains as well as mutating the individuals CDRs (complementarity-determining regions)
Key steps of recombinant antibody method----
1) Antibody gene2) Amplification & cloning3) Expression4) Functional Abs5) Antibody selection
Advantages of rAbs
No animals used if antibodies come from synthetic human antibody libraries
Less purified antigen is required compared to Hybridoma technology Production process gives complete control over the state of the
antigen rAbs to toxic, fragile, or highly conserved antigens can be generated Production time is weeks instead of months Nucleic acid sequence of rAb is easily accessible for further
manipulation rAb fragments can be produced cheaply in bacterial and yeast
expression systems
Disadvantages of rAbs
Technically challenging Improved methods of generating antibody libraries are
protected intellectual property. High-throughput equipment to automate selection
procedures can be expensive Most libraries available to researchers are made of
antibody fragments. An extra step is required to convert these fragments to full length antibodies if full length antibodies are required.
Thanks to All