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