This document summarizes antibody engineering techniques. It discusses DNA sequencing of antibody genes to determine sequences, modeling antibody combining sites, and changing antibody structures through altering CDR orientations or residues. The limitations of hybridoma technology include inability to alter antibody properties and issues with murine antibodies in humans. Recent technologies involve recombinant antibody methods using DNA manipulation to make new heavy and light chain combinations or mutate CDRs, allowing control over antibody properties.

1. Chapter-5:
Antibody engineering (Part-
2)
Presented By
Md. Abu Taher
Department Of Microbiology
Noakhali Science And technology
University, Bangladesh

2. 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

3. Antibody Engineering
 What?
 Why?
 Supplementary !
Why supplementary?

4. 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 ?

5. Sequencing Process through Sanger method

6. DNA Sequencing-
Photographic View
Comparison between template (original
strand/sequence of AB) and newly
synthesized strand

7. 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?

8. Changing the Structure
 How?
 Increasing copy number?
 Which Ab is selected or produced?
 How to assesse the mutation?

9. 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.

10. Hybridoma Technology
Hybridoma 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 mouse
2) Isolation of B cells from the spleen
3) Cultivation of myeloma cells
4) Fusion of myeloma and B cells
5) Separation of cell lines
6) Screening of suitable cell lines
7) in vitro (a) or in vivo (b) multiplication
8) Harvesting

11. 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

12. Recent Technologies
The recent technologies are based on-
1) Desired conformation of Ab and their functions
2) DNA manipulation and mutagenesis
3) Criteria of bacteriophage replication
The techniques are –
1) Recombinant antibody method
2) Antibody engineering

13. Recombinant Antibody Method
Recombinant 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 gene
2) Amplification & cloning
3) Expression
4) Functional Abs
5) Antibody selection

14. 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

15. 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.

16. Thanks to All