1. ANTIBODY ENGINEERING
Joud Shafiq

2. ANTIBODY ENGINEERING
ANTIBODIES
METHODOLOGIES
APPLICATIONS

3. IMMUNOGLOBULIN
NH2
Light chain
220 a.a
Heavy chain
~500 a.a

4. Variable
Regions
Antigen Binding Site
Constant Regions
Effector
Functions

5. LIGHT CHAIN GENES
Variable domain
CDR3

6. HEAVY CHAIN GENES
HUMAN
H.C Variable region= V+D+J gene segments

7. ANTIBODY ENGINEERING

8. MONOCLONAL ANTIBODIES
Y Y
Mouse splenocytes
Mouse myeloma
Y Y
hybridoma
Y Y Y
Monoclonal
Antibodies

9. PROBLEMS HAMA Response. Loss of effector functions.
Reduce the size of antibody.

10. SOLUTIONS Production of human Mab. Make mouse Ab more “human-like”
Production of small Ab fragments.

11. CH1
CH1 c
HINGE c
CH2
CH2
CH3
CH3

12. Fab Fab PROTEASE DIGESTION F(ab`)2 c c c c CH1 CH1 HINGE CH1 CH1 CH3

13. .HAMA Response. Loss of effector functions.
Making the antibody more “human-like”. (the cutting and pasting)
Chimeric Ab’s
Humanized Ab’s
Hybridoma
Production of human Mab’s.
Transgenic/SCID Mice
Phage Display

14. VARIABILITY PLOT CHIMERIC HUMAN/MOUSE
CONSTANT
FR1
FR2
FR3
FR4
CDR1
CDR2
CDR3
CONSTANT
Human
Chimeric
CONSTANT
FR1
FR2
FR3
FR4
CDR1
CDR2
CDR3

15. Human
Mouse
Chimeric

16. HUMANIZED ANTIBODIES Mouse CONSTANT FR1 FR2 FR3 FR4 CDR1 CDR2 CDR3

17. Human
Mouse
Humanized

18. CHIMERIC/HUMANIZED .Reduce the HAMA response.
.Maintain effector functions.
.Increase the half life of the antibody.
.Maintain binding affinity.
.Easy to construct.

20. ANTIBODY ENGINEERING

21. .HAMA Response. Loss of effector functions.
Making the antibody more “human-like”. (the cutting and pasting)
Chimeric Ab’s
Humanized Ab’s
Hybridoma
Production of human Mab’s.
Transgenic/SCID Mice
Phage Display

22. MONOCLONAL ANTIBODIES
Y Y
Mouse splenocytes
Mouse myeloma
Y Y
hybridoma
Y Y Y
Monoclonal
Antibodies

23. HUMAN HYBRIDOMAS . Unstable hybridomas.
.Small number of human B-cells in peripheral blood.
. Obtaining antigen-reactive human B-cells.
Heteromyeloma
fusion
In vitro immunization

24. MICE SCID Transgenic Reconstituted with human peripheral blood
Containing human immunoglobulin genes.

25. PHAGE DISPLAY

26. Reduce the size of Antibody.
Production of antibody fragments.
Simplifying the expression/production of antibodies.

27. ANTIBODY FRAGMENTS

28. EXPRESSION SYSTEMS
Bacterial
Fungal
Mammalian cell lines