ANTIBODY ENGINEERING Joud Shafiq

ANTIBODY ENGINEERING ANTIBODIES METHODOLOGIES APPLICATIONS

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

Variable Regions Antigen Binding Site Constant Regions Effector Functions

LIGHT CHAIN GENES Variable domain CDR3

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

ANTIBODY ENGINEERING

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

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

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

CH1 CH1 c HINGE c CH2 CH2 CH3 CH3

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

.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

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

Human Mouse Chimeric

HUMANIZED ANTIBODIES Mouse CONSTANT FR1 FR2 FR3 FR4 CDR1 CDR2 CDR3

Human Mouse Humanized

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

ANTIBODY ENGINEERING

.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

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

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

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

PHAGE DISPLAY

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

ANTIBODY FRAGMENTS

EXPRESSION SYSTEMS Bacterial Fungal Mammalian cell lines