690 likes | 2k Views
ANTIBODY ENGINEERING. J o ud 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.
E N D
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
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
PROTEASE DIGESTION F(ab`)2 CH1 CH1 c HINGE c CH1 CH1 Fab c c Fab CH3 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 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 CONSTANT Human Humanized 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.
.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.
Reduce the size of Antibody. Production of antibody fragments. Simplifying the expression/production of antibodies.
EXPRESSION SYSTEMS Bacterial Fungal Mammalian cell lines