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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 YY Mouse splenocytes Mouse myeloma YY hybridoma Monoclonal Y Y Y 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. c CH1 CH1 HINGE CH2 CH3 CH2 CH3 c PROTEASE DIGESTION F(ab`)2 c Fab c CH1 HINGE CH1 CH1 CH1 c c Fab .HAMA Response. Making the antibody more “human-like”. (the cutting and pasting) •Loss of effector functions. Chimeric Ab’s Humanized Ab’s Hybridoma Transgenic/SCID Mice Phage Display Production of human Mab’s. CHIMERIC HUMAN/MOUSE Mouse FR1 FR2 CDR1 CDR2 FR3 FR4 CONSTANT CDR3 CONSTANT Human Chimeric FR1 FR2 FR3 CDR1 CDR2 FR4 CDR3 CONSTANT Human Mouse Chimeric Mouse HUMANIZED ANTIBODIES FR1 FR2 CDR1 CDR2 FR3 FR4 CONSTANT CDR3 CONSTANT Human Humanized FR1 FR2 FR3 CDR1 CDR2 FR4 CDR3 CONSTANT Human Mouse Humanized CHIMERIC/HUMANIZED .Reduce the HAMA response. .Maintain binding affinity. .Maintain effector functions. .Easy to construct. .Increase the half life of the antibody. ANTIBODY ENGINEERING .HAMA Response. Making the antibody more “human-like”. (the cutting and pasting) •Loss of effector functions. Chimeric Ab’s Humanized Ab’s Hybridoma Transgenic/SCID Mice Phage Display Production of human Mab’s. MONOCLONAL ANTIBODIES YY Mouse splenocytes Mouse myeloma YY hybridoma Monoclonal Y Y Y 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. Simplifying the expression/production of antibodies. Production of antibody fragments. ANTIBODY FRAGMENTS EXPRESSION SYSTEMS Bacterial Mammalian cell lines Fungal
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