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ANTIBODY ENGINEERING

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.

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ANTIBODY ENGINEERING

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  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. PROTEASE DIGESTION F(ab`)2 CH1 CH1 c HINGE c CH1 CH1 Fab c c Fab CH3 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 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 CONSTANT Human Humanized 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.

  19. ANTIBODY ENGINEERING

  20. .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

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

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

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

  24. PHAGE DISPLAY

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

  26. ANTIBODY FRAGMENTS

  27. EXPRESSION SYSTEMS Bacterial Fungal Mammalian cell lines

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