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RECOMBINANT ANTIBODIES AND THE PHAGE DISPLAY TECHNOLOGY

Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s P rogrammes at the University of Pécs and at the University of Debrecen Identification number : TÁMOP-4.1.2-08/1/A-2009-0011.

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RECOMBINANT ANTIBODIES AND THE PHAGE DISPLAY TECHNOLOGY

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  1. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identificationnumber: TÁMOP-4.1.2-08/1/A-2009-0011

  2. RECOMBINANT ANTIBODIES AND THE PHAGE DISPLAY TECHNOLOGY Éva Csősz Molecular Therapies - Lecture 7 Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identificationnumber: TÁMOP-4.1.2-08/1/A-2009-0011

  3. TÁMOP-4.1.2-08/1/A-2009-0011 The aim of lecture 7 is to present the possibilities for therapeutic antibody production, to highlight the pros and cons of the different production methods. In this lecture the production of antibodies in the bodyand by different techniques like in hybridoma cells or the generation of high antibody diversity by phage display technology will be discussed. Chapters in lecture 7. 7.1. Introduction VI.I.1. The structure of antibodies and their production in the body VI.I.2. Antigen-antibody binding 7.2. The production of therapeutic antibodies VI.II.1. The production of antibodies in hybridoma cells. VI.II.2. Humanized antibodies VI.II.3. Production of human antibodies 7.3. Generation of antibodies by phage display VI.III.1. The phage display technology VI.III.2. Generation of phage libraries 7.4. Administration of therapeutic antibodies

  4. TÁMOP-4.1.2-08/1/A-2009-0011 The structure of antibodies Supervariable region NH3+ NH3+ NH3+ NH3+ VH VH VL VL Fab region CH1 CH1 Light chain:constant region, variable region CL CL COO- COO- Hinge region CH2 Heavy chain:constant region, variable region Disulfide bonds Fc region CH3 COO- COO-

  5. TÁMOP-4.1.2-08/1/A-2009-0011 The structure of antibody heavy chain kb. 85 gene kb. 27 gene kb. 6 gene VH1 VH2 VH3 VH4 VHn DH1 DHn JH1 JH2 JH3 JHn Cµ C C C  C α Heavy chain VH4 DH1 JH2 C  IgG

  6. TÁMOP-4.1.2-08/1/A-2009-0011 The structure of antibody light chain approx. 35 kappa gene approx. 5 kappa gene VL1 VL2 VL3 VL4 VLn JL1 JL2 JL3 JLn C approx. 30 lambda gene approx. 4 lambda gene VL2 JL3 C kappa light chain

  7. TÁMOP-4.1.2-08/1/A-2009-0011 Production of antibodies in B cells B cell Antibody

  8. Y Y Y Y TÁMOP-4.1.2-08/1/A-2009-0011 Clonal selection and clonal expansion Recombination Junctional diversity Somatic hipermutation B cell BCR Clonal selection Antigene/epitope B cell Clonal expansion Specific antibody Plazma cell

  9. TÁMOP-4.1.2-08/1/A-2009-0011 Polyclonal antibodies Y Y Y B cell B cell antibody Y antigene epitope antibody B cell

  10. TÁMOP-4.1.2-08/1/A-2009-0011 Monoclonal antibodies B cell antibody antigene epitope

  11. TÁMOP-4.1.2-08/1/A-2009-0011 Production of antiodies in hybridoma cells Antigene Myeloma cells HGPRT  antibody production  Mouse immunization Fusion of spleen and myeloma cells, generation of hibridoma cells Spleen cell isolation HGPRT antibody production Culturing of the hibridoma cells Y Y Y Y antibody isolation Y Y Y

  12. TÁMOP-4.1.2-08/1/A-2009-0011 Humanized antibodies Human antibody Mouse antibody Humanized antibody / chimera antibody

  13. TÁMOP-4.1.2-08/1/A-2009-0011 Production of human antibodies in genetically modified mice Mouse immunoglobulin gene Human immunoglobulin gene Human or humanized antibody production

  14. E. coli TÁMOP-4.1.2-08/1/A-2009-0011 The structure of M13 phage M13 bacteriophage 5 db p6 DNS - 6.4 kb 5 db p9 5 db p3 5 db p7 2700 db p8 F-pilus 900 nm

  15. TÁMOP-4.1.2-08/1/A-2009-0011 Specific elution of immobilized phage particles Specific elution Immobilized protein / affinity matrix

  16. TÁMOP-4.1.2-08/1/A-2009-0011 Enzyme phage display matrix

  17. TÁMOP-4.1.2-08/1/A-2009-0011 Substrate phage display I. matrix

  18. TÁMOP-4.1.2-08/1/A-2009-0011 Substrate phage display II. matrix matrix

  19. TÁMOP-4.1.2-08/1/A-2009-0011 Enzyme-substrate phage display I. matrix

  20. TÁMOP-4.1.2-08/1/A-2009-0011 Enzyme-substrate phage display II. matrix matrix

  21. TÁMOP-4.1.2-08/1/A-2009-0011 Generation of phage libraries Various sequences Phagemid Recombinant phagemid

  22. TÁMOP-4.1.2-08/1/A-2009-0011 Generation of protease substrate phage library Generation of various sequences Protease substrate sequence Protease substrate hGH gene M13 gIII gene hGH gene Protease substrate M13 gIII gene phagemid vector phagemid vector Phage library

  23. TÁMOP-4.1.2-08/1/A-2009-0011 Substrate phage display –engineering of protease substrate sequences Protease sensitive sequences Protease hGH receptor Protease Sequencing matrix low pH Protease resistent sequences

  24. TÁMOP-4.1.2-08/1/A-2009-0011 In vivo phage display – mapping vascular endothelial cells Phage particles bind to the vascular endothelial cell surface proteins Biopsy Intravenous injection of phage library Removal of bound phages Identification of phage- bound proteins/peptides Propagation of bound phages

  25. TÁMOP-4.1.2-08/1/A-2009-0011 mRNA Whole blood Limfocytes cDNA (immunized donor) antibody genes phagemid E. coli cells Generation of antibody libraries from whole blood antibody specific primer contain 108 differnt antibody genes

  26. TÁMOP-4.1.2-08/1/A-2009-0011 The mechanism of antibody dependent cell mediated cytotoxicity (ADCC) Y Y Y Antibody against tumor cells Y Y Fc receptor Y Y Tumor cell Killer cell (NK cell or monocyte)

  27. TÁMOP-4.1.2-08/1/A-2009-0011 Administration of therapeutic antibodies with immunosupressant activity • Monoclonal antibody • Adalimumab • Infliximab • Golimumab • Cetrolizumab pegol • Psoriasis • Rheumatoid arthritis • Crohn disease • Spondilitis TNFalpha Inhibition of organ rejection after transplantation, especially in case of kidney transplantations. IL2 receptor alpha chain Basiliximab Human-mouse chimera antibody

  28. TÁMOP-4.1.2-08/1/A-2009-0011 Forms of therapeutic antibodies Bispecific antibody (approx. 300 kDa) IgG - scFv (Fab – scFv)2

  29. TÁMOP-4.1.2-08/1/A-2009-0011 Forms of small-sized therapeutic antibodies Fab F(ab’)2 scFv Tandem scFv Diabody Triabody Bispecific antibody S-S Fv S-S scFv2 dsFv Nanobody

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