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

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 Programmes at the University of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Éva Csősz Molecular Therapies - Lecture 7 RECOMBINANT ANTIBODIES AND THE PHAGE DISPLAY TECHNOLOGY

VI.I.1. The structure of antibodies and their production in the body 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 body and 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

The structure of antibodies 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- Figure 1. The structure of antibodies. Hinge region CH2 Heavy chain: constant region, variable region Disulfide bonds Fc region CH3 COO- COO- 4

The structure of antibody heavy chain 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 Figure 2. The structure of antibody heavy chain. Each heavy chain is a combination of V (variable), J ( junction), D (diversity) and C (constant) genes. 5

The structure of antibody light chain 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 Figure 3. The structure of antibody light chain. Each light chain is a combination of V (variable), J ( junction) and C (constant) genes. 6

Production of antibodies in B cells TÁMOP-4.1.2-08/1/A-2009-0011 Production of antibodies in B cells B cell Antibody Figure 4. Production of antibodies by B cells. 7

Somatic hipermutation TÁMOP-4.1.2-08/1/A-2009-0011 Clonal selection and clonal expansion Y Recombination Junctional diversity Somatic hipermutation B cell BCR Clonal selection Antigene/epitope Figure 5. The clonal selection and clonal expansion provides the appearance of a high number of specific antibody producing B cells and plasma cells in a very short period of time. B cell Clonal expansion Specific antibody Plazma cell 8

Y Y Y Y Polyclonal antibodies B cell B cell antibody antigene epitope TÁMOP-4.1.2-08/1/A-2009-0011 Polyclonal antibodies Y Y Y B cell Figure 6. Polyclonal antibodies – a combination of immunoglobulins originated from different B cells recognising different epitopes of the antigene. B cell antibody Y antigene epitope antibody B cell 9

Monoclonal antibodies TÁMOP-4.1.2-08/1/A-2009-0011 Monoclonal antibodies B cell Figure 7. Monoclonal antibodies – immunogloulins originated from one type of B cells recognizing one epitope. antibody antigene epitope 10

Production of antiodies in hybridoma cells 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 Figure 8. Production of antiodies in hybridoma cells. Y Y Y Y antibody isolation Y Y Y 11

Humanized antibodies Human antibody Mouse antibody TÁMOP-4.1.2-08/1/A-2009-0011 Humanized antibodies Figure 9. Humanized antibodies. Human antibody Mouse antibody Humanized antibody / chimera antibody 12

Production of human antibodies in genetically modified mice TÁMOP-4.1.2-08/1/A-2009-0011 Production of human antibodies in genetically modified mice Mouse immunoglobulin gene Figure 10. Production of human antibodies in genetically modified mice. Human immunoglobulin gene Human or humanized antibody production 13

The structure of M13 phage TÁMOP-4.1.2-08/1/A-2009-0011 The structure of M13 phage M13 bacteriophage 5 db p6 5 db p9 DNS - 6.4 kb E. coli 5 db p3 5 db p7 2700 db p8 F-pilus 900 nm Figure 11. The structure of M13 phage. 14

Specific elution of immobilized phage particles TÁMOP-4.1.2-08/1/A-2009-0011 Specific elution of immobilized phage particles Specific elution Figure 12. Specific elution of immobilized phage particles. Immobilized protein / affinity matrix 15

Enzyme phage display matrix Figure 13. Enzyme phage display. 16 TÁMOP-4.1.2-08/1/A-2009-0011 Enzyme phage display Figure 13. Enzyme phage display. matrix 16

Substrate phage display I. TÁMOP-4.1.2-08/1/A-2009-0011 Substrate phage display I. Figure 14. Substrate phage display I. matrix 17

Substrate phage display II. TÁMOP-4.1.2-08/1/A-2009-0011 Substrate phage display II. Figure 15. Substrate phage display II. matrix matrix 18

Enzyme-substrate phage display I. TÁMOP-4.1.2-08/1/A-2009-0011 Enzyme-substrate phage display I. matrix Figure 16. Enzyme-substrate phage display I. 19

Enzyme-substrate phage display II. TÁMOP-4.1.2-08/1/A-2009-0011 Enzyme-substrate phage display II. Figure 17. Enzyme-substrate phage display II. matrix matrix 20

Generation of phage libraries TÁMOP-4.1.2-08/1/A-2009-0011 Generation of phage libraries Various sequences Phagemid Figure 18. Generation of phage libraries. Recombinant phagemid 21

Generation of protease substrate phage library TÁMOP-4.1.2-08/1/A-2009-0011 Generation of protease substrate phage library Generation of various sequences Protease substrate sequence hGH gene Protease substrate M13 gIII gene hGH gene Protease substrate M13 gIII gene phagemid vector Figure 19. Generation of protease substrate phage library. phagemid vector Phage library 22

Substrate phage display –engineering of protease substrate sequences TÁMOP-4.1.2-08/1/A-2009-0011 Substrate phage display –engineering of protease substrate sequences Protease sensitive sequences Protease hGH receptor Figure 20. Substrate phage display –engineering of protease substrate sequences. Protease Sequencing matrix low pH Protease resistent sequences 23

In vivo phage display – mapping vascular endothelial cells 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 Figure 21. In vivo phage display – mapping vascular endothelial cells. Identification of phage- bound proteins/peptides Propagation of bound phages 24

contain 108 differnt antibody genes TÁMOP-4.1.2-08/1/A-2009-0011 Generation of antibody libraries from whole blood Whole blood (immunized donor) antibody genes Limfocytes antibody specific primer phagemid Figure 22. Generation of antibody libraries from whole blood. mRNA E. coli cells cDNA contain 108 differnt antibody genes 25

The mechanism of antibody dependent cell mediated cytotoxicity (ADCC) 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 Y Fc receptor Y Figure 23. The mechanism of antibody dependent cell mediated cytotoxicity (ADCC). Tumor cell Killer cell (NK cell or monocyte) 26

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 Figure 24. Administration of therapeutic antibodies with immunosupressant activity. Inhibition of organ rejection after transplantation, especially in case of kidney transplantations. IL2 receptor alpha chain Basiliximab Human-mouse chimera antibody 27

Forms of therapeutic antibodies TÁMOP-4.1.2-08/1/A-2009-0011 Forms of therapeutic antibodies Bispecific antibody (approx. 300 kDa) Figure 25. Forms of therapeutic antibodies. IgG - scFv (Fab – scFv)2 28

Forms of small-sized therapeutic antibodies 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 Figure 26. Forms of small-sized therapeutic antibodies. S-S Fv S-S dsFv scFv2 Nanobody 29