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Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University.

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Presentation on theme: "Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University."— Presentation transcript:

1 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 /1/A

2 RECOMBINANT ANTIBODIES AND THE PHAGE DISPLAY TECHNOLOGY Éva Csősz Molecular Therapies - Lecture 7 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 /1/A

3 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 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 TÁMOP /1/A

4 Heavy chain: constant region, variable region Light chain: constant region, variable region Hinge region Supervariable region Disulfide bonds COO - NH 3 + VHVH VHVH VLVL VLVL C H3 CLCL CLCL C H2 C H1 F ab region Fc region TÁMOP /1/A The structure of antibodies

5 V H1 V H2 D Hn D H1 V Hn V H4 V H3 J H1 CC CµCµ J Hn J H3 J H2 C  CC C α J H2 D H1 V H4 C  IgG Heavy chain kb. 85 genekb. 27 genekb. 6 gene TÁMOP /1/A The structure of antibody heavy chain

6 VL1VL1 VL2VL2 VLnVLn VL4VL4 VL3VL3 JL1JL1 CC JLnJLn JL3JL3 JL2JL2 JL3JL3 VL2VL2 CC kappa light chain approx. 35 kappa geneapprox. 5 kappa gene approx. 30 lambda gene approx. 4 lambda gene TÁMOP /1/A The structure of antibody light chain

7 B cell Antibody TÁMOP /1/A Production of antibodies in B cells

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

9 Y Y Y Y B cell antibody antigene epitope antibody TÁMOP /1/A Polyclonal antibodies

10 B cell antigene epitope antibody TÁMOP /1/A Monoclonal antibodies

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

12 Mouse antibody Human antibody Humanized antibody / chimera antibody TÁMOP /1/A Humanized antibodies

13 Mouse immunoglobulin gene Human immunoglobulin gene Human or humanized antibody production TÁMOP /1/A Production of human antibodies in genetically modified mice

14 5 db p9 5 db p7 5 db p3 5 db p db p8 DNS kb M13 bacteriophage E. coli 900 nm TÁMOP /1/A The structure of M13 phage F-pilus

15 Immobilized protein / affinity matrix Specific elution TÁMOP /1/A Specific elution of immobilized phage particles

16 matrix TÁMOP /1/A Enzyme phage display

17 matrix TÁMOP /1/A Substrate phage display I.

18 matrix TÁMOP /1/A Substrate phage display II.

19 matrix TÁMOP /1/A Enzyme-substrate phage display I.

20 matrix TÁMOP /1/A Enzyme-substrate phage display II.

21 Phagemid Recombinant phagemid Various sequences TÁMOP /1/A Generation of phage libraries

22 hGH geneProtease substrateM13 gIII gene phagemid vector hGH gene Protease substrate M13 gIII gene phagemid vector Generation of various sequences Phage library Protease substrate sequence TÁMOP /1/A Generation of protease substrate phage library

23 hGH receptor matrix Protease low pH Protease resistent sequences Protease sensitive sequences Sequencing Protease TÁMOP /1/A Substrate phage display –engineering of protease substrate sequences

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

25 Limfocytes mRNA cDNA antibody specific primer Whole blood (immunized donor) antibody genesphagemid E. coli cells contain 10 8 differnt antibody genes TÁMOP /1/A Generation of antibody libraries from whole blood

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

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

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

29 Fv Tandem scFvDiabodyTriabody Bispecific antibody F(ab’) 2 Fab scFv S-S scFv 2 dsFv S-S Nanobody TÁMOP /1/A Forms of small-sized therapeutic antibodies


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