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    Human antibodies  from transgenic animals Human antibodies from transgenic animals Presentation Transcript

    • HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS S. Kumarakurubaran, CENTRE FOR PLANT MOLECULAR BIOLOGY
    • HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 2
    • IntroductionMonoclonal antibodies has been extensively used from its discoveryThese monoclonal antibodies are used in the field of medicine for therapeutic applicationsThe research in the field of immunology has flourished to produce human antibodies from transgenic animals HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 3
    • CELL CULTURE Itis defined as the culturing up of individual cells or by disaggregated cell mass, either totipotent or pleuripotent, in a suitable culture medium HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 4
    • THREE ROUTES FOR DESTINATION ANIMALCELL CULTURE TRANSGENIC ANIMALS IMMUNOLOGY OF MAbs HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 5
    • HISTORY Alexis carrel(1912)-chick embryo tissue extract increases cell proliferation in vitro Fibrin clot plasma used as solid support Ross harrison(1907)cultured embryonic nerve cell of frog Using Hanging drop method Carcinoma cell line HE LA, myeloma cells identified HUMAN ANTIBODIES FROM TRANSGENIC 6 ANIMALS
    • Contd…. 1966 Alec Issacs discovered filtrates have secretions called interferon Chinese hamster ovary cells were cultured High density culturing methods were found Stem cell technology was discovered 1996 Dolly was produce by egg enucleation HUMAN ANTIBODIES FROM TRANSGENIC 7 ANIMALS
    • CHARACTERISTICS OF ANIMALCELL Grow only to a limited number of generations Neurons cannot divide to grow Skin cells grow only for a few generation Process of contact inhibition HUMAN ANTIBODIES FROM TRANSGENIC 8 ANIMALS
    • SUBSTRATESPlastics-polystyrenes, PTTES, TPS, PVCGlass, methyl cellulosesMetallic surfaceAdhesives-d lysine, gelatin,Feeder layer for glial cells HUMAN ANTIBODIES FROM TRANSGENIC 9 ANIMALS
    • MEDIApH ,osmotic pressure, gas phasesNATURAL MEDIAPlasma clotsBiological fluidsSYNTHETIC MEDIASerum containingSerum free HUMAN ANTIBODIES FROM TRANSGENIC 10 ANIMALS
    • COMPOSITION Serum Proteins Carbohydrates Co factors Examples-MEM,CMRL media HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 11
    • CELL LINES Finite cell lines-Epidermal cells, Infinite cell lines- Cancerous cells HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 12
    • MODEL ORGANISMMICE-short oestrous cycle, and gestation period-relatively short generation time-several litters per pregnancy-Convenient in vitro fertilization-successful in vitro embryo culture-production and maintenance of ES lines HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 13
    • HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 14
    • Transgenic animals Method of introducing transgeneTransfection Transient-transgene gradually lost from daughter cells Stable-retained and expressed in all the cells derived from transfected cells HUMAN ANTIBODIES FROM 15 TRANSGENIC ANIMALS
    • Transfection methods Calcium phosphate precipitation DEAE-dextran mediated transfection Lipofection Retroviral vector infection Microinjection Embryonic stem cell transfer Mark.M.Davis (2004) HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 16
    • HUMAN ANTIBODIES FROM Gardener et al., 2003 TRANSGENIC ANIMALS 17
    • Gardener et al., 2003HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 18
    • VECTORS Simian virus 40 Bovine papilloma virus with transforming region of pBR322 Polyoma virus Vaccinia virus P element vectors Baculovirus vectors HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 19
    • Gene construct Promoter/enhancer element RSV, SV40, β-casein Initiation codon, Gene of interest Reporter gene scorable marker β- galactosidase, lux selectable marker-thymidine kinase Termination signal, Poly A tail HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 20
    • HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 21
    • HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 22
    • HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 23
    • HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 24
    • HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 25
    • HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 26
    • HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 27
    • HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 28
    • Milstein and Kohler 1975 HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 29
    • HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 30
    • HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 31 Nil Lonberg (2005)
    • HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS Nil Lonberg (2005 32
    • Nil Lonberg (2005)HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 33
    • MONOCLONAL ANTIBODYDerived from single cell line have identical structure binds same epitope,Specific antigen is repeatedly injected and and spleen cells are extracted to obtain MAbs HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 34
    • ApplicationsHighly sensitive (magic bullets) epitopes helps in in vitroImmuno diagnostics (detection and quantification)Used in treatment of cancer, delivers drugs specifically to target tumor cells. HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 35
    • Negative effects Antibodies recognized as murine antibodies and mounts immune response and eliminates from the body. Causes serious allergenicity to the level of lethality. Repeated use of mouse antibodies induces host to produce antibodies that neutralizes mouse antibody called HAMA HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 36
    • Chimerised & human antibodies Variable region is obtained from non human rat antibody Constant region is obtained from human sequence Chimerics exhibit lower immunogenicity to the host Still the chimeric sequence requires changes this is done by r-DNA by engineering specific amino acid sequence HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 37
    • Contd.. Humanised to make more compatible Utilizes recombinant DNA technology Epitopes are substituted with human gene sequences Constant regions are derived from human HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 38
    • Human monoclonal antibodies from SCID mouse by injecting human B and T cells and grafting mouse by transplanting spleens and lymph nodes The transplanted mouse is immunized with target antigens to produce human antibodies Phage derived combinatorial antibody library, phage display technique-mimics in vitro affinity maturation HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 39
    • Hybridoma technology First discovered by Milstein and Kohler 1975 Specific antigen is given repeatedly to the host The spleen producing B cells are isolated B cells are fused with myeloma cells that are immortal and this is called hybridoma cells This produces specific antibody indefinitely precisely can be isolated easily HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 40
    • HUMAN ANTIBODIES FROM William Fodor 41 TRANSGENIC ANIMALS 2004
    • CHARACTERISTICSPOLYCLONAL ANTIBODY MONOCLONAL ANTIBODY Contains many  Contains single specific antigenic determinants determinant Produces various  Single class of antibody classes is produced Reproducibility and  Highly reproducible standardization is difficult HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 42
    • CONCLUSION HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 43
    • HUMAN ANTIBODIES FROM 44 TRANSGENIC ANIMALS