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HUMAN ANTIBODIES
 FROM TRANSGENIC
     ANIMALS


                                  S. Kumarakurubaran,




     CENTRE FOR PLANT MOLECULAR
              BIOLOGY
HUMAN ANTIBODIES FROM
 TRANSGENIC ANIMALS     2
Introduction

Monoclonal antibodies has been extensively
  used from its discovery
These monoclonal antibodies are used in the
  field of medicine for therapeutic applications
The 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 ANIMAL
CELL

 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 phases
NATURAL MEDIA
Plasma clots
Biological fluids
SYNTHETIC 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 ORGANISM
MICE
-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 transgene


Transfection
 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., 2003
HUMAN 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 ANTIBODY
Derived 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
Applications
Highly sensitive (magic bullets) epitopes
  helps in in vitro
Immuno 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
CHARACTERISTICS
POLYCLONAL 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

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Human antibodies from transgenic animals

  • 1. HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS S. Kumarakurubaran, CENTRE FOR PLANT MOLECULAR BIOLOGY
  • 2. HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 2
  • 3. Introduction Monoclonal antibodies has been extensively used from its discovery These monoclonal antibodies are used in the field of medicine for therapeutic applications The research in the field of immunology has flourished to produce human antibodies from transgenic animals HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 3
  • 4. 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
  • 5. THREE ROUTES FOR DESTINATION  ANIMALCELL CULTURE  TRANSGENIC ANIMALS  IMMUNOLOGY OF MAbs HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 5
  • 6. 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
  • 7. 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
  • 8. CHARACTERISTICS OF ANIMAL CELL  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
  • 9. 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
  • 10. MEDIA pH ,osmotic pressure, gas phases NATURAL MEDIA Plasma clots Biological fluids SYNTHETIC MEDIA Serum containing Serum free HUMAN ANTIBODIES FROM TRANSGENIC 10 ANIMALS
  • 11. COMPOSITION  Serum  Proteins  Carbohydrates  Co factors  Examples-MEM,CMRL media HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 11
  • 12. CELL LINES  Finite cell lines-Epidermal cells,  Infinite cell lines- Cancerous cells HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 12
  • 13. MODEL ORGANISM MICE -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
  • 14. HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 14
  • 15. Transgenic animals  Method of introducing transgene Transfection  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
  • 16. 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
  • 17. HUMAN ANTIBODIES FROM Gardener et al., 2003 TRANSGENIC ANIMALS 17
  • 18. Gardener et al., 2003 HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 18
  • 19. 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
  • 20. 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
  • 21. HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 21
  • 22. HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 22
  • 23. HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 23
  • 24. HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 24
  • 25. HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 25
  • 26. HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 26
  • 27. HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 27
  • 28. HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 28
  • 29. Milstein and Kohler 1975 HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 29
  • 30. HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 30
  • 31. HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 31 Nil Lonberg (2005)
  • 32. HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS Nil Lonberg (2005 32
  • 33. Nil Lonberg (2005) HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 33
  • 34. MONOCLONAL ANTIBODY Derived 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
  • 35. Applications Highly sensitive (magic bullets) epitopes helps in in vitro Immuno diagnostics (detection and quantification) Used in treatment of cancer, delivers drugs specifically to target tumor cells. HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 35
  • 36. 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
  • 37. 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
  • 38. 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
  • 39. 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
  • 40. 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
  • 41. HUMAN ANTIBODIES FROM William Fodor 41 TRANSGENIC ANIMALS 2004
  • 42. CHARACTERISTICS POLYCLONAL 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
  • 43. CONCLUSION HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 43
  • 44. HUMAN ANTIBODIES FROM 44 TRANSGENIC ANIMALS