The document discusses the production of human monoclonal antibodies from transgenic animals. It introduces the use of monoclonal antibodies in medicine and the research goal of producing human antibodies in transgenic animals to avoid immune responses. The key methods discussed are inserting human antibody genes into animals using transfection techniques and generating transgenic mice that can produce human antibodies for therapeutic and research applications.

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
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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
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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
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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
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ANIMALS

9. SUBSTRATES
Plastics-polystyrenes, PTTES, TPS,
PVC
Glass, methyl celluloses
Metallic surface
Adhesives-d lysine, gelatin,
Feeder layer for glial cells
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ANIMALS

10. MEDIA
pH ,osmotic pressure, gas phases
NATURAL MEDIA
Plasma clots
Biological fluids
SYNTHETIC MEDIA
Serum containing
Serum free
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ANIMALS

11. COMPOSITION
 Serum
 Proteins
 Carbohydrates
 Co factors
 Examples-MEM,CMRL media
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TRANSGENIC ANIMALS 11

12. CELL LINES
 Finite cell lines-Epidermal
cells,
 Infinite cell lines- Cancerous
cells
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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
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TRANSGENIC ANIMALS 13

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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
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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)
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TRANSGENIC ANIMALS 16

17. HUMAN ANTIBODIES FROM Gardener et al., 2003
TRANSGENIC ANIMALS 17

18. Gardener et al., 2003
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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
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TRANSGENIC ANIMALS 20

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29. Milstein and Kohler 1975
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TRANSGENIC ANIMALS 31
Nil Lonberg (2005)

32. HUMAN ANTIBODIES FROM
TRANSGENIC ANIMALS
Nil Lonberg (2005 32

33. Nil Lonberg (2005)
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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.
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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
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TRANSGENIC ANIMALS 42

43. CONCLUSION
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