CENTRE FOR PLANT MOLECULAR BIOLOGY

HUMAN ANTIBODIES FROM TRANSGENIC

ANIMALS

S. Kumarakurubaran,

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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

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CELL CULTURE

It is defined as the culturing up of individual cells or by disaggregated cell mass, either totipotent or pleuripotent, in a suitable culture medium

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THREE ROUTES FOR DESTINATION

ANIMAL CELL CULTURETRANSGENIC ANIMALS IMMUNOLOGY OF MAbs

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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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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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CHARACTERISTICS OF ANIMAL CELL

Grow only to a limited number of generations

Neurons cannot divide to grow Skin cells grow only for a few generationProcess of contact inhibition

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SUBSTRATES

Plastics-polystyrenes, PTTES, TPS, PVC

Glass, methyl cellulosesMetallic surfaceAdhesives-d lysine, gelatin,Feeder layer for glial cells

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MEDIApH ,osmotic pressure, gas phasesNATURAL MEDIANATURAL MEDIAPlasma clotsBiological fluids

SYNTHETIC MEDIASYNTHETIC MEDIASerum containingSerum free

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COMPOSITION

SerumProteinsCarbohydratesCo factorsExamples-MEM,CMRL media

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CELL LINES

Finite cell lines-Epidermal cells,

Infinite cell lines- Cancerous cells

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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 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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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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Gardener et al., 2003

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Gardener et al., 2003

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VECTORS

Simian virus 40 Bovine papilloma virus with transforming

region of pBR322 Polyoma virus Vaccinia virus P element vectors Baculovirus vectors

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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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Milstein and Kohler 1975

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HUMAN ANTIBODIES FROM TRANSGENIC ANIMALS 31Nil Lonberg (2005)

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Nil Lonberg (2005

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Nil Lonberg (2005)

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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

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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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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

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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

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Contd..

Humanised to make more compatible Utilizes recombinant DNA technology Epitopes are substituted with human gene

sequences Constant regions are derived from human

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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

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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

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William Fodor 2004

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CHARACTERISTICS

POLYCLONAL ANTIBODY

Contains many antigenic determinants

Produces various classes

Reproducibility and standardization is difficult

MONOCLONAL ANTIBODY

Contains single specific determinant

Single class of antibody is produced

Highly reproducible

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CONCLUSION

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