ANTIBODY ENGINEERING

ARCHANA GAUTAM M.Sc. 3rd Sem.

BBAU lucknow

ANTIBODY ENGINEERING

• ANTIBODIES

• CHIMERIC /HUMANIZED

• METHODOLOGIES

• APPLICATIONS

Cont.... • In recent years, antibodies have become increasingly

accepted as therapeutics for human diseases, particularly for cancer, viral infection and autoimmune disorders.

• Monoclonal antibodies (Mabs) have been used as diagnostic and analytical reagents since hybridoma technology was invented in 1975.

• “man-made antibodies.” was named by Cesar Milstein, who was one of the inventors of monoclonal antibody technology.

• Until the late 1980’s, antibody technology relied primarily on animal immunization and the expression of engineered antibodies.

PROBLEMS

• HAMA Response.

• Loss of effector functions.

• Reduce the size of antibody.

LIGHT CHAIN GENES

Variable domain

CDR3

HEAVY CHAIN GENES

HUMAN

H.C Variable region= V+D+J gene segments

• Production of human Mab.

• Make mouse Ab more “human-like”

• Production of small Ab fragments.

SOLUTIONS

Human Mouse

Chimeric

HUMANIZED ANTIBODIES

FR1 FR2 FR3 FR4

CDR1 CDR2 CDR3

CONSTANT

CONSTANT

CONSTANTFR1 FR2 FR3 FR4

CDR1 CDR2 CDR3

Mouse

Human

Humanized

Human Mouse

Humanized

CHIMERIC/HUMANIZED.Reduce the HAMA response.

.Maintain effector functions.

.Increase the half life of the antibody.

.Maintain binding affinity.

.Easy to construct.

ANTIBODY ENGINEERING

.HAMA Response.

•Loss of effector functions.

Making the antibody more “human-like”. (the cutting and pasting)

Production of human Mab’s.

Chimeric Ab’s Humanized Ab’s

Hybridoma

Phage Display

Transgenic/SCID Mice

MONOCLONAL ANTIBODIES

Mouse splenocytes

Mouse myeloma

hybridoma

Y Y YMonoclonal

Y Y

Antibodies

Y Y

HUMAN HYBRIDOMAS

. Unstable hybridomas.

.Small number of human B-cells in peripheral blood.

. Obtaining antigen-reactive human B-cells.

Heteromyeloma

fusion

In vitro immunization

MICE

SCIDTransgenic

Reconstituted with human peripheral blood

Containing human immunoglobulin genes.

PHAGE DISPLAY

Reduce the size of Antibody. Production of

antibody fragments.

Simplifying the expression/production of antibodies.

ANTIBODY FRAGMENTS

EXPRESSION SYSTEMS

Fungal Mammalian cell lines

Bacterial

Application

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