ANTIBODY ENGINEERING Joud Shafiq. ANTIBODY ENGINEERING ANTIBODIES METHODOLOGIES APPLICATIONS.
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Transcript of ANTIBODY ENGINEERING Joud Shafiq. ANTIBODY ENGINEERING ANTIBODIES METHODOLOGIES APPLICATIONS.
ANTIBODY ENGINEERING
Joud Shafiq
ANTIBODY ENGINEERING
• ANTIBODIES
• METHODOLOGIES
• APPLICATIONS
Heavy chain
Light chain
220 a.a
~500 a.a
NH2
IMMUNOGLOBULIN
Antigen Binding Site
Variable
Regions
Constant Regions
Effector
Functions
LIGHT CHAIN GENES
Variable domain
CDR3
HEAVY CHAIN GENES
HUMAN
H.C Variable region= V+D+J gene segments
ANTIBODY ENGINEERING
MONOCLONAL ANTIBODIES
Mouse splenocytes
Mouse myeloma
hybridoma
Y Y YMonoclonal
Y Y
Antibodies
Y Y
PROBLEMS
• HAMA Response.
• Loss of effector functions.
• Reduce the size of antibody.
• Production of human Mab.
• Make mouse Ab more “human-like”
• Production of small Ab fragments.
SOLUTIONS
c cCH1CH1
CH2 CH2
CH3CH3
HINGE
PROTEASE DIGESTION
c cCH1CH1
c cCH1CH1
HINGE
F(ab`)2
FabFab
.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
CHIMERIC HUMAN/MOUSE
FR1 FR2 FR3 FR4
CDR1 CDR2 CDR3
CONSTANT
CONSTANT
CONSTANTFR1 FR2 FR3 FR4
CDR1 CDR2 CDR3
Mouse
Human
Chimeric
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