Transcript Antibody Engineering ppt

ANTIBODY
ENGINEERING
Joud Shafiq
ANTIBODY ENGINEERING
• ANTIBODIES
• METHODOLOGIES
• APPLICATIONS
IMMUNOGLOBULIN
NH2
Light chain
220 a.a
Heavy chain
~500 a.a
Variable
Regions
Antigen
Binding Site
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
YY
Mouse
splenocytes
Mouse
myeloma
YY
hybridoma
Monoclonal
Y Y Y
Antibodies
PROBLEMS
• HAMA Response.
• Loss of effector
functions.
• Reduce the size of
antibody.
SOLUTIONS
• Production of
human Mab.
• Make mouse Ab
more “human-like”
• Production of small
Ab fragments.
c
CH1
CH1
HINGE
CH2
CH3
CH2
CH3
c
PROTEASE DIGESTION
F(ab`)2
c
Fab
c
CH1 HINGE
CH1
CH1
CH1
c
c
Fab
.HAMA Response.
Making the
antibody more
“human-like”. (the
cutting and pasting)
•Loss of effector
functions.
Chimeric Ab’s
Humanized Ab’s
Hybridoma
Transgenic/SCID
Mice
Phage Display
Production of
human Mab’s.
CHIMERIC HUMAN/MOUSE
Mouse
FR1
FR2
CDR1 CDR2
FR3
FR4
CONSTANT
CDR3
CONSTANT
Human
Chimeric
FR1
FR2
FR3
CDR1 CDR2
FR4
CDR3
CONSTANT
Human
Mouse
Chimeric
Mouse
HUMANIZED ANTIBODIES
FR1
FR2
CDR1 CDR2
FR3
FR4
CONSTANT
CDR3
CONSTANT
Human
Humanized
FR1
FR2
FR3
CDR1 CDR2
FR4
CDR3
CONSTANT
Human
Mouse
Humanized
CHIMERIC/HUMANIZED
.Reduce the HAMA
response.
.Maintain binding
affinity.
.Maintain effector
functions.
.Easy to construct.
.Increase the half
life of the antibody.
ANTIBODY
ENGINEERING
.HAMA Response.
Making the
antibody more
“human-like”. (the
cutting and pasting)
•Loss of effector
functions.
Chimeric Ab’s
Humanized Ab’s
Hybridoma
Transgenic/SCID
Mice
Phage Display
Production of
human Mab’s.
MONOCLONAL ANTIBODIES
YY
Mouse
splenocytes
Mouse
myeloma
YY
hybridoma
Monoclonal
Y Y Y
Antibodies
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
SCID
Transgenic
Reconstituted
with human
peripheral blood
Containing
human
immunoglobulin
genes.
PHAGE DISPLAY
Reduce the
size of
Antibody.
Simplifying the
expression/production
of antibodies.
Production of
antibody
fragments.
ANTIBODY FRAGMENTS
EXPRESSION SYSTEMS
Bacterial
Mammalian cell
lines
Fungal