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