Alpbach 2007 Van der Maarel - ProteomeBinders

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Alpbach 2007 Van der Maarel - ProteomeBinders

Camelid-Derived Antibody Fragments

For Biomedical

Research and Therapy

Silvère M. van der Maarel

Center for Human and Clinical Genetics


Heavy chain antibody repertoires

Llama: Large Laboratory Animal for

Monovalent Antibody-derivatives.

VHH: variable domain of a heavy

chain of HCAb.

The VHH is the minimal intact

antigen-binding fragment that can

be generated from HCAb.


Camelid heavy chain IgG

Phage display:

• fast

• easy source

• high affinity

• high specificity

• stable

• uniform

• small

• genetic modification


Optimal combinations for selection


Production and purification of individual VHH

Production:

• Standard heterologous

protein production in E. coli

• Purification by IMAC

VHH:

• On average 20kDa

• Variable yield


Applications

VHH antibody fragments can be used for:

• antibody arrays

• immunohistochemistry

• immunocytochemistry

• immunoprecipitation

• chromatin immunprecipitation

• column chromatography


One anitgen....

Selections for skeletal muscle Actin (ACTA1)


Immunomodulation of protein aggregation disorders

• antibody arrays

• immunohistochemistry

• immunocytochemistry

• immunoprecipitation

• chromatin immunprecipitation

• column chromatography

• immunomodulation

Oculopharyngeal muscular dystrophy


Genetic defect in OPMD

• Autosomal dominant OPMD is caused by expansions of the polyalanine tract

in PABPN1

• Mutant PABPN1 is sequestered in intranuclear inclusions in muscle, which

are a hallmark of the disease

• Administration of anti-aggregation drugs results in clearance of inclusions in

cell models, decreased toxicity, and attenuates muscle weakness in vivo

• Antibodies specific for PABPN1 which interfere with inclusion formation

provide potential for a specific intervention in PABPN1 aggregation

Ala10

WT-PABPN1

Ala12-17

MUT-PABPN1


Basic characterization of VHH 3F5


Preventive properties of VHH 3F5

Intracellular expression of VHH 3F5:

• prevents nuclear aggregation of

mutant PABPN1

• no detectable toxic side-effects

20


Curative properties of VHH 3F5

Intracellular expression of VHH 3F5:

• reduction of already existing aggregates

• no detectable toxic side-effects


New directions: disorders of proteinaceous aggregation

Cell culture

• Stable vs. transient expression

• Promoter, optimal tag use, compartimentalization

• Develop better readout systems for intrabody-antigen

interaction

Animal models

• Stability and immunogenicity

• Methods of delivery

• Methods of detection (optical imaging, MRI, PET)


Acknowledgements

LUMC

Rinse Klooster

Sabine Baars

Antoinetta Impagliazzo

Alexandra van Remoortere

Kim Rutgers

Paula de Galan

Marjolein van der Meer

Tsion Abraham

Ntambua Tshimbalanga

Johan den Dunnen

Gert Jan van Ommen

Rune Frants

UU

Theo Verrips

Peter Verheesen

Mohammed El Khattabi

Margriet Roelse

David Lutje Hulsik

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