My unique single-domain antibodies

2. Johannes Passecker 05.02.08 My unique single-domain antibodies

3. Antibodies • 5 classes of immunoglobins • ~ 150 kilo Dalton (kDa) in size • Main structure is very conserved: • two identical 50kD heavy chains • two 25kD light chains

4. Classes of antibodies IgD found on B-cells IgE binds to allergens IgG fight against invading pathogens Able to cross placenta IgA mostly in mucosal areas IgM - found on the surface of B cells eliminates pathogens in early humoral immune response Monomer (IgD, IgE, IgG) Dimer (IgA) Pentamer (IgM)

5. Binding sites Where are my sleeping pills? • hypervariable regions (HV) • HV1, HV2 and HV3 • hypervariable loops of the HV regions (also called complementary determining regions or CDR ) • Binds the antigens by electrostatic and hydrophobic interactions, hydrogen bonds and Van der Waals forces

6. Another class of ABs • Tylopoda species have however different antibodies in their serum • Camelidae family • Lamini and the Camelini genus • The dromedar and the two-hump camel Naturally occurring antibodies devoid of light chains , Hamers-Casterman C. Et al. , Nature. 1993 Unique single-domain antigen binding fragments derived from naturally occurring camel heavychain antibodies, Serge Muyldermans and Marc Lauwereys, J. of Mol. Recognition, 1999

7. Heavy Chain Antibodies • No light chains • VHH domain of heavy chain ~ 15 kDa • Only 3 CDRs instead of 6 • Longer 3rd HV loop • more resistant to heat and pH E. De Genst et al. / Developmental and Comparative Immunology 30 (2006) 187–198

8. Production of Nanobodies • Immunization of camelids with Freunds adjuvant • serum is fractionated by the use of protein G and A columns • Selection of VHH by two-step PCR and agarose gel seperation • PCR fragments are ligated into a phage display vector and transformed into an expression host • Expression of VHH domains by the host E. De Genst et al. / Developmental and Comparative Immunology 30 (2006) 187–198

9. Benefits of Nanobodies • a smaller size (MW of 15,000 instead of 30,000 for a scFv) • a good expression level in bacteria or yeast • a good specificity and affinity for the antigen • a higher thermo and chemical stability than corresponding Fv derivatives • a strictly monomeric behaviour

10. Applications Buy shares of Ablynx • For use in affinity chromatography • Protection against virus infections • Detection of cell architecture and dynamics and non-invasive imaging for early detection of diseases e.g. Alzheimer D. • Better suited as enzyme inhibitor than regular Fv domains • Useful for treatments of many diseases – most progressed teatments against – Thrombosis and Athritis • Use in Anti-venom treatment • Main patent holder: Ablynx, Belgium IPO in Nov. 2007

11. Short Reminder – the AD Hypothesis http://www.alzforum.org/res/adh/cur/knowntheamyloidcascade.asp - Author: Dennis Selkoe

12. 2 approaches for Treatment of AD • Inhibition of Aβ aggregate formation • Passive immunization therapy

13. AD Treatment Pipeline • Bapineuzumab (Humanized mAB against Aβ) • Wyeth and Elan (Phase III started in 2007, Phase II not finished!!) • Alzhemed™ ( designed to cross the blood-brain barrier and inhibit Aβ formation) • Neuochem Inc. (considered to be failed in Phase III) • Flurizan™ (γ-secretase activity modulator) • Myriad Inc. (start of Phase III early 2007) • LY450139 (γ-secretase inhibitor) • Eli Lilly (Phase III clinical trials started mid 2007)

14. A camelid antibody fragment inhibitsthe formation of amyloid fibrils byhuman lysozyme • VHH domain raised against wild-type human lysozyme inhibits the in vitro aggregation of its amyloidogenic variant • transmission of long-range conformational effects • reducing the ability to form an amyloidogenic protein • effective method of preventing its aggregation -> Implications for AD or Parkinson Disease Aggregation of D67H lysozyme Blue: absence of AB Pink:presence of AB (1:0,5) Green: presence of AB (1:1) Red: wild type Lysozyme – non aggregated form Dumoulin M, et al. A camelid antibody fragment inhibits the formation of amyloid fibrils by human lysozyme, Nature. 2003 Aug 14;424(6950):783-8

15. Passive Immunization • Active immunization – risk of meningitis • Passive Imm.: Antibodies capable of binding monomeric/low molecular weight forms of Aβ (in the periphery) or aggregated states of Aβ (in the brain) reduce the amyloid burden in animal studies Weiner HL, Frenkel D (2006) Nat Rev Immunol 6:404–416

16. 3 hypothesis on how passive immunization in AD works • microglial activation • antibodies bind to amyloid plaques, triggering microglia activation and infiltration of tissue* • catalytic dissolution • Abs act as chaperones catalyzing the structural change of the Aβ peptide from the – β-strand to an alternative conformation less prone to aggregation ‡ * Schenk D et al. (1999) Nature 400:173–177. Bard F et al. (2000) Nat Med 6:916–919. ‡ Solomon B et al. (1997) Proc Natl Acad Sci USA 94:4109–4112.

17. 3 hypothesis on how passive immunization in AD works (2) • the peripheral sink hypothesis • antibodies bind to Aβ in the bloodstream, shifting the distribution of Aβ between the brain and the peripheral circulatory system and thereby leading to a net efflux of Aβ from the central nervous system to plasma, where it is degraded DeMattos et al. (2001) Proc Natl Acad Sci USA 98:8850–8855.