My unique singledomain antibodies Johannes Passecker 05. 02. 08
Antibodies 5 classes of immunoglobins ~ 150 kilo Dalton (k. Da) in size Main structure is very conserved: two identical 50 k. D heavy chains two 25 k. D light chains
Classes of antibodies Monomer (Ig. D, Ig. E, Ig. G) Dimer (Ig. A) Pentamer (Ig. M) Ig. D found on B-cells Ig. E binds to allergens Ig. G fight against invading pathogens Able to cross placenta Ig. A mostly in mucosal areas Ig. M - found on the surface of B cells eliminates pathogens in early humoral immune response
Binding sites Where are my sleeping pills? hypervariable regions (HV) HV 1, HV 2 and HV 3 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
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
Heavy Chain Antibodies No light chains VHH domain of heavy chain ~ 15 k. Da Only 3 CDRs instead of 6 Longer 3 rd HV loop more resistant to heat and p. H E. De Genst et al. / Developmental and Comparative Immunology 30 (2006) 187– 198
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
Benefits of Nanobodies a smaller size (MW of 15, 000 instead of 30, 000 for a sc. Fv) 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
Applications Buy shares of Ablynx For use in affinity chromatography Protection against virus infections Detection of cell architecture and dynamics and noninvasive 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
Short Reminder – the AD Hypothesis http: //www. alzforum. org/res/adh/cur/knowntheamyloidcascade. asp Author: Dennis Selkoe
2 approaches for Treatment of AD Inhibition of Aβ aggregate formation Passive immunization therapy
AD Treatment Pipeline Bapineuzumab (Humanized m. AB 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) LY 450139 (γ-secretase inhibitor) Eli Lilly (Phase III clinical trials started mid 2007)
A camelid antibody fragment inhibits the formation of amyloid fibrils by human lysozyme VHH domain raised against D 67 H lysozyme wild-type human Aggregation of lysozyme inhibits the in vitro aggregation of its amyloidogenic variant Blue: absence of AB transmission of long-range conformational effects reducing the ability to form protein effective method of preventing its aggregation -> Implications for AD or Parkinson Disease Pink: presence of AB (1: 0, 5) Green: presence of AB (1: 1) an amyloidogenic 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
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
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.
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 De. Mattos et al. (2001) Proc Natl Acad Sci USA 98: 8850– 8855.
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