Presentation on theme: "Johannes Passecker 05.02.08 5 classes of immunoglobins ~ 150 kilo Dalton (kDa) in size Main structure is very conserved: two identical 50kD heavy."— Presentation transcript:
5 classes of immunoglobins ~ 150 kilo Dalton (kDa) in size Main structure is very conserved: two identical 50kD heavy chains two 25kD light chains
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)
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 Where are my sleeping pills?
Tylopoda species have however different antibodies in their serum Camelidae family ▪ Lamini and the Camelini genus ▪ The dromedar and the two-hump camel Unique single-domain antigen binding fragments derived from naturally occurring camel heavychain antibodies, Serge Muyldermans and Marc Lauwereys, J. of Mol. Recognition, 1999 Naturally occurring antibodies devoid of light chains, Hamers- Casterman C. Et al., Nature. 1993
No light chains VHH domain of heavy chain ~ 15 kDa Only 3 CDRs instead of 6 Longer 3 rd HV loop more resistant to heat and pH E. De Genst et al. / Developmental and Comparative Immunology 30 (2006) 187–198
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
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
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 Buy shares of Ablynx IPO in Nov. 2007
http://www.alzforum.org/res/adh/cur/knowntheamyloidcascade.asphttp://www.alzforum.org/res/adh/cur/knowntheamyloidcascade.asp - Author: Dennis Selkoe
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)
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 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 Blue: absence of AB Pink:presence of AB (1:0,5) Green: presence of AB (1:1) Red: wild type Lysozyme – non aggregated form Aggregation of D67H lysozyme
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
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.
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.