2. Johannes Passecker 05.02.08

3.  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. 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.  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?

6.  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

7.  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

8.  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.  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.  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

11. http://www.alzforum.org/res/adh/cur/knowntheamyloidcascade.asphttp://www.alzforum.org/res/adh/cur/knowntheamyloidcascade.asp - Author: Dennis Selkoe

12.  Inhibition of Aβ aggregate formation  Passive immunization therapy

13.  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.  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

15.  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.  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.  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.