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This article is aimed at a brief introduction to phage display technology for production of single-domain Abs (dAbs), popularly also called ‘nanobodies’, and then to discuss their diagnostic applications.

Publicada em: Ciências
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  1. 1. WELCOMEWELCOME Credit Seminar On Role of nanobodies in diagnosis of bacterial diseases
  2. 2. Outline Nanobodies Properties Selection and production Potential applications
  3. 3. Conventional antibody and their engineering CH2 CH3 Camel Paratope VHH Hinge DOMAIN ANTIBODY (NANOBODY) (Ag binding site) Fig. 3 Camelid heavy chain antibody (HCAb) nanobody/single domain Abs & their derivatives that can be produced by rDNA techniques . and CHOs CAMELID HCAb Human framework regions HUMANIZED NANOBODY BISPECIFIC NANOBODY BIFUNCTIONAL NANOBODY Ag1 binding paratope Ag2binding paratope Nanobody “magic bullet” Enzyme/Radionuclide/toxin conjugate Paratope
  4. 4. Heavy chain antibodies (hcAbs) Camel sera also contain antibodies devoid of light chains and CH1 (Hamers-Casterman et al. 1993) 90 KDa Also in Camelidae family (e.g., llamas and alpacas) Immunoglobulin new antigen receptor, (Ig-NAR) discovered in cartilaginous fish (Greenberg et al.1995)
  5. 5. Nanobodies (Nb) Single domain antibodies (SdAb)  The recombinant antigen-specific, single-domain of the Heavy chain of the Heavy-chain antibody (VHH) with dimensions in the nanometer range.
  6. 6. VHH domain of a camelid heavy chain antibody 6 Muyldermans, 2013
  7. 7. Selection of nanobodies Phage display Ribosomal display mRNA display Microbial display
  8. 8. Phage display Physically links genotype to phenotype Ff filamentous phage, Lambda and T7 (M13, fd-phage)  PIII or pVIII protein preferred
  9. 9. Phage display 11 Muyldermans, 2001
  10. 10. Biopanning
  11. 11. Microbial display 13 immune library displayed on the surface of Pichia pastoris Works well for affinity maturation (Ryckaert et al., 2010)
  12. 12. Ribosomal display 14 Very large libraries Least aggregation
  13. 13. mRNA/cDNA display 15 (Doshi, et al. 2014)
  14. 14. Affinity maturation • Process to improve antibody affinity for an antigen. • In vivo, by somatic hypermutation and clonal selection. • In vitro, in the laboratory affinity maturation can be obtained by mutation and selection.
  15. 15. Expression 17 Bacteria- Periplasmic Nb production 1 to 10 mg/l Fungi - from <1 to >100 mg/l Mammalian cell lines - when eukaryotic protein folding machinery is required Plants - free of possible contamination with human and animal pathogens Nb-Fc antibodies in soyabean as feed for oral passive immunisation De Meyer et al.., 2014
  16. 16. Immunogenicity • Nanobodies tested to date do not appear to show any unexpected levels of immunogenicity • VHH domains from which Nanobodies are derived show a high degree of homology with human VH domains
  17. 17. Potential applications 19 Nanobodies as Diagnostic Tools Nanobodies as Research Tools Nanobodies as Therapeutics
  18. 18. Nanobodies as Diagnostic Tools 20 Pathogen detection In vivo molecular imaging
  19. 19. Pathogen and toxin detection • Distinguish between Brucella and Yersinia infections in livestock, mAbs have failed. (Abbady, et al. 2012) • Anti-caffeine Nbs for the quantification of caffeine in hot beverages. (Ladenson et al. 2006) • Nanobodies against three toxins, staphylococcal enterotoxin B and botulinum toxin A complex toxoid, have been isolated by panning a semi-synthetic shark dAb display library. (Liu et al., 2007). • Sandwich assays using these dAbs as the reporter antibody were developed to demonstrate their utility for future sensor applications (Liu et al., 2007).
  20. 20. 22
  21. 21. Molecular imaging 23 Chakravarty et al., 2014 Fast extravasation, good tumour penetration, and rapid renal clearance of excess tracer Radiolabelled Nbs
  22. 22. Nanobodies as Therapeutics 24  Pathogen targeting  Bacteria/Phages Viruses Fungi/ Parasites Crossing blood brain barrier Inhibition of enzymes, cell surface and other soluble proteins Cancer therapeutics  Auto immune diseases  Others
  23. 23. Targeting bacteria and phages 25 Transformed lactobacilli with VHH against Streptococcus mutans, in dental caries. Reducing the bacterial resistance to antibiotics by VHHs with beta-lactamase inhibitory effect. Preventing the infection of Lactococcus lactis cultures by the p2 bacteriophage by Nbs against a phage tail protein Engineering (an interesting approach for vaccinating microorganisms of biotechno-logical interest) (Wesolowski et al. 2009)
  24. 24. Targeting fungi, protozoans, toxins 26 Against a cell wall protein of Malassezia furfur, a fungus implicated in dandruff –used in shampoo formulation Trypanosomes (oligomannose cryptic epitope) Scorpion, snake venom (Baral et al. 2006)
  25. 25. Crossing Blood Brain Barrier 27 Nanobody FC5, binding to a putativ a(2,3)-sialoglycoprotein receptor Apolipoprotein E (ApoE) binds to low density lipoprotein receptor- related protein 1 (LRP1) inducing transcytosis Transferrin receptor (TrfR) Shifting the isoelectric point (pI) of (Rissiek et al. 2014)
  26. 26. Cancer therapeutics 28 platform A : naked nanobodies platform B: nanobodies fused to effector domains platform C : nanobodies decorating the surface of nanoparticles Kijanka et al 2015
  27. 27. Nanobodies as Research Tools 29 Protein purification and immunoprecipitation crystallization and structural determination of challenging targets
  28. 28. Protein purification and immunoprecipitation 30 As protein purification ligands Stability ensures a high column regeneration capacity Nbs with an anti-human IgG specificity for depletion of IgGs from blood (BAC BV/Life Technologies) A new protein affinity tag of only four amino acids (EPEA) Nguyen-Duc et al.., 2013
  29. 29. Crystallization chaperones • Determining protein structures by X-ray crystallography is difficult for ‘high hanging fruits’ such as membrane proteins and large protein complexes. • Nanobodies facilitated crystallization of many proteins
  30. 30. SUMMARY 32 Single domain antibodies from heavy chain antibodies An accessible and streamlined protocol available Recombinant Nbs are well expressed, highly robust, and easy to engineer Prefer to interact with cavities on the surface of their antigen Applications in research, diagnostic and therapeutics