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.

1. WELCOMEWELCOME
Credit Seminar
On
Role of nanobodies in diagnosis
of bacterial diseases

2. Outline
Nanobodies
Properties
Selection and production
Potential applications

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. 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. 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. VHH domain of a camelid heavy chain
antibody
6
Muyldermans, 2013

9. Selection of nanobodies
Phage display
Ribosomal display
mRNA display
Microbial display

10. Phage display
Physically links genotype to phenotype
Ff filamentous phage, Lambda and T7 (M13, fd-phage)
 PIII or pVIII protein preferred

11. Phage display
11
Muyldermans, 2001

12. Biopanning

13. Microbial display
13
immune library
displayed on the surface
of Pichia pastoris
Works well for affinity
maturation
(Ryckaert et al., 2010)

14. Ribosomal display
14
Very large libraries
Least aggregation

15. mRNA/cDNA display
15
(Doshi, et al. 2014)

16. 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.

17. 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

18. 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

19. Potential applications
19
Nanobodies as Diagnostic Tools
Nanobodies as Research Tools
Nanobodies as Therapeutics

20. Nanobodies as Diagnostic Tools
20
Pathogen detection
In vivo molecular imaging

21. 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).

22. 22

23. Molecular imaging
23
Chakravarty et al., 2014
Fast extravasation, good
tumour penetration, and
rapid renal clearance of
excess tracer
Radiolabelled Nbs

24. 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

25. 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)

26. 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)

27. 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)

28. 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

29. Nanobodies as Research Tools
29
Protein purification and immunoprecipitation
crystallization and structural determination of
challenging targets

30. 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

31. 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

32. 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