This document discusses nanobodies, which are single domain antibody fragments derived from heavy chain antibodies in camelids. It provides background on the structure of nanobodies and their advantages over conventional antibodies like small size, high solubility, and ability to recognize cryptic epitopes. The document outlines strategies for generating nanobody libraries by phage display and their various applications. These include use as diagnostic tools for pathogen detection and molecular imaging as well as therapeutics for targeting viruses, bacteria, and toxins. Nanobodies may also help deliver drugs across the blood brain barrier.

2. Introduction
Heavy chain antibody
Nanobodies/ single domain antibody
Conventional antibody vs Nanobodies
Structure of single domain of a camelid HcAb
Unique properties of VHH
Strategy to generate nanobodies
Different display system used in Nbs library
Expression system
Potential applications of Nanobodies
Conclusion
Future Perspectives
Outlines

3. Introduction
 Core for many Diagnostic and Therapeutic applications
 Capturing and/or as detection agents
 Natural therapeutics in vertebrates
 Can be obtained in monoclonal form in nearly unlimited
amounts.

4. Conventional Antibody and their
engineering
[Smolarek et. al., 2012]
• Limitations of Mabs
• High cost of production in
eukaryotic system
• Inefficient tissue penetration
• Cloning of fab fragment not
efficient
• ScFv less soluble and have
tendency to aggregate
ScFv

5. Heavy Chain antibody (HcAb)

6. Heavy Chain Antibody (HcAb)
 Camel sera contain antibodies devoid
of light chains and CH1
 90 KDa
 Immunoglobulin new antigen receptor,
(Ig-NAR) discovered in cartilaginous
fishes (shark rays and rat fish)
[Greenberg et.al., 1995]
[Hamers-Casterman et. al., 1993]

7. Conventional antibody vs HcAb
Camilidae family
 IgG1- Heterodimer
 IgG2 and IgG3 – Homodimer
 CH1 domain absent
 Hinge region – elongated and
Composed of a repeated sequence
of proline, lysine and glutamine
 Antigen binding fragment - VHH
[Muyldermans et. al., 1994]
150kDa 90kDa
15kDa

8. Nanobodies/
Single domain antibodies
• Single domain antibodies (SdAb): 15 kDa, 4nm long and 2.5 nm in diameter
• Easy in cloning, small size and high efficiency of expression
[Hamers et. al., 1993]

9. Expression of heavy and light chains in
endoplasmic reticulum (ER)
[Handershan, et. al., 1987]

10. Why CH1 is missing in HcAb
 Mutation G>A at the 3’-border of the exon encoding the CH1 domain in
the splicing site leads to its removal during splicing together with
downstream and upstream introns
 Removal of exon encoding CH1 during mRNA processing
[Nguyen, et. al., 1999]

11. VHH domain of a camelid heavy
chain antibody
[Muyldermans, 2013]
• CDR3 loop longer – compensate
for the smaller antigen binding
sites.
• CDR 3 HcAb – 16-18 A.A.
• CDR 3 Human– 9-12 A.A.
• CDR 3 Camel – 24 A.A.
recognize Lysozyme
• Second di sulphide bond linking
CDR1 and CDR3
• Recognized increased variety of
epitopes

12. Continue….
[Czerwinski et. al., 2005; Mudyldermans, 1994]
 VHH of hcAb contain several amino acid substitution
 Amino acid Changes in region engaged in the hydrophobic interaction with a light chain
 Val, Gly, Leu and Trp replaced by Phe, Glu, Arg and Gly respectively
 These changes provide high solubility and stability to VHH

13. Unique properties of VHH
 Weak Immunogenic
 Easy to clone and expression
 Recognize a wide range of epitopes
 Small size
 High solubility and stability at high temperature
[Deschant et. al., 2010]
[Harmsen et al., 2007]
[Landenson et. al., 2006]
[Lafaye et. al., 2009]
[Landenson et. al., 2006]

14. Strategy to generate nanobodies
pMECS Phagemid
[Smolarek et. al., 2012]

15. Phage Display
[Wesolowski et. al., 2009]

16. Biopanning
Solid phase panning of protein using phage display
[Wesolowski et. al., 2009]

17. Expression and Purification of Nbs
 SdAbs generated following
transfection of Phagemid in E.coli
 SdAbs purified from periplasmic
lysate by affinity chromatography
 Yield- 0.5-1mg/100 ml of E.coli
culture
[Harmsen et. al., 2007]

18. Yeast Surface display
 Immune library
displayed on the
surface of Pichia
pastoris
 VHh expressed on
the outer glucan
layer of cell wall
[Ryckaert et.al., 2010]

19. mRNA/cDNA display
[Doshi et. al., 2014]

20. Nb Expression system
 Bacteria - E. coli, where depending on the selected vector and bacterial strain.
Periplasmic space or remain in cytoplasm as a soluble products. Yields of
production 1 to 10 mg/l
 Fungi - From 1 to 100 mg/l, higher in Pichia pastoris
 Mammalian cell lines - For production of VHH-Fc Ab (increased functional
activity and prolonged serum half life) require a eukaryotic protein folding
machinery and have been produced 100mg/l in mammalian cell culture.
 Plants – Alternative to expensive production and are free from possible
contamination with human and animal pathogens
 Vhh-Fc antibodies in soyabean as feed for oral passive immunization
against ETEC infection
 Anti rotavirus Nbs produced in Rice [De Meyer et al., 2014]

21. Potential Applications
 Nanobodies as Diagnostic Tools
 Nanobodies as Therapeutics
 Nanobodies as Research Tools

22. [Siontorou, 2013]

23. Nanobodies as Diagnostic Tools
 Implementation as
sensitive detection probes
 Pathogen detection
 In vivo molecular imaging

24. Implementation as sensitive
detection probes
 Detection probes
 High probe accessibility
 Stability
 Sensitivity
 Cost effective large scale production
 Small Ab – poor accessibility in ELISA
 His-tag or fusion to an fc chain (VHH-Fc probe)
[Saerens et. al., 2005; Harmsen et.al., 2012]

25. 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]

26. Continue….
 Staphylococcus aureus β -hemolysin-neutralizing single-domain
antibody were isolated from phage display library of Indian desert
camel in LUVAS, Hisar
[Jangra and Singh, 2010]
 Nanobodies against toxins, staphylococcal enterotoxin B and botulinum toxin A
complex toxoid, have been isolated by panning a semi-synthetic shark sdAb
display library.
 Sandwich assays using these sdAbs as the reporter antibody were developed to
demonstrate their utility for future sensor applications
[Liu et al., 2007]

27. Strategies to select the influenza specific VHHs and to apply the detection functions
[Zhu et. al., 2014]
virus

28. Continue….
 Nanobody based agglutination reagent has been evaluated for
HIV diagnosis
 Fusion of HIV-1 p24 ag with red blood cells specific VHH (p24-VHH)
 Agglutination occur upon addition of anti-p24 HIV ab and p24-VHH
 This approach could be used for diagnosing variety disease
[Habib et. al., 2013]

29. In vivo molecular imaging
 Fast extravasation,
 Good tumour penetration
 Rapid renal clearance of
excess tracer
 Radiolabelled Nbs – to
discriminate between moderate
and high epidermal growth factor
receptor expression on tumours
[Chakravarty et. al., 2014]

30. Nanobodies as Therapeutics
 Unique physiochemical and pharmacokinetic property – Immunotherapy or drug
delivery
 Rapid blood clearance or short serum half life – Fusion with other therapeutic
compound, pentamerization and IgFc enhance the serum half life
 Small, monomeric format of Nbs - Generate bispecific Nbs or larger pluripotent
constructs to combat toxins and pathogenic infections
 Nbs that cross the blood–brain barrier
 Attachment of Nbs to lentiviral particles or even on the surface of bacteria
 Creation of Nbs that are secreted by bacteria will create novel therapeutic
opportunities.

31. Nanobodies as Therapeutics
 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

32. Targeting bacteria
 Blocking attachment of bacteria to host cells and/or for more effective
delivery of pro-drugs.
 Nbs inhibit the β-lactamase activity leads to increase antibiotic activity
 Therapeutic effect of llama derived VHH fragments against
Streptococcus mutans on the development of dental caries have been
shown.
[Conrath et. al., 2001; Krüger et.al., 2006]

33. Therapeutic effect of VHH fragments against
Streptococcus mutans
llama
S. mutans
fused fungal
glucose
oxidase
Immunization
Fungal glucose oxidase
produces hydrogen
peroxide (H2O2) in the
presence of glucose
and oxygen
Fusion protein VHH-
glucose oxidase
produced in yeast
system
Rat challenged with
S. mutants infection
Daily application of
the sdAb monomer as
well as the VHH-
glucose oxidase fusion
protein
Reduced level of
bacterial recovery
21 days after
infection
[Krüger et. al., 2006]

34. VHH against E. coli F4 fimbriae
A VHH (K609) against E. coli F4 fimbriae, applied at high doses, reduced
E. coli induced diarrhoea in piglets.
Applied Orally Proteolytically degraded
Pepsin, trypsin and
chymotrypsin
>100-fold increase resistance
to in vitro degradation by
gastric and jejunal fluid.
VHH K609
DNA-shuffling strategy involving
the random recombination of gene
segments
Variant VHH (K922)
[Harmsen et. al., 2006]

35. Targeting Phages & Fungi
 VHHs recognizing the tail of infectious phages
were also shown to protect Lactococcus bacterial
strains of which are important in the dairy
industry, against infection by phages.
 Application of VHH is in antidandruff shampoo.
VHH recognize a surface protein of Malassezia
furfur.
[Hultberg et. al., 2007]
[Dolks et. al.,2005]

36. Targeting Parasites
 Trypanosomes – Variable surface Glycoprotien (VSG)
 sdAbs better penetrate the VSG coat to gain access to conserved
epitopes [Stijlemans et. al., 2004]
Purified VSG sdAb Oligomannose cryptic epitope
 Fluorochrome-conjugate sdAb detect parasite in direct blood smear
Prodrug Active drug
 Genetic fusion to a truncated form of human apolipoprotein L-I yielded
an immunotoxin with lytic activity against a range of different
trypanosomes infections. [Baral et. al.,2006]
immunization
Penetrate
VSG
VHH-β-lactamase
(cephalosporin) (potent toxic phenylenediamine
mustard (PDM))

37. Continue….
 VHH inhibit invasion of protozoa by recognizing Duffy antigen
receptor for chemokines (DARC) from human erythrocytes. it
can inhibit binding of Plasmodium vivax to red blood cells .
[smolareck et. al., 2010]
 Immunization of two dromedaries with T. solium extracts
yielded an sdAb recognized the 14 kD diagnostic glycoprotein
Ts14.
[Deckers et. al., 2009]

38. Targeting toxins
 IgGs isolated from the serum of an immunized dromedary can neutralize toxins
of the scorpion Androctonus australis
 VHHs recognizing the most important toxin components, AahII and AahI’. the
bivalent VHHs consisting of two identical anti-AahI’ VHH domains linked together
or bispecific VHHs containing anti-AahI’ and anti-AahII VHHs can neutralize
scorpion toxins. [Hmila et. al.,2008]
 VHH against SpvB salmonella toxin - that block SpvB enzymatic activity
[Koch-Nolte et. al., 2006]
 Bacterial toxin specific sdAbs including cholera toxin, staphylococcal enterotoxin
B, and botulinum neurotoxin A has been successfully isoalted
[Wesolowski et.al.,2009]

39.  In Mouse model nbs administered by intranasal route that
reduced virus replication
 Up to 48 h before challenge (Prophylactic) – Fully protected
 up to 72 h after challenge (Therapeutic) – Protective and delay time to
death
 Bivalent 60 fold more effective than monovalent Nbs
llama
Immunized
H5 haemagglutinin
Phage display and
panning
VHHs
Neutralization
of virus
Blocking of HA to
Sialic acid residue
Bivalent and trivalent NBs > Monovalent Nbs
Neutralizing
activity
[Hultberg et. al., 2011].
[Iban˜ez et. al., 2011]
Targeting Viruses
VHH against Influenza virus (H5N1)

40. Rabies virus Nbs
 Fatality is 100%.
 PEP (passive immunization with polyclonal IgG)
 Alternatives VHHs
 VHHs phage library was constructed
 Five Nbs derived neutralize rabies prototype strain (CVS-11) and street
isolates.
 IC50= 7-325nm
 Improved by producing bivalent Nbs
[Hultberg et. al., 2011]

41. FMD virus VHHs
 Highly contagious and can cause devastating disease in livestock
 FMDV neutralizing sdAbs - immunized llama
 Half life of VHH enhanced fusion with VHH specific for pig IgG
 I/m route of bispecific VHH reduced viraemia in infected piglets
 I/v route 50mg/kg in pigs prevent transmission
[Harmsen et.al., 2008]

42. Crossing Blood Brain Barrier
 Therapeutic applications of biologics in neuroinflammatory diseases have to face an
important biological barrier.
 The obstacle for effective delivery of therapeutic drugs, especially antibodies, is the
blood brain barrier.
 The delivery of conventional antibodies to the brain is especially tedious because of Fc-
receptor mediated efflux to the blood.
 Nanobodies (lacking an Fc-part ) - promising alternative to brain targeting monoclonal
antibodies.
 Nbs with a high isoelectric point (pI) spontaneously cross the blood brain barrier,
penetrate cells and bind to intracellular proteins.

43. Strategies to delivery of nanobodies to
the brain.
1. Nanobody FC5, binding to a putative a (2,3)-
sialoglycoprotein receptor, can potentially be used as
shuttling- antibody to deliver other therapeutic
proteins
2. Apolipoprotein E (ApoE) binds to low density
lipoprotein receptor-related protein 1 (LRP1) inducing
transcytosis, which can be exploited as shuttle for
therapeutic nanobodies.
3. In a similar fashion, transferrin receptor (TrfR) could
be targeted for the transfer of therapeutic nanobodies.
4. Finally, shifting the isoelectric point (pI) of therapeutic
nanobodies to a basic level facilitates crossing of the
BBB by these nanobodies.
[Rissiek et al. 2014]

44. Anti cancer therapy
 VHHs can bind antigens on the surface of tumor cells and their immunogenicity is low
 Carcino embryonic antigen (CEA, also named CD66e) is highly expressed on cancer
cells of epithelial origin.
 Specific sdAb which was anti-CEA derived from an immunized dromedary (cAb-CEA5)
was fused to a -lactamase, an enzyme that can convert non-toxic prodrugs into
potent cytotoxic agents. The tumour mouse had decreased significantly.
Application of VHH in ADEPT
(Antibody Dependent Enzyme Prodrug Therapy)
[Smolareck et.al .,2012]

45. Continue….
 sdAbs against endoglin (CD105), (an endothelial cell surface protein associated
with tumor neovascularization) were developed for cancer therapy.
 VHH recognizes the antigen with high affinity
 sdAbs recognizing mucin MUC1, (an epithelial cell surface protein over-expressed
during lactation and in breast cancer)
VHH bind with endolgin Inhibit angiogenesis in tumour
(anti-angiogenic agent )
[Rahbarizadeh et.al., 2005]
[Ahmadvand et. al., 2008]

46. Auto Immune diseases
 Anti TNF α VHHs can be used to inhibit with functions if TNF α in a
murine model
 Anti-TNF alpha VHH can be used in Rheumatoid arthritis therapy
 Lactococcus lactis strain can be used as a carrier for Anti TNF α
VHHs: antibody fragments were secreted by bacteria directly in the colon.
It act as a therapeutic agent for patients suffering from chronic colon
inflammation
 Major advantage is to avoid proteolysis of orally administered VHHs
[Smolarek et.al., 2012]

47. Nanobodies as Research Tools
 Protein purification
 Crystallisation chaperon

48. Protein purification
 VHH recognizing GFP serve as a tool for purification of GFP-fused
proteins.
 VHH used as a protein purification ligands because of their stability,
monomeric nature, and easy directional immobilisation to solid
supports.
 Stability ensures a high column regeneration capacity
 VHHs with an anti-human IgG specificity were also developed for
depletion of IgGs from blood (BAC BV/Life Technologies)
[Thomas De Meyer et. al., 2014]

49. Crystallisation Chaperon
Specific nanobodies can:
 Increase the solubility of insoluble targets
 Increase the stability of unstable (solubilized membrane) proteins
 Stabilize individual components or parts of multiprotein complexes
 Produce affinity columns aimed to purify difficult proteins to homogeneity.
 Nanobodies facilitated crystallization of many proteins
 Nbs bound to these membrane proteins may increase the polar surface
thus enabling better growing crystals and may allow the resolution of their
structure by molecular replacement

50. Continue…..
 The structure of the instable active state of the β2 adrenergic receptor
has been elucidated by growing crystals in the presence of active state-
specific VHHs
 VHH that inhibits prion oligomerisation has been used to obtain
structural information on the disordered N-terminal prion protein
region.
[Rasmussen et. al., 2011]
[Abskharon et. al., 2014]

51. Conclusion and future perspectives
 Nanobodies are smallest fully active fragments.
 Small size, high affinity, ability to bind epitopes inaccessible for regular
antibodies or their fragments, amazing stability, and feasibility of
expression – suggest that they may be considered as a new magic
bullet of medicine.
 In addition, they may be employed in biotechnology, therapy, imaging
and laboratory practice.
 Nbs developed to combat cancer, diseases such as viral infections and
toxin envenoming could be treated with future Nbs based therapeutics.

52. Thank
You....