.

1. Single chain fragment variable (ScFv)
antibody
Presented
by :
Sivasankar . P
CONVENTIONAL CAMEL SHARK

2. OVERVIEW
HISTORY
INTRODUCTION
PRODUCTION
APPLICATION
CONCLUSION

3.  In 1890- Emil von behring- antibodies
 In1975-Kohler & Milstein-mAb
 1st mAb -Muromonab
 Nanobody
HISTORY
Kohler & Milstein

4. INTRODUCTION
 Camels immunoglobulin -lacks light chain.
 Variable heavy chain fragment –VHH fragment
 Binds specific antigen
 Mol wt 12-15kDa
 Cartilaginous fish-VNAR fragments
 Alternative to monoclonal antibodies
 Produced in microbial system
 Currently in research further has to be developed

5.  Single domain antibody also
called nano body is an antibody
fragment consisting of a single
monomeric variable heavy chain
antibody . It is also able to bind
to a specific antigen
 Patent holder : Ablynx
Definition

6. Conventional antibody Camel antibody nanobody

7. Advantages
 Small
 Sequence homology
 Nano to picomolar affinity
 Enhanced tissue penetration
 Crosses blood brain barrier
 Stable under high pH & temp
 High solubilty & rapid clearance

8.  Alternative delivery route
 Easy formating
 Customized half life extension

9. • Produced in microbial system
• Challenging intractable target
• Cell specificity

10. Antibody formats
Drug Trade Type Source Target Use
Abciximab Reopro Fab chimeri
c
CD41 Platelet aggregation
TRBS07 Ektomab Tri fuc ----- GD2 melanoma
Trastuzum
ab
Herceptin mAb Human NCA90 Osteomyelitis -imaging
Monotax ------ ScFv Human EPCAM cancer

11. Production

12. Various steps of production
 Phase1: immunization of lama with desired antigen
 Phase2: PBMC isolation :
collection of peripheral mononuclear cell and isolation of mRNA
 Phase3: cDNA library construction , screening, sequencing:
constructed library will be screened to identify high affinity antigen
specific clones and identified positive clones are sequenced
 Phase 4: single domain antibody production :
purified antibody will be provided from upto 5 identified unique
positive clones

13. Application
Used as various diagnostic and therapeutic tools:
Diagnostic aspects:
1.Screening of cysticercosis in pigs
2.Detection of breast carcinoma
3.Immuno imaging
Therapeutic aspects :
1.Botulism toxin neutralization
2.Tumour targeting by psedomonas exotoxin
3.Phtothermal therapy of cancer
4.RSV viral infection in infants
5.Acute coronary syndrome

14. Screening of cysticercosis in pigs
 Ts-14 is a glycoprotein in
Taenia solium used as target
antigen
 For screening test ELIZA
usually practiced in pigs
 While using ELIZA cross
reaction occurs between
species and detects only
genus . Not species specific
 While using nanobodies it
detects the species and it is
species specific and no cross
reaction occurs (Deekers.et al.,2009)

15. Diagnosis of Breast carcinoma
 Human epidermal growth factor receptor 2. HER-2 is a transmembrane
protein expressed in 20-30 % of breast cancer 20-24% of gastric cancer
as well as ovarian and colon cancer
 The nanobody conjugated with iodine-131 and the results are:
 Highest tumour uptake
 Fast clearance
 Lower accumulation in non target organs except kidney & bladder
 Based on above results it shows better tumour targeting properties
 Used to determine HER-2 status in breast cancer patients

16. Immuno imaging
 It is the technology aims at studying diseased patients using positron
emission tomography in combination with radio labelled
immunoglobulin against particular antigen.
 Being smallest fragment shows fast and specific targeting in vivo.
 These studies currently under investigation
Monoclonal antibody mediated
immuno imaging
Nano body mediated immuno
imaging
Residence time in blood is few days
to weeks.
Peak contrast occurs 2-4 days after
injection so long lived isotopes used
.
20-40 mSv per scan
Fastly cleared from the body
Imaging within 1-3 hours , so short
lived isotopes can be used
Less than 5 mSv per scan
Vaneycken.et
al.,2012

17. Targeting myeloma cells as antigen
Vaneycken.et al.,2012

18. Botulism toxin neutralization
 Botulism toxin is a holotoxin made up of three protein subunit
one is metalloproteinase 50 kda
Breaks the SNARE protein
Failure of neurotransmitter release
Respiratory failure and flaccid paralysis
 The nanobody created against the metlloproteinase neutralizes
them even at 1 nM concentration
Tremblay. et al.,2010

19. Break down of SNARE proteins by toxin

20. Tumour targeting by psuedomonas exotoxin
 Vascular endothelial growth factor receptor 2 is a molecule connected
with tumour angiogenesis. it is well expressed in tumour cells and very
less in normal cell
 The nanobody conjugated with recombinant pseudomonas exotoxin and
directed against the tumour antigen causes death of the cancerous cell
by protein synthesis inhibition
 It is in vitro study not performed
in animals
Behdani et al ., 2013

21. Photothermal therapy of cancer
 The nanobody is conjugated
with gold nano particles and
directed against the respective
tumour antigen
 while applying electromagnetic
waves heat created in the
tumour cells & destroyed
Natarajan.et al.,2008

22. Acute coronary syndrome
Atherosclerosis
Plaque formation
Attracts platelets causes adhesion and aggregation
Coronary lumen size reduction
Ischemic myopathy

23. Blocking of vWF by nanobody
 Von willebrand factor a glycoprotein in blood involved in platelet
aggregation
 The factor is blocked by ALX-0081 nanobody results in
antithrombotic effect
 The drug is in phase 2 study

24. Respiratory syncytial virus
 The virus in infants causes wheezing and asthma in
infants and 3,00,000 childrens hospitalized per
year
 The virus contains antigen glycoprotein- F is
essential for viral entry and fusion of host
membrane
 ALX -0171 is the nanobody based drug targeted
against the viral antigen
 Daily inhalation of the drug in infected lamb
markedly reduced the gross lung viral lesion
demonstrates strong therapeutic effect
 Well tolerated by multiple phase 1 studies in adults,
phase 2 is under trial.

25. Nebulization of nanobody Therapeutic effect of nanobody to
pnemonia

26. Various drugs in phase

27. CONCLUSION
 Nanobodies are llama derived proteins with similar specificities
and affinities as monoclonal antibodies and because they are
naturally occuring single domain binding structures they also
have several biophysical properties and characteristics
 So, the nanobody platform has proven that they are robust and
currently in clinical development.

28. Reference :
1.Tremblay M,Cheu ling kuo et al.(2010).Camelid single domain antibodies as
a neuronal cell intra antibody binding agents and inhibitors of clostridium
botulinam neurotoxin. Toxicon56(2010) 990-998
2.Pruszynski M, Koumarianou E et al.(2013).Targeting breast carcinoma with
radio iodinated anti HER- 2 nanobody. Nuclear medicine & biology40
(2013)52-59
3.Vaneycken I, Dhuyvetter M et al.(2011). Immuno imaging using
nanobodies. Current opinion in biotechnology (2011) 22:877-881
4.Kolkma ja ,Law D A et al.(2010).Nanobodies from Ilamas to therapeutic
proteins .Drug discovery today-tecnologis vol 7.no:2,(2010)
5.Harmsen M M, De Hard H et al.(2007) Properties production & application
of camelid single domain antibodies fragments. Appl microbial
biotecnol(2007) 77:13-22

29. THANK YOU