Camel Nanotechnologies and its Medical Applications

1
Camel Nanotechnologies and its Medical
Applications

• Prof. Dr. Serge Muyldermans

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Overview of the todays presentation

• Structural properties of functional Heavy-chain
  antibodies in Camelidae Nanobodies
• Natural generation of Heavy-chain antibodies
• Selection of antigen-specific Nanobodies
• Nanobodies in medical applications

3
Camel Nanotechnology and its medical Applications

• Structural properties of functional Heavy-chain
  antibodies in Camelidae Nanobodies
• Natural generation of functional Heavy chain
  antibodies
• Selection of antigen-specific Nanobodies
• Nanobodies in medical applications

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Pros Cons of antibodies

• Antibodies are of medical interest because
• Limitless diversity
• High specificity
• High affinity
• Yet, far from being perfect because
• Immunogenicity
• Unwanted effector function
• Solubility
• Stability
• Yield
• Size
• SOLUTION Look in nature on how to make
  antibodies smaller

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Artilodactyla
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Serum immunoglobulins
Serum from camelids IgG purification on Protein
A /Protein G
Protein G 2.7 - 3.5 Protein A
IgG1 IgG2 IgG3
H2L2 conv. Ab
H2 HCAb
H
L
Hamers et al., Nature, 1993
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Camelid antibodies
Fv
Fab
Classical antibody (IgG1)
Single domain antigen binding fragment (15 kDa)
NANOBODY
Camel Heavy-Chain antibody (IgG2 IgG3)
Monomeric Prolate particle Diameter 2.4 nm
Height 4 nm
Hamers et al., Nature, 1993
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Camel Nanotechnology and its medical Applications

• Structural properties of functional Heavy-chain
  antibodies in Camelidae Nanobodies
• Natural generation of functional Heavy chain
  antibodies
• Selection of antigen-specific Nanobodies
• Nanobodies in medical applications

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VH and VHH differences
VH
W47
V37
G44
L45
CDR1
CDR2
CDR3
VHH
F37
E44
R45
G47
Vu et al., Mol. Immunol., 1997
Desmyter et al., Nat.Struct.Biol., 1996
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IgH locus
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IgH locus of camelids
Cg1
Cm1
Cm2
Cm3
Cm4
Ca1
Ca2
Ca3
h
Cg2
Cg3
Sequential arrangement of C-genes (over 200 kb)
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V-D-J rearrangement produces VHH
Nguyen et al., EMBO J., 2000
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HCAbs lack CH1 domain
Dedicated H-chain gene for conventional Ab
Poly-A
Poly-A
AGTGTGG GTAAGT
VH
CH1
hinge
CH2
CH3
M1
M2
Dedicated H-chain gene for Heavy-chain Ab
Poly-A
Poly-A
CGTGTGG ATAAGT
VHH
CH1
hinge
CH2
CH3
M1
M2
Nguyen et al., Mol. Immunol., 1999 Zou et al.,
J.Immunol., 2005
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Camel Nanotechnology and its medical Applications

• Structural properties of functional Heavy-chain
  antibodies in Camelidae Nanobodies
• Natural generation of functional Heavy chain
  antibodies
• Selection of antigen-specific Nanobodies
• Nanobodies in medical applications

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Animalarium CVRL, Dubaï, UAE
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Dromedary immunization
Lauwereys et al., EMBO J., 1998
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Selection of antigen-specific VHH
Ghahroudi et al., FEBS Letters, 1997
Lauwereys et al., EMBO J., 1998
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Nb properties versus scFv and Fab
Nb gt scFv Fab Nb gt scFv Fab Nb gt Fab gt
scFv Nb gt Fab gt scFv Nb Fab scFv Nb Fab
scFv Nb gt scFv Fab Nb ? scFv Fab

• Efficient identification of Ag binders
• Good expression yields
• Good stability
• Good solubility
• Antigen specific
• High affinity for the Ag
• Easy tailoring
• Nbs target unique epitopes

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Tailoring into pluripotent constructs
Bivalent Conrath et al., JBC 2001 Bispecific
Conrath et al., JBC 2001 Pentavalent Zhang et
al., JMB 2004 Decavalent/bispecific Stone et
al., J Imm Meth 2007 Immuno-enzyme (ADEPT)
Cortez-Retamozo et al., Can Res
2004 Immuno-toxin Baral et al., Nat Med
2006 Chromobody Rothbauer et al., Nat Meth
2006 HCAb Hmila et al., Mol Immunol
2008 Scorpion (bispecific Fc effector function)
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Camel Nanotechnology and its medical Applications

• Structural properties of functional Heavy-chain
  antibodies in Camelidae Nanobodies
• Natural generation of functional Heavy chain
  antibodies
• Selection of antigen-specific Nanobodies
• Nanobodies in medical applications

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Blood retention vs Ab size
Residence time in blood
Several minutes
Several weeks
Fab
IgG
scFv
Size
Nb
diabody
Renal cut off
trimers, tetramers, ...
Secondary binding
anti-HSA or Pegylation
FcRn
Engineering of CHO of IgG or mutagenesis of
interacting AA (e.g. H310, H435)
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Tumor targeting

• OBJECTIVE
• Search for maximal tumor load and fastest blood
  clearance
• POSSIBILITIES
• scFv have minimal blood retention and
  consequently insufficient tumor load
• IgG have much longer blood retention, reasonable
  tumor laod but insufficient tumor penetration
• Minibody, scFv-Fc and HCAb are best performers
  (bivalecy size)

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Fused micro PET/CT images

• Mouse with tumor expressing EGFR
• 2.5 mCi 99mTc-7C12
• 1h p.i.
• CTSPECT 30 min

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Application in therapy (ADEPT principle)
ADEPT Ab dependent enzyme prodrug therapy
?
CEA
Retamozo et al., Cancer Res., 2004
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Therapeutic effect of ADEPT
Control
PDM (Toxine)
Lys3bL 150mg CCM/kg
1800
CEA5bL 100mg CCM/kg
1500
CEA5bL 150mg CCM/kg
CEA5bL 200mg CCM/kg
1200
tumor volume (mm³)
900
600
300
0
0
15
30
45
60
75
90
Treatment
Days after tumor implantation
Retamozo et al., Cancer Res., 2004
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Nbs against African trypanosomes
Mammalian host
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Antigenic variation
Hypervariable immunodominant
More conserved regions
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Trypanolytic Nbs
Stijlemans et al., J.Biol.Chem., 2004 Baral et
al., Nat Med., 2006
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Tech transfer spin off

• Foundation of Ablynx NV (December 2001)
• 70 M from venture capitalists (3 rounds)
• Sofinnova, Alto Partners, Abingworth, SR-one
• GIMV, KBC Private equity,
• (Gilde, VIB, Privak Biotech)
• November 2007 Introduction at EURONEXT
• Research collaborations
• Proctor Gamble (July 2004 April 2006)
• Genencor (2004), Centocor (2006)
• Novartis (2006), Kirin (2006)
• WYETH pharmaceuticals (212 M, anti-TNF)
• BOEHRINGER Ingelheim (265 M, alzheimer)
• Merck Serono Boehringer (XXXXXM)
• Achievements
• Phase Ib finished for ALX081 (anti thrombotic,
  December 2008)
• Phase I initiated for ALX681 (December 2008)
• Phase I announced for anti-TNF (Wyeth)
  December2009

Ablynx 2002 5 man 2003 10 man 2004 20
man 2005 40 man 2006 70 man 2007 90
man 2008 190 man
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Camel group partners
Prof. L. Wyns (ULTR) Prof. P. De Baetselier (CIMM)

• VIB-6 collaborations
• Loris Remy, Decanniere K.
• Magez Stefan, Stijlemans B., Toya BN.
• Revets Hilde (Ablynx), Cortez-Retamozo V., Huang
  L., De Groeve K., Kindt A.

• Postdocs
• Saerens Dirk (from mid 2005)
• Ghloamreza Hassanzadeh (from 2006)
• Conrath Katja (till mid 2006)
• De Genst Erwin (till 2006)
• Pardon Els (till mid 2006)
• PhD students
• Nguyen Viet Khong (till 2001)
• Pellis Mireille
• Vincke Ceçile
• Deschacht Nick
• Nguyen Throng
• Scientists joining Ablynx
• T. Laeremans, M. Lauwereys, K. Silence
• H.Revets (2006)

• Non-VIB collaborations
• Ablynx
• D. Altschuh (Strasbourg, QSAR)
• K. Andersson (Uppsala, QSAR)
• M. Brüggemann (Cambridge, camel-mouse)
• C. Cambillau (CNRS,Marseilles)
• C. Dobson (Oxford) A. Matagne (Ulg, folding)
• F. Frederix, . (IMEC, biosensors)
• R. Harrison (Liverpool, viper toxin)
• M. El Ayeb B. Bouhaouala (Institut Pasteur
  Tunis)
• H. Leonhardt U. Rothbauer (LMU, München)
• M. Przybylski (Constanz, peptibodies)
• M. Sara (Vienna, biosensors)

Dubaï (Dr Wernery Kinne)