muy lder mans
TRANSCRIPT
Camel Antibodies and
Nanobodies: Improved Variants
of Monoclonal Antibodies
Serge Muyldermans
Overview of the today’s presentation
1. What are Camel antibodies and Nanobodies
2. Advantages of Nbs over other Ag-binding fragments
3. Humanising Nbs, making bivalent, bispecific
Slide3-6-08
Camel antibodies and nanobodies
3. Humanising Nbs, making bivalent, bispecific Nbs and Nb-fusions to optimise potency
4. Therapeutic potential of Nbs & their derivatives
2
1. What are Camel antibodies and Nanobodies
2. Advantages of Nbs over other Ag-binding fragments
Camel Antibodies and Nanobodies: Improved Variants of Monoclonal Antibodies
3. Humanising Nbs, making bivalent, bispecific Nbs and Nb-fusions to optimise potency
4. Therapeutic potential of Nbs & their derivatives.
Artilodactyla
Tylopoda Camelidae
Camelus dromedariusCamelus bactrianusLama glamaLama guanocoLama alpacaLama vicugna
Suiformes
Suidae
Hyppopotamidae
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Camel antibodies and nanobodies
Ruminantia
Suiformes
Tragulidae
Cervidae
Giraffidae
Antilocapridae
Bovidae
Tayassuidae
AntilopinaeCephalophinaeHippotraginaeBovinaeCaprinae
4
Camel & Llama antibodies
Fab
Fv
scFv
CH3
CH2
FcClassical antibody
(IgG1)
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Camel antibodies and nanobodies
Single domain antigen binding fragment (15 kDa)NANOBODY
MonomericProlate particle:
Diameter 2.4 nm Height 4 nm
CH2
CH3
Fc
(IgG1)
Camel Heavy-Chain antibody
(IgG2 & IgG3)
Hamers-Casterman et al., Nature, 19935
Origin of camel HCAb
H-chain gene for conventional Ab
CH1 hinge CH2 CH3
Poly-A Poly-A
M1 M2
VH
AGTGTGG GTAAGT
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Camel antibodies and nanobodies
CH1 hinge CH2 CH3
Poly-A Poly-A
M1
H-chain gene for Heavy-chain Ab
VHH
CGTGTGG ATAAGT
Nguyen et al., Mol. Immunol., 1999Zou et al., J.Immunol., 2005
M2
6
VH and VHH differences
VH
V3
7
G4
4
L4
5
W4
7
VH
N
C
CDR1 CDR2 CDR3
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Camel antibodies and nanobodies
CDR1 CDR2 CDR3
F3
7
E4
4
R4
5
G4
7
VHHVHH
N
C
Desmyter et al., Nat.Struct.Biol., 1996
Vu et al., Mol. Immunol., 19977
V-D-J rearrangement produces VHH
~ 50 VH genes
Ig-promotor Leader signal peptide
CDR1 CDR2
RSS
6 J -genesD-genes
IgG1CDR3
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Camel antibodies and nanobodies
6 JH-genesD-genes
IgG2IgG3
~ 40 VHH-genes(VH3) •Position of Cys in CDR1
•CDR2 length
7 subfamilies
Nguyen et al., EMBO J., 20008
1. What are Camel antibodies and Nanobodies
2. Advantages of Nbs over other Ag-binding fragments
Camel Antibodies and Nanobodies: Improved Variants of Monoclonal Antibodies
3. Humanising Nbs, making bivalent, bispecific Nbs and Nb-fusions to optimise potency
4. Therapeutic potential of Nbs & their derivatives.
Animalarium : CVRL, Dubaï, UAE
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Camel antibodies and nanobodies
10
Selection of antigen-specific VHH
Isolate lymphocytesCollectblood
Extract mRNA
RT-PCR
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Camel antibodies and nanobodies
Produce solubleantigen-specific VHH
Select Ag-specificVHHs by panning
Make libraryof ~ 107
transformants
Immunize camel
Ghahroudi et al., FEBS Letters, 1997
Lauwereys et al., EMBO J., 1998
Monoclonal
antibody fragments
11
Nb properties versus scFv and Fab
� Identification of Ag binders
� Expression yields
� Stability
� Solubility
Nb > scFv = Fab
Nb > scFv = Fab
Nb > Fab > scFv
Nb > Fab > scFv
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Camel antibodies and nanobodies
� Solubility
� Antigen specificity
� Affinity for the Ag
� Easy tailoring & intrabody
� Targeting unique epitopes
Nb > Fab > scFv
Nb = Fab = scFv
Nb = Fab = scFv
Nb > scFv = Fab
Nb ≠ scFv = Fab
12
VHH targets unique epitopes
Hen egg-white lysozyme
Nb-Lys3(camel VHH)
D1.3(mouse Fv)
Serum HCAbs
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Camel antibodies and nanobodies
• Non-canonical CDR loops
• Protruding CDR3 loop
• CDR3 inserts into catalytic siteof lysozyme
De Genst et al., Proc.Natl.Acad.Sci.US, 2006Desmyter et al., Nat.Struct.Biol., 1996
13
Nb-GFP specificity
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Camel antibodies and nanobodies
14 Rothbauer et al., Mol Cell Prot, 2008
Supportive Nb engineering
Human in vivo imagingReporter tool
Generic Nb
N
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Camel antibodies and nanobodies
Capturing tool
CXHumanized NbTagged Nb
X-body & Directional
immobilization
15
Nbs in biosensor & µ-array
50
100
150
200
Re
sp
. D
iff.
RU
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Camel antibodies and nanobodies
-50
0
0 100 200 300 400 500 600 700 800
Time s
16
1. What are Camel antibodies and Nanobodies
2. Advantages of Nbs over other Ag-binding fragments
Camel Antibodies and Nanobodies: Improved Variants of Monoclonal Antibodies
3. Humanising Nbs, making bivalent, bispecific Nbs and Nb-fusions to optimise potency
4. Therapeutic potential of Nbs & their derivatives.
Humanising Nbs towards human VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFS xxxxx WVRQAPGKGLEWVS
Q---------S--A------------Y-Y- xxxxx -F------ER-G-A* * * * * * * * * * *1 5 10 15 20 25 30 35 40 45 49
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Camel antibodies and nanobodies
human VH consensuscamel VHH
xIxxxxxxTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
xIxxxxxx--------- -----Q--A---V-------KP----I----A* * * * * * * * * *
50 a 55 60 65 70 75 80 abc 85 90 94
* *103 113
xxxxxxxxxxx WGQGTLVTVSS
xxxxxxxxxxxxxxxxx -----Q-----
Saerens et al., J.Mol.Biol., 2005Conrath et al., J.Mol.Biol., 2005
18
Tailoring into pluripotent constructs
Bivalent constructs
CH2
Bispecific constructs
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Camel antibodies and nanobodies
Linker = hinge
good serum stabilitygood functionalitygood expression
CH2
CH3
Fc
Conrath et al., J.Biol.Chem., 2001
�Immuno-toxin
Nb-enzyme conjugate
Bifunctional constructs
19
Molecular imaging: In vivo cell staining
GFP β-actin
GFP Lamin
GFP PCNA
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Camel antibodies and nanobodies
Nb mRFP
+ Anti-GFPchromobody
+ Anti-GFPchromobody Nb mRFP
GFPHistone H2B
Rothbauer et al., Nat.Meth., 200620
Molecular imaging: In vivo cell staining
Nb mRFP
+ chromobody:
anti-cytokeratin 8
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Camel antibodies and nanobodies
Nb mRFPanti-Lamin
Rothbauer et al., Nat.Meth., 200621
Nb-GFP capturing GFP fusions
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Camel antibodies and nanobodies
22 Rothbauer et al., Mol Cell Prot, 2008
1. What are Camel antibodies and Nanobodies
2. Advantages of Nbs over other Ag-binding fragments
Camel Antibodies and Nanobodies: Improved Variants of Monoclonal Antibodies
3. Humanising Nbs, making bivalent, bispecific Nbs and Nb-fusions to optimise potency
4. Therapeutic potential of Nbs & their derivatives
Application in therapy (ADEPT principle)
ADEPT= Ab dependent enzyme prodrug therapy
�ProdrugCCM
�CYTOTOXICAGENT
( PDM )
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Camel antibodies and nanobodies
Healthy cellTumor cell
Nb-CEA
ENZYME
( ββββ-LACTAMASE)
CEA
Retamozo et al., Cancer Res., 200424
Therapeutic effect of ADEPT
1200
1500
1800
Control
PDM (Toxine)
Lys3:βL + 150mg CCM/kg
CEA5:βL + 100mg CCM/kg
CEA5:βL + 150mg CCM/kg
CEA5:βL + 200mg CCM/kg
tumor volume (mm³)
cAb-Lys3::βL+ 150 mgCCM/kg
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Camel antibodies and nanobodies
0 15 30 45 60 75 900
300
600
900
1200 CEA5:βL + 200mg CCM/kg
Days after tumor implantation
tumor volume (mm³)
cAb-CEA5:: βL+ 200 mg CCM/kg
Retamozo et al., Cancer Res., 2004
Treatment
25
Nbs against African trypanosomes
Mammalian host
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Camel antibodies and nanobodies
26
Antigenic variation
Hypervariableimmunodominant
Trypanosomosis
0
250
500
(2)(3)
(4)
Para
sit
es /
ml b
loo
d (
x1
06) Trypanosomosis
0
250
500
(2)(3)
(4)
Para
sit
es /
ml b
loo
d (
x1
06)
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Camel antibodies and nanobodies
More conserved regions
0 10 20 30 40 50(1)
Days post infection
0 10 20 30 40 50(1)
Days post infection
27
SRA mechanism of resistance
N
C
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Camel antibodies and nanobodies
SRA interacting domain
C
28 Pays et al., Nat. Reviews, 2006
Only Nbs access conserved epitopes
Rabbit Poly Ab Nb-An33
AnTat 1.1
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Camel antibodies and nanobodies
Hypervariableregions
More conserved regions
MITat 1.4
Nb-An33 binds conserved carbohydrate
Stijlemans et al., J.Biol.Chem., 200429
Nb::Apo L1 against trypanosomosis
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Camel antibodies and nanobodies
NbAn33
Baral et al., Nat. Med., 200630
Nb-Tr apo L1 mediated trypanolysisIn vitro trypanolysis In vivo trypanolysis
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Camel antibodies and nanobodies
Targeted
Non-targeted
Control
Baral et al., Nat. Med., 200631
Nb-apoL-I therapy during chronic infection & to remediate associated pathology
Targeted
Non-targeted
Control
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Camel antibodies and nanobodies
Targeted
Non-targeted
Control
Infection associated pathologies (day 25)
Baral et al., Nat. Med., 200632
Acknowledgments
Non-VIB collaborations
CVRL (Dubai, UAE)
ABLYNX
K. Andersson (Uppsala) & D. Altschuh (Strasbourg, QSAR)
M. Brüggemann (Cambridge, camel-mouse)
C. Cambillau (CNRS,Marseilles)
C. Dobson (Oxf. & Cam.) & A. Matagne (Ulg, folding)
F. Frederix, (IMEC) M. Sara (Vienna, Biosensors)
Project Leader
Conrath Katja (till 2006)
Postdocs
De Genst Erwin,
Hassanzadeh Gholamreza (NSF),
Saerens Dirk (2005)
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Camel antibodies and nanobodies
F. Frederix, (IMEC) M. Sara (Vienna, Biosensors)
R. Harrison (U. Liverpool, viper toxin)
M. El Ayeb & B. Bouhaouala (Institut Pasteur Tunis)
H. Leonhardt (U. München, chromobodies)
M. Przybylski (U. Constanz, peptibodies)
E. Pays (ULB, Gosselies, BE, Trypanosomes)
A. Bossuyt & T. Lahoutte (UZ-VUB)
VIB collaborationsProf. L. Wyns (ULTR), Loris Remy, Decanniere K. Prof. P. De Baetselier (CIMM), Magez Stefan,
Stijlemans B., Baral T.N., Cortez-Retamozo V.,Huang L., De Groeve K., Kindt A.
PhD students
Nguyen Viet Khong (2001)
Pellis Mireille
Vincke Ceçile
Deschacht Nick
Govaert Jochen
Scientists joining Ablynx
M. Lauwereys, K. Silence,
T. Laeremans, H. Revets
33