Camel Antibodies and Nanobodies: Improved Variants of Monoclonal Antibodies

Serge Muyldermans

Overview of the todays presentation

1. What are Camel antibodies and Nanobodies 2. Advantages of Nbs over other Ag-binding fragments 3. Humanising Nbs, making bivalent, bispecific Nbs and Nb-fusions to optimise potency 4. Therapeutic potential of Nbs & their derivatives

Camel antibodies and nanobodies Slide 2 3-6-08

Camel Antibodies and Nanobodies: Improved Variants of Monoclonal Antibodies

1. 2. 3. 4. What are Camel antibodies and Nanobodies Advantages of Nbs over other Ag-binding fragments Humanising Nbs, making bivalent, bispecific Nbs and Nbfusions to optimise potency Therapeutic potential of Nbs & their derivatives.

Artilodactyla
Camelus dromedarius Camelus bactrianus Lama glama Lama guanoco Lama alpaca Lama vicugna

Tylopoda

Camelidae Suidae Hyppopotamidae Tayassuidae Tragulidae Cervidae Giraffidae Antilocapridae Bovidae

Suiformes

Ruminantia

Antilopinae Cephalophinae Hippotraginae Bovinae Caprinae

Camel antibodies and nanobodies Slide 4 3-6-08

Camel & Llama antibodies

Fv Fab
CH2

Fc
CH3

Classical antibody (IgG1)

scFv

CH2

Fc
CH3
Camel antibodies and nanobodies Slide 5 3-6-08

Camel Heavy-Chain antibody (IgG2 & IgG3)

Single domain antigen binding NANOBODY fragment (15 kDa)

Monomeric Prolate particle:

Diameter 2.4 nm Height 4 nm
Hamers-Casterman et al., Nature, 1993

Origin of camel HCAb

H-chain gene for conventional Ab
AGTGTGG GTAAGT

Poly-A

Poly-A

VH CH1 hinge CH2 CH3 M1 M2

H-chain gene for Heavy-chain Ab

CGTGTGG ATAAGT

Poly-A

Poly-A

VHH CH1 hinge CH2 CH3 M1 M2

Camel antibodies and nanobodies Slide 6 3-6-08

Nguyen et al., Mol. Immunol., 1999 Zou et al., J.Immunol., 2005

N VH and VHH differences

VH

CDR1 VH

CDR2

CDR3

VHH

VHH CDR1

V37 G44 L45 W47

CDR2
F37 E44 R45 G47

CDR3

Camel antibodies and nanobodies Slide 7 3-6-08

Vu et al., Mol. Immunol., 1997 Desmyter et al., Nat.Struct.Biol., 1996

V-D-J rearrangement produces VHH

Ig-promotor Leader signal peptide CDR1 CDR2 RSS

~ 50 VH genes

CDR3

IgG1
6 JH-genes

D-genes

IgG2 IgG3

~ 40 VHH-genes
(VH3)

7 subfamilies

Position of Cys in CDR1 CDR2 length

Camel antibodies and nanobodies Slide 8 3-6-08

Nguyen et al., EMBO J., 2000

Camel Antibodies and Nanobodies: Improved Variants of Monoclonal Antibodies

1. 2. 3. 4. What are Camel antibodies and Nanobodies Advantages of Nbs over other Ag-binding fragments Humanising Nbs, making bivalent, bispecific Nbs and Nbfusions to optimise potency Therapeutic potential of Nbs & their derivatives.

Animalarium : CVRL, Duba, UAE

Camel antibodies and nanobodies Slide 10 3-6-08

Selection of antigen-specific VHH

Collect blood Isolate lymphocytes

Extract mRNA

RT-PCR Select Ag-specific VHHs by panning

Immunize camel

Produce soluble antigen-specific VHH

Make library of ~ 107 transformants

Camel antibodies and nanobodies Slide 11 3-6-08

Monoclonal antibody fragments

Ghahroudi et al., FEBS Letters, 1997 Lauwereys et al., EMBO J., 1998

Nb properties versus scFv and Fab

Identification of Ag binders Expression yields Stability Solubility Antigen specificity Affinity for the Ag Easy tailoring & intrabody Targeting unique epitopes
Camel antibodies and nanobodies Slide 12 3-6-08

Nb > scFv = Fab Nb > scFv = Fab Nb > Fab > scFv Nb > Fab > scFv Nb = Fab = scFv Nb = Fab = scFv Nb > scFv = Fab Nb scFv = Fab

VHH targets unique epitopes

Hen egg-white lysozyme

Serum HCAbs

D1.3 (mouse Fv)

Nb-Lys3 (camel VHH)

Non-canonical CDR loops Protruding CDR3 loop CDR3 inserts into catalytic site of lysozyme

Camel antibodies and nanobodies Slide 13 3-6-08

De Genst et al., Proc.Natl.Acad.Sci.US, 2006 Desmyter et al., Nat.Struct.Biol., 1996

Nb-GFP specificity

Camel antibodies and nanobodies Slide 14 3-6-08

Rothbauer et al., Mol Cell Prot, 2008

Supportive Nb engineering
Reporter tool Human in vivo imaging

Generic Nb
N

Tagged Nb
C

Humanized Nb

X-body & Directional immobilization

Capturing tool
Camel antibodies and nanobodies Slide 15 3-6-08

Nbs in biosensor & -array

RU 200

150

100 Resp. Diff.

50

-50 0 100 200 300 400 Tim e 500 600 700 800 s

Camel antibodies and nanobodies Slide 16 3-6-08

Camel Antibodies and Nanobodies: Improved Variants of Monoclonal Antibodies

1. 2. 3. 4. What are Camel antibodies and Nanobodies Advantages of Nbs over other Ag-binding fragments Humanising Nbs, making bivalent, bispecific Nbs and Nbfusions to optimise potency 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

xIxxxxxxTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR xIxxxxxx--------- -----Q--A---V-------KP----I----A * * * * * * * * * * 50 a 55 60 65 70 75 80 abc 85 90 94 human VH consensus camel VHH

xxxxxxxxxxx WGQGTLVTVSS xxxxxxxxxxxxxxxxx -----Q----* * 103 113

Camel antibodies and nanobodies Slide 18 3-6-08

Saerens et al., J.Mol.Biol., 2005 Conrath et al., J.Mol.Biol., 2005

Tailoring into pluripotent constructs

Bivalent constructs Bispecific constructs

CH2

Fc
CH3

Bifunctional constructs
Linker = hinge
good serum stability good functionality good expression

Immuno-toxin

Nb-enzyme conjugate
Camel antibodies and nanobodies Slide 19 3-6-08

Conrath et al., J.Biol.Chem., 2001

Molecular imaging: In vivo cell staining

GFP -actin

GFP

Lamin

GFP

PCNA

+ Anti-GFP chromobody

Histone H2B

GFP Nb mRFP

+ Anti-GFP chromobody

Nb

mRFP

Camel antibodies and nanobodies Slide 20 3-6-08

Rothbauer et al., Nat.Meth., 2006

Molecular imaging: In vivo cell staining

+ chromobody: anti-cytokeratin 8

Nb

mRFP

anti-Lamin

Nb

mRFP

Camel antibodies and nanobodies Slide 21 3-6-08

Rothbauer et al., Nat.Meth., 2006

Nb-GFP capturing GFP fusions

Camel antibodies and nanobodies Slide 22 3-6-08

Rothbauer et al., Mol Cell Prot, 2008

Camel Antibodies and Nanobodies: Improved Variants of Monoclonal Antibodies

1. 2. 3. 4. What are Camel antibodies and Nanobodies Advantages of Nbs over other Ag-binding fragments Humanising Nbs, making bivalent, bispecific Nbs and Nbfusions to optimise potency Therapeutic potential of Nbs & their derivatives

Application in therapy
CYTOTOXIC AGENT ( PDM )

(ADEPT principle)

ADEPT= Ab dependent enzyme prodrug therapy

Prodrug CCM

ENZYME ( -LACTAMASE)

Nb-CEA

Tumor cell Healthy cell

CEA

Camel antibodies and nanobodies Slide 24 3-6-08

Retamozo et al., Cancer Res., 2004

Therapeutic effect of ADEPT

cAb-Lys3::L + 150 mgCCM/kg Control PDM (Toxine) Lys3:L + 150mg CCM/kg CEA5:L + 100mg CCM/kg 1500 1200 900 600 300 0 0 15 30 45 60 75 90 cAb-CEA5:: L + 200 mg CCM/kg CEA5:L + 150mg CCM/kg CEA5:L + 200mg CCM/kg

tumor volume (mm)

1800

Treatment

Days after tumor implantation

Camel antibodies and nanobodies Slide 25 3-6-08

Retamozo et al., Cancer Res., 2004

Nbs against African trypanosomes

Mammalian host

Camel antibodies and nanobodies Slide 26 3-6-08

Antigenic variation
Parasites / ml blood (x106)

Trypanosomosis
500

250

(4) (2)

(3)

Hypervariable immunodominant

0 0 10 20 30 40 50

(1)

Days post infection

More conserved regions

Camel antibodies and nanobodies Slide 27 3-6-08

SRA mechanism of resistance

SRA interacting domain

Camel antibodies and nanobodies Slide 28 3-6-08

Pays et al., Nat. Reviews, 2006

Only Nbs access conserved epitopes

Rabbit Poly Ab Nb-An33

AnTat 1.1

MITat 1.4 Hypervariable regions

Nb-An33 binds conserved carbohydrate

More conserved regions

Camel antibodies and nanobodies Slide 29 3-6-08

Stijlemans et al., J.Biol.Chem., 2004

Nb::Apo L1 against trypanosomosis

NbAn33

Camel antibodies and nanobodies Slide 30 3-6-08

Baral et al., Nat. Med., 2006

Nb-Tr apo L1 mediated trypanolysis

In vitro trypanolysis In vivo trypanolysis

Targeted Non-targeted Control Camel antibodies and nanobodies Slide 31 3-6-08

Baral et al., Nat. Med., 2006

Nb-apoL-I therapy during chronic infection & to remediate associated pathology

Targeted Non-targeted Control

Infection associated pathologies (day 25)

Targeted Non-targeted Control

Camel antibodies and nanobodies Slide 32 3-6-08

Baral et al., Nat. Med., 2006

Acknowledgments
Project Leader Conrath Katja (till 2006) Non-VIB collaborations CVRL (Dubai, UAE) ABLYNX Postdocs K. Andersson (Uppsala) & D. Altschuh (Strasbourg, QSAR) De Genst Erwin, M. Brggemann (Cambridge, camel-mouse) Hassanzadeh Gholamreza (NSF), C. Cambillau (CNRS,Marseilles) Saerens Dirk (2005) C. Dobson (Oxf. & Cam.) & A. Matagne (Ulg, folding) F. Frederix, (IMEC) M. Sara (Vienna, Biosensors) PhD students R. Harrison (U. Liverpool, viper toxin) Nguyen Viet Khong (2001) M. El Ayeb & B. Bouhaouala (Institut Pasteur Tunis) Pellis Mireille H. Leonhardt (U. Mnchen, chromobodies) Vincke Ceile M. Przybylski (U. Constanz, peptibodies) Deschacht Nick E. Pays (ULB, Gosselies, BE, Trypanosomes) Govaert Jochen A. Bossuyt & T. Lahoutte (UZ-VUB) Scientists joining Ablynx M. Lauwereys, K. Silence, T. Laeremans, H. Revets
Camel antibodies and nanobodies Slide 33 3-6-08

VIB collaborations Prof. 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.