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