The binding modes of sequence-based epitope and paratope mimetics :

real or imaginary mimicry ?

Danièle Altschuh

Tel. +33 (0)3 68 85 48 32E-mail daniele.altschuh@unistra.fr

Biosensor groupUMR 7242, CNRS – University of StrasbourgESBS, Parc d'innovation, Boulevard Sébastien Brant.BP 10413, 67412 ILLKIRCH CEDEX

1. QuestionIs functional mimicry based on structural mimicry ?

2. MethodologyPeptide synthesis and microarraysSurface Plasmon Resonance biosensing

3. Mimicry of a linear protein binding siteViral epitope

4. Mimicry of a structured protein binding siteParatope (antibody binding site)

Protein A – protein B  Mimic A – protein B  Protein A –mimic B 

Mutational analysis

Identical perturbations similar effects on binding affinity ?

Structural analysis (X-ray crystallography, NMR)

Similar interfaces architectures ?

Mutational analysis

12

34

12

3

4

1 2

3

4

12 3

4

Mutational analysis

Surface plasmon resonance for monitoring binding activity (Biosensor group)

• Detection of low affinity binding activity

• Quantitative information : kinetics, affinity stoichiometry

Peptide synthesis (Pepscan Therapeutics, The Netherlands; University of Montpellier, France)

• Peptide micro-arrays : Ala-scan, X-scan

• High throughput peptide synthesis and analysis (Pepscan Therapeutics)

• Constrained loops (Pepscan Therapeutics)

1

prismglassgold

Sensor Surface

Flow

Intensity

Angle

1 2

prismglassgold

2

Sensor Surface

Flow

RU

Time

1

2

Sensorgram

Surface plasmon resonance (SPR) biosensing

The BIACORE technology (GE Healthcare ‐ Biacore, Uppsala, Sweden)

The variation in SPR signal (position of the resonance angle) is expressed in arbitrary units called resonance unit : RU

It is expressed as a function of time. The resulting graph is called  a sensorgram.

1000 RU = 1ng protein/mm2

Biacore® SPR technology: http://www.biacore.com/technology

10

RU

Time (s)

Analyte injection phase

Post-injection phase Regeneration

The phases of a sensorgram

14000

15000

16000

17000

0 120 240 360Time (s)

Res

pons

e (R

U)

Glass

GoldLinker

Carboxylated dextran(non cross-linked)

Ligand

The sensor surface

SPR: from signal to information

• Detection of bindingIn particular low affinity

• Quantification of bindingKinetic (ka or kon, kd or koff), Equilibrium (Ka, Kd), thermodynamic (∆H, T∆S) parameters

• Binding mode Multivalent, heterogeneous…

• Molecular propertiesActive concentration, Multimerisation - Aggregation

t

R

R =ka ∗ A0 ∗ Rmax ∗ 1− e− ka ∗A0+ kd( )t( )

ka ∗ A0 + kd

Peptide 137 ‐ 151

Fab 57P 

(Recombinant antibody fragment ) VL

CL

VH

CH1

VL

CL

VH

CH1

CH3

CH2

CH3

CH2

VL

CL

VH

CH1

Antibody Fab

Tobacco mosaic virus protein (TMVP)

Image made with VMD (developed with NIH support by the Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana‐Champaign)

PDB code: 1ei7

Phage display (12-mer library)

135 140 145 150I R G T G S Y N R S S F E S S S G L V

H Y S R S S F D S P M L

T Y N R S S Y L A S W P

V L E P N R S S F L V A

Y P T P M L N R S S F T

N K T S F S P P P L S I

H A E D W S H R S S F G

I Q Q R A S F G P H G F

V Q P E P L P Q R A S F

TMVP(12mers)5

TMVP(12mers)2

TMVP(12mers)6

TMVP(12mers)11

TMVP(12mers)7/8

TMVP(12mers)9

TMVP(12mers)13

TMVP(12mers)14

TMVP(12mers)4 Q S A Q Y T I N R V M M

TMVP(12mers)12 Y P T G P K H W L L L T

Consensus N R S S F

K’A = 2.9 x 106 M-1

K’A = 1.8 x 106 M-1

Response %111081111121515225887817768100765649139111415

Peptide sequence122-136123-137124-138125-139126-140127-141128-142129-143130-144131-145132-146133-147134-148135-149136-150137-151138-152139-153140-154141-155142-156143-157144-158

125 130 135 140 145 150 155. . . . . . .

RSAINNLIVELIRGTGSYNRSSFESSSGLV WTSGPATRSAINNLIVELIRGTSAINNLIVELIRGTGAINNLIVELIRGTGSINNLIVELIRGTGSYNNLIVELIRGTGSYNNLIVELIRGTGSYNR LIVELIRGTGSYNRSIVELIRGTGSYNRSSVELIRGTGSYNRSSFELIRGTGSYNRSSFELIRGTGSYNRSSFESIRGTGSYNRSSFESSRGTGSYNRSSFESSSGTGSYNRSSFESSSGTGSYNRSSFESSSGLGSYNRSSFESSSGLVSYNRSSFESSSGLV WYNRSSFESSSGLV WTNRSSFESSSGLV WTSRSSFESSSGLV WTSGSSFESSSGLVWTSGPSFESSSGLV W TSGPAFESSSGLV W TSGPAT

Overlapping SPOTs peptides (15-mers)

Ala scan SPOT peptides

138 140 142 144 146 148 150

Peptide name G S Y N R S S F E S S S G L V % reactivity

WT G S Y N R S S F E S S S G L VG137A A S Y N R S S F E S S S G L VS138A G A Y N R S S F E S S S G L VY139A G S A N R S S F E S S S G L VN140A G S Y A R S S F E S S S G L VR141A G S Y N A S S F E S S S G L VS142A G S Y N R A S F E S S S G L VS143A G S Y N R S A F E S S S G L VF144A G S Y N R S S A E S S S G L VE145A G S Y N R S S F A S S S G L VS146A G S Y N R S S F E A S S G L VS147A G S Y N R S S F E S A S G L VS148A G S Y N R S S F E S S A G L VG149A G S Y N R S S F E S S S A L VL150A G S Y N R S S F E S S S G A VV150A G S Y N R S S F E S S S G L A

100999078141090522172

12388

114115

68134

. . . . . . .

G S Y N R S S F E S S S G L V

X-scan SPOT peptides (Fab57P)

Replaced amino-acid

Alanine A / / / /

Arginine R / / / /

Asparagine N / / / /

Aspartic acid D / / / /

Glutamine Q / / / /

Glutamic acid E / / / /

Glycine G / / / /

Histidine H / / / /

Isoleucine I / / / /

Leucine L / / / /

Lysine K / / / /

Methionine M / / / /

Phenylalanine F / / / /

Proline P / / / /

Serine S / / / /

Threonine T / / / /

Tryptophan W / / / /

Tyrosine Y / / / /

Valine V / / / /

Replacing amino-acid Y139 N140 R141 S142 S143 F141 E145 S146 L150S143 S143 G143

75-100 %

16-75 %

0-15 %

Reactivity

Y139 N140 R141 S142 S143 F141 E145 S146 L150S143 S143 G143

AntibodyfragmentAntibodyfragment

TMVPTMVP

AntibodyfragmentAntibodyfragment

PeptidePeptide

• Choulier et al. (2001) J. Immunol. Methods 249, 253‐264

• Choulier et al. (2002)  J. Immunol. Methods 259, 77‐86

137 138 139 140 141 142 143 144 145 146 147 148 149 150 151G S Y N R S S F E S S S G L V

Epitope identification ( Ala-scan, X-scan, phage display)

S 142  A, E, N

S 146  A

E 145  A

Experimental design

1.Immobilization Cys extended peptide antigen using thiol coupling chemistryProtein antigen using amine coupling chemistry

2. Reference surface Empty or unrelated peptides

3. Regeneration 10 to 100 mM HCl or NaOH

Antigen

FabFab

Comparative binding kinetics of Fab 57P

with protein and peptide antigen surfaces

0

100

200

300

0 400 800 1200

Protein S142A

Peptide C-S142A

Fab

Ag

Time (s)

Res

pons

e (R

U)

.

WT

S142

A

S142

E

S142

N

E145

A

S146

A

kd(10‐3 s‐1)

ka(104 M‐1 s‐1)

WT

S142

A

S142

E

S142

N

E145

A

S146

A

The peptide mimics some, but not all, binding properties of the parent protein

• Similar equilibrium affinities (Ka)• Similar effects on Ka of 4/5 replacements

• Different binding kinetics• Different effect on Ka of 1/5 replacements

Peptide mimic

Protein

Antibodies Antibody fragments CDR-derived synthetic binder

StrategyPeptides corresponding to the CDRs (Complementarity Determining Regions) of anti-gastrin antibodies are covalently coupled on a chemical scaffold

BINDING GASTRIN (COOP-CT-2004-512691)

Coordinators: Peter Timmerman, Rob MeloenPepscan Systems, Lelystad, The Netherlands (Website: www.pepscan.com)

Antibody (IgG)~ 150,000 da (~1360 residues, ~12400 atoms)

Light chain~ 25,000 da (~210 residues, ~ 2000 atoms)

Heavy chain~ 50,000 da (~470 residues, ~4200 atoms)CH3

CH2

-S-S-

-S-S-

-S-S-

CH 1

CL

VH

VL

Antibodies

CDR: Complementarity Determining Region

H1, H2, H3: CDR1, CDR2, CDR3 of the heavy chainL1, L2, L3: CDR1, CDR2, CDR3 of the light chain

L1

L2

L3

H3

H2

H1

VL VH

VL

CL

VH

CH1

VL

CL

VH

CH1

CH3

CH2

CH3

CH2

VL VH

Antibody Fv

Images made with VMD (developed with NIH support by the Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana‐Champaign)

Reference Antibody Antigen Disease Antibody CDRs

In vitro neutralisation assay

Casset et al. 2003 ST40 CD4 AIDS aas from 5 CDRs cyclic

Inhibition of viral particle production

Tsumoto et al., 2002 8D4 NS3 protease domaine of HCV

Hepatitis C H1Linear, cyclic

inhibition of NS3 protease activity

Park et al., 2000Berezov et al., 2001

rhumAb 4D5

(Herceptin)

HER2 receptor (neu, c-erbB2)

Breast ovarian cancer

variants of CDR-H3

Inhibition of cell proliferationDownmodulation of surface receptorsInhibition of cell transformation

Feng et al., 2005Qin et al., 2006Chang et al., 2007

Z12 TNF-alpha rheumatoid arthritis Various CDRs Inhibition of TNF-induced cytotoxicity

Feng et al., 1998 44aacb Mac-1 cell adherence H1, H2, H3 Inhibition of cell adherence

Levi et al., 1993Jackson et al., 1999Levi et al., 2000Heap et al., 2005

F58 gp120 of HIV-1 AIDS CDR-H3-cyclic

Neutralization of HIV-1

----- ----- ----- ----- ----- -----

Experimental design

1. Immobilization Cys extended G17 using thiol coupling chemistry

2. Reference surface Empty or unrelated peptides

3. Regeneration 10 to 100 mM HCl or NaOH

Gastrin

Antibodies scFvs Synthetic peptides

Aggregates / multimers (Binding stoichiometry )

Rmax (analyte)RLigand

Rmax

RLigand=

MMAnalyte

MMLigand

Rmax

RLigand

Rmax

RLigand=

2 x MMAnalyte

MMLigand

RmaxRLigand

Rmax

RLigand=

MMAnalyte

MMLigand

Bivalent analyte binding model to fit kinetic data

Langmuir and complex kinetics

Langmuir binding

Bivalent binding model

No fit

H H

L L

SS SSSS

Antibodies ScFvs Synthetic binders

Rodrigo Barderas, Ignacio Casal Laboratorio de Proteomica FuncionalCentro de Investigaciones Biologicas (CSIC)Madrid (Spain)

Barderas R, Shochat S, Timmerman P, Hollestelle MJ, Martínez-Torrecuadrada1 JL, Höppener JWM, Altschuh D, Meloen R, Casal JI (2007)Designing antibodies for the inhibition of gastrin activity in tumoral cell linesInt. J. Cancer 122: 2351-9

0

0 100 200 300 400 500 600 700 800

RU

Time900s

20

40

ka1 (1/Ms) kd1 (1/s) ka2 (1/RUs) kd2 (1/s) Rmax (RU) Chi2

3.58e6 5.56e‐4 55.3 250 33.3 0.201

mAb (0.3 ‐ 11 nM) injected on a gastrin surfaceG17

mAb

‐5

0

5

10

15

20

25

0 50 100 150 200Time (sec)

Repo

nse SPR (RU)

Injection Post-injection

1,24356

SPR affinity evaluations for mAbs : 

KD’ in the 0.1 nM ‐ 1 µMrange

0

10

20

30

40

0 50 100 150 200 250

RU

sTime

ka (1/Ms)   kd (1/s)   Rmax (RU)       KA (1/M)     KD (M)    Chi2

2.76e4       5.79e‐4      54.8 4.77e7       2.1e‐8     1.36

0

100

200

300

400

0 50 100 150 200 250

RU

sTime

ka (1/Ms)   kd (1/s)   Rmax (RU)    KA (1/M)   KD (M)   Chi21.29e5        0.108          575 1.19e6     8.38e‐7   14.3

G17

scFvscFvs (10 ‐ 1000 nM) injected on gastrin surfaces

SPR affinity evaluations for scFvs : 

KD in the 10 nM to 1µM range

H H

L L

SS SSSS

Antibodies ScFvs Synthetic binders

Peter Timmerman 1,2, Rob Meloen 1,3

1 Pepscan Therapeutics B.V., Zuidersluisweg 2, 8243 RC Lelystad, The 

Netherlands2Van 't Hoff Institute for Molecular Sciences, Faculty of Science, University 

of Amsterdam, The Netherlands3Academic Biomedical Centre, University Utrecht, The Netherlands

Timmerman P, Barderas R, Desmet J, Altschuh D, Shochat S, Hollestelle MJ, Hoppener JW, Monasterio A, Casal JI, Meloen RH. 2009. A combinatorial approach for the design of complementarity determining regions (CDR)‐derived peptidomimetics with in vitro anti‐tumoral activity. J. Biol. Chem. 2009, 284, 34126‐34134

Timmerman, Geifman Shochat S., Desmet J., Rodrigo Barderas R., Casal J.I., Meloen R.H., Altschuh D. 2010. Binding of CDR‐derived peptides is mechanistically different from that of high‐affinity parental antibodies. J. Molec. Recognit. 23: 559‐68

VH2

VH2 VH3

VL3

1 (bicyclic)

C

CC

C

CC

VH2 VH3

VL3

2 (tricyclic)

C C

C

CH3

CH3

H3C

syntheticplatform (’T3’)

bromomethylated aromatic scaffold

Small linear peptides are attached onto organic scaffolds making them conformationally constrained. This technology applies to single, double as well as triple loops, depending on the scaffold (T2, T3 or T4) that is used.

There is no need for side‐chain protection in the cyclization reaction of cysteine‐containing linear peptides onto bromomethylated aromatic scaffolds. The reaction is extremely fast and clean (y>95%) and runs to completion in <15 min. at room temperature.

The CLIPS technology (Pepscan Systems; www.pepscan.com)

Chemically Linked Peptides on Scaffolds

Timmerman P, Beld J, Puijk WC, Meloen RH (2005)  Rapid and quantitative cyclization of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces. ChemBioChem. 6: 821‐824.

Simulation of SPR binding curvesRe

spon

se (R

U)

Time (s)

0

200

400

600

800

1000

KD 1 µM

KD 10 µM

KD 100 µM

KD 1000 µM

0 30 60 90 120

Injection of synthetic binder at 50 µM on a gastrin surface with Rmax 1000 RU

0 100 200Time (s)

2000

4000

RU

0 100 200

Time (s)

2000

4000

RU

Illustration of “inconsistent” binding curves

Time (s)

Res

pons

e (R

U)

Ligand surface

Reference surface

950RU < Rmax < 1450RU

[SB] = 50µM

G17

SB

[SB] = 50µM

5b

1d

3 - 46 µM

1 - 19 µM

0

10

20

30

s0 100 200

Time

Response

RU

s0 100 200

Time

Response

RU

-20

0

20

5c

2 - 27 µM

4 - 62 µM

0

40

80

120

s0 100 200

TimeR

esponse

RU

s0 100 200

Time

Response

RU

0

20

40

60

2a

Illustration of “consistent” SPR binding curves

SPR affinity evaluations for synthetic binders : KD in the 1 to 5 x 10‐4 M rangeSPR affinity evaluations for synthetic binders : KD in the 1 to 5 x 10‐4 M range

BxPc3-cell bioassay

-5

15

35

55

75

95

% o

f inh

ibiti

on

cyclic+ T3

cyclic+ T3

cyclic+ T3

cyclic+ T2

cyclic+ T2

200 µM

100 µM

50 µM

25 µM

12.5 µM

negativecontrol

linearcyclic- T3

cyclic+ T3

3a 3b 1d 1h 2a 2b 4a 5c 1x

G17-dependent BxPc3 cell proliferation assays

Timmerman P, Barderas R, Desmet J, Altschuh D, Shochat S, Hollestelle MJ, Hoppener JW, Monasterio A, Casal JI, Meloen RH (2009) A combinatorial approach for the design of complementarity determining regions (CDR)-derived peptidomimetics with in vitro anti-tumoral activityJ. Biol. Chem. 284, 34126

Rodrigo Barderas, Ignacio Casal Laboratorio de Proteomica FuncionalCentro de Investigaciones Biologicas (CSIC)Madrid (Spain)

Reference Antibody Antigen Disease Antibody CDRs

In vitro neutralisation assay

Casset et al. 2003 ST40 CD4 AIDS aas from 5 CDRs cyclic

Inhibition of viral particle production

Tsumoto et al., 2002 8D4 NS3 protease domaine of HCV

Hepatitis C H1Linear, cyclic

inhibition of NS3 protease activity

Perosa et al., 2004 16D7 anti-human anti CD4 mAb HP2/6

autoimmune disease L2

Park et al., 2000Berezov et al., 2001

rhumAb 4D5

(Herceptin)

HER2 receptor (neu, c-erbB2)

Breast ovarian cancer

variants of CDR-H3

Inhibition of cell proliferationDownmodulation of surface receptorsInhibition of cell transformation

Feng et al., 2005Qin et al., 2006Chang et al., 2007

Z12 TNF-alpha rheumatoid arthritis Various CDRs Inhibition of TNF-induced cytotoxicity

Feng et al., 1998 44aacb Mac-1 cell adherence H1, H2, H3 Inhibition of cell adherence

Levi et al., 1993Jackson et al., 1999Levi et al., 2000Heap et al., 2005

F58 gp120 of HIV-1 AIDS CDR-H3-cyclic

Neutralization of HIV-1

----- ----- ----- ----- ----- -----

Timmermann et al. 2009 189DB3 Gastrin 17 Pancreatic cancer L3,H3,H2Linear, cyclic

Inhibition of cell proliferation

AntibodyAntibody

GastrinGastrin

MimicMimic

GastrinGastrin

FDMWGYAEEEEELWPGpEFDMWGYAEEEEELWPGAFDMWGYAEEEEELWPGCFDMWGYAEEEEELWPGDFDMWGYAEEEEELWPGE

……

FDMWGYAEEEEELWPGpEFDMWGYAEEEEELWPApEFDMWGYAEEEEELWPCpEFDMWGYAEEEEELWPDpEFDMWGYAEEEEELWPEpE

pE-1

GP

FL A

F

Y

W

G-2 P-3 W-4

L-5

OD

450n

m(A

U)

0.0

1.0

2.0

3.0

W

A-11 W-14

Gastrin 17 pE G P W L E E E E E A Y K W M D F C…

…

Mapping of G17 residues recognized by antibodies

X-scan of G17 using micro-arrays (Pepscan Therapeutics, The Netherlands)

E1  A G2  A P3  A

Fractional occupancy of binding sites

FDMWGYAEEEEELWPGE

SB 1h

SB 1d

Synthetic binders (SB)

G17 sequence:

FDMWGYAEEEEELWPGEG17 sequence:

Ala‐scan of gastrin (G17)

Antibodies and synthetic binders recognize different residues on G17Antibodies and synthetic binders recognize different residues on G17

Antibodies (mAb)

Binding as WT

Binding < WT

No binding

Binding << WT mAb 189DB3

%R m

ax

G17HELInsulin

1d 1h 1m 1n 1o 1p 1q 1r 1s 1t 1v 1w0

100

200 PAR10C3

PAR10D10D1.3/random

189DB3

nd * ** *

Selectivity of recognition

Salt-sensitivity of proteins recognition (SPR)

1d 1h 1m 1n 1o 1p 1q 1r 1s 1t 1v 1w

0

100

200%R m

ax

nd

400 mM NaCl

150 mM NaCl

SBs recognize different proteins using different binding modesSBs recognize different proteins using different binding modes

• Synthetic binders can be developed (based on antibody CDRs)

• Anti gastrin antibodies and their CDR-derived synthetic binders use different binding modes

• Random sequences may be as good a starting point as CDR sequences of high-affinity

antibodies

Paratope mimicry

SPR Susana SHOCHATBiosensor group, CNRS and University of Strasbourg, France

Antibodies Rodrigo BARDERAS, Ignacio CASALCellular assays Laboratorio de Proteomica Funcional, CSIC, Madrid, Spain

Project coordination Peter TIMMERMAN1,2, Rob MELOEN1,3

Synthetic binders1 Pepscan Therapeutics B.V, Lelystad, The Netherlands2Van 't Hoff Institute for Molecular Sciences, University of

Amsterdam3Academic Biomedical Centre, University Utrecht,

Epitope mimicry

SPR Laurence CHOULIER, Nathalie RAUFFER-BRUYEREIBMC – CNRS, Strasbourg, France

Antibodies Myriam BEN KHALIFA, Thierry VERNETInstitut de Biologie Structurale –CEA, Grenoble, France

Spot peptides Claude GRANIERUniversity of Montpellier, France

Journal Home:           http://www.frontiersin.org/immunology

Frontiers Special Topic

Synthetic binders based on the sequence of natural binders: real or imaginary mimicry ?

Researchers are invited to submit on or before the abstract submission date a max. 1 page abstract/outline of work related to the focus of the special topic directly to the host editor for inclusion as an elaborated full article.