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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 [email protected]
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.