THEJOURNAL
OF BIOLOGICAL
CHEMISTRY
0 1994 by The American Society for Biochemistry and Molecular Biology, Inc
Vol. 269, No. 52, Issue of December 30, pp. 32788-32795, 1994
Printed in U.S.A.
Building Synthetic Antibodies as Adhesive Ligandsfor Integrins*
(Received for publication, June 10, 1994, and in revised form, September 12, 1994)
Jeffrey W.Smith+§,Dana HuS, Arnold Satterthwaitl, Sally Pinz-Sweeneyll, and
Carlos F. Barbas IIInll
From the Devartments o f PVascular (VB-1) andllMolecular
Biology (MB-ll), The Scripps Research Institute,
..
La Jolla, Caiifornia 92037
An antibody engineering strategy was employed to
ligand binding to integrins and maintained essentially
build high affinityligands and antagonists of integrins the same specificityas the parentantibody.
a,,& and cyIrn& Previously, we inserted the integrin recognition motif, RGD, into the antigen binding site of a
human antibody and selected the optimal flanking seIntegrin adhesion receptors are ap heterodimers that bind
quencesfrom a phage-display library (Barbas, C. F., extracellular matrix and plasma-adhesive proteins(1-3). InteLanguino, L. R., and Smith, J. W. (1993)Proc. Nutl. Acad.
grins direct numerouscell-matrix and cell-cell contacts and are
Sci. U. S. A. 90,10003-10007). The resulting antibody,
linked to organismal development and t o the progression of
Fab-9, blocked the function of integrin a,& but also
bound to the ligand binding site of platelet integrin several human diseases. The extensive primary structurehoa common gloanb&In thisreport, the antibody engineering effort has mology among all integrins indicates they have
been extended by 1)redesigning Fab-9 to achieve spec- bal fold and probably operate via similar mechanisms. Perhaps
ificity for platelet integrin aIm& 2) building non-RGD- the most important aspect of integrin function is the ligand
containing antibodies that bind the ligand binding site binding event. Ligand binding triggers cell adhesion and cellof both &-integrins, and 3) testing the hypothesis that cell contact and often generates a cascade of intracellular sigpeptides derived from complementaritydetermining re- nals thatchange cellular behavior (2,4). Complications arise in
gions (CDR) can be used to emulate the activity of the the studyof integrins because their natural ligandsoften bind
in a non-dissociable manner (5, 6) and because most adhesive
parent synthetic antibody.Thesegoalswereaccomplished by subjecting the original antibody, Fab-9,to a proteins bind multiple integrins on the cell surface.
“motif optimization” (MTF). A phage library was conSince the studyof integrin function is complicated by native
structed in which the residues flanking the RGD motif ligands that are
difficult to work with andoften yield data that
in Fab-9 weremaintained, but the RGDX sequence was are difficult to interpret,we have made an
effort to “build high
randomized. Thislibrary was panned on purified arm& affinity, well behaved, integrin ligands. We accomplished the
to identify high affinity binders.
Four function-blocking first step in this process by building Fab-9 (7). Fab-9 was obantibodies lacking RGD, but with specificity for aIrnp3, tained by redesigning a human antibody against the human
were characterized. The antibody with the highest pref- immunodeficiency virus coat protein gp120 (8) using a synerence for aIrn&,MTF-10, had an adhesion sequence of thetic strategy to insert
an RGD motif into thesequence of the
KGDN. This sequenceis similar in primary structure to heavy chain CDR3 (Fig. 1).By incorporating this motif into a
the active sequence within the disintegrin barbourin,
phage library where the
6 residues flanking theRGD sequence
which alsoantagonizes aIm& (Scarborough, R. M., Rose,
were randomized, a series of antibodies was obtainedby “panJ. W., HSU,M. A., Phillips, D. R., Fried, V.A, Campbel1,A
M., Nannizzi, L., and Charo, I. F. (1991)J.Biol. Chem. 266, ning” this library on purified integrin a,& (7). Fab-9, themost
potent of these antibodies, binds toav& nearly 1000-fold better
9359-9362).MTF-10 had a 70-fold higher affinity for am&
a,& and asp1.However, Fab-9 did not appear to distinthan
than a&,. Through our selection strategy, we also identified several antibodies that lack RGD but still blocked guish the two &-containing integrins, a,& and aIIbPs.
One of the objectives of this study was to determine whether
ligand binding to both integrins withhighaffinity.
Therefore, the RGD sequence is not necessary for a high further rounds of engineering and selection on the RGD motif
affinity interaction with the ligand binding site of p3- within HCDR3l of Fab-9 could produce an antibody with specAlthough murine antibodiesthat
integrins. Further investigation showed that the activ- ificity for either a,& or aIIb,Ps.
ity of inhibitory antibodies could be emulated by syn- preferentially recognize the ligand binding site of aIB& over
thetic peptides derived from the protein sequences of a,& have been raised via immunization (9, 10) andextensive
the antibody’s HCDR3. CDR-derived peptides blocked screening of snake venoms has identified barbourin, a ligand
that can distinguish aIIbPs
and av& (ll), theapplication of the
* This work was supported in part by National Institutes of Health synthetic antibody approach toward this end could supplant
Grants CA56483 and AR42750 and Grant 3RT-0258 from the Tobacco- conventional immunization, where the generationof functionrelated Disease Research Program of the University of California (to
J. W. S.). This is Manuscript 8365-VB from The Scripps ResearchInsti- blocking antibodies is largely fortuitous, and also obviate the
tute. The costsof publication of this article were defrayedin part by the need for cumbersome screening of biological samples for spebe hereby marked cific inhibitors. A second objective of this study was to deterpayment of page charges. This article must therefore
“uduertisement”in accordancewith18U.S.C.Section1734solely
to mine if the CDR of an antibody is an appropriate vehicle for the
indicate this fact.
6 An Established Investigator supportedby the American Heart Association and Genentech. Tiwhom correspondence should be addressed:The abbreviations used are: HCDR3, heavy chain complementarity
Dept. of Vascular Biology(VB-l),The Scripps ResearchInstitute, 10666 determining region three; CDR, complementarity determining region;
SPR, surface plasmon resonance; MTF,motif optimization; ELISA, enNorth Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-554-7107; Fax:
zyme-linked immunosorbent assay; Fmoc, N-(9-fluorenyl)methoxycar619-554-6402.
I/ A Scholar of the American Foundation for AIDS Research andthe bonyl; HPLC, high pressure liquid chromatography; PCR, polymerase
recipient of an InvestigatorAward from the Cancer Research Institute. chain reaction.
32788
Integrins
Against
Antibodies
Synthetic
32789
Ligand Binding and Inhibition
Studies-The ability of synthetic antibodies to antagonize the activity of aV& and u ~ was
~ ~compared
&
in
purified ligand-receptor binding assays. The method for these binding
studies has been documented (7, 21). Ideally, the same ligand would
have been usedfor inhibition studies, but fibrinogen
does not bind a,p3
when Ca2+ is present (22). In contrast, Ca2+ is essential for the rapid
binding of fibrinogento
anb& Similar complications arisewhen
vitronectin is considered becauseit does not bind identically tothe two
integrins (22). Therefore, we chose to use the optimal ligand for each
integrin, i.e. vitronectin for a,& and fibrinogen for arrb&.
Radiolabeled
MATERIALSANDMETHODS
ligands were used
at concentrations of 1nM, and binding was challenged
Library
Construction
and
Phage
Selection of Antibodieswith unlabeled antibody. Following a 3-h incubation, free ligand was
Oligonucleotides were purchased from OperonTechnologies (Alameda, removed by washing, and bound ligand was detected
by gamma countCA). PCR mutagenesis was performed to construct
a library of variants
ing. The data from this analysis were highly reproducible with the
within theHCDR3 of Fab-9 (7).PCR amplification was performed with difference betweendata points typicallybelow 11%. Data areexpressed
the Fab-9 cDNA as a template using the following two oligonucleotide
as the average of triplicate data points, and all experiments were reprimers: 1) CTCCTCCTCCTCCTCGACGTCCATATAATAGCAATTCC- peated at least four times.
TMNNMNNMNNMNNCCCAAACGAGCACCCCACTCTCGCACA
Surface Plasmon Resonance-Surface plasmon resonance (SPR) is a
AATA and 2) GCAAWAACCCTCACTAAAGGG, where N isA, C, G, or
means of assessing ligand affinities in real time (23, 24). SPR was
T and M is A orC. The PCR product was digested withXhoI and AatII performed usingthe BIAcore plasmon resonance instrument
from Pharand gel-purified. This product, 700 ng, was then ligated t o the Fab-9
macia Biotech Inc. Integrins were immobilized on the biosensor chip
construct in thepComb vector (3 pg) digested with the same restriction
withN-hydroxysuccinimide and N-ethyl-N"(3-diethylaminopropyl)carenzymes.
Introduction
of thisconstruct
by electroporation
into
bodiimide according tothe methods outlinedby Pharmacia. The sensor
Escherichia coli XL-1 Blue yielded a library of 3 x lo7 independent
clones, which vary only in theRGDX region. Subsequent steps were as surface wasfirst activated with N-hydroxysuccinimideand N-ethyl-N"
(3-diethylaminopropyl) carbodiimide. Integrins were
coupled by injectpreviously described to produce phage-displaying antibody fragments
ing 30 plof a 100pg/ml sample of purified integrin ontothe sensor chip
on their surfaces (7, 12).
surface. Ethanolamine was used t o block unreacted moieties on the
To select antibodies withspecificity for each integrin from the phage
sensor chip surface.
library, integrinsa,& or aIIbp3were immobilized in Costar 3690microThe association and dissociation rate constants( k , and k - , ) for syntiter plates in 50mM Tris-HC1, 150 mM NaCl, 1 mM MgCl,, 1 mM CaCl,,
thetic antibodies were obtained
from BIAcore measurements asfollows.
and 1 mM MnCl,, pH 7.4. Selection of phage-bearing antigen-specific
antibody fragments was performed by panning for six rounds as de- To derive k - l , a pulse-containing ligand was passed throughthe sensor
chip. Atthe endof the association phase, theflow was changedt o buffer
scribed (12)but with thefollowing modification. To select for antibodies
without ligand and the change in response unit (RU) was measured as
with specificity for one or the other integrin, the second integrin was
a function of time. The dissociation rate constant k-, is derived from
used as a soluble competitor at 20 pg/ml during panning of the phage
with the immobilized target integrin. For example,to select antibodies Equation 1.
specific for aIIbPa,this receptor wasimmobilized in microtiterwells, and
k-, = (In RU,IRU,)It - t,
0%. 1)
avp3 was added, in solution, to the phage library during the panning
step.
RU, is the initial response unit due to binding of antibody, and RU, is
Following selection of high affinity binders, soluble Fab was
prothe response unit remainingfollowing dissociation. Time is designated
duced by excision of the gIII fragment by digestion of the vector with
as t.
SpeI and NheI and subsequent religation as described (7, 12). Positive
To obtain the association rate constant k,, an antibody was passed
clones identified by simple ELISA and subsequent purification of isothrough
the sensor chip containing unoccupied integrin, and the related clones was performedby affinity chromatography with goat antisponse unit as a result of binding is again measured as a function of
human Fabcolumns. Dideoxy sequencing of double-stranded DNA was
k , , is calculated as shownin
time.Theassociationrateconstant,
performed with the primer GGGAAGTAGTCCTTGACCAGGC.
PeptideSynthesis-Syntheticpeptidesderived
from HCDR3 se- Equation 2.
quences of antibodies Fab-9, MTF-10, and MTF-32 were synthesized.
k, = [((dRU/dt)/RU)-k-,/L
2)(Eq.
Peptides from other antibodies were not synthesized or tested
for economic reasons. Synthesis was performed on Rink's amide resin (0.5
L is the concentration
of ligand. Measurements ofdRUldt are obtained
mmoVg, NovaBiochem) with anAdvanced Chemtech 350 multiplepepa t several ligand concentrations. The overall
K, of each binding event is
tide synthesizer using Fmoc synthesis (13). Amino acids were coupled
derived by simple division as shown in Equation 3.
with
diisopropylcarbodiimide
and 1-hydroxybenzotriazole monohydrate. The side-chainFmoc amino acids were protected by pentamethKD= k - , l k ,
(Eq. 3)
ylchroman-6-sulfonate for arginine, trityl for Cys, and butyl for Ser,
Duplicate measurementsof k - , yielded identical values in all cases. In
Thr, Asp, and Tyr. Each peptide was blocked at the N terminus by
acetylation andat the C terminus by amidation. Peptides werecleaved the caseof k,, the difference was typically lessthan 13% across several
from the resin with 10% trifluoroacetic acid/dichloromethane, and
pro- ligand concentrations.
Cell Adhesion and Platelet Aggregation Studies-The ability of retecting groups were removed with 5% water, 5% anisole,
2.5% ethanedicombinant antibodies t o support cell adhesion was examined with M21
thiol, 7.5% phenol in trifluoroacetic acid (14). Peptides were precipia&
tatedwithdiethyletherandpurified
on a C-18 column (Vydac melanoma cells, which adhere to vitronectin primarily through
(25). Cell adhesion was measured essentially as described (22,
26). The
201TP1022, 2.2 x 25 cm) with a 0-100% water, acetonitrile gradient
containing 1% trifluoroacetic acid. Cyclizations of cysteine peptides (1 wells of Titertek microtiter plates were coated with
a range of antibody
mg/ml) were carried to completion
in 20% dimethyl sulfoxide, H,O at pH
for 18 h a t 4 "C. M21 cells were harvestedfrom tissue culture flasks and
4.0 in 1 dayaccordingto Tam's procedure(15).Products from this placed in microtiter wells coated with the given antibody. Cells were
reaction were purified via HPLC.Fast atom bombardment mass spec- allowed to adhere to immobilized antibody for 1 h. Non-adherent cells
troscopy confirmed the molecular weight of the linear and cyclic pep- were removed by gentle washing, and bound cells were detected by
tides. The cyclic disulfide peptides were negative for free thiol when
colorimetric assay for acid phosphatase (26). Astandard curve with
cells
tested with Ellman's reagent (16).
in suspension showed that all absorbance values were directly
proporProtein Purification-Integrin aIbp3was purified from human plate- tional to cell number.
lets by afflnity chromatographyon KYGRGDS-Sepharose (17). Integrin
Platelet aggregation was performed with washed
human platelets
a,& was purified from a n octylglucoside extract of human placenta by
(27). Platelets were drawn into acid/citrate/dextrose and then purified
monoclonal antibody affinity chromatography (18). Vitronectin was pu- by gel filtration on Sepharose CL-2B. Aggregation was measured in
rified from human plasmaby heparin-agarose affinity chromatography Tyrode's buffer, containing 2mM Ca" and 100 pglml purified fibrinogen.
(19). Fibrinogen was purified
from plasma by cold-ethanol precipitation Platelets were stimulated with 20 p~ ADP. Aggregation was measured
(20). The Fab fragmentsof antibodies were affinity-purified from bacas a function of light transmission in a Scienco aggregometer. All agterial lysates by antibody affinity chromatography using goat anti-hu- gregation assays were performedat leasttwice with virtually identical
man IgG-Sepharose. Proteins were radiolabeled with Iodogen (Pierce). results between experimental repetitions.
display and identification of inhibitory peptide motifs. The prototypical RGD peptide, with sequence GRGDSP, does not
greatly distinguish among integrins that bind to RGD. Here,
we test the hypothesis that peptides with specificity for particular integrins can be designed based on the amino acid sequences of HCDRs in the antibodies selected from phage-display libraries.
Synthetic Antibodies Against Integrins
32790
Motil Position
Orlglnal Ubrary
t234587891011
--VGCXXXRGDXXXCYY
-?.-SI
Fab-9
--VGCSFGRGDIRNCYY
‘-
I
S
S
I
-VGCSFGXXXXRNCYY-
-
TARI.EI
Amino acidsequences of HCDRJ of synthetic antibodies
The protein sequencesof the randomized section of HCDR3 for Fab-9
and the MTF antibodies are shown. The residues randomized for the
motif optimization are shown inbold. The protein sequence was derived
from the nucleic acid sequence obtained from purified bacteriophage as
described under “Materials andMethods.” Antibodies MTF-2, -10, -32,
and -40 show substantial specificity for anh&Antibodies MTF-1, -12,
-15 (selected by panning on a”&)and MTF-7, -13, -14, and -20 (selected
by panning on all,@,)were derivedvia the competitive selection scheme
described under “Materials and Methods” but either showed substantially lower binding affinity than Fab-9 or did not distinguish between
a”&and allh&
by more than 3-fold in ELISA.
Synthetic
antibody
MTF Ubrary
-S.5-
FIG.1.Engineering and selection
of synthetic anti-integrin antibodiee. Many RGD motifs are displayed at theapex of a flexible loop
(42-44). Since antibodyCDRs (particularly HCDR3) often exist
a s flexible loops, we placed an integrin recognition sequence into this region.
To optimize antibody binding affinity, a phage library was constructed
in which the residues flanking the
RGD were randomized. The resulting
antibodies are expressed as afusion protein on the surface of bacteriophage (12). The optimal antibody
from our initial selection, Fab-9,
bound a,& and allh&with high affinity. In this report,a similar strategy was used to obtain specificity for platelet integrin allh&and to
identify inhibitory motifs that lack RGD. The sequences flanking the
RGDI from Fab-9 were maintained to preserve the optimal presentation, but positions 5-8 were randomized in anew phage library termed
MTF. Antibodies specific for al& were selected from this library by
competing with purified a,& in solution and subsequently panningon
purified all,,&.
RESULTS
Engineering Antibody Specificity by Motif OptimizationOne of the goals of this study was to determine whether antibodies that discriminate between the ligand binding pocket of
the two PI-integrins could be designed. To accomplish this task,
a “motif optimization”was performed starting with the
scaffold
of Fab-9, which binds to both receptors. The motif optimization
involved the construction of a modified phage library. Residues
corresponding to 1-4 and9-11 of the integrin bindingmotif in
Fab-9 (Fig.1)were maintained in thenew phage library. However, residues a t positions 5-9, corresponding to RGDI of Fab-9,
were randomized by oligonucleotide doping and PCR a s described under “Materials andMethods.” The complexity of this
library ensureda 99% probability that allof the possible amino
acid sequences were represented in the randomized 4-residue
motif. This phage library was screened
for antibodies thatcould
distinguish a,& from allhPR
using three rounds of competitive
selection as described under“Materialsand
Methods.” The
phage antibodies resulting from this selection were initially
tested for specificity by ELISA (not shown). Antibodies that
displayed a substantial preference for the target integrin in
ELISA were expressed as soluble Fab fragments in E. coli (7)
and then screened more rigorously by measuring their ability
to antagonize receptor activity in purified ligand-receptor binding assays.
Threeantibodies wereobtained that showed substantial
binding preference for allh&:MTFS, MTF-10, and MTF-32
(Table I). The abilityof these antibodies to interfere
with ligand
binding was measured with purified ligand-receptor binding
assays. MTF-2, -10, and -32 had lower IC,, values for allhPJ
than a,& in purified ligand-receptor binding assays. The
IC,, of
MTF-2 was 20-fold lower for alrh&than for a,& (Fig. 2 A ) .
MTF-10 exhibited a 50-fold greater ability to antagonize cyllh&
(Fig. 2B). In the sameassay, MTF-32 displayed a 15-fold preference for q r h & (not shown). All of the MTF antibodies exhibited a higher IC,, than the parent Fab-9, indicating that by
gaining specificity for aIIhP3,
some affinity for the integrin was
sacrificed. Interestingly, no antibodies were identified with the
competitive selection of thephagelibrarythat
displayed
CDR sequence
1-2-3-4-5-6-7-8-9-10-11’
Fab-9
MTF-2
MTF-10
MTF-32
MTF-40
C-S-F-G-R-G-D-I-R-N-C
C-S-F-G-R-T-D-Q-R-I-C
C-S-F-G-K-G-D-N-R-I-C
C-S-F-G-R-R-D-E-R-N-C
C-S-F-G-R-N-D-S-R-N-C
MTF- 1
MTF-12
MTF-15
MTF-7
MTF-13
MTF-14
MTF-20
C-S-F-G-R-V-D-D-R-N-C
C-S-F-G-R-A-D-R-R-N-C
C-S-F-G-R-S-V-D-R-N-C
C-S-F-G-K-R-D-M-R-N-C
C-S-F-G-R-W-D-A-R-N-C
C-S-F-G-R-Q-D-V-R-N-C
C-S-F-G-R-D-D-G-R-N-C
Numbers indicate motif positions.
greater than 3-fold binding specificity for a&
This suggests
that within thecontext of the Fab-9 scaffold, it isunlikely that
the adhesion motifs can be manipulated to gain substantial
specificity for a,& over allh&.
This is not surprising
because we
are unawareof any RGD-based ligands thatbind to a,& but not
to allh&’
One antibody, MTF-40, survived the competitive selection
process for q I h & and showed preference for this integrin by
SPR (see below) but did not display specificity in the purified
ligand-receptor binding assays. Plasmon resonance analysis of
the affinity for this Fab showed that in Ca2+ it has a 10-fold
higher affinity for allh&than a,& (see below) and also the
highest affinityfor allhB3(1 x
M) of any of the antibodies
derived from the phage libraries. Itlikely
is that thepreference
of this antibody for aIlhPRwas not detected in the inhibition
assays because it also retained very high affinity for a,&. The
association rate of the native ligands for both integrins is so
slow (22) that the ligand binding assaynot
is sensitive enough
to detect small differences in binding affinity if the affinity of
the antibody is very high. Asimilar scenario is evident for the
parent Fab-9, which showed no preference in ligand competition assays but has obvious
an
preference for a,& when binding
is measured directly with plasmon resonance (Table 11).
Since sequences corresponding to RGDI in Fab-9 were randomized in the MTF phage library (Fig.l), another advantage
of this selection scheme is that it has the potential identify
to
amino acid sequences other than RGD that could bind to the
integrin ligand binding pocket. In fact, none of the MTF antibodies contain the exact RGD motif, although in three of four
cases Arg and Asp are conserved. No functional substitutions
for Asp were observed in anyof the selected clones. In MTF-10,
the arginine was replaced by lysine, a substitution that has
been found in other ligandswith specificity for a,,&, including
barbourin (11).Four other antibodies lacking RGD were also
identified from this phage library that bound equally well to
a,& and allhPI.These antibodies did not display a substantial
preference for either a,& or all,,&in ELISA. This group of
antibodies also had IC,, values 20-50-fold higher than Fab-9,
consistent with a substantially lower binding affinity for integrin. The CDR sequences of these antibodies a t positions 5-9
were RWDA, RADR, and KRDM (Table I). One antibody with
32791
Synthetic Antibodies Against Integrins
A
.-GI
_I
MTF-llb-Ill0
TABLE
I1
The affinities of synthetic antibodies for a,& and alIbP3as measured
by surface plasmon resonance
The association (k, or ken) and dissociation (k-l or k,) rate constants
between synthetic antibodies and the two P,-integrins were determined
in real time with surface plasmon resonance. Data collection and analysis were performed with purified integrins and Fab fragments as described under "Materials and Methods." Measurements were made in
either 2 mM Ca2+or 0.2 mM Mn2+,both of which have been found to
saturate theligand binding response of the p,-integrins.
(2)
;:I
lo
nl
-- 1 1
Cation
I
-10
I
y
-9
-8
Log[MTF-Z]
B
(10)
MTF-llb-lllo
KGDN
2
Y
c
0
0
L
O
x
n
I
-7
Receptor Antibody KonW's-l)
Fab-9
1.3 x loL
Ca2+(2 mM)
cyII&
MTF-2 7.8 x lo4
MTF-10 1.7 x lo5
MTF-32 6.8 x lo5
MTF-40 7.7 x lo5
Fab-9
1.4 x lo6
aVp3 MTF-2 1.4 X lo4
MTF-103.4 x lo3
MTF-32 1.5 x lo5
MTF-40 1.0 x lo5
Fab-9
4.0 x lo3
aIm& MTF-2
1.8 x lo4
Mn2+( 0 . 2 m ~ )
MTF-10
4.3
x lo4
MTF-32 1.1x lo5
MTF-40 4.1 x lo4
Fab-9
2.4 x lo5
a,&
MTF-2
3.8
x lo4
MTF-103.6 x lo4
MTF-32 1.3 x lo5
MTF-40 1.1 x lo5
KO,&")
7.0 x
1.3 X
1.2 x
2.0 x
8.4 X
2.3 x
1.8x
10-4
10-3
10-3
10-3
10-4
10-3
10-3
1.7 x 10-3
1.0 x 10-3
1.1 x 10-3
6.6 x
1.7 x 10-4
3.6 x 10-4
1.2 x 10-4
1.5 x 10-4
6.5 x 10-5
1.0 x 10-3
1.8 x 10-3
8.7 x 10-4
9.6 x 10-4
KdU"
5.0 x
1.7 x
7.0 x
2.9 x
1.1x
10-9
10.'
10-5
10-9
10-9
1.6x 10-9
1.3 x 10-7
5.0 x 10-7
6.7 x 10.'
1.1 x
1.6 x 10"
1.0 x 10-8
8.3 x 10.'
1.0 x 10-9
3.6 x 10-9
2.8 x 10"'
2.6 x lo-'
5.0 x lo-'
6.7 X 10-9
8.8 x lo-'
lute on and off rates. SPR also enabled a measure of the effect
of different divalent ions on k, and k - l . Because divalent cations, like Ca2+and Mn2+,are known t o dramatically influence
10
\i#l
ligand binding to &-integrins (221, wemeasured the binding of
I
0
-11
-10
-9
-8
-7
the antibodies in the presence of both ions. In prior studies, it
log[MTF-IO]
was determined that ligand binding to either integrin could be
FIG.2. Motif optimization of Fab-9 generates antibodies with maximally supported by either 2 mM Ca2+or 200 V M Mn2+and
specificity for orm&. Antibodies with binding preference for arIbP3 that these ions had different effects on ligand association (22,
were generated by redesign of Fab-9 as described under "Materials and
28). These conditions were adopted for SPR analysis, and an
Methods" and Fig. 1. Following selection by panning and initial screening byELISA, the MTF antibodies were tested in ligand inhibition SPR "sensogram" showing the association and dissociation of
studies. The ability of two of these antibodies, MTF-2 (A) and MTF-10 MTF-10 with integrin aIm& is shown in Fig. 3. The marked
( B ) ,to block ligand binding is shown. Ligand binding to avp3(0)was influence of the type of divalent ion present on the binding of
measured with '251-vitronectin,and ligand binding to aImp3(0)
was MTF-10 is apparentfrom the slopes of the sensograms in Fig.
assessed with '251-fibrinogen.All data are expressed as the percent of
control binding in the absence of inhibitor. Data points are the average 3. The association of MTF-10 with cyIIb/.?, in Ca2+(Fig. 3 A ) is
of triplicate points in which the error was less than 11%of the total more rapid than inMn2+(Fig. 3B), leading to a correspondingly
specific binding. Nonspecific binding was determined by competition greater slope of the binding isotherm. The quantitative differwith RGD peptides and was normally less than 10%of the total bound ence between association in the presence of the two ions is
counts.
actually more dramatic than displayed because a higher concentration of MTF-10 had to be used in buffer containing Mn2+
the sequence RVDD had anIC,, value comparable with that of to obtain valid binding isotherms. The association rate conFab-9 but also failed to distinguish the two P,-integrins. In stant (k,) is derived from the slope of this phase as described
conjunction with the sequences of the other MTF antibodies under "Materials and Methods." To measure dissociation, the
(Table I), these data indicate that position 6 in the adhesion sensor chip was placed in buffer lacking free antibody (arrows),
motif (Fig. l), which corresponds to the Gly position in the
and antibody dissociation from arm& is readily observed as a
RGD, is highly permissive. This glycine can be substituted by descending phase in the sensogram.
Val, Ala, Asn, Arg,
Thr, Gln, Asp, Ser, and Trp. Consequently, a
The rate constants for the other MTF antibodies in these two
full steric spectrum of side chainsis tolerated in this position. ions is shown in Table 11. Regardless of the antibody tested,
Although several ligands for both a,P3 and uIIbP3have been association of antibodies with uIIbPswas always faster in ea2+.
identified, there hasbeen no systematic searchfor other poten- However, sequence changes in residues 5-9 did influence the
tial ligands. Our data indicate that such a search should not be magnitude of the cation preference as much as 5-fold.For MTFlimited to proteins containing RGD. The potential ligands for 40, the ratio of k,Mn2+/k,Ca2+
is 0.05, but for MTF-2,the ratio is
the &-integrins should be expanded to include proteins with 0.23. In contrast to aIIb&,
integrin a,P3 usually binds its ligands
(K/R)XD sequences.
better when Mn2+is present (22). In general, this trendis also
Measuring Ligand Association and Dissociation Constants maintained with the MTF antibodies. Fab-9 appears to be the
for Synthetic Antibodies-To further characterize the binding one exception because it binds faster in Ca". The type of divabetween the MTF antibodies and integrins, SPR was used (23, lent ion present can affect k , as much as 10-fold. However,the
24). In contrast to ligand competition assays, where ligand on rates of both MTF-32 and MTF-40 fora$, were very similar
affinity is a parameter and where association and dissociation regardless of the type of ion present. Antibody dissociation rate
cannot be distinguished, SPR allows a direct measure of abso- constants also varied with the type of divalent ion present, but
"1
I
32792
Synthetic Antibodies Against Integrins
600 I
Control
3
a
5xlOSM
1
',
2dO
4d0
do
Time (s)
860
71300
'
20xlOgM
50xIO9M
Fab-9
Control
5x1OW
c
3
a
100
20x10-W
50
'0
500
1000
1500
2000
2500
3000
Time (s)
FIG.3. Measuring the binding of MTF-10to integrins with surface plasmon resonance. Surface plasmon resonance was used to
measure on and off rates between MTF-10and a&.
The binding was
measured in buffer containing 2 m Ca2+( A )or 200 PM Mn2+( B ) .In A,
a solution containing 5 p g h l of MTF-10 waspassed through the sensor
chip containing immobilized aIB&. Because the association of antibody
occurs more slowly in Mn2+,in B , the concentration of solution phase
MTF-IO was increased to 25 pg/ml to obtain a valid binding isotherm.
Arrows denote the time at which solution phase was changed to buffer
lacking free MTF-10 to allow for dissociation of bound antibody from
cur,,,&. RU, response unit.
MTF-IO
FIG.4.Fab-9and MTF-10 block platelet aggregation.The ability
of Fab-9 (A) and MTF-10 ( B ) to block platelet aggregation was measured with washed human platelets. Human platelets (1 x 10') were
mixed with 100 pg/ml of purified fibrinogen and 2.0 mM Ca2+in Tyrode's
buffer.Thesewereplaced
in a glass aggregation tube. The indicated concentration of antibody was added, and platelets were stimulated with 20 PM ADP. Aggregationwas measured using a Scienco
aggregometer.
rI
there was no obvious correlation between the influence of ions
on k , and k-, for a given antibody. Consequently, divalent ions
can influence the ligand on-rate without necessarily influencing itsoff-rate. These data suggest thatligand association and
dissociation with integrinsare independently regulated by
divalent ions.
A Comparison of Fab-9 and MTF-10 in Platelet Aggregation
and Cell Adhesion-All of the initial selection and characterization of the MTF antibodies relied upon the use of purified
integrins. To test the activity of the MTF antibodies in a biological assay, we measured their ability to block platelet aggregation (Fig. 4). Washed platelets were placed in Tyrode's buffer
containing 2 m~ Ca2+ and100 pg/ml fibrinogen, and aggregation was stimulated by addition of 20 p~ ADP. Fab-9 wasused
as a reference, and it blocked 50% of the aggregation at a
concentration near 5 nM and completely blocked aggregation at
a concentration of 20 nM (Fig. 4A). Only slightly more MTF-10
was requiredfor inhibiting aggregation, with half-maximal inhibition a t 20 nM Fab and complete inhibition a t 100 r m (Fig.
4B).MTF-2 and MTF-40 were slightly better at blocking aggregation than MTF-10 with IC,, concentrations of 15 and 10
nM, respectively (not shown). The IC,, for the MTF antibodies
in platelet aggregation studies agree with theaffinity of each
antibody as measured by SPR. The ability of these synthetic
antibodies to abrogate platelet aggregation validates their potential as anti-thrombotic agents.
The antibody most selective for aIh& is MTF-10. To assess its
ability to interact with the sister integrin a,& in a biological
membrane, cell adhesion assays were performed using M21
melanoma cells because they adhere toRGD ligands primarily
via a,P3 (25). M21 cells were tested for adherence to a concentration rangeof immobilized Fab-9 orMTF-10. As shown in Fig.
5 , the cells were able to adhere
to Fab-9 butwere unable to bind
to MTF-10. Thus, the specificity exhibited by MTF-10 in puri-
Fab-9
." n
0.8
0.6
0.4
0.2
-
MTF- 10
V
0.0
[Antibody] (ug/rnl)
FIG.5. MTF-10 fails to support a,&-mediated cell adhesion.
The ability of Fab-9 (0)and MTF-10 (D) to support a&,-mediated cell
adhesion was compared with M21 melanoma cells. A range of each
antibody was immobilizedin microtiter wells for 18 h at 4 "C. Nonspecific binding sites on the plate were blocked with 0.1% bovine serum
albumin, and asuspension of cells were addedto each well. Followinga
I-h incubation period, non-adherent cells were removed
by gentle washing and aspiration. The relative number of cells in each well wasquantified with a colorimetric assay for lysosomal acid phosphatase using
the absorbance at 405 nm (26).
fied receptor assays isalso evident incell-based assays. Importantly, antibody MTF-10 will support the adhesion of Chinese
hamster ovary cells expressing recombinant human aIIbP3.2
Synthetic Peptides Derived from Antibody HCBR3 Protein
Sequences Faithfully Antagonize p3 Zntegrins-Phage display of
random peptide libraries is becoming standard technology in
protein biochemistry (29,30). Engineering synthetic
antibodies
by manipulating HCDR3 offers a unique vehicle for the expression and analysisof random peptide sequences.To determine if
the CDR is an appropriate vehicle for identifying inhibitory
peptide motifs from phage libraries, we measured theability of
synthetic peptides derivedfrom the sequences of Fab-9, MTF-2,
and MTF-40 to block ligand binding to selected integrins. PepJ. Smith, C. Barbas 111, and J. Loftus, manuscript in preparation.
Integrins
Against
Antibodies
Synthetic
A.
32793
Lin-F9
100 I90c
80 70-
T
.
I
.
,
r
60
x
u
D
-
50 -
!j
40
2
30
-
-
2ol
.
-I
1
10
’
0‘
I
-9
-7
-8
-5
-6
[Peptide]
6. GRGDSP
loor a
90
-
80
-
70 60
-
50
-
40
-
30 -
-9
-7
-8
-5
-6
[Peptide]
Peptide #40
C.
2
120
110
100
90
L
80
2
,“
v
’O
v
c
50
g
40
r-
60
-11
-9
-10
-8
-7
-6
-5
-4
[Peptide]
D.
=
Peptide # 2
120
110
100
90
;
0”
e
tides derived from the sequence of MTF-10, which has a KGD
sequence, werenot testedbecause peptides containingthis motif have beenpreviously shown to inhibit thefunction of a&,
(31, 32).
The RGD motif we originally inserted into the CDRcontained 2 flanking cysteine residues t o provide the opportunity
for constraining theflexibility of the RGD loop by the formation
ofa disulfide bond. Since it is notevident whether thisdisulfide
bond actually forms in theCDR of the antibody, both cyclic and
linear forms of each peptide were synthesized and tested. Cyclization was achieved as described under“Materialsand
Methods” and confirmed by HPLC and mass spectroscopy. In
the case of Fab-9, the linear peptides were synthesized with
cysteines (sequence of CSFGRGDIRNC) and with glycine bequence of GSFGRGDIRNG) to eliminate thepotential for peptide cyclization during the inhibition assay. Importantly, both
the linear (Cys and Gly forms) and cyclic forms of these peptideshadnearlyidentical
efficacy in all assays (datanot
shown). Consequently, we conclude that cyclization is not a
determinant in binding affinity for peptides derived from the
Fab-9 sequence.
The ability of synthetic peptides basedon the Fab-9sequence
to block ligandbinding t o av&, alIbP3,
and a,& in purified
ligand-receptor binding assays is shown in Fig. 6. In examining
the peptides for activity, there aretwo criteria for judging their
efficacy: affinity and specificity. The proto-typical RGD peptide,
GRGDSP, was used as a positive control in inhibition assays
because it antagonizes all three integrins (Fig. 6B). Interestingly, the CDR-derived peptide (Lin-9) inhibited ligand binding
t o a,& and aIIb&
butdidnot antagonize(Fig.
6A). Thus,
Lin-9 is the first
RGD-containing peptide, that we are awareof,
with specificity for a,& over CY&.It is important to note that
GRGDSP did antagonize
Infact, of thethreeintegrins
tested, a,& had the highest apparentaffinity for this peptide.
We conclude that the synthetic
peptide derived fromthe CDR3
of Fab-9 emulates the activity of the whole antibody and, like
Fab-9, maintains specificity for a,& over the related integrin
:
Synthetic peptides based on the sequence of HCDRB in antibodies MTF-2 and MTF-40 (sequence of GCSFGRTDQRNCY,
peptide 2; and GCSFGRNDSRNCY, peptide40) were also
tested for their ability to block ligand bindingto a,p3 and arrb&.
These twoexamples
were chosen because 1) MTF-2 and
MTF-40 have binding preference for aIIbP3
and 2) they provide
a good comparison to see if CDR-derived peptides maintain the
Antibody
samerank-order potency as theFabfragments.
MTF-40 has a much higher affinity than antibody MTF-2 for
aIIbp3,and peptide 40 completely blocked ligand binding to
anb& but only inhibited 40% of the ligand binding to a,P3 over
the range of concentration tested (Fig. 6C).We were unable to
test higher concentrations of peptide because several nonspe-
80
almP3(W. B , the same concentration range of peptide GRGDSP was
tested in an identical assay. All data are expressed as the percent of
E
50
control binding in the absence of inhibitor and are theaverage of triplicate data points. This is representative of four experiments in which
m0 40
nearly identical results were obtained in each repetition. C, peptides
u
30
derived from HCDR3 of MTF-40 (sequence = GCSFGRNDSRNCY)were
6m 2 0
.also synthesized in linear (filled symbols) and cyclic forms (open symA
’0
b o k ) .The ability of these peptides toblock ligand bindingto a,& (V,0)
01’
’
-11 -10 --89
-7
-6
-5 - 4
and allbPa
(0,V ) were compared. All data are expressed as the percent
of control binding in the absence of inhibitor and are the average of
LPsptidsJ
triplicate values. This experiment is representativeof three repetitions
FIG.6. Synthetic peptides derived from synthetic antibodies in which nearly identical results were obtained. D, similar measureinhibit integrin function and display the specificity
of the whole ments were madefor peptides derivedfrom HCDR3 of MTF-2 (sequence
antibody. Peptides derived from the CDFU protein sequence of Fab-9 = GCSFGRTDQRNCY). Linear (filled symbols) and cyclic (open sym(GSFGRGDIRNG and CSFGRGDIRNC) were synthesized in linear and bols) peptides were tested for the ability to block ligand binding to a,P3
cyclic forms as described under “Materials andMethods.” A, the linear (V,0)
and allbP3(0,VI. Again, the data are the average of triplicate
form of this peptide, Lin-9, was tested for its ability to block lZ5I- points, and the experiment is representative of three repetitions with
vitronectin binding t o a$, (V), avP5(0)and ‘251-fibrinogenbinding to similar results.
v
70
60
0
,
8
8
1
32794
Synthetic Antibodies AgainstIntegrins
cific peptides began to disrupt ligand binding in this assayat
concentrations of 5 x
M. Peptide 2 also abolished fibrinogen
binding to aImP3but did not interfere with ligand binding to
a$, over the range tested (Fig. 6D). These data show that
synthetic peptides derived from CDR3 sequences of MTF-2 and
MTF-40 have the same integrinpreference as the parent antibodies. The peptides alsohave the same rank-order
potency as
IC,,of peptide 40 is 5 x
the antibodies. For integrinthe
M, and that of peptide 2 is 2 x
M. Like peptides based
on the Fab-9 sequence, the activity of peptide 2 or 40 was not
enhanced by cyclization (data not shown).
only a 20-fold lower IC,, for a$, than aIrbP3
(32). Collectively,
these data suggest that itwill be substantially more challenging to obtain a n RGD-based antagonist with a large degree of
selection for a&. Since we exhausted allof the potential natural amino acid sequences with the MTF selection strategy, it is
likely that a$,-specific antagonists will require access to functional moieties not present in the20 amino acids and will have
to be organically synthesized. This does not exclude the possibility that antibodies can be further engineered to gain selection for a,&. For example, the HCDR3 of Fab-9 can be recombined with libraries of light chains to build in other contact
sites specific for a$,. Alternatively, five other CDRs are presDISCUSSION
ent in the antibody and could be genetically manipulated to
Numerous integrin ligands have been identified. These in- achieve the same end.
clude extracellular matrix proteins, plasma adhesive proteins,
It is somewhat surprising that our
selection strategy did not
cell-bound adhesive ligands, and venom proteins from snakes and identify the sequences flanking the RGD that are present in
ticks (2).All of these ligands have been optimized through millions natural extracellular matrix and
blood-borne adhesive proteins
of years of evolution. Here, we showthat integrin ligands and an(35). This discrepancy suggests that the integrin-ligand bindtagonists that maintaina high degree of specificity can be built by
ing pocket could accommodate more proteins than originally
using antibodies as a scaffold. We believe this is a significant step
thought. Our datashow that theRGD motif is certainly not an
toward the futuredesign of biologicalligands with desired binding
absolute requirement for occupation of the ligand binding site
characteristics. The synthetic approach described here may evenof the P,-integrins. Extending the definition of &-ligands to
tually obviate the need to randomly screen biological samples for
sequences of ( w R ) X D should be considered, and this would
inhibitors of protein-protein interactions and could also eliminate
vastly increase the number of potential natural ligands for
the cumbersome manipulations associated with conventional
integrins. This is illustrated by a computer search of the Prohybridoma technology.
tein Identification Resource Data base using the RTDQ seInterestingly, important differences were observed in the
way that the synthetic antibodies and native ligands bind to quence of antibody MTF-32. Of 56,849 sequences in the data
integrins. Native adhesive ligands for the integrins typically base, 3,026 contained RTDQ. Similar numbers were obtained
have verylow association rate constants but ultimately
bind in for RNDS, the active sequence within MTF-40. Consequently,
a non-dissociable manner (5, 6, 22). In contrast, our engineer- the pool of potential p,-ligands is much larger than originally
ing efforts generated synthetic antibodies that rapidly bound believed. The sequences we identified at motif positions 5-9
the ps integrins, with at least one interaction greater than 1x were also distinct from antibodies raised by immunization that
lo6 “‘s-’. This rate is 600-fold faster than the association of bind the ligand binding site of aIm/3,(9, 10). Antibodies derived
RYD or RHD motif in CDR3 and
vitronectin for a$, (22). However, unlike vitronectin, all of the from immunization all have an
antibody ligands bound in a completely dissociable manner. are specific for aI1,&,but our selection scheme did not identify
The inability of the synthetic antibodies to bind in a non-dis- any antibodies with this sequence. It is likely that antibodies
sociable manner indicates that the native ligands
probably derived from immunization, where in vivo selection of the anhave ancillarycontact surfaces aside from RGD that contribute tigen binding siteoften relies upon the collective binding affinity of six CDRs, also derive binding affinity from secondary
to the stabilized binding state. These hypothetical secondary
contact points are also a likely means to obtain anotherlevel of contacts with integrin that are contributed by other CDRs in
the antibody. It is unlikely that the antibodies described here
specificity between integrins and their naturalligands.
Attempts to build synthetic antibodies that could distinguish make contacts outside of HCDR3 because these contacts would
the two &integrins produced three antibodies in the MTF be entirely fortuitous. The parent antibody, hivl2, does not
series thatshow a substantial preference for a,&,, e.g. MTF-10 bind either integrin.
Much recent effort has been placed on screening for ligand
has a 70-fold lower KD for aIIbpsthan av&. Interestingly, the
same selection strategy to find antibodies with preference for motifs using random peptide libraries displayed on the surface
of phage (29), and some studies haveidentified integrin binding
a$, failed. Fab-9, which was originally identified by panning
on pure a,& does show preference for this integrin inplasmon peptides from phage libraries (36). Our approach differed substantially in that the
CDR of an antibody was used asa vehicle
resonance binding studies, but we did not identify antibodies
withgreater specificity by optimizing the motif in Fab-9. for presentation of the peptide motif. Therefore, we were unpeptides derived from the selected
Clearly, the method that must be used to identify specific an- sure as to whether synthetic
tibodies leaves a slight chance that antibodies with a similar sequences would behave faithfully as high fidelityantagonists.
preference for a,$, could be missed. However, it is unlikely that In fact, HCDR3-derived peptides do block integrin function.
any motifs with greater than 10-fold selectivity for a,& were More importantly though, these peptides mimic thetarget
present. The complexity of the library we screened was suffl- specificity of the parent antibody. Even though both p,-integrins and the highly related integrin a,& bind t o the protocient to ensure that all potential amino acid sequences were
present in the
motif optimization, leading to the
conclusion that typical RGD sequence, GRGDSP, the RGD peptide derivedfrom
within thecontext of the adhesion motif shown in Fig. 1,the set Fab-9 bound only the P,-integrins. Similar target specificity
Interestingly, no was observed for peptides derived from MTF-2 and MTF-40.
of all sequences that bind a$, also bind aIIbP3.
natural ligands have been identified that bind a$, substan- Like the parent antibodies, peptides 2 and 40 showed specificover a,&. It is unclear whether the
selection of the
tially better than aIIbp3.
Although peptidomimetics and cyclic ity for aIIb,p3
antibody CDR contribute more
peptides have been reported to have a high degreeof specificity proper flanking residues in the
(33, 34), we are unaware of any such compounds that contacts with integrinor whether these flanking residues have
for aIIbp3
have good preference for a,P3. Despite the identification of sev- conformationally constrained the angle that defines the orientation of the side chains of the Arg and Asp residues. Surpriseral snake venom peptides that selectively antagonize aI&,
only one such protein, cerastin, hasspecificity for a,& but has ingly, cyclizing the CDR peptides provided no advantage over
Synthetic Antibodies Against Integrins
32795
A,, and Shattil, S.J. (1989) J. Biol. Chem. 264, 259-265
the linear peptide sequences in terms of affinity or selectivity.
10. 'Ibmiyama, Y., Tsubakio, T., Piotrowicz, R. S., Kurata, Y.,Loftus, J. C., and
The selection of the optimal flanking sequences may add SO
Kunicki, T. J. (1992) Blood 79, 2302-2312
many additional linear contact points for the integrin that CY- 11. Scarborough, R. M., Rose, J. W., Hsu, M. A,, Phillips, D. R., Fried, V. A.,
Campbell, A. M., Nannizzi, L., and Charo, I. F. (1991) J. Biol. Chem. 266,
clization provides no additional advantage. Alternatively, the
9359-9362
minimal energy conformation of the linear peptides may ap- 12. Barbas, C. F., 111, Bain, J. D., Hoekstra, D. M., and Lerner,R. A. (1992) Proc.
Natl. Acad. Sci. U. S. A . 89, 44574461
proximate the same structure as thecyclic form, in which case
no difference in theaffinity of the two forms would be expected. 13. Fields, G . B., and Noble, R. L. (1990) Int. J . Pept. Protein Res. 35, 161-214
Fields, C. G., and Fields, G. B.(1990) Int. J. Pept. Protein Res.36,
14. King, D. S.,
Resolution of this issue must await an analysis of peptide con255-266
15.
Tam,
J.
P.,
Wu,
C. R., Liu, W., and Zhang, J. W. (1991)J. Am. Chem. Soc. 113,
formation by NMR.
6657-6662
The findings presented here show that CDRs are a viable 16. Stewart,
J.M., and Young, J. D. (1984) Solid Phase Peptide Synthesis,2nd Ed.,
design template for obtaining inhibitory peptides that mainpp. 116-134, Pierce Chemical Co., Rockford, IL
tain thesame specificity as theparent antibody. There are now 17. Lam, S. C-T., Plow, E. F., Smith, M. A,, Andrieux, A,, Ryckwaert, J-J.,
Marguerie, G., and Ginsberg, M. H. (1987) J . Biol. Chem. 262,947-950
examples in the literature describing the use of CDR-derived 18. Smith,
J. W., and Cheresh, D. A. (1988) J. Biol. Chem. 263,18726-18731
synthetic peptides to emulate the activity of the whole antibody 19. Yatohgo, T., Izumi, M., Kashiwagi, H., and Hayasbi, M. (1988) Cell Struct.
Funct. 13, 281-292
(9, 37, 38). By optimizing with phage display, the affinity and
20. Doolittle, R. E , Schubert, D., andSchwartz, S. A. (1967) Arch. Biochem.
specificity of such CDR-derived peptides could be improved.
Biophys. 118,456-521
Consequently, monoclonalantibodies could be used as a start- 21. Smith, J. W., Vestal, D. J., Irwin, S.V., Burke, T. A., and Cheresh,D. A. (1990)
J. Biol. Chem. 266, 11008-11013
ing point for obtaining antagonist peptides.
22. Smith, J. W., Piotrowicz, R. S., and Mathis, D. (1994)J. Biol.Chern. 269,
Finally, a major advantage to building human antibodies
9fiO-967
". ".
that bind the integrin ligand binding site is that they could 23. Altschuh, D., Dubs, M-C., Weiss, E., Zeder-Lutz, G., and Van Regenmortel, M.
H. V. (1992) Biochemistry 31,6298-6304
have immense therapeutic application, particularly because
24. Fagerstam, L. G., Frostell-Karlsson. A,, Karlsson, R., Persson, B., and
they should circumvent a host immune response. The two p3Ronnberg, I. (1992) J. Chromatogr. 697,397-410
integrins have been implicated in numerous diseases. An an- 25. Cheresh, D. A,, and Spiro, R. C. (1987) J. Biol. Chem. 262,17703-17711
tagonist of the platelet integrin would arrest platelet aggrega- 26. Pratner, C. A., Plotkin, J., Jaye,D., and Frazier,W. A. (1991) J. Cell Biol. 112,
1031-1040
tion and could find wide application in treating thrombotic 27. Marguerie, G. A,, Edgington, T. S., and Plow, E. F. (1980)J . Bid. Chem. 265,
154-161
episodes. In fact, antagonists of aIIbp3have been effective at
blocking platelet function in vivo (34,39).The fact that some of 28. Smith, J. W., and Cheresh, D. A. (1991) J. Biol. Chem. 266,11429-11432
29. Barbas, C. F., I11 (1993) Cum Opin. Biotechnol, 4, 526-530
the synthetic antibodies antagonize both &-integrins with high 30. Cull, M. G., Miller, J. F., and Schatz,P. J. (1992)Proc. Natl. Acad.Sci. U. S. A.
89,1865-1869
affinity may be extremely useful in combating certain types of
M., Pierschbacher, M. D., Ruoslahti, E., Marguerie, G., andPlow, E.
metastasis. For example, it is now thought that melanoma is 31. Ginsberg,
(1985) J. Bzol. Chem. 260,3931-3936
often carried through the vasculature by adhesion to circulat- 32. Scarborough, R. M., Rose, J. W., Naughton, M. A,, Phillips, D. R., Nannizzi, L.,
Arfsten, A., Campbell, A. M., and Charo, I. F. (1993) J. Biol. Chem. 268,
ing platelets (40,41). This cell-cell bridge is thought to involve
105a-1065
a,& on the tumor cell and qrn&
on the platelet, and blocking 33. Isoai,
A., Ueno, Y., Giga-Hama, Y., Goto, H.. and Kumagai, H. (1992) Cancer
both ends of this adhesion event with the synthetic antibodies
Lett. 66, 259-264
described here would seem to present a superb therapeutic 34. Zablocki, J. A., Miyano, M., Garland, R. B., Pireh, D., Schretzman, L.,Rao, S.
N., Lindmark, R. J., Panzer-Knodle, S. G., Nicholson, N. S., Taite, B. B.,
strategy,
Salyers, A. K., King, L. W., Campion, J. G., and Feigen, L. P. (1993) J. Med.
Acknowledgments-Weacknowledge Yverre Bobay, Doug Cababa,
Terri Jones, and Kim Green for superb technical assistance. We thank
Shari Olsen for secretarial support.
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