From Scios Inc., Sunnyvale, California 94086
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ABSTRACT |
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Peptides or small molecules that can block the
interaction of the integrin Mac-1 with its receptor, intercellular
adhesion molecule-1 (ICAM-1), have not previously been developed. We
studied this interaction by measuring the adherence of
ICAM-1-expressing Chinese hamster ovary (CHO) cells to immobilized,
purified Mac-1. Nucleotide sequence information was obtained for the
complementarity determining regions (CDRs) of three antibodies (44aacb,
MY904, and 118.1) shown to block Mac-1-mediated cell adherence.
Peptides were synthesized based on the predicted amino acid sequences
of the CDRs and tested for the ability to block cell adhesion to Mac-1.
Peptides derived from CDR1 of 44aacb, CDR2 of 118.1, and CDRs 1 and 3 of MY904 heavy chains were found to possess blocking activity at
10-100 µM. This may indicate that one or two CDRs contribute disproportionately to the antibody binding affinity. The
binding of ligands to Mac-1 has been shown to require a region of the
-chain known as the I- or A-domain. We have recombinantly produced
Mac-1 I-domain, and show that it is also capable of supporting the
adherence of ICAM-1-expressing CHO cells. The adherence of ICAM-1-CHO
cells to the I-domain is inhibited by 44aacb and 118.1 and by the CDR
peptides from 44aacb and 118.1. By using phage display of peptide
libraries based on the 118.1 CDR peptide with five residues randomized,
we were able to identify a novel peptide inhibitor of Mac-1 with
substitutions at all five positions. These peptides provide lead
structures for development of Mac-1 antagonists.
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INTRODUCTION |
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The extravasation of white blood cells to sites of inflammation and the phagocytosis of opsinized microorganisms by these cells is clearly crucial to host defense. However, the mounting of an inappropriately large mobilization of phagocytic cells is thought to contribute to organ damage in sepsis, in adult respiratory distress syndrome, and following reperfusion of ischemic tissue (1, 2). Activated neutrophils are recruited into tissues or are sequestered in the microcirculation of the lung and liver; in either case tissue is damaged upon their degranulation and release of enzymes and activated oxygen species. Interruption of neutrophil extravasation or oxidative burst may be an effective means of damage control in these situations.
Mac-1 is a cell surface glycoprotein contributing to several myeloid
cell functions including adherence to and transmigration across the
endothelium, binding and phagocytosis of opsinized particles, and the
oxidative burst (3-5). It is a heterodimer of two transmembrane
proteins, CD11b (M) and CD18 (
2), the
latter also being part of the related integrins LFA-1, p150/95, and
D
2. The major adhesion partner for Mac-1
is ICAM-11 (6), which is a
member of the Ig supergene family and contains five extracellular Ig
domains of the C2 type, characteristic of Fc receptors and proteins
involved in cell adhesion (7). However, the complexity of the functions
involving Mac-1 results in part from the fact that an array of ligands
besides ICAM-1 is also recognized by this molecule, including iC3b,
fibrinogen, and factor X (3).
The precise residues of Mac-1 and ICAM-1 mediating their interaction
are not known, although the domains responsible have been elucidated.
Ig domain 3 of ICAM-1 has clearly been implicated in binding to Mac-1,
whereas domain 1 mediates binding to the related adhesion molecule,
LFA-1 (8). CD11b contains a 200-amino acid "inserted domain" or
"I domain," so called due to its presence only in the other
2 integrins and in the VLA
1 and
2 subunits and
its absence in most other integrins. Antibodies to this domain can
block ICAM-1 binding, as well as that of iC3b and fibrinogen (3).
Mutations within the I-domain of Mac-1 have been shown to prevent
binding of ICAM-1 and iC3b (9-11). The Mac-1 I-domain has been
expressed recombinantly, and there are some data suggesting that it
interacts with fibrinogen, iC3b, and soluble ICAM-1 (4, 12). Whether
the I-domain can support the adherence of ICAM-1-expressing cells
independent of other domains of Mac-1 has not been previously demonstrated.
The lack of information on the precise sequences within ICAM-1 and Mac-1 that interact has precluded modeling of small molecule inhibitors. One approach to overcoming this problem that has proven successful in other cases has been to use CDR sequences from antibodies directed at the active site as lead structures. Antibodies useful in this regard have been either developed as anti-idiotypic to the ligand (13) or simply chosen by virtue of their ligand blocking activity (14). As well as providing lead inhibitors, in some cases CDRs have shared sequence similarity with a portion of the known ligand, implicating that sequence in receptor binding (14-16).
We have produced the Mac-1 I-domain recombinantly and show that it supports the adherence of ICAM-1-expressing cells. Several antibodies that block the adherence of ICAM-1 to both Mac-1 and to the I-domain have been sequenced to allow determination of CDR structures. Peptides based on these CDR structures are shown to block the adherence of ICAM-1 to both Mac-1 and the I-domain.
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MATERIALS AND METHODS |
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Purification of Mac-1-- Peripheral blood leukocytes were purified from "Buffy coats" (Stanford Medical School Blood Center, Stanford, CA). Buffy coats were diluted 1:1 with HBSS and layered on Histopaque (Sigma) gradients as described (17). Both mononuclear and polymorphonuclear cell layers were collected, and red blood cells were lysed one or two times as necessary. Mac-1 was purified from lysates of the remaining leukocytes by immunoaffinity chromatography essentially as described by Diamond et al. (6) and purity assessed using SDS-polyacrylamide gel electrophoresis (see Fig. 1A).
Construction of Mac-1 I-domain-- The construction of the I-domain was based on a plasmid construct reported by Michishita et al. (11). The I-domain of human Mac-1 from the glycine residue at position 111 to the alanine at position 318 was generated using synthetic oligonucleotides and PCR. Eight overlapping oligonucleotides were synthesized and combined in a stepwise PCR procedure to generate the final 603-base pair fragment. The 5'-most oligo included a BamHI site present naturally in the Mac-1 gene sequence, whereas the 3'-most oligo included an added EcoRI site. The internal EcoRI site in this I-domain region was eliminated via a single base change from A to T at the third position in the glutamate codon at position 179 (a silent mutation). Each pair of oligonucleotides sharing partial complementarity was annealed and subjected to PCR (3' at 94 °C, followed by 10 cycles of 1' at 94 °C, 2' at 55 °C, and 3' at 72 °C) using 250 µM dNTPs and VENT polymerase (New England Biolabs, Beverly, MA). The PCR products were then mixed, melted for 3' at 94 °C, and subjected to PCR as described above. After assembly of all of the oligos, the resulting BamHI-EcoRI fragment was cloned into the pGEX-2T (Pharmacia Biotech Inc.) vector at the BamHI and EcoRI sites, resulting in an in-frame fusion with an N-terminal domain encoding glutathione S-transferase.
Purification of Recombinant I-domain--
The glutathione
S-transferase-I-domain fusion protein was expressed in
Escherichia coli cells (strain JM101). Overnight cultures of
E. coli JM101 were diluted 1:10 with L broth medium
containing ampicillin and grown for 1 h at 37 °C.
Isopropyl--D-thiogalactoside (1 mM) was
added to induce expression of the fusion protein, and after 3 h of
growth, bacteria were pelleted and frozen at
80 °C. Pellets
derived from a 1-liter culture were then thawed and resuspended in 18 ml of cold PBS, pH 7.4, phenylmethylsulfonyl fluoride was added at 1 mM, and the samples were disrupted on ice at 10,000 p.s.i.
in a French Press (SLM Instruments, Inc., Rochester, NY). 20% Triton
X-100 was then added to a final concentration of 1%, and the lysate
was incubated on ice for 30 min with occasional rocking. After
centrifugation at 12,000 × g for 10 min, the
supernatant was incubated with glutathione-Sepharose 4B (1.5 ml,
Pharmacia) for 1 h at room temperature. The beads were then washed
once in PBS plus 0.35 M NaCl and washed four times with PBS
and were resuspended in 5 ml of PBS. 200 units of human thrombin
(Enzyme Research Laboratories, South Bend, IN) was added to cleave the
I-domain from the fusion protein, and the mixture was incubated for
2 h at room temperature. NaCl and MgCl2 were added to
the cleaved soluble recombinant I-domain at final concentrations of
0.35 M and 1 mM, respectively, and the samples
were then passed through a 700-µl benzamidine-Sepharose 6B
(Pharmacia) column to remove the thrombin. The flow-through was assayed
for thrombin activity using Chromozym TH (Boehringer Mannheim,
Indianapolis, IN) as a substrate, and the clearance was greater than
150-fold. Protein concentration was determined using the Bio-Rad
protein assay (Bio-Rad). Typical yields of the recombinant I-domain
were 3-5 mg/liter bacterial culture.
Mass Spectrometric Analysis of I-domain-- Electrospray ionization was performed on a Finnigan SSQ 7000 mass spectrometer (San Jose, CA) in the positive ion mode. Liquid chromatography/mass spectroscopy was performed using a capillary reversed phase column with a flow rate into the mass spectrometer of 5 µl/min. To map tryptic peptides, electrophoresis of I-domain protein was carried out in 12% polyacrylamide followed by transfer to Immobilon (70 V, 60 min; Ref. 18). In situ tryptic digestion was carried out according to the method of Wong et al. (19). Capillary HPLC was performed using a Valco tee to split the 200 µl/min flow from an HP 1090 HPLC PV5, driving the capillary system (Vydac C18 0.32 × 250-mm column, maintained at 40 °C, Microtech Inc., Sunnyvale, CA) at 5 µl/min. The gradient was as follows: 0 min, 100% A (0.1% trifluoroacetic acid in H2O); 40 min, 30% B (0.09% trifluoroacetic acid in acetonitrile); 50 min, 60% B; 51 min, 0%B; 72 min, 0% B.
Adherence Assays--
The adherence of human neutrophils to
wells coated with human serum was carried out exactly as described
previously (17). CHO cells were stably transfected with an expression
vector encoding human ICAM-1 (20), and their adherence was assessed as
follows. ICAM-1-CHO cells were grown in RPMI 1640 with 10% fetal
bovine serum and used at 80% confluence. Cells were loaded with
calcein-acetoxymethyl ester (Molecular Probes, Eugene, OR) at 5 µM in HBSS++ for 30 min at 37 °C. The cells were then
detached from the flask with PBS containing 5 mM EDTA (15 min, 37 °C), washed in HBSS++ containing 0.1% HSA (Sigma), and
resuspended in HBSS++ containing 0.5% HSA at 2 × 106/ml. 96-well plates were coated with purified Mac-1 or
I-domain in HBSS++ (50 µl/well) for 2 h at 37 °C. In the case
of Mac-1, -octylglucoside at a final concentration of 0.15% was
present in the coating solution. For each lot of Mac-1, the optimal
protein concentration for coating the wells (5-13 µg/ml) was
determined as that which best supported the adherence of ICAM-1-CHO
cells but not of vector-CHO cells. Wells were washed twice with HBSS++ containing 0.1% HSA and blocked with HBSS++ supplemented with 0.5%
HSA for 30 min at 37 °C. Test compounds were preincubated in blocked
wells for 15 min at 37 °C in 50 µl, and then 50 µl of cells were
added for an additional 60-min incubation. Nonadherent cells were
removed by gently inverting plates and blotting on paper towels. Wells
were washed twice with HBSS++ containing 0.1% HSA. Adherent cells were
quantitated in a 96-well fluorescence plate reader (IDEXX Labs,
Westbrook, ME). HSA lots were tested in this assay to find those that
gave a minimal background adherence and a maximal adherence to Mac-1.
All incubations in HBSS++ were carried out without CO2.
Monoclonal Antibodies--
Hybridoma cells producing the
anti-Mac-1 monoclonal antibodies LM2/1 (murine IgG1), 44aacb (murine
IgG2a ) and MY904 (murine IgG1
) were from ATCC (21, 22). The
hybridoma cell line secreting mAb 118.1 (murine IgG1
) was generated
from a Balb/C mouse immunized according to Diamond et al.
(3). Spleen cells were mixed in a 5:1 ratio with FOX-NY murine myeloma
cells (ATCC 1732 CRL) and fused by slow addition of polyethylene glycol
1500 (0.5 ml/108 cells, Boehringer Mannheim). After 1 min
at 37 °C, the cell suspension was slowly diluted in RPMI,
centrifuged at 200 × g for 7 min, and resuspended in
selection medium (RPMI 1640, 20% fetal bovine serum, Pen/Strep, AAT
media supplement (Sigma), and STM Mitogen (RIBI ImmunoChem Research
Inc.)). After 9 days of growth in 96-well culture plates, the hybridoma
supernatants were screened for antibodies binding to purified Mac-1 in
an enzyme-linked immunosorbent assay adapted from Diamond and Springer
(23). Serum-free conditioned media from the hybridoma lines were
harvested from confluent roller bottles, and antibodies were purified
by protein A chromatography using PROSEP-A affinity resin
(BioProcessing, Ltd.) following the manufacturer's suggested
protocol.
Cloning and Sequencing of the Variable Regions of 44aacb, MY904, and 118.1-- mRNA was isolated from 44aacb, MY904, and 118.1 hybridoma cells (108 cells each, grown in RPMI 1640, 10% fetal bovine serum, 10 mM HEPES) using the Mini RiboSepTM mRNA Isolation Kit (Becton Dickinson, Bedford, MA). Following first strand cDNA synthesis using a cDNA synthesis kit (Amersham Corp.), VH and VL regions were amplified (Vent DNA polymerase, New England Biolabs, Beverly, MA) using the following primers: VH regions, upstream primer 5'-GCAGAATTCSARGTSCARTTRCARCA and downstream primer 5'-GCAGAATTCGGGGCCAGTGGATAGAC; VL regions, downstream primer 5'-GCAGAATTCGGTGGGAAGATGGATACAGTT coupled to upstream primers 5'-GCAGAATTCACMCARTCHCCAGTNAT (44aacb), 5'-GCAGAATTCGAYATYGTBCTGACNCA (MY904), or 5'-GCAGAATTCGAYGTBGTKATGACMCA (118.1) (S = C or G; R = A or G; M = A or C; Y = C or T; K = G or T; H = A, C, or T; B = C, G, or T; and N = A, C, T, or G). The sequences of the downstream primers were based on the conserved nucleotide sequences of the 5'-ends of the constant regions of the heavy and light chains. The upstream primers were designed from either the experimentally determined N-terminal amino acid sequences of the three antibodies or deduced N-terminal "consensus" amino acid sequences of murine immunoglobulin variable regions from GenBankTM. The mouse codon usage was also considered when degenerate primers were designed. An EcoRI site was introduced at the 5'-end of all the primers to facilitate cloning. PCR was performed for 30 cycles of 1 min at 94 °C, 1 min at 55 °C, and 1 min at 72 °C, and bands were resolved by electrophoresis in a 1% agarose gel.
The correctly sized PCR products were digested with EcoRI, gel-purified, and ligated into EcoRI-digested pUC9 vector DNA. The ligated DNA was then transformed into E. coli JM101 by electroporation, and clones were selected on LB plates containing ampicillin. For each VH or VL region, three or four positive clones were picked, DNA was prepared using the Wizard miniprep system (Promega, Madison, WI), and the PCR product carried in the plasmid DNA was sequenced.Construction of Phage Libraries-- Two peptide libraries were constructed in which the library inserts were directly linked to the N terminus of the C-terminal domain of M13 gene III as described (24). The GYXDXYXGXIXYN and GXIXPXYXGXTYN libraries were prepared using synthetic oligonucleotides containing core sequences GGA TAT NNS GAT NNS TAC NNS GGT NNS ATT NNS TAC AAC and GGA NNS ATT NNS CCT NNS TAT NNS GGT NNS ACC TAC AAC, respectively. The nucleotides were made double-stranded by extension with Klenow polymerase (New England Biolabs) from a 5' primer. The resulting products were gel purified using MERmaid spin kit (Bio101 Inc., Vista, CA) and inserted into the BstEII and BamHI sites of the phagemid vector pAL53.2 The library-containing vectors were then transfected into E. coli Top10F' cells using 10-12 electroporations. The yields of the two primary libraries were 0.5-1 × 107.
Panning of Phage Libraries--
Libraries were packaged into
phage particles by infection of the transfected E. coli
Top10F' library with M13KO7 helper phage, resulting in expression of
less than one copy of the peptide-gene III protein per phage (24).
Phage expressing Mac-1 binding peptides were selected by panning on
96-well enzyme-linked immunosorbent assay plates (Corning) coated with
Mac-1 (0.5 µg/well in HBSS++ containing 0.15% -octylglucoside for
2 h at 37 °C). After the wells were washed and blocked with
HBSS++ supplemented with 0.5% HSA, 10 µl of partially purified phage
(~5 × 1011 cfu) was added to the wells in a total
volume of 100 µl of the same buffer and incubated at 37 °C for
2 h while shaking. The unbound phage particles were then removed
by washing with 100 µl of HBSS++/0.05% Tween six times quickly on
ice. Bound phage were eluted with 200 µl of 100 mM
citrate buffer, pH 3.0/0.1% Tween for 30 min at room temperature with
shaking and neutralized with 25 µl of 1 M Tris base. The
wells were then washed three times with 100 µl of PBS, and eluates
and washes were combined. The eluted phage were titered and amplified
and carried through three more rounds of enrichment by panning on
Mac-1. The stringency of the PBS wash conditions was increased in
successive rounds as follows: nine quick washes at room temperature in
round 2, followed by two additional washes for 10 min each with shaking in round 3 and three washes for 10 min each with shaking in round 4. Phage were randomly selected for sequencing after round 4.
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RESULTS |
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Production of Recombinant Mac-1 I-domain-- Purified Mac-1 has been shown to support the adherence of ICAM-1-expressing L-cells (6). To determine whether the I-domain of Mac-1 could similarly support the adherence of cells expressing ICAM-1, this domain was expressed recombinantly in E. coli. Analysis of the purified protein by SDS-polyacrylamide gel electrophoresis showed a major band migrating as expected and in some cases a minor band with slightly slower migration (Fig. 1B). Analysis of the purified protein by mass spectrometry showed a major peak of 24,103 Da, in agreement with the molecular mass predicted for this construct. Also revealed was a minor peak of 25,102 Da, present at approximately 10-20% the level of the major peak in different preparations. To determine the source of the additional 1000 Da, the proteins in the I-domain preparation were separated by SDS-polyacrylamide gel electrophoresis and subjected to both N-terminal sequence analysis and tryptic peptide mapping. Both the 24- and 25-kDa proteins had the expected N-terminal sequence of GSNLRQQP. The 24- and 25-kDa bands showed a similar tryptic map pattern with the exception of the C-terminal peptide (IFANSS; mass, 638 Da), which disappeared from the digest of the 25-kDa band. A new peptide (mass, 1634 Da) was evident in the map of the 25-kDa protein. The mass of the new peptide was 997 Da larger than the predicted C-terminal peptide, similar to the mass difference observed in the intact molecules, and thus we conclude that the added mass is contained at the C terminus. The 1634-Da peptide could be accounted for by translation of the usual stop codon in the pGEX-2T expression vector as a tryptophan residue, translation of 10 additional vector residues prior to the next stop codon, and proteolytic trimming of three C-terminal vector residues, resulting in the tryptic peptide IFANSSWLTDDLPR.
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Adherence Assays Based on Purified Mac-1 or the Mac-1 I-domain-- Purified recombinant I-domain was tested for the ability to support the adherence of ICAM-1-CHO cells. As shown in Fig. 2A, the I-domain did support the adherence of ICAM-1-CHO cells, in a concentration-dependent manner. For this particular lot of I-domain, 10-30 µg/ml in the solution used to coat the wells resulted in good adherence of ICAM-1-CHO cells and low background adherence of vector-transfected CHO cells. The adherence of ICAM-1-CHO cells was blocked by the anti-Mac-1 antibody 44aacb. However, at high levels of I-domain, vector-transfected CHO cells were also adherent, and this adherence was blocked by 44aacb. These results indicate that the I-domain of Mac-1 interacts not only with ICAM-1 but also with an unknown receptor on CHO cells. This interaction with a CHO cell receptor is not unique to the recombinant I-domain, because vector-CHO cells also adhered to high levels of purified Mac-1, and this adherence was similarly blocked by 44aacb (data not shown).
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Characterization of Anti-Mac-1 Antibodies-- The anti-Mac-1 antibodies 44aacb and MY904 block the Mac-1-mediated adherence of neutrophils (17, 22). The antibody 118.1 was generated as described under "Materials and Methods" and also shown to block Mac-1-mediated neutrophil adherence (data not shown). 44aacb, MY904, and 118.1 were found to bind to Mac-1 in an enzyme-linked immunosorbent assay format with EC50 values of 12.0, 9.3, and 10.7 nM, respectively. Preincubation of these antibodies with Mac-1 (Fig. 3A) resulted in inhibition of the adherence of ICAM-1-CHO cells with IC50 values of 50-500 pM in four assays. The three antibodies also blocked I-domain-mediated adherence (Fig. 3B) with IC50 values of 1-3 nM in four assays. LM 2/1, which does not block neutrophil adherence, does not block ICAM-1-CHO adherence to Mac-1 (Fig. 3A).
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Characterization of CDRs of Anti-Mac-1 Antibodies-- mRNA isolated from hybridoma cells producing 44aacb, MY904, and 118.1 was used to determine the nucleotide sequence encoding the CDRs of each of the antibodies. The deduced amino acid sequences are shown in Table I. Peptides were synthesized corresponding to the underlined portions of the sequences in Table I. These peptides were tested for inhibition of ICAM-1-CHO cell adherence to Mac-1 (Table I). One or two HC CDRs from each antibody blocked Mac-1-mediated adherence with an IC50 at or below 106 µM. Of the light chain CDR peptides made for antibodies 44aacb and MY904, none possessed blocking activity. Two of the active HC CDR peptides were also tested for blocking activity in the adherence assay using the I-domain as a substrate. HC CDR1 from 44aacb and HC CDR2 from 118.1 were found to possess comparable blocking activity in this assay (data not shown).
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Identification of Additional Blocking Peptides through Phage
Display--
Variants of the 118.1 HC CDR2 blocking peptide were
identified by displaying libraries based on this peptide on phage and selecting for those phage that bound to Mac-1. To select residues for
randomization, the structure of 118.1 was modeled on the
three-dimensional structure of the antibody D11.15, an anti-lysozyme
antibody (Brookhaven Protein Data Bank 1JHL). D11.15 was chosen for
this purpose because of significant homology in the residues
immediately flanking the hypervariable domains of 118.1 and D11.15. The
antigen binding loop of 118.1 HC CDR2 was predicted to consist of
GYIDPYYGGITYN, with PYYG making the -turn. Two libraries, each with
five randomized residues in this region were expressed as N-terminal
fusions with M13 gene III. To select for high affinity peptides, a
monovalent phage display system was utilized in which one copy or less
of the gene III protein is expressed as a fusion peptide on each phage
(24). Phage expressing these libraries were panned on Mac-1 for four
rounds with increasingly stringent wash conditions. Sequencing of phage
eluted after four rounds revealed a consensus from one of the
libraries, in which all five randomized residues differ from the
original sequence:
GYRDGYAGPILYN.
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DISCUSSION |
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Peptides that block the binding of ICAM-1 to Mac-1 have been derived from the CDRs of anti-Mac-1 antibodies. Antibodies were chosen for this purpose based on their ability to block Mac-1-mediated adherence of ICAM-1-expressing CHO cells. The specificity of these antibodies was further verified by showing that they also block ICAM-1-CHO cell adherence to the recombinant I-domain of Mac-1. The amino acid sequences of the three HC CDRs and three light chain CDRs of each of the antibodies were deduced from the nucleotide sequences encoding the antibodies, and peptides were made from 15 of the 18 total CDR sequences. The adherence of ICAM-1-CHO cells to Mac-1 was blocked by four of these peptides, two from MY906 and one each from 44aacb and 118.1. This may result from one or two of the CDRs contributing most of the binding affinity of the antibody, or it may reflect a subpopulation of peptides taking on an active conformation out of their native context.
Because these peptides compete with ICAM-1 for binding to Mac-1, they
were analyzed for homology with ICAM-1 (Ref. 25; Lasergene, DNAstar,
Madison, WI). Some similarity was found between HC CDR2 from 118.1 and
residues Val238 to Ala249 of ICAM-1 (Table
II). These residues in ICAM-1 constitute
the loop between -strands D and E in immunoglobulin domain 3. Mutations in loops between strands C and D and strands E and F of
domain 3 have been shown to inhibit adhesion to Mac-1 (8).
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A variant of HC CDR2 from 118.1 was identified using a monovalent display system on the phage M13 and selecting for Mac-1 binding. Interestingly, although all five randomized residues differed from the original 118.1 sequence, this peptide was found to possess similar Mac-1 blocking activity to the parental 118.1 peptide, indicating that there is considerable room for variation, and presumably improvement, within this CDR sequence. The selection of this CDR variant through binding of phage to Mac-1 demonstrates that this peptide does bind to Mac-1 and provides evidence that its blocking activity can be attributed to a specific interaction with Mac-1.
Evidence has suggested that protein-protein interaction involves
multiple contact sites, and until recently little progress had been
made in disrupting such interactions with small peptides. However,
several examples now exist of peptides that can block protein-protein
interaction; for example, an 8-amino acid peptide has been identified
that can can block the activation of FGFR1 by basic fibroblast growth
factor (26). Further, the interaction of the two cell surface proteins,
CD4 and MHC class II, can be blocked by a heptapeptide based on a
protruding loop from one of the immunoglobulin-like domains of CD4.
Thus even in this latter example, where many contact sites from two Ig
domains of CD4 are thought to interact with MHC class II, binding
appears to depend on a single -turn-containing loop (27). In the
case of antibody-antigen interaction, varying numbers of hypervariable
regions may act together to provide the net affinity. In each of the
three antibodies we have examined, we have identified one or two CDRs
as candidates for important binding sites.
Peptides that block the interaction of ICAM-1 with Mac-1 have not
previously been reported. Peptides derived from factor X and related
peptides derived from filamentous hemagglutinin have been shown to
prevent factor X binding to cells, presumably via Mac-1 (28, 29).
However, the I-domain is not the primary binding site for factor X
(12), and these peptides have not been shown to affect ICAM-1 binding.
There is precedent, however, for peptide inhibition of
ICAM-1-2-integrin interaction. A peptide from ICAM-2 has
been shown to bind to LFA-1 and inhibit endothelial cell adhesion (at
100 µg/ml), presumably via ICAM-1 (30). Peptides from ICAM-1, domains
1 and 2, have also been shown to inhibit ICAM-1-mediated adhesion to
unknown ligands (at 100 µM) (31).
In conclusion, we have utilized sequence information from anti-Mac-1 antibodies to derive the first peptide Mac-1 antagonists. These findings support a growing body of evidence that protein-protein interactions may depend disproportionately on one site or pocket.
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ACKNOWLEDGEMENTS |
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We thank Pat Hummel for preparation of antibodies, Rodney Jue for carrying out protein sequencing, and Judy Miller for DNA sequencing. The antibody WS02025 was generously provided by C. Wayne Smith, Baylor College of Medicine, Houston, TX.
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FOOTNOTES |
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* This work was supported by Scios, Inc.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Scios Inc., 820 West
Maude Ave., Sunnyvale, CA 94086. E-mail: Endemann{at}aol.com.
1 The abbreviations used are: ICAM-1, intercellular adhesion molecule-1; CDR, complementarity-determining region; HBSS, Hanks' buffered salt solution; HBSS++, HBSS with calcium and magnesium; HC, heavy chain; PBS; phosphate-buffered saline; CHO, Chinese hamster ovary; PCR, polymerase chain reaction; HPLC, high pressure liquid chromatography; HSA, human serum albumin.
2 D. L. Damm, B. L. Garrick, K. McFadden, D. D. Lesikar, A. B. Lucas, and R. T. White, manuscript in preparation.
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REFERENCES |
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