Mimotopes of Human Platelet Glycoprotein Ib/Ix

The present invention relates to a peptide capable of functionally mimicking the binding site for a monoclonal antibody, the monoclonal antibody recognizing an epitope within the human platelet glycoprotein Ib/IX complex.

Throughout this application various publications are referenced, many in parenthesis. Full citations for these publications are provided at the end of the Detailed Description. The disclosures of these publications in their entireties are hereby incorporated by reference in this application.

The platelet glycoprotein Ib/IX (GPIb/IX) receptor for von Willebrand factor (vWf) is believed to consist of a 1:1 heterodimeric complex (Du et al. 1987) between GPIb (160 kDa) and GPIX (17 kDa) in a noncovalent association. GPIb in turn consists of a disulfide-linked 140 kDa alpha chain (GPIb alpha) and a 22 kDa beta chain (GPIb beta) (Fitzgerald and Phillips 1989).

In the 1980's, Miller et al. developed a series of monoclonal antibodies (mab) directed against the GP Ib/IX complex receptor for vWf. In particular, monoclonal antibody C-34 was characterized in detail and it was determined that mab C-34 recognized an epitope within the platelet glycoprotein Ib/IX complex (Miller et al. 1990). In this and subsequent work, Miller et al. showed that monoclonal antibodies C-34, AS-2 and AS-7 were potent inhibitors of the ristocetin-induced aggregation of normal platelets that was dependent upon von Willebrand factor. Miller et al. also showed that the epitopes for all three monoclonal antibodies lay within the GPIb/IX complex. However, in further experiments, while Miller et al. were able successfully to localize monoclonal antibody binding sites for AS-2 and AS-7 to the amino-terminal 45 kDa of GPIb alpha, they were unable to localize the binding region for C-34.

Attempts to define the binding sites for various monoclonal antibodies have led to the development of epitope libraries. Parmley and Smith developed a bacteriophage expression vector that could display foreign epitopes on its surface (Parmley and Smith 1988). This vector could be used to construct large collections of bacteriophage which could include virtually all possible sequences of a short (e.g. six-amino-acid) peptide. They also developed biopanning, which is a method for affinity-purifying phage displaying foreign epitopes using a specific antibody (see Parmley and Smith 1988; Cwirla et al. 1990; Scott and Smith 1990; Christian et al. 1992; Smith and Scott 1993).

After the development of epitope libraries, Smith et al. then suggested that it should be possible to use the bacteriophage expression vector and biopanning technique of Parmley and Smith to identify epitopes from all possible sequences of a given length. This led to the idea of identifying peptide ligands for antibodies by biopanning epitope libraries, which could then be used in vaccine design, epitope mapping, the identification of genes, and many other applications (Parmley and Smith 1988; Scott 1992).

Using epitope libraries and biopanning, researchers searching for epitope sequences found instead peptide sequences which mimicked the epitope, i.e., sequences which did not identify a continuous linear native sequence or necessarily occur at all within a natural protein sequence. These mimicking peptides are called mimotopes. In this manner, mimotopes of various binding sites/proteins have been found. LaRocca et al. (1992) expressed a mimotope of the human breast epithelial mucin tandem repeat in Escherichia coli. Balass et al. (1993) identified a hexapeptide that mimics a conformation-dependent binding site of the acetylcholine receptor. Hobart et al. (1993) isolated a mimotope that mimics the C6 epitope (the epitope for the sixth component of complement).

The sequences of these mimotopes, by definition, do not identify a continuous linear native sequence or necessarily occur in any way in a naturally-occurring molecule, i.e. a naturally occuring protein. The sequences of the mimotopes merely form a peptide which functionally mimics a binding site on a naturally-occurring protein. For example, the mimotope of Balass et al. (1993) mimics the binding site of the acetylcholine receptor.

Many of these mimotopes are short peptides. The availability of short peptides which can be readily synthesized in large amounts and which can mimic naturally-occurring sequences (i.e. binding sites) offers great potential application.

A need continues to exist, therefore, for the elucidation of useful mimotopes.

This need is met by the mimotopes of the subject invention. The invention thus provides an isolated peptide that functionally mimics a binding site for a monoclonal antibody, the monoclonal antibody recognizing an epitope within the human glycoprotein Ib/IX complex. This isolated peptide is a mimotope. A peptide functionally mimics a binding site for a monoclonal antibody if the monoclonal antibody can bind to the peptide.

The invention further provides an isolated molecule capable of binding to the peptide, which molecule can be an antibody, a second peptide, a carbohydrate, a DNA molecule, an RNA molecule, or any chemically synthesized molecule, for example. This isolated molecule is an anti-mimotope. Anti-mimotopes that bind to a receptor can be used to mediate the functional activity of that receptor.

The invention thus also provides a method for modulating the adhesion, aggregation, or agglutination of platelets, each of which is dependent on von Willebrand factor interaction with platelets through the glycoprotein Ib/IX complex receptor. The methods provide for exposure of platelets to the molecule (anti-mimotope) in order to modulate adhesion, aggregation, or agglutination of the platelets.

The invention further provides an isolated peptide capable of binding to monoclonal antibody C-34, as well as an isolated molecule capable of binding to such peptide. Also provided is a method for modulating the adhesion, aggregation, or agglutination of platelets by exposing the platelets to the molecule (anti-mimotope).

In a preferred embodiment, the isolated peptide capable of binding to monoclonal antibody C-34 includes an amino acid sequence corresponding to SEQ ID NO:38: WNWRYREYV.

These and other features and advantages of this invention will be evident from the following detailed description of preferred embodiments when read in conjunction with the accompanying drawings in which:

FIG. 1 illustrates the ristocetin-induced full aggregation of platelets in the presence of von Willebrand factor;

FIG. 2 illustrates the inhibition of ristocetin-induced aggregation of platelets by 20 .mu.g/ml of monoclonal antibody C-34;

FIG. 3 illustrates the continued inhibition of ristocetin-induced aggregation of platelets by 20 .mu.g/ml of mab C-34 in the presence of 0.14 .mu.M of the synthetic peptide mimotope having SEQ ID NO: 1: AWNWRYREYV;

FIG. 4 illustrates the partial neutralization of the inhibition of ristocetin-induced aggregation of platelets by 20 .mu.g/ml of mab C-34 in the presence of 0.27 .mu.M of the synthetic peptide mimotope having SEQ ID NO: 1: AWNWRYREYV;

FIG. 5 illustrates the partial neutralization of the inhibition of ristocetin-induced aggregation of platelets by 20 .mu.g/ml of mab C-34 in the presence of 0.55 .mu.M of the synthetic peptide mimotope having SEQ ID NO: 1: AWNWRYREYV;

FIG. 6 illustrates the partial neutralization of the inhibition of ristocetin-induced aggregation of platelets by 20 .mu.g/ml of mab C-34 in the presence of 1.1 .mu.M of the synthetic peptide mimotope having SEQ ID NO: 1: AWNWRYREYV; and

FIG. 7 illustrates the complete neutralization of the inhibition of ristocetin-induced aggregation of platelets by 20 .mu.g/ml of mab C-34 in the presence of 2.3 .mu.M of the synthetic peptide mimotope having SEQ ID NO: 1: AWNWRYREYV.

The invention provides an isolated peptide that functionally mimics a binding site for a monoclonal antibody, the monoclonal antibody recognizing an epitope within the human glycoprotein Ib/IX complex. This peptide is called a mimotope.

In one preferred embodiment, the monoclonal antibody is designated C-34, and the peptide includes an amino acid sequence selected from the group consisting of:

SEQ ID NO:1: AWNWRYREYV

SEQ ID NO:2: KWNWRNKKYV

SEQ ID NO:3: LSTWRYFEYV

SEQ ID NO:4: YLGWRYSEYV

SEQ ID NO:5: TQMWRAREYL

SEQ ID NO:6: WRQREYWDPV

SEQ ID NO:7: EGSWRYRKGG

SEQ ID NO:8: GYHWWRNWEY

SEQ ID NO:9: KGFLWRARNW

SEQ ID NO:10: MNWKHWRARH

SEQ ID NO:11: FKWREWRGKL

SEQ ID NO:12: PDRQVRLWVR

SEQ ID NO:13: RVLRHWHPRT

SEQ ID NO:14: GRRVWMLNHG

SEQ ID NO:15: KKGRHHVTRV

SEQ ID NO:16: GGVCKCWQCL

SEQ ID NO:17: FSHSYGSAIR

SEQ ID NO:18: MHGHRRPGLA

SEQ ID NO:19: MSKKPHLGLR

SEQ ID NO:20: TMWVELYSLK

SEQ ID NO:21: FVDPGRAGRG

SEQ ID NO:66: KRAWWKQKWV

Most preferably, the peptide includes an amino acid sequence corresponding to concensus sequence SEQ ID NO: 38: WNWRYREYV.

Each of these peptides, represented by SEQ ID NOs 1 to 21 and 66, mimics the binding site within GPIb/IX for mab C-34. Mab C-34 thus binds to each of these peptides. However, the sequences of each of these peptides do not identify a continuous linear native sequence or necessarily occur at all within the sequence of any chain (i.e. GPIb alpha, GPIb beta, GPIX) of the GPIb/IX complex, thus the peptides are mimicking the mab C-34 binding site and are therefore mimotopes. The peptide of the subject invention also includes fragments of the above exemplified peptides which retain the ability to functionally mimic the binding site for a monoclonal antibody, such as C-34. The peptide having an amino acid sequence corresponding to SEQ ID NO:38 is an example of such a fragment, being a fragment of the peptide which includes the amino acid sequence corresponding to SEQ ID No:1.

According to the subject invention, the monoclonal antibody (whose binding site is mimicked by the peptide of the invention) recognizes an epitope within the human glycoprotein Ib/IX complex.

The invention also provides an isolated molecule capable of binding to the peptide. This isolated molecule is called an anti-mimotope. The anti-mimotope molecule can be any suitable molecule, such as, for example, an antibody, a second peptide, a carbohydrate, a DNA molecule, an RNA molecule, or a chemically synthesized molecule. Such peptides, proteins, or other biological, synthetic, or semi-synthetic molecules that are capable of binding to the mimotope can be identified by: raising antibodies against the mimotope; selecting from bacteriophage, chemical, hybridoma cell, or other types of libraries, cells, or chemical syntheses that might produce a set or subset of molecules having high affinity for the mimotope sequence; or designing molecules intended to have a high affinity for the mimotope sequences using computer-assisted or other theoretical approaches. Suitable anti-mimotopes can also be developed using in vitro evolution of nucleic acids capable of binding to the peptide mimotope (see Joyce 1994).

Such anti-mimotopes could serve as anti-thrombotic drugs. For example, the binding of mab C-34 to GPIb/IX inhibits ristocetin-induced aggregation of platelets. The mimotope peptide mimics the binding site in GPIb/IX, and the anti-mimotope molecules bind to the mimotope peptide. Therefore, the anti-mimotopes, which could be peptides, should themselves complement the mimotope peptide. As such, the anti-mimotopes should be capable of binding to the original epitope for mab C-34 within the platelet glycoprotein Ib/IX complex, thereby inducing similar effects as does mab C-34, i.e. the inhibition of ristocetin-induced aggregation of platelets that is dependent upon von Willebrand factor.

The invention thus provides a method of modulating the adhesion, aggregation, or agglutination of platelets, the method comprising selecting platelets and exposing the platelets to the anti-mimotope molecule of the subject invention. Such exposure affects von Willebrand factor interaction with platelets through the glycoprotein Ib/IX receptor, thereby modulating the adhesion, aggregation, or agglutination of the platelets.

The invention also provides an isolated peptide capable of binding to monoclonal antibody C-34, the peptide including an amino acid sequence selected from the group consisting of:

SEQ ID NO:1: AWNWRYREYV

SEQ ID NO:2: KWNWRNKKYV

SEQ ID NO:3: LSTWRYFEYV

SEQ ID NO:4: YLGWRYSEYV

SEQ ID NO:5: TQMWRAREYL

SEQ ID NO:6: WRQREYWDPV

SEQ ID NO:7: EGSWRYRKGG

SEQ ID NO:8: GYHWWRNWEY

SEQ ID NO:9: KGFLWRARNW

SEQ ID NO:10: MNWKHWRARH

SEQ ID NO:11: FKWREWRGKL

SEQ ID NO:12: PDRQVRLWVR

SEQ ID NO:13: RVLRHWHPRT

SEQ ID NO:14: GRRVWMLNHG

SEQ ID NO:15: KKGRHHVTRV

SEQ ID NO:16: GGVCKCWQCL

SEQ ID NO:17: FSHSYGSAIR

SEQ ID NO:18: MHGHRRPGLA

SEQ ID NO:19: MSKKPHLGLR

SEQ ID NO:20: TMWVELYSLK

SEQ ID NO:21: FVDPGRAGRG

SEQ ID NO:23: FRCCVFSCCLLS

SEQ ID NO:24: GFRCLVSLGGCF

SEQ ID NO:25: YSLWGLPVGDVV

SEQ ID NO:26: LPLLWFNGAGFF

SEQ ID NO:27: VWGLFRGLENGS

SEQ ID NO:28: SLWRQWRGLFW

SEQ ID NO:29: TLSLFGGRDKGF

SEQ ID NO:30: IGPAVSCLFRVC

SEQ ID NO:31: MSLFPLSFCRLI

SEQ ID NO:32: ALFSSVWGDVTL

SEQ ID NO:33: GWFGPFWVRGSG

SEQ ID NO:34: FWVSVGGVEGVV

SEQ ID NO:35: LGAFGGAGFLWR

SEQ ID NO:36: CRGIVFLFVGWL

SEQ ID NO:37: FWLVKGAGAWRF

SEQ ID NO:39: QVRLWARAGAGQ

SEQ ID NO:40: GLAVTFGSVLEG

SEQ ID NO:41: VRWMCVIRLGVR

SEQ ID NO:42: RLWGPGVSRPVL

SEQ ID NO:43: CGSSLFRGPRCP

SEQ ID NO:44: LGISSLSFLQLR

SEQ ID NO:45: TWGWDGVSYLFL

SEQ ID NO:46: TRSLFDDFVSLR

SEQ ID NO:47: CYASLFRSRLCA

SEQ ID NO:48: DGSVRVVWVRLL

SEQ ID NO:49: LSGFPVALVRFA

SEQ ID NO:50: LGGGLLVGSVFP

SEQ ID NO:51: VWARGVFRDRFF

SEQ ID NO:52: TGLLAGPVWRWT

SEQ ID NO:53: WLGGIFSCLVCG

SEQ ID NO:54: WFLRDVGCGSCL

SEQ ID NO:55: SRCGVFTWCSRS

SEQ ID NO:56: RCLVGYRCWGGV

SEQ ID NO:57: GFRCLVMGGGCA

SEQ ID NO:58: CGFDLVCARLFG

SEQ ID NO:59: DSGVRWFFGFLG

SEQ ID NO:60: ILDGCFFLGRCP

SEQ ID NO:61: CVRWLVSAGCSG

SEQ ID NO:62: CVGCWLVCDVLL

SEQ ID NO:63: CLFVFAAGFACG

SEQ ID NO:64: SCALFGSCFGIS

SEQ ID NO:65: CWGGVGVCGLLV

SEQ ID NO:66: KRAWWKQKWV

SEQ ID NO:67: CVGGVASRCGVL

SEQ ID NO:68: SGAVLAGPFGVW

SEQ ID NO:69: CRAFDRVGVCVW

SEQ ID NO:70: RCLVGYVVGGVW

SEQ ID NO:71: VCLVYRSVDCWA

SEQ ID NO:72: WRVFVFTCVVWA

SEQ ID NO:73: LWREWRGLFAVL

SEQ ID NO:74: SGAVLAGPLWRL

SEQ ID NO:75: FVVRGGTFLFVR

SEQ ID NO:76: CRGIVFLFVGWL

SEQ ID NO:77: TGLLAGPVWRWT

SEQ ID NO:78: DSGVRWFFGFLG

SEQ ID NO:79: CAWHRLSFCGLV

SEQ ID NO:80: CFGSALVLAVLA

SEQ ID NO:81: WFWDMSGEWGGL

Further provided is a fragment of any of the above peptides wherein the fragment retains the ability to bind to monoclonal antibody C-34. Such a fragment is exemplified by SEQ ID NO:38, which is a fragment of SEQ ID NO:1.

The invention also provides an isolated molecule capable of binding to the above peptides, also known as an anti-mimotope. Suitable molecules include an antibody, another peptide, a DNA or RNA molecule, a carbohydrate, or a chemically synthesized molecule.

As above, the invention thus provides a method of modulating the adhesion, aggregation, or agglutination of platelets, the method comprising selecting platelets and exposing the platelets to the anti-mimotope molecule. Such exposure affects von Willebrand factor interaction with platelets through the glycoprotein Ib/IX receptor, thereby modulating the adhesion, aggregation, or agglutination of the platelets.

In one preferred embodiment, the invention provides an isolated peptide capable of binding to monoclonal antibody C-34 and including an amino acid sequence corresponding to SEQ ID NO:38: WNWRYREYV.

The invention is described in further detail as follows.

The C-34 Epitope

As reported by Miller, et al. (1990), platelets from patients with platelet-type von Willebrand disease (PT-vWD) were used as immunogens for the production of murine mabs. One such mab, C-34, inhibited ristocetin-induced aggregation of patient or normal platelets, but not aggregation induced by other aggregating agents. As demonstrated by crossed-immunoelectrophoresis, mab C-34 recognized an epitope within the GPIb/IX complex. In indirect immunofluorescence studies on fresh platelets, the ratio of any of four different anti-GPIb mabs to one another was near unity (0.88-1.14) both for normals and for patients. In contrast, the ratio of the binding of mab C-34 to such a mab (AP-1) was 0.31.+-.0.02 (means.+-.SE) for normal platelets and significantly increased to 0.54.+-.0.01 for patient platelets (p<0.001). In immunoprecipitations on NP-40 lysates of .sup.3 H-labeled platelets, saturating concentrations of mab C-34 produced much fainter bands than did AS-2 or other anti-GPIb mabs. In contrast to the other anti-GPIb mabs, C-34 did not bind to the purified .sup.125 I-labeled glycocalicin fragment of GPIb or to the glycocalicin derivative identified by crossed-immunoelectrophoresis. In immunoprecipitation studies of .sup.3 H-labeled platelets subjected to digestion with trypsin or with chymotrypsin, C-34 identified neither the glycocalicin nor the amino-terminal 45 kDa fragment of GPIb alpha that were immunoprecipitated by mab AS-2 or by mab AS-7.

Thus, using three independent techniques (immunoprecipitation of platelet glycoproteins following radiolabeling of intact platelets and subsequent proteolytic digestion of these glycoproteins; immunoprecipitation of radiolabeled purified glycocalicin; crossed immunoelectrophoresis of platelet glycoproteins)(Miller et al. 1990), it has been shown that while C-34 recognizes an epitope within the GPIb/IX complex, this epitope does not appear to reside within glycocalicin.

While these studies reported a relatively simple method that succeeded in epitope mapping mabs AS-2 and AS-7 to the 45 kDa region of GPIb alpha, this work demonstrated that mab C-34 cannot be mapped to any single tryptic or chymotryptic domain of glycocalicin. Additionally, mab C-34 does not produce immunoprecipitation patterns similar to those of a mab recognizing GPIX.

Biopanning of Mab C-34 With Bacteriophage Display Libraries

Scott and Smith (1990) presented a method of defining peptide ligands by using randomly synthesized peptide inserts in bacteriophage. Related methods were published by Cwirla et al. (1990) and by Devlin et al. (1990). Since that time a literature has arisen in which both the original hexapeptide inserts and larger inserts have been used in identifying epitopes recognized by monoclonal antibodies. This technique has great potential for the detection of critical epitopes within the platelet vWF receptor known as GPIb/IX. The studies disclosed herein focus on monoclonal antibody C-34, but can be applied to other monoclonal antibodies having binding sites (epitopes) within GPIb/IX by the methods disclosed herein for mab C-34.

A well-balanced decapeptide (10-mer) library from Dr. Bruce Malcom of Alberta, Canada and a dodecapeptide (12-mer) library from Clontech Laboratories (Palo Alto, Calif.) were used. In the dodecapeptide library, a reduced frequency of adenosines at the first two positions of each codon causes a characteristic underrepresentation of the following amino acids indicated by their one-letter codes: I,M,T,N,K,Y,H,Q,D, and E. The libraries have both been constructed into a Fuse 5 vector (Scott and Smith 1990) by the insertion of a mixture of synthetic oligonucleotides, with the random decapeptides (or modified-random dodecapeptides) fused to the minor viral coat protein pIII of the bacteriophage. The libraries each have a complexity of approximately 3.times.10.sup.8 independent clones, and a titer of 10.sup.12 to 10.sup.14 per ml. While the Malcom library constitutes only a partial decapeptide library, it is complete as a hexapeptide library.

The strategy for using these libraries largely follows the review recently presented by Scott (1992) and employs, with modifications, the detailed methodology for use of this system as described recently by Smith and Scott (1993). The strategy used herein is as follows.

Specifically, in the first round of biopanning a 60 mm streptavidin-coated petri dish is filled with blocking solution (0.5% BSA, 0.1M NaHCO.sub.3, 0.1 .mu.g/ml streptavidin, 0.2% NaN.sub.3) for 2 hours, then washed three times with TBS-0.5% Tween. Next, 1 .mu.l of the library (about 1.times.10.sup.11 phage) that has been incubated overnight at 4.degree. C. with 1 .mu.g of biotinylated Mab is diluted with 1 ml of TBS-Tween, and this mixture is then added to the petri dish and rocked for 15 minutes at room temperature. The petri dish is washed 10 times with TBS-Tween, and bound phage is eluted by pipetting 800 .mu.l of 0.1N HCl (pH adjusted to 2.2 with glycine)--1 mg/ml BSA into the dish. The eluate is then pipetted into a microfuge tube containing 48 .mu.l of 2M Tris, to bring the pH up to about 8.

The eluate is concentrated and washed twice in TBS using an Amicon Centricon-30 filter (Amicon, Inc., Beverly, Mass.). This final product is titered out by making dilutions from a small amount of concentrated eluate in TBS-0.1% gelatin and adding 1 .mu.l of each dilution made to 19 .mu.l of TBS-gelatin, then adding 20 .mu.l of starved K91 E. coli cells and incubating for 10 minutes at room temperature. After adding 200 .mu.l of NZY medium containing 0.2 .mu.g/ml tetracycline (Tc) and incubating at 37.degree. C. for 1 hour, the mixture is plated out on NZY agar plates containing 40 .mu.g/ml tetracycline and allowed to grow up overnight at 37.degree. C.

After titering, the entire concentrated eluate from the first round of biopanning (about 50 .mu.l) is added to an equal volume of fresh starved K91 cells, and amplification performed as described by Smith and Scott (1993). Following the first PEG/NaCl precipitation, the resulting pellet is dissolved in 1 ml TBS. Phage is then precipitated a second time with PEG/NaCl, allowed to stand at least 1 hour at 4.degree. C., and the precipitate collected following centrifugation at 4.degree. C. After careful removal of all the supernatant, the pellet is dissolved in 100 .mu.l TBS. This amplified product can then be titered.

Second and third biopannings are carried out exactly as in the first, but using either an equal or a decreased amount of biotinylated Mab for each subsequent round, with 5 .mu.l of amplified eluate. The second round of biopanning is concentrated and amplified as in the first round. The third round is stopped after eluting the bound phage from the petri dish. This eluate is not concentrated or amplified. Titerings are done before and after each round, and the percent yield is calculated as the number of bacteriophage obtained in an elution fraction relative to the initial number of bacteriophage (Christian et al. 1992). A yield should generally be greater than 10.sup.-5 to exceed background, with values of 10.sup.-4 to 10.sup.-1 typically observed. Increasing percent yields in subsequent rounds of biopanning are, in particular, suggestive that clones of increasing affinity are being selected.

In some experiments, an immunological screening assay, as described by Christian, et al. (1992) may be performed using NZY and Tc agar plates containing about 500 well-separated colonies. The colonies are transferred to nitrocellulose membrane filters (Biorad Laboratories, Hercules, Calif.), and the filters are immediately washed twice in TNT Buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20), blocked for 30 minutes at room temperature with gentle agitation in 20% normal goat serum in TNT buffer, then incubated for 2 hours at room temperature in primary mab that has been diluted 1:1000 in blocking buffer. The filters are washed sequentially for 10 minutes at room temperature each wash, in washing buffer A (TNT Buffer+0.1% BSA), washing buffer B (TNT Buffer+0.1% BSA+0.1% NP-40), and then again washing buffer A, and incubated in a secondary peroxidase-conjugated goat anti-mouse IgG for 11/2 hours at room temperature. The filters are washed as before, then put in a final wash of TN (10 mM Tris, pH. 7.5, 150 mM NaCl). Color development is observed after putting filters in ABTS substrate.

Small cultures of individual colonies are then grown up overnight, by either: a) selecting the colonies that were positive from the immunological screening; or b) skipping the screening step and randomly selecting colonies (about 100). Each colony is inoculated into 2 ml of NZY medium containing 20 .mu.g/ml tetracycline, and these small cultures grown up overnight at 37.degree. C., with vigorous shaking. The next day cultures are centrifuged to pellet the cells, and the supernatant is removed. To 1 ml of the supernatant is then added 150 .mu.l PEG/NaCl, and the phage are precipitated overnight at 4.degree. C. Following subsequent centrifugation and removal of supernatant, the pellet is dissolved in 1 ml TBS.

For DNA sequencing, 400 .mu.l of the dissolved pellet is extracted once with phenol, and the resulting aqueous phase (about 300 .mu.l) is added to 500 .mu.l TE and 80 .mu.l 3M sodium acetate buffer. Then 1 ml ethanol is added and the SS DNA is allowed to precipitate overnight at 4.degree. C. Each sample is then microfuged for 30 minutes at 4.degree. C., the DNA pellet washed once in 70% ETOH, dried, and resuspended in 7 .mu.l H.sub.2 O. This template can be stored at -20.degree. C. until ready to use.

Due to the quite GC-rich Sfi 1 cloning site flanking the insertion region, sequencing reactions are carried out using the Sequenase 7-deaza dGTP DNA sequencing kit (Amersham-US Biochemicals, Arlington Heights, Ill.) with .sup.32 P-DATP and an antisense primer located approximately 40 nucleotides 3' to the insert site. Samples are run on a standard 6% sequencing gel using an IBI STS 45 sequencing apparatus (Eastman Kodak Company, Rochester, N.Y.).

The GCG software (Genetics Computer Group, Inc., Madison Wis.) is helpful for aligning sequences obtained from multiple clones in order to find consensus sequences. Certainly in the case of new mabs for which binding sites are sought, but even in the case of mab C-34, there is an interest in searching for sequences not only in GPIb alpha, but also in GPIb beta, GPIX, and in fact other platelet proteins that have been deposited in the available databases (Swiss Prot, Gen Bank, EMBL, etc.). Indeed, this analysis may provide important new information suggesting that a particular monoclonal antibody's epitope may be comprised of multiple components of the GPIb/IX complex that must accordingly be in close spatial proximity.

At this point, an ELISA assay can be used to evaluate individual clones, if the number of clones is high. In brief, phage having undergone two PEG precipitations, and subsequently adjusted for titer, can be incubated overnight with biotinylated mab, following which the mab-phage mixture can be added to wells of microtiter plates that have been previously coated with formalin-fixed platelets (or other suitable immobilized target recognized by the mab). Following a series of washing steps, avidin-peroxidase is added, the wells washed again, chromogenic substrate added, and the wells eventually read on an ELISA plate reader. The relative decrease in strength of signal in this assay provides guidance as to the most promising clones for further study. Concensus peptides identified in this manner can be chemically synthesized and characterized with respect to ability to bind original antibody. Peptides showing high binding affinity for the antibody can then be used as immunogens in mice and/or rabbits.

Epitope Mapping Studies of mab C-34

The two phage display libraries discussed above were employed in mapping studies with mab C-34. Results with the balanced, 10-mer peptide library were quite definitive with respect to strong concensus development among clones selected after two or three rounds of biopanning. Not only is there an evident concensus towards the 9-mer sequence SEQ ID NO: 38: W N W R Y R E Y V, but the 10-mer peptide including this sequence (SEQ ID NO: 1) with an amino-terminal alanine appeared to have the greatest selective advantage in the biopanning, since clones bearing this sequence were found the most frequently.

The series of cloned sequences is included in alignment form below. Double-underlines represent concensus amino acids and single-underlined amino acids represent significant homology to the concensus.

Results with the second peptide display library that is partially restricted in its amino acid repertoire revealed a series of clones which bind to C-34 without any appearance of the mimotope concensus sequence SEQ ID NO:38. The series of cloned sequences from the second library is included in alignment form below. SEQ ID NO:22 is the native sequence of GPIb alpha from amino acid 484 to 499, and represents a possible natural epitope sequence revealed by the clones isolated from the second library. The ' represents potential chymotrypsin cleavage sites. As above, double-underlines represent the possible native sequence (SEQ ID NO:22) within this second library and single-underlined amino acids represent significant homology to the possible native sequence.

The following cloned sequences were also obtained from the second peptide display library:

SEQ ID NO:39: QVRLWARAGAGQ

SEQ ID NO:40: GLAVTFGSVLEG

SEQ ID NO:41: VRWMCVIRLGVR

SEQ ID NO:42: RLWGPGVSRPVL

SEQ ID NO:43: CGSSLFRGPRCP

SEQ ID NO:44: LGISSLSFLQLR

SEQ ID NO:45: TWGWDGVSYLFL

SEQ ID NO:46: TRSLFDDFVSLR

SEQ ID NO:47: CYASLFRSRLCA

SEQ ID NO:48: DGSVRVVWVRLL

SEQ ID NO:49: LSGFPVALVRFA

SEQ ID NO:50: LGGGLLVGSVFP

SEQ ID NO:51: VWARGVFRDRFF

SEQ ID NO:52: TGLLAGPVWRWT

SEQ ID NO:53: WLGGIFSCLVCG

SEQ ID NO:54: WFLRDVGCGSCL

SEQ ID NO:55: SRCGVFTWCSRS

SEQ ID NO:56: RCLVGYRCWGGV

SEQ ID NO:57: GFRCLVMGGGCA

SEQ ID NO:58: CGFDLVCARLFG

SEQ ID NO:59: DSGVRWFFGFLG

SEQ ID NO:60: ILDGCFFLGRCP

SEQ ID NO:61: CVRWLVSAGCSG

SEQ ID NO:62: CVGCWLVCDVLL

SEQ ID NO:63: CLFVFAAGFACG

SEQ ID NO:64: SCALFGSCFGIS

SEQ ID NO:65: CWGGVGVCGLLV

SEQ ID NO:67: CVGGVASRCGVL

SEQ ID NO:68: SGAVLAGPFGVW

SEQ ID NO:69: CRAFDRVGVCVW

SEQ ID NO:70: RCLVGYVVGGVW

SEQ ID NO:71: VCLVYRSVDCWA

SEQ ID NO:72: WRVFVFTCVVWA

SEQ ID NO:73: LWREWRGLFAVL

SEQ ID NO:74: SGAVLAGPLWRL

SEQ ID NO:75: FVVRGGTFLFVR

SEQ ID NO:76: CRGIVFLFVGWL

SEQ ID NO:77: TGLLAGPVWRWT

SEQ ID NO:78: DSGVRWFFGFLG

SEQ ID NO:79: CAWHRLSFCGLV

SEQ ID NO:80: CFGSALVLAVLA

SEQ ID NO:81: WFWDMSGEWGGL

Comparison of Consensus Sequence to Native Sequences

Considerable effort was extended in trying to relate the consensus sequence of the above peptide (SEQ ID NO:38) to native sequences within GPIb alpha or other known proteins in the Swiss Protein or NCBI data banks. No such relation was found. This sequence accordingly represents a "mimotope"--i.e., a peptide which mimics a native epitope (a binding site for a monoclonal antibody), despite a lack of apparent homology at the primary amino acid sequence level (for mimotopes, see: Motti et al. 1994, Larocca et al. 1992, Lenstra et al. 1992, Balass et al. 1993, Hobart et al. 1993, and Luzzago et al. 1993). As noted after reviewing SEQ ID NOs: 1-21 and 66 above, not all selected clones appear to be part of this concensus group, and it is possible that with further sequencing clues as to the native epitope may be derived.

By using the second peptide display library that is partially restricted in its amino acid repertoire, another series of clones ("C34b" series) binding to C-34 without appearance of the mimotope concensus peptides were obtained. Following sequencing of these clones, a FASTA analysis (Pearson and Lipman 1988; Pearson 1990) was performed upon this group of clones by moving a 7-amino acid window along the sequence of GPIb alpha, advancing one amino acid at a time, and determining the group score as a function of position in the GPIb alpha molecule.

The results do not, in general, offer compelling matches in the sense of concensus development among the clones. However, the possible native GPIb alpha sequence revealed by this analysis is represented by SEQ ID NO:22.

Synthesized Peptide

A peptide including the concensus sequence (SEQ ID NO: 38) was chemically synthesized (Genosys Biotechnologies, The Woodlands, Tex.). The synthesized peptide had an amino acid sequence corresponding to SEQ ID NO:1: AWNWRYREYV. A modification of this peptide with a biotin attached to the amino-terminal alanine was also synthesized. One mg of the chemically synthesized biotinylated peptide was dissolved in one ml of water containing 20 .mu.l of DMSO. Since C-34 at a final concentration of 20 .mu.g/mL is a potent inhibitor of ristocetin-induced aggregation in citrated platelet-rich plasma (PRP), the synthetic peptide's potency was assessed by examining whether the peptide could neutralize the inhibitory activity of C-34 in this setting. Accordingly, approximately 10 .mu.g of C-34 was incubated at 22.degree. C. for 30 minutes with varying concentrations of test or control peptide, following which the mixture was added to PRP in a final volume of approximately 0.5 ml for an additional 10 minutes at 22.degree. C. As can be seen from the resulting aggregation curves (FIGS. 1-7), the synthesized peptide fully neutralized the C-34, producing half-maximal neutralization of the C-34 at about 1.0 .mu.g/ml, which is approximately 0.55 .mu.M for the biotinylated peptide. A similar pattern of C-34 antibody neutralization was observed when the non-biotinylated form of the peptide (having SEQ ID NO:38) was used, with half-maximal neutralization at approximately 3.0 .mu.M. The peptide (native or biotinylated) by itself did not induce platelet aggregation, nor did it appear to have non-specific effects, inasmuch as it had no influence on ADP-induced aggregation.

More specifically, FIG. 1 shows the ristocetin-induced full aggregation of platelets in the presence of von Willebrand factor. FIG. 2 shows the inhibition of ristocetin-induced aggregation of platelets by 20 .mu.g/ml of mab C-34. FIGS. 3-7 show varying degrees of neutralization of the inhibition of ristocetin-induced aggregation of platelets by 20 .mu.g/ml of mab C-34 in the presence of 0.14, 0.27, 0.55, 1.1, and 2.3 .mu.M of the synthetic biotinylated peptide mimotope having SEQ ID NO:1, respectively. In FIG. 3, 0.14 .mu.M of the peptide does not neutralize the C-34 inhibition; in FIG. 7, 2.3 .mu.M of the peptide fully neutralizes the C-34 inhibition, and FIGS. 4-6 show varying degrees of neutralization of the C-34 inhibition.

Additional Use of Synthesized Peptide

The chemically synthesized peptide can be conjugated to bovine serum albumin and used for raising polyclonal antibodies in rabbits. Standard procedures can be used to immunize the rabbits and to collect serum, as described below. Polyclonal antibody can be tested for its ability to bind to normal platelets, as well as to the wild-type and valine 233 mutant forms of recombinant GPIb alpha. For polyclonal antibody that shows a high affinity binding to platelets, functional studies can then be undertaken. These studies include adhesion, aggregation, agglutination, and vWF binding. F(ab)'.sub.2 and Fab fragments of the polyclonal antibody can be made if steric hindrance appears to be preventing an accurate evaluation of more specific modulating effects of the antibody (Becker and Miller 1989, Kupinski and Miller 1986, and Miller et al. 1986). Polyclonal antibody to the synthetic peptide that recognizes or stabilizes a conformation associated with heightened or diminished affinity for binding vWF can be obtained at .gtoreq.95% purity and conjugated to bovine serum albumin or to another carrier protein, for the production of murine monoclonal antibodies.

Production of Antibodies to Synthesized Peptides

Mice: Monoclonal antibody production can be carried out using BALB/c mice. Immunization of the B-cell donor mice can involve immunizing them with antigens mixed in TiterMax.TM. adjuvant as follows: 50 .mu.g antigen/20 .mu.l emulsion.times.2 injections given by an intramuscular injection in each hind flank on day 1. Blood samples can be drawn by tail bleeds on days 28 and 56 to check the titers by ELISA assay. At peak titer (usually day 56) the mice can be subjected to euthanasia by CO.sub.2 inhalation, after which splenectomies can be performed and spleen cells harvested for the preparation of hybridomas by standard methods.

Rabbits: Polyclonal antibodies can be raised in New Zealand white rabbits. Preimmune serum can be collected from rabbits sedated with ketamine/rompun (ketamine HCl at 20 mg/kg IM and xylazine HCl at 4 mg/kg IM) via the auricular artery. Ten to fifteen percent of the total blood volume can be collected at each bleeding. The hair over the ear can be shaved with a #40 clipper blade, wiped with 70% alcohol, and a sterile 22 gauge butterfly can be used for blood collection. The antigen can be mixed with either RIBI adjuvant or TITER-MAX.TM. adjuvant and used according to the manufacturer's instructions. The back can then be shaved, wiped with 70% alcohol, and a sterile 25 gauge needle with the antigen/adjuvant mixture therein can be used to administer subcutaneously and intramuscularly as recommended by the manufacturer's instructions. Immune serum samples can be collected as described for preimmune samples. When sufficient titers are reached, the animal can be anesthetized with sodium pentobarbital (60 mg/kg BW) via the lateral ear vein until deep anesthesia is achieved. Blood can be immediately collected via cardiac puncture into plastic centrifuge tubes and allowed to clot; afterwards, the blood can be centrifuged and the serum aspirated and frozen at -70.degree. C. For euthanasia, while under sodium pentobartital anesthesia at a dosage of 60 mg/kg, the rabbit can be exsanguinated via cardiac puncture.

Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

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