WO1998038214A1 - Heparan sulfate/heparin interacting protein compositions and methods of use - Google Patents

Abstract

Disclosed are novel polynucleotides encoding mammalian heparan sulfate/heparin interacting proteins and methods for their use in a variety of diagnostic and therapeutic regiments, including recombinant production of the encoded heparin/heparan sulfate interacting proteins, peptide epitopes, synthetic derivatives, and mutagenized forms thereof. Also disclosed and claimed are methods and kits for their use in the modulation of blood coagulation, and the interaction with antithrombin-3. Methods and compositions for enrichment of particular heparin species are also disclosed.

Description

HEPARAN SULFATE/HEPARIN INTERACTING PROTEIN

COMPOSITIONS AND METHODS OF USE

1. BACKGROUND OF THE INVENTION

The present application is a continuation-in-part of co-pending U.S. Patent Application

Serial No. 08/810,609 filed February 28, 1997, the entire text of which is specifically incorporated by reference herein without disclaimer. The government owns rights in the present invention pursuant to grant numbers HD25235 and CA16672 from the National Institutes of

Health.

1.1 FIELD OF THE INVENTION

The present invention relates generally to the field of molecular biology. More particularly, the invention discloses and claims novel nucleic acid sequences encoding a heparin/heparan sulfate

(Hp/HS) interacting protein, as well as the encoded protein, peptide epitopes, synthetic derivatives, and mutagenized forms thereof. Also disclosed and claimed are methods and kits for their use in the modulation of blood coagulation, and the interaction with antithrombin-3. Particular aspects of the invention relate to the use of the instant heparin/heparan sulfate interacting protein (HIP) nucleic acid and amino acid segments in the preparation of medicaments for a variety of diagnostic and therapeutic regimens.

1.2 DESCRIPTION OF RELATED ART

1.2.1 HEPARAN SULFATE PROTEOGLYCANS Heparan sulfate proteoglycans (HSPGs) located either on cell surfaces or in extracellular matrices are found in nearly all mammalian tissues (Fransson, 1987; Gallagher et al, 1986; Hook et al, 1984; Jalkanen, 1987; Yanagishita and Hascall, 1992). Functionally, HSPGs and a variety of Hp/HS-binding proteins have been shown to participate in a diverse range of biological processes such as cell attachment, growth factor binding, cell proliferation, migration, morphogenesis, and viral pathogenicity (Hassell et al, 1986; Poole, 1986; Templeton, 1992).

Several lines of evidence indicate that HSPGs play an important role during the initial attachment of the apical plasma membrane of trophectodermal cells of the blastocyst to the apical plasma membrane of the uterine epithelium. In mice, HSPGs are expressed on the cell surfaces of two-cell stage and post-implantation stage embryos (Dziadek, 1985). Furthermore, blastocyst attachment to laminin, fibronectin, and isolated mouse uterine epithelial cells in inhibited by HP. Embryo attachment also is inhibited by the treatment of embryos with Hp HS lyases or inhibitors of proteoglycan biosynthesis (Farach et al, 1987; 1988). Immunological studies of murine embryo implantation sites indicated that the core protein of the basement membrane HSPG, perlecan, and Hp/HS chains are located between the apical cell surfaces of trophectodermal cells and uterine epithelial cells during the peri-implantation stage (Carson et al, 1993). Expression of perlecan on the external trophectodermal surface correlates with acquisition of attachment competence in vitro as well. Externally disposed H/HS-binding sites have been described on the cell surface of mouse uterine epithelial cells (Wilson et al, 1990). Furthermore, using a heterologous cell adhesion assay, the inventors demonstrated that Hp/HS-like glycosaminoglycans participate in the initial attachment between two human cells lines, JAR and

RL95, used to mimic the initial attachment of the human embryonic trophectoderm to human uterine epithelial cells, respectively (Rohde and Carson 1993). As is the case for mouse uterine epithelia, the human uterine epithelial cell line, RL95, has specific, high affinity cell surface Hp/HS-binding sites, which are sensitive to mild trypsin digestion of intact cells. Three tryptic peptides that retained Hp/HS binding specificity were isolated from such trypsinates and partially amino-terminal sequenced (Raboudi et al, 1992).

1.2.2 HEPARIN SULFATE PROTEOGLYCAN EXPRESSION

A number of studies described above have demonstrated that HSPGs are expressed on the surfaces of mouse blastocysts and human trophoblastic cell lines where they function in cell adhesion events. In these studies, it was further demonstrated that adhesive activity resides in the constituent HS chains of the HSPGs. Consistent with these observations, specific

Hp/HS-binding sites were identified on the surfaces of both mouse uterine epithelial cells and human uterine epithelial cell lines (Wilson et al, 1990; Raboudi et al, 1992). Hp HS-binding sites have been described on the surfaces of a number of cell lines (Kjellen et al, 1980; Biswas,

1988; Redini et al, 1989; Watt et al, 1993); however, identification of these proteins has been elusive. N-CAM represents one well described cell surface Hp/HS-binding protein (Cole and

Glaser, 1986) but is not expressed in the uterus. Recently, Hp-binding epidermal growth factor-like growth factor was identified at mouse implantation sites (Das et al, 1994) and is one potential ligand for embryonic HSPGs. Several other candidate proteins have been described that display Hp/HS-binding activity (Lankes et al, 1988; Kohnke-Godt and Gabius, 1991) but have not been well characterized.

Hp/HS proteoglycans (HSPGs) expressed by different cells are able to interact with Hp/HS-binding effector proteins and perform important roles in extra-cellular matrix structure and function, cell adhesion growth, and differentiation (Ruoslahti and Yamaguchi, 1991; Jackson et al, 1991). Hp/HS-binding effector proteins comprise a variety of proteins that include growth factors (Ruoslahti and Yamaguchi, 1991), extracellular matrix components (Kallunki and Tryggvason, 1992; Vlodvasky et al, 1991), cytokines (Bernfield et al, 1992), and cell adhesion molecules (Cole and Glaser, 1986).

1.2.3 COAGULATION CONDITIONS AND DISORDERS

One of the most important inhibitors of coagulation is the antithrombin III-heparan (or AT-III-heparin) complex (Schrier and Leung, 1996). AT-III-antagonizes the action of the serine protease procoagulants, factors Xlla, XIa, IXa, Xa, and Ila, by forming stoichiometric complexes with them. Heparan (or heparin) binds to lysyl residues on AT-III, producing a conformational change that makes the active site on AT-III more accessible to the serine protease procoagulants. This action of heparan (or heparin) enhances thrombin inactivation by 750-fold and factor Xa inactivation to an even greater degree. Both thrombin and factor Xa, however, are protected from inactivation by AT-III-heparan when they are bound to platelets or endothelial cells. Heparan sulfate proteoglycans on the luminal surfaces of endothelial cells appear to activate

AT-III in a manner identical to that of administered heparin. These proteoglycans are intercalated into the plasma membrane of endothelial cells with the protein moiety inserting into the membrane core while the polysaccharide moiety, which is analogous to heparin, protrudes from the cell surface into the bloodstream. A number of diseases and disorders are characterized by excessive bleeding, including, but not limited to, thrombocytopenia. Thrombocytopenia may be caused by a number of factors, including abnormal platelet production, accelerated platelet removal resulting from immunologic or nonimmunologic mechanisms, sequestration of platelets in the spleen, or combinations of these mechanisms. The clinical presentation of thrombocytopenia varies depending on such factors as the presence or absence of pancytopenia and the etiology of the disorder. The hallmark of thrombocytopenia is petechiae, which reflect bleeding probably at the level of the capillary or postcapillary venule. Petechiae usually occur at sites of increased intravascular pressure. For example, they develop over the lower extremities, presumably because of the elevated hydrostatic pressure in the veins of the legs; they appear on the oral mucosa of the cheeks because the masseters generate enormous force on the mucosa during chewing; and they are found at sites where constricting items of clothing, such as brassiere straps, produce an increase in intravascular pressure (Schrier and Leung, 1996).

Administration of heparin to patients can lead to a form of thrombocytopenia termed heparin thrombocytopenia. Several prospective studies of many patients have made the clinical picture and pathogenesis of heparin thrombocytopenia reasonably clear. Clinically, about one percent of patients receiving porcine heparin and five percent of patients receiving bovine heparin will experience thrombocytopenia six to 10 days after administration is begun (Boshkov et al, 1993). Thrombocytopenia is usually mild, although a relatively small subset of patients with heparin-induced thrombocytopenia progressed to an extremely dangerous and even fatal disorder characterized by arterial thrombosis, including stroke and myocardial infarction as well as peripheral arterial occlusion, skin necrosis, and even purpura fulminans (Boshkov et al, 1993). There have also been reports of venous thromboembolism as part of this picture. This thrombotic variant of heparin-induced thrombocytopenia thus presents an extraordinary clinical challenge, because the major antithrombotic agent (heparin) is the cause of the disorder and cannot be used, and in addition, the thrombocytopenic patient has a potential for bleeding.

Heparin overdose may not be obvious. It causes subcutaneous hemorrhages and deep tissue hematomas. Intravenous protamine at a dose of 1 mg/100 U of administered heparin can terminate the disorder. However, because the half-life for the disappearance of protamine is faster than that for heparin, a heparin rebound may occur, requiring a second administration of protamine. Furthermore, the low-molecular-weight heparin preparations cause as much bleeding as standard unfractionated heparin (Thomas, 1992). Enoxaparin and Fragmin are the only low-molecular-weight heparins approved by the FDA, but it is likely that several more will be approved for specific indications. The ability of protamine to reverse the actions of these low-molecular-weight heparins is highly variable, and in some cases ineffective. For example, protamine does not completely reverse the actions of enoxaparin (Schrier and Leung, 1996). 1.3 DEFICIENCIES IN THE PRIOR ART

There is therefore, a continuing need in the art for the isolation and characterization of nucleic acid segments encoding such polypeptides, and particularly mammalian Hp/HS interacting proteins. In addition, the discovery of more effective heparin inhibitory compositions, and new treatments for bleeding disorders and diseases, including, but not limited to, thrombocytopenia, would represent a significant advance in the art.

2. SUMMARY OF THE INVENTION The present invention overcomes these and other limitations in the prior art by providing novel nucleic acid sequences encoding mammalian Hp HS interacting proteins (HIPs), and in particular, human and murine-derived sequences. The invention also provides methods of inhibiting heparin, for example inhibiting heparin binding to antithrombin-3, as well as methods of treating various bleeding disorders and diseases, including heparin induced thrombocytopenia. The present invention first provides a method of identifying a heparin component that binds to antithrombin-3. In certain aspects, the method comprises contacting a heparin sample suspected of containing a heparin component that binds to antithrombin-3 with an antithrombin-3 competitive heparan sulfate/heparin interacting protein under conditions effective to allow binding of the heparin component, and detecting the binding of the heparin component to the heparan sulfate/heparin interacting protein. As used herein, the term "antithrombin-3 competitive heparan sulfate/heparin interacting protein" refers to a heparan sulfate/heparin interacting protein that competes with antithrombin-3 for binding to heparin. In certain cases the heparin component so identified is analyzed for the ability to bind to antithrombin-3 under conditions effective for a standard heparin preparation to bind to antithrombin-3. The invention also provides a method for purifying a heparin species that binds to antithrombin-3, which in certain embodiments comprises contacting a heparin sample suspected of containing a heparin species that binds to antithrombin-3 with a heparan sulfate/heparin interacting protein under conditions effective to allow binding of the heparin species to the heparan sulfate/heparin interacting protein, and collecting the heparin species bound to the heparan sulfate/heparin interacting protein. Particularly preferred aspects of the present invention concern heparin components, samples or fractions that bind to antithrombin-3, thus inducing coagulation, but that do not bind, or bind less effectively, to the PF4 protein. Thus, the invention also provides a method of identifying a heparin component that binds to antithrombin-3, but does not bind to the PF4 protein. In certain aspects, the method comprises contacting a heparin sample suspected of containing a heparin component that binds to antithrombin-3, but does not bind to the PF4 protein with a heparan sulfate/heparin interacting protein under conditions effective to allow binding of the heparin component, collecting the heparin component bound to the heparan sulfate/heparin interacting protein, and comparing the binding of the heparin component to antithrombin-3 and the PF4 protein, wherein the binding of the heparin component to antithrombin-3 and the lack of binding of the heparin component to the PF4 protein is indicative of a heparin component that binds to antithrombin-3, but does not bind to the PF4 protein.

Also provided are methods for purifying a heparin species that binds to antithrombin-3, but does not bind to the PF4 protein, which in particular embodiments comprises contacting a heparin sample suspected of containing a heparin species that binds to antithrombin-3, but does not bind to the PF4 protein with a heparan sulfate/heparin interacting protein under conditions effective to allow binding of the heparin species to the heparan sulfate/heparin interacting protein, collecting the heparin species bound to the heparan sulfate/heparin interacting protein, and comparing the binding of the heparin component to antithrombin-3 and the PF4 protein, wherein the binding of the heparin component to antithrombin-3 and the lack of binding of the heparin component to the PF4 protein is indicative of the purification of a heparin component that binds to antithrombin-3, but does not bind to the PF4 protein.

In certain aspects, the purification method further comprises removing any remaining heparin components that bind to PF4, for example by contacting the purified, or partially purified, heparin species with PF4 protein. In an exemplary aspect, the PF4 protein is immobilized on a column matrix before contacting with the heparin species, while in other aspects the heparin/PF4 complex is separated from the heparin species or component by binding to an antibody that specifically recognizes and binds to the heparin/PF4 complex.

The invention also provides a method for neutralizing heparin, comprising contacting a heparin sample with a heparan sulfate/heparin interacting protein composition under conditions effective to allow binding of heparin to the heparan sulfate/heparin interacting protein composition. In a preferred aspect, the method is further defined as a method for neutralizing low molecular weight heparin, wherein the heparin sample comprises low molecular weight heparin.

Thus, also provided in the present invention are methods for inhibiting the binding of heparin to antithrombin-3, which in preferred aspects comprise contacting a heparin composition that binds to antithrombin-3 with a heparan sulfate/heparin interacting protein under conditions effective to allow binding of the heparin composition to the heparan sulfate/heparin interacting protein, thereby inhibiting the binding of heparin to antithrombin-3.

The invention further provides a method for promoting blood coagulation, comprising contacting a heparin-containing blood sample with a heparan sulfate/heparin interacting protein composition under conditions effective to allow binding of heparin within the blood sample to the heparan sulfate/heparin interacting protein composition. In certain preferred embodiments of the methods provided herein, the heparin sample, the low molecular weight heparin sample, the heparin composition that binds to antithrombin-3 or the heparin-containing blood sample is contacted with the heparan sulfate/heparin interacting protein composition in vitro.

The invention also provides methods of identifying a candidate substance that alters the binding of heparin to a heparin-binding molecule, which in certain aspects comprises admixing heparin, a heparan sulfate/heparin interacting protein and a candidate substance under conditions effective to allow binding of heparin to the heparan sulfate/heparin interacting protein, and determining the binding of heparin to the heparan sulfate/heparin interacting protein in the presence of the candidate substance and in the absence of the candidate substance, wherein the ability of a candidate substance to alter the binding of heparin to the heparan sulfate/heparin interacting protein is indicative of a candidate substance that alters the binding of heparin to a heparin-binding molecule. Thus provided are methods for identifying a candidate substance that inhibits the binding of heparin to a heparin-binding molecule, comprising determining the ability of the candidate substance to decrease the binding of heparin to the heparan sulfate/heparin interacting protein, and methods for identifying a candidate substance that increases the binding of heparin to a heparin-binding molecule, comprising determining the ability of the candidate substance to increase the binding of heparin to the heparan sulfate/heparin interacting protein. In preferred aspects of the instant methods, the heparan sulfate/heparin interacting protein comprises the amino acid sequence of SEQ ID NO:2. In certain embodiments of the present methods, the heparan sulfate/heparin interacting protein is encoded by the nucleic acid sequence of SEQ ID NO:l. In particularly preferred aspects of the presently disclosed methods, the heparan sulfate/heparin interacting protein is a recombinant heparan sulfate/heparin interacting protein prepared by expressing the nucleic acid sequence of SEQ ID NO: 1.

The heparan sulfate/heparin interacting protein compositions disclosed herein are contemplated for use in a number of different embodiments, exemplified by, but not limited to, use in inhibiting the binding of heparin to antithrombin-3; use in the preparation of a medicament for inhibiting the binding of heparin to antithrombin-3; use in promoting blood coagulation; use in the preparation of a medicament for promoting blood coagulation; use in neutralizing heparin; use in the preparation of a medicament for neutralizing heparin; use in neutralizing low molecular weight heparin; use in the preparation of a medicament for neutralizing low molecular weight heparin; use in treating a disease characterized by excessive bleeding; use in the preparation of a medicament for treating a disease characterized by excessive bleeding in a human patient; use in treating heparin overdose; use in the preparation of a medicament for treating heparin overdose in a human patient; use in treating heparin induced thrombocytopenia; and use in the preparation of a medicament for treating heparin induced thrombocytopenia in a human patient. In preferred uses, the heparan sulfate/heparin interacting protein comprises the amino acid sequence of SEQ ID NO:2. In other preferred uses, the heparan sulfate/heparin interacting protein is encoded by the nucleic acid sequence of SEQ ID NO: 1. In yet other preferred uses, the heparan sulfate/heparin interacting protein is a recombinant heparan sulfate/heparin interacting protein prepared by expressing the nucleic acid sequence of SEQ ID NO:l. Thus, the present invention also contemplates the use of a heparin sulfate/heparin interacting protein in the preparation of a medicament for: inhibiting the binding of heparin to antithrombin-3; promoting blood coagulation; neutralizing heparin; neutralizing low molecular weight heparin; treating a disease characterized by excessive bleeding in a human patient; treating heparin overdose in a human patient; and treating heparin induced thrombocytopenia in a human patient. 3. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1. Overlapping clones and RT-PCR™ product for HIP. The full length sequence, including restriction enzyme sites for Bsu36l (B), EcoRV (E), Hrødlll (H), and Pstl (P) and positions of start codon, ATG, and stop codon, TAG, is shown at the top of the figure (1). Also shown is the RT- PCR™ 224 product (2), clone 23-1 and clone 42-1 (3), clone 35-2 (4) and clone

36-1 (5). The lines indicate the size and position of each clone and RT-PCR™ product. The scale in basepairs (bp) is shown at the bottom of the figure (6).

FIG. 2A and FIG. 2B. Binding of anti-ΗIP to intact RL95 cells is saturable and specific. FIG. 2A. Shown are monolayers of RL95 cells grown in a 24-well tissue culture plate to 90% confluency. Cells were incubated at 4°C for 45 min with anti-ΗIP (•) or nonimmune rabbit IgG

(D) as described. The data represent the average ± S.Ε. of duplicate determinations. The average ± S.Ε. obtained for specific binding (anti-ΗIP binding) minus the average binding obtained with nonimmune rabbit IgG (nonspecific) is also shown (Δ). Binding is both specific and saturable between 5 and 10 μg of anti-ΗIP/ml. The amount of I-Protein A bound to the RL95 cell surface (cpm x 10^" ) is shown on the vertical axis, and the antibody concentration

(μg/ml) is shown on the horizontal axis. FIG. 2B. 25 μg of anti-ΗIP or nonimmune rabbit IgG was preincubated without (striped boxes; anti-ΗIP lane 1 ; rabbit IgG lane 3) or with (open boxes; anti-ΗIP lane 2; rabbit IgG lane 4) 100 μl of peptide affinity matrix for 2 h before incubation with RL95 cells. The data are the averages ± S.Ε. for duplicate determinations in each case. The amount of I-Protein A bound to the RL95 cell surface (cpm x 10^" ) is shown on the vertical axis, and the lane number is shown on the horizontal axis.

FIG. 3. Factor Xa-dependent chromogenic reaction was performed as described in Section 5.3.1. Bulk (▼), RT-Ηp (♦), LA-Ηp (•), and ΗA-Ηp (A) were added to the assay at the indicated concentrations (uronic acid content; ng/ml, horizontal axis). The concentration of 36.25 ng/ml (uronic acid content) of unfractionated Ηp is equivalent to 0.01 IU/ml of bulk Ηp. Results are means of duplicate determinations with standard deviations less than 15%. OD₄₀₅ is shown on the vertical axis.

FIG. 4A and FIG. 4B. Effect of HIP peptide on blood coagulation. FIG. 4A. The factor Xa (FXa)-dependent chromogenic reaction was performed as described in Section 5.3.1. The FXa-dependent chromogenic reaction was performed in the presence of 0.1 IU/ml of antithrombin-3 (AT-III) and 0.07 IU/ml Hp and indicated HIP peptide concentrations (μg/ml; horizontal axis). FXa-activity is expressed as the percentage of the AT-III complex inhibitable enzymatic activity (percent FXa activity shown on vertical axis). Data shown represents means of duplicate determinations with standard deviations less than 15%. FIG. 4B. The thrombin- dependent chromogenic reaction was performed as described in Section 5.3.1. The thrombin- dependent chromogenic reaction was performed in the presence of 0.1 IU/ml of antithrombin-3 (AT-III) and 0.07 IU/ml Hp and indicated HIP peptide concentrations (μg/ml; horizontal axis). Thrombin-activity is expressed as the percentage of the AT-III complex inhibitable enzymatic activity (percent thrombin activity shown on vertical axis). Data shown represents means of duplicate determinations with standard deviations less than 15%.

4. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS 4.1 SOME ADVANTAGES OF THE INVENTION

Those of ordinary skill having the benefit of this disclosure will appreciate that the invention provides a number of advantages, including the following:

The HIP peptide has been shown to selectively bind a subset of species of the polysaccharide, Hp, with high affinity and selectivity. This subset of Hp represents the form that bind with highest affinity to antithrombin-3, a protease inhibitor that prevents blood coagulation when complexed with Hp. The HIP peptide when added to in vitro clinical assays for blood coagulation activity has been shown to block Hp's anticoagulant activity, presumably by preventing Hp from binding to and activating antithrombin-3. The HIP peptide and parent protein represents an important means for the isolation and identification of anticoagulant species of Hp (constituting approximately 5% of the total Hp population) thereby generating preparations of this anticoagulant factor that are enriched for the desired activity and depleted of undesired side effects. Because the HIP peptide can be synthesized in large quantities and used to generate affinity resins for isolation of anticoagulant Hp, and such resins can be used multiple times, the methods and compositions of the present invention represent a monumental improvement over current procedures, which are expensive, tedious, produce relatively low yields and involve complicated fractionation schemes.

In another important embodiment, the peptides of the present invention may be used directly to stimulate blood coagulation, e.g., at would sites or as pretreatments for bandages, and minimize bleeding.

The peptide and parent protein contains a sequence of amino acids that are of such a composition and arrangement as to allow species of Hp with a certain structural motif, i.e., one identical to or overlapping the antithrombin-3 binding motif, to selectively bind tightly. Hp bound to HIP peptide affinity matrices can be dissociated by increasing the salt concentration. This salt can be subsequently removed by dialysis. The interaction of the peptide and Hp is of sufficient strength in solution to prevent anticoagulantly active Hp from being able to dissociate and bind to antithrombin-3. As a result, the peptide and, presumably, the parent protein neutralize Hp's anticoagulant activity by preventing formation of the biologically active Hp: antithrombin-3 complex.

In another important application of the technology disclosed and claimed herein, methods are provided for using HIPs as a convenient method of purifying anticoagulant species of Hp from heterogeneous mixtures. Currently, it is not possible to isolate anticoagulant-active species from non-active species due to limitations in the art. Furthermore, high levels of Hp must be used to provide enough of the quantitatively minor active species. Purification of these species using the peptide affinity resins disclosed herein may be performed cheaply in a short time period and can be readily scaled up to production levels. A further illustrative embodiment of the technology is the use of the peptides disclosed to promote blood coagulation. This may be desirable in cases of excessive bleeding, e.g., injury, trauma, or surgery. The peptide may either be applied directly as a mixture or used to coat or pretreat bandages or other wound coverings. The peptide would neutralize species of Hp released by mast cells at the wound/invasion site which would inhibit coagulation and promote bleeding. The peptide has been shown to display this activity in in vitro blood coagulation assays.

4.2 HPSG ' s AND THEIR ROLE IN EMBRYONIC ATTACHMENT The present inventors have found that HSPGs and their corresponding binding sites on cell surfaces may be important in the initial stage of mouse embryo attachment to uterine epithelium. Upon hatching from the zona pellucida, the embryo initially attaches to the uterus through the adhesion of the apical surfaces of the trophectodermal cells of the blastocyst. HSPGs are expressed by mouse embryos at the two-cell and post-implantation stages (Dziadek et al, 1985). Expression of HSPGs on trophectodermal cell surfaces of mouse blastocysts increases

4-5 fold at the peri-implantation stage (Carson et al, 1993: Farach et al, 1987), and HS expressed on mouse embryo surfaces is required for embryo attachment to isolated mouse uterine epithelial cells, fibronectin, and laminin (Farach et al, 1987). Similarly, studies have shown that the initial attachment of JAR cells, a human trophoblastic cell line, to RL95 cells, a human uterine epithelial cell line, is Hp/HS-dependent, and enzymatic removal of HS from cell surfaces of both JAR and RL95 cells markedly inhibits JAR-RL95 cell-cell adhesion (Rohde and Carson, 1993). Therefore, HSPGs and their binding proteins also may play an important role in the initial attachment of human trophoblast cells to uterine epithelial cells.

Specific, saturable cell surface Hp/HS-binding sites have been identified on mouse uterine epithelial cells and human uterine epithelial cell lines (Wilson et al, 1990; Raboudi et al,

1992). Since mouse uterine epithelial cells have only been available by primary cell culture, there is a practical limitation to isolating Hp/HS-binding sites from this source. Effort has been placed on the study of Hp/HS-binding proteins expressed on the cell surfaces of a human uterine epithelial cell line, RL95 (Raboudi et al, 1992). Mild tryptic digestion of RL95 cell surfaces removed most of cell surface Hp/HS-binding activity. Three major tryptic peptide fragments, ranging in M_t between 6,000 and 14,000, were released from cell surfaces and retained Hp/HS-binding activity. Partial amino-terminal amino acid sequences from each of these three peptides were obtained (Raboudi et al, 1992).

The inventors have employed an approach of reverse transcription^'polymerase chain reaction (RT-PCR™) to identify transcripts encoding cell surface Hp/HS-binding peptides. Predicted peptide sequence from one of the RT-PCR™ products revealed an antigenic sequence that also has features of a Hp HS-binding motif suggested by others (Cardin and Weintraub, 1989). Polyclonal antibodies directed against the synthetic peptide corresponding to this motif recognize a novel Hp/HS-binding protein, named Hp/HS interacting protein (HIP), expressed on RL95 cell surfaces with an apparent M_r of 24,000 determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Rohde et al, 1996). This peptide selectively binds Hp/HS, recognizes certain forms of HP and cell surface HS expressed by JAR and RL95 cells, and supports the attachment of human trophoblast cell lines and a variety of other mammalian adherent cell lines. Complete cDNA sequence of HIP has been isolated by screening cDNA libraries using the partial cDNA sequence of RT-PCR™ product of HIP. HIP cDNA sequence contains a single open reading frame encoding 159 amino acids with a calculated molecular mass of 17,754 Da and a predicted pi of 11.75. This protein is approximately 80% homologous at both the nucleotide and amino acid level to a rodent protein designated as ribosomal protein L29. Transfection of HIP into NIH-3T3 cells results in expression of an M_t 24,000 protein that can be detected on the cell surface.

4.3 i /p-ENCODiNG NUCLEIC ACID SEGMENTS

The invention discloses and claims novel nucleic acid sequences encoding a mammalian HIP, and in particular murine and human HIP. As used herein, a gene encoding HIP or a "hip gene" is used to refer to a gene or DNA coding region that encodes a protein, polypeptide or peptide that is capable of binding or interacting with Hp and/or HS. A preferred nucleic acid sequence encoding a HIP gene is the nucleotide sequence of SEQ ID NO:l, which is the DNA sequence of a HIP isolated from human uterine cell line RL95, or a nucleic acid sequence which is complementary to the sequence of SEQ ID NO:l, or a nucleic acid sequence which hybridizes to the nucleic acid sequence of SEQ ID NO:l under conditions of moderate to high stringency. It is expected that the gene encoding HIP will vary in nucleic acid sequence from cell line to cell line, or from strain to strain, but that the variation in nucleic acid sequence will not preclude hybridization between sequences encoding HIP of each strain under moderately strict to strict hybridization conditions. As used herein, HIP means a purified and isolated protein including a strain variant or an active fragment thereof, derived from mammalian sources, and in particular human or murine cells.

Preferably, HIP is a recombinant protein encoded by a nucleic acid sequence isolated from human uterine cell line RL95, and most preferably comprises the amino acid sequence of SEQ ID NO:2, or a variant or an active fragment thereof.

Alternatively, HIP means a protein selected from the group consisting of: polypeptides that are immunologically reactive with antibodies generated against HIP and also immunologically reactive with HIP encoded by a nucleic acid sequence contained in SEQ ID NO: 1 , or a biologically- active variant thereof; polypeptides that are capable of eliciting antibodies that are immunologically reactive with HIP encoded by a nucleic acid sequence contained in SEQ ID NO: 1 , or a biologically- active variant thereof; and polypeptides that selectively bind a subset of a species of Hp with high affinity and selectivity, this species being the subset of Hp molecules that bind with the highest affinity to antithrombin-3, a protease inhibitor that prevents blood coagulation when complexed with heparin, and in particular, polypeptides whose antibodies are immunologically reactive with the human HIP disclosed in SEQ ID NO:2, or a protein encoded by the nucleic acid sequence of

SEQ ID NO: 1.

As used herein, a strain variant of HIP means any polypeptide encoded, in whole or in part, by a nucleic acid sequence which hybridizes under strict hybridization conditions to a nucleic acid sequence of SEQ ID NO: 1 encoding the HIP of human cell line RL95, the amino acid sequence of which is disclosed in SEQ ID NO:2. One of skill in the art will understand that strain variants of

HIP include those proteins encoded by nucleic acid sequences which may be amplified using a contiguous nucleic acid sequence from SEQ ID NO: 1.

As used herein, an active fragment of HIP includes HIP which is modified by conventional techniques, e.g., by addition, deletion, or substitution, but which active fragment exhibits substantially the same structure and function as HIP as described herein. For example, portions of the protein not required to block the formation of a biologically-active Hp:antithrombin-3 complex may be deleted or altered; additions to the protein may be made to enhance the protein's antigenicity according to conventional methods.

It will, of course, be understood that one or more than one gene encoding HIPs or peptides may be used in the methods and compositions of the invention. The nucleic acid compositions and methods disclosed herein may entail the administration of one, two, three, or more, genes or gene segments. The maximum number of genes that may be used is limited only by practical considerations, such as the effort involved in simultaneously preparing a large number of gene constructs or even the possibility of eliciting a significant adverse cytotoxic effect. In using multiple genes, they may be combined on a single genetic construct under control of one or more promoters, or they may be prepared as separate constructs of the same of different types. Thus, an almost endless combination of different genes and genetic constructs may be employed. Certain gene combinations may be designed to, or their use may otherwise result in, achieving synergistic effects on formation of an immune response, or the development of antibodies to gene products encoded by such nucleic acid segments, or in the production of diagnostic and treatment protocols. Any and all such combinations are intended to fall within the scope of the present invention. Indeed, many synergistic effects have been described in the scientific literature, so that one of ordinary skill in the art would readily be able to identify likely synergistic gene combinations, or even gene-protein combinations. It will also be understood that, if desired, the nucleic segment or gene could be administered in combination with further agents, such as, e.g., proteins or polypeptides or various pharmaceutically active agents. So long as genetic material forms part of the composition, there is virtually no limit to other components which may also be included, given that the additional agents do not cause a significant adverse effect upon contact with the target cells or tissues. Other aspects of the present invention concern isolated DNA segments and recombinant vectors encoding HIP, and the creation and use of recombinant host cells through the application of DNA technology, that express hip gene products. As such, the invention concerns DNA segment comprising an isolated gene that encodes a protein or peptide that includes an amino acid sequence essentially as set forth by a contiguous sequence from SEQ ID NO:2. These DNA segments are represented by those that include a nucleic acid sequence essentially as set forth by a contiguous sequence from SEQ ID NO: 1. Compositions that include a purified protein that has an amino acid sequence essentially as set forth by the amino acid sequence of SEQ ID NO:2 are also encompassed by the invention.

As used herein, the term "DNA segment" refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding HIP refers to a DNA segment that contains HIP coding sequences yet is isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment is obtained. Included within the term "DNA segment" are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, viruses, and the like.

Similarly, a DNA segment comprising an isolated or purified HIP gene refers to a DNA segment including HIP coding sequences and, in certain aspects, regulatory sequences, isolated substantially away from other naturally occurring genes or protein encoding sequences. In this respect, the term "gene" is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides or peptides. Such segments may be naturally isolated, or modified synthetically by the hand of man.

"Isolated substantially away from other coding sequences" means that the gene of interest, in this case, a gene encoding HIP, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or polypeptide coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man. In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incoφoratingDNA sequences that encode a HIP species that includes within its amino acid sequence an amino acid sequence essentially as set forth in SEQ ID NO:2. In other particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that include within their sequence a nucleotide sequence essentially as set forth in SEQ ID NO: 1.

The term "a sequence essentially as set forth in SEQ ID NO:2" means that the sequence substantially corresponds to a portion of SEQ ID NO:2 and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of SEQ ID NO:2. The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein below (see Section 4.8). Accordingly, sequences that have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91% and about 99%; of amino acids that are identical or functionally equivalent to the amino acids of SEQ ID NO:2 will be sequences that are "essentially as set forth in SEQ ID NO:2". In certain other embodiments, the invention concerns isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO:l. The term "essentially as set forth in SEQ ID NO:l" is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:l and has relatively few codons that are not identical, or functionally equivalent, to the codons of SEQ ID NO: 1. Again, DNA segments that encode proteins exhibiting

HIP-like activity will be most preferred.

It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5 ' or 3' portions of the coding region or may include various upstream or downstream regulatory or structural genes. Naturally, the present invention also encompasses DNA segments that are complementary, or essentially complementary, to the sequence set forth in SEQ ID NO: 1. Nucleic acid sequences that are "complementary" are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules. As used herein, the term "complementary sequences" means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ ID NO: 1 , under relatively stringent conditions such as those described herein.

The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, antibody tags, isolation sequences (such as a hexahistidine sequence) and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, nucleic acid fragments may be prepared that include a short contiguous stretch identical to or complementary to SEQ ID NO:l, such as about 14 nucleotides, and that are up to about 10,000 or about 5,000 base pairs in length, with segments of about 3,000 being preferred in certain cases. DNA segments with total lengths of about 2,000, about 1,000, about 500, about 200, about 100 and about 50 base pairs in length (including all intermediate lengths) are also contemplated to be useful.

It will be readily understood that "intermediate lengths", in these contexts, means any length between the quoted ranges, such as 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,

31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through the 200-500; 500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000; 5,000- 10,000 ranges, up to and including sequences of about 12,001, 12,002, 13,001,

13,002 and the like.

The nucleic acid sequences disclosed herein also have a variety of other uses, for example as probes or primers in nucleic acid hybridization embodiments. As such, it is contemplated that nucleic acid segments that comprise a sequence region that consists of at least a 20 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 20 nucleotide long contiguous sequence of SEQ ID NO:l will find particular utility. Longer contiguous identical or complementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediate lengths) and even up to full length sequences will also be of use in certain embodiments. The ability of such nucleic acid probes to specifically hybridize to HIP-encoding sequences will enable them to be of use in detecting the presence of complementary sequences in a given sample. However, other uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.

Nucleic acid molecules having sequence regions consisting of contiguous nucleotide stretches of about 10-14, about 15-20, about 30-40, about 50-60, or even about 60, 70, 80, 90, or 100-200 nucleotides or so, identical or complementary to SEQ ID NO:l, are particularly contemplated as hybridization probes for use in, e.g., Southern and Northern blotting. This would allow HIP structural or regulatory genes to be analyzed, both in diverse cell types and also in various host cells, or from various cell lines or tissue types. The total size of fragment, as well as the size of the complementary stretch(es), will ultimately depend on the intended use or application of the particular nucleic acid segment. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the contiguous complementary region may be varied, such as between about 17 and about 100 nucleotides, or more preferably between about 20 and about 60 nucleotides, but larger contiguous complementarity stretches may be used, according to the length complementary sequences one wishes to detect.

The use of a hybridization probe of about 17 to about 25 or 30 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having contiguous complementary sequences over stretches greater than about 20 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of 21 to 45 contiguous nucleotides, or even longer where desired.

Hybridization probes may be selected from any portion of any of the sequences disclosed herein. All that is required is to review the sequence set forth in SEQ ID NO:l and to select any continuous portion of the sequence, from about 14-35 nucleotides in length up to and including the full length sequence, that one wishes to utilize as a probe or primer. The choice of probe and primer sequences may be governed by various factors, such as, by way of example only, one may wish to employ primers from towards the termini of the total sequence.

The process of selecting and preparing a nucleic acid segment that includes a contiguous sequence from within SEQ ID NO:l may alternatively be described as preparing a nucleic acid fragment. Of course, fragments may also be obtained by other techniques such as, e.g., by mechanical shearing or by restriction enzyme digestion. Small nucleic acid segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCR™ technology of U.S. Patent 4,683,202 (incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.

Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of the entire hip gene or gene fragments. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of 50°C to

70°C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating HIP genes.

Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate HIP-encoding sequences from related species, functional equivalents, or the like, less stringent hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ conditions such as about 0.15 M to about 0.9 M salt, at temperatures ranging from 20°C to 55°C. Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.

In certain embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In preferred embodiments, one will likely desire to employ a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known that can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid- containing samples.

In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantitated,by means of the label.

Thus, methods for detecting HIP-encoding nucleic acid sequences are also provided by the invention. The hip nucleic acid sequences disclosed, and particularly those in SEQ ID NO:l are useful as diagnostic probes to detect the presence of HIP-encoding polynucleotides in a test sample, using conventional techniques. In one such method, hip nucleic acid segments may be used in Southern hybridization analyses or Northern hybridization analyses to detect the presence of hip nucleic acid segments within a laboratory specimen, clinical sample, cell line, or from virtually any aqueous sample suspected of containing such polynucleotide. In a preferred embodiment, the nucleic acid sequence of SEQ ID NO: 1 is preferable as a probe for such hybridization analyses.

It will also be understood that this invention is not limited to the particular nucleic acid and amino acid sequences as disclosed in SEQ ID NO:l and SEQ ID NO:2, respectively. Recombinant vectors and isolated DNA segments may therefore variously include the HIP coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include HIP coding regions or may encode biologically functional equivalent proteins or peptides that have variant amino acids sequences.

The DNA segments of the present invention encompass biologically functional equivalent HIP proteins and peptides, in particular those HIP proteins isolated from mammalian sources, and particularly human and murine species. DNA segments isolated from humans which are shown to bind Hp/HS are particularly preferred for use in the methods disclosed herein. Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, e.g. , to introduce improvements to the antigenicity of the protein or to test mutants in order to examine activity at the molecular level. If desired, one may also prepare fusion proteins and peptides, e.g. , where the HIP coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetecti on purposes (e.g., proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively).

Recombinant vectors form further aspects of the present invention. Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA segment, whether encoding a full length protein or smaller peptide, is positioned under the control of a promoter. The promoter may be in the form of the promoter that is naturally associated with a HIP gene, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment, for example, using recombinant cloning and/or PCR™ technology, in connection with the compositions disclosed herein. In connection with expression embodiments to prepare recombinant HIP and peptides, it is contemplated that longer DNA segments will most often be used, with DNA segments encoding the entire HIP or functional domains, epitopes, ligand binding domains, subunits, etc. being most preferred. However, it will be appreciated that the use of shorter DNA segments to direct the expression of HIP peptides or epitopic core regions, such as may be used to generate anti-HJP antibodies, also falls within the scope of the invention. DNA segments that encode peptide antigens from about 15 to about 100 amino acids in length, or more preferably, from about 15 to about 50 amino acids in length are contemplated to be particularly useful.

The hip gene and DNA segments may also be used in connection with somatic expression in an animal or in the creation of a transgenic animal. Again, in such embodiments, the use of a recombinant vector that directs the expression of the full length or active HIP protein is particularly contemplated. Expression of hip transgene in animals is particularly contemplated to be useful in the production of anti-HIP antibodies.

4.4 RECOMBINANT EXPRESSION OF HIP Recombinant clones expressing the hip nucleic acid segments may be used to prepare purified recombinant HIP (rHIP), purified rHIP-derived peptide antigens as well as mutant or variant recombinant protein species in significant quantities. Additionally, by application of techniques such as DNA mutagenesis, the present invention allocs the ready preparation of so- called "second generation" molecules having modified or simplified protein structures. Second generation proteins will typically share one or more properties in common with the full-length antigen, such as a particular antigenic/immunogenic epitopic core sequence. Epitopic sequences can be provided on relatively short molecules prepared from knowledge of the peptide, or encoding DNA sequence information. Such variant molecules may not only be derived from selected immunogenic/ antigenic regions of the protein structure, but may additionally, or alternatively, include one or more functionally equivalent amino acids selected on the basis of similarities or even differences with respect to the natural sequence.

A particular aspect of this invention provides novel ways in which to utilize recombinant HIPs or HIP-derived peptides, nucleic acid segments encoding these peptides, recombinant vectors and transformed host cells comprising hip or hip-derived DNA segments, recombinant vectors and transformed host cells comprising hip or /zzp-derived DNA segments, and recombinant vectors and transformed host cells comprising mammalian hip-deή\ed DNA segments. As is well known to those of skill in the art, many such vectors and host cells are readily available, one particular detailed example of a suitable vector for expression in mammalian cells is that described in U. S. Patent 5,168,050, incorporated herein by reference. However, there is no requirement that a highly purified vector be used, so long as the coding segment employed encodes a protein or peptide of interest (e.g., a HIP from human or murine sources, and particularly a HIP from human uterine cell line RL95), and does not include any coding or regulatory sequences that would have an adverse effect on cells. Therefore, it will also be understood that useful nucleic acid sequences may include additional residues, such as additional non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various regulatory sequences. After identifying an appropriate epitope-encoding nucleic acid molecule, it may be inserted into any one of the many vectors currently known in the art, so that it will direct the expression and production of the protein or peptide epitope of interest (e.g., a HIP from human or murine sources, and particularly a HIP from human uterine cell line RL95) when incoφorated into a host cell. In a recombinant expression vector, the coding portion of the DNA segment is operatively positioned under the control of a promoter. It is understood in the art that to operatively position a coding sequence "under the control" of such a promoter, one positions the 5' end of the transcription initiation site of the transcriptional reading frame of the protein between about 1 and about 50 nucleotides "downstream" of (i.e., 3' of) the chosen promoter. The promoter may be in the form of the promoter which is naturally associated with a HIP- encoding nucleic acid segment, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment, for example, using recombinant cloning and/or PCR™ technology, in connection with the compositions disclosed herein. Direct amplification of nucleic acids using the PCR™ technology of U.S. Patents 4,683,195 and 4,683,202 (herein incoφoratedby reference) are particularly contemplated to be useful in such methodologies.

In certain embodiments, it is contemplated that particular advantages will be gained by positioning the HIP-encoding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a hip or hip-like gene segment in its natural environment. Such promoters may include those normally associated with other HS/Hp interacting protein-encoding genes, and/or promoters isolated from any other bacterial, viral, eukaryotic, or mammalian cell. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the particular cell containing the vector comprising the HIP- encoding nucleic acid segment. For the expression of HIP and HIP-derived epitopes, once a suitable clone or clones have been obtained, whether they be native sequences or genetically-modified, one may proceed to prepare an expression system for the recombinant preparation of HIP or HIP-derived peptides. The engineering of DNA segment(s) for expression in a prokaryotic or eukaryotic system may be performed by techniques generally known to those of skill in recombinant expression. It is believed that virtually any expression system may be employed in the expression of HIP or HIP-derived epitopes.

The use of recombinant promoters to achieve protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al, (1989). The promoters employed may be constitutive or inducible, and can be used under the appropriate conditions to direct high level or regulated expression of the introduced DNA segment. In certain embodiments, the expression of recombinant HIPs is carried out using prokaryotic expression systems, and in particular bacterial systems such as E. coli, Salmonella or related Enter obacteriaceae. Such prokaryotic expression of nucleic acid segments of the present invention may be performed using methods known to those of skill in the art, and will likely comprise expression vectors and promoter sequences such as those provided by tac, trp, lac, lacUV5 or T7 promoters.

The DNA sequences encoding the desired HIP or HIP-derived epitope (either native or mutagenized) may be separately expressed in bacterial systems, with the encoded proteins being expressed as fusions with β-galactosidase, ubiquitin, Schistosoma japonicum glutathione S- transferase, S. aureus Protein A, maltose binding protein, and the like. It is believed that bacterial expression will ultimately have advantages over eukaryotic expression in terms of ease of use and quantity of materials obtained thereby.

For eukaryotic expression, the some of the preferred promoters are those such as CMV, RSV LTR, the SV40 promoter alone, and the SV40 promoter in combination with the SV40 enhancer. Another eukaryotic promoter system contemplated for use in high-level expression is the Pichia expression vector system (Pharmacia LKB Biotechnology). Baculovirus-based, glutamine synthase-based or dihydrofolate reductase-based systems may also be employed. In preferred embodiments it is contemplated that plasmid vectors incoφorating an origin of replication and an efficient eukaryotic promoter, as exemplified by the eukaryotic vectors of the pCMV series, such as pCMV5, will be of most use.

Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA. This ability to act over a large distance had little precedent in classic studies of prokaryotic transcriptional regulation. Subsequent work showed that regions of DNA with enhancer activity are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins.

The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.

Additionally any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of a transgene. Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.

Table 1 - Promoter and Enhancer Elements

Table 2 - Inducible Elements

Where eukaryotic expression is contemplated, one will also typically desire to incoφorate into the transcriptional unit which includes nucleic acid sequences encoding HIP or HIP-derived peptides, an appropriate poly adenylation site (e.g., 5'-AATAAA-3') if one was not contained within the original cloned segment. Typically, the poly- A addition site is placed about 30 to 2000 nucleotides "downstream" of the termination site of the protein at a position prior to transcription termination.

Particular aspects of the invention concern the use of plasmid vectors for the cloning and expression of recombinant peptides, and particular peptide epitopes comprising either native, or site-specifically mutated HIP epitopes. The generation of recombinant vectors, transformation of host cells, and expression of recombinant proteins is well-known to those of skill in the art. It is proposed that transformation of host cells with DNA segments encoding such epitopes will provide a convenient means for obtaining HIP or HIP-derived peptides. Genomic sequences are suitable for eukaryotic expression, as the host cell will, of course, process the genomic transcripts to yield functional mRN A for translation into protein.

In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli may be typically transformed using vectors such as pBR322, or any of its derivatives (Bolivar et ah, 1977). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophagemay also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of endogenous proteins.

In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts.

For example, bacteriophage such as λGEM™-l 1 may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392.

Those promoters most commonly used in recombinant DNA construction include the β-lactamase (penicillinase) and lactose promoter systems (Chang et al, 1978; Itakura et al., 1977; Goeddel et al, 1979) or the tryptophan (trp) promoter system (Goeddel et al., 1980). The use of recombinant and native microbial promoters is well-known to those of skill in the art, and details concerning their nucleotide sequences and specific methodologies are in the public domain, enabling a skilled worker having the present disclosure to construct particular recombinant vectors and expression systems for the puφose of producing compositions of the present invention. In addition to the preferred embodiment expression in prokaryotes, eukaryotic microbes, such as yeast cultures may also be used in conjunction with the methods disclosed herein. Saccharomyces cerevisiae, or common bakers' yeast is the most commonly used among eukaryotic microorganisms, although a number of other species may also be employed for such eukaryotic expression systems. For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used (Stinchcomb et al., 1979; Kingsman et al, 1979; Tschemper et al, 1980). This plasmid already contains the trp gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC44076 or PEP4-1 (Jones, 1977). The presence of a trp lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Suitable promoting sequences in yeast vectors include the promoters for

3-phosphoglyceratekinase (Hitzeman et al, 1980) or other glycolytic enzymes (Hess et al, 1968; Holland et al, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination. Other promoters, which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3 -phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization, such as the GAL1-10 promoter, controlled by the GAL4 protein. Any plasmid vector containing a yeast-compatible promoter, an origin of replication, and termination sequences is suitable.

In addition to microorganisms, cultures of cells derived from multicellular organisms may also be used as hosts in the routine practice of the disclosed methods. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture. However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years. Examples of such useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and Wl 38, BHK, COS-7, 293 and MDCK cell lines. Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expression vectors are often provided by viral material. For example, commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40). The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al, 1978). Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the Hindlll site toward the BgR site located in the viral origin of replication. Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems. The origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from S V40 or other viral (e.g. , Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient. The present invention also concerns engineered or recombinant host cells for expression of an isolated hip gene. As used herein, the term "engineered" or "recombinant" cell is intended to refer to a cell into which a recombinant gene, such as a gene encoding a HIP, has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced gene. Engineered cells are thus cells having a gene or genes introduced through the hand of man. Recombinantly introduced genes will either be in the form of a single structural gene, an entire genomic clone comprising a structural gene and flanking DNA, or an operon or other functional nucleic acid segment which may also include genes positioned either upstream and/or downstream of the promoter, regulatory elements, or structural gene itself, or even genes not naturally associated with the particular structural gene of interest.

Where the introduction of a recombinant version of one or more of the foregoing genes is required, it will be important to introduce the gene such that it is under the control of a promoter that effectively directs the expression of the gene in the cell type chosen for engineering. In general, one will desire to employ a promoter that allows constitutive (constant) expression of the gene of interest. Commonly used constitutive eukaryotic promoters include viral promoters such as the cytomegalovirus (CMV) promoter, the Rous sarcoma long-terminal repeat (LTR) sequence, or the SV40 early gene promoter. The use of these constitutive promoters will ensure a high, constant level of expression of the introduced genes. The inventors have noticed that the level of expression from the introduced genes of interest can vary in different clones, or genes isolated from different strains or bacteria. Thus, the level of expression of a particular recombinant gene can be chosen by evaluating different clones derived from each transfection experiment; once that line is chosen, the constitutive promoter ensures that the desired level of expression is permanently maintained. It may also be possible to use promoters that are specific for cell types used for engineering, such as the insulin promoter in insulinoma cell lines, or the prolactin or growth hormone promoters in anterior pituitary cell lines.

It is contemplated that virtually any host cell may be employed for this purpose, but certain advantages may be found in using a bacterial host cells such as E. coli, and in particular, E. coli strains ATCC69791, BL21(DE3), JM101, XLl-BlueJ, RR1, LE392, B, X1776 (ATCC No. 31537), and W3110 (F-, lambda-, prototrophic, ATCC273325), other Enterobacteriaceae species such as Salmonella typhimurium, B. subtilis and Serratia marcescens, or even other Gram-negative hosts including various Pseudomonas species may be used in the recombinant expression of the genetic constructs disclosed herein. Expression in eukaryotic cells is also contemplated such as those derived from yeast, insect, or mammalian cell lines, and in particular, human and murine cell lines. It is contemplated that virtually any of the commonly employed host cells can be used in connection with the expression of the HIP and HIP-derived epitopes in accordance herewith.

Examples include cell lines typically employed for eukaryotic expression such as 239, AtT-20, HepG2, VERO, HeLa, CHO, WI 38, BHK, COS-7, RIN and MDCK cell lines. These recombinant host cells may be employed in connection with "overexpressing" HIP proteins, that is, increasing the level of expression over that found naturally in cell lines such as RL95. It is thus contemplated that the HIP or epitopic peptides derived from native or recombinant

HIPs may be "overexpressed",/.e., expressed in increased levels relative to its natural expression in human cells, or even relative to the expression of other proteins in a recombinant host cell containing HIP-encoding DNA segments. Such overexpression may be assessed by a variety of methods, including radiolabeling and/or protein purification. However, simple and direct methods are preferred, for example, those involving SDS/PAGE and protein staining or Western blotting, followed by quantitative analyses, such as densitometric scanning of the resultant gel or blot. A specific increase in the level of the recombinant protein or peptide in comparison to the level in natural HIP-producing cells is indicative of overexpression, as is a relative abundance of the specific protein in relation to the other proteins produced by the host cell and, e.g., visible on a gel. Host cells that have been transformed could be used in the screening of natural and artificially derived compounds or mixtures to select those that are capable of complexing with the HIP and HIP-derived proteins of the present invention. This could be useful in the search for compounds that inhibit or otherwise disrupt, or even enhance the ability of HIP or HIP-derived peptides to bind Hp/HS. It is contemplated that effective pharmaceutical agents could be developed by identifying compounds that complex with the particular HIP epitopes, including, for example, compounds isolated from natural sources, such as plant, animal and marine sources, and various synthetic compounds. Natural or man-made compounds that may be tested in this manner could also include various minerals and proteins, peptides or antibodies. 4.5 HIP PROTEIN AND PEPTIDE COMPOSITIONS

The present invention is also directed to protein or peptide compositions, free from total cells and other peptides, which comprise a purified protein or peptide which incoφorates an epitope that is immunologically cross-reactive with one or more of the antibodies of the present invention. As used herein, the term "incoφorating an epitope(s) that is immunologically cross-reactive with one or more anti-HIP antibodies" is intended to refer to a peptide or protein antigen which includes a primary, secondary or tertiary structure similar to an epitope located within a HIP polypeptide. The level of similarity will generally be to such a degree that monoclonal or polyclonal antibodies directed against the HIP polypeptide will also bind to, react with, or otherwise recognize, the cross-reactive peptide or protein antigen. Various immunoassay methods may be employed in conjunction with such antibodies, such as, for example, Western blotting, ELISA, RIA, and the like, all of which are known to those of skill in the art.

The identification of HIP epitopes such as those derived from hip or / -like gene products and/or their functional equivalents, suitable for use in vaccines is a relatively straightforward matter. For example, one may employ the methods of Hopp, as taught in U.S. Patent 4,554,101, incoφorated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. The methods described in several other papers, and numerous computer programs are available for use in predicting antigenic portions of proteins (see e.g., Jameson and Wolf, 1988; Wolf et al, 1988). Computerized peptide sequence analysis programs (e.g., DNAStar® software, DNAStar, Inc., Madison, WI) may also be useful in designing synthetic HIP peptides and peptide analogs in accordance with the present disclosure. The amino acid sequence of these "epitopic core sequences" may then be readily incoφorated into peptides, either through the application of peptide synthesis or recombinant technology.

Preferred peptides for use in accordance with the present invention will generally be on the order of about 5 to about 25 amino acids in length, and more preferably about 8 to about 20 amino acids in length, as well as peptides of intermediate length. Thus, peptides of about 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23 and 24 amino acids in length will find use in certain aspects of the present invention. It is proposed that shorter antigenic peptide sequences will provide advantages in certain circumstances, for example, in the preparation of vaccines or in immunologic detection assays. Exemplary advantages include the ease of preparation and purification, the relatively low cost and improved reproducibility of production, and advantageous biodistribution.

However, in other embodiments of the invention, peptides of moderate length, up to and including the full length sequence, are contemplated for use. Thus, peptides of about 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,

56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158 or 159 amino acids or so find utility in particular aspects of the present invention.

It is proposed that particular advantages of the present invention may be realized through the preparation of synthetic peptides which include modified and/or extended epitopic/immunogeniccore sequences which result in a "universal" epitopic peptide directed to HIP and HIP-related sequences, or other domains which bind Hp/HS or related proteoglycans. It is proposed that these regions represent those which are most likely to promote T-cell or B-cell stimulation in an animal, and, hence, elicit specific antibody production in such an animal.

An epitopic core sequence, as used herein, is a relatively short stretch of amino acids that is "complementary" to, and therefore will bind, antigen binding sites on HIP epitope-specific antibodies. Additionally or alternatively, an epitopic core sequence is one that will elicit antibodies that are cross-reactive with antibodies directed against the peptide compositions of the present invention. It will be understood that in the context of the present disclosure, the term "complementary" refers to amino acids or peptides that exhibit an attractive force towards each other. Thus, certain epitope core sequences of the present invention may be operationally defined in terms of their ability to compete with or perhaps displace the binding of the desired protein antigen with the corresponding protein-directed antisera.

In general, the size of the polypeptide antigen is not believed to be particularly crucial, so long as it is at least large enough to carry the identified core sequence or sequences. The smallest useful core sequence expected by the present disclosure would generally be on the order of about 5 amino acids in length, with sequences on the order of 8 or 25 being more preferred. Thus, this size will generally correspond to the smallest peptide antigens prepared in accordance with the invention. However, the size of the antigen may be larger where desired, so long as it contains a basic epitopic core sequence.

The peptides provided by this invention are ideal targets for use as vaccines or immunoreagents for the promotion or prevention of blood coagulation, and in particular, the modulation of binding of Hp to antithrombin-3 by the use of HIP and HIP-encoding genes, or cells which express either hip or hip-like gene product(s). In this regard, particular advantages may be realized through the preparation of synthetic peptides that include epitopic/immunogenic core sequences. These epitopic core sequences may be identified as hydrophilic and/or mobile regions of the polypeptides or those that include a T cell motif. It is known in the art that such regions represent those that are most likely to promote B cell or T cell stimulation, and, hence, elicit specific antibody production.

It will be further understood that certain of the polypeptides may be present in quantities below the detection limits of the Coomassie brilliant blue staining procedure usually employed in the analysis of SDS/PAGE gels, or that their presence may be masked by an inactive polypeptide of similar M_r. Although not necessary to the routine practice of the present invention, it is contemplated that other detection techniques may be employed advantageously in the visualization of particular polypeptides of interest. Immunologically-basedtechniques such as Western blotting using enzymatically-, radiolabel-, or fluorescently-tagged antibodies described herein are considered to be of particular use in this regard. Alternatively, the peptides of the present invention may be detected by using antibodies of the present invention in combination with secondary antibodies having affinity for such primary antibodies. This secondary antibody may be enzymatically- or radiolabeled, or alternatively, fluorescently-, or colloidal gold-tagged. Means for the labeling and detection of such two-step secondary antibody techniques are well-known to those of skill in the art.

The preparation of epitopes which produce antibodies which inhibit the interaction of a Hp/HS-specific gene product and Hp/HS with HIP, or antibodies which modulate or alter the interaction of HIP with Hp or the binding of Hp species to antithrombin-3 or compositions which are structurally similar to Hp/HS are particularly desirable. To confirm that a protein or peptide is immunologically cross-reactive with, or a biological functional equivalent of, one or more epitopes of the disclosed peptides is also a straightforward matter. This can be readily determined using specific assays, e.g., of a single proposed epitopic sequence, or using more general screens, e.g., of a pool of randomly generated synthetic peptides or protein fragments. The screening assays may be employed to identify either equivalent antigens or cross-reactive antibodies. In any event, the principle is the same, i.e., based upon competition for binding sites between antibodies and antigens.

Suitable competition assays that may be employed include protocols based upon immunohistochemical assays, ELIS As, RIAs, Western or dot blotting and the like. In any of the competitive assays, one of the binding components, generally the known element, such as the HIP- derived peptide, or a known antibody, will be labeled with a detectable label and the test components, that generally remain unlabeled, will be tested for their ability to reduce the amount of label that is bound to the corresponding reactive antibody or antigen.

As an exemplary embodiment, to conduct a competition study between a HIP and any test antigen, one would first label HIP with a detectable label, such as, e.g., biotin or an enzymatic, radioactive or fluorogenic label, to enable subsequent identification. One would then incubate the labeled antigen with the other, test, antigen to be examined at various ratios (e.g, 1 :1, 1 :10 and 1 : 100) and, after mixing, one would then add the mixture to an antibody of the present invention. Preferably, the known antibody would be immobilized, e.g., by attaching to an ELISA plate. The ability of the mixture to bind to the antibody would be determined by detecting the presence of the specifically bound label. This value would then be compared to a control value in which no potentially competing (test) antigen was included in the incubation.

The assay may be any one of a range of immunological assays based upon hybridization, and the reactive antigens would be detected by means of detecting their label, e.g., using streptavidin in the case of biotinylated antigens or by using a chromogenic substrate in connection with an enzymatic label or by simply detecting a radioactive or fluorescent label. An antigen that binds to the same antibody as HIP, for example, will be able to effectively compete for binding to and thus will significantly reduce HIP binding, as evidenced by a reduction in the amount of label detected. The reactivity of the labeled antigen, e.g., a HIP composition, in the absence of any test antigen would be the control high value. The control low value would be obtained by incubating the labeled antigen with an excess of unlabeled HIP antigen, when competition would occur and reduce binding. A significant reduction in labeled antigen reactivity in the presence of a test antigen is indicative of a test antigen that is "cross-reactive", i.e., that has binding affinity for the same antibody. "A significant reduction", in terms of the present application, may be defined as a reproducible (i. e., consistently observed) reduction in binding.

In addition to the peptidyl compounds described herein, the inventors also contemplate that other sterically similar compounds may be formulated to mimic the key portions of the peptide structure. Such compounds, which may be termed peptidomimetics, may be used in the same manner as the peptides of the invention and hence are also functional equivalents. The generation of a structural functional equivalent may be achieved by the techniques of modeling and chemical design known to those of skill in the art. It will be understood that all such sterically similar constructs fall within the scope of the present invention. Syntheses of epitopic sequences, or peptides which include an antigenic epitope within their sequence, are readily achieved using conventional synthetic techniques such as the solid phase method (e.g., through the use of a commercially-available peptide synthesizer such as an Applied Biosystems Model 430A Peptide Synthesizer). Peptide antigens synthesized in this manner may then be aliquotedin predetermined amounts and stored in conventional manners, such as in aqueous solutions or, even more preferably, in a powder or lyophilized state pending use.

In general, due to the relative stability of peptides, they may be readily stored in aqueous solutions for fairly long periods of time if desired, e.g, up to six months or more, in virtually any aqueous solution without appreciable degradation or loss of antigenic activity. However, where extended aqueous storage is contemplated it will generally be desirable to include agents including buffers such as Tris or phosphate buffers to maintain a pH of about 7.0 to about 7.5. Moreover, it may be desirable to include agents which will inhibit microbial growth, such as sodium azide or Merthiolate. For extended storage in an aqueous state it will be desirable to store the solutions at 4°C, or more preferably, frozen. Of course, where the peptides are stored in a lyophilized or powdered state, they may be stored virtually indefinitely, e.g., in metered aliquots that may be rehydrated with a predetermined amount of water (preferably distilled) or buffer prior to use . 4.6 METHODS OF NUCLEIC ACID DELIVERY AND DNA TRANSFECTION

In certain embodiments, it is contemplated that the nucleic acid segments disclosed herein will be used to transfect appropriate host cells. Technology for introduction of DNA into cells is well-known to those of skill in the art. Four general methods for delivering a nucleic segment into cells have been described: chemical methods (Graham and VanDerEb, 1973); physical methods such as microinjection (Capecchi, 1980), electroporation (Wong and Neumann, 1982; Fromm et al, 1985) and the gene gun (Yang et al, 1990); viral vectors (Clapp, 1993; Eglitis and Anderson, 1988); and receptor-mediatedmechanisms (Curiel et al , 1991 ; Wagner et al. , 1992). In certain embodiments of the invention, the nucleic acid encoding the gene may be stably integrated into the genome of the cell. In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.

4.6.1 INFECTION WITH VIRAL VECTORS

In certain embodiments of the invention, the nucleic acid encoding a selected gene may be stably integrated into the genome of the cell. In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.

4.6.1.1. ADENOVIRAL VECTORS

A particular method for delivery of the expression constructs involves the use of an adenovirus expression vector. Although adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors. "Adenovirus expression vector" is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to ultimately express a tissue-specific transforming construct that has been cloned therein. The expression vector comprises a genetically engineered form of adenovirus.

Knowledge of the genetic organization or adenovirus, a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus and Horwitz, 1992). In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification.

Adenovirus is particularly suitable for use as a gene transfer vector because of its midsized genome, ease of manipulation, high titer, wide target-cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The El region (El A and EIB) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off (Renan, 1990). The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNA's issued from this promoter possess a 5'-tripartite leader (TPL) sequence which makes them preferred mRNA's for translation.

In a current system, recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure. Generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (El A and EIB; Graham et al, 1977). Since the E3 region is dispensable from the adenovirus genome (Jones and Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions (Graham and Prevec, 1991). In nature, adenovirus can package approximately 105% of the wild-type genome (Ghosh-Choudhury et al, 1987), providing capacity for about 2 extra kb of DNA. Combined with the approximately 5.5 kb of DNA that is replaceable in the El and E3 regions, the maximum capacity of the current adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone.

Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells. As stated above, the preferred helper cell line is 293.

Recently, Racher et al (1995) disclosed improved methods for culturing 293 cells and propagating adenovirus. In one format, natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100- 200 ml of medium. Following stirring at 40 φm, the cell viability is estimated with trypan blue.

In another format, Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/1) is employed as follows. A cell inoculum, resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask and left stationary, with occasional agitation, for 1 to 4 h. The medium is then replaced with 50 ml of fresh medium and shaking initiated. For virus production, cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25% of the final volume) and adenovirus added at an MOI of 0.05. Cultures are left stationary overnight, following which the volume is increased to 100% and shaking commenced for another 72 h.

Other than the requirement that the adenovirus vector be replication defective, or at least conditionally defective, the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.

As stated above, the typical vector according to the present invention is replication defective and will not have an adenovirus El region. Thus, it will be most convenient to introduce the transforming construct at the position from which the El -coding sequences have been removed. However, the position of insertion of the construct within the adenovirus sequences is not critical to the invention. The polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors as described by Karlsson et al (1986) or in the E4 region where a helper cell line or helper virus complements the E4 defect. Adenovirus growth and manipulation is known to those of skill in the art, and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 10 to 10 plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al. ,

1963; Top et al, 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al, 1991; Gomez-Foix et al, 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al, 1991; Rich et al, 1993). Studies in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld et al, 1991; Rosenfeld et al, 1992), muscle injection (Ragot et al, 1993), peripheral intravenous injections (Herz and Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle et al, 1993). Recombinant adenovirus and adeno-associated virus (see below) can both infect and transduce non-dividing human primary cells.

4.6.1.2. AAV VECTORS Adeno-associated virus (AAV) is an attractive vector system for use in the cell transduction of the present invention as it has a high frequency of integration and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells, for example, in tissue culture (Muzyczka, 1992) or in vivo. AAV has a broad host range for infectivity (Tratschin, et al, 1984; Laughlin, et al, 1986; Lebkowski, et al, 1988; McLaughlin, et al, 1988). Details concerning the generation and use of rAAV vectors are described in U.S. Patent

No. 5,139,941 and U.S. Patent No. 4,797,368, each incoφorated herein by reference.

Studies demonstrating the use of AAV in gene delivery include LaFace et al (1988); Zhou et al (1993); Flotte et al (1993); and Walsh et al. (1994). Recombinant AAV vectors have been used successfully for in vitro and in vivo transduction of marker genes (Kaplitt, et al, 1994; Lebkowski, et al. , 1988; Samulski, et al. , 1989; Yoder, et al. , 1994; Zhou, et al. , 1994; Hermonat and Muzyczka, 1984; Tratschin, et al, 1985; McLaughlin, et al, 1988) and genes involved in human diseases (Flotte, et al, 1992; Luo, et al, 1994; Ohi, et al, 1990; Walsh, et al, 1994; Wei, et al, 1994). Recently, an AAV vector has been approved for phase I human trials for the treatment of cystic fibrosis. AAV is a dependent parvovirus in that it requires coinfection with another virus (either adenovirus or a member of the heφes virus family) to undergo a productive infection in cultured cells (Muzyczka, 1992). In the absence of coinfection with helper virus, the wild type AAV genome integrates through its ends into human chromosome 19 where it resides in a latent state as a provirus (Kotin et al, 1990; Samulski et al, 1991). rAAV, however, is not restricted to chromosome 19 for integration unless the AAV Rep protein is also expressed (Shelling and

Smith, 1994). When a cell carrying an AAV provirus is superinfected with a helper virus, the AAV genome is "rescued" from the chromosome or from a recombinant plasmid, and a normal productive infection is established (Samulski, et al, 1989; McLaughlin, et al, 1988; Kotin, et al, 1990; Muzyczka, 1992). Typically, recombinant AAV (rAAV) virus is made by cotransfecting a plasmid containing the gene of interest flanked by the two AAV terminal repeats (McLaughlin et al. , 1988; Samulski et al, 1989; each incoφorated herein by reference) and an expression plasmid containing the wild type AAV coding sequences without the terminal repeats, for example pIM45 (McCarty et al, 1991; incoφorated herein by reference). The cells are also infected or transfected with adenovirus or plasmids carrying the adenovirus genes required for AAV helper function. rAAV virus stocks made in such fashion are contaminated with adenovirus which must be physically separated from the rAAV particles (for example, by cesium chloride density centrifugation). Alternatively, adenovirus vectors containing the AAV coding regions or cell lines containing the AAV coding regions and some or all of the adenovirus helper genes could be used (Yang et al, 1994; Clark et al, 1995). Cell lines carrying the rAAV DNA as an integrated provirus can also be used (Flotte et al, 1995).

4.6.1.3. RETROVIRAL VECTORS The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990). In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al, 1983). When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al, 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al, 1975).

Concern with the use of defective retrovirus vectors is the potential appearance of wild- type replication-competent virus in the packaging cells. This can result from recombination events in which the intact sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome. However, new packaging cell lines are now available that should greatly decrease the likelihood of recombination (Markowitz et al. , 1988; Hersdorffer et al, 1990).

4.6.1.4. OTHER VIRAL VECTORS Other viral vectors may be employed as expression constructs in the present invention.

Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988), sindbis virus and heφesviruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988; Horwich et al, 1990). With the recent recognition of defective hepatitis B viruses, new insight was gained into the structure-function relationship of different viral sequences. In vitro studies showed that the virus could retain the ability for helper-dependent packaging and reverse transcription despite the deletion of up to 80% of its genome (Horwich et al, 1990). This suggested that large portions of the genome could be replaced with foreign genetic material. Chang et al. recently introduced the chloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virus genome in the place of the polymerase, surface, and pre-surface coding sequences. It was cotransfected with wild-type virus into an avian hepatoma cell line. Culture media containing high titers of the recombinant virus were used to infect primary duckling hepatocytes. Stable CAT gene expression was detected for at least 24 days after transfection (Chang et al, 1991). 4.6.1.5. MODIFIED VIRUSES

In still further embodiments of the present invention, the nucleic acids to be delivered are housed within an infective virus that has been engineered to express a specific binding ligand. The virus particle will thus bind specifically to the cognate receptors of the target cell and deliver the contents to the cell. A novel approach designed to allow specific targeting of retrovirus vectors was recently developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification can permit the specific infection of hepatocytes via sialoglycoprotein receptors.

Another approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin (Roux et al, 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro (Roux et al, 1989).

4.6.2. TRANSFECTION

In order to effect expression of a gene construct, the expression construct must be delivered into a cell. As described herein, the preferred mechanism for delivery is via viral infection, where the expression construct is encapsidated in an infectious viral particle. However, several non-viral methods for the transfer of expression constructs into cells also are contemplated by the present invention. In one embodiment of the present invention, the expression construct may consist only of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned which physically or chemically permeabilize the cell membrane.

4.6.2.1. LIPOSOME-MEDIATED TRANSFECTION

In a further embodiment of the invention, the expression construct may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated is an expression construct complexed with Lipofectamine (Gibco BRL). Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful (Nicolau and Sene, 1982; Fraley et al, 1979; Nicolau et al, 1987). Wong et al. (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells.

In certain embodiments of the invention, the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al, 1989). In other embodiments, the liposome may be complexed or employed in conjunction with nuclear non- histone chromosomal proteins (HMG-1) (Kato et al, 1991). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1.

4.6.2.2. ELECTROPORATION

In certain embodiments of the present invention, the expression construct is introduced into the cell via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge. Transfection of eukaryotic cells using electroporation has been quite successful. Mouse pre-B lymphocytes have been transfected with human kappa-immunoglobulin genes (Potter et al, 1984), and rat hepatocytes have been transfected with the chloramphenicol acetyltransferase gene (Tur-Kaspa et al, 1986) in this manner.

4.6.2.3. CALCIUM PHOSPHATE PRECIPITATION OR DEAE-DEXTRAN TREATMENT

In other embodiments of the present invention, the expression construct is introduced to the cells using calcium phosphate precipitation. Human KB cells have been transfected with adenovirus 5 DNA (Graham and Van Der Eb, 1973) using this technique. Also in this manner, mouse L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with a neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes were transfected with a variety of marker genes (Rippe et al, 1990).

In another embodiment, the expression construct is delivered into the cell using DEAE- dextran followed by polyethylene glycol. In this manner, reporter plasmids were introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985).

4.6.2.4. PARTICLE BOMBARDMENT

Another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.

4.6.2.5. DIRECT MICROINJECTION OR SONICATION LOADING

Further embodiments of the present invention include the introduction of the expression construct by direct microinjection or sonication loading. Direct microinjection has been used to introduce nucleic acid constructs into Xenopus oocytes (Harland and Weintraub, 1985), and

LTK" fibroblasts have been transfected with the thymidine kinase gene by sonication loading (Fechheimer et al, 1987).

4.6.2.6. ADENOVIRAL ASSISTED TRANSFECTION In certain embodiments of the present invention, the expression construct is introduced into the cell using adenovirus assisted transfection. Increased transfection efficiencies have been reported in cell systems using adenovirus coupled systems (Kelleher and Vos, 1994; Cotten et al, 1992; Curiel, 1994). 4.6.2.7. RECEPTOR MEDIATED TRANSFECTION

Still further expression constructs that may be employed to deliver the tissue-specific promoter and transforming construct to the target cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis that will be occurring in the target cells. In view of the cell type-specific distribution of various receptors, this delivery method adds another degree of specificity to the present invention. Specific delivery in the context of another mammalian cell type is described by Wu and Wu (1993; incoφorated herein by reference).

Certain receptor-mediated gene targeting vehicles comprise a cell receptor-specific ligand and a DNA-binding agent. Others comprise a cell receptor-specific ligand to which the DNA construct to be delivered has been operatively attached. Several ligands have been used for receptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al, 1990; Perales et al, 1994; Myers, EPO 0273085), which establishes the operability of the technique. In the context of the present invention, the ligand will be chosen to correspond to a receptor specifically expressed on the neuroendocrine target cell population.

In other embodiments, the DNA delivery vehicle component of a cell-specific gene targeting vehicle may comprise a specific binding ligand in combination with a liposome. The nucleic acids to be delivered are housed within the liposome and the specific binding ligand is functionally incoφorated into the liposome membrane. The liposome will thus specifically bind to the receptors of the target cell and deliver the contents to the cell. Such systems have been shown to be functional using systems in which, for example, epidermal growth factor (EGF) is used in the receptor-mediated delivery of a nucleic acid to cells that exhibit upregulation of the EGF receptor.

In still further embodiments, the DNA delivery vehicle component of the targeted delivery vehicles may be a liposome itself, which will preferably comprise one or more lipids or glycoproteins that direct cell-specific binding. For example, Nicolau et al. (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, incoφorated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes. It is contemplated that the tissue-specific transforming constructs of the present invention can be specifically delivered into the target cells in a similar manner. 4.7 SITE-SPECIFIC MUTAGENESIS

Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA. The technique, well-known to those of skill in the art, further provides a ready ability to prepare and test sequence variants, for example, incoφorating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 14 to about 25 nucleotides in length is preferred, with about 5 to about 10 residues on both sides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known in the art, as exemplified by various publications. As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage are readily commercially-available and their use is generally well-known to those skilled in the art. Double-stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double-stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as

E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement. The preparation of sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained. For example, recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants. Specific details regarding these methods and protocols are found in the teachings of Maloy et al, 1994; Segal, 1976; Prokop and Bajpai, 1991 ; Kuby, 1994; and Maniatis et al, 1982, each incoφorated herein by reference, for that purpose.

As one illustrative example of the protocols which are known to those of skill in the art for making mutants, the PCR™-based strand overlap extension (SOE) (Ho et al, 1989) for site- directed mutagenesis is particularly preferred for site-directed mutagenesis of the nucleic acid compositions of the present invention. The techniques of PCR™ are well-known to those of skill in the art, as described hereinabove. The SOE procedure involves a two-step PCR™ protocol, in which a complementary pair of internal primers (B and C) are used to introduce the appropriate nucleotide changes into the wild-type sequence. In two separate reactions, flanking PCR™ primer

A (restriction site incoφorated into the oligo) and primer D (restriction site incoφorated into the oligo) are used in conjunction with primers B and C, respectively to generate PCR™ products AB and CD. The PCR™ products are purified by agarose gel electrophoresis and the two overlapping PCR™ fragments AB and CD are combined with flanking primers A and D and used in a second PCR™ reaction. The amplified PCR™ product is agarose gel purified, digested with the appropriate enzymes, ligated into an expression vector, and transformed into E. coli JM101, XL1- Blue™ (Stratagene, LaJolla, CA), JM105, or TGI (Carter et al, 1985) cells. Clones are isolated and the mutations are confirmed by sequencing of the isolated plasmids.

4.8 BIOLOGICAL FUNCTIONAL EQUIVALENTS

Modification and changes may be made in the structure of the peptides of the present invention and DNA segments which encode them and still obtain a functional molecule that encodes a protein or peptide with desirable characteristics. The following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second- generation molecule. The amino acid changes may be achieved by changing the codons of the DNA sequence, according to Table 3.

Table 3

Amino Acids Codons

Alanine Ala A GCA GCC GCG GCU

Cysteine Cys C UGC UGU

Aspartic acid Asp D GAC GAU

Glutamic acid Glu E GAA GAG

Phenylalanine Phe F UUC UUU

Glycine Gly G GGA GGC GGG GGU

Histidine His H CAC CAU

Isoleucine He I AUA AUC AUU

Lysine Lys K AAA AAG

Leucine Leu L UUA UUG CUA CUC CUG CUU

Methionine Met M AUG

Asparagine Asn Ν AAC AAU

Proline Pro P CCA CCC CCG CCU

Glutamine Gin Q CAA CAG

Arginine Arg R AGA AGG CGA CGC CGG CGU

Serine Ser S AGC AGU UCA UCC UCG UCU

Threonine Thr T ACA ACC ACG ACU

Naline Val V GUA GUC GUG GUU

Tryptophan Tφ w UGG Amino Acids Codons

Tyrosine Tyr Y UAC UAU

For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incoφorate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (- 3.9); and arginine (-4.5).

It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. , still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101, incoφorated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (- 1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

4.9 ANTIBODY COMPOSITIONS AND FORMULATIONS THEREOF

A further aspect of the invention is the preparation of immunological compositions, and in particular anti-HIP antibodies for detection, isolation, and purification of HIP and HIP-derived peptides in a variety of laboratory and clinical samples. These antibodies may be specific for native, site-specifically mutated, full-length, or truncated variants of the HIP or peptide. Alternatively the antibodies of the present invention may be specific for HIP compositions engineered by the hand of man, such as, synthetic HIP peptides, analogs, or peptidomimetics.

The invention also encompasses HIP and HIP-derived peptide antigen compositions together with pharmaceutically-acceptable excipients, carriers, diluents, adjuvants, and other components, such as additional peptides, antigens, or formulations, as may be employed in the formulation of particular vaccines. The nucleic acid sequences of the present invention encode HIP and are useful to generate pure recombinant HIP for administration to a host. Such administration is useful for the production of anti-HIP antibodies by the host. Antibodies may be of several types including those raised in heterologous donor animals or human volunteers immunized with HIPs, monoclonal antibodies (mAbs) resulting from hybridomas derived from fusions of B cells from HIP-immunized animals or humans with compatible myeloma cell lines, so-called "humanized" mAbs resulting from expression of gene fusions of combinatorial determining regions of mAb-encoding genes from heterologous species with genes encoding human antibodies, or HIP-reactive antibody-containing fractions of plasma from humans known to express HIP. It is contemplated that any of the techniques described above might be used for the vaccination of subjects for the puφose of antibody production.

Also disclosed in a method of generating an immune response in an animal. The method generally involves administering to an animal a pharmaceutical composition comprising an immunologically effective amount of a HIP or HIP-derived peptide composition disclosed herein.

Preferred peptide compositions include the human HIP sequence disclosed in SEQ ID NO:2 and antigenic or epitopic fragments thereof. Preferred animals include mammals, and particularly humans. Other preferred animals include murines, bovines, equines, porcines, canines, and felines. The composition may include partially or significantly purified HIP peptide epitopes, obtained from natural or recombinant sources, which proteins or peptides may be obtainable naturally or chemically synthesized, or alternatively produced in vitro from recombinant host cells expressing DNA segments encoding such epitopes. Smaller peptides that include reactive epitopes, such as those between about 10 and about 50, or even between about 50 and about 100 amino acids in length will often be preferred. The antigenic proteins or peptides may also be combined with other agents, such as other HIPs, HIP-derived peptides, or /zip-containing nucleic acid compositions, if desired.

Further means contemplated by the inventors for generating an immune response in an animal includes administering to the animal, or human subject, a pharmaceutically-acceptable composition comprising an immunologically effective amount of a nucleic acid composition encoding a HIP epitope, or an immunologically effective amount of an attenuated live organism that includes and expresses such a nucleic acid composition.

By "immunologically effective amount" is meant an amount of a composition that is capable of generating an immune response in the recipient animal. This includes both the generation of an antibody response (B cell response), and/or the stimulation of a cytotoxic immune response (T cell response). The generation of such an immune response will have utility in both the production of useful bioreagents, e.g., CTLs and, more particularly, reactive antibodies, for use in diagnostic embodiments, and will also have utility in various prophylactic or therapeutic embodiments. Immunoformulations of this invention, whether intended for vaccination, treatment, or for the generation of antibodies, may comprise native or synthetically-derived antigenic peptide fragments from these proteins. As such, antigenic functional equivalents of the proteins and peptides described herein also fall within the scope of the present invention. An "antigenically functional equivalent" protein or peptide is one that incoφorates an epitope that is immunologically cross-reactive with one or more epitopes derived from disclosed HIPs, and particularly the HIP of human or murine origins. Antigenically functional equivalents, or epitopic sequences, may be first designed or predicted and then tested, or may simply be directly tested for cross-reactivity.

The identification or design of suitable HIP epitopes, and/or their functional equivalents, suitable for use in immunoformulations, vaccines, or simply as antigens (e.g., for use in detection protocols), is a relatively straightforward matter. For example, one may employ the methods of

Hopp, as enabled in U.S. Patent 4,554,101, incoφorated herein by reference, that teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. The methods described in several other papers, and software programs based thereon, can also be used to identify epitopic core sequences, for example, Chou and Fasman (1974a,b; 1978a,b; 1979); Jameson and Wolf (1988); Wolf et al. (1988); and Kyte and Doolittle (1982) address this subject.

The amino acid sequence of these "epitopic core sequences" may then be readily incoφorated into peptides, either through the application of peptide synthesis or recombinant technology.

It is proposed that the use of shorter antigenic peptides, e.g., about 25 to about 50, or even about 15 to 25 amino acids in length, that incoφorate epitopes of the HIP will provide advantages in certain circumstances, for example, in the preparation of vaccines or in immunologic detection assays. Exemplary advantages include the ease of preparation and purification, the relatively low cost and improved reproducibility of production, and advantageous biodistribution.

In another aspect, the present invention contemplates an antibody that is immunoreactive with a polypeptide of the invention. As stated above, one of the uses for HIPs and HIP-derived epitopic peptides according to the present invention is to generate antibodies. Reference to antibodies throughout the specification includes whole polyclonal and monoclonal antibodies (mAbs), bispecific antibodies, and parts thereof, either alone or conjugated with other moieties. Antibody parts include Fab, Fab', F(ab)₂ and F(ab')₂ fragments and single chain antibodies. The antibodies may be made in vivo in suitable laboratory animals or in vitro using recombinant DNA techniques. In a preferred embodiment, an antibody is a polyclonal antibody.

Means for preparing and characterizing antibodies are well known in the art (see, e.g., Harlow and Lane, 1988; incoφorated herein by reference). The methods for generating mAbs generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies. Antibodies, both polyclonal and monoclonal, specific for HIP and HIP-derived epitopes may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art. A composition containing antigenic epitopes of particular HIPs can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against the particular HIP peptide or peptides used to immunize the animal. Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.

The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen, as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, also may be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs (described in detail below).

One of the important features provided by the present invention is a polyclonal sera that is relatively homogenous with respect to the specificity of the antibodies therein. Typically, polyclonal antisera is derived from a variety of different "clones," i.e., B-cells of different lineage. mAbs, by contrast, are defined as coming from antibody-producing cells with a common B-cell ancestor, hence their "mono" clonality .

When peptides are used as antigens to raise polyclonal sera, one would expect considerably less variation in the clonal nature of the sera than if a whole antigen were employed. Unfortunately, if incomplete fragments of an epitope are presented, the peptide may very well assume multiple (and probably non-native) conformations. As a result, even short peptides can produce polyclonal antisera with relatively plural specificities and, unfortunately, an antisera that does not react or reacts poorly with the native molecule.

Polyclonal antisera according to present invention is produced against peptides that are predicted to comprise whole, intact epitopes. It is believed that these epitopes are, therefore, more stable in an immunologic sense and thus express a more consistent immunologic target for the immune system. Under this model, the number of potential B-cell clones that will respond to this peptide is considerably smaller and, hence, the homogeneity of the resulting sera will be higher. In various embodiments, the present invention provides for polyclonal antisera where the clonality, i.e., the percentage of clone reacting with the same molecular determinant, is at least 80%. Even higher clonality - 90%, 95% or greater - is contemplated. As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimideester, carbodiimide and bis-biazotizedbenzidine. mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, incoφorated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g. , a purified or partially purified protein, polypeptide or peptide. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately about 5 x 10⁷ to about 2 x 10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized, to produce antibody-secreting hybridomas. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).

For example, following immunization spleen cells are removed and fused, using a standard fusion protocol with plasmacytoma cells to produce hybridomas secreting mAbs against HIP. Hybridomas which produce mAbs to the selected antigens are identified using standard techniques, such as ELISA and Western blot methods. Hybridoma clones can then be cultured in liquid media and the culture supernatants purified to provide the HIP-specific mAbs.

Any one of a number of myeloma cells may be used, as are known to those of skill in the art

(Goding, 1986; Campbell, 1984). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NSl/l.Ag 4 1, Sp210-Agl4, FO, NSO/U, MPC-11,

MPC11-X45-GTG 1.7 and S194/5XX0 Bui; for rats, one may use R210.RCY3, Y3-Ag 1.2.3,

IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (also termed P3-NS-1- Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.

Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2: 1 ratio, though the ratio may vary from about 20:1 to about 1 :1, respectively, in the presence of an agent or agents

(chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol

(PEG), such as 37% (vol./vol.) PEG, by Gefter et al. (1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986). Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10^" to g about 1 x 10 . However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine.

Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine. The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.

This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.

The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific mAb produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration. The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.

It is proposed that the mAbs of the present invention will also find useful application in immunochemical procedures, such as ELISA and Western blot methods, as well as other procedures such as immunoprecipitation, immunocytological methods, etc. which may utilize antibodies specific to HIPs. In particular, HIP antibodies may be used in immunoabsorbent protocols to purify native or recombinant HIPs or HIP-derived peptide species or synthetic or natural variants thereof. The antibodies disclosed herein may be employed in antibody cloning protocols to obtain cDNAs or genes encoding HIPs from other species or organisms, or to identify proteins having significant homology to HIP. They may also be used in inhibition studies to analyze the effects of HIP in cells, tissues, or whole animals. Anti-HIP antibodies will also be useful in immunolocalization studies to analyze the distribution of HIPs or to determine the cellular or tissue- specific distribution of HIPs under different physiological conditions. A particularly useful application of such antibodies is in purifying native or recombinant HIPs, for example, using an antibody affinity column. The operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure. In still further embodiments, the present invention concerns immunodetection methods and associated kits. It is contemplated that the proteins or peptides of the invention may be employed to detect antibodies having reactivity therewith, or, alternatively, antibodies prepared in accordance with the present invention, may be employed to detect HIP or peptides. The kits may also be used in antigen or antibody purification, as appropriate. In general, the preferred immunodetection methods will include first obtaining a sample suspected of containing a HIP-reactive antibody, such as a biological sample from a patient, and contacting the sample with a first HIP or peptide under conditions effective to allow the formation of an immunocomplex (primary immune complex). One then detects the presence of any primary immunocomplexesthat are formed.

Contacting the chosen sample with the HIP or peptide under conditions effective to allow the formation of (primary) immune complexes is generally a matter of simply adding the protein or peptide composition to the sample. One then incubates the mixture for a period of time sufficient to allow the added antigens to form immune complexes with, i.e., to bind to, any antibodies present within the sample. After this time, the sample composition, such as a tissue section, ELISA plate, dot blot or western blot, will generally be washed to remove any non-specifically bound antigen species, allowing only those specifically bound species within the immune complexes to be detected.

The detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches known to the skilled artisan and described in various publications, such as, e.g., Nakamura et al (1987), incoφorated herein by reference. Detection of primary immune complexes is generally based upon the detection of a label or marker, such as a radioactive, fluorescent, biological or enzymatic label, with enzyme tags such as alkaline phosphatase, urease, horseradish peroxidase and glucose oxidase being suitable. The particular antigen employed may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of bound antigen present in the composition to be determined.

Alternatively, the primary immune complexes may be detected by means of a second binding ligand that is linked to a detectable label and that has binding affinity for the first protein or peptide. The second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies and the remaining bound label is then detected.

For diagnostic puφoses, it is proposed that virtually any sample suspected of containing the antibodies of interest may be employed. Exemplary samples include clinical samples obtained from a patient such as blood or serum samples, bronchoalveolar fluid, ear swabs, sputum samples, middle ear fluid or even perhaps urine samples may be employed. Furthermore, it is contemplated that such embodiments may have application to non-clinical samples, such as in the titering of antibody samples, in the selection of hybridomas, and the like. Alternatively, the clinical samples may be from veterinary sources and may include such domestic animals as cattle, sheep, and goats.

Samples from feline, canine, equine and other animal sources may also be used in accordance with the methods described herein.

Another aspect of the invention are immunodetection kits containing antibodies of the present invention and suitable immunodetection reagents such as a detectable label linked to a protein, peptide or the antibody itself. Alternatively, the detectable label may be linked to a second antibody which binds to an antibody of the invention. Related embodiments include diagnostic and therapeutic kits which include pharmaceutically-acceptableformulations of the antibodies disclosed herein. Such kits are useful in the detection of HIP or HIP related peptides in clinical samples, and other samples. In related embodiments, the present invention contemplates the preparation of kits that may be employed to detect the presence of HlP-specific antibodies in a sample. Generally speaking, kits in accordance with the present invention will include a suitable protein or peptide together with an immunodetection reagent, and a means for containing the protein or peptide and reagent.

The immunodetection reagent will typically comprise a label associated with a HIP or peptide, or associated with a secondary binding ligand. Exemplary ligands might include a secondary antibody directed against the first HIP or peptide or antibody, or a biotin or avidin (or streptavidin) ligand having an associated label. Detectable labels linked to antibodies that have binding affinity for a human antibody are also contemplated, e.g., for protocols where the first reagent is a HIP peptide that is used to bind to a reactive antibody from a human sample. Of course, as noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present invention. The kits may contain antigen or antibody- label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit.

The container means will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antigen may be placed, and preferably suitably allocated.

Where a second binding ligand is provided, the kit will also generally contain a second vial or other container into which this ligand or antibody may be placed. The kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.

4.10 IMMUNOASSAYS

As noted, it is proposed that native and synthetically-derivedpeptides and peptide epitopes of the invention will find utility as immunogens, e.g. , in connection with vaccine development, or as antigens in immunoassays for the detection of reactive antibodies. Turning first to immunoassays, in their most simple and direct sense, preferred immunoassays of the invention include the various types of enzyme linked immunosorbent assays (ELISAs), as are known to those of skill in the art. However, it will be readily appreciated that the utility of HIP-derived proteins and peptides is not limited to such assays, and that other useful embodiments include RIAs and other non-enzyme linked antibody binding assays and procedures. In preferred ELISA assays, proteins or peptides incoφorating HIP, rHIP, or HIP-derived protein antigen sequences are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity, such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, one would then generally desire to bind or coat a nonspecific protein that is known to be antigenically neutral with regard to the test antisera, such as bovine serum albumin (BSA) or casein, onto the well. This allows for blocking of nonspecific adsoφtion sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

After binding of antigenic material to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen/antibody) formation. Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/ Tween™. These added agents also tend to assist in the reduction of nonspecific background. The layered antisera is then allowed to incubate for, e.g., from 2 to 4 hours, at temperatures preferably on the order of about 25°C to about 27°C. Following incubation, the antisera-contacted surface is washed so as to remove non-immunocomplexedmaterial. A preferred washing procedure includes washing with a solution such as PBS/Tween™, or borate buffer.

Following formation of specific immunocomplexes between the test sample and the bound antigen, and subsequent washing, the occurrence and the amount of immunocomplex formation may be determined by subjecting the complex to a second antibody having specificity for the first. Of course, in that the test sample will typically be of human origin, the second antibody will preferably be an antibody having specificity for human antibodies. To provide a detecting means, the second antibody will preferably have an associated detectable label, such as an enzyme label, that will generate a signal, such as color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the antisera- bound surface with a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions that favor the development of immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween™). After incubation with the second enzyme-tagged antibody, and subsequent to washing to remove unbound material, the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol puφle or 2,2'-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H₂O₂, in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectrum spectrophotometer.

4.11 IMMUNOPRECIPITATION

The anti-HIP antibodies of the present invention are particularly useful for the isolation of HIP antigens by immunoprecipitation. Immunoprecipitation involves the separation of the target antigen component from a complex mixture, and is used to discriminate or isolate minute amounts of protein. For the isolation of cell-surface localized proteins such as HIP, peptides must be solubilized from the bacterial cell wall by treatment with enzymes such as lysozyme, lysostaphinor mutanolysin, or alternatively, into detergent micelles. Nonionic salts are preferred, since other agents such as bile salts, precipitate at acid pH or in the presence of bivalent cations.

In an alternative embodiment the antibodies of the present invention are useful for the close juxtaposition of two antigens. This is particularly useful for increasing the localized concentration of antigens, e.g., enzyme-substrate pairs.

In a related embodiment, antibodies of the present invention are useful for promoting the binding of Hp/HS to hip gene products. Such binding is readily measured by monitoring ligand binding using well-known procedures. Detection of the binding may be accomplished by using radioactive ly labeled antibodies or alternatively, radioactively-labeled Hp/HS. Alternatively, assays employing biotin-labeled antibodies are also well-known in the art as described (Bayer and Wilchek, 1980).

4.12 WESTERN BLOTS

The compositions of the present invention will find great use in immunoblot or western blot analysis. The anti-HIP antibodies may be used as high-affinity primary reagents for the identification of proteins immobilized onto a solid support matrix, such as nitrocellulose, nylon or combinations thereof. In conjunction with immunoprecipitation, followed by gel electrophoresis, these may be used as a single step reagent for use in detecting antigens against which secondary reagents used in the detection of the antigen cause an adverse background. This is especially useful when the antigens studied are immunoglobulins (precluding the use of immunoglobulins binding bacterial cell wall components), the antigens studied cross-react with the detecting agent, or they migrate at the same relative molecular weight as a cross-reacting signal. Immunologically-based detection methods in conjunction with Western blotting (including enzymatically-, radiolabel-, or fluorescently-tagged secondary antibodies against the toxin moiety) are considered to be of particular use in this regard.

4.13 AFFINITY CHROMATOGRAPHY

Proteins of amino acid sequence derived from, or similar to, HIP are expected to have affinity for Hp and/or HS and can be purified from other constituents of host cells which express HIP by chromatography on matrices containing Hp or HS, so-called "affinity chromatography." HIPs may also be purified by methodologies not relying on affinity for Hp/HS such as ion exchange chromatography, size exclusion chromatography, metal chelation chromatography, or the like. Buffer, detergent, and other conditions may be dissimilar from those optimal for "affinity chromatography." In a preferred embodiment, an affinity matrix comprising Hp/HS or a related proteoglycan may be used for the isolation of HIPs from solution, or alternatively, isolation of intact bacteria expressing HIPs, or even membrane fragments of bacteria expressing HIPs. Affinity chromatography is generally based on the recognition of a protein by a substance such as a ligand or an antibody. The column material may be synthesized by covalently coupling a binding molecule, such as an activated dye, for example to an insoluble matrix. The column material is then allowed to adsorb the desired substance from solution. Next, the conditions are changed to those under which binding does not occur and the substrate is eluted. The requirements for successful affinity chromatography are: that the matrix must specifically-adsorbthe molecules of interest; that other contaminants remain unadsorbed; that the ligand must be coupled without altering its binding activity; that the ligand must bind sufficiently tight to the matrix; and that it must be possible to elute the molecules of interest without destroying them.

A preferred embodiment of the present invention is an affinity chromatography method for purification of antibodies from solution wherein the matrix contains HIPs or peptide epitopes derived from HIPs such as those derived from the mammalian sources, covalently-coupled to a Sepharose such as CL6B or CL4B. This matrix binds the antibodies of the present invention directly and allows their separation by elution with an appropriate gradient such as salt, GuHCl, pH, or urea. Another preferred embodiment of the present invention is an affinity chromatography method for the purification of HIPs and peptide epitopes from solution. The matrix binds the amino acid compositions of the present invention directly, and allows their separation by elution with a suitable buffer as described above.

In a second preferred embodiment, the invention contemplates the formulation of an affinity chromatography matrix for the purification and/or enrichment of particular species of Hp from solution. In this embodiment, the matrix contains HIPs or peptide epitopes derived from HIPs such as those disclosed herein, covalently-coupled to a Sepharose such as CL6B or CL4B, or other solid support. The matrix-bound HIP binds with high affinity to the species of Hp which interacts with antithrombin-3. Elution of the bound Hp, using an appropriate gradient such as salt, GuHCl, pH, or urea, permits the recovery of a Hp fraction enriched in this particular species of Hp.

4.14 LIPOSOMES AND NANOCAPSULES

In certain embodiments, the inventors contemplate the use of liposomes and/or nanocapsules for the introduction of particular peptides or nucleic acid segments into host cells. Such formulations may be preferred for the introduction of pharmaceutically-acceptable formulations of the nucleic acids, peptides, and/or antibodies disclosed herein. The formation and use of liposomes is generally known to those of skill in the art (see for example, Couvreur et al, 1977 which describes the use of liposomes and nanocapsules in the targeted antibiotic therapy of intracellular bacterial infections and diseases). Recently, liposomes were developed with improved serum stability and circulation half-times (Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987).

Nanocapsules can generally entrap compounds in a stable and reproducible way (Henry- Michelland et al, 1987). To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 μm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be are easily made, as described (Couvreur et al, 1977; 1988).

Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles, MLVs). MLVs generally have diameters of from 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.

In addition to the teachings of Couvreur et al. (1988), the following information may be utilized in generating liposomal formulations. Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the preferred structure. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and drugs.

Liposomes interact with cells via four different mechanisms: Endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsoφtionto the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. It often is difficult to determine which mechanism is operative and more than one may operate at the same time.

4.15 PHARMACEUTICALCOMPOSITIONS

It is expected that to achieve an "immunologically effective formulation" it may be desirable to administer HIPs to the human or animal subject in a pharmaceutically acceptable composition comprising an immunologically effective amount of HIPs mixed with other excipients, carriers, or diluents which may improve or otherwise alter stimulation of B cell and/or T cell responses, or immunologically inert salts, organic acids and bases, carbohydrates, and the like, which promote stability of such mixtures. Immunostimulatory excipients, often referred to as adjuvants, may include salts of aluminum (often referred to as Alums), simple or complex fatty acids and sterol compounds, physiologically acceptable oils, polymeric carbohydrates, chemically or genetically modified protein toxins, and various particulate or emulsified combinations thereof. HIPs or peptides within these mixtures, or each variant if more than one are present, would be expected to comprise about 0.0001 to 1.0 milligrams, or more preferably about 0.001 to 0.1 milligrams, or even more preferably less than 0.1 milligrams per dose. It is also contemplated that attenuated organisms may be engineered to express recombinant

HIP gene products and themselves be delivery vehicles for the invention. Particularly preferred are attenuated bacterial species such as Mycobacterium, and in particular M. bovis, M. smegmatis, or BCG. Alternatively, pox-, polio-, adeno-, or other viruses, and bacteria such as E. coli, Salmonella, Shigella, Listeria, Streptococcus species may also be used in conjunction with the methods and compositions disclosed herein.

The naked DNA technology, often referred to as genetic immunization, has been shown to be suitable for protection against infectious organisms. Such DNA segments could be used in a variety of forms including naked DNA and plasmid DNA, and may administered to the subject in a variety of ways including parenteral, mucosal, and so-called microprojectile-based "gene-gun" inoculations. The use of hip nucleic acid compositions of the present invention in such immunization techniques is thus proposed to be useful as a strategy for the production of anti-HIP antibodies in a mammal.

It is recognized by those skilled in the art that an optimal dosing schedule of a vaccination regimen may include as many as five to six, but preferably three to five, or even more preferably one to three administrations of the immunizing entity given at intervals of as few as two to four weeks, to as long as five to ten years, or occasionally at even longer intervals.

The pharmaceutical compositions disclosed herein may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, enclosed in hard or soft shell gelatin capsule, compressed into tablets, or incoφorated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incoφorated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1 % of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit. The amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; a sweetening agent, such as sucrose, lactose or saccharin; or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compounds, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incoφorated into sustained-release preparation and formulations. The active compounds may also be administered parenterally or intraperitoneally.

Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absoφtion of the injectable compositions can be brought about by the use in the compositions of agents delaying absoφtion, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incoφorating the active compounds in the required or effective amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incoφorating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absoφtion delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incoφorated into the compositions.

For oral prophylaxis the polypeptide may be incoφorated with excipients and used in the form of non-ingestiblemouthwashes and dentifrices. A mouthwash may be prepared incoφorating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incoφorated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate. The active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries. The active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.

The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The composition can be formulated in a neutral or salt form. Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to

1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate or effective dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

4.16 THERAPEUTIC AND DIAGNOSTIC KITS COMPRISING HP/HS COMPOSITIONS Therapeutic kits comprising, in suitable container means, a HIP composition of the present invention in a pharmaceutically acceptable formulation represent another aspect of the invention. The HIP composition may be native HIP, truncated HIP, site-specifically mutated HIP, or HIP- encoded peptide epitopes, or alternatively antibodies which bind native HIP, truncated HIP, site- specifically mutated HIP, or HIP-encoded peptide epitopes. In other embodiments, the HIP composition may be nucleic acid segments encoding native HIP, truncated HIP, site-specifically mutated HIP, or HIP-encoded peptide epitopes. Such nucleic acid segments may be DNA or RNA, and may be either native, recombinant, or mutagenized nucleic acid segments.

The kits may comprise a single container means that contains the HIP composition. The container means may, if desired, contain a pharmaceutically acceptable sterile excipient, having associated with it, the HIP composition and, optionally, a detectable label or imaging agent. The formulation may be in the form of a gelatinous composition, e.g. , a collagenous-HIP composition, or may even be in a more fluid form that nonetheless forms a gel-like composition upon administration to the body. In these cases, the container means may itself be a syringe, pipette, or other such like apparatus, from which the HIP composition may be applied to a tissue site, skin lesion, wound area, or other site where promotion or prevention of coagulation of blood may be desirable. However, the single container means may contain a dry, or lyophilized, mixture of a HIP composition, which may or may not require pre-wetting before use.

Alternatively, the kits of the invention may comprise distinct container means for each component. In such cases, one container would contain the HIP composition, either as a sterile DNA solution or in a lyophilized form, and the other container would include a suitable matrix, which may or may not itself be pre- wetted with a sterile solution, or be in a gelatinous, liquid or other syringeable form which may be used for topical or intra- wound delivery.

The kits may also comprise a second or third container means for containing a sterile, pharmaceutically acceptable buffer, diluent or solvent. Such a solution may be required to formulate the HIP component into a more suitable form for application to the body, e.g., as a topical preparation, or alternatively, in oral, parenteral, or intravenous forms. It should be noted, however, that all components of a kit could be supplied in a dry form (lyophilized), which would allow for "wetting" upon contact with body fluids. Thus, the presence of any type of pharmaceutically acceptable buffer or solvent is not a requirement for the kits of the invention. The kits may also comprise a second or third container means for containing a pharmaceutically acceptable detectable imaging agent or composition.

The container means will generally be a container such as a vial, test tube, flask, bottle, syringe or other container means, into which the components of the kit may placed. The matrix and gene components may also be aliquoted into smaller containers, should this be desired. The kits of the present invention may also include a means for containing the individual containers in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials or syringes are retained.

Irrespective of the number of containers, the kits of the invention may also comprise, or be packaged with, an instrument for assisting with the placement of the ultimate matrix-gene composition within the body of an animal. Such an instrument may be a syringe, pipette, forceps, or any such medically approved delivery vehicle.

5. EXAMPLES

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

5.1 EXAMPLE ^-IDENTIFICATION AND CLONING OF THE GENE ENCODING HIP 5.1.1 EXPERIMENTAL PROCEDURES 5.1.1.1 MATERIALS

Sodium chloride, sodium citrate, Tris base, glycine, bovine serum albumin (BSA), phenylmethylsulfonyl fluoride were purchased from Sigma. Sodium dodecyl sulfate (SDS), β-mercaptoethanol, and Tween-20 were purchased from Bio-Rad. Trichloroacetic acid, acetone, paraformaldehyde, calcium chloride were purchased from Fisher. Formamide and restriction enzymes were purchased from Boehringer Mannheim. All chemicals used were reagent grade or better.

5.1.1.2 CELL CULTURE

RL95, JAR, NIH-3T3, and other cell lines were grown in a 1 :1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum, 15 mM HEPES, pH 7.4, 50 units of penicillin/ml, and 50 μg of streptomycin sulfate/ml (Irvine Scientific, Santa Ana, CA).

5.1.1.3 REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION™

Based on human codon bias (Aota et al, 1988), degenerate oligonucleotide primers (Genosys, The Woodlands, TX) were designed based on amino-terminal amino acid sequence determined for each tryptic peptide (Raboudi et al, 1992) as follows: peptide 1, GGVKKPL

(SEQ ID NO:3): 5'-CCGAGCTCACMGGVGGVGTAARAARCC-3' (SEQ ID NO:4); peptide 2, GAKAKG (SEQ ID NO:5): 5'-CCGAGCTCGGVGCYAARGCYAARGGV-3' (SEQ ID NO:6); peptide 3, VLNIQI (SEQ ID NO:7): 5'-CCGAGCTCGTSCTSAAYATHCARATH-3' (SEQ ID NO:8) (M = A/C, V = G/A/C, R = A/G, Y = C/T, S = G/C, and H = A/T/C). A Sαcl restriction enzyme site (underlined) was added 5' to each primer. CCGCGGCCGCdT₁₈ (SEQ ID

NO: 9) with a 5' Notl restriction enzyme site (underlined) was used as a primer for reverse transcription (RT) and another primer for polymerase chain reaction (PCR™). Two cytidine nucleotides were added 5' to each primer to maintain hybridization of terminal regions during RT-PCR™. Complete degenerate oligonucleotide primers for each tryptic peptide also were made and used in RT-PCR™. Total RNA from RL95 cells was isolated using the method of RNA isolation of Xie and Rothblum (1991). RT-PCR™ was performed on a DNA Thermal Cycler (Perkin-Elmer) using the protocol described in the GeneAmp RNA PCR™ kit (Perkin-Elmer) with the concentration of MgCl₂ adjusted to 1 mM. The thermal cycle profile described in the 3'-rapid amplification of cDNA ends protocol (Frohman, 1990) was followed. The cDNA pools from RT were amplified by PCR™. RT-PCR™ products were cloned into the pCR II vector using the TA Cloning System kit (Invitrogen, San Diego, CA). Plasmids containing RT-PCR™ products were isolated either following the method described by Sambrook et al. (1989) or using "Magic Minipreps" DNA Purification System kit (Promega, Madison, WI). All of RT-PCR™ products were sequenced using either the dideoxy-mediated chain termination method (Sambrook et al, 1989) following the procedure provided in the Sequenase™ version 2 kit (Amersham Coφ.) or automatic sequencing using fluorescently labeled sequencing primers (T7 and SP6) provided in the Applied Biosystems cycle sequencing kit and analyzed on an Applied Biosystem model 373A automated sequencer (Perkin-Elmer).

5.1.1.4 NORTHERN BLOT ANALYSIS

Northern blot analysis of RNA was performed using method described by Sambrook et al (1989). Total RNA was extracted (Xie and Rothblum, 1991), and poly(A⁺) RNA was isolated using Oligotex-dT mRNA kit (Qiagen Inc., Chatsworth, CA). Both total RNA (20 μg) and poly (A⁺) RNA (3.5 μg) were separated on 1% (wt/vol) agarose gel and transferred to a nylon membrane (Hybond-N, Amersham Coφ.). A cDNA probe (Clone 23-1 in FIG. 1 depleted of poly (A ) tail) was labeled with P by utilizing the random oligonucleotide primer method (Sambrook et al, 1989). Hybridization was performed at 42°C overnight in a solution containing 50% (vol/vol) formamide, 5χ sodium chloride-sodium citrate (SSC), 5x Denhardt's, 50 mM NaH₂PO₄, 0.1% (wt/vol) SDS, and 100 μg/ml denatured, sonicated salmon sperm DNA.

The blot then was washed using 2χ SSC, 0.1% (wt/vol) SDS with several changes during 1 h at 25°C and washed once again using 0.5 SSC and 0.1% (wt/vol) SDS for 2 h at 42°C before exposure to Kodak XAR film with an intensifying screen at -70°C.

For the analysis of HIP mRNA expression and distribution among human cell lines, total RNA (20 μg/each) from AFb-11, normal fibroblast; HeLa, cervical epithelium; HEC, uterine epithelium; HL60, leukemic; HUVEC, normal umbilical vein endothelium; Ishikawa, uterine epithelium; MDA-231, breast epithelium; JAR, trophoblastic epithelium; NCI-H69, lung small cell; RL95, uterine epithelium; TU138, lung fibroblast; and NIH-3T3, mouse embryonic fibroblast cell lines were isolated, separated on 1% (wt./vol.) agarose gel, and subjected to Northern blot analysis using P-labeled cDNA for clone 23-1 as a probe as described above.

5.1.1.5 cDNA LIBRARY SCREENING

HeLa cell cDNA libraries constructed in the Lambda gtl 1 vector from Stratagene (La Jolla, CA) and Clontech (Palo Alto, CA) were used for cDNA library screening following the protocol provided by the manufacturer. Briefly, nitrocellulose filters (Schleicher and Schuell) lifted off the plated cDNA library were prehybridized in 0.8 M NaCl, 20 mM 1 ,4-piperazinediethanesulfonic acid (Pipes), pH 6.5 50% formamide, 0.5% (wt./vol.) SDS, and 100 μg/ml denatured, sonicated salmon sperm DNA for >4 h at 42°C and hybridized in a fresh solution of the same composition containing the P-labeled probe (2-4 x 10 cpm/ml) for library screening, at 42°C overnight. After hybridization, the blots were briefly washed with O.lx SSC,

0.1%) (wt/vol) SDS at room temperature and then washed with same mixture at 65°C for 1 h. The blots were exposed to Kodak XAR films with an intensifying screen at -70°C overnight. Positive plaques were isolated and subjected to subsequent screenings under the same conditions.

5.1.1.6 SOUTHERN BLOT ANALYSIS ANDDNA SEQUENCING

Positive phage DNA from cDNA library screening was purified from the phage lysate (Sambrook et al, 1989) and digested with EcoRI. The cDNA inserts were separated on 1% (wt/vol) agarose gel, and transferred to a nylon membrane by standard method (Sambrook et al , 1989). Blots were hybridized with ³²P-labeled probe in 6x SSC, 0.5% (wt/vol) SDS, 50% (vol/vol) formamide, and 100 μg/ml denatured, sonicated salmon sperm DNA overnight. The blots were washed consecutively with 2χ SSC and 0.5%) (wt/vol) SDS for 5 min at room temperature, 2x SSC and 0.1% (wt/vol) SDS for 15 min at room temperature, and O.lx SSC and 0.5% (wt/vol) SDS for 1 h at 37°C. Then the blots were washed with O.lx SSC and 0.5% (wt/vol) SDS for 1 h at 68°C prior to the exposure to Kodak film with an intensifying screen at - 70°C. Phage with positive cDNA inserts was digested with EcoRI, separated on a 1% (wt/vol) agarose gel, purified by phenol/chloroform extraction and ethanol precipitation, and then subcloned into the EcoRI site of pBluescript II SK- (Stratagene). Subcloned inserts were further analyzed using Southern blot analysis as described above. Clones with positive inserts were identified and both strands of the cDNA sequences were determined.

5.1.1.7 CONSTRUCTION OF CDNA EXPRESSION VECTOR AND TRANSFECTION

The entire HIP cDNA (clone 36-1 in FIG. 1) was digested from HIP cDNA-containing pBluescript using Λfotl, separated on 1% (wt/vol) agarose gel, purified by phenol/chloroform extraction and ethanol precipitation, and subcloned into the Notl site of mammalian expression vector pOPRSVI (LacSwitch Inducible Mammalian Expression System, Stratagene; La Jolla,

CA). The clone that contained the entire cDΝA in the correct orientation was determined by PCR™ using oligonucleotide primers derived from both vector and HIP cDΝA sequences and selected for transfection. ΝIH-3T3 cells (30-40% confluence in 100-mm cell culture plates) were transfected with 18 μg/plate HIP expression vector DNA using the calcium phosphate method (Sambrook et al, 1989) and grown for 48-72 h. The transfected cells were then harvested and used for Western blot and immunocytochemical analyses.

5.1.1.8 SDS-PAGE AND WESTERN BLOTTING

Cells on cell culture plates were washed three times with PBS and solubilized in sample extraction buffer: 4 M urea, 1% (wt/vol) SDS, 50 mM Tris, pH 7.0, 1% (vol/vol) β-mercaptoethanol, and 0.01% (vol/vol) phenylmethylsulfonyl fluoride. Solubilized samples were concentrated by precipitation with 10% (wt/vol) trichloroacetic acid at 4°C. Trichloroacetic acid precipitates were centrifuged at 1,200 x g for 10 min at 4°C, washed sequentially with 10%

(wt vol) trichloroacetic acid and 100%) acetone, and air-dried. The pellets were dissolved in equal volumes of sample extraction buffer and sample buffer (Laemmli, 1970), heated for 2 min at 90°C. Total homogenates of RL95 cells (50 μg), parental NIH-33 cells (200 μg), and

NIH-3T3 cells transiently transfected with HIP cDNA (200 μg) were resolved by SDS-PAGE on a 15% (wt/vol) acrylamide resolving gel as described (Porzio and Pearson, 1977). After a brief rinse in transfer buffer (100 mM Tris base and 100 mM glycine, pH 9.2) the gel was transferred to a nitro-cellulose membrane at 4°C for 5 h at 40 V in a Transblot apparatus (Bio-Rad). The transferred blot was blocked with 1% (wt/vol) BSA in PBS, 0.01%> (wt/vol) sodium azide, and 0.05% (wt/vol) Tween-20 (P.A.T.) overnight at room temperature. The blot then was incubated with primary antibody diluted in 0.1% (wt/vol) BSA in P.A.T. three times 5 min each, and incubated for at least 4-6 h with 6 μCi of ¹²⁵I-protein A (30 μCi/μg) in 70 ml of 0.1% (wt/vol) BSA in P.A.T.

For analysis of HIP expression and distribution in different human cell lines, total proteins (100 μg/each) from HeLa, cervical epithelium; 2774, ovary epithelium; NCI-H69, lung small cell; AN3-CA, lymph endometrium; RKO, intestine epithelium; Ter9113, trophoblastic; Ter9117, trophoblastic; Glioma2, glial; Tul38, lung fibroblast; NeuroBl, neural; HL60, leukemic, and JAR, trophoblastic epithelium human cell lines were extracted and separated on

SDS-PAGE, and HIP expression was determined by Western blot analysis as described above.

5.1.1.9 IMMUNOCYTOCHEMICAL ANALYSIS

Transiently transfected and parental NIH-3T3 cells were grown on coverslips for 24 h in Dulbecco's modified Eagle's medium/Ham's F12 supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum. The cells were briefly rinsed with PBS and fixed with 2.5%

(wt/vol) paraformaldehyde in PBS for 15 min at room temperature. Cells were rinsed with PBS and soaked in PBS for 10 min. Aldehyde groups were blocked by incubation with 2 ml of 50 mM ammonium chloride in PBS for 15 min at room temperature. Immunostaining with primary and secondary antibodies and mounting of coverslips were performed as described previously

(Julian et al, 1994). Briefly, coverslips were then incubated with primary antibody in PBS for

1 h at 37°C. After three rinses of 5 min each with PBS, the cells were incubated with the second antibody, fluorescein-conjugated donkey anti-rabbit IgG (Amersham Coφ.), for 40 min at 37°C.

Unbound antibody was removed by several 5 -min rinses at room temperature with PBS prior to mounting for fluorescence microscopy.

5.1.1.10 COMPUTER ANALYSIS

Nucleotide and protein sequence analyses were carried out using GCG and MicroGenie programs and the data bases from GenBank (release 90.0), EMBL (release 43), and SWISS-PROT (release 31). 5.1.2 RESULTS

5.1.2.1 CHARACTERIZATION OF RT-PCR™ PRODUCTS

RT-PCR™ products obtained from each pair of primers were isolated, subcloned, and sequenced. Sequences of all of the RT-PCR™ products were determined. Among all of the

RT-PCR™ products, one product (RT-PCR™ 224 in FIG. 1) displayed several interesting features as follows. 1) There was a single open reading frame. 2) The amino-terminal peptide sequence used to design the primer was contained in the predicted peptide sequence. 3) A poly-adenylation signal was found adjacent tot he poly(A⁺) tail. 4) The predicted polypeptide sequence of the cDNA contained an antigenic peptide sequence, CRPKAKAKAKAKDQTK

(SEQ ID NO: 10), with features associated with Hp/HS-binding motifs (Cardin and Weintraub, 1989). Computer analysis predicted that his motif was α-helical and hydrophilic and likely to be exposed on the external surface of the protein where it may bind Hp/HS The sequence of this 270-bp RT-PCR™ product was compared with available sequences in GenBank. The highest similarities found by this analysis were 64% (in 265 -bp overlap) similarity to Ratus norvegicus

(rat) mRNA for ribosomal protein YL43. These features and homology analysis suggested that the parental mRNA corresponding to this RT-PCR™ product encodes a novel protein related to L29.

5.1.2.2 ISOLATION AND CHARACTERIZATION OF A CDNA ENCODING HIP

A HeLa cell cDNA library was screened using the cDNA of HIP RT-PCR™ product (RT-PCR™ 224 depleted of poly (A⁺) tail in FIG. 1A) as a probe. As a result, 20 positive clones with identical inserts to that of 23-1 (or 42-1) (FIG. 1A) were obtained from approximately 5 x 10⁵ plaque-forming units. The cDNA sequence of inserts of 23-1 and 42-1 was determined by primer walking. The determined cDNA sequence contains an incomplete open reading frame encoding 117 amino acid residues. Re-screening of a 5'-stretched HeLa cDNA library (Clontech) using 23-1 cDNA as a probe resulted in the isolation of 12 positive clones from 3.0 x 10⁵ plaque-forming units. The inserts ranged between 400 and 650 bp. Restriction analysis revealed that they had the same restriction enzyme maps, indicating that they were likely to be derived from the same mRNA transcript. Sequencing of the two largest inserts, 35-2 and 36-1, revealed identical sequences except that 1) 36-1 contains 26 more bp of 5 '-untranslated region than 35-2; 2) 35-2 contains an extra 6 bp of nucleotides (bp - of SEQ ID NO:l) at the 3' end of 3 '-untranslated region; and 3) there is a nucleotide replacement (C replaced with T at nucleotide position 145 of SEQ ID NO:l). This nucleotide replacement does not affect the coding of the amino acid, leucine. The nucleotide sequence of HIP cDNA is shown in SEQ ID

NO:l, and the predicted amino acid sequence of HIP is shown in SEQ ID NO:2 (accession number, U49083).

Further analysis of the sequence showed that HIP cDNA contains a single open reading frame of 477 bp, starting with an ATG codon at position 28 with characteristic purines at positions -3 and +4 relative to the start ATG codon (Kozak, 1987), and ending with a stop codon TAG at position 506. This open reading frame encodes a protein of 159 amino acid residues with a calculated molecular mass of 17,754 Da. The HIP peptide sequence used for antibody production corresponds to amino acids 119-134 of SEQ ID NO:2. Following the open reading frame, there are 121 bp of a 3 '-untranslated region that contains a polyadenylation signal at nucleotide position 613. The predicted protein sequence has high content of positively charged amino acid residues (K + R = 29.6%) and a predicted pi of 11.75.

A comprehensive search of the GenBank, EMBL, and SWISS-PROT data bases revealed that the nucleotide sequence of HIP has 80.5% identity in 549-bp overlap to a rat mRNA for ribosomal protein related to yeast ribosomal protein YL43, 11.6% identity in 603-bp overlap to R. norvegicus (rat) mRNA for ribosomal protein L29 and 16.6% identity in 640-bp overlap to Mus musculus (murine) large ribosomal subunit protein mRNA. A BLAST homology search using GenBank revealed two human nucleotide sequences, designated as a putative human ribosomal protein L29, in GenBank (accession number U 10248 and Z49148) showing the essentially the same nucleotide sequence as that of HIP cDNA (Law et al, 1996). The predicted amino acid sequence of HIP has 80.3% identity in 157-amino acid residue overlap to a rat 60 S ribosomal protein, L29; however, the region encoding the peptide sequence (HIP peptide) used for antibody production and Hp/HS-binding activity studies is not conserved among human and rat or mouse. Consequently, the antibodies are specific to human and do not cross-react with L29 of rat or mouse. 5.1.2.3 NORTHERN BLOT ANALYSIS OF RNA FROM RL95 CELLS

The expression of HIP mRNA was examined by Northern blot analysis using a P-labeled clone 23-1. A single predominant transcript of 1.3 kb was detected either using total RNA or poly(A ) RNA from RL95 cells. Further studies using a variety of human cell lines (see below) indicated that the 1.3-kb transcript is the major HIP mRNA found in most cases.

5.1.2.4 TRANSFECTION OF HIP cDNA INTO NIH-3T3 CELLS AND EXPRESSION OF HIP

To demonstrate that the cloned cDNA sequence encodes the protein recognized by antibodies generated against the HIP peptide and to verify cell surface expression of HIP, clone

36-1 was subcloned into a Rous sarcoma virus-based mammalian expression vector and used to transfect NIH-3T3 cells. Transiently transfected NIH-3T3 cells were fixed with paraformaldehyde, and expression of HIP was detected using anti-HIP. Immunostaining demonstrated cell surface expression of transfected HIP protein in a portion of transfected cells, whereas other cells that presumably were not transfected during the transient assay were negative. Negative staining also was observed using paraformaldehyde-fixed parental-3T3 cells. Similar controls as described in Example 2 using antibodies directed against cytokeratins were done for the cell surface staining of the transiently HIP-transfected NIH-3T3 cells, and no reactivity was observed under the fixing conditions performed. Therefore, the immunostaining of the transfected cells was cell surface staining. Western blot analysis of total protein extracted from transiently transfected NIH-3T3 cells using anti-HIP-peptide detected a newly expressed protein with an apparent M_v of 24,000, i.e., the same molecular weight as that of HIP detected in RL95 cells. In contrast, the M_τ 24,000 component was not detectable in the parental NIH-3T3 cells. Collectively, these data demonstrate that the isolated cDNA sequence encodes the protein recognized by the anti-HIP peptide antibody and that this protein can be expressed on cell surfaces.

5.1.2.5 EXPRESSION AND DISTRIBUTION OF HIP

Expression and distribution of HIP in different human cell lines and normal tissues were examined using both Northern blot analysis and Western blot analysis. Northern blot analysis using the HIP cDNA probe detected a single transcript of 1.3 kb in most human cell lines tested. HIP is expressed highly in most human cell lines tested. HIP is expressed highly in most human epithelial cell lines including RL95, JAR, HeLa, HEC, and Ishikawa, as well as AFb-11, human fibroblastic cells, moderately in HU-VEC (endothelial cells), and relatively low in HL60, a human leukemic cell line. HIP mRNA was not detectable in MDA-231, a human breast cancer cell line, or mouse NIH-3T3 cells. Western blot analyses on several human cell lines also revealed a similar distribution pattern of HIP as that shown in Northern blot analyses (Table 4). Again, consistent with Northern blot analysis, HIP was not detectable in cell lines of MDA-231 (Table 4) and NCI-H69. Table 4 provides a summary of results showing differential HIP expression among a panel of human cell lines examined. Collectively, these results indicate that

HIP mRNA and protein are expressed differentially in human cell lines.

5.1.3 DISCUSSION

In the present study, the inventors have isolated and sequenced a full-length cDNA encoding HIP, a novel human Hp/HS-binding protein expressed on cell surfaces. This cDNA encodes a protein of 159 amino acids with high content of basic amino acids. There is not a potential transmembrane domain present in the predicted amino acid sequence of HIP, although, HIP is associated with the cell surface. Therefore, it is likely that HIP is a peripheral membrane protein, perhaps bound to other proteins, lipids, or polysaccharides. The predicted amino acid sequence of HIP does not contain a classical hydrophobic amino-terminal signal peptide (Blobel and Dobberstein, 1975); however, there are multiple reports of the lack of a signal peptide in the sequences of membrane or secreted proteins (Kikutani et al, 1986; Bettler et al, 1989; Brown et al, 1987). The predicted protein sequence of HIP predicts a molecular mass of 17,754 Da. This is significantly less than expected for M_τ 24,000 protein recognized by anti-HIP -peptide on SDS-PAGE. The anomalous molecular mass may be due to the highly basic character of HIP

(predicted pi = 11.75). Other highly basic proteins, e.g. histones, migrate relatively slowly on SDS-PAGE (von Holt et al, 1989; Weber and Osborn, 1975), apparently due to an inordinately high amount of SDS binding. Alternatively, post-translational modifications may increase the size of HIP. No consensus sites for glycosylation are evident in this sequence, but other modifications are possible. Transfection of full-length cDNA of HIP into NIH-3T3 cells resulted in the expression of a protein with M_τ of 24,000 determined by SDS-PAGE, further demonstrating that the cloned cDNA sequence encodes the same protein recognized by the antibody and contains the predicted peptide sequence. Northern blot and Western blot analyses revealed that both the 1.3-kb mRNA and M_r 24,000 protein are expressed of HIP protein also has been observed in normal human tissues examined (Rohde et al, 1996).

Table 4

HIP Expression In Human Cell Lines- a

Cell type Cell line Protein RNA

Neural Neuroblastoma ++ ND

Glial U251 ++ ND

U343 +/- ND

Muscle Rhabdomyosarcoma ++++ ND

Epithelial

Choriocarcinoma JAR ++++

JEG +++ +++

BeWo

Uterine RL95 +++

HEC

Ishikawa ++ ++++

Breast MCF-7 ++

MDA-231

AR75-1

Prostate A5153-4 ++ ND

Intestine RKO ++ ND

Cervical HeLa ++ +++

Ovary 2774 +/- ND

Lung NCI-H69

Endothelial HUVEC + + Cell type Cell line Protein RNA

Fibroblast AFb-11 + ++

Tul38 - +/-

Lymphoid HL60 +/- +

Embryonic Terato 9117 +++ ND

Terato 9113-Clone l +++ ND

Terato 9113 -Clone 6 + ND

'Total RNA (20 μg) and total protein (50-100 μg) from different human cell lines were extracted and subjected to Northern blot analysis using clone 23-1 cDNA probe and Western blot analysis using anti-HIP-peptide antibody, respectively, as described. The relative expression level of HIP mRNA in different cells is designated semi-quantitatively as following examples: JAR or Ishikawa, ++++; HeLa or RL95, +++; AFb-11, ++;

HL60, +; and MDA-231 or NCI-H69, -. Similarly, the level of HIP expression is designated as in the following examples: JAR or Ter9113, +++; RKO or NeuroBl, ++; HeLa, +; and NCI-H69, -. N.D., not determined.

Sequence comparison with available data bases revealed that HIP has a relative high similarity (80%) to rodent L29, a ribosomal protein, at both the nucleotide and protein sequence level. It is possible that HIP is the human homologue of rodent L29. It is noteworthy that in the mouse, L29 is a member of 15-18 genes or pseudogenes (Rudert et al, 1993). It is not clear what functions, if any, these sequences serve in rodents. Several lines of evidence indicate that

HIP does not function simply as a ribosomal protein. First, HIP can be detected on cell surfaces of cells transfected with HIP cDNA or RL95 cells. Second, HIP is expressed in a nonconstitutive fashion in different human cell lines and normal tissues. Constitutent ribosomal proteins would be expected to be expressed at stoichiometric levels in different cells and with respect to the cellular content of rRNA species. While there is precedent for limited modulation of ribosomal proteins in some cases (Nomura et al, 1982; Rudert et al, 1993), these proteins are never essentially absent as in the case for both HIP mRNA and protein in cells like MDA-231 and

NCI-H69. Collectively, these data strongly argue that HIP is not critical to ribosomal function.

Cell surface localization and Hp/HS-binding activity suggest that HIP may play a role in Hp/HS-involved cell-cell or cell-matrix interactions or have other functions yet to be determined. In the studies of rodent L29, the identification and localization of this protein was based on sequence homology analysis and standard procedures of ribosomal protein isolation (Ostvold et al, 1992; Svoboda et al, 1992; Rudert et al, 1993). The distribution of the protein was only examined in one study by Northern blot analysis and in situ hybridization (Rudert et al. 1993). No studies of the expression of the L29 protein are reported. Thus, it is of interest to re-examine the expression of rodent L29 considering the possibility that it may not be a "housekeeping" protein. Considering the existence of the high number of sequences closely related to L29 (Rudert et al, 1993), it will be important to use probes specific for each gene in such studies.

HIP may be expressed both at cell surfaces and intracellularly. Several reports indicated that some proteins are present both at cell surfaces or secreted as well as inside the cell (Terada et al, 1995). These examples include certain growth factors (Abraham et al, 1986; Jaye et al,

1986), cytokines (Matsushima et al, 1986), and lectins (Cooper and Barondes, 1990). Why these proteins are expressed in both locales is unclear. In the present case, it is not known if intracellular HIP is contained within vesicles or organelles or in the cytoplasm. The following Example demonstrates that almost all of the cell-associated HIP is formed in a 100,000 x g sedimentable fraction and, therefore, is not present in a freely soluble form. Several mechanisms for sorting of cytoplasmic and secreted proteins have been postulated, including that cell lysis, death, or leakage might be responsible for the release of these proteins (D'Amore, 1990) or that the release might be induced by plasma membrane evaginations (Cooper and Barondes, 1990).

Previous studies have suggested that HSPGs and their corresponding binding sites may play an important role in the initial attachment of mouse embryo to uterine epithelium. In the present study, a novel cell surface Hp/HS-binding protein from a human uterine epithelial cell line has been cloned and expressed. The following Example describes expression of this protein in normal human lumenal epithelium, a location where HIP could participate in embryo attachment.

5.2 EXAMPLE 2 - EXPRESSION OF HIP AND PREPARATION OF HIP ANTIBODIES

Previous studies established that uterine epithelial cells and cell lines express cell surface Hp/HS-binding proteins (Wilson et al, 1990; Raboudi et al, 1992). The HIP antibodies of the present invention specifically recognize a protein with an apparent _r of 24,000 by SDS-polyacrylamide gel electrophoresis that was highly enriched in the 100,000 x g particulate fraction of RL95 cells. This molecular weight is similar to that of the protein expressed by 3T3 cells transfected with HIP cDNA. HIP was solubilized from this particulate fraction with NaCl concentrations >0.8 M demonstrating a peripheral association consistent with the lack of a membrane spanning domain in the predicted cDNA sequence. HIP was not released by heparinase digestion suggesting that the association is not via membrane-bound HS proteoglycans. NaCl-solubilized HIP bound to Hp^'agarose in physiological saline and eluted with NaCl concentrations of 0.75 M and above.

125

Furthermore, incubation of I-HP with transblots of the NaCl-solubilized HIP preparations separated by two-dimensional gel electrophoresis demonstrated direct binding of HP to HIP. Indirect immunofluorescence studies demonstrated that HIP is expressed on the surface of intact RL95 cells surfaces at 4°C was saturable and blocked by preincubation with the peptide antigen. Single cell suspensions of RL95 cells formed large aggregates when incubated with antibodies directed against HIP but not irrelevant antibodies. Finally, indirect immunofluorescence studies demonstrate that HIP is expressed in both lumenal and glandular epithelium of normal human endometrium throughout the menstrual cycle. In addition, HIP expression increases in the predecidual cells of post-ovulatory day 13-15 stroma. Collectively, these data indicate that HIP is a membrane-associated HP -binding protein expressed on the surface of normal human uterine epithelia and uterine epithelial cells lines.

5.2.1 EXPERIMENTAL PROCEDURES 5.2.1.1 MATERIALS

Tissue culture media components were obtained from Irvine Scientific (Santa Ana, CA)

125 and Life Technologies, Inc. I-Protein A was from ICN Radiochemicals (Irvine, CA). Tris, glycine, bovine serum albumin, urea, phenylmethylsulfonyl fluoride, polyhema, EDTA, magnesium chloride, Hp, and hemoglobin were purchased from Sigma. Sodium dodecyl sulfate, β-mercaptoethanol, acrylamide, bisacrylamide, and Tween-20 were purchased from Bio-Rad (Richmond, California). Sodium azide, trichloroacetic acid, acetone, sucrose, paraformaldehyde, ammonium chloride, and calcium chloride were purchased from Fisher. Sodium chloride and methanol were purchased from EM Science (Gibbstown, NJ). Tissue culture plates (100 mm) were purchased from Falcon (Lincoln Park, NJ), and 24-well tissue culture plates were purchased from Corning (Corning, NY). Nitrocellulose membrane (0.45 μm) was purchased from Intermountain Scientific Coφ. (Bountiful, UT). Dithiothreitol was purchased from Boehringer Mannheim. Ethanol was purchased from AAPER Alcohol and Chemical Co. (Shelbyville, KY). Rabbit anti-Na⁺/K -ATPase was purchased from Chemicon International, Inc. (Temecula, CA). Rabbit antibodies to human factor VIII and laminin were purchased from Dakopatt's (Glostrup,

Denmark) and Collaborative Research (Bedford, MA), respectively. All chemicals used were reagent grade or better.

5.2.1.2 CELL CULTURE Cells (RL95-2 or HEC-la) were cultured in Dulbecco's minimal essential medium/Ham's

F12, 1 :1 supplemented with 100 units/ml penicillin and 10 μg/ml streptomycin sulfate and 10% (vol/vol) heat-inactivated fetal bovine serum at 37°C in a humidified atmosphere of 95%) air:5%> CO₂ (vol/vol). For collection of conditioned medium, the same medium was used except that the fetal bovine serum was omitted. Medium was collected after a 24-h incubation. In most studies, RL95 cells were used; however, in some studies HEC-la (purchased from the American Type

Culture Collection) or Ishikawa cells (obtained from Dr. Erlio Guφide, Mt. Sinai School of Medicine, New York) were cultured under the same conditions and used for preparation of whole cell extracts or conditioned media. Human endometrial tissue was obtained from routine biopsy specimens.

5.2.1.3 PEPTIDE SYNTHESIS AND ANTIBODY GENERATION

A synthetic peptide of the following sequence was constructed on a Vega 250 peptide synthesizer using FMOC methodology (Chang and Meienhofer, 1978), CRPKAKAKAKAKDQTK (SEQ ID NO: 10). This synthetic peptide was conjugated to the keyhole limpet hemocyanin protein, using the Imject Maleimide Activated Carrier Proteins kit

(Pierce) and was used for rabbit immunization following standard protocols (University of Texas M.D. Anderson Cancer Center, Bastrop, TX). Western blot analysis and immunocytochemical studies were conducted using polyclonal antibodies affinity purified with the synthetic peptide linked to maleimide-activated BSA (Pierce) conjugated to cyanogen bromide-activated Sepharose (Sigma), using the manufacturer's protocol. 5.2.1.4 SDS-PAGE AND WESTERN BLOTTING

Cells or particulate subcellular fractions were initially solubilized and SDS-PAGE and Western blotting performed as described previously (Porzio and Pearson, 1977; Laemmli, 1970;

195 Julian et al. , 1994) using affinity purified rabbit anti-HIP as primary antibody and I-protein A

(30 μCi/μg) as the detection system.

5.2.1.5 MEMBRANE PREPARATIONS

RL95 cells were grown to 70% confluency on a 100-mm tissue culture plate. Membranes were isolated by differential centrifugation. Briefly, cells were washed three times with PBS and released from the plate by incubation with 10 mM EDTA in PBS at 37°C for 15-30 min. Cells were pelleted at 1000 x g for 10 min at 4°C and resuspended in homogenizing buffer [0.25 M sucrose, 5 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.25 mM dithiothreitol, and a mixture of protease inhibitors (Farach et al, 1987)] and homogenized on ice. The homogenate was centrifuged at 1000 x g for 10 min at 4°C. The 1000 x g supernatant was centrifuged at

10,000 x g for 20 min at 4°C. The 10,000 x g supernatant was centrifuged at 100,000 x g for 1 h at 4°C.

Subcellular fractions were prepared from RL95 cells and analyzed by SDS-PAGE and Western blotting as described above. Approximately 50 μg of protein was added per lane. Analyzed were total RL95 homogenate; 1000 x g/10-min supernatant; 10,000 x g/20-min supernatant; 100,000 x g/l.O-h. supernatant; 100,000 x g/4-h supernatant; 1000 x g/10-min pellet; 10,000 x g/20-min pellet; 100,000 x g/l.O-h pellet; and 100,000 x g/4-h pellet.

RL95 cell surface components were radioiodinated with 1 mCi/ml Na ⁵I (carrier-free; Amersham Coφ., Arlington Heights, IL) in PBS for 30 min on ice by overlaying the cell layers with glass coverslips coated with 10 μg of IODO-GEN™ (Pierce; Rockford, IL) (Markwell,

1982). After this period, the coverslips were removed, and the cell layers were rinsed several times with PBS containing 1 mM Nal. Cells were scraped from the tissue culture dish with a rubber policeman and subsequently homogenized and subjected to the subcellular fractionation scheme described above. Equal protein loads of each fraction was applied to SDS-PAGE, and the location of the ^I2T-labeled cell surface components determined by autoradiography of the dried gels.

5.2.1.6 NACL EXTRACTION OF MEMBRANES High speed (100,000 x g) membrane fractions were divided into equal parts and extracted either with 0.15, 0.4, 0.8, 1.2, or 1.6 M NaCl in 0.25 M sucrose, 1 mM EDTA, 0.25 mM dithiothreitol, and 5 mM Tris (pH 7.4), incubated overnight at 4°C, and centrifuged the next day at 100,000 x g for 1 h. Supematants were precipitated overnight at 4°C by the addition of trichloroacetic acid to a final concentration of 10% (wt/vol). Pellets were dissolved in 0.2 ml of sample extraction buffer and then precipitated. Half of each extract or pellet was used for protein determination, and the other half was used for SDS-PAGE and Western blot analysis as described above.

5.2.1.7 HP AGAROSE AFFINITY CHROMATOGRAPHY High speed (100,000 x g) membrane preparations were extracted overnight at 4°C with

0.4 M NaCl in 5 mM Tris (pH 8.0) and centrifuged at 100,000 x g for 1.5 h. The 0.4 M NaCl-insoluble pellet was subsequently extracted with 0.8 M NaCl in 5 mM Tris (pH 8.0) at 4°C for 4 h and centrifuged 1.5 h at 100,000 x g. The protein eluted between 0.4 and 0.8 M NaCl was diluted to 0.15 M NaCl, applied to a 0.5-ml pellet of prerinsed Hp-agarose (Sigma, St. Louis, MO), and incubated overnight batchwise with constant rotary agitation at 4°C A stepwise elution from Hp-agarose was performed with NaCl concentrations of 0.15, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75 and 2.0 M in 5 mM Tris (pH 8.0). All fractions were trichloroacetic acid-precipitated and prepared for SDS-PAGE and Western blotting as described above.

5.2.1.8 ^{1 5}I-HP OVERLAY OF TWO-DIMENSIONAL GELS

High speed (100,000 x g) particulate preparations were subjected to differential salt extraction with 0.4 M NaCl followed by 0.8 M NaCl as described above. The proteins then were separated by two-dimensional gel electrophoresis (O'Farrell et al, 1977) and transferred to nitrocellulose as described above for Western blotting. The unblocked nitrocellulose was incubated with ¹²⁵I-Bolton-Hunter reagent-derivatized HP (Raboudi et al, 1992) in 0.15 M NaCl overnight at 4°C. The blot then was washed 3 times with PBS before drying for autoradiography. The same blot was blocked and then probed with HIP antibody and binding subsequently visualized with a peroxidase ABC system using a diaminobenzidene substrate kit as described by the manufacturer's instmctions (Vector Labs, Burlingame, CA). A parallel gel mn under exactly the same conditions was silver-stained as described (Wray et al, 1981) to visualize the migration positions of all proteins on the gel.

5.2.1.9 IMMUNOCYTOCHEMISTRY

Cells were grown on coverslips for 48 h in Dulbecco's modified Eagle's medium/Ham's F12 with 10% (vol/vol) fetal bovine serum. After a brief rinse in PGS, the cells were fixed with

2.5% (wt/vol) paraformaldehyde in PBS for 15 min at room temperature, rinsed twice in PBS, and aldehyde groups blocked by incubation with 50 mM ammonium chloride in PBS for 15 min at room temperature. Incubation with primary and secondary antibodies and mounting of coverslips were as described (Julian et al, 1994). For staining of endometrium, human endometrium was rapidly frozen in O.C.T. (Miles; Elkhart, IN) and sections prepared at -20°C on a Reichert-Jung cryostat. These sections were fixed in 100% methanol for 10 min at room temperature, rehydrated in PBS for 5 min at room temperature, and immediately used for immunostaining. The stage of the menstmal cycle was identified in all cases by standard histological examination (Noyes et al, 1950) and serum hormone profiles. In all cases, the affinity purified HIP primary antibody was used at a concentration of 25 μg/ml and the secondary antibody, fluorescein-conjugated donkey anti-rabbit Ig (Amersham Coφ.), at a 1 :10 dilution. Rabbit antiserum to human factor VIII was used at a 1 :30 dilution. Rabbit antisemm to mouse laminin was used at a 1 :50 dilution.

5.2.1.10 BINDING OF ANTI-HIP TO RL95 CELL SURFACES

RL95 cells were grown to 90% confluency in 24-well tissue culture plates with

Dulbecco's modified Eagle's medium Ham's F12 containing 10% (vol/vol) fetal bovine serum.

Cells were rinsed three times with Hanks' -buffered saline and preincubated for 15 min at 4°C in

0.5 ml of binding buffer (PBS containing 2 mM CaCl₂, 2 mM MgCl₂, 0.1% (wt/vol) hemoglobin, 1 mM Nal, and 0.02% (wt vol) NaN₃). The binding buffer was removed, and 0.2 ml of binding buffer containing anti-HIP or nonimmune rabbit IgG was incubated for 45 min at 4°C in duplicate wells. IgG was added at the following concentrations, 0, 10, 50, 100, and 200 μg/ml. Cells were rinsed 3 times with binding buffer at 4°C for 5 min and incubated with binding buffer containing I-protein A (1 x 10 cpm/well) for 30 min at 4°C. After rinsing 3 times with ice-cold binding buffer, cells were solubilized with 1% (wt/vol) SDS and 0.5 M NaOH, and the amount of I-protein A bound to RL95 cell surface was determined. To determine nonspecific binding of anti-HIP or nonimmune rabbit IgG, antibody was preincubated for 2 h at 4°C with or without 100 μl of peptide affinity matrix and then centrifuged.

5.2.1.11 AGGREGATION OF RL95 CELLS BY ANTI-HIP

RL95 cells were grown in tissue culture plates to 70% confluency and detached with 10 mM EDTA in PBS without calcium and magnesium. Cells were resuspended in media (Dulbecco's modified Eagle's medium/Ham's F12 containing 1% (vol/vol) penicillin/streptomycin and 0.1% (wt/vol) BSA) at a concentration of 3.5 x 10⁶ cells/ml. To prevent adhesion, wells were precoated with 1 mg of polyhema in 100% ethanol at 37°C overnight until dry and then rinsed 3 times with media before use. The following components were added to each well: 1 ml of the media, 100 μl of buffer (PBS plus 0.02% (wt/vol) sodium azide) with or without control IgG antibody, or anti-HIP protein at 25 μg/ml and 7 x 10⁵ cells (200 μl). Plates were incubated for 3 h at 37°C on a rotary shaker at 1700 φm, Afterward, cellular aggregation was viewed and photographed with a Nikon Diaphot inverted microscope using phase microscopy.

5.2.2 RESULTS

5.2.2.1 SUBCELLULAR DISTRIBUTION OF HIP An antibody was generated to a synthetic peptide sequence predicted from the full-length

HIP cDNA sequence. The sequence was predicted to be hydrophilic and likely to be exposed on the external surface of the intact protein. The antibodies routinely used for the studies described below were affinity purified on a column composed of the BSA-conjugated HIP peptide linked to agarose. These antibodies reacted primarily with a protein with the _r of 24,000 as estimated by SDS-PAGE and Western blotting The molecular weight was similar to that observed for HIP protein expressed by 3T3 cells transfected with full-length HIP cDNA.

Subcellular fractionation was used as an initial step to partially purify HIP for subsequent analytical studies. Fractionation of RL95 cells and subsequent Western blot analysis determined that HIP was most highly enriched in the 100,000 x g pellet; however, HIP was detected in other particulate fractions as well. Lower molecular weight components immunologically related to HIP were detected in the 1000 x g/20 min and 10,000 x g/20 min particulate fraction. These components were presumed to be partially degraded forms of HIP. In contrast, HIP appeared to be quantitatively depleted from the 100,000 x g soluble fraction. A similar distribution of HIP was observed in JAR and HEC-la cells, human trophoblastic and uterine adenocarcinoma cell lines, respectively. The high speed particulate fraction was used further as the most convenient source of HIP.

5.2.2.2 NACL SOLUBILIZATION OF HIP The 100,000 x g particulate fraction was subjected to incubations with increasing concentrations of NaCl and then analyzed by Western blot analysis At NaCl concentrations greater than 0.8 M, HIP was eluted from the membrane fraction into a 100,000 x g soluble fraction. Analyses of the corresponding supematants for each salt wash indicated that HIP is partially eluted with 0.4 M NaCl but is completely eluted with NaCl concentrations of 0.8 M or greater. The solubilization of HIP with salt indicates that HIP was peripherally associated with the particulate fractions of cells. Conditioned media from RL95 cell were centrifuged at 100,000 x g, the supernatant was trichloroacetic acid-precipitated, and equal portions of all fractions were analyzed for the presence of HIP by Western blot analysis. HIP was not detected in secretions from RL95 cells, indicating that this protein was not secreted or released from RL95 cells to a significant extent.

5.2.2.3 HIP BINDS HP

The 0.8 M NaCl eluate from the 100,000 x g particulate fraction was diluted to 0.15 M

NaCl and incubated with Hp-agarose. Elution of the Hp-agarose with increasing concentrations of NaCl demonstrated that HIP bound to Hp and was released at NaCl concentrations greater than 0.75 M. Staining with Coomassie Blue indicated that multiple proteins were present in the Hp-binding fractions. Therefore, a HP overlay assay was employed to demonstrate the ability of HIP to bind HP directly (Raboudi et al, 1992). Samples were subjected to two-dimensional SDS-PAGE, transferred to nitrocellulose, and sequentially probed with I-HP and anti-HIP. Profiles of total proteins were visualized in a parallel sample by silver staining. Many proteins co-isolated with HIP by this procedure, however, only a subset of these proteins retained the

125 ability to bind I-HP. Collectively, the binding and the elution of HIP from Hp-agarose and the

125 coincident binding of I-HP and anti-HIP indicated that HIP is a HP -binding protein.

5.2.2.4 CELL SURFACE LOCALIZATION OF HIP

Anti-HIP was used to determine if this protein was expressed on the external surface of intact cells. Initially, concentration dependence and saturability of anti-HIP binding was examined. The binding of anti-HIP to intact RL95 cells was both specific and saturable as compared with binding of nonimmune rabbit IgG (FIG. 2A and FIG. 2B). Furthermore, when anti-HIP protein was pre-absorbed with peptide affinity matrix, its binding was reduced to the level observed with nonimmune rabbit IgG. Next, anti-HIP was used to examine the distribution of this protein on HEC-la cell surfaces. Immunostaining of methanol-permeabilized, paraformaldehy de-fixed HEC-la cells with anti-cytokeratins demonstrated a strong positive signal. In contrast, fixed nonpermeabilized cells displayed only background staining comparable with that observed when primary antibody was omitted. Staining of fixed, nonpermeabilized cells with anti-HIP was uniformly distributed on the surfaces of all cells in these cultures including points of cell-cell contact. Similar results were obtained with RL95 cells. Collectively, these data indicated that reactivity with anti-HIP was reflective of cell surface staining and not due to permeabilization in human uterine epithelial cell lines. It was further reasoned that if HIP was on RL95 cell surfaces then non-fixed, single cell suspensions of living RL95 cells could be aggregated by anti-HIP. Incubation of single cell suspensions of RL95 cells with anti-HIP greatly enhanced cell aggregation. Parallel controls, including PBS, PBS containing 0.02% sodium azide and an antibody to the cytoplasmic tail of the mucin, MUC1 (Surveyor et al, 1995), did not enhance RL95 cell-cell aggregation. Collectively, the data indicated that HIP is located on the extracellular surface of the plasma membrane of human uterine epithelial cell lines.

Studies also were performed to determine if HIP was expressed by other human uterine epithelial cell lines as well as normal human uterine epithelium in situ. Western blots of several human uterine epithelial cell lines as well as human endometrium displayed a prominent band corresponding to the molecular weight of HIP. A 1.3-kilobase (kb) transcript was detected in all three cell lines by Northern analyses using HIP cDNA as a probe (Liu et al, 1996).

5.2.2.5 HIP EXPRESSION IN HUMAN ENDOMETRIUM Expression and localization of HIP was examined in methanol-fixed frozen sections of human endometrium taken at various stages throughout the menstmal cycle. In all cases, strong reactivity of lumenal and glandular epithelia was detected. Through the proliferative and until post-ovulatory day 7 of the cycle, HIP reactivity was not detected in underlying stroma cells. Nonimmune IgG failed to react with these tissues. Furthermore, the epithelial identity of the HIP-positive cells was confirmed by demonstration of reactivity with antisera to cytokeratins and

Muc-1 in serial sections. Strong reactivity was detected at both the apical and basal aspects of these cells. Some variation in the intensity of signal between these glandular structures was noted. It is unclear if this variation reflects differences between glands or regional differences in HIP expression of individual glands that normally extend from the uterine lumen (functionalis) to deep within the endometrium (basalis). By post-ovulatory day 13, additional staining for HIP was detected within the underlying stroma. As expected, the underlying stroma extracellular matrix also displayed strong expression of the decidual marker, laminin (Kisalus et al, 1987), at this time. In contrast, laminin expression was confined to basal lamina in stromal tissue of late proliferative stage uteri. The heparan sulfate proteoglycan, perlecan, also has been reported to be expressed by decidualizing stroma cells (Kisalus and Herr, 1988); however, stromal staining for perlecan was much less intense than that of basal lamina. As mentioned above, HIP was not detected in stromal cells through the entire proliferative phase of the cycle. These data demonstrated that HIP is a protein normally expressed by uterine epithelia. 5.2.3 DISCUSSION

The predicted pi of HIP, >10, is consistent with its behavior on isoelectric focusing gels. Alternatively, HIP may be post-translationally modified. No consensus sites for glycosylation are indicated by the predicted sequence; however, other modifications are possible. Subcellular fractionation studies indicate that HIP is most highly enriched in the high speed particulate fraction and is quantitatively depleted from the high speed supernatant, i.e., cytosolic fraction. The inventors have detected various plasma membrane markers in this fraction including Na⁺/K⁺-ATPase and radioiodinated cell surface components; however, rearrangement of peripheral membrane components like HIP may occur during such fractionation making inteφretation of subcellular locale by this approach problematic. The ability of NaCl to release

HIP from the particulate fraction is consistent with the lack of a potential membrane spanning domain in the predicted sequence of HIP and demonstrates that HIP is a peripheral membrane protein. Digestion of membranes with a mixture of Hp/HS lyases did not release HIP into the 100,000 x g soluble fraction. This suggests that HIP is not retained by membrane-bound HSPGs. Therefore, it is possible that other membrane components bind and retain HIP. Alternatively, it is possible that HIP binds to a region of HS close to the protein core and protects HS from enzymatic digestion. Characterization of the HIP-binding sites is necessary to define the nature of the HIP-membrane interaction.

Several lines of evidence indicate that HIP is displayed on cell surfaces. Antibodies to this protein bind specifically and in a saturable manner to intact RL95 cells under conditions where endocytosis should not occur. Assuming a 1 : 1 stoichiometry of IgG binding to HIP and protein A to antibody, it can be calculated that there is an average of approximately 1.5 x 10 molecules of HIP displayed on the surface of each RL95 cell. If each IgG binds to two HIP molecules and each protein A tetramer binds four IgG molecules then this estimate may be as high as 1.2 x 10 HIP molecules per cell surface. In either case, these numbers are well below the number of [ HjHP-binding sites (9 x 10 ) previously determined for RL95 cells (Raboudi et al, 1992). Consequently, even given potential inaccuracies in both estimates, it seems that HIP can only be one of multiple cell surface Hp/HS-binding proteins displayed on RL95 cell surfaces. It is possible that many HIP molecules are occupied by HS at the cell surface and masked from antibody binding. HS lyase pretreatment of cells did not expose additional anti-HIP-binding sites; however, if, as discussed above, HIP binding "protects" HS chains from digestion then HS lyases might not be expected to expose more HIP.

Antibodies to HIP also display staining patterns on intact RL95 cells that are consistent with those of cell surface components, e.g. enrichment at cell peripheries and regions of cell-cell contact. Similar patterns of immunoreactivity with anti-HIP are detected on human trophoblastic and breast cancer cell lines. Furthermore, these same antibodies specifically aggregate RL95 cells in suspension, a property expected for antibodies reacting with epitopes displayed on the cell surface. Studies with an impermeant chemical cross-linking reagents destroyed antibody reactivity with HIP, but larger cell associated bands were not observed. Collectively, these data strongly argue that at least a fraction of the population of HIP is displayed on RL95 cell surfaces where these proteins may directly participate in Hp/HS binding.

HIP is detected in several human uterine epithelial cell lines and in human endometrium by Western blotting of total protein extracts. Moreover, anti-HIP strongly reacts with uterine epithelial cells in sections of human endometrium through post-ovulatory day 7 of the cycle. By post-ovulatory day 13, HIP is also detected in the predecidual cells of the uterine stroma. The

HSPG, perlecan, is expressed by human decidual cells (Kisalus and Herr, 1988). It is possible that HS chains of perlecan also serve as ligands for HIP in basal lamina and in the decidual extracellular matrix. In any event, these observations indicate that HIP is expressed by normal human endometrium. Potential functions could involve binding to basal lamina or intercellular HSPGs expressed by uterine epithelia or HSPGs expressed by blastocysts during implantation.

5.3 EXAMPLE 3 - METHODS FOR MODULATING BLOOD COAGULATION

The glycosaminoglycans Hp and HS appear to exert their biological functions through regulatory interactions with specific target proteins. Because of the microheterogeneity of the sulfate substitutions of the polysaccharides it has been speculated that specific monosaccharide sequences in Hp bind to selective domains in target proteins (Lindahl et al, 1980; Esmon and Owen, 1981; Casu et al, 1981; Esmon et al, 1982; Tollefsen 1989). A Hp hexasaccharide can theoretically occur in more than 10⁵ different stmctural forms which allows for sufficient stmctural variations expected for an information molecule. On the other hand, the biosynthesis of a structurally heterogeneous glycosaminoglycan seems to result from a series of incomplete enzymatic reactions raising concern about the genetic control of glycosaminoglycan fine structure (Kjellen and Lindahl, 1991). At least one protein, antithrombin-3 (AT-III), binds to a specific sequence present in some Hp molecules but not in others (Lindahl et al, 1980; Esmon and Owen, 1981; Casu et al, 1981). Other proteins such as the FGFs bind to Hp with suφrisingly high affinity; however, attempts to identify specific binding sites in Hp for different growth factors have given inconclusive and sometimes conflicting results. This example describes the synthesis of a peptide derived from a cell surface Hp/HS interacting protein (HIP), which binds to a Hp sequence that is similar or identical to that specifically recognized by AT-III. These data suggest that HIP may play a physiological role as a modulator of blood coagulation.

5.3.1 MATERIALS AND METHODS

5.3.1.1 MATERIALS

[ H]Hp (0.44 mCi/mg) was purchased from DuPont-New England Nuclear (Wilmington, DE). Dulbecco's phosphate buffered saline (PBS) was obtained from GIBCO (Grand Island,

NY). Imject activated immunogen conjugation kits were purchased from Pierce (Rockford, IL). COATEST Hp and COATEST Antithrombin kits were purchased from Helena Laboratories (Beaumont, TX). Antithrombin-3 (AT-III)-agarose, sodium chloride, potassium chloride, and sodium phosphate were purchased from Sigma Chemical Co. (St. Louis, MO). All chemicals used were reagent grade or better.

5.3.1.2 PEPTIDE SYNTHESIS AND HIP PEPTIDE AFFINITY MATRIX

The synthetic HIP peptide, derived from a segment of the predicted amino acid sequence of HIP (Liu et al, 1996), CRPKAKAKAKAKDQTK (SEQ ID NO: 10), was synthesized on a Vega 250 peptide synthesizer using Fmoc methodology (Chang and Meienhofer, 1978). This synthetic peptide was conjugated to maleimide-activated BSA (Pierce, Rockford, IL) through the sulfhydryl group of cysteine in the peptide following the coupling procedures provided by the manufacturer. HIP peptide affinity matrix was formed by cross-linking the BAS-conjugated HIP peptide to cyanogen bromide activated Sepharose (Sigma Chemical Co., St. Louis, MO) in the presence of JV-acetylated-Hp. Inclusion of acetylated-Hp was adopted to produce a more stable affinity matrix by shielding the Hp binding sites of the HIP peptide-BSA complex from cross- linking to the Sepharose beads. Acetylation of Hp was performed following the method previously described (Hook et al, 1976).

5.3.1.3 HIP PEPTIDE AFFINITY CHROMATOGRAPHY

The HIP peptide affinity matrix was packed into a one ml FPLC column (Pharmacia, Sweden) and conditioned by repeated washing in a lO mM phosphate buffer pH 7.4 with a gradient of 0.15-4.0 M NaCl. Commercial [³H]Hp was resuspended in 0.15 M NaCl-lO mM phosphate, pH 7.4 and loaded into the column. The column was eluted with a linear gradient from 0.15 M to 4.0 M NaCl in a 10 mM phosphate buffer pH 7.4 at a rate of 0.5 ml/min.

Fractions of 0.5 ml were collected and aliquots were analyzed for radioactivity. Large scale fractions were performed on a 5 ml HIP peptide affinity matrix. [ H]Hp resuspended in 0.15 M NaCl-lO mM phosphate, pH 7.4 was loaded into the column. The column was washed sequentially with 0.15 M NaCl-lO mM phosphate, pH 7.4 (run-through Hp, RT-HIP-Hp) and 0.45 M NaCl- 10 mM phosphate, pH 7.4 (low affinity Hp, LA-HIP-Hp) extensively. Finally, the column was eluted with 3.0 M NaCl- 10 mM phosphate, pH 7.4 (high affinity Hp, HA-HIP-Hp); radioactivity in an aliquot (50 μl) from each fraction (1 ml/fraction) was determined by liquid scintillation counting. Each of these affinity classes of Hp was collected, extensively dialyzed against doubly-distilled, deionized H₂O), lyophilized and used for further analysis. Hp oligosaccharides were obtained by partial deaminative cleavage of the polysaccharide with nitrous acid at pH 1.5 (Shively and Conrad 1976). The reaction was allowed to proceed for

10 min and interrupted by the addition of ammonium sulfamate in a four-fold molar excess over nitrite. The resulting mixture was separated by gel permeation chromatography on a 1.5 x 170 cm Sephadex G-50 (superfine) column and eluted with 0.5 M NH₄HNO₃ at a flow rate of 6.5 ml/hr. Fractions of 2.0 ml were collected and uronic acid concentration of individual fraction was determined by the carbazole method (Bitter and Muir, 1962). The peaks corresponding to terra-, hexa-, octa- and deca-saccharides, as determined by elution positions relative to an internal [ H]-octasaccharide standard, were pooled and reductively labeled with [ H]NaBH₄. Each size- fractioned mixture was repurified by a second round of gel permeation chromatography performed under the identical conditions to eliminate possible carryover from adjacent peaks. The eluted fractions were analyzed for radioactivity and peak fractions representing size- homogenous oligosaccharides were collected. Estimated specific activity of the radiolabeled oligosaccharides was 2 to 20 x 10 dpm/nmol. The [ H] -labeled Hp oligosaccharides were loaded onto the HIP affinity matrix and the column was extensively washed with 1 M NaCl-lO mM phosphate, pH 7.4. Bound Hp oligosaccharides were eluted with 3 M

NaCl-lO mM phosphate, pH 7.4. Radioactivity in each fraction was determined by liquid scintillation counting.

5.3.1.4 DETERMINATION OF SIZE AND CHARGE DENSITY OF HP SPECIES The size of the Hp fractions was analyzed on a 1 x 30 cm Superose 12 column

(Pharmacia, Sweden) equilibrated and eluted with 2 M guanidine hydrochloride, 0.01%) (wt vol) octylglucoside, 20 mM Tris-acetate, pH 7.0, and 0.02% (wt/vol) sodium azide at a flow rate of 0.7 ml/min at room temperature with a back pressure of approximately 300 psi. Fractions were collected every 0.5 min and the radioactivity was determined by liquid scintillation counting. The charge density of Hp fractions was analyzed by anion exchange liquid chromatography on a 0.5 x 5 cm Mono Q column (Pharmacia, Sweden). Samples were loaded into a column equilibrated with 0.5 M urea, 0.01% (wt/vol) octyl glucoside, 20 mM Tris-acetate, pH 7.0, and 0.02% (wt vol) sodium azide, and the column was eluted with a gradient of 0 to 4 M NaCl in the same buffer. The column was pumped at a flow rate of 1 ml/min at room temperature with a back pressure of approximately 450 psi. Fractions were collected every

0.5 min and the radioactivity determined by liquid scintillation counting.

5.3.1.5 ANTITHROMBIN 3 AND BFGF AFFINITY CHROMATOGRAPHY

AT-III-agarose (Sigma, St. Louis, MO) was resuspended in 10 mM phosphate, pH 7.4, packed into a 1 ml FPLC column, and conditioned by repeated gradient washing of 0 to 2.0 M

NaCl- 10 mM phosphate, pH 7.4. Unfractionated [³H]Hp or HA-HIP-Hp were loaded onto the AT-III-agarose column. Then the column was washed with 10 mM phosphate, pH 7.4 and eluted with a gradient of 0 to 2.0 M NaCl at a flow rate of 0.5 ml/min. Fractions of 0.5 ml were collected and analyzed for radioactivity. Recombinant bFGF was coupled to CNBr-activated Sepharose 4B in the presence of a five-fold molar excess of JV-acetylated Hp to protect potential Hp-binding sites as described (Hook et al, 1976). The bFGF affinity chromatography of uunnfrfraaccttiioonnaatteedd [[³HH]]HHpp aanndd HHAA--HHIIPP--HHpp wwaass performed using the procedures described above for affinity chromatography on AT-III-agarose.

5.3.1.6 COAGULATION ASSAYS

COATEST Hp (for determination of Hp in plasma), and COATEST Antithrombin (for determination of AT-III-Hp cofactor activity), were employed to examine the effect of HIP peptide on the anticoagulatory activity of Hp and AT-III, respectively. Coagulation assays were performed following the procedures provided by the manufacturer. The principle for determination of Hp concentration in plasma is as follows:

( 1 ) Hp + AT-III (excess) → [Hp-AT-III]

(2) [Hp-AT-III] + FXa (excess) → [Hp-AT-III-FXa] + FXa (remaining), and

(3) S-2222 + FXa (remaining) → Peptide +pNA (yellow)

S-2222 (Bz-Ile-Glu-Gly-Arg-pNA) is a chromogenic substrate susceptible to FXa, and pNA stands for ^-nitroaniline. Briefly, standard samples including normal human plasma, 0.1 U/ml of AT-III and varying concentrations of Hp from 0.01 to 0.07 IU/ml (Elkins-Sinn. Inc., Cherry Hill, NJ), were incubated at 37°C for 3 to 4 min, then FXa (0.355 nkat) kept room temperature was added and mixed well. The mixture was incubated at 37°C for exactly 3 min. The reaction was stopped by adding 20%> (vol./vol.) acetic acid. To test effect of HIP peptide, different concentrations of HIP peptide were added to the standard sample containing 0.07 IU/ml of Hp, and the Hp activity was analyzed as described above. The principle for determination of AT-III-Hp cofactor activity in plasma is as follows:

( 1 ) AT-III + Hp (excess) → [AT-III-Hp] (2) [AT-III-Hp] + Thrombin (excess) → [AT-III-Hp-Thrombin] + Thrombin

(residual), and

(3) S-2238 + Thrombin (residual) → Peptide + pNA (yellow)

S-2238 (H-D-Phe-pip-Arg-pNA) is a chromogenic substrate susceptible to thrombin.

Briefly, standard samples containing normal human plasma at different dilutions (creating varying concentrations of plasma AT-III) were incubated, in the presence of Hp, at 37°C for 3 to 6 min then, thrombin was added (1.77 nkat), mixed well and the solution further incubated at 37°C for 30 sec. The S-22238 substrate was added and incubated at 37°C for 30 sec. The reaction was stopped with 20% (vol/vol) acetic acid. To test the effect of HIP peptide, different concentrations of HIP peptide were added to the standard sample containing 100% normal AT-III concentration ([Hp] « 0.05 IU/ml), and the assay was carried out as described above.

5.3.1.7 DETERMINATION OF ANTICOAGULANT ACTIVITIES OF FRACTIONATED HP

Hp (1,000 IU/ml, Elkins-Sinn. Inc., Cherry Hill, NJ) was fractionated in a HIP peptide affinity column using stepwise elution as described above resulting in three affinity classes of Hp: RT-HIP-Hp, mn through Hp from HIP-peptide affinity chromatography; LA-HIP-Hp, low affinity (0.45 M NaCl eluate) Hp from HIP-peptide affinity chromatography; and HA-HIP-Hp, high affinity (3.0 M NaCl eluate) Hp from HIP-peptide affinity chromatography. The anticoagulant activity of each Hp fraction was determined by FXa-dependent coagulation assay (COATEST Hp) as described above. Comparison of specific activities was based upon the uronic acid content determined by carbazole assay (Bitter and Muir 1962).

5.3.2 RESULTS

5.3.2.1 A SUBSET OF HP CHAINS BIND TO HIP PEPTIDE WITH HIGH AFFINITY

Commercial [ H]Hp was fractionated by HIP peptide affinity chromatography using salt gradient elution. Most [ H]Hp appeared in the run-through fractions (0.15 M NaCl-PBS)

(RT-HIP-Hp) while another portion of [³H]Hp bound to the column and eluted with 0.3 M

NaCl-PBS elution (LA-HIP-Hp). A small percentage of [³H]Hp (« 1 - 5% of total [³H]Hp) bound to HIP peptide affinity matrix tightly and eluted at 2.2 M NaCl (HA-HIP-Hp). Large scale preparations of three affinity classes of Hp for further study were obtained using a stepwise elution procedure with 0.15 M, 0.45 M, and 3.0 M NaCl. Re-chromatography of each affinity class of [ H]Hp, after dialysis resulted in near quantitative elution (>90%) at the salt concentration used for the initial fractionation. These results suggest that HIP-peptide binds to a particular stmctural motif present only in a subset of Hp molecules. To determine the minimum size of the stmctural motif recognized by HIP, the ability of [ H]Hp oligosaccharides of different sizes to bind to the HIP peptide-affinity matrix was studied. A small percentage of both octa- and deca-saccharides bound to HIP peptide affinity matrix and eluted at >1.0 M NaCl whereas no high affinity binding of tetra- and hexa-saccharides to HIP peptide affinity matrix was detected. Since in generating the radiolabeled oligosaccharides, the glucosamine residue at the reducing end is converted into an anhydromanitol, the inventors conclude that the motif recognized by HIP is contained in a hexa- or hepta-saccharide of intact Hp molecules.

5.3.2.2 HA-HIP-HP MOLECULAR WEIGHT AND NEGATIVE CHARGE DENSITY

To determine the average size and charge density of different HIP-binding Hp fractions, each affinity class of Hp was subjected to gel permeation and ion exchange liquid chromatography. HA-HIP-Hp has the largest median size when analyzed on a column of

Superose 2, LA-HIP-Hp has an intermediate median size, and RT-HIP-Hp is enriched in smaller size classes of Hp chains. Comparison of elution positions with polysaccharide standards indicate that the median molecular weight (MW) of HA-HIP-Hp is about 40,000, i.e., approximately four times greater than that of unfractionated Hp (median MW of 10,000). Thus, HA-HIP-Hp is enriched in larger species of Hp chains. Results of anion exchange chromatography indicate that HA-HIP-Hp also has a slightly higher average negative charge density than either LA-HIP-Hp or unfractionated Hp, indicating that the Hp chains selectively recognized by HIP peptide may have an unusually high sulfate density. Collectively, these results indicate that HA-HIP-Hp chains differ, both in terms of size and charge, from unfractionated and other HIP peptide affinity classes of Hp.

5.3.2.3 HA-HIP-HP IS HIGHLY ENRICHED IN ANTICOAGULANT ACTIVITY

Since HIP peptide recognizes a specific stmctural motif in Hp, it was of interest to determine if similar stmctural features were required for Hp binding to other Hp-binding proteins such as AT-III or basic fibroblast growth factor (bFGF) (Maccarana et al, 1993; Faham et al,

1996; Lindahl et al, 1980; Casu et al, 1981; Afha et al, 1984). Therefore, HA-HIP-Hp was subjected to both AT-III and bFGF affinity chromatography. HA-HIP-Hp was not enriched in species that specifically bound to bFGF affinity matrix and was eluted as a single homogeneous peak at 0.86 M NaCl. AT-III affinity chromatography of unfractionated [³H]Hp resulted in several distinct fractions, i.e., one eluting at the run-through and another eluting at 0.6 M NaCl with two smaller peaks at 0.4 M NaCl and 0.86 M NaCl. Thus HA-HIP-Hp is highly enriched in Hp species that bind to AT-III with highest affinity. These observations suggest that the same or similar Hp motif(s) are recognized by both HIP peptide and AT-III. Therefore, the stmctural requirements for high affinity Hp binding to HIP peptide and bFGF appear to be distinct. Since the anticoagulant activity of Hp primarily is exerted via binding to and activation of

AT-III, it was of interest to determine the specific anticoagulant activity of Hp fractions obtained by HIP-peptide affinity chromatography. To this end, the inventors used a FXa-dependent coagulation assay kit. As shown in FIG. 3, HA-HIP-Hp displayed the highest anticoagulant activity and was at least 10-fold more potent than unfractionated Hp on a mass basis. Since the median size of HA-HIP-Hp is about four times that of unfractionated Hp, anticoagulant activity of HA-HIP-Hp is at least 40-fold higher on a molar basis. LA-HIP-Hp also had higher anticoagulant activity than that displayed by unfractionated Hp, but lower than that of HA-HIP-Hp. RT-HIP-Hp had marginal anticoagulant activity. Collectively, these results demonstrate that HA-HIP-Hp contains Hp molecules with exceptionally high anticoagulant activity.

5.3.2.4 HIP ANTAGONIZES AT-III:HP ACTIONS ON BLOOD COAGULATION

Based on the above observations, it was hypothesized that HIP peptide competes with AT-III for Hp binding and might modulate the anticoagulant activity of the polysaccharide. To test this, both factor Xa (FXa) and thrombin-dependent coagulation assays were employed. As shown in FIG. 4 A, the addition of HIP peptide at a concentration of 150 μg/ml in the presence of the AT-III Hp complex restored FXa-dependent coagulation activity. And as shown in FIG. 4B, the addition of HIP peptide at a concentration of 900 μg/ml in the presence of the AT-III Hp complex restored thrombin-dependent coagulation activity.

5.4 EXAMPLE 4 - FURTHER STUDIES UTILIZING HIP

5.4.1. TREATMENT OF HEPARIN INDUCED THROMBOCYTOPENIA

Platelet Factor IV (PF4) is a platelet granular protein. When heparin binds to PF4, the heparin/PF4 complex forms a potential neoantigen. "Heparin" is actually a mixture of sulfated mucyl polysaccharides, representing thousands of different sizes of polymers with different degrees of sulfation. In several percent of all people who receive heparin, between about 1% and about 3%, the heparin/PF4 complex is treated as a neoantigen, and antibodies are formed against the complex. The antigen binding regions of the IgG interact with the heparin/PF4 neoantigen, either on the platelet surface or in the vicinity of the platelet surface, and the F_c portion of the IgG interacts with neighboring F_c receptors on platelets. This interaction with the platelet surface receptor activates the platelets, which through a variety of signaling mechanisms leads ultimately to platelet aggregation.

The aggregation of platelets leads to two problems. First, the aggregated platelets are not effective in participating in the coagulation cascade, thus leading to reduced coagulation and excessive bleeding. Second, the platelets clump in microcirculation, which leads to an increased thrombotic risk, i.e. increased risk of heart attacks, stroke, and venous thrombocytopenia. As heparin is usually administered to prevent these thrombotic risks, it is a paradoxical and dangerous, yet common phenomenon. A heparin species, component or molecule that does not interact well with platelet factor IV, but that still has the capability of binding to and activating antithrombin-3, would work better than standard heparin in terms of potentiating anticoagulation without the thrombotic complications.

The HIP compositions of the present invention are used to isolate a heparin species, component or molecule that is capable of interacting with AT3, causing it to become a very effective anticoagulant, but that does not bind effectively to PF4. This can be accomplished in two ways. First, the heparin fraction that interacts with PF4 can be removed from cmde heparin preparations in vitro by affinity chromatography using the HIP attached to a column matrix, either as a pass through or using differential elution from the affinity column. The heparin preparation is passed over the HIP affinity column and the PF4 heparin binding fraction or component binds to the HIP. The remaining heparin sample is administered for its antithrombotic effects ~ the anticoagulant aspects without the thrombotic risk. This aspect encompasses in vitro preparative procedures. Second, the HIP can be co-administered with the heparin composition, in an effective ratio, in vivo to neutralize the species of heparin that interacts with PF4.

After elution from or passing through the affinity column, the differential binding of the heparin species or component to PF4 and AT3 is determined. In a preferred aspect, isolated AT3 and PF4 are attached to microtiter plates, the heparin from the affinity column is labeled, either prior to running the column or after the column purification, and serial dilutions are contacted with the AT3 or PF4 on the microtiter plates. The heparin fraction that binds to either AT3 and/or PF4 is detected. Furthermore, the labeled heparin is checked for binding to antibodies from people with heparin-induced thrombocytopenia. The heparin species isolated is also tested for the ability to activate AT3 and prevent coagulation by using a standard coagulation test as described herein.

To prove the clinical effectiveness of the heparin species to prevent heparin-induced thrombocytopenia, while activating AT3 and inhibiting blood clotting, the binding of the heparin species to an anti-heparin/PF4 antibody from a patient with heparin-induced thrombocytopenia is tested. IgG is isolated from people with heparin-induced thrombocytopenia and added to the heparin species under conditions effective for binding of the anti-heparin/PF4 antibody to a control heparin composition, and the binding of the antibody to the heparin species is detected, for example by a detectable radio- or fluorescent label. The lack of interaction of the heparin species and the anti-heparin/PF4 antibody is indicative of a clinically effective heparin species or component.

A variety of different heparin types and fractions are tested, both unfractionated heparin, the most commonly used variety, or low molecular weight heparin. Although the only heparins that are presently used clinically are bovine and porcine, all types of presently known animal heparins are evaluated.

5.4.1.1. BINDING OF HEPARIN SPECIES WITH ANTIBODIES TO PF4/HEPARIN COMPLEX

Clinical heparin preparations were fractionated by HIP peptide affinity chromatography to obtain low affinity, high affinity or unfractionated heparin. A commercially available assay kit for the detection of antibodies to heparimplatelet factor-4 complexes (HIT assay; American

Bioproducts Company, Parsippany, NJ) was used. Soluble platelet factor-4 :heparin complexes were used to compete for antibody binding to platelet factor-4 :heparin complexes isolated from patients displaying heparin-induced thrombocytopenia which were linked to the ELISA plate. Soluble heparin at concentrations of 1-100 ng per ml was mixed with 0.1 μg of purified platelet factor-4 and the standard human serum containing antibodies to platelet factor-4 :heparin complexes provided with the kit. All other aspects of the assay were performed exactly following the manufacturer's instmctions. The binding was compared to the antibody binding observed in the absence of soluble competitor complexes.

The results show that all three heparin preparations are equally active in competing for antibody binding to a solid phase platelet factor-4: heparin complex. Therefore, high and low affinity heparin preparations are not more reactive than bulk heparin with antibodies to platelet factor-4 :heparin complexes elicited in patients with heparin-induced thrombocytopenia. As shown herein, both the low and high affinity fractions are at least 10-fold enriched for anticoagulant activity. Thus similar anticoagulant benefit can be achieved with low and high affinity heparins with greatly reduced reactivity with antibodies typically elicited in heparin- induced thrombocytopenia.

5.4.1.2. HIGH AND LOW AFFINITY HEPARINS IN VIVO

High and low affinity heparins fractionated by HIP peptide affinity chromatography are more effective anticoagulants in vivo. Further tests of the efficacy of high and low affinity heparins include injection into an animal model, e.g., rabbits, and subsequent determination of blood clotting parameters using commercially-available assays and semm obtained at different intervals following these injections. In parallel, serum heparin concentrations are also determined using commercially available assays.

5.4.1.3. HIGH AND Low AFFINITY HEPARINS IN HUMANS

High and low affinity heparins fractionated by HIP peptide affinity chromatography are also more effective anticoagulants in humans. High and low affinity heparins represent subsets of FDA-approved heparin already in routine clinical use. Following satisfactory results in the animal trials, high and low affinity heparins are tested similarly in humans using clinical in vitro assays for semm heparin concentrations, blood clotting parameters and routine patient monitoring for signs of heparin-induced thrombocytopenia. 5.4.2. PROTAMINE REPLACEMENT

The HIP compositions of the present invention are also used in protamine like heparin neutralization. In a preferred aspect this is an ex vivo situation that is used after various extracaφorial processes, including, but not limited to, cardiovascular surgery, hemodialysis, and cardiac bypass surgery. Any indication wherein protamine is administered, including heparin overdose, is contemplated for the use of the instant HIP compositions.

As protamine is known not to work very well in neutralizing the low molecular weight heparins, which are becoming more and more popular, the instant HIP compositions are expected to find widespread acceptance. The binding of low molecular weight heparin species or components is tested versus unfractionated heparin. As the "low molecular weight" heparins are expected to continue to get smaller in the near future, a number of low molecular weight fractions are tested, from heparins having a molecular weight of about 1000 to 2000 Daltons, to heparins as small as a pentasaccharide, which is the critical minimal heparin unit for AT3 binding. In other words, the instant HIP compositions are tested for their relative capacity to bind unfractionated, low molecular weight, and pentasaccharide heparins.

The preferred HIP species have both in vivo and ex vivo uses. Preferred ex vivo uses include, but are not limited to, putting a HIP affinity column in the line that returns blood to patients, at the end of bypass surgery or at the end of hemodialysis. The in vivo evaluation of the HIP species requires both toxicity studies in animals and half-life studies. These animal studies can be performed in mice, rats, rabbits, pigs or primates. The animal studies are conducted following FDA guidelines, and are generally well known to those of skill in the art.

As an example of in vivo animal testing for half-life determination, a small number of monkeys are injected with a labeled HIP composition, and the half-life of the HIP composition is determined. Half-lives of minutes, to 1-2 hours is preferred in certain aspects of the invention, however, HIP compositions having a half-life of 3, 4 or 6 hours, or even longer, are expected to find utility in particular aspects of the invention, for example in subjects having with liver failure and renal failure.

Once the half-life study is complete, toxicity and dose-response studies are performed.

Approximately six monkeys and rabbits are evaluated for toxicity of the HIP composition by injecting the animals with various doses of the HIP compositions. A variety of blood tests are conducted, including clotting studies such as prothrombin time and partial thromboplastin time, CBC, platelet counts, electrolyte analysis and semm chemistries, including liver function and renal function studies.

5.4.2.1. HIP AND HIP PEPTIDE NEUTRALIZE HEPARIN

As shown herein, both HIP peptide and HIP are able to neutralize anticoagulant species of heparin. Therefore, both HIP and HIP peptide are useful alternatives for protamine-based therapies currently used to neutralize heparin and low molecular weight heparins. This is tested by injecting animals with heparin and various doses of HIP or HIP peptide, and monitoring the effects on blood clotting parameters measured as described above. HIP and HIP peptide are contemplated for use to neutralize heparin in semm and restore normal semm clotting parameters. The HIP peptide had no obvious side effects when injected intravenously into mice at levels up to 40 mg/kg body weight.

5.4.2.2. HIP AND HIP PEPTIDE NEUTRALIZE Low MOLECULAR WEIGHT HEPARINS IN VITRO

HIP and HIP peptide are tested for their ability to neutralize low molecular weight heparin in clinical in vitro assays using exactly the same procedures described herein above. In this case, clinically-used low molecular weight heparin preparations are substituted for heparin normally used in these assays. These studies demonstrate that HIP and HIP peptide offer unique therapies for neutralization of low molecular weight heparins for which protamine is not effective.

5.4.2.3. HIP AND HIP PEPTIDE NEUTRALIZE LOW MOLECULAR WEIGHT HEPARINS IN VIVO

The efficacy of HIP and HIP peptide in neutralizing low molecular weight heparins is confirmed in animal models as described above by injecting animals with clinically-used doses of low molecular weight heparin sufficient to reduce serum clotting parameters using the in vitro assays described above. Some groups of animals are also injected with doses of HIP or HIP peptide up to 40 mg/kg body weight to demonstrate neutralization of the anticoagulant effect of low molecular weight heparin. Following the successful outcome of these studies, similar tests are performed in humans. All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Texas A&M University System

(B) STREET: 310 Wisenba er

(C) CITY: College Station

(D) STATE : Texas

(E) COUNTRY: USA

(F) POSTAL CODE (ZIP) : 77843-3369

(ii) TITLE OF INVENTION: HEPARAN SULFATE/HEPARIN INTERACTING PROTEIN COMPOSITIONS AND METHODS OF USE

(iii) NUMBER OF SEQUENCES: 10

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO)

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/810,609

(B) FILING DATE: 28-FEB-1997

(2) INFORMATION FOR SEQ ID NO : 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 634 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 145

(D) OTHER INFORMATION : /note= "Y = C or T"

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 28..504

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 40

(D) OTHER INFORMATION : /note= "Xaa = Leu"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1: TCGGGAGCCG CGGCTTATGG TGCAGAC ATG GCC AAG TCC AAG AAC CAC ACC 51

Met Ala Lys Ser Lys Asn His Thr 1 5

ACA CAC AAC CAG TCC CGA AAA TGG CAC AGA AAT GGT ATC AAG AAA CCC 99 Thr His Asn Gin Ser Arg Lys Trp His Arg Asn Gly lie Lys Lys Pro 10 15 20

CGA TCA CAA AGA TAC GAA TCT CTT AAG GGG GTG GAC CCC AAG TTC YTG 147 Arg Ser Gin Arg Tyr Glu Ser Leu Lys Gly Val Asp Pro Lys Phe Xaa 25 30 35 40

AGG AAC ATG CGC TTT GCC AAG AAG CAC AAC AAA AAG GGC CTA AAG AAG 195 Arg Asn Met Arg Phe Ala Lys Lys His Asn Lys Lys Gly Leu Lys Lys 45 50 55

ATG CAG GCC AAC AAT GCC AAG GCC ATG AGT GCA CGT GCC GAG GCT ATC 243 Met Gin Ala Asn Asn Ala Lys Ala Met Ser Ala Arg Ala Glu Ala lie 60 65 70

AAG GCC CTC GTA AAG CCC AAG GAG GTT AAG CCC AAG ATC CCA AAG GGT 291 Lys Ala Leu Val Lys Pro Lys Glu Val Lys Pro Lys lie Pro Lys Gly 75 80 85

GTC AGC CGC AAG CTC GAT CGA CTT GCC TAC ATT GCC CAC CCC AAG CTT 339 Val Ser Arg Lys Leu Asp Arg Leu Ala Tyr lie Ala His Pro Lys Leu 90 95 100

GGG AAG CGT GCT CGT GCC CGT ATT GCC AAG GGG CTC AGG CTG TGC CGG 387 Gly Lys Arg Ala Arg Ala Arg lie Ala Lys Gly Leu Arg Leu Cys Arg 105 110 115 120

CCA AAG GCC AAG GCC AAG GCC AAG GCC AAG GAT CAA ACC AAG GCC CAG 435 Pro Lys Ala Lys Ala Lys Ala Lys Ala Lys Asp Gin Thr Lys Ala Gin 125 130 135

GCT GCA GCC CCA GCT TCA GTT CCA GCT CAG GCT CCC AAA CGT ACC CAG 483 Ala Ala Ala Pro Ala Ser Val Pro Ala Gin Ala Pro Lys Arg Thr Gin 140 145 150

GCC CCT ACA AAG GCT TCA GAG TAGATATCTC TGCCAACATG AGGACAGAAG 534

Ala Pro Thr Lys Ala Ser Glu 155

GACTGGTGCG ACCCCCCACC CCCGCCCCTG GGCTACCATC TGCATGGGGC TGGGGTCCTC 594

CTGTGCTATT TGTACAAATA AACCTGAGGC AGGATTTGTC 634

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 159 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2:

Met Ala Lys Ser Lys Asn His Thr Thr His Asn Gin Ser Arg Lys Trp 1 5 10 15

His Arg Asn Gly lie Lys Lys Pro Arg Ser Gin Arg Tyr Glu Ser Leu 20 25 30

Lys Gly Val Asp Pro Lys Phe Leu Arg Asn Met Arg Phe Ala Lys Lys 35 40 45

His Asn Lys Lys Gly Leu Lys Lys Met Gin Ala Asn Asn Ala Lys Ala 50 55 60

Met Ser Ala Arg Ala Glu Ala lie Lys Ala Leu Val Lys Pro Lys Glu 65 70 75 80

Val Lys Pro Lys lie Pro Lys Gly Val Ser Arg Lys Leu Asp Arg Leu 85 90 95

Ala Tyr lie Ala His Pro Lys Leu Gly Lys Arg Ala Arg Ala Arg lie 100 105 110

Ala Lys Gly Leu Arg Leu Cys Arg Pro Lys Ala Lys Ala Lys Ala Lys 115 120 125

Ala Lys Asp Gin Thr Lys Ala Gin Ala Ala Ala Pro Ala Ser Val Pro 130 135 140

Ala Gin Ala Pro Lys Arg Thr Gin Ala Pro Thr Lys Ala Ser Glu 145 150 155

(2) INFORMATION FOR SEQ ID NO : 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Gly Gly Val Lys Lys Pro Leu 1 5

(2) INFORMATION FOR SEQ ID NO : 4:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 11..12

(D) OTHER INFORMATION :/note= "M = A or C"

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 14..17

(D) OTHER INFORMATION :/note= "V = G, C or A"

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 22..25

(D) OTHER INFORMATION :/note= "R = A or G"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: CCGAGCTCAC MGGVGGVGTA ARAARCC 27

(2) INFORMATION FOR SEQ ID NO : 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Gly Ala Lys Ala Lys Gly 1 5

(2) INFORMATION FOR SEQ ID NO : 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 11..26

(D) OTHER INFORMATION : /note= "V = G, C or A"

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 14..20 (D) OTHER INFORMATION :/note= "Y = C or T/U"

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 17..23

(D) OTHER INFORMATION: /note= "R = A or G"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CCGAGCTCGG VGCYAARGCY AARGGV 26

(2) INFORMATION FOR SEQ ID NO : 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS :

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7:

Val Leu Asn lie Gin lie 1 5

(2) INFORMATION FOR SEQ ID NO : 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 11..14

(D) OTHER INFORMATION :/note= "S = G or C"

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 17..18

(D) OTHER INFORMATION : /note= "Y = C or T/U"

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 20..26

(D) OTHER INFORMATION :/note= "H = A, C or T/U"

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 23..24

(D) OTHER INFORMATION : /note= "R = A or G" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: CCGAGCTCGT SCTSAAYATH CARATH 26

(2) INFORMATION FOR SEQ ID NO : 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9: CCGCGGCCGC TTTTTTTTTT TTTTTTTT 28

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Cys Arg Pro Lys Ala Lys Ala Lys Ala Lys Ala Lys Asp Gin Thr Lys 1 5 10 15

Claims

WHAT IS CLAIMED IS:

1. A method of identifying a heparin component that binds to antithrombin-3, comprising:

a) contacting a heparin sample suspected of containing a heparin component that binds to antithrombin-3 with a heparan sulfate/heparin interacting protein under conditions effective to allow binding of said heparin component; and

b) detecting the binding of said heparin component to said heparan sulfate/heparin interacting protein.

2. A method for purifying a heparin species that binds to antithrombin-3, comprising:

a) contacting a heparin sample suspected of containing a heparin species that binds to antithrombin-3 with a heparan sulfate/heparin interacting protein under conditions effective to allow binding of said heparin species to said heparan sulfate/heparin interacting protein; and

b) collecting the heparin species bound to said heparan sulfate/heparin interacting protein.

3. A method of identifying a heparin component that binds to antithrombin-3, but does not bind to the PF4 protein, comprising:

a) contacting a heparin sample suspected of containing a heparin component that binds to antithrombin-3, but does not bind to the PF4 protein with a heparan sulfate/heparin interacting protein under conditions effective to allow binding of said heparin component; b) collecting the heparin component bound to said heparan sulfate/heparin interacting protein; and

c) comparing the binding of said heparin component to antithrombin-3 and the PF4 protein, wherein the binding of said heparin component to antithrombin-3 and the lack of binding of said heparin component to the PF4 protein is indicative of a heparin component that binds to antithrombin-3, but does not bind to the PF4 protein.

4. A method for purifying a heparin species that binds to antithrombin-3, but does not bind to the PF4 protein, comprising:

a) contacting a heparin sample suspected of containing a heparin species that binds to antithrombin-3, but does not bind to the PF4 protein with a heparan sulfate/heparin interacting protein under conditions effective to allow binding of said heparin species to said heparan sulfate/heparin interacting protein;

b) collecting the heparin species bound to said heparan sulfate/heparin interacting protein; and

c) comparing the binding of said heparin component to antithrombin-3 and the PF4 protein, wherein the binding of said heparin component to antithrombin-3 and the lack of binding of said heparin component to the PF4 protein is indicative of the purification of a heparin component that binds to antithrombin-3, but does not bind to the PF4 protein.

5. A method for neutralizing heparin, comprising contacting a heparin sample with a heparan sulfate/heparin interacting protein composition under conditions effective to allow binding of heparin to said heparan sulfate/heparin interacting protein composition.

6. The method of claim 5, further defined as a method for neutralizing low molecular weight heparin, and wherein said heparin sample comprises low molecular weight heparin.

7. A method for inhibiting the binding of heparin to antithrombin-3, comprising contacting a heparin composition that binds to antithrombin-3 with a heparan sulfate/heparin interacting protein under conditions effective to allow binding of said heparin composition to said heparan sulfate/heparin interacting protein, thereby inhibiting the binding of heparin to antithrombin-3.

8. A method for promoting blood coagulation, comprising contacting a heparin-containing blood sample with a heparan sulfate/heparin interacting protein composition under conditions effective to allow binding of heparin within the blood sample to said heparan sulfate/heparin interacting protein composition.

9. The method of any one of claims 5-8, wherein said contacting occurs in vitro.

10. A method of identifying a candidate substance that alters the binding of heparin to a heparin-binding molecule, comprising:

a) admixing heparin, a heparan sulfate/heparin interacting protein and a candidate substance under conditions effective to allow binding of heparin to said heparan sulfate/heparin interacting protein; and b) determining the binding of heparin to said heparan sulfate/heparin interacting protein in the presence of the candidate substance and in the absence of the candidate substance, wherein the ability of a candidate substance to alter the binding of heparin to said heparan sulfate/heparin interacting protein is indicative of a candidate substance that alters the binding of heparin to a heparin-binding molecule.

11. The method of claim 10, further defined as a method for identifying a candidate substance that inhibits the binding of heparin to a heparin-binding molecule, comprising determining the ability of the candidate substance to decrease the binding of heparin to said heparan sulfate/heparin interacting protein.

12. The method of claim 10, further defined as a method for identifying a candidate substance that increases the binding of heparin to a heparin-binding molecule, comprising determining the ability of the candidate substance to increase the binding of heparin to said heparan sulfate/heparin interacting protein.

13. The method of any preceding claim, wherein said heparan sulfate/heparin interacting protein comprises the amino acid sequence of SEQ ID NO:2.

14. The method of any preceding claim, wherein said heparan sulfate/heparin interacting protein is encoded by the nucleic acid sequence of SEQ ID NO: 1.

15. The method of any preceding claim, wherein said heparan sulfate/heparin interacting protein is a recombinant heparan sulfate/heparin interacting protein prepared by expressing the nucleic acid sequence of SEQ ID NO:l.

16. A heparan sulfate/heparin interacting protein for use in inhibiting the binding of heparin to antithrombin-3.

17. A heparan sulfate/heparin interacting protein for use in the preparation of a medicament for inhibiting the binding of heparin to antithrombin-3.

18. A heparan sulfate/heparin interacting protein for use in promoting blood coagulation.

19. A heparan sulfate/heparin interacting protein for use in the preparation of a medicament for promoting blood coagulation.

20. A heparan sulfate/heparin interacting protein for use in neutralizing heparin.

21. A heparan sulfate/heparin interacting protein for use in the preparation of a medicament for neutralizing heparin.

22. A heparan sulfate/heparin interacting protein for use in neutralizing low molecular weight heparin.

23. A heparan sulfate/heparin interacting protein for use in the preparation of a medicament for neutralizing low molecular weight heparin.

24. A heparan sulfate/heparin interacting protein for use in treating a disease characterized by excessive bleeding.

25. A heparan sulfate/heparin interacting protein for use in the preparation of a medicament for treating a disease characterized by excessive bleeding in a human patient.

26. A heparan sulfate/heparin interacting protein for use in treating heparin overdose.

27. A heparan sulfate/heparin interacting protein for use in the preparation of a medicament for treating heparin overdose in a human patient.

28. A heparan sulfate/heparin interacting protein for use in treating heparin induced thrombocytopenia.

29. A heparan sulfate/heparin interacting protein for use in the preparation of a medicament for treating heparin induced thrombocytopenia in a human patient.

30. The heparan sulfate/heparin interacting protein of any one of claims 16-29, wherein said heparan sulfate/heparin interacting protein comprises the amino acid sequence of SEQ ID NO:2.

31. The heparan sulfate/heparin interacting protein of any one of claims 16-29, wherein said heparan sulfate/heparin interacting protein is encoded by the nucleic acid sequence of SEQ ID NO:l .

32. The heparan sulfate/heparin interacting protein of any one of claims 16-29, wherein said heparan sulfate/heparin interacting protein is a recombinant heparan sulfate/heparin interacting protein prepared by expressing the nucleic acid sequence of SEQ ID NO: 1.

33. Use of a heparin sulfate/heparin interacting protein in the preparation of a medicament for inhibiting the binding of heparin to antithrombin-3.

34. Use of a heparin sulfate/heparin interacting protein in the preparation of a medicament for promoting blood coagulation.

35. Use of a heparin sulfate/heparin interacting protein in the preparation of a medicament for neutralizing heparin.

36. Use of a heparin sulfate/heparin interacting protein in the preparation of a medicament for neutralizing low molecular weight heparin.

37. Use of a heparin sulfate/heparin interacting protein in the preparation of a medicament for treating a disease characterized by excessive bleeding in a human patient.

38. Use of a heparin sulfate/heparin interacting protein in the preparation of a medicament for treating heparin overdose in a human patient.

39. Use of a heparin sulfate/heparin interacting protein in the preparation of a medicament for treating heparin induced thrombocytopenia in a human patient.

40. A use in accordance with any one of claims 33-39, wherein said heparan sulfate/heparin interacting protein comprises the amino acid sequence of SEQ ID NO:2.

41. A use in accordance with any one of claims 33-39, wherein said heparan sulfate/heparin interacting protein is encoded by the nucleic acid sequence of SEQ ID NO: 1.

42. A use in accordance with any one of claims 33-39, wherein said heparan sulfate/heparin interacting protein is a recombinant heparan sulfate/heparin interacting protein prepared by expressing the nucleic acid sequence of SEQ ID NO: 1.