WO1992017499A1

WO1992017499A1 - A unique hexapeptide derived from thrombospondin and its uses

Info

Publication number: WO1992017499A1
Application number: PCT/US1992/002825
Authority: WO
Inventors: Ralph L. Nachman; Adam S. Asch
Original assignee: Cornell Research Foundation, Inc.
Priority date: 1991-04-08
Filing date: 1992-04-07
Publication date: 1992-10-15

Abstract

The present invention relates to a unique hexapeptide having the amino acid sequence Cys-Ser-Val-Thr-Cys-Gly, antibody to the hexapeptide, and the use of the hexapeptide and its antibody.

Description

A UNIQUE HEXAPEPTIDE DERIVED FROM THROMBOSPONDIN AND ITS USES

Partial funding for the research which led to the making of the present invention was provided by grants from the National Institutes of Health. Accordingly, the United States federal government has certain statutory rights to the present invention in accordance with the provisions of 35 USC §200 et seq.

Thrombospondin is a large multi-functional trimeric glycoprotein of approximately 450 Kd, and a major component of platelet alpha-granules secreted in response to thrombin or ionophore stimulation. When stimulated, thrombospondin is expressed on the activated platelet surface where it supports irreversible platelet aggregation. Thrombospondin has a broad biological distribution, is expressed by several cell types, and is incorporated into the extracellular matrix of growing cells in a regulated fashion. Thrombospondin binds a variety of molecules including heparin, sulfated glycolipids, collagen, fibronectin, fibrinogen, histidine rich glycoprotein, plasminogen, tissue and urinary type plasminogen activators.

In culture, the molecule is synthesized by a variety of cells including monocytes and macrophages. Thrombospondin is a trimeric glycoprotein composed of three identical, disulfide- linked glycopeptide chains which, in rotary shadowed images obtained by electron microscopy, resemble a bola with a central 9~

disulfide knot, three linear domains, and three large globular domains at the ends.

In addition to its cytoadhesive activity, thrombospondin has several other functions. As a surface for macromolecular assembly, the molecule may mediate or modulate certain aspects of coagulation and fibrinolysis. Thrombospondin binds to the A- alpha and B-beta chains of fibrinogen. During fibrin formation, the molecule is incorporated into the growing clot and modulates the structure of the fibrin network, leading to a finer and more highly branched fibrin network. In the presence of coagulation factor XIII, thrombospondin may participate in an amide linkage and become crosslinked to other thrombospondin molecules or to fibrin via several reactive glutamyl and lysyl residues. As a transient component of the fibrin clot, the molecule may regulate the strength of the fibrin clot by affecting fibrin filament thickness and may influence the efficiency of fibrinolysis. Like fibrin, the molecule can serve as a surface upon which plasminogen activation occurs, since it forms a ternary complex with plasminogen and tissue type plasminogen activator (t-PA). Compared with fluid phase plasminogen activation by t-PA, a 40- fold increase in the catalytic efficiency of activation has been observed with plasminogen bound to thrombospondin as compared to plasminogen-t-PA complexes formed in the absence of thrombospondin. In addition, formation of the thrombospondin- plasminogen complex is enhanced in the presence of t-PA or urinary-type plasminogen activator (u-PA) in a manner that b

appears to be dependent upon the generation of small amounts of plasmin.

In addition to its role as a modulator of localized proteolysis, thrombospondin plays an important role in the regulating of basic cellular functions. Most cell types examined in vitro - including endothelial, smooth muscle, glial, melanoma, type II pneumocytes, keratinocytes, and fibroblasts - synthesize thrombospondin and incorporate it into their extracellular matrices. In tissue sections, thrombospondin has been demonstrated in basement membranes of the dermis-epidermis, of renal peritubular connective tissues, in small blood vessels, in the subendothelium of the aorta, and in atherosclerotic lesions. In the early development of mouse embryos, thrombospondin is deposited in the basement membrane of surface ectoderm, pharynx and neuroectoderm. Its observed distribution, and its interactions with several known matrix components such as thrombin, fibrinogen, fibronectin, histidine-rich glycoprotein, plasminogen, urokinase-type plasminogen activator, and collagen types l-V, suggests that it plays a major role in matrix biology. Thrombospondin may also affect matrix events by localizing and regulating plasmin in matrices which are undergoing rapid remodeling such as those found associated with wound healing, development, or neoplastic growth. The plasminogen-plasminogen activator complex bound to thrombospondin is relatively resistant to inhibition by protease inhibitors such as alpha-2 macroglobulin and PAI. Thrombospondin is a substrate for several 1

proteases, including plasmin, and may be modified enzymatically in the matrix or by cells in the vicinity. Thrombospondin incorporation into the matrix of growing smooth muscle cells is also heparin-inhibitable. Embryonic lung fibroblasts and bovine aortic smooth muscle cells endocytose thrombospondin in a heparin inhibitable fashion, and degrade it in a process which is saturable (Km of 6x10-8M with a V_maχ of 1.4x10⁵ molecules/cell/min). It is not presently clear whether this is a mechanism for the removal of matrix thrombospondin by growing or migrating cells, or whether processed thrombospondin may exhibit unique functions distinct from the intact molecule.

Thrombospondin incorporation into the matrix appears to be cell cycle dependent since recently-plated and rapidly proliferating subconfluent cells (aortic endothelial cells, smooth muscle cells and fibroblasts) synthesize and secrete more thrombospondin into the culture medium than senescent.density- arrested, cell cultures. The synthesis of thrombospondin by bovine aortic smooth muscle cells is enhanced by platelet- derived growth factor (PDGF). PDGF-stimulated glial cells also show a 10-fold increase in levels of thrombospondin synthesis. A role for thrombospondin in regulating the behavior or growth of cells is suggested by data that thrombospondin and epidermal growth factor act synergistically to stimulate mitogenesis in quiescent rat vascular smooth muscle cells and that antibodies to thrombospondin arrest proliferating smooth muscle cells in the G1 phase of the cell cycle. f

It is believed that the increased synthetic rates are due to a rapid but short-lived increase in levels of thrombospondin mRNA. The thrombospondin message can be rapidly induced by TGF-beta, FGF, and PDGF in several cell types including vascular smooth muscle cells and fibroblasts. Thrombospondin mRNA is superinduced by cycloheximide, as is expression of the c-fos and c-myc oncogene products. The similarities between expression of thrombospondin and oncogene products are indicative to shared growth factor-like functions between these materials. Thrombospondin has an autocrine role in the regulation of smooth muscle cell proliferation through a synergistic effect with EGF; it is not known whether thrombospondin directly binds with EGF or other growth factors, but it has been suggested that thrombospondin reverses the inhibitory effect of heparin on smooth muscle cell growth by blocking heparin-smooth muscle cell interactions. Furthermore, monoclonal antibodies to thrombospondin inhibits smooth muscle cell growth. Together, this data indicates that thrombospondin is an important regulator of cell growth and further supports the concept that thrombospondin has growth factor-like properties.

Immunolocalization studies show that thrombospondin is localized primarily at sites of active cell migration and proliferation; thrombospondin is more widely distributed in the matrices of wounded or actively developing tissues than in more stable fully differentiated tissues. Thrombospondin is also prevalent in human fetal skin and cartilage; in adult tissues e

thrombospondin is restricted to some basement membranes, loose connective tissues, and areas of injury, especially in the clefts of atherosclerotic blood vessels. These data suggest that thrombospondin is a modulator of developmental and reparative cell processes.

Recent sequencing of cDNA coding for thrombospondin has provided an opportunity to correlate structure and function of the thrombospondin domains. While in vitro mutagenesis experiments are likely to provide a good deal of information regarding the functional aspects of domains of the molecule, our own experiments with synthesized oligopeptides have yielded important information concerning the interaction of a domain of thrombospondin with one of its cellular receptors leading to the discovery of the present invention. Malarial infected erythrocytes express a histidine rich protein on knob-like structures that serve as a site of attachment to endothelial cells along the lumen of the smaller blood vessels, by which means the parasites are protected from the lethal immune activity of the spleen. This process of attachment, called cytoadherence, is responsible for some of the fatal complications of infection with the causative organism, Plasmodium falciparum. The Plasmodium organism is known to produce a malarial histidine rich protein which we speculated might interact with thrombospondin or a thrombospondin-like molecule. Recent data developed in our laboratory demonstrated certain homologies between thrombospondin and several malarial proteins expressed at various stages in the malarial life cycle. Thus, we speculated that thrombospondin, as a multi-functional cell adhesive protein with growth factor-like characteristics, might possess the functional binding diversity needed by Plasmodium during its life cycle in the infected host to bind receptors on liver and endothelial cells. The identification of the specific domain that is the most likely to be responsible for these interactions led to the synthesis of the unique hexapeptide according to the present invention. The specific hexapeptide according to the present invention has the amino acid sequence [Seq ID No. 1]:

Cys-Ser-Val-Thr-Cys-Gly which corresponds to the region of homology of thrombospondin and the malarial circumsporozoite protein, and is found twice within each thrombospondin monomer at amino acid positions

429-434 and 486-491 , and a total of six times within the intact trimeric molecule. Further data suggest that a similar area of homology exists in the erythrocyte stage of malaria.

An indication of the advantages and uses to which the hexapeptide and its antibody according to the present invention may be put will become apparent from the following detailed description of the present invention which is to be taken in conjunction with the accompanying examples and drawings in which : Figure 1 is a dose responsive curve of the inhibition of the binding of histidine rich glycoprotein to insolubolized thrombospondin;

Figure 2 is a graphic representation of the metastatic potential of cells which express glycoprotein IV and those that do not express glycoprotein IV on their surfaces.

Figure 3 is a graphic representation of the profound inhibition of thrombospondin binding to U937 tumor cells by the peptide according to the present invention; Figure 4 is a graphic representation showing the peptide according to the present invention bound to glycoprotein IV on the surface of U937 cells.

Figure 5 is a graphic representation of thrombospondin binding to salivary protein; and Figure 6 is a graphic representation of the inhibition of platelet thrombospondin expression by the peptide according to the present invention in which 6A1 and 6A2 depict the results of flow cytometry, and 6B-1 and 6B-2 depict platelet aggregometry of platelet rich plasma. Inhibition of binding of histidine rich glycoprotein (HRGP) to thrombospondin requires the specific stereospecificity found in this peptide, and was demonstrated, as described in the following Example I:

EXAMPLE I 1

Thrombospondin was prepared from human platelets by a conventional technique. Briefly, outdated normal human platelets were washed free of contaminating plasma proteins by three centrifugations in saline containing buffer. The platelets were then stimulated with 10 micromolar ionophore A23187 to cause the platelets to release intracellular stores of thrombospondin. The released thrombospondin was further purified by affinity chromatography on heparin-Sepharose followed by high performance liquid chromatography using Mono-Q Sepharose. Thrombospondin was coated in wells of a conventional 96 well

ELISA plate at a concentration of 10 μg/ml. HRGP was prepared from normal human plasma by heparin affinity chromatography, and varying concentrations of HRGP in a total volume of 200 μl were added alone (or in the presence of the indicated concentrations of the hexapeptide Cys-Ser-Val-Thr-Cys-Gly according to the present invention) to thrombospondin-coated wells, and the binding was determined by an ELISA system using monospecific antibody to HRGP followed by alkaline phosphatase conjugated goat anti-rabbit IgG and para-nitrophenylphosphate was used as a colorimetric substrate. Color generation was quantitated using a Titertex spectrophotometer at 405 nanometers.

Using this protocol, the data collected from Example I, and depicted in figure 1 , clearly demonstrate a dose dependent inhibition of the binding of histidine rich glycoprotein to insolubilized thrombospondin on a conventional 96 well microtiter plate in which varying amounts of HRGP were added alone or in the presence of the indicated concentrations of the hexapeptide to thrombospondin coated wells in the plate and binding determined by a monospecific antibody to HRGP. When graphed, as in figure 1 , a dose response curve of the data indicates functional inhibition at the level of 5 - 10 μm with maximum inhibition at the 50μm level; no inhibition was demonstrated with thrombospondin-plasminogen binding as a control. In addition, a control peptide of a scrambled sequence [Seq ID No. 2], specifically Thr-Val-Ser-Gly-Cys-Cys, had no inhibitory effect.

On the basis of these studies, an antibody was made to the hexapeptide. The hexapeptide was prepared containing a carboxy terminus aminocaprolylcysteine amide (ACM) which provided the cysteine residues protection from oxidation and allowed for the selective conjugation to the carboxy terminus cysteine residue as well as a spacer for presenting the peptide from the Keyhole limpet hemocyanin (KLH) carrier backbone. In addition, this prevented intra- and inter-chain disulfide bond formation. Thus, the peptide used for immunization had the following sequence:

ACM ACM O

[Cys-Ser-Val-Thr-Cys-Gly-NH(CH2)5-C-CysNH2]KLH

Antisera raised in rabbits blocked the binding of HRGP to thrombospondin on conventional 96 well microtiter plates. In addition, when tested using Western blot procedures, the antisera //

so prepared reacted with both the isolated circumsporozoite protein and the purified thrombospondin. The adhesion of erythrocytes infected with Plasmodium falciparum to human endothelial cells is mediated by a receptor on the latter cells identified as glycoprotein IV. We have identified this glycoprotein as a cellular receptor for thrombospondin on several cell types.

These studies suggested to us that the antibody to the hexapeptide might interfere with malarial infection in experimental animals, and latency experiments were subsequently performed in ten mice to confirm any interference.

In five experimental animals, inoculation of sporozoites (utilizing the rodent parasite, Plasmodium berghii) was preceded by incubation with antibodies to the hexapeptide prepared as above, and the latency to parasitemia was compared with control inoculations. Of the mice tested, 60% demonstrated a latency of 24 hours when compared to the control group.

This suggests that the hexapeptide according to the present invention, or congeners coupled to the proper immunogenic carrier may function as a potential vaccine (or a potential treatment) to prevent infection with the malarial organism in mammals including man. Cogeners may include tandem repeats of the peptide sequence up to 40 amino acid residues in length, or the protein coupled to a branching carboxyterminal polysine network. The carrier for immunizing patients using such a protocol would most likely to be a conventional tetanus toxoid or another immunogenic vaccine carrier. The optimal ratio of peptide to carrier in such uses has yet to be determined, however, it is likely to be in the ratio of approximately 20-50 peptide:1 carrier.

In addition to the potential use of the peptides according to the present invention for inhibition of malarial adhesion, the peptides also have potential for the inhibition of tumor cell metastasis.

The function of thrombospondin on carcinoma cells appears, in part, to mediate adhesion of the cells to endothelial or subendothelial metastatic sites. Experimental evidence suggests that antibodies to thrombospondin can inhibit the metastasis of human tumor cells in animal models. Our own data generated in making the present invention demonstrates that the adhesion of tumor cells to surfaces rich in thrombospondin is mediated by interaction with tumor cell glycoprotein IV. The adhesion of human melanoma cell line (C-32) to human endothelial cells is mediated by a receptor on the latter cells identified as glycoprotein IV, and the metastatic potential of cells that express glycoprotein IV is markedly greater than that of cells that do not (figure 2). In one study, for example, the tail veins of male nude mice, aged 6 to 8 weeks old, were injected with 1 x 10⁶ glycoprotein IV-rich or glycoprotein IV-poor C32 human melanoma cells. After three months the animals were sacrificed, the lungs were insufflated with 15% India ink solution followed by bleaching with Fekete's solution, and the pulmonary nodules were counted. Glycoprotein IV-rich innocula resulted in a 14-fold increase in the number of pulmonary metastasis as compared to those found when glycoprotein IV-poor innocula was used. The homology between thrombospondin and the proteins expressed by malaria that mediate malarial adhesion to endothelium, tumor cells, and possibly to hepatic cells which resulted from our experiments suggest that an agent such as the peptide according to the present invention might potentially interfere with thrombospondin's interaction with glycoprotein IV and thus may be useful to inhibit tumor cell metastatic behavior. Utilizing cell line U937, inhibition of thrombospondin binding to cell line U937 was studied, as described in the following Example II, to confirm that the peptide would interfere with thrombospondin's interaction with glycoprotein IV.

The binding of ^{1 25}I-TSP to U937 cells was examined in the presence of increasing concentrations of the peptide according to the present invention, and according to the following example:

EXAMPLE II Thrombospondin was labelled to a specific activity of 8 x Λ cpm/μg for these studies, and cell binding was performed in triplicate in Tris-buffered saline with 2 mM CaCl2 utilizing 5 x 10⁵ cells in a total volume of 0.2 ml at 4° C for 40 minutes. Cell bound radioactivity was determined by centrifuging the cells through silicone oil, and removing and counting the cell pellets in a gamma counter. Nonspecific binding was determined by cold inhibition studies. The data collected from this example is graphically represented in figure 3 and illustrates the profound inhibition of thrombospondin binding to U937 cells by the hexapeptide according to the present invention.

In addition, the peptide itself is capable of binding to glycoprotein IV on these cells as measured by radioactive binding studies as depicted in figure 4. In this study, the peptide according to the present invention was radiolabelled to a specific activity of 4.6 x 10⁴ cpm/μg and the direct binding measured as described previously.

This data suggests that the hexapeptide according to the present invention or its congeners may be useful as anti metastatic agents. These agents would function by blocking the binding of tumor cells to thrombospondin at metastatic sites within the body, or by serving as an immunogen for the development of antibodies that serve the same purpose and inhibit thrombospondin-tumor cell interactions.

Thrombospondin is also found in inflammatory tissues such as those that would be present in various wounds. Based upon the investigational findings collected during the making of the present invention, it would be reasonable to assume that thrombospondin may also be present in inflammatory oral lesions such as those found in acute gingivitis. To test this premise, formalin fixed sections of normal and inflamed gingiva were subjected to immunohistochemical identification using monospecific antisera to thrombospondin. The inflamed gingiva /

contained significant deposits of thrombospondin in the acutely inflamed regions and in the adjacent mucosal epitheliuim. No thrombospondin was identified in normal gingival tissue.

A major defense mechanism in the oral cavity for clearance of bacterial from gingival surfaces and in saliva is a salivary agglutinin that clumps bacteria and leads to clearance of aggregated bacteria by phagocytes and leukocytes. In view of the data collected in the present invention, it would be reasonable that thrombospondin in inflamed tissue would interact with the salivary protein. Thus, a study was conducted whereby purified salivary agglutinin from parotid secretions were coated on ELISA plates and incremental amounts of thrombospondin were added; binding of the thrombospondin was detected by monospecific antisera to thrombospondin in a conventional ELISA protocol using alkaline phosphatase conjugated goat anti-rabbit antibody and the colorimetric substrate para-nitrophenylphosphate. Color generation was measured over time using a Titertek spectrophotometer at 405 nanometers. Thrombospondin interacted strongly with the salivary protein. Subsequent testing with the hexapeptide according to the present invention demonstrated that this binding reaction was partially inhibited by the presence of the hexapeptide, and no inhibition was observed using an unrelated peptide. The data from this example is graphically presented in figure 5. It has subsequently been demonstrated that thrombospondin (at a level of 0.5 μg/ml) will It

inhibit the aggregation of Streptococcus sanguis by purified salivary agglutinin.

These studies demonstrate that the domain of thrombospondin containing the hexapeptide of the present invention plays an important role in determining the binding functions of this adhesive salivary glycoprotein. In the oral cavity, thrombospondin present in inflamed tissues, such as those which might be seen in peridontal disease, interferes with the normal antibacterial agglutinating function of salivary protein. Thus, thrombospondin could potentiate bacterial growth on inflamed gingiva. However, the thrombospondin present in such tissues can be blocked by the hexapeptide according to the present invention. This peptide (Cys-Ser-Val-Thr-Cys-Gly) if provided a dental patient in a mouthwash and/or toothpaste formulation could potentially prevent gingival infection and limit the development of periodontal disease.

In blood platelets, thrombospondin serves to support the secretion-dependent phase of aggregation by stabilizing the interaction of fibrinogen with its receptor complex on the activated platelet surface. Our studies into thrombospondin demonstrated the endogenous thrombospondin binding to the platelet surface is mediated by glycoprotein IV. Thus, an agent, such as the hexapeptide according to the present invention, which inhibits expression of thrombospondin on the activated platelet surface, and thereby inhibits platelet aggregation (see figure 6), might be useful as a therapeutic anti-platelet agent. More specifically, figures 6A-1 and 6A-2 depict the results of flow cytometry data and reveal normal thrombospondin expression on the platelet surface following stimulation with the calcium ionophore A23187 in the presence of a control peptide of the formula Thr-Val-Ser-Gly-Cys-Cys at 200μM, and inhibition of thrombospondin surface expression in the presence of the peptide according to the present invention, Cys-Ser-Val-Thr-Cys-Gly, at 200 μM. Figures 6B-1 and 6B-2 depicts data generated by platelet aggregometry of platelet rich plasma, and reveals a diminished response to A23187 in the presence of 200 μM of the peptide according to the present invention, compared with the control aggregation in the presence of 200 μM of the same control peptide used for the flow cytometry tests. Thus the peptide according to the present invention when coupled to a conjugate, such a albumin, may be useful as a clinical agent to inhibit platelet aggregation in patients undergoing coronary bypass, or post-transluminal angioplasty.

Clinically useful doses of the peptide, peptide-conjugates or peptide antibodies need to be more particularly defined in clinical trials. However, in vitro experimental data suggests that significant inhibition of thrombospondin binding occurs at peptide concentrations less than 200 micromolar. It is also likely that peptide conjugates, because of their relative large size, will be more potent clinical agents than the purified peptide alone. The determination of the dosage levels for the various uses that the peptide according to the present invention may be ι <

put, is well within the capabilities of pharmacologists to determine given the targeted use, route of administration, age and weight of the patient, severity of the condition to be corrected, and other factors conventionally taken int consideration in the pharmaceutical sciences in determining optimum dose levels.

As described above, the hexapeptide according to the present invention is potentially useful as a pharmaceutical for treating warm-blooded animals suffering from many different pathologic conditions when administered in amounts sufficient to treat or correct the specific targeted condition. The specific amounts of the peptide given in such instances may be adapted to provide the optimum therapeutic response sought. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic solution. The peptide may be administered in the form of the free peptide, a conjugated peptide, or as a nontoxic pharmaceutically acceptable salt thereof (collectively referred to as "its modifications"). By the term "pharmaceutically acceptable salt" is meant those acid-addition salts of the parent compound which do not significantly adversely affect the pharmaceutical properties (e.g. toxicity, effectiveness, etc.) of the parent compound, such as are conventionally used in the pharmaceutical art. The hexapeptide or its modifications may be administered parenterally, e.g. by subcutaneous, intramuscular, or intravenous 11 injection. Solutions or suspensions of the active hexapeptide or its modifications can be prepared in water suitably mixed with a surfactant such as hydroxyproplycellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms. As the hexapeptide or its modifications have a natural tendency to adhere to glass or plastic surfaces, these preparations may also contain an agent, such as gelatin or albumin, to competitively inhibit this effect.

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.

These forms must also be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms such as bacterial and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, polyol (for example, glycerol, proplylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Compositions suitable for intramuscular or subcutaneous injection may also contain minor amounts of salts, acids, buffers, and bases. Suitable pharmaceutically acceptable buffering and tonicity agents are readily determinable by persons skilled in the art. {Lt>

The hexapeptide and its modifications according to the present invention may also be suitable for oral administration, for example with an inert diluent or with an assimilable edible carrier, or it may be encapsulated within a hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the hexapeptide or its modification may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, mouthwashes, wafers and the like.

In addition to the hexapeptide or its modifications, the tablets, troches, pills, and the like may also contain binders such as gum tragacanth, acacia, corn starch or gelatin; colorants; excipients such as dicalcium phosphate; disintegrating agents such as corn starch, potato starch, alginic acid and the like; lubricating agents such as magnesium stearate; sweetening agents such as sucrose lactose or saccharin; and flavoring agents such as peppermint, oil of wintergreen or cherry flavorings.

Thus while we have illustrated and described the preferred embodiment of our invention, it is to be understood that this invention is capable of variation and modification, and we therefore do not wish or intend to be limited to the precise terms set forth, but desire and intend to avail ourselves of such changes and alterations which may be made for adapting the invention of the present invention to various usages and conditions.

Accordingly, such changes and alterations are properly intended m

to be within the full range of equivalents and therefore within the purview of the following claims. The terms and expressions which have been employed in the foregoing specification are used therein as terms of description and not of limitation, and thus there is no intention in the use of such terms and expressions of excluding equivalents of features shown and described or portions thereof, it being recognized that the scope of the invention is defined and limited only by the claims which follow.

Having thus described our invention and the manner and process of making and using it in such full, clear, concise, and exact terms so as to enable any person skilled in the art to which it pertains, or to with which it is most nearly connected, to make and use the same:

Claims

1. The peptide whose structure is:

ACM ACM O

[Cys-Ser-Val-Thr-Cys-Gly-NH(CH2)5-C-CysNH2]KLH wherein ACM is aminocaprolylcysteine amide and KLH is keyhole limpet hemocyanin.

2. An antibody which is reactive with a hexapeptide selected from the group consisting of;

Cys-Ser-Val-Thr-Cys-Gly;

ACM ACM

Cys-Ser-Val-Thr-Cys-Gly;

ACM ACM O

[Cys-Ser-Val-Thr-Cys-Gly-NH(CH2)5-C-CysNH2]KLH; and cogeners thereof wherein ACM is aminocaprolylcysteine amide and KLH is keyhole limpet hemocyanin.

3. The antibody according to Claim 2 which is substantially reactive to the hexapeptide Cys-Ser-Val-Thr-Cys-Gly.

4. The antibody according to Claim 2 which is substantially reactive to the hexapeptide (ACM)Cys-Ser-Val-Thr-Cys(ACM)- Gly.

5. A method for inhibiting malarial parasite cytoadherence to host cell binding receptors which comprises providing the host cell with a sufficient amount of a peptide of the group:

Cys-Ser-Val-Thr-Cys-Gly;

ACM ACM

Cys-Ser-Val-Thr-Cys-Gly; £ϊ

wherein ACM is aminocaprolylcysteine amide; and cogeners thereof connected to an immunogenic carrier to inhibit said malarial parasite cytoadherence.

6. A method for inhibiting tumor cell adhesion to host cell binding receptors which comprises providing the host cell with a sufficient amount of a peptide of the group:

Cys-Ser-Val-Thr-Cys-Gly;

ACM ACM

Cys-Ser-Val-Thr-Cys-Gly; wherein ACM is aminocaprolylcysteine amide; and cogeners thereof connected to an immunogenic carrier to inhibit said tumor cell adhesion to host cell binding receptors.

7. A method for inhibiting the bacterial agglutinating inhibition function of salivary protein brought about by the presence of thrombospondin which comprises providing the oral cavity with a sufficient amount of a peptide of the group:

Cys-Ser-Val-Thr-Cys-Gly;

ACM ACM

Cys-Ser-Val-Thr-Cys-Gly; wherein ACM is aminocaprolylcysteine amide; and cogeners thereof connected to an immunogenic carrier to bring about said inhibition.

8. A method for inhibiting thrombospondin binding to glycoprotein IV on the surface of a host animal cell which comprises providing an agent which binds with glycoprotein IV on the cell surface prior to exposure of the cell to thrombospondin.

9. A method according to Claim 8 which comprises providing a peptide of the formula:

Cys-Ser-Val-Thr-Cys-Gly to the host cell, and allowing the peptide to bind with the glycoprotein IV on the surface of the host cell prior to exposure of the cell to thrombospondin.

10. A hexapeptide selected from the group consisting of:

Cys-Ser-Val-Thr-Cys-Gly; (ACM)Cys-Ser-Val-Thr-Cys(ACM)-Gly; and cogeners thereof, in which ACM is aminocaprolylcysteine amide.

11. The hexapeptide according to Claim 10 wherein the peptide is Cys-Ser-Val-Thr-Cys-Gly.

12. A hexapeptide according to Claim 10 wherein the hexapeptide is attached to an immunogenic carrier.

13. The hexapeptide according to Claim 10 wherein the peptide is (ACM)Cys-Ser-Val-Thr-Cys(ACM)-Gly.