Monoclonal antibodies recognizing glycoproteins involved in platelet function. Their application as diagnostic and therapeutic agents.

Abstract

The invention relates to monoclonal antibodies, or the idiotypic fragments of these monoclonal antibodies, recognizing antigens involved in the steps of platelet physiology and in particular in the reactions of adhesion and / or aggregation and / or platelet secretion, and / or adhesion of endothelial cells and / or monocyte-platelet interaction, characterized in that they have the ability to modulate platelet or endothilial functions induced by a low concentration of agonists, for example in the presence of 2.5 to 5 µM of ADP, 2 µg / ml of collagen, 0.016 U / ml of thrombin and in that their ability to inhibit platelet functions is considerably reduced, or even absent, in the presence of high concentrations of agonists, for example in the presence of thrombin concentrations greater than 0.5 U / ml or collagen concentrations greater than 10 µg / ml. It also concerns the diagnostic and therapeutic application of these antibodies.

Description

Inonoclonal antibodies recognizing κ-lycoproteins that have a role in platelet functions. Their application as a diagnostic and therapeutic approach. This application is a request for a certificate of addition to French patent application No. 89 / 10016 of July 25, 1989, hereinafter referred to as the main application. The invention relates to monoclonal antibodies directed against proteins involved in platelet functions and their application as a diagnostic and therapeutic agent. The mechanisms of platelet adhesion, aggregation, and secretion, which are involved in the various stages of platelet physiology, involve numerous proteins and glycoproteins in vivo. These proteins and glycoproteins are present on platelets, either permanently or transiently, and / or on other cells, such as endothelial cells and, in some cases, monocytes. Some of these proteins and glycoproteins have been identified, and antibodies, including monoclonal antibodies that recognize them, have been prepared. In particular, antibodies usable as platelet activation markers for diagnostic tests have already been described. For example, US patent 4,783,330 concerns IgG1 antibodies that recognize activated platelets but do not recognize inactivated platelets, azurophilic granules of monocytes, or granulocytes. Preferably, these monoclonal antibodies recognize a glycoprotein with a molecular weight of approximately 140,000 Da (gpl40) on activated human platelets. The inventors have now focused more specifically on pathologies related to platelet physiology and their treatment using controlled methods. With this in mind, they have developed new antibodies directed against specific epitopes of proteins, glycoproteins, or protein complexes involved in platelet physiology. These antibodies have been selected for their specific ability to interfere with platelet functions. The invention therefore relates to novel antibodies directed against proteins, glycoproteins, or protein complexes specifically involved in platelet adhesion, aggregation, and / or secretion reactions that can lead to thrombus formation or are involved in inflammatory or wound healing processes. These antibodies offer the significant advantage, within the scope of the invention, of being able to modulate platelet functions as described above. The invention further relates to the preparation and selection of these monoclonal antibodies and their use for diagnosis or as therapeutic agents. They can thus be applied to the treatment of pathologies such as cardiovascular diseases, atherosclerosis, retinopathies, diabetes and also, for some of them, in the treatment of tumor metastases. Within the framework of the above definitions, the invention relates to monoclonal antibodies, or the idiotypic fragments of such monoclonal antibodies, recognizing antigens involved in at least one of the steps of platelet physiology, and in particular in platelet adhesion and / or aggregation and / or secretion reactions, or in endothelial cell adhesion, or in both types of action, characterized in that they have the capacity to modulate platelet or endothelial functions induced by a low concentration of agonists, for example, in the presence of 2.5 to 5 pM of ADP, or 2 pg / ml of collagen, or 0.016 U / ml of thrombin, and in that their capacity to inhibit platelet functions is considerably reduced or even absent in the presence of high concentrations of agonists, for example, in the presence of thrombin concentrations greater than 0.5 U / ml or collagen concentrations greater than 10 pg / ml. According to a particular aspect of the invention, monoclonal antibodies meeting the above definitions are characterized in that they have the ability to modulate the interaction between platelets and monocytes when the platelets are in a stimulated state, for example when they have been stimulated with thrombin. The idiotypic fragments referred to above are Fab, Fab', F(ab')z fragments prepared according to classical methods, notably by enzymatic digestion for example with pepsin or papain, depending on the desired result. These idiotypic fragments are further characterized in that they lack all or part of their constant fraction Fc. Monoclonal antibodies thus defined, or their idiotypic fragments, have the particularly interesting property of binding to an epitope of a protein, glycoprotein or protein complex, in such a way that the normal physiological activity of that protein, glycoprotein or complex is modulated. In the following, the terms protein, glycoprotein, and protein complex will sometimes be grouped under the term antigen. This modulation of platelet functions, or of endothelial cell functions, or, for certain antibodies of the invention, of the platelet / monocyte interaction referred to above, corresponds, within the framework of the definitions of the invention, to a partial inhibition of certain functions such as adhesion, aggregation, or secretion reactions, or several of these reactions, which occur following stimulation from, for example, lesions in the vascular endothelium, or to an inhibition of reactions at the level of endothelial cells. This limitation must be such that the action of the chosen antibodies or their fragments, possibly in combination with other substances, prevents thrombus formation. The modulating properties of the antibodies according to the invention may vary depending on the nature of the antibody and the recognized antigen. In any case, the antibodies of the invention no longer inhibit platelet function in the presence of high concentrations of thrombin (greater than 0.5 U / ml) or collagen (greater than 10 pg / ml). As a result, these antibodies can be used in the treatment of pathologies such as those mentioned above, without the disadvantage of excessively blocking platelet physiological processes, which could lead to hemorrhagic phenomena. According to a first preferred aspect of the invention, monoclonal antibodies or their idiotypic fragments are characterized in that they recognize an epitope of a protein, glycoprotein or protein complex selected from the group of glycoproteins GMPl4O, complex gpllb-IIIa, complex gpIa-IIa, complex gpIc-IIa or glycoprotein pifa. The 'antigens above' are involved at different levels of platelet physiology. GMP140 was described by McEver et al in Cell (vol 56 - 1033.1044, 24 March 1989). The other antigens have been described in various publications such as McEver et al, (1984) J. Biol. Chem. 259:9799-9804; Hsu-Lin, SC et al, (1984) J. Biol. Chem. 259: 9121-9126; Kunicki, TJ et al, (1988) J. Biol. Cher. 263: 4516-4519 and Premier, M.E. et al, (1988), J. Biol. Chem. 263, 7660-7665. According to another preferred aspect of the invention, a class of antibodies or their idiotypic fragments is further characterized in that these antibodies or fragments recognize an epitope contributing to the functions normally performed by the antigen which contains it. According to another preferred aspect of the invention, some monoclonal antibodies are sensitive to the chemical reduction of the antigen they recognize. Consequently, they are inactivated when the normally recognized antigen is chemically reduced. The functional effect of the epitope with respect to the antigen that contains it indicates that this antigen cannot perform its functions in platelet physiology if the epitope is not present or if it is inactivated. In an advantageous embodiment of the invention, monoclonal antibodies or their idiotypic fragments are characterized in that they recognize an epitope present on GMP140, at a site exposed on the surface of activated platelets, said antibodies or their idiotypic fragments being capable of interfering with platelet aggregation and / or secretion reactions. Particularly preferred antibodies recognizing GMP140 are LYP20 antibodies or antibodies with equivalent immunological and functional properties. In general, for monoclonal antibodies according to the invention, the "equivalent immunological and functional properties" will be evaluated by a test aimed at verifying that the recognized antigen is identical to that which is recognized by the comparison antibody (in this case LYP20) and a test determining the ability of isolated antibodies to modulate platelet functions induced by a low concentration of agonists, for example in the presence of 2.5 to 5 pM of ADP, or 2 pgXml of collagen, or 0.016 U / ml of thrombin and in that their ability to inhibit platelet functions is considerably reduced or absent in the presence of high concentrations of agonists, for example in the presence of thrombin concentrations greater than 0.5 U / ml or collagen concentrations greater than 10 pg / ml. The LYP20 antibody (also known as P20) also has the ability to recognize GNP140 present on the surface of endothelial cells. Furthermore, this antibody no longer recognizes GMP140 when it is in its chemically reduced form, that is, when its molecular weight increases from 128 kDa to 130 kDa. Specific antibodies according to the invention, which have the property of recognizing an epitope present on the GMP140 glycoprotein, also advantageously have the ability to recognize GMP140-like glycoproteins present on cells other than platelets, for example, monocytes. Advantageously, LYP20 also has the ability to recognize a GMP140-like antigen present on monocytes, particularly on transformed U937 monocytes. In another advantageous embodiment of the invention, monoclonal antibodies or their idiotypic fragments are characterized in that they recognize an epitope present at the level of glycoprotein IIa, the Ia-IIa complex, the Ic-Ia complex, the glycoprotein GP110-120, said antibodies or their idiotypic fragments having the ability to interfere with platelet physiological reactions. These antibodies recognize an epitope or determinant carried by glycoprotein IIa. Consequently, they recognize protein complexes involving glycoprotein IIa or its transformation or degradation products. In a particularly preferred manner, these antibodies are LYP22 antibodies or antibodies with equivalent immunological and functional properties. Analogous tests to those described above are used to select antibodies with equivalent immunological and functional properties, taking as a basis for comparison. LYP22. The monoclonal antibody LYP22 (also known as P22) has the particular advantage of targeting proteins that function as collagen receptors on platelets and are therefore important for platelet adhesion to the walls of damaged blood vessels. This antibody also recognizes an epitope located on a beta (ss) subunit of VLA lymphocytes. Furthermore, the monoclonal antibody LYP22 recognizes antigens present on endothelial cells, these antigens having the same characteristics as those found on platelets. LYP20 and LYP22 antibodies have the advantage of inhibiting platelet activity by approximately 60% in the presence of low concentrations of agonists such as, for example, 2.5–5 pM ADP, 2 pg / ml collagen, and 0.016 U / ml thrombin. This corresponds to a modulation of platelet activity, as it is not completely inhibited. Other antibodies of particular interest for the realization of the invention are monoclonal antibodies whose idiotypic fragments are characterized in that they recognize an epitope of the IIb-IIIa complex on the surface of stimulated platelets, said antibodies or their idiotypic fragments having the ability to inhibit platelet functions and being devoid of this ability when in the presence of high concentrations of agonists as described above. Particularly preferred antibodies are LYP18 antibodies, or antibodies with equivalent immunological and functional properties. Tests similar to those described above are used to select antibodies with equivalent immunological and functional properties, using as a basis for comparison, LYP18. These antibodies, and in particular the LYP18 antibody, have the advantage of detecting both antigens present on resting platelets and antigens expressed after cell stimulation. The invention also relates to a mixture of monoclonal antibodies or of idiotypic fragments of these antibodies or even a mixture of antibodies and fragments, characterized in that it comprises at least two different antibodies or fragments described above. Particularly preferred mixtures of antibodies and / or fragments of these antibodies are, for example, mixtures comprising LYP20 and LYP22 and / or their idiotypic fragments. Another preferred mixture is that of LYP20 and LYP18 and / or their idiotypic fragments, or yet another mixture of LYP20. LYP22 and LYP18 or their idiotypic fragments. Antibodies or their idiotypic fragments meeting the definition of the invention are particularly interesting for their platelet function modulation properties. These antibodies and their idiotypic fragments can therefore be used as antiplatelet agents in patients at risk of platelet function disorders, and in particular problems related to thrombosis formation. In this respect, the antibodies of the invention and their fragments allow for the avoidance of complete inhibition of platelet function, which generally leads to a risk of dangerous bleeding for the treated patient. The antibodies or their fragments defined in the preceding pages can therefore be used separately or in mixtures in order to target the inhibition of platelet functions as needed and / or to obtain levels of inhibition of platelet functions adapted to the situation that one wishes to treat. The invention also relates to monoclonal antibodies or their idiotypic fragments as described above characterized in that they are labeled, the labeling preferably being carried out with a radioactive substance, in particular a halogenated or metallic radioelement, for example 1251, 1311, 1111n or 99T, or by a fluorescent substance. The labeling substance must be chosen according to the intended use of the antibodies, in vitro or in vivo. If these antibodies are used in vivo, the chosen substance must be physiologically acceptable to humans and suitable for detection by means such as imaging. Different labeling protocols can be used, depending on the chosen marker. For example, the techniques described in the publication Mason, DW et al (1980) Biochem J 187, 1-9 will be used. For techniques relating to the use of antibodies after labeling, see, for example, the publication McGregor JL et al (1986) Eur J. Bioche 159, 443-449. The invention relates in this respect to a method for labeling antibodies or their fragments as defined above, characterized in that it comprises the following steps - bringing the antibody or its fragments to be labeled into contact with a specific marker, for example a radioisotope, under conditions allowing coupling of the marker and the antibody, - where appropriate, purification for example by gel filtration, to eliminate components that have not reacted. If labeling of antibodies or their fragments is performed indirectly, for example with technetium-99, known chelating agents will be used to complex the marker and achieve coupling with the antibody or fragments. Usable chelating agents include polyaminocarboxylic acids, notably DTPA (diethylenetriaminepentaacetic acid), diethylenetriaminepentaacetic acid (EDTA) analogs, diaminodithiol compounds, triaminothiols (e.g., thioacetamide / pentanoil), polycyclic amines, dithiosemicarbozones, and metallothiones or anhydrides of these acids, particularly cyclic anhydrides. These chelating agents are prepared using known methods. For technetium labeling, see, for example, the publication by Eckelman et al., Nucl. Med. Biol. vol 16 no 2, pp 171-176, 1989). Also within the scope of the invention is the epitope recognized by the monoclonal antibodies defined above or recognized by the idiotypic fragments of these antibodies, this epitope being present at the level of an antigen involved in the steps of platelet physiology and in particular in the reactions of adhesion and / or aggregation and / or platelet secretion and / or in the adhesion reactions of endothelial cells, said antigen being chosen more particularly from among the proteins GMP140, the glycoprotein GPIIa or the GPIIb-IIIa complexes, GPIa-IIa, GPIc-IIa, characterized in that it is a conformational epitope, necessary for the activity in the above reactions of the antigen which contains it. A particular epitope of the invention is an epitope sensitive to the reduction of the antigen that contains it. The invention relates more specifically to the distinct epitopes recognized by each of the LYP20 and LYP22 antibodies. Another epitope covered by the invention is an epitope meeting the general definitions above, characterized as follows: a) in that it is recognized by complex IIb IIIa of human platelets. b) in that it is not recognized by the complex IIb-IIIa of dog platelets, c) in that it is recognized by the IIb-IIIa complex analogue (IIb-IIIa like complex), present on endothelial cells. Preferably, this epitope is the epitope recognized by the LYP18 antibody. The epitopes recognized by the different antibodies or idiotypic fragments of these antibodies are further characterized as follows A first protocol for characterizing them includes - the proteolytic degradation of the glycoprotein believed to contain the tepitope to obtain small fragments, - localization of the monoclonal antibody determinant by SDS PAGE and Western blot, using small glycoprotein fragments resulting from proteolysis, - obtaining the amino acid sequence of the fragment(s) recognized by the monoclonal antibody, - the production of overlapping peptides around the amino acid sequence believed to contain the epitope, - the selection of the peptide(s) capable of inhibiting the binding of the antibody with its receptor during an inhibition reaction test using the monoclonal antibody and the purified glycoprotein or washed platelets. This technique can be implemented with reference to the publications of Foster PA et al (1988) J Biol Chem 263, 5230-5234 and Geysen MH et al Ciba Foundation Symposium 119, 130-149. Obtaining epitopes is further characterized by the following steps: - the use of monoclonal antibodies having an inhibitory effect and capable of binding in Western blot, - isolation of the complete glycoprotein gene from a lambda gtll cDNA library of a cell line expressing the antigen recognized by the antibody, - gene sequencing, - the isolation of cDNA of different sizes, recognized by the monoclonal antibody, - the determination, from the isolated cDNA, of the determinant of the monoclonal antibody according to a process conforming to the first protocol. This protocol can be carried out by reference to the publication Bahou WF et al (1989) J Clin Invest 84, 56-61. The invention also relates to a composition for diagnosing platelet activation in a subject likely to have a pathology associated with platelet physiology disorders, particularly for detecting thrombopathy, characterized in that it comprises at least two different antibodies or their idiotypic fragments as defined above, preferably antibodies directed against distinct proteins or glycoproteins, in particular a mixture of antibodies selected from LYP20, LYP22, and LYP18 or their idiotypic fragments. For detection purposes, these antibodies or antibody fragments are labeled. Other compositions for the diagnosis of platelet activation, meeting the above definitions, are compositions characterized in that they further comprise monoclonal antibodies or their idiotypic fragments directed against thrombospondin and preferably antibodies of the LYP9 and / or LYP11 type. LYP9 and LYP11 antibodies are antibodies directed against thrombospondin obtained by a method such as that described in the publication of Clezardin et al (Eur J Biochem. 154, 95-102 (1986)). The diagnostic compositions described above can be used to detect in vitro in a biological sample such as blood, the presence of platelet antigens, significant of a thrombotic state. These compositions can also be used for in vivo diagnostics, when monoclonal antibodies or their fragments have been previously labeled under physiological conditions, allowing their detection with suitable devices and in particular through gamma scintigraphy and NMR methods. Another advantage of the diagnostic compositions described above is that they allow for selective diagnosis of the presence of certain particular antigens characteristic of specific stages of platelet physiology. A class of monoclonal antibodies or fragments according to the invention can also be used, in the form of an in vitro diagnostic composition, for the detection of metastatic melanomas and their differentiation from benign melanocytes. These monoclonal antibodies are preferably the LYP18 antibodies of the invention or antibodies exhibiting similar immunological and functional properties, particularly their ability to recognize the complex analog IIb-IIIa (IIb-IIIa "like11). The IIb-IIIa "like" complex is characterized by an identical molecular weight, in reduced or unreduced form, to that of the platelet glycoprotein ITb-IIIa. This IIb-IIIa "like" complex also presents an IIb-ss "like" fragment (reduced form) like reduced IIb platelets (platelet II-ss). The invention also relates to a pharmaceutical composition characterized in that it comprises, as an active ingredient, a monoclonal antibody or its idiotypic fragments, processed under physiological conditions or a mixture of antibodies and fragments as described above or a mixture of these different antibodies and / or idiotypic fragments, in combination with an acceptable pharmaceutical vehicle. According to a first preferred aspect of the invention, such pharmaceutical compositions can be used for the treatment of various pathologies resulting from disorders of platelet physiology or related to these platelet functions. One use of pharmaceutical compositions comprising the antibodies of the invention or their fragments may be the treatment of cardiovascular diseases. According to another particular aspect of the embodiment of the invention, the pharmaceutical compositions are characterized in that the antibody(ies) or their idiotypic fragments are - either coupled with an anticoagulant substance or a substance with thrombolytic activity, this substance being for example tpa, streptokinase or urokinase, - either modified at the level of one of their idiotypic chains so as to replace one of the variable fragments or at least part of these fragments with one of the aforementioned anticoagulant substances. When the idiotypic fragment of the antibody is coupled to a substance having the activity described above, the bond between the fragment and this substance is a covalent bond resulting from the implementation of a protocol such as that described by Bode et al. (1985) Science 229, 765. Other usable techniques are those described by Frantz and Robertson in Infect and Immunity, 33, 193-198 (1981) or that described in Environment Microbiology (October 1981) vol. 42 no. 4, 611-614. PE Kauffmann. In practice, the following compounds, cited as a non-limiting example, will be advantageously used as coupling agents: glutaric aldehyde, ethyl chloroformate, water-soluble carbodiimides [N-ethyl-N'(3-dimethylamino-propyl) carbodiimide, HCl], diisocyanates, bis-diazobenzidine, di- and trichloro-s-triazines, cyanogen bromides, benzoquinone, as well as the coupling agents mentioned in Scand. J. Immunol., 1978, vol. 8, p. 7-23 (Avrameast Ternynck, Guesdon). In the case where the pharmaceutical composition is composed of an antibody or a mixture of these antibodies and / or their idiotypic fragments coupled or modified by an anticoagulant or thrombolytic substance, this composition, associated with a physiologically acceptable vehicle, is administered to the patient at a dose comparable to that which would be used for one of the constituents of the composition taken in isolation. The invention further relates to the use of antibodies or their idiotypic fragments taken separately or in mixture, for the manufacture of a drug intended for the treatment of pathologies related to disorders of platelet physiology. The invention also relates to the use of antibodies according to the invention to modulate, or even inhibit, the interaction between platelets and monocytes. In this respect, it relates to an agent capable of modulating the interactions between monocyte-like cells and stimulated platelets, characterized in that it comprises at least one monoclonal antibody according to the invention. A particularly advantageous agent is characterized in that it comprises the LYP20 antibody. According to another preferred aspect of the invention, the above-mentioned pharmaceutical compositions can be used in a different application from the treatment of cardiovascular diseases, to block the proliferation of melanoma cells and thus thwart the metastatic invasion of these cells. In this respect, the invention relates to pharmaceutical compositions comprising the monoclonal antibody LYP18 or an antibody having the property of blocking the proliferation of melanoma cells, for inhibiting the proliferation of tumor melanomas in vivo. The invention also relates to the application of LYP18 antibodies or their idiotypic fragments, to inhibit the growth of malignant melanoma cells. The invention also relates to the use of LYP18 antibodies or their idiotypic fragments, or antibodies having melanoma recognition properties of the type described for LYP18, for the in vitro diagnosis of malignant melanomas, in particular for differentiating these tumors from other tumors such as lymphomas or carcinomas. In this respect, the invention relates to a composition for the in vitro diagnosis of melanoma tumor cells, characterized in that it comprises LYP18 antibodies. The invention also relates to a method for preparing monoclonal antibodies according to the invention, characterized in that it comprises the following steps a / fusion of mouse splenic cells with myeloma cells, said mice having been previously immunized with the antigen against which antibodies are to be produced, the splenic cells being in excess relative to the myeloma cells, in the presence of a fusion promoter, for example PEG, b / culture of hybridomas formed by fusion, c / cloning and subcloning of hybridomas producing the desired antibodies, d / production of ascites in mice, e / recovery and purification of said antibodies. For the production of LYP18 or LYP20 antibodies, human blood platelets treated with chymotrypsin at a concentration of 0.2 mg / ml / 109 platelets for 30 minutes at 370C are used as antigen. In this case, the myeloma cells used for fusion are, for example, Sp2*gl4 cells. Other myeloma cells for fusion are P3x6 Ag8. For details, refer to the obtaining protocol in the example given for LYP20. For the preparation of LYP22 antibodies, a mixture of GMP1QO antigens, glycoproteins Ia, Ic, IIa, and GP110-115 is used to immunize mice. In this case, the myeloma cells used for fusion are advantageously P3x63Ag8 cells. Other myeloma cells for fusion are Sp2 / Agl4. Refer to the examples for details of the production protocol. Other features and advantages of the invention will become apparent in the examples and figures that follow. Figure 1 Elution profile of Lubrol-treated platelet extracts obtained from a Mono~2 column Sepharose Rapid Flow on-FPLC The eluted fraction from the Sepharose WGA column was dialyzed against 0.1% Lubrol in a buffer Tris. Peak 1 was eluted at 0.15 M NaCl, peak 2 at 0.25 M NaCl, peak 3 at 0.32 M NaCl, peaks 4, 5, 6 eluted between 0.6 - 0.75 M NaCl. SDS-PAGE analysis of peaks eluted by anion exchange chromatography. Each fraction was concentrated and processed under non-reducing (NR) and reducing (R) conditions. The samples were subjected to 7.5% SDS-PAGE electrophoresis and the gels were silver-stained. Firuse 2 Comparison of data obtained in SDS-PAGE of GPIIb-IIIa, antigen 512 and P20. Platelet lysates labeled with I251 were incubated with non-immune IgG (C), P20 with S12, and with an anti-GPIIb / IIIa P18 antibody. Samples were denatured under non-reducing and reducing (R) conditions, placed on a 5–15% SDS-PAGE gradient, and autoradiographed. Line A shows the total labeled, non-reduced lysate. Figure 3 Immunoblot results of the antigen corresponding to P20. Platelet lysates were denatured under non-reducing (NR) and reducing (R) conditions and placed on a 5–15% SDS-PAGE gradient before transfer to Immobilon strips. Strips P20 and P21 were incubated with P20 and P21, respectively, while strip C was incubated with non-immune IgG (c). Western blotting was performed using the peroxidase-conjugated secondary antibody. Figure 4 Binding of P20 monoclonal antibody to unstimulated and stimulated platelets. Washed platelets at a concentration of 10 / ml were incubated for 30 minutes at 250C in a complete Tyrode solution with different concentrations (0.1-4 pgXml) of P20 labeled with 1251. Washed platelets showed increased binding of labeled P20 with 1251 after stimulation with 1U / ml of alpha-thrombin. Figure 5 Effect of P20 on platelet adhesion. The monoclonal antibody P20 (50 pg / ml) partially inhibits (60%) aggregation induced by 2.5 M ADP, a dose of 40 g / ml inhibits aggregation induced by 2 g / ml of collagen (63% inhibition) P20 (90 g / ml) also inhibits (50%) aggregation induced by 0.016 U / ml of thrombin. Figure 6 Binding of the monoclonal antibody LYP18 to megakaryocytes present in a bone marrow biopsy (frozen sections to be cut then treated with peroxidase until stained and stained with ethylene blue (x200) Figure 7 Binding of the monoclonal antibody LYP18 to metastatic malignant melanomas of the lymph nodes. A and box (left): frozen sections treated with peroxidase until stained and then stained with methylene blue as an additional dye (x100; box x200). LYP18 does not bind to neo-melanocytes of the nevi- B (right): frozen sections treated with alkaline phosphatase for initial staining and then stained with hexatoxyline (x200). Figure 8 Growth of M3Dau tumors in the presence of LYP18, G7A5, and IgG2a antibodies Figure 9 Effect of LYP18 injection on tumor growth. EXAMPLE 1 - Isolation and characterization of platelet alveocroteins by FPLC chromatography followed by a gas phase sequence. In this experiment, glycoproteins present in small quantities on the surface of platelets were isolated. This was achieved using rapid liquid chromatography (FPLC) and gas sequencing, allowing for the study of the structure, immunological characteristics, and functional properties of the glycoproteins present in small quantities on platelets. Platelet membranes solubilized in sodium deoxycholate were applied to a column of wheat germ (WGA) and lectin (Lens Culinaris). LCH) mounted sequentially with chromatography FPLC. Glycoproteins eluted from lectin columns in a non-ionic detergent were then separated on an anion exchange column (Mono-Q). Peaks eluted from the Mono-Q column at different NaCl concentrations were subjected to one- and two-dimensional electrophoresis on SDS polyacrylamide gels and analyzed by Western blot on Inmobilon PVDF membranes. Glycoproteins present on the Inmobilon membranes were directly sequenced using an automated gas sequencer. The WGA column allowed the binding of a mixture of antigens including GPIb, the GPIIIb (or GPIV) GPIIb-IIIa in addition to a number of proteins such as GP150 / 155(GPIa) (unreduced / reduced molecular weight) GP15 / 13u(GPIc), GP130 / 135(GPIIa), GP128 / 132(GMP14G and GP115 / 120 (still identified as GP110 / 115). These glycoproteins, separated by FPLC, were then identified using a monoclonal antibody (mAb) or polyclonal antibodies. GP128 / 132 was identified as corresponding to GMP140 or PADGEM by a LYP21 monoclonal antibody. GP15Q / 155 and the GP130 / 135 have been identified as a complex GPIa-IIa and GPIc-IIa by a monoclonal antibody LYP22. The LCH column primarily bound the GPIIb-IIIa complex, identified by an anti-GPIIb-IIIa monoclonal antibody (LYP4). The N-terminal portions of a limited number of these separated glycoproteins were microsequenced using a gas sequencing system. The combined use of FPLC and gas sequencing allows for the rapid isolation and characterization of platelet glycoproteins. Furthermore, the identification of N-terminal amino acid sequences enables the synthesis of oligonucleotide probes to screen and isolate different cDNA clones of glycoproteins from databases. Materials and methods Membrane preparation Concentrated human platelets were obtained at the Beynost blood transfusion center. The platelet membranes were prepared according to the method described by Cooper et al (1979, Proc. Natl. Acad. Sci. 76: 1069-11073), using 10 units of human platelet concentrate (50 x 10⁹ platelets / unit). The sedimented red blood cells were separated from the platelets by centrifugation at 160 g for 25 minutes at 250°C. The platelets were isolated and washed using the method of Massini et al. modified by McGregor et al. (respectively, 1974, Biochiica and Phyica Acta 372: 109-121; 1979, Thromb. Res. 16: 437-452). The washed platelets were resuspended (5 x 10⁹ platelets / l) in 15 mM Tris(Hydroxymethylaminomethane)-HCl (Boehringer). Mannheim, Meylan, France) at pH 7.4 with 5mM EDTA (BDH, Poole, Great Britain) and 1 M leupeptin (Boehringer Mannheim). These platelets were then subjected to a Branson sonication device set to discontinuous mode with a 50% duty cycle and an ultrasonic intensity level of 5. The ultrasonically treated platelets were centrifuged at 4°C for 20 minutes at 9000 g in a fixed-angle SS34 rotor using a Sorvall RC2B centrifuge (DuPont de Nemours, Newtown, Connecticut, USA). The supernatant was then removed and centrifuged for one hour at 4°C at 100,000 g in a variable-angle TST 55.5 rotor using a Kontron 2060 centrifuge (Kontron, Zurich, Switzerland). The pelleted merranes were resuspended in buffer containing 10 mM Tris-HCl at pH 8.2, in 5 mM EDTA and 1 ijM of leupeptin. Extraction with sodium deoxycholate (DOC): Platelet membranes were treated for 30 minutes at 40°C with 1% (w / w) sodium deoxycholate (Merck, Darmstadt, Germany) in 10 MM Tris-HCl at pH 8.2 with 5 mM EDTA and 1 pM leupeptin. Antigens from the platelet membranes were then ultracentrifuged at 40°C for 30 minutes at 100,000 g in a variable-angle rotor. TST 55.5 using a Kontron 2060 centrifuge. The soluble supernatant containing the antigens from the ultracentrifuged membranes was collected. Lectin affinity chromatography Wheat germ lectin (WGA) bound to Sepharose 6MB (Pharmacia, Uppsala, Sweden) was placed in an HR10 / 10 glass column (100 m x 10 mm) (Pharmacia). The column was then connected to a rapid full-flow liquid chromatography (FPLC) system (Pharmacia). The column was equilibrated in a starting buffer containing 10 mM Tris-HCl at pH 8.2 with 0.5% DOC and 5 mM EDTA. Antigens from the solubilized platelet membranes were loaded onto the column at a flow rate of 0.5 mL / min, and the column was washed with starting buffer until an optical density equal to the baseline value was achieved. Glycoproteins bound to the WGA column were eluted with a sugar, 2.5% N-Acetyl Glucosamine (Sigma, St. Louis, MO, USA). The elution peaks were dialyzed overnight against 10 mM Tris-HCl at pH 7.4 with 0.1% Lubrol PX (Sigma). Anion exchange chromatography: Glycoproteins eluted from the lectin column and resuspended in lubrol PX were placed on a MONO Q anion exchange column HR5 / 5 (50 mm x 5 mm) (Pharmacia). The starting buffer contained 0.1% Lubrol PX in 20 mM Tris-HCl at pH 7.4, and the final buffer consisted of 0.1% Lubrol PX in Tris-HCl at pH 7.4 containing 1 M sodium chloride (Merck). A growing medium was established for 25 minutes at a flow rate of 1 mL / min. D-immunoaffinity chromatography: Glycoproteins eluted from the column MONO Q antibodies were identified on immunoaffinity columns using the Sepharose-4B-bound monoclonal antibodies LYP21 (directed against GMP140) and LYP22 (directed against gpIIa). These antibodies were obtained after immunization of mice with a mixture of glycoproteins isolated by affinity on WGA column and anion-exchange chromatography. Glycoproteins IIb-IIIa were identified on immunoaffinity column using the monoclonal antibody LYP4 described by McGregor et al (1986, Eur. J. Biochem. 159: 443-440). EXAMPLE 2 - Inhibition of platelet aqreq induced by collagen, with anti-gpIa-IIa monoclonal antibodies. A glycoprotein (GP) Ia-IIa (VLA-2) has been shown to play an important role in platelet adhesion to collagen. Platelets from a patient with a GPia deficiency do not undergo aggregation in response to collagen. A monclonal antibody, LYP22, was obtained from mice immunized with a mixture of platelet membrane glycoproteins isolated by wheat germ agglutinin affinity chromatography coupled with anion-exchange chromatography. Subsequent immunoprecipitation of a surface-iodinated platelet lysate solubilized in a detergent showed that LYP22 precipitated two glycoproteins with estimated molecular weights under non-reducing and reducing (NR / R) conditions of 140 / 155 kDa and 120 / 135 kDa, respectively. The changes in molecular weights of these two glycoproteins due to reduction are consistent with those previously described for GPIa and GPIIa, respectively. When platelets solubilized with Lubrol were contacted with LYP22 on a Sepharose affinity column, two other glycoproteins (120 / 128 kDa (GPIc) and 100 / 105 kDa) were detected in much smaller quantities using Coomassie blue.Studies of the binding of the iodine-125 (12,5I)-labeled LYP22 antibody to platelets showed that the binding is on the order of 7708 ± 928 molecules per resting platelet and 8940 ± 86 molecules per platelet activated by thrombin (Kd = 65.6 nM ± 1.43 nM). These data suggest that the surface expression of LYP22 binding sites is not increased after platelet activation. To determine the effect of LYP22 on platelet function, aggregation studies with washed human platelets were performed. LYP22 partially inhibits platelet aggregation induced by a low dose of collagen (lpg / ml) but has no effect on other agonists. However, LYP22 has no effect on platelet adhesion to collagen, fibronectin, or fibrinogen in the presence of 2 mM magnesium. These results show that a / LYP22 is directed against the GPIa-IIa complex, GPIc-IIa and indirectly against a degradation product of GPIIa or of a glycoprotein attached to GPIIa. b / The GPIIa-TIa and Ic-IIa complex is involved in collagen-induced platelet aggregation, via GPIIa. c / P22 has an effect on platelet adhesion when in the presence of collagen 10pg / ml. EXAMPLE 3 - Inhibition of platelet functions of human platelets by a monoclonal antibody LYP20 directed against a membrane glycoprotein whose expression depends on activation. A limited number of membrane glycoproteins (Ib, IIb-IIla, fa-IIa, Ic-IIa, IIIb) play an important role in platelet aggregation and adhesion. The monoclonal antibodies LYP20 and LYP21 were used. LYP20 antibodies were obtained after immunization of a BALB / C mouse with a concentrate of washed human blood platelets treated with chymotrypsin (0.2 µg / ml / 10⁹ platelets for 30 minutes at 370°C). The monoclonal antibodies LYP21 were obtained with a mixture of 4 platelet membrane glycoproteins eluted from a wheat germ agglutinin affinity column. Immunoprecipitation using surface-labeled and then lysed platelets, activated with thrombin-, showed that LYP20 and LYP21- precipitate a 128 kDa (unreduced) and 132 kDa (reduced) protein which is called GP128. LYP20 and LYP21 antibodies recognize distinct epitopes on gp128, with the antigenic determinant of LYP20 depending on the presence of intact disulfide bonds. Washed resting platelets bound 2400 ± 270 molecules per iodine-125 labeled LYP20 antibody and 12,200 ± 1,184 molecules per antibody after activation by 1 µL of thrombin. These data show that LYP20 and LYP21 antibodies are directed against a platelet surface protein (gp128) whose expression depends on platelet activation and secretion. gp128 migrates in conjunction with the platelet antigen immunoprecipitated by an antibody directed against the glycoprotein GMPl40 or PADGEM. This demonstrates that gp128 is related to or identical with GMPl40. PADGEM. In order to determine the potential role of the GP128 (also known as GMP14d) in platelet function: studies concerning aggregation with washed human platelets were conducted. F(ab')2 fragments of LYP20, but not of LYP21, partially inhibit platelet aggregation and release induced by 2.5M ADP, 2 pg / ml collagen, and 0.016 U / ml thrombin. These results show that a / LYP20 and LYP21 are directed against different epitopes of the platelet surface protein (GP128) whose expression depends on platelet activation b / the GP128 is related to or identical to the GMP140 or PADGEM c / GP128 exposed to the surface of activated platelets can play an important role in platelet aggregation. EXAMPLE 4 - Preparation of anticormonoclonal drugs targeted against the GMP 140 protein - Material Adenosyl diphosphate (ADP), aprotinin, fibronectin (FN), lactoperoxidase and leupeptin, lubrol WX, N-acetyl glucosamine, orthophenyldiamine (OPD), phenylmethylsulfonylmethane (PMSF), prostaglandin E1 (PGE1) and Triton X-100 were obtained from Sigma (St. Louis, MO, USA). 51-Chromium, 12S-Iodine, and 14C-Serotenine were obtained from Amersham International (UK). Horseradish peroxidase conjugated to anti-goat mouse antibodies, gelatin, HRP color developing reagent, and Tween 20 were obtained from BioRad (Richmont, CA, USA). Sodium deoxycholate (DOC), dithicthreitol (DTT), leupeptin, and PMSF were obtained from Merck (Darstadt, Germany). Polyethylene glycol (PEG) and sodium dodecyl Sulfate (SDS) was obtained from BDH (Poole, UK). Mono-Q-Sepharose Rapid Flow Gel (Mono-Q Sepharose fast flow gel (registered trademark), the protein At Sepharose, wheat germ agglutinin (WGA) and a complete FPLC system were obtained from Pharmacia (Uppsala, Sweden). Bovine serum albumin (BSA) and rabbit anti-mouse IgG antibodies are sourced from Miles Laboratories (Naperville, IL, USA). The collagen comes from Hormon-Chemie - (Munich, (West Germany). Thrombine was obtained from Hoffman-La Roche (Basel, Switzerland) and alpha-thrombin, fibrinogen (FG) were obtained from IMCO. Pansorbin was obtained from Calbiochem (La Jolla, CA, USA). All cell culture reagents were obtained from Flow Laboratories (Les Ulis, France) and Intermed (Lyon, France). 2,4,6,10-teramethalpentadecane (Pristanej) was obtained from Aldrich Chemical Company (Milwaukee, WI, USA). Polyvinylidene difluoride membranes (Immobilon) were obtained from Millipore Corporation (Bedford, MA, USA). D-Phenyl-L-propyl- L-arginine chloromethyl ketone (PPACK) comes from Calbiochem-Behring (Paris, France). - Methods a) Preparation of plates and marking Platelet concentrates (Center for Blood transfusions from Beynost) were washed according to the technique of Massini et al (1974), Biochim biophys. Acta 372, 109-121. The surface proteins were iodized according to the method described by Philipps et al. (1977) J. Clin. Invest. 60, 535-545, using lac-toperoxidase. The platelets used in the fixation, aggregation and adhesion studies were obtained from the blood (1 ACD volume: 6 blood volumes) of normal volunteers. The platelets were isolated and washed according to the technique of Mustard et al. (1972) Br. J. Eaematol. 22, 193-204 with slight modifications 20 ng / ml PGE1 and 10- 6 M PPACK were added to the first wash. b) Isolation of platelet membrane glycoproteins Platelets washed according to the method of Massini et al. cited above were resuspended at 40 x 10⁹ cells / ml in 0.02 M Tris-HCl at pH 8.6, with 0.005 M EDTA, 1% (w / v) DOC, 17 U / ml aprotinin, and 1 M leupeptin. Solubilization was carried out at 4°C under constant stirring for one hour. The lysate was centrifuged at 100,000 g for one hour at 40°C. The supernatant was diluted in 5 volumes of 0.02 M Tris-HCl at pH 8.6, 1 M EDTA, and 0.5% (w / v) DOC (buffer A) and loaded onto a Sepharose 6B-WGA column. The column was pre-equilibrated in buffer A and coupled to a rapid full-phase liquid chromatography (FPLC) system. The column was washed at 0.4 mL / minute with buffer A until unbound material was removed. The specifically bound fraction was eluted with 2.5% (w / v) N-acetylglucosanine. The collected fraction was dialyzed overnight against a mixture of 0.02 M Tris-HCl at pH 7.4, 0.005 M EDTA, and 0.1% Lubrol. PX at 4 C. The dialysis sample was subjected to anion exchange chromatography using a fast-flow Mono-Q column, connected to the FPLC system and pre-equilibrated in 0.02 M Tris-HCl at pH 7.4 containing 0.18 tw / v) of lubrol WX (buffer B). The sample was applied to the column at a flow rate of 2 ml per minute, and the different fractions were eluted using a NaCl (0 to 1 M) gradient in buffer B. Each fraction was collected, concentrated, and analyzed by SDS-PAGE using the Laemmli (1970) technique (Nature 227, 680-685). The protein concentration was determined by the method of Lowry (1951) modified by Markwell (1978). Anal. Biochem. 87, 206-210. For more details concerning the isolation of glycoproteins, see example 1. c) Production of non-non-clonal antibodies c.1) Preparation of the LYP21 antibody BALB / c mice (6-week-old females), obtained from IFFA CREDO (L'Arbresle, France), were intraperitoneally immunized with 100pg of a mixture of 4 glycoproteins (isolated as described above and corresponding to glycoproteins GMP140, Ia, Ic, IIa, and GP110-115), with an equal volume of complete Freund's adjuvant. Two weeks later, 100pg of the antigen was injected into the mice in the presence of incomplete Freund's adjuvant. We verified that the mice's serum reacted to the antigen using the technique ELISA. In this regard, the technique is briefly recalled: plastic wells were coated with 100 µL of antigen (a mixture of 4 glycoproteins) diluted to 2 pg / ml in bicarbonate buffer (25 mM Na₂CO₃, 25 mM NaHCO₃, pH 9.6) and left for 3 hours at 370°C. The wells were washed and saturated with 0.5% BSA in Tris buffer (15 mM Tris-HCl, pH 7.4, 2 mM). CaCl2, 0.05% (w / v) (Tween) for 30 minutes at room temperature. Mouse serum (100 µL) was incubated in the wells for 3 hours at 37°C. After 3 washes in Tris buffer, a goat mouse antibody (secondary antibody, diluted to 1 / 3000) conjugated to peroxidase was added to each well and reacted for 30 minutes at 370C. The wells were then washed again and filled with a 100-inch substrate solution (4 mg OPD in 10 ml of 0.1M citrate buffer, at pH 5, with 0.7 µl of 30% H2O2), The reaction was stopped with 100 wu of 4t1H2SO, 4N. The optical density (492 nmi) was measured with a plate reader. Four weeks after the first injection, the immunized mice underwent a booster dose of 100 pg of antigen mixture. Splenic cells were fused four days later, according to the method of Kohler and Milstein (1975) Nature 256, 177-181, with P3X63Ag8 myeloma cells in a 10:1 ratio, in the presence of PEG. The hybridomas were divided and distributed with peritoneal macrophages from non-immune mice of the same strain as the immunized mice, these macrophages being used as feeder cells, in 24-well plates in hypoxanthine / aminopterin / thymidine (HAT) medium 24 hours later. The hybridoma supernatants were screened using the technique ELISA described above for mouse serum. Positive supernatants were tested for their ability to bind to labeled platelet membrane surface antigens, according to the protocol described in the "immunoprecipitation" section, as well as for their ability to bind to unlabeled platelet membrane antigens, transferred onto Immobilon membranes according to the protocol described in "Immunoblot". Positive wells were cloned and subcloned by limited dilution and screened again using the same techniques before the onset of ascites in pristane-treated BALB / c mice. Monoclonal immunoglobulins were purified by the method described by Ey et al. (1978) I-n. unochemistry 15, 429-436 using protein A-sepharose affinity chromatography. F(ab')2 fragments of a monoclonal antibody were prepared according to the technique described by Lamoyi et al. (1986) Methin Enzym, vol 121, p 652-663, Acad. Press INC. c.2) Preparation of the LYP20 antibody The LYP20 monoclonal antibody was obtained after immunization of BALB / C mice with chymotrypsin-treated and washed human blood platelets. Chymotrypsin treatment (0.2 mg / ml / 10⁹ platelets) was administered for 30 minutes at 370°C. The platelets were then washed according to the technique of Massini et al. Chymotrypsin-treated platelets (1 x 10⁸ platelets / 50 ml) were mixed with an equal volume (50 ml) of complete adjuvant and injected intraperitoneally into mice. BALB / C. Mice were immunized twice over four weeks with the treated platelets. These mice were then kept at rest for one month before undergoing a booster with chymotrypsin-treated platelets. Three days later, splenic cells were harvested and fused with Sp2 / Ag14 cells using the previously described technique to prepare LYP2α antibodies. Fusion supernatants obtained with chymotrypsin-treated platelets were selected for their ability to inhibit platelet function. These LYP21 or LYP20 antibodies were purified by Protein A Sepharose CL64B column chromatography as previously described by Ey PL et al 1978, Immunochemistry 15, 429-436. Immunoprecitation Immunoprecipitation was performed according to the method described by McEver et al. (1984) J. Biol. Chem, 259, 9799-9804. Platelets were activated (1 U / ml of platelet concentrate for 10 minutes, followed by 10⁻⁶ M PPACK for 10 minutes) prior to iodination, solubilized in 0.01M Tris-HCl, 0.15M NaCl, 1 / 1M EDTA, containing 1% Lubrol PX (Bulk S), and microcentrifuged. Labeled supernatants were incubated with either hybridone supernatant, ascitic fluid, or purified antibody for 2 hours at room temperature with constant stirring. 10 µL of purified anti-rabbit mouse antibody, diluted 1 / 10 in Buffer S, was added to each sample and placed on a rocker shaker at room temperature for 30 minutes. Sepharose protein A (10% in buffer S) was incubated (1 volume of lysate to 2 volumes of protein A) with each sample on a rocker shaker for 30 minutes at room temperature. Centrifugation was then performed at 9,000 g for 2 minutes in an Eppendorf centrifuge. Each pellet was washed 4 times in buffer S. After the final wash, each pellet was resuspended in 0.4 ml of Laemmli buffer (1970) Nature 227, 680-685, and then heated at 1000°C for 5 minutes. A fraction of each sample was reduced in the presence of 0.04 M DTT. All samples were then microcentrifuged, and the supernatant was stored at -700°C. SDS-PAGE and immunoblotting One-dimensional gel electrophoresis was performed according to the Laemmli method. SDS-PAGE cn two dimensions (non-reduced / reduced) was performed according to the method of Phillips et al. (1977) J. Clin. Invest. 60, 535-545. The gels were silver-stained according to the method of Merril et al. (1982i Electrophoresis, 3, 17-23. Immobilon gels and membranes were equilibrated in 0.025M Tris, 0.192M glycine, at pH 8.3, for 30 minutes before performing a western blot. The proteins were transferred by electrophoresis according to the method of Towbin (1979) Proc. Acad. Sci. USA 76, 4350-4354, from gel to Immobilon membrane using a BioRad miniblot apparatus at 100 V for one hour. After transfer, the membrane was incubated for 30 minutes in TBS buffer (0.02 M Tris, 0.05 M NaCl, pH 7.5) containing 0.05% (w / v) Tween-20 and 3% (w / v) gelatin to block nonspecific binding sites. Immobilon bands were cut and placed in a tube containing either hybridoma supernatant, ascitic fluid, or purified antibody. Each tube was shaken on a rocker for one hour at room temperature. The strips were then washed three times in TBS buffer containing 0.05% (w / v) Tween-20 and 1% (w / v) gelatin. They were then incubated for 30 minutes in the presence of horseradish peroxidase conjugated to goat-mouse antibodies, diluted 1 / 3000 in TBS-Tween buffer containing 1% gelatin. All strips were washed once in TBS-Tween buffer and twice in the TBS. The substrate was prepared for use, using 60 mg of color developing reagent. HRP was mixed with 20 ml of methanol at 40C, 100 nl of TBS and 60p1 of 30% H2O2. The reaction was stopped by rinsing the strips with distilled water. Study of platelet fixation The number of LYP20 and LYP21 monoclonal antibodies labeled with I-25 bound to unstimulated and stimulated platelets (by ADP and thrombin) were tested according to the method by McGregor et Al. (1986) Eur. J. Biochem. 159, 443-449. When thrombin (1U / ml) was used, its activity was blocked with 10⁻⁶ M PPACK before the addition of other reagents. Unstimulated and stimulated platelets were incubated with different concentrations of labeled P20 or P21 monoclonal antibodies (0.1 to 4 pg / ml) for 30 minutes at 250°C. Then, 50 pg / ml of the suspension was resuspended and spread onto 400 µl Eppendorf tubes containing 300 µl of 20% (w / v) sucrose and 2% (w / v) BSA diluted in Tyrode's solution. The tubes were centrifuged in an Eppendorf microcentrifuge for 6 minutes. The ends of the centrifuge tubes were cut off and the amount of iodine-125 (12I) labeled antibodies fixed was determined with a gamma counter. Non-specific binding was achieved by adding 100-fold concentrated unlabeled antibodies. The number of binding sites and the degree of dissociation (CD) were determined by Scatchard analysis. Platelet Aggregation and Secretion Aggregation studies were performed with washed platelets treated with serotonin 14C according to the method of Çreenberg et al. (Lab. Invest. 1975, vol. 32, p. 476). Platelets, adjusted to 2 x 10⁸ platelets / ml, aggregated with 2.5 µM ADP, 2 µg / ml of collagen, or 0.016 U / ml of thrombin in the presence or absence of either the P20 or P21 monoclonal antibody (complete monoclonal antibody or F(ab)'2 fragment). After each aggregation, the suspension was microcentrifuged, and 100 µL of supernatant were passed through a counter to measure the level of secretion. Platelet adhesion Adhesion studies were performed on washed platelets treated with slCr (0.1 mCi per 5 x 10⁹ platelets) according to the method described by Cazenave et al. (Lab. Invest n 34 p 471, 1976), PAGE 1 (20 ng / l) was added to Tyrode's solution throughout the procedure. The plastic wells were coated with 100 µl of BSA (0.5%), fibronectin (2 µg / ml), fibrinogen (2 g / l), or collagen (2 g / ml) in 25 mM bicarbonate buffer at pH 9.5 for 3 hours at 370°C. The wells were blocked for 90 minutes at room temperature with 1% BSA. Platelets adjusted to a count of 5 x 10⁸ cells / ml were pre-incubated with either the P20 monoclonal antibody or the P21 monoclonal antibody (10 µg / ml) at 370°C for one hour. The suspension of pre-incubated platelets (100 µl) was added to each well and reacted at 370°C for one hour. All wells were thoroughly washed in Tyrode's solution to remove any adhering nDn platelets. The wells were then separated, and individual mouse counts were performed on a gamma counter. RESULTS isolate.ent glycoproteins from platelet membranes Platelet extracts obtained after detergent treatment were solubilized in 1% DOC and subjected to water affinity chromatography (WGA) in the presence of 0.5% DOC at pH 8.6. No glycoproteins of the GPIIb-IIIa complex were present in the eluted fraction, as shown by the analyses. SDS-PAGE and ELISA. After dialysis versus 0.1% of Lubrol WX, this fraction was loaded onto a fast-flow anion-exchange Q column and eluted in Nazi. The first peak contained four proteins, as shown by the SDS-PAGE result. The molecular weights were 85, 120, 130, and 150 kDa under non-reducing conditions and 85, 125, 140, and 155 kDa after reduction. No GPIb glycoprotein was present in this fraction, as shown by detection after silver staining of the gel. However, GPIb was present in peak ne 3. The first fraction was selected to immunize mice in such a way as to produce monoclonal antibodies specific for the four platelet membrane antigens. Monoclonal antibody production Hybridoma supernatants were screened with a mixture of 4 glycoproteins obtained from a platelet concentrate treated according to the above procedure using the ELISA technique. Several supernatants exhibited a reaction to the antigen mixture. The selected antibodies P20 and P21 were purified on Sepharose protein A and these two antibodies were characterized as IgGl since they were eluted at p.i 6. Identification of the Plauetter antigen recognized by P20 and P21 P20- and P21-labeled platelet lysates were compared by SDS-PAGE to platelet lysates immunoprecipitated with the S12 antibody directed against GMP-140 and described by McEver in the publication already cited. Another comparison was made with the monoclonal antibody specific to the GPIIb-IIIa complex. Autoradiograms (Fig. 2) clearly show that the antigen recognized by P20 is neither GPIfb nor the GPIIIa. The apparent molecular weight of this antigen was estimated at approximately 130 kDa (unreduced) and 140 kDa (reduced). The antigen recognized by P20 migrates along with the immunoprecipitated antigen using an S12 McEver antibody that does not inhibit platelet function. The same results were obtained with P21. This result was confirmed by an analysis Western blot. The P20 monoclonal antibody binds specifically to the antigen under non-reductive denaturing conditions. The apparent molecular weight of the spot was estimated at 128 kDa. However, P21 recognized the denatured antigen under both non-reductive and reducing conditions (Fig. 3). Binding of the P20 antibody to washed platelets Washed platelets (from 4 donors) bound 2,400 ± 266 molecules of labeled P20 antibody per unstimulated platelet (K = 2.3 nM ± 0.54) and 12,200 ± 1,184 molecules of labeled P20 antibody per cell after thrombin stimulation (K = 5 nM ± 0.61) (Fig. 5). The labeled P21 antibody did not recognize either resting or stimulated platelets. Effect of P20 and P21 antibodies on plague adhesion Neither P20 nor P21 have a significant effect on the adhesion of platelets present on plastic wells coated with fibronectin, fibrinogen or collagen (Fig. 5). Effect of P20 and P21 on plasmid aqreq and secretion P20 partially inhibits ADP-induced (60%), collagen-induced (63%), and thrombin-induced (50%) aggregation compared to the control. Fab fragments of P20 enable the obtaining of these results. P20 also reduces collagen- and thrombin-induced platelet secretion. P21 has no effect on platelet aggregation. DISCUSSION Monoclonal antibodies directed against GMP-140 of solubilized platelets were produced. These 'P20 and P21 antibodies tested by immunoprecipitation showed that they exhibit specificity for the platelet membrane antigen also recognized by McEver's S12 antibody. However, unlike the S12, P20 and antibody P21 antibodies are directed against a conformational epitope of GMP-140. Furthermore, P20 modulates platelet function. Another characteristic of these antibodies is their recognition of denatured GMP-140, as demonstrated by the positive results of Western blot. P20 recognizes only unreduced GMP-140, while P21 remains active against reduced GMP-140. These results suggest that the P20 binding site contains disulfide bonds. In contrast, the P21 binding site likely does not contain disulfide bonds. We can conclude at this stage that P20 and P21 are specific to different epitopes on platelet GMP-140. Washed resting platelets bound approximately 2,000 molecules of iodine-labeled p20 antibody. After activation of the platelets with alpha-thrombin, they bound approximately 12,000 molecules of labeled p20 antibody. Activation with ADP did not increase p20 antibody binding. The plates were probably slightly activated during the washing steps, although PGE1 and PPACK were added at the first wash level. The labeled P21 antibodies did not bind to either resting platelets or platelets stimulated with thrombin. These results proved positive. It is concluded that P21 does not bind to whole platelets, whether active or not, as the epitope recognized by P21 is inaccessible on the native GMP-140 membrane. The P21 binding site could be expressed on the cytoplasmic face of GFIP-14Q, this sequence remaining cytoplasmic after activation and expression of alpha-granules at the membrane. It could also be that P21 binding is not possible due to the conformation of native GMP-140, which masks the P21 epitope. However, P21 reacts with whole platelets solubilized in Triton X-100 and subjected to cross-immunoeietroporesis, conditions that are apparently not denaturing. P20 partially inhibits platelet aggregation and secretion. S12 is known to have no effect on platelet aggregation. This confirms that S12 and P20 are directed against different epitopes. The inhibitory effect of the P20 antibody on thrombin-induced platelet aggregation against collagen is entirely consistent with the localization of the GMP-140 is located within the alpha granule membrane. After activation with thrombin or collagen, the alpha granule membrane fuses with the platelet plasma membrane, and GMP-140 becomes a surface receptor. Furthermore, the presence of P20 affects serotonin C14 secretion. EXAMPLE 5: Detection of the presence of qpIIb-IIIalike glycoprotein in normal and neoplastic tissues using LYP18 antibodies Materials and methods Tissue studies Normal, abnormal, and benign skin tissue samples were obtained from the Department of Pathology and the Dermatology at Stanford University Medical Center. Sixteen normal tissue samples from selected organs (Table 1) and 96 tumor biopsies (Tables 2, 3, 4) were obtained from the Department of Pathology. The tumor biopsies included 21 cases of malignant melanoma with lymph node metastases, 20 cases of lymphomas not belonging to the Hodgkin lymphoma category (1 small lymphocytic, 5 dividing follicular, 3 mixed follicular, 3 diffuse small cell, 2 diffuse large cell, 1 immunoblastic large cell, 3 uncleaved small cell, and 2 lymphoblastic), 4 cases of Hodgkin lymphoma with sixty-cell content, 2 cases of thymoma, and 28 cases of carcinomas including 19 cases of primary carcinomas (breast 6, ovary 4, oropharynx 3, lung 2, colon 2, stomach 1, thyroid 1), 9 cases of metastatic carcinomas in the lymph nodes (3 adenocarcinomas, 3 squamous cell carcinomas.3 poorly differentiated), 7 neuroendocrine tumors (4 undifferentiated small cell lung carcinomas, 2 pancreatic islet tumors and one medullary thyroid carcinoma), 1 case of dysgerminoma of the ovaries, 10 cases of sarcomas (5 osteosarcomas, 3 Ewing sarcomas and 2 malignant histiocytomas) and 3 cases of neuroblastomas. 13 cases of benign skin lesions were obtained in the dermatology department including 9 nevis (1 junctional, 1 compound, 2 dermal, 2 dysplastic and 3 congenital), 3 lentigines and 1 dermofibrone. Monoclonal antibodies The non-clonal antibodies LYP2 and LYP18 were selected to perform the following experiments. LYP2 (IgGz) and LYP18 (IgG2a) do not cross during cross-immunoelectrophoresis with platelet glycoproteins IIb or IIIa separated by EDTA but recognize a determinant present on the intact calcium-dependent IIb-IIIa glycoprotein complex (McGregor et al, Eur J Biochem 1986, 159: 443-449). LYP2 and LYP18 were purified from ascitic fluid on a Protein A-Sepharose 4B column as described in the previously cited McGregor et al. reference, at concentrations ranging from 1.0 to 1.5 mg / mL and using a 1:200 dilution. The monoclonal antibody directed against platelet glycoprotein 111e (IgG1) was obtained from Dakopatts (Santa Barbara, CA) as a culture supernatant and then used at a 1:50 dilution. The purified monoclonal antibody Leu 9 (CD7)(IaG2a) was used as a control antibody at a dilution of 1:200 and was obtained from Becton-Dickinson (Mountain View, CA). Immunohistochemical staining All normal and abnormal tissues, as well as specimens with benign skin lesions, were directly frozen, processed in the same manner, and stained as described by Bindl (Am J Cilin Patio) 1986.85: 490-493). In short, serial frozen, air-dried sections fixed with acetone (4-5 µm) were incubated for 30 minutes in a humidified chamber with the various microclonal antibodies. The sections were then serially incubated (with washes in phosphate saline between incubations) in a Coplin container containing biotinylated goat anti-mouse immunoglobulins (Jackson SmmunoResearch Laboratories, West Grove, PA). These sections were then treated with streptavidin-conjugated horseradish peroxidase (Jackson), each incubation lasting 30 minutes at 40°C.The stain was developed with 3 mg / ml 3,3-diaminobenzidine (Sigma Chemical Co., St. Louis, MO) and 0.3% hydrogen peroxide for 5 minutes at room temperature. After staining the reaction product with 0.5% copper sulfate in 1N NaCl for 5 minutes, the sections were washed, stained with 2% methylene blue, dehydrated, and covered with a coverslip. For the study of the pigmented melanocytic lesion, alkaline phosphatase conjugated with streptavidin (Jackson) was substituted for the peroxidase, and the stain was performed in Fast Red Salt (FRS, Sigma) for 2d minutes, as previously described. Results LYP18 strongly stains megakaryocytes in bone marrow, platelets, as well as capillaries covering endothelial cells, venules, and arterioles in normal tissues. The LYP18 monoclonal antibody also exhibits a strong staining reaction with the glomerular epithelium of endometrial glands and weakly stains smooth muscle cells. However, a large number of normal tissues from different organs were not stained with the LYP18 monoclonal antibody (Table 1). The LYP2 monoclonal antibodies stained megakaryocytes and platelets with the same intensity as LYP18 but did not bind to other normal tissues. The control monoclonal antibody Dako, directed against gpfIIa, strongly stained megakaryocytes, platelets, glomerular epithelium, and endometrial glands, but weakly stained endothelial and smooth muscle cells. Among normal cells of a given type, no significant variation was observed in the intensity of the number of cells stained by these three monoclonal antibodies. Of the 21 melanoma cases studied, 16 (75%) resulted in a reaction with the LYP18 antibody. Monoclonal antibodies LYP18 produce variable staining in melanomas. With the exception of one case of very weak staining, no cell-to-cell variation in stain intensity was observed in individual cases. LYP18 exhibits a fine, diffuse cytoplasmic staining pattern with enhanced staining at the peripheral membrane. The staining intensities and percentage of melanomas associated with the Dako anti-gpIIIa antibody are identical to those obtained with LYP18. LYP2 does not bind to metastatic malignant melanoma. On the other hand, normal melanocytes present in the skin, including those in dermatofibromas (1) and activated melanocytes forming a junctional nevi (1), dermal nevi (2), dysplastic nevi (2), and congenital nevi (3), do not allow the binding of LYP18, LYP2, or Dako anti-gpIIIa antibodies (Table 2). Among the 75 non-melanocytic tumors, only 3 (4%) stained for LYP18 (see Table 3): a metastatic papillary thyroid carcinoma, a primary endometriotic adenocarcinoma of the ovaries showing focal positivity, and a weakly stained osteosarcoma. 96% (72 of the 75) did not show LYP18 binding. Dako anti-gpIIIa antibodies bound to various tumors, similarly to LYP18. LYP2 was not associated with the different tumors studied. Discussion These studies demonstrate the presence of IIb-IIIa-like glycoproteins on metastatic malignant melanoma cells and the absence of these glycoprotein complexes on benign melanocytes. Frozen sections of melanoma biopsies were not recognized by other ronoclonal antibodies (LYP2) also directed against the platelet IIb-IIIa complex, but they did recognize the anti-gpIIIa monoclonal antibody Dako, suggesting that gpIIIa is one of the components of the integrin complex in metastatic malignant melanomas. The lack of recognition by LYP2 could indicate structural differences between the IIb-IIIa-like glycoproteins of melanomas and the platelet complex. The presence of IIb-IIIa-like glycoproteins, also known as cytoadhesins, on malignant melanoma cells but not on benign melanocyte cells, can be interpreted as aberrant antigen expression occurring during the malignant transformation process. Cytoadhesins present on melanomas may confer properties to malignant cells that facilitate metastatic invasion and may also explain their interaction with extracellular matrix adhesive proteins and other blood cells. The reason why a quarter were not recognized by LYP18 is unclear. It is possible that the tumor cells did not synthesize these glycoproteins or that the antigens were expressed but at an undetectable level. If the first hypothesis is correct, it may imply that the expression of IIb-IIIa-like glycoproteins is not an essential factor for the establishment of metastases in malignant melanomas. Among the various non-melanoma neoplasms, only 4% showed a link with LYP18. The reactivity of LYP18 with 3 out of 75 malignant non-melanocytic neoplasms could be due to the synthesis of IIb-fIIa-like antigens by these tumors. Another hypothesis is that this same epitope is present on a different molecule or that nonspecific staining occurred. The lack of LYP18 reactivity with a large majority of the non-melanocytic tumors studied suggests that aberrant expression of IIb-IIIa-like glycoproteins is not universal among malignant cells, including metastatic cells. EXAMPLE 6 In vivo inhibition of human melanoma growth by the LYP1S antibody directed against the β-protein complex IIb-IIIa. In this experiment, the binding of the monoclonal antibody LYP18, directed against glycoprotein IIb-IIIa of human platelets, to a highly tumorigenic human melanoma cell line (M3Dau) was tested. The results of this test show that M3Dau melanoma cells bind specifically to 'tI-labeled LYP18 antibodies. To study the biological role of glycoproteins Given the presence of IIb-IIIa-like glycoproteins on the surface of melanomas, an M3Dau melanoma cell line was incubated with LYP18, or with a control monoclonal antibody directed against other melanoma cell surface antigens, and then subcutaneously implanted in nude mice. The results show that LYP18 significantly inhibits tumor growth in vivo. LYP18 is not directly cytotoxic to melanoma cells. These results demonstrate that the gpIIb-IIIa-like glycoprotein is present on melanoma cells and plays an important role in tumor cell growth. Therefore, the use of monoclonal antibodies directed against specific tumor cytoadhesive receptors for tumor treatment is a promising avenue for considering. MATERIALS AND METHODS - Cells M3Dau cell lines were obtained from an acromial skin metastasis of a patient with malignant melanomas and immunofluorescence studies were performed as previously reported. - Immunoprecipitation by LYP18 Confluent melanoma cells were surface-labeled with 1251 or metabolically labeled with 35S methionine for 24 hours. Melanoma cell lysates extracted with Triton X100 were immunoprecipitated with LYP18 or with non-immune mouse IgG and then analyzed by SDS-PAGE as previously described. LYP18 does not bind to gpIIb or gpIII on EDTA-separated platelets, according to a cross-immunoelectrophoresis result, but binds to a calcium-mediated determinant present on the intact gpIIb-IIIa complex. - Binding of LYP18 to melanoma cells Human melanoma cells growing to confluence were detached from culture plates with trypsin-EDTA or EDTA and then resuspended in RPMI 1640 medium containing 2 mg / ml BSA. Increasing concentrations of LYP18 12 sI were added and incubated overnight at 40°C. Non-specific fixation of labeled LYP18 was achieved using a 100-fold excess of unlabeled LYPi8. The number of binding sites per cell and the dissociation constant were obtained by analyses using a computer program designated by "double-reciprocal plots and least-squares analysis". - Effect of LYP18~ on the growth of tumor melanomas in mice. 200 microliters of M3Dau l.lOS cells were subcutaneously grafted onto the abdomen of nude mice after pre-incubation for 20 minutes with purified LYP18 antibody, or with a GoAs antibody (directed against a melanoma cell surface antigen different from gpIIb-IIIa-like), or with non-immune mouse IgG2a (same isotype as LYP18) at a final concentration of 35 g / ml. Tumor sizes at different time points were expressed as the mean of the sum of opposite diameters. In studies conducted after transplantation, LYP18 was injected 2, 14, and 21 days after tumor implantation. In these experiments, 5 mice were used for each test. Results The LYP18 antibody exhibits binding capacity on M3Dau melanoma cells. From surface-labeled melanoma cell lysates, LYP18 immunoprecipitated two proteins with the same apparent molecular weight as the platelet membrane glycoproteins gpIIb and gpIIIa, with characteristic changes in mobility upon reduction. Metabolic labeling studies confirmed that gpIIb-IIIa-like proteins were synthesized by these cells. These results clearly demonstrate that the M3Dau cell line synthesizes and expresses surface proteins that are immunologically linked to platelet glycoproteins gpIIb-IIIa. To determine the number of gpIIb-IIIa-like molecules expressed on the surface of melanoma cells, binding studies of LYP18 labeled with 1251 were performed. The binding of LYP18 to M3Dau melanosis cells was specific, concentration-dependent, and saturable with approximately 337,000 ± 61,200 molecules per cell (mean ± standard deviation) and a dissociation constant (kd) of 6.8 nmol / l. M3Dau melanoma cells proliferate after subcutaneous injection in nude mice and produce tumor cells at the implantation sites. They have been used as a model to study the local invasion of tumor cells through the basement membrane. The biological role of gpIIb-IIIa-like in MRDau cells was investigated by incubating melanoma cells with a saturating amount of LYP18 (7 pg / 10⁶ cells in 200 µl aliquots), injecting the mixture subcutaneously into nude mice, and monitoring tumor growth in vivo. Controls were performed with the two antibodies described above, non-immune G7As and igGza from mice of the same isotype as LYP18, at the same concentration as LYP18. Tumor growth of M3Dau in control mice is palpable after 20 days and measurable after 26 days. With the addition of LYP18, the growth of M3Dau tumors is not visible or palpable after 26 days and becomes measurable after 40 days (Figure 8). Increasing the incubation time to more than 50 days results in small, visible tumors with slower growth than those in the controls, visible in only one of the five mice (Figure 8). A similar degree of tumor growth inhibition by LYP18 was observed even when more melanoma cells (2 x 10⁶ cells) were used. The lack of effect caused by anti-melanoma G,A5 antibodies suggests that the inhibition observed with LYP18 in vivo was not due to complement-dependent cytotoxicity or corpuscularization. The investigation into the potential for LYP18 to have a direct cytotoxic effect on melanoma cells was conducted by pre-incubating these cells with an excess of LYP18 (70 pg / ml) and monitoring cell viability and growth. No loss of cell viability was detected in melanomas with trypan blue exclusion. Furthermore, cells treated with the control antibody and with LYP18 exhibits a similar degree of thymidine-3H retention and cell growth in vitro. Post-transplantation studies have determined whether LYP18 inhibits tumor growth when injected after tumor implantation. LYP18 injected two days after tumor cell transplantation shows a 50% inhibitory effect on tumor growth. However, LYP18 injection on day 14 or 21 shows no significant reduction in tumor growth compared to the result obtained with the control (Figure 9). DISCUSSION This study demonstrates that glycoproteins IIb- IIIa-like cells are synthesized and present on a human melanoma cell line and play a fundamental role in tumor cell growth. The use of affinity chromatography on RGDS-Sepharose revealed two proteins with molecular weights similar to that of platelet glycoproteins gpilb-IIa in the M21 melanoma cell line. Another monoclonal antibody, LYP2, also directed against the platelet gpIIb-IIIa complex, does not bind to melanoma cells. M3Dau or precipitates any of the proteins from these cells. The lack of recognition of melanoma antigens by LYP2 could indicate a structural difference between the gpIIb-IT'a-like glycoproteins of the M3Dau cell line and their counterparts on platelets. The growth of tumor cells represents a complete interaction between the tumor cells and their stromal matrix. The stroma of a tumor is composed of fibrin, fibronectin, laminin, collagen and other related tissues, in addition to blood vessels and inflammatory cells. The monoclonal antibody LYP18, which binds to gpfb-IIla-like cells, may block critical interactions between melanoma cells and stromal matrix cells, which are necessary for the survival and growth of M3Dau cell lines. This binding could promote macrophage access and activation, leading to rapid destruction of melanomas in vivo. Post-transplant studies indicate that LYP18 is more effective when injected at the time of tumor implantation, suggesting that LYP18 interferes with one or more early stages of tumor establishment. Immunocytochemical studies performed on frozen tissue biopsies using LYP18 show the presence of gpfb-IIla-like cells in metastatic malignant melanomas but not in benign human melanocytes. EXAMPLE 7 Inhibition of transformed monocytes, by LYP20. Initially, transformed monocytes US37 were incubated for 30 minutes with platelets stimulated with thrombin (0.0 U / ml). At the end of this incubation, stimulated platelet rosettes were detected around the U937 cells. In a second step, the monoclonal antibody LYP20 (60 pg / ml) was added to platelets stimulated with thrombin for 5 minutes. PPALK was then added to inhibit thrombin. The platelets were then incubated with transformed U937 monocytes for 30 minutes. The results obtained under microscopy show that LYP20 inhibits rosette formation by approximately 50%. Therefore, LYP20 inhibits the interaction between platelets and transformed monocytes by approximately 50%. Table I: LYP18 reactivity to frozen sections of normal tissues Tissue Staining of Cell Types * Bone marrow megakaryocytes Tonsils O Thymus o Rate 0 Lymph node 0 Peripheral blood 0 Liver o kidneys glomerular epithelium Skin 0 Stomach 0 Thyroid o Lung O Prostate 0 Breasts 0 Uterus endometrial glands * All tissues show LYP18 reactivity with endothelial cells and with platelets in tissue sections. Table 2: Binding of LYP 18 to normal elanones and ielanocytes on frozen sections. Lesions Number of reactions / Number tested Melanomas 16 / 21 (75%) N. Melanocytes Normal skin 0 / 2 Lentigines 0 / 3 Dermatofibroma 0 / 1 Nevi 0 / 9 Table 3: LYP18 reaction with frozen sections of different tumors Histopathological types Number of reactions / Number tested Non-Hodgkin lymphomas 0 / 20 initial stage 0 / 9 intermediate stage 0 / 5 advanced stage 0 / 6 Hodgkin's Disease (HD) 0 / 4 Thymomes 0 / 2 Carcinomas metastatic Malpighian 0 / 3 glandular 1 / 3 Neuroendocrine tumors well differentiated 0 / 3 poorly differentiated 0 / 4 Sarcomas osteosarcoma 1 / 5 sarcoe Erwing 0 / 3 MFH 0 / 2 Others neuroblastoma 0 / 3 dysgermonome 3 / 75

Claims

CLAIMS 1 / An agent capable of modulating the interactions between monocyte-like cells and stimulated platelets, characterized in that it comprises at least one monoclonal antibody according to claim 2 of the main application. 2 / Agent according to claim 1, characterized in that it comprises the LYP20 antibody 3 / - Composition for the in vitro diagnosis of the presence of melanoma tumor cells, characterized in that it comprises LYP18 antibodies according to claim 6 of the main application.