MXPA00007178A - MONOCLONAL ANTIBODY ANTI&agr;v-INTEGRIN AND ITS USE TO INHIBIT&agr;v&bgr;6-INTEGRIN ATTACHMENT TO FIBRONECTIN - Google Patents

Abstract

The invention relates to a novel monoclonal antibody and a hybridoma cell line producing said antibody. The monoclonal antibody, a preferred embodiment of which is named 14D9.F8, has the following properties:reacting only with the&agr;v-chain of human&agr;v-integrins, and inhibiting selectively the attachment to the integrin substrate fibronectin of the&agr;v&bgr;6-integrin bearing cell.

Description

- - MONOCLONAL ANTIBODY BEFORE INTEGRINES ALPHA V AND ITS USE FOR INHIBIT INTEGRINAALFA v BETA 6 LINKED TO FIBRONECTIN TECHNICAL FIELD OF THE INVENTION The invention relates to a new monoclonal antibody and an ibidoma cell line that produces said antibody. This monoclonal antibody, one of whose preferred representatives is called 14D9. FB, has the following properties: it reacts only with the V chain of the human intagrins ^ V and selectively inhibits the binding to the integrin substrate of the integrin carrier cell a-v 6. The object of the invention is, therefore, a monoclonal antibody that has these properties. Also a subject of the invention is a hibphone cell line a, designated 271.1409. F8 and deposited under accession number DSM ACC2331, which produces a monoclonal antibody, and also a monoclonal antibody having the properties - indicated above and which can be obtained by this hybridoma cell line. Finally, a pharmaceutical composition containing an antibody as defined above is also the object of the invention. BACKGROUND OF THE INVENTION Integrins constitute a superfamily 1 of recep-REF. 121083 Cell surface adhesion targets, receptors that control the binding of cells to the extracellular solid environment, both to the extracellular matrix (ECM) and to other cells. Adnesion is of fundamental importance for a cell; provides anchoring, elements necessary for migration and signs for growth and differentiation. The integpnas are directly involved in numerous normal and pathological conditions, and as such are primary objectives in the therapeutic intervention. Integrins are integral transmem-bránicas proteins, heterozyme, whose specificity of - junction depends on which chain among the existing 15 is combined with one of the 8 chains f-. Integrins are classified into four subfamilies that overlap and that contain the chains p1, ¡3.2, | $ 3 or and a particular cell can express several different integrins of each subfamily. In this last decade it has been shown that integrins are important receptors involved in cell adhesion. On integrins, for example, E. Ruoslahti (J. Clin, Invest., 1991, 87) and R.O. Hynes (Cell, 1992, 59), and, therefore, may be an adequate target for therapeutic intervention. With the exception of the erythrocytes, all the human cells have one or more integrins. Its functions are regulated on many levels, but its specificity for a ligand depends mainly on which chain or. is associated with which -chain ^ in the heterozymer, and the activation state of the - integrins (Hynes, 1992; Diamond and Sppnger, 1994). The cell type in which integrins act (Chan and He ler, 1993) and -the form of the integrin binding variant used (De_l ^ wel et al., 1993) may also affect specificity. The history of integrin research has shown that reagents that can specifically block the function of integrins are decisive factors in the analysis - functional. From the CSAT antibody, function blocker, which has defined for the first time a 1 chain of integrin - (Neff et al., 1982), to numerous subsequent fundamental examples (for example, P1D5, P1B5 (ayner and Carter, 1987), -P4Clo (Cárter et al., 1990, and LM509 (Cheresh and Spiro, 1987: in this field it is absolutely dependent on such reagents.) It has recently been shown that the integrins of the V-series constitute a large subfamily with both classical and new functions, as well as classically intervening in the -union and extension of cells (Pytela et al., 1985; Cheresh, 1991), integrins or V are also involved in cellular locomotion (Seftor et al., 1992), in the reciprocalization of receptors (Panetti and McKeown Longo, 1993a; Panetti and McKeown Longo, 1993b), as virus co-receptors (ic ham et al., 1993), in the conduction of cascades of extracellular proteases (de Boer et al., 1993), and as regulators of the progression of tumors (Felding Haberman et al., 1992). the specificities of the five well-known integrins of the series "^^^ (Zhang et al., 1993), £ LV £ 3 (Pytela et al, 1985; Cheresh et al., 1987); < j_Vß5 (Cheresh et al., 1989 ),? Vp6 (Busk et al., 1992) and &, V? 8 (Moyle et al., 1991), and all of them seem to recognize only ligands 5 carrying the RGD peptide sequences (NH2-arg? N? na-gl? c_? na-aspartic acid-COOH), including those in vitronectin (t? 1, v? l P 3, aVp5), fibronectin (o, V li 1, < p 3, o- V? 5 , ^ - 5), and von Illebrand factor, fibpndigen and osteopontin (o-V 3) (for example 1991, Busk and Col., 1992, Zhang et al., -10 1993, Denhardt and Guo, 1993, Smith and Cheresh, 1990). It is known that the integrins CL V | 33 and ¡V ^ 5 participate in the angiogenesis of tumors, whereas the integrin V 6 is - a co-stimulant of the cell proliferation of the colon carcinoma and increases during the epithelial inflammation proliferation. 15 matopa and epithelial carcinogenesis (Breuss et al., J. Cell, Sci. 108: 2241-2251, 1995). Its expression seems to characterize the epithelium neo-tuperprol íferante (Zambruno et al .., J. Cell, Biol., 129, 853-865 (1995)). The increased expression of integrins can produce a model condition similar to psopa ^ 0 sis. Antagonists and specific agonists of the integrins < / 55 can therefore present a great therapeutic potential. The RGD peptides block the binding of V 6 integrins to fibronectin, but they also block other receptors for fibronectin located on the cell surface, including of ^ 5 ^ 1 (Pytela, R. et al., Cell (1985) 40, 191-198) and a, Vp3 (Pytela, R. Proc. Nati, Acad. Sci. USA 82, 5766-70 (1985)). Antibodies have been described that target all integrins c_, V (Lehmann et al., Cancer Res. 54, 2102-2107 (1994) and the co-cher s-H ß 3 or a.V6 5 (Weinacker et al. , J. Biol. Chem., 269, 6940-6948 (1994), but there is no known reagent that selectively inhibits the cvVß6 series .. A series of antibodies that bind human integrins of the * -V series have been described previously, including -Mab 14D9.F8"non-inhibitory" (Mitjans et al., J. Cell, Sci.108, 2825-2838 (1995).) The present invention provides a new mono-clonal antibody that reacts only with the chain of the human a-integrins and selectively blocks the binding to the integrin substratum of the carrier cell of the integrin a, V fi 3 and dV ß 6, and is called 14D9.F8 GENERAL, MATERIALS, GRAPHICS, TABLES Purification and protein sources, cell lines and their cultures, ELISA assays, adhesion assays, FACS analysis and production The ion and characterization of the antibodies employed have already been described in detail elsewhere (Mitjans. F. et al., J. Cell Sci.108, 2825-2838 (1995)). The Mabs (monoclonal antibodies) 14D9.F8, 11D1 and 17E6 were purified from the serum free hybridoma supernatants by affinity chromatography of - Protein A and subsequent removal of the endotoxins, and had a purity > 99% by SDS-PAGE (Mitjans, F. et al., J. Cell, Sci. 108, 2825-2838 (1995)). The mouse monoclonal 11 D 1 is an IgG, and is directed against integpna 5. The molecular weight that is assigned to the mouse IgG is 155 KDa. The preparation of -the < aV (transgenic truncated recombinant? 6 was carried out - essentially as described by Ueinacker, A. et al., in J. Biol. Chem. 269, 6940-48 (1994) .P3d10 (Agrez, M. and col., J. Cell, Biol. 127, 547-556 (1994)), E. Wayner (University of Minnesota, Minneapolis), and 487, J. Marshall (IRC-, London) were generous donations. Table 1 shows the selectivity of the antibodies used and the integrin profile of the cell lines used is indicated in Table 2. The cell line was deposited in the Deutache Samlung fur Mikroorganismen, Braunschweig, RFA, with the access number DSM ACC2331 DNA and amino acid sequences also include slightly modified or altered sequences such as mutants and variants that can be obtained intentionally or through chemical or physical processes, usually all mutants and vandals. that are included show the> properties and functions described The term antibody also includes, as a rule, fragments of antibodies such as Fab ', F (ab'), or single chain Fvs. These fragments can be produced by conventional classical techniques.

Table 1 The derivation and characterization of these antibodies is defined in the cited literature and in the present description.

Table 2 The mtegpna expression profiles of these lines have been defined by surface marking and FACS analysis or immunoprecipitation [FACS = Fluorescence Activated Cell Sorter (flow cytometric analysis)].

Figure 1 The Mab 14D9. F8 joins integer chains c-yV in d _? _ Verses contexts: the integrin .5wVj! > 3 soluble and recombinant (A), the integpna ciV ß5 of placenta (B), the integrin ^ -Vß? soluble and recornbi-nante (C), and integrin o- 11 b ß 3 platelets (D) all at a concentration of 1 μg ml ") were absorbed onto 96 well plates for ELISA and investigated with the indicated concentrations of Mabs 14D9.F8 (full squares), 17E6 (full circles), 4B7 (full triangles pointing upwards), 11D1 (full triangles pointing downwards), AP3 (full diamonds), E7P6 (open circles) was used as a hybrid supernatant a, representing the point of 5 ug / ml unadilution of the supernatant of 1: 5. The bound antibody was detected with anti-mouse IgG conjugated with peroxidase Error margin = DS (n = 3) Figure 2 Mab 14D9.F8 inhibits cellular adhesion af íbpomect i -na but not to vi tronect ina: colon carcinoma HT-29 (A, B), carcinoma of -pulmon UCLA-P3 (C, D), melanoma WM- 164 (E, F) were bound to plates coated with 5 ug / ml solutions of fibronectin (A, C, E) or vitronectin (B, D, F) in the presence of Indications of Mabs 14D9.F8 (filled squares), 17E6 (open circles), P1F6 (full triangles pointing downwards), P4C10 - full triangles pointing upwards), LM 609 (diamonds - full). After one hour the unbound cells were separated by washing and the bound cells were detected by titration - with lysosomal hexosaminidase. To normalize the binding, the maximum number of cells bound was used as 100%. The actual junctions to fibronectin and vitronectin were respectively 27% and 53% for HT-29, 42% for UCLA-P3 and 84% and 50% for WM 164. Error margins = DS (n = 3) . Figure 3 Mab 14D9.F8 selectively acts on the integrin ml ß 6. The SW480 cells transfected in mock form (Mock) or those transfected with human integrin ßd throughout their length (beta 6) were allowed to bind to plates coated with -Fibronectin (FN) for one hour and in the presence of 10 iig / ml of the antibodies described in figure 2. Error margin = DS (n = 3). References 1. Weinacker, A., Chen. A., Agrez, M., Cone. R.I., Nishimura, S., Wayner E., Pytela, R. and Sheppard, D. (1994) J. Biol. Chem. 269, 6940-6948. 2. Agrez, M., Chen, A., Cone, R.I., Pytela, R. and Sheppard, D. (1994) J. Cell Biol. 127, 547-556. 3. Mitjan's, F., Sander, D., Adam, J., Sutter, A., Marinez, JM, Jaggle, C, Moyano, J., Kreysch, H.-G., Piulats, J. and Goodman, SL (1995) J. Cell Sci, 108, 2825-2838. 4. Cheresh, D.A. and Spiro, R.C. (1987) J. Biol. Chem. 262, 17703-17711. 5. Houghton, A. N., Eisinger, M., Albino, A.P., Cairncross, J. G. and Oíd, L. J. (1982) J. EXP MED. 156, 1755-1766 6. Carter, W., Wayner, E., Bouchard, T. and Kaur, P. (1990) J. Cell Sci 110, 1387-1404. 7. Furihata, K., Nugent, D.J., Bissonette, A., Aster, R.H. and Kunicki, T. J. (1987) J. Clin. Invest. 80, 1624-1630 8. Ebert, E. C (1996) Dig. Dis. Sci. 41, 1551-1556. 9. Koretz, K. Bruderlein, S. Henne, C, Fietz, T., Laque, M. and Moller, P. (1994) Virchows Arch. 425, 229-236. DETAILED DESCRIPTION Monoclonal antibody 14D9.F8 binds strongly to - the integrins ^ V p 1, scV P 3, ot Vf > 5 and otV 6. In the ELISA assay on purified human integrins, the 14D9.F8 and 17E6 antibodies revealed identical reaction profiles and similar concentrations of antibodies were needed to achieve 50% of uncid binding (Figure 1). ) .14D9.F8 did not react with 0 * 11 b P 3, indicating that it pointed to the V chain, as confirmed previously from the FACS analysis (Mit-20 Jans, F. et al., J. Cell Sci .108, 2825-2838 (1995) .14D9.F8 had a slight effect on cell binding mediated by r V p 5, exerted slight and moderate effects on the unions mediated by V 3 and V 1 (Mitjans , F. et al., J. Cell Sci. 108, 2825-2838 (1995) .However, when testing the ability of 14D9, F8 to affect the cell attachment of carcinoma cells HT-29 and UCLA-P3 to fibronectin were found to be extremely active (FIG. 2). 14D9.F8 abolished the cellular adhesion of HT-29 to fibronectin with an IC- of about 0.3 nM, whereas on vitro-nectin a concentration of up to 600 nM was rather stimulating. The reasons for this phenomenon are not clear. P1F6 completely excluded the binding of HT-29 to vitronectin, whereas the binding to fibronectin was insensitive to P1F6, but was --analyzed by 17E6. LM609 did not react with HT-29 or UCLA.P3 cells (Mit ans, F. et al., J. Cell Sci. 108, 2825-2838 - - (1995) and had no effect on the binding of the latter. Therefore, an integral p-V other than x? 83 or <7> P5 modulates the adhesion of HT-29 to fibronectin, and this integrin is blocked selectively. The amino acids involved in the adhesion to vitronectin are inactivated by the Mab P1F6 and this integrin is weakly inhibited by 14D9.F8, to demonstrate that this was not a peculiarity of the HT-cells. 29 inhibition assays were repeated using UCLA-P3, a carcinoma cell from a different source (puj_ món) which expresses high levels of < - > 'V ß 5 &< - (, VR 6, and obtained similar results (Figure 2C, D) Mab 14D9.F8 abolished cell adhesion af íbronect ina, adhesion mediated mainly by mole électros sensitive to 17E6 with some contribution of molecules sensitive to P4C10 (probably »lf5 or CVÍH). It inhibited up to 30% of the cell binding on vitronectin, but P1F6 abolished the cell-binding. If, as was thought, P1F6 is selective for - V 5 (Weinacker et al., J. Biol. Chem. 269, 6940-6948 (1994)), then these data suggest that the integrin on UCLA-P3 - which intermediate the binding to fibronectin is • V ß.5, and this molecule is inhibited by 17E6 4 times stronger than by 14D9. F8 In ELISA and FACS antibodies 17E6 and 14D9.F8 bind d V f > 3, "(? P 5 and V f * 6 with a very similar dependence of concentration.) This denouement is unexpected, the melanoma cell lines uniformly expressing C? V /? 5, c V p 3 oo (.V p1, but they do not express V ^ 6 ociV ^ 8 (Mar-shall, JF et al., Semin. Cancer, Bio., 7, 129-138 (1996).) Such -as indicated in Figure 2.EF on the cells of WM164 melanoma, fibronectin binding was insensitive to V inhibitors, while vitronectin binding was blocked by 17E6, 50% by 14D9.F8 and up to 80% by LM609.Addition of P1F6 to LM609 or 14D9. F8 completely inhibited the binding of WM164 to vi tro-nectin, indicating that this binding was mediated by molecules sensitive to both LM609 and P1F6, and that the target sensitive to LM609, but not to P1F6, was also inhibited by 14D9.F8 Many carcinoma cells express V 6 (Sheppard, D (1996) Bioessays 18, 655-660) .As a hypothesis, it was suggested that the adhesion of carcinoma cells to fibropectin I was widely mediated by (? V (36, and that 14D9.F8 selectively inhibited this molecule, but did not inhibit V3 and 9? 5. Pa-1 r confirming this hypothesis was measured the binding of the SW480 cell line, transfected with human $ 6 along its length, in the presence of 14D9.F8 SW480 cells transfected with 6 were slightly affected by P3D10 on fibronectin, while 14D9, F8, 17 E6 and L230 were potent inhibitors of the union (Figure 3A However, the SW480 transfected in a fictitious manner, where only 0-5 S1 was used as an adhesion receptor, could be strongly blocked with Mab P3D10 (Figure 3B) (Weinacker, A. et al. Biol. Chem 269, 6940-6948 (1994) and 17E6, L230 and 14D9.F8 showed only a slight additional activity, possibly due to the inhibition of C? 1 (Marshall, JF et al., J. Cell Sc?. 108, 1227-1238 (1995). Again, cell adhesion on vitronectin was strongly inhibited by P1F6, eg ro was not affected by 14D9. F8 (figure 3C). The set of these data indicates that the -14D9.F8 selectively inhibits the A V ß 6 and dV £ 3 more strongly than the # V f >; 1, and does not affect the ¿7 > V / J 5, despite the fact that it binds - strongly to all these integrins. The molecular details of this process are unknown, but a usual interpretation of the data reveals that - after ligand binding, substantial alterations occur in the conformation of integpna (Schwartz, M.A. et al.

Ann. Rev. Cell Dev. Biol. 11, 549-599 (1995)). For this reason - the possibility is favored that when 14D9.F8 is linked to the chain ot v deaCV dy cj Vf 3, it avoids a movement of the chains against the V chains that is necessary to allow the binding of the ligand . By sequence comparison, potential sequences are observed that exhibit the necessary conservation between ß 3 fi 6, that vary in (35) and that could be involved in this process (observations that have not been pu blished.) THERAPEUTIC USE AND DIAGNOSIS The antibody according to the invention can be administered to human patients for treatment, Therefore, an object of the invention is to provide a pharmaceutical formulation which, as an active ingredient, comprises at least one antibody or antibody fragment as defined above and In the claims, associated with one or more pharmaceutically acceptable vehicles, excipients or diluents, the antibody of the invention is usually injected intravenously or parenterally, usually the limits of the dosage for the administration of the antibody. (or its fragments) are large enough to produce the desired suppression of the tumor and the effect tumor destroyer. The dosage will depend on the age, condition, sex and extent of the disease in the patient, and may vary between 0.1 mg / kg and 200 mg / kg, preferably between 0.1 mg / kg and 100. mg / kg per dose, in one or more doses administered per day, lasting one or several days. Preparations for parenteral administration include aqueous, non-aqueous, aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are: propylene glycol, polyethylene glycol, vegetable oils, such as olive oil; injectable organic compounds, such as ethyl oleate, and other solvents known in the art and suitable for this purpose. The antibodies of this invention can be used in a composition comprising a physiologically acceptable vehicle. Examples of these suitable vehicles are saline solutions, -PBS, Ringer's solution or Ringer's solution with lactate. Preservatives and other aggregates such as antibiotics, antioxidants and chelate formers may also be present in pharmaceutical formulations. The antibody (or a fragment thereof) can also be conjugated with cytokines such as IL-2, according to all known methods, to maintain its toxicity. The pharmaceutical formulations of the present invention are suitable for the treatment of carcinomas. For diagnostic purposes the antibody can be conjugated, for example, with a radiopaque ink or can be labeled in radioactive form. A preferred method of labeling is that of iodogen. The antibody is preferably administered co or scFv fragments for diagnostic purposes. This gives better results, making unnecessary the subtraction of the environment, which surrounds it. EXAMPLES: Example 1: Animal materials. Mice used for antibody production (BALB / c, females, 8 weeks old) and for tumor models ("nude mice": BALB / c nu / nu, females, homozygous, no thymus, 4 at 5 weeks of age) came from Cpffa (Bar-celona, Spain). The nude mice were kept in a sterile retina, in microallative cages; they were given food and water-sterilized at their discretion. All manipulations were performed in a laminar flow hood. Proteins Fibronectin (Ruoslahti et al., 1982) and the vitro nectin (Yatohgo et al., 1988) were purified from human plasma, fresh and frozen. All manipulations were performed at 20SC and all -washes were performed with PBS free of calcium and magnesium, unless otherwise indicated ("PBS": 137 mM NaCl, 2.8 M --KCl, 8.1 mM Na ., HP04; 1.5mM KH., P04; PH = 7.4). PBS + 1- is PBS plus 1 mM MgCl ~ and 1 mM CaCl ?. The chemicals (from Merck KGaA, Darmstadt) were of the highest purity obtainable, unless otherwise indicated. Cyclic peptides such as RGDfV were synthesized according to known classical techniques (for example, FEBS Let.291, pp. 50-54, 1991).

Cell lines and tumor cultures. The American Type Culture Collection (ATCC) provided the HT29 human carcinoma, the human lung adenocarcinoma UCLA-P3 (Cheresh et al., 1989) (Dr. DA Cnereshí Scppps) and the melanoma WM 164 (Dr. M. Her -lyn et al., 1990). All cells were cultured at 37 ° C - in air 92.5% with 7.5% C02 > in 90% of RPMI 1640 and 10% of calf ifetal serum (FCS) plus 2 mM of L-glutamine, and were consequently free of microplasma, as determined by a patented assay (Mycotect Kit, Gibco). Antibodies The mergers for obtaining monoclonal antibodies (Mab), selection in ELISA, subcloning and preservation of the cultures were carried out using classical techniques (Harlow and Lane, 1988), unless otherwise indicated. Example 2 Immunization. Monoclonal antibodies against o were produced. V p 3 by an intrapeptonal injection (íp) of J, V £ > 3 pl? Centapa purified and immobilized in Sepharose (80 ug of?? Vp3 in 80? L of Sepharose in 200 l of PBS), or of live M21 cells (1 x 10 in 0.5 ml of PBS), every two weeks for twelve weeks. Four days after the last injection, fusion induced by PEG was performed, using Fpendly Myeloma (Ventrex) as a fusion partner. Antibodies against a surface protein-associated to a 200 kDa melanoma were produced, immunizing with whole M21 cells (1 x 10 cells in 0.5 ml of -PBS).

Selection. ELISA was used in receptors and M21 cells fixed. For ELISA of receptors, 96-well ELISA plates (Dynatech) were coated with purified ΔV] 33 (1 μg / ml in PBS, 16 hours, 4SC); was blocked (with 1.5% of skimmed milk in PBS, 1 hour, 4-C) and incubated with hibpdoma supernatant. The bound immunoglobulins were detected with anti-mouse Ig conjugated to alkaline phosphatase (Dako), using as substrate p-nitrophenium phosphate. For cell ELISA, UCLAP-3 cells were fixed in 96-well tissue culture plates (with 4% paraformaldehyde in PBS, 15 minutes, 20 [deg.] C.) and blocked (with 3% BSA, PBS, 1 hour , 4 ^ 0), before incubation with hybpdoma supernatants and were detected as in ELISA of receptors. - The positive hybrids were subcloned three times by limited dilution and adapted to the RPMI medium. The immunoglobulin was determined using antibodies against heavy chain, specific for subclasses (Zymed) or antibodies against light chain (Promega). Other mouse antibodies, used in the studies, were donations from our colleagues: LM609 for ^ V r 3, P4C10 for integrin JB 1 (Carter et al., 1990) (Telios) and AP3 for the chain β 3 (Fuphata et al. ., 1987) (ATCC). Example 3: Purification and adjustment of the antibody. For the large-scale purification, the supernatant antibody was collected from cultures in their exponential phase, grown in bottles in rotations. tion. The antibodies purified on protein-A-Sepharose CL-4B (Pharmacia) were dialyzed against PBS before a sterile filtration (pore 0.45 m) and storage at -70 ° C - (Harlow and Lane, 1988). The purified antibodies were released from endotoxins by passing them through Kupmover-II columns (Kupta-Vater, Tokyo). This reduces endotoxin levels of 250 IU of endotoxin per ml of antibody to 0.2 IU / mg in the Limulus assay (Melvaer and Fystro, 1982). The fragments - F (ab ') "and F (ab') of 17E6 (mouse IgGl) were prepared by classical techniques of digestion with pepsin and separation in - columns of proteins A (Pharmacia), followed by digestion with papain and gel filtration separation (Harlow and Lane, 1988). Purifications of integrins The c? V p > S was purified from human placenta (Smith and Cheresh, 1988). The placenta was minced in and washed with -2- volumes of an ice cold solution (0.05% w / v digitionine, 2 mM CaCl 2, 2 mM PMSF, pH = 7.4) and then filtered. The retentate was treated with -4 volumes of ice cold buffer B (100 mM octi 1- ß-D-glucopyranoside (0G), 1 mM CaCl 2 and 2 mM PMSF in PBS) and centrifuged (max. 12,000 g, 45 min., 4 ° C). The supernatant was recirculated through a P3DI0 column (16 hours, 4 ° C). after washing with buffer C (0.1% NP-40 in PBS, -10 hp) and buffer D (0.1% NP-40, 2 mM CaCl2 and 10 mM Na acetate, pH = 4,5, -10 cv), the bound material was eluted with buffer E - (buffer D carried to pH 3.1). The eluent was neutralized with 3 M Tris (pH 8.8), dialyzed against buffer C and concentrated ten times using Aquacide II (Calbio chem). The purified receptors were stored at -70 ° C. The < Vß3 and. { jlV p B6 recombinants were purified from the five upper cells infected with baculovirus by affinity chromatography on LM609 (? V £ 3) and 17E6 (GAV¡J6) and with elution, concentration and storage similar to that of the £ 7 > Vf 5. The IIb / 33 was prepared from human platelets (Pytela et al., 1986). Old concentrates of platelets were mixed with a volume of Tyrodes solution; centrifuged (max.l200g) and released (1 hour at 205C) with lysis solution (50 mM OG, 1 mm MgCl, 1 mm CaCl ?, 1 uM --MnXl2, 2 mM PMSF, 150 mM NaCl, 50 mM Tris hydrochloride, pH = 7.4). After centrifugation (max 32000 g, 30 minutes, 4 [deg.] C.) the supernatant was recirculated (16 hours, 4-C) through a column of Sepharose C1-4B conjugated with GRGDSPK. The column was washed with lysis solution (-10 hp) and eluted with GRGDSPK (3mg / ml in 90% buffer solution 1 and 10% DMSO) The peak was concentrated - about 5 times, dialyzed against the modified h sis solution (0.1% NP-40 replaced by 0G) and stored at 70 ^ 0 Integrin preparations were -95% pure, determined by anti-integpna ELISA, using specific monoclonal antibodies the chains £ 7 * and p, and by SDS-PAGE.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (10)

1. The invention having been described as above, the content of the following claims is claimed as property: 1.- Mopoclonal antibody characterized in that it has the following properties: - it reacts only with the t V chain of the integrins < V manas e - selectively inhibits the binding to the integrin fibro-nectin substrate of the carrier cell of the integrin 0ÍV &6.
2. 2. Monoclonal antibody of claim 1, characterized in that the tumor whose growth of cells is inhibited, is a lung carcinoma cell UCLA-P3.
3. 3. The monoclonal antibody of claim 1, characterized in that the tumor whose growth of cells is inhibited is a colon carcinoma cell HT-29.
4. 4. Fragment of the mopoclonal antibody of claims 1 to 3, characterized in that it has the following properties - it reacts only with the chain Qt. V of human $ V integrins e - selectively inhibits the binding to the integrin substrate fibronectin of the integrin carrier cell xvp 6.
5. 5.- Hybridoma cell line capable of producing a monoclonal antibody according to claims 1 to 3, characterized because it has the denomination of 271.14D9.F8 and is deposited with access number DSM ACC2331.
6. 6. - Monoclonal antibody according to claims 1 to 3, characterized in that it can be obtained by the hibpdoma cell line DSM ACC 3221.
7. 7.- Pharmaceutical composition based on a monoclonal antibody according to one of claims 1 to 4 or 6, characterized because it comprises at least one of these antibodies together with a pharmaceutically acceptable carrier.
8. 8. Process for producing a monoclonal antibody according to one of claims 1 to 3, characterized by the immunization of a ra-ton with the M21 human tumor cell line.
9. 9.- Procedure for producing the monoclonal antibody of claim 6, characterized by the immunization of a mouse with the M21 cell line, the selection of clones that bind to the pu infected integrins by ELISA, and the obtaining by classical techniques of the cell line defined in claim 5, which produces said antibody.
10. 10. - Monoclonal antibody according to one of claims 1 to 4 or 6, characterized in that it is used to obtain a remedy against tumors, preferably a colon or lung carcinoma cell, or for the location and diagnostic determination of tumor growth.