MXPA96003080A - Monoclonal antibodies immuno-estimulan - Google Patents

Abstract

The present invention relates to a monoclonal antibody that has immunostimulatory effects, binds specifically to lymphoblastoid B cells and induces the proliferation and activation of peripheral blood lymphocytes, and when injected into animals bearing tumors causes an antitumor effect

Description

IMMUNO-STIMULATING MONOCLONAL ANTIBODIES FIELD OF THE INVENTION The present invention is generally in the field of immunotherapy and more specifically concerns monoclonal antibodies useful within the framework of therapy for a variety of indications, for example, in the treatment of cancer. PREVIOUS TECHNIQUE The following is a list of prior art references that are considered important for the subsequent description. 1. Clark EA, and Ledbetter, JA, Amplification of immuno-response by antagonistic antibodies, Current Immunology (Inmunol., Today), 7: 267-270, 1986. 2. Meuer, SC, Huspey, RE, Cantrell, DA, Hodgdon, JC Schlornman, S.F., Smith, K.A. and Reinberg, E. L., Triggering of the T3-T1 antigen receptor complex results in the proliferation of clonal T cells through the interleukin-2 dependent autocrine pathway, Proc. Nati Acad. Sci. (USA), 31: 1509-1513, 1984. 3. Van auve, J.P., De Mey, JR., And Groser, J.G., OKT3; an anti-human T lymphocyte antibody and potent mitogenic properties, J. Immunol. , 124: 2708-2713, 1980. 4. Jung, G., Martin, D.E., and Muller-Eberhard, J.H. , Induction of cytotoxicity in human mononuclear peripheral blood cells by monoclonal antibodies 0KT3, J. Immunol. , 139: 639-644, 1987. Van Lier, R., Blocmena, E., Brouwer,., Van Heijm, J., Wenreich, S., and Aarden, L., Studies on monocyte dependence on the proliferation of T cells induced by monoclonal antibodies directed against region I and II of CD2 antigen, Immunology, 67; 333-338, 1989. Ellenhorn, JD, Hirsh, R. Schreiber, H. and Bluestone, JA, In vivo administration of an anti-CD3 that prevents progressive tumor growth, Science, (Washington, DC) 242: 569- 571 1988. Gallinger, S., Hoskins, D.W., Mullen, J.B.M., Wong, A.H.C., and Roder, J.C. Comparison of cellular and anti-CD3 immunotherapies in the treatment of experimental hepatic metastases MCA-38-LD in C57BL / 6 mice, Cancer Research (Cancer Res.), 50: 2476-2480, 1990. Ledbetter, JA, Martin, PJ , Spooner, CE., Wofsy, D. Tsu, TT, Beatty, PG and Gladstone, P., Antibodies to the rise of Tp67 and Tp44 and sustenance of the proliferation responses of activated T cells. J. Immunol. , 135: 2331-2336. 1985. Moretta, A. Goggi, A., Pende, D., Tripodi, G., Orengo., A., Pella, N., Augugliaro, R., Bottino, C, Ciccone, E., and Moretta, I ., CD69-mediated pathway of lymphocyte activation: The anti-DC69 monoclonal antibodies trigger the cytolytic activity of different projectiles of lymphoid except for the cytolytic T lymphocyte expressing a / β cell receptors, J. Exp. Med., 174: 1393-1398, 1991. 10. Van Lier, R.A. Brouwer, M. and Aarden, L.A., Signals involved in the activation of T cells. Proliferation induced through the synergistic action of anti-CD28 and anti-CD2 monoclonal antibodies, Eur. J. Immunol. , 13: 167-172, 1988. 11. Jenkins, M.K., Taylor, P.S., Norton, S.D., and Urdahl, K.B. CD28 delivers a co-stimulatory signal involved in the production of antigen-specific IL-2 by human T cells, J. "Immunol., 147: 2461-2466, 1991. 12. Townsend, SE and Allison, JP, Rejection of tumor after co-stimulation of CD28 + cells by B7-transferred melanoma cells, Science (Science) (Washington, DC), 259: 368-370, 1993. 13. Hardy, B., Dotan, D. and Novogrodsky , A., A Monoclonal Antibody to Human B 1-Lymphoblast Cells Activates * Human and Murine T Lymphocytes, Cell Immunology (Cell Immunol.), 118: 22-29, 1989. The knowledge provided herein and the foregoing references is to enable the The reader must obtain an appreciation of the prior art, but the knowledge must not be constructed as an indication that these references are in any way relevant to the issuance of patentability of the invention as defined in the appended claims. The knowledge of the previous references will be done by indicating the number of the previous list. BACKGROUND OF THE INVENTION Cancer in its different forms is a major cause of death in humans. One third of all individuals develop cancer and 20% die of cancer (494, 00 in 1988). The most widely used therapeutic treatments for cancer are surgery, radiation and chemotherapy. In recent years, another therapeutic treatment based on the use of biological response modifiers (BMR) has also been proposed. The BMRs used include mainly cytokines (e.g., interleukin-2 (IL-2) and Interferon-a (INF-O!) Activated mononuclear cells (e.g., active killer cells with lymphocyte (LAK)) and antibodies. BRMs act directly on tumors and indirectly by improving specific and non-specific immunotoxic and cytotoxic mechanisms. To date, no substantial clinical success has been obtained using BRMs, mainly due to the toxicity of the natural effects. Active immunization against tumor cancer has also been proposed but has been ineffective. In addition, several moclonal antibodies (mAbs) were evaluated for use in the diagnosis and therapy of cancer but mAbs not yet proven to be effective in a standard therapeutic procedure in patients with cancer. Several mABs capable of binding to determinants on the surface of T cells have been found to induce proliferation, activation or differentiation of those cells * 1 *. The binding of mAbs directed against the CD3 / TCR complex in * 2"4 * T cells, the binding of mAbs directed in the CD (5) receptor antigen in T cells as well as the binding of both of the above types of antibodies to T cells, has been shown to lead T cells to proliferation, to the expression of the IL-2 receptor in T cells resulting in the enrichment of the cytolytic process in these cells.The anti-DC3 mAbs were shown to stimulate an activity anti tumor in vivo in an animal model (6"7). Several other mAbs directed against T-lymphocyte antigens have also been reported, which after binding to the cells cause their activation such as the mAbs directed against CD (8), against CD < 9) and Against CD28 (10"11) Anti-CD28 mAbs were reported to have decreased the growth rate of a murine melanoma although they were successfully used to completely eliminate tumors in mouse-1 **. A human lymphoblastoid cell line called "Daudt" was shown to stimulate murine lymphocytes and peripheral T cells.OVERVIEW OF THE INVENTION The present invention provides, in the first of its aspects, a monoclonal immuno-imulating antibody, which binds lymphoblastoid B cells and induces the proliferation and activation of peripheral blood lymphocytes, monoclonal antibodies are characterized in that when they are injected into an animal that carries a tumor, obtains an antitumor effect. An antitumor effect is a biological effect that can be manifested by a decrease in tumor size, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition. An anti tumor effect can also be manifested by the ability of the mAb in preventing the occurrence of tumor in the first place. Given their properties, the mAbs of the invention can be used both in the treatment of acute cancer as well as in the prophylaxis of cancer. The monoclonal antibodies of the invention can be obtained by first immunizing an animal with an immunogen, with lymphoblastoid B cells, lymphoblastoid lysate cells or preparations of its membranes. Following immunization and the development of an immune reaction in immunized animals, lines are developed and selected for such secret antibodies that bind to the immunogen. The selected mAbs are then subjected to further selection to be able of inducing proliferation and activation of peripheral lymphocytes of the blood. To obtain the mAbs of the invention, the antibodies selected as mentioned above, are then subjected to further selection so that they are capable of causing an anti-tumor effect. To select such antibodies, an animal model can typically be used. Sometimes the effect can also be tested in in vitro models. Such a model can be, for example, any laboratory animal in which cancer has been induced. Specifically relevant, particularly to choose a mouse for therapeutic use in humans, are the models that are allowed to develop tumors of human origin. Examples of animal models of the latter type are immunocompromised mice, for example, a SCID mouse or a nude mouse that has been injected or implanted with tumor cells or tissue obtained from human patients with cancer. In such selection, mAb's ability to reduce tumor size, increase survival time, etc. it is determined in comparison to the control (an animal that carries a similar tumor not treated with the mAb or treated with a non-relevant mAb). The selection may also involve testing, in a suitable model, the ability of the mAb to prevent the occurrence of cancer. For example, animals that have a genetic predisposition to develop cancer can be injected with the mAb and then the incidence of tumor and survival in the group treated with the mAb can be compared to a control group, otherwise treated and maintained in the same way. Instead of using animals that have a genetic predisposition, the ability of the mAb to prevent cancer can be tested in other animals treated with the mAb prior to the experimental administration of the malignant cells in the animal. To achieve such anti-tumor effect, the subject has to be administered with an effective amount of the mAb of the invention. The term "effective amount" should be interpreted as an amount of a mAb required to achieve a therapeutic effect. The effective amount required to achieve the final therapeutic result may depend on a number of factors including, for example, the type of tumor and the severity of the condition of the patients (ie, the cancerous state), and whether the mAb it is co-administered together with another agent that acts together with the mAb in an additive or synergistic manner (with respect to co-administration, see below). Obtaining an immortalized cell line that secretes mAbs of the invention can be performed by a number of means known per se, such as by fusion with an immortalized cell line to give a hybridoma; by EBV transformation, by genetically energizing a cell line, for example, a line of CHO cells, which can achieved by a variety of means known per se; etc. In general, the way to obtain cell lines secreting moclonal antibodies is currently a routine procedure known to artisans and the description of these goes beyond the present writing. Typically, when mAb is intended for treatment in a human, the immunogen will be of human origin. However, there is sometimes cross-species cross-reactivity and it is also occasionally possible to use mAbs obtained in humans followed by immunization with a non-human-derived immunogen, such as, for example, primate derivative. The antibodies of the invention should be construed to encompass monoclonal antibodies, which may be IgG or IgM antibodies, may be Fab fragments of such antibodies, F (ab ') 2 fragments, single chain antibodies, and the like. In addition, antibodies derived from a non-human origin, eg, murine, can be "humanized" by various genetic engineering means (a "humanized" antibody is an antibody in which the major portions have been replaced by portions of human origin). Antibodies according to the invention, while being useful for a variety of therapeutic indications, are used, according to a preferred embodiment of the invention, for the treatment of cancer. It has been found that a monoclonal antibody according to the invention is activated in the reduction of tumorigenicity of a variety of tumors. A representative hybridoma cell line according to the present invention was deposited in Collection Nationale de Cultures de Microorganimes (CNCM), Pasteur Institute, 25, Rué du Docteur Roux, 75724, Paris, Codex 15, under the deposit entry No. 1 1397, January 28, 1994. The cells of this line are here sometimes referred to as "BAT-1 cells", and the respective monoclonal antibody is sometimes referred to herein as "BAT-1 mAb". The use of a monoclonal antibody has the characteristics of the BAT-1 mAb, specifically the BAT-1 mAb itself, is a preferred embodiment of the invention. The monoclonal antibody BAT-l was selected based on its binding to the cells of the human lymphoblastoid B line Daudi. The BAT mAb was found to bind a protein substance (hereinafter referred to as BAT-1 linker protein) having an apparent molecular weight of 48-50K Daltons, as determined by SDS PAGE.The BATI linker protein forms another aspect of the present invention The BAT-1 linker protein can be isolated, by means known per se using the mAbs of the invention The BAT-1 linker protein can then be used for the immunization of animals of which the mAbs of the invention can be released later. The present invention also provides a pharmaceutical composition comprising, as an active ingredient, an effective amount of a mAb of the invention, and a pharmaceutically acceptable carrier. A further aspect of the invention is a method of treating a mismatch or disorder, particularly cancer, comprising administering to a subject in need thereof an effective amount of mAb. Administration of the mAb is typically by means of parental administration, for example, intravenously (i.v.), intraperitoneally (i.p.), or intramuscularly (i, m). The vehicle for administration can be one of such known per se, for example a saline solution or any appropriate physiological solution. The mAb of the invention was found, as already noted above, to be active in reducing tumorigenicity of a variety of tumors. The efficacy of the mAb of the invention in the reduction of tumorigenicity is correlated with its ability to induce lymphocyte cytotoxic activity. To trigger this activity, it is sometimes advantageous to administer the mAb of the invention together with other agents that can act additively or synergistically with the mAb. Examples include various cytokines such as IL.-1 (interleukin-1), IL-2, IL-6 and IFN-o; (interferon-a) The mAb of the invention can be useful in therapy of a variety of diseases other than cancer when the activation of other effects of the mAb on the cytotoxic activity of the immune system can have a therapeutic effect, such as, for example, in early stages of HIV infection (the cause of the acquired immune deficiency virus - AIDS), in several autoimmune disorders, or in some cases of genetic or acquired immune deficiency. In the treatment of cancer the antibodies can be administered either followed by the primary or secondary detection of tumors in the subject or, in preventive therapies of the subject having a high risk of developing cancers, such as individuals exposed to radiation or those who They have a genetic disposition. Similarly, in patients with AIDS, the mAb can be administered to infected individuals, who have not yet developed any of the symptoms of the disease, or individuals in the early stages of the HIV infection process. In the following, the invention will be illustrated by several examples describing experiments demonstrating anti-cancer therapeutic activity. It is understood, however, that this means for purposes of illustration only and should not be considered imitative. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the flow cytometry analysis of the binding of monoclonal antibodies BAT to T-lymphocytes purified. The binding of BAT antibodies was evaluated by a secondary antibody carrying an FITC anti-mouse antibody, fluorescent marker. Fig. La - background reading without BAT antibody; Fig. Ib - anti-CD3 antibody; Fig. 1c-BAT-1 Fig. Id-BAT-5 Fig. 1f-BAT-2; Figure 2 shows a flow cytometric analysis of human peripheral blood monocytes (PBM) (right plate) and of Jurkat T cells (right plate) double label with the primary antibody being either a BAT mAb or a CD3 mAb and a second conjugated FITC anti-mouse IgG antibody. Fig. 2A - cells without firing; Fig. 2A (a) - PBM cells treated with BAT mAb; Fig. 2A (b) - PBM cells treated with anti-CD3 mAb; Fig. 2A (c) - Jurkat T cells treated with BAT mAb; Fig. 2A (d) - Jurkat T cells treated with anti-CD3 mAb; Fig. 2B - fired cells; Fig. 2B (a) - PBM cells fired to small (Rl) and large (R2) cells; Fig. 2B (b) - cells (Rl) treated with BAT mAb Fig. 2B (c) - cells (Rl) treated with anti-CD3; Fig. 2B (d) - cells (R2) treated with BAT mAb; Fig. 2B (e) - cells (R2) treated with anti-CD3; Figure 3 shows a flow cytometric analysis of the surface expression of the BAT and CD3 linker protein in human PBM double-labeled with mAB BAT FITC-conjugated and anti-CD3 PE-conjugated. Fig. 3A- non-fired cells; Fig. 3A (a) - simulated test control used as a negative control; Fig. 3A (b) - anti-CD3 with PE alone; Fig. 3A (c) - mAb BAT with FITC; Fig. 3a (d) - Double labeling with antibodies mAb BAT-FIT and anti-CD3-PE; Fig. 3B (a) - PBM cells fired to small (Rl) and large (R2) cells; Fig. 3B (b) - cells (Rl) double labeled with ItlAB BAT-FITC and anti-CD3-PE; Fig. 3B (c) - cells (R2) doubly labeled with mAB BAT-FITC and anti-CD3-PE; Figure 4 shows Western blood analysis of binding of BAT-l antibodies to different lysates of Daudi cells. Fig. - 4 (1) - lysates of Daudi cells (untreated); Fig. - 4 (2) lysates treated with neuraminidase (0.2 μ / ml); Fig. - 4 (3) lysates treated with neuraminidase (0.4 μ / ml); Fig. - 4 (4) lysates treated with Endo Hf (100 μ / ml); Figure 5 is a representation of the results of [3 H] Thymidine incorporation in cells cultured for six days in the presence of increasing concentrations of a panel of BAT mAbs: BAT-1 (-A-), BAT 2 (-X-), BAT-3 (- • -), BAT-5 (- * -), BAT-6 (-f-), BAT-7 (-M-). Figure 6 is a graphical representation of an experiment in which the induction of cytotoxic activity was tested in PBM cultured for several time intervals with 2.5 μg / ml of BAT mAbs. HT-29 (right) or RC 29 (right) cells were used as target cells. The ratio of projectile to target was 20: 1. Control (-0-, BAT-1 (- • -), BAT-2 (-X-), BAT-3 (-A-) Figure 7 shows lungs of C57BL mice inoculated with B-16 melanoma cells: The upper row shows mouse lungs inoculated with cells only, 24 days after inoculation, the lower row shows mouse lungs inoculated with B-16 cells followed 14 days later by iv injection of 10 μg of mAbs BAT-l, 24 days after the inoculation Figure 8 is a graphic representation of a summary of experiments of the type shown in Figure 7, which shows the number of metastases in the mouse lungs which were inoculated with tumor cells and were already B-16 melanoma cells, Lewis 3LL lung carcinoma cells or MCA 105 fibrosarcoma cells, one month after inoculation. The results summarize experiments 3-4 carried out with each type of tumor. The untreated (-) or treated (+) mouse with BAT-l 810 μg / mouse) 2 weeks after tumor administration. Metastasis (O), - without metastasis (•). Figure 9 shows mouse lungs inoculated with Lewis 3LL lung carcinoma cells. Similar to Figure 7, the upper row shows lungs inoculated with only tumor cells, and the lower row shows mouse lungs inoculated with cells, 14 days later by i.v. of 10 μg of mAbs BAT-l. Figure 10 shows an experiment similar to that shown in Figure 9 where the tumor cells are MCA 105 fibroblastoma cells. MATERIALS AND METHODS Production of monoclonal antibodies The BALB / c mouse was immunized with the membrane preparation of Daudi cells. The Daudi cell membranes were prepared by the hypotonic shock method loaded with glycerol (Jett, M. Seed, TM, and Jamieson, GA, (Journal of Chemical Biology (J. Biol. Chem.), 252,: 2134, ( 1977)). 50-80xlOß cells suspended in PBS and incubated at 37 ° C were gradually loaded with 30% glycerol. After 5 minutes of incubation on ice, these were centrifuged and resuspended in cold Tris lysate buffer (containing 10 nM Tris-HCl, 1 mM MgCl 2, 1 mM CaCl 2, pH 7.4) mixed for 5 minutes at 4 ° C and centrifuged at 700 g. The supernatants were removed and centrifuged at 3300 g (10 min, 4 ° C). The pills were washed once and the two supernatants containing membrane fraction were pooled. 260 μl of the membrane preparation (3 mg / ml) were emulsified with 260 μl of the complete Freund auxiliary and injected i.p. in BALB / c mouse. Three weeks later the mouse spleens were removed. The bazocytes were fused with the NS-0 myeloma cell line at a ratio of 10: 1. The fusion was performed using polyethylene glycol and the hybridomas grew in the selective medium according to Kohler and Milstein (Kohler, G., and Milstein, C, Nature (Nature), (London) 256: 495, (1975). used an enzyme-linked cell immunosorbent assay (ELISA) to screen supernatants from growing hybridomas (Glassy, MC- and Surh, CD, Journal of Immunological Methods (J. Immunol. Me-thod, 81: 115 (1985 )) which bind to the Daudi cells.The positive hybridoma supernatants were then further selected for their ability to induce the proliferation of human PBM using the above-described assay of [3H] Thymidine. positive were subcloned by limited dilution, repeatedly tested, expanded and grown in culture. The mAbs were purified from the culture medium by 50% ammonium precipitation, followed by extensive dialysis against PBS. In addition, purification was performed by affinity chromatography on Sepharose (a brand of Pharmacia, Sweden) linked to anti-mouse antibody columns. Culture medium All cells were suspended in R1640 medium supplemented with 10% fetal calf serum (FSC), Na-pyruvate (1.1 mg / ml), L-glutamine (0.3 mg / ml) and antibodies (Penicillin 200 u / ml and streptomycin 10 μg / ml) and incubated in a humid incubator with 5% C02. The IL-2 units used are Cetus units (1 cetus unit equals 3 international units). Cell Preparations Human peripheral blood mononuclear cells (PBM) were obtained from healthy adult donors by ficoll-hypaque density centrifugation (Histopaque, a trademark of Sigma, St. Louis, Missouri, U.S.A). PBMs were decongested from monocytes by G10 Sephadex columns (a trademark of Fharmacia, Sweden). The T cells were separated by the SRBC enrosetate method. Decongestion of CD3 positive and Leul9 positive cells was performed by the immunomagnetic technique. The PBM cultures were incubated for 5-6 days with BAT mAbs or the control was washed three times with PBS. The unconjugated antibodies either with CD- or with Leul9 (CD56) were added to the cells in the complete Rmedium and incubated for one hour at 4 ° C. Magnetic pills coated with anti-mouse antibody were added for 30 minutes. The cells bound to the pills were removed from the magnet and the unbound cells were analyzed for cytotoxic activity and stained by flow cytometry. Cytotoxicity assay The cytotoxicity assays were made as follows: 2-4 × 10 6 blank cells were mixed with 200 μCi of chromos slCr for 1 hour in serum-free medium. These were washed three times with the complete medium and finally resuspended in R10% FCS and chromated at 104 cells per deposit. The projectile cells were cultured, the lymphocytes prepared from normal peripheral blood were incubated for various periods of time with the different mAbs, isotypic control IgG or with IL-2. Before the test the cells were washed three times in the Rmedium, stained for cell viability using 1% trypan blue, the projectile cells were mixed at several target-projectile ratios in microdosed dishes in incubated for 3 hours at 15-37 ° C in C02 at 5%. The culture supernatants were harvested and contained in a 0-scintillation counter. the maximum isotope release (MR) is produced by incubation of the target cells with Triton (a brand of Sigma, St. Louis Missouri, USA) x 100. Spontaneous release (SR) was mediated by the incubation of the targets with the half alone The percentage of cell lysis is calculated by (ER-SR / MR-SR) xl00, where ER is the experimental release of the projectile. Induction of cytotoxicity in subpopulations of human lymphocytes PBM cells (4.106 / ml) were cultured for 6 days in the presence of BAT mAbs. Hereafter, the cells were washed three times and decongestated from CD3 and Leul9 cells by means of magnetic pills coated with anti-mouse f (ab ') 2 and the toxicity was tested against K562 and Daudi cells. Flow cytometry Cell surface antigens were detected by flow cytometry using FACS 440 (Becton-Dickinson). For each analysis 106 cells were used, the cells were stained for sequential incubation with an optimal concentration of murine mAb to human CD3, IL-2 receptor, or the BAT 1-9 mAb that was produced. Goat F (ab ') 2 goat anti-mouse conjugated with FITC was used as a second antibody in this classification by races. Each incubation was carried in PBS pH 7.4 containing 1% BSA and 5% Na-acid for 30 min. to 4 ° C and was followed by three washes with the same regulator. The stained cells 104 were analyzed. Detection of the BAT mAb binding determinant (s) Detection of the BAT mAb binding determinants in lyzed B lymphoblastoid Daudi cells was done using the Western blood technique. Briefly, 50x106 cells / ml suspended in PBS were gradually loaded with 30% glycerol and the membranes were separated by sequential centrifugations. Samples of the membrane preparations were separated by SDS-PAGE (12%) and then transferred to nitrocellulose stains which were immersed in 1% low-fat milk in PBS. The detection of the BAT mAb that binds protein in nitrocellulose stains was done by incubating the stains with BAT mAb for 2 hours at room temperature, followed by 30 minutes of incubation with anti-mouse IgG (Fab ') 2 antibody conjugated to horseradish peroxidase. The cells were then wiped and bands with O-dianizidine substrate were detected. Mouse tumor models Three mouse models were used: Melanoma B16, Lewis lung carcinoma (3LL) and fibrosarcoma induced with methylclorantrene (MCA 105). From 50-200 xlO6 cells were injected i.v. to C57BL mice (8 weeks old). Two months later, BAT-l (i.v.), 1-10 μg / mouse and 10 days were injected then they were sacrificed and the established lung metastasis was counted. EXAMPLES Example 1 a. Binding characteristics Nine monoclonal antibodies (mAbs designated BAT 1-9, obtained by immunization with B lymphoblastoid cells, were first selected by binding to Daudi cells and then to induce proliferation of peripheral blood lymphocytes. by ELISA as by Ochteriony assays, BAR 1,2,3,6,7 and 9 were found to be of the IgG class while BAT 4 and 5 were of 1 IgM class, Bat 8 was IgG2a. Mabs for purified peripheral human T cells were analyzed by FACS analysis using indirect immunofluorescence staining.Figure 1 demonstrates the FACS analysis of such an experiment.As can be seen, BAT mAbs bind to peripheral blood CD3 T cells. of binding varied from BAT-2 44%, BAT-5 38%, BAT 1-32% to somewhat weaker binding of BAT-4 (13%). Purified peripheral blood B lymphocytes from the same blood donors ngre, did not bind these mAbs (data not shown). b. Binding of BAT-l mAbs to human lymphocyte subpopulations As seen in Figure 2, further FACS analysis showed that BAT-l binds human CD3 + PBM as well as the Jukat cell line. The finding that BAT-l binds CD3 + PBM cells was further corroborated using FACS analysis of double-labeled cells (Fig. 3). As seen in Table 1 below, BAT-1, in addition to its binding to cells carrying CD3, also binds to Leu 19 / NK cells. Table 1 Binding of mAb BAT-l to subpopulations of human lymphoid. c. Binding of BAT mAbs to various cell types The binding of BAT mAbs to various cell types was determined. As can be seen in Table 2 below, mAbs BAT bind to K562, a line of eri-roleukemia cells, and MCF7, a human mammary carcinoma cell line in addition to its binding to PBL, the line of Daudi and Jukat cells. The degree of binding varied between the BAT mAbs and the cell types used. MAbs 2-9 were linked to mouse renal carcinoma (MR28) only with very little affinity. Only BAT-8 was linked to MEL cells (murine citroleukemia), also at very low affinity. Table 2 Linking BAT nAbs to various cell types Binding was assayed by ELISA and expressed as: + (0.1-0.2 OD) ++ (0.2-0.3 OD) +++ (more than 0.3 OD) Example 2 Analysis of BAT mAb binding site and purification of binding protein BAT-l To determine the molecular weight of the membrane protein that interacts with mAb BAT-l, a membrane preparation of Daudi cells was solubilized and the protein was separated by SDS-PAGE. The transferred nitrocellulose stains were incubated with BAT-1 mAb and bands were detected by further incubation with horseradish peroxidase conjugated antibody to anti-mouse IgG (Fab ') 2 and detection using 0-dianizidine substrate. The molecular size of the BAT-1 binding protein was found to be 48-50 KDa - (Fig. 4). The BAT mAb binding protein was purified using BAT mAb conjugated to Sepharose (a trademark of Pharmacia, Sweden). This binding protein can also be prepared by cloning using biology techniques. Admission of BAT-1 in vivo to the mouse in the induction of BAT antibodies. EXAMPLE 3 Functional Characteristics of BAT mAbs a. mAbs BAT induced the incorporation of thymidine Peripheral human cells were cultured for 6 days in the presence of increasing concentrations of a panel of BAT mAbs and pulsed with [3 H] Thymidine 20 hours before harvest them. As seen in Figure 5, a gradual increase in BAT mAb concentrations resulted in a modest but significant increase in [3 H] Thymidine incorporation in PMB cells. However, a high dose of antibodies caused a decrease in admission by the cells. In control experiments, the corresponding isotype antibodies did not increase [3 H] thymidine in the PBL cells indicating that the agonistic effect of the BAT mAbs was dependent on their specific binding properties. For example, mAb of the IgGI isotype, which was raised in our laboratory against ovarian carcinoma cells, did not cause an increase in the admission of [3H] Thymidine in contrast to mAbs BAT 1,2,3,6,7 and 9, which also belong to the IgGl class. b. BAT mAbs Induce Cytotoxicity in Human PBM To cultures of human peripheral blood mononuclear cells incubated with BAT mAbs for various periods of time, they were tested for their ability to lyse tumor cell lines. As can be seen in Table 3, below, human PBM cells incubated for a week with a panel of BAT mAbs were cytotoxic against human cytoleukemia cells (NK sensitive) and renal cell carcinoma lines RC-29 (NK resistant) . The kinetics of the increase in cytotoxic activity of human PBM that was stimulated with BAT mAbs. As seen in Figure 6, the maximal cytoxicity towards human colon carcinoma (HT-29) «and renal cell carcinoma (RC-29) was reached after 7 days of incubation of human PBM with BAT mAbs. Table 3 BAT mAbs induce cytoxicity in human PBM The percentage of lysis of target cells using the projectile ratio: white 5: 1. c. Characterization of the subpopulation and lymphocytes involved in BAT- induced cytoxicity In order to assess whether the increase in the cytotoxic activity of OBM induced by BAT mAbs, it is due to the activation of NK cells, T cells or both, NK cells and T were purified and their toxicity induced by BAT was determined. for the purification of Nk and T cells the monoclonal antibodies Leul9 and anti CD3 were incubated with human PBM cells, followed by incubation with magnetic pills coated with anti-mouse IgG. This allowed the decongestion of the subpopulations of cells which bind to the corresponding antibody. As can be seen in Table 4 below, the number of units increased both CD3 and Leul9 decongested cell cultures. BAT 6 and 8 were used in these experiments and the targets were erythroleukemia (K562) and human lymphoma (Daudi).

Table 4 * Binding of BAT-l mAb to human lymphocyte subpopulations.

Ejmeplo 4 Synergism between BAT and mAb and II. -2 in the induction of cytoxicity Induction of cytoxicity in human PBM was studied under the incubation of PBM cells with mAbs BAT in combination with IL-2. IL-2 was added in suboptimal combinations (lU / ml) together with increasing concentrations of mAbs BAT-2. Cytoxicity was tested after one week in culture against tumor cell line K562 and HT29. As shown in table 5 below. The low concentration of BAT-2 synergized with IL-2 in the induction of cytoxicity of PBM cells against both types of target cells.

INF-Q! previously it was shown to improve the expression of MHC-1 class antigens. Therefore, the administration of lNF-a is possible to enhance the antitumor effect of BAT which is mediated by cytotoxic cells directed against several tumor cells (which support MHC class I antigens). Table 5 Synergistic effect of mAb BAT-2 in the induction of cytoxicity in human PBL.

Example 5: Immunostimulatory effects of mouse BAT-1: a. In vitro studies BAT-l mAb demonstrated stimulatory properties in murine bazocitos similar to that seen in human PBL. They may include: (i) increased proliferation of in vitro bazocyte as measured by the incorporation of 3HTimidine (Table 6); (ii) the synergistic stimulating effect by the incubation of bazocitos with a combination of BAT-l and IL-2 (Table 6); Table 6 C57BL murine bazocitos were incubated during 5 days in vi tro with several concentrations of BAT-l and in combination with interleucin-2 (lu and 10 u per ml). (iii) Cytoxicity increased in cultures of murine bazocytes in the presence of BAT-1 and increased also in cytotoxicity under incubation in the presence of IL-2 (Table 7). Table 7 Induction of cytotoxicity in cultures of C57BL bazocytes for 5 days in vivo in the presence of several * • * concentrations and combinations of BAT-l with low IL-2. a B16 melanoma cells were used as target; 15 the ratio of projectile to target was 50: 1. The murine tumor target cells that were susceptible to the annihilating effect by activated bazoites BAT-l included: B16 melanoma, Lewis lung carcinoma (3LL), fibrosarcoma (MCA 105), renal cell carcinoma (MR 28) and lymphoma (YAC) (Table 8).

Table 8 BAT-l Induced cytoxicity in bazocytes against mouse tumor cells (Study in vivo) a Bazocitos cultured in vi tro, for 5 days, in the absence of BAT-l mAb (lμg / ml). Cytotoxicity was determined at a projectile / target ratio of 60: 1. b. In vivo studies As seen in Table 9 below, BAT-1 showed immunostimulating effects under in vivo administration.

These included: (i) Stimulation of [3H] Thymidine incorporation in mouse bazocytes injected 10 days earlier with BAT-l (Table 9). The maximum stimulation (10 times) was achieved under the administration of BAT at a dose of.10 μg / mouse. (ii) Induction of cytotoxicity in mouse bazocytes injected with BAT-l (Table 9B). BAT-1 was administered at different doses for 10 days before the assay that induced toxicity to murine melanoma cells (B16-F10), renal carcinoma cells (MR-28) and lymphoma cells (YAC). The maximum effect was achieved under the administration of BAT at a dose of 10 μg / mouse. Table 9 Proliferation and cytotoxicity of mouse bazocytes injected with BAT mAb The mouse C57BL and BALB / c were injected I.V. with BAT mAb in different concentrations. 10 days later, cytotoxicity and [3H] Thymidine incorporation were determined in isolated bazocytes. The spleens of the C57BL mouse were tested on B16 melanoma target cells and those of the BALB / c mouse were tested on MR-29 and YAC cells (projectile ratio: 50: 1 target). Each group contained between 6-16 mice in 3-4 separate experiments. Cytotoxicity was given and the [3H] Thymidine incorporation was done in triplicate and the meaning was calculated, the results are expressed in the mean ± standard error in n mice, p-values for the differences between the control and the animals treated with BAT.

EXAMPLE 6 Immunotherapeutic effect of BAT-1 against tumors of mice. As seen in Table 10, administration of BAT-1 to mice inoculated with melanoma 14 days after the inoculation of the tumor cells, the number of lung metastases in the lungs of mice with tumors B16, 3LL and MCA. BAT-l abolishes lung methothesis in mice inoculated with B16 melanoma, using an established lung metastasis model. C57BL mice were injected (i.v.) with 50 x 103 with B16 melanoma cells (Table 10). 24 days after the Injection, numerous metastases were developed in the lungs (practically reached confluence) as seen in figure 7 above. Against this, as seen in figure 7, bottom row, the mouse lung that was injected with BAT-l (10 μg / mouse) 2 weeks after inoculation with melanoma B16 were practically free of metathesis. Figure 8 summarizes the results of 6 separate experiments performed under conditions similar to the previous one. Table 10 Antitumor effect of mAB BAT BAT-induced reduction in pulmonary metastasis The tumor was inoculated on day () The BAT mAb (10 μg / mouse) was injected i.v. on day 14 Pulmonary metasthesis was obtained 24 days post tumor inoculation using a Zeiss stereomicroscope. d Number of mice e Mean ± standard deviation of the weight of the lungs of the (n) mice. a2. Antitumor effect of mAb BAT-l injected at different times in relation to inoculation with B16 tumor As seen in Table 11, -the injections given to the mice of melanoma cells and treated 10-14 days later by the administration of mAb BAT-l were found free of meta-synthesis and have normal lung weights. A marked decrease in the number of metastasises in mouse lungs although not complete was noted 5 days after tumor inoculation and as late as 19 days post tumor administration. The injection of BAT-l on the same day as the inoculation of the tumor cells had no therapeutic effect.

Table 11 Administration of BAT at different times in relation to the inoculation of melanoma B16 Day and month management in relation to tumor inoculation on day zero. b. BAT-l cancels pulmonary metasthesis in mice inoculated with Lewis lung carcinoma (3LL) using an established model of pulmonary metastasis. The experimental conditions were similar to those described for melanoma B16 (see above), except that 2xl05 3LL cells were injected. Figure 9, upper row, shows lungs of mice inoculated with 3LL with numerous metastases. Figure 9, lower row shows lungs of mice that have been inoculated with tumor cells followed 14 days later by treatment with BAT-l, which as seen, are almost free of metasthesis. c. BAT cancels lung metasthesis in mice inoculated with MCA fibrosarcoma (MCA-105) using an established model of pulmonary metastasis. The experimental conditions were similar to those described for the Lewis 3LL lung carcinoma model. Figure 10, upper column, shows the lungs of mice inoculated with MCA 105 with numerous metastases. Figure 10, bottom row shows mice that have been inoculated with the tumor followed by treatment with BAT-l, which as they are seen are almost free of metastasis. . Example 7 BAT-1 cures mice bearing B16 and 3LL tumors Mice inoculated with B16 melanoma cells or 3LL cells as described above died 25-35 days after tumor inoculation. Against this, as can be seen in Figure 11, all mice were injected with BAT-l (10 μg / mouse) 14 days after the tumor inoculation survived more than 100 days. Most of the animals that were followed for up to 5 months showed no signs of disease and were free of metastases under pathological examination. Example 8 Adaptive transfer of mouse bazocytes treated with BAT-1 mAb Mouse bazocytes that were treated with BAT-1 mAb alone or given the first injections of B16 melanoma cells were then treated with BAT-1 mAb, were transferred into a recipient mouse . The recipient mouse was inoculated with B16 melanoma cells or with 3LL tumor cells. As seen in Table 12 below, the adaptive transfer of the bazocitos of the mouse injected with MAB BAT-l induced tumor radiation in the mouse that carries the tumor that receives the transferred bazocitos. The most effective treatment was obtained when 108 mouse spleens that were inoculated with B16 melanoma cells and 14 days later injected with BAT-1 were adoptively transferred to recipient mice carrying tumors. As seen in Table 12, in this case there was complete elimination of melanoma tumor cells in recipients carrying B16 'tumors and pronounced tumor regression in recipients carrying 3LL tumors, as presented in the number of metastases as well as also in the weight of the lung.

Table 12 Adaptive transfer of tumor induced regression tumor. Bazocytes were transferred from the mice 20 days after i.v. injection. of BAT (10 μg / mouse). Conditions as in b except that the mice were inoculated with B16 melanoma 14 days before the administration of BAT Mean number of metastases ± standard deviation of three mice. Average number of lung weight (gr) ± standard deviation of three mice.

These results indicated that single bazoites from mice inoculated with B16 and treated with BAT-1 exhibited anti-tumor effects against B16 and 3LL tumors. Therefore, it seems that BAT-l improves non-specific cellular trigger mechanisms. In addition, the presence of tumors potentiated the improvement of the induced generation of BAT-1 from such projectile cells.

Claims (14)

1. R E I V I ND I CA C I O N S 1. - A monoclonal antibody or its antigen fragment, being the monoclonal body i. a monoclonal antibody secreted by the hybridoma cell line in the Collection Natioale de Cultures de Microorganismes (CNCM), under the entry number 1-1397, or ii a monoclonal antibody which binds to an antigen to which the antibody binds from sane with (i).
2. 2. The monoclonal antibody or fragment according to claim 1, secreted by the cell line deposited under accession number 1-1397.
3. 3. A line of immortalized cells that secrete the monoclonal antibody according to claim 1 or 2.
4. 4. An immortalized cell line according to claim 3, wherein a line of hybridoma cells is.
5. 5. - The hybridoma cell line according to claim 4, the hybridoma cell line being deposited in the CNCM under the entry No. 1-1397.
6. 6. - A method for the treatment of a disease or disorder, comprising administering to a subject in need an effective amount of an antibody in accordance with the claim 1 or 2, to affect the immune system of the individual.
7. 7. - The method according to claim 6, wherein the cytotoxic activity of the immune system of the individual is stimulated.
8. 8. The method according to claim 6, for the treatment of cancer.
9. 9. A pharmaceutical composition comprising, as an active ingredient, an effective amount of a monoclonal antibody according to claim 1 or 2, and a pharmaceutically acceptable carrier.
10. 10. - The pharmaceutical composition according to claim 9, also comprising an agent other than said antibody, capable of improving the activity of cytotoxic lymphocytes, in a synergistic manner to that of the antibody.
11. 11. A protein substance to which the antibody of claim 1 or 2 binds specifically.
12. 12. - A substance according to claim 11, having an apparent molecular weight, as established by gel electrophoresis of about 48-50 Daltons.
13. 13. - A protein substance to which the antibody of claim 1 binds specifically.
14. 14. - A protein substance according to claim 13, which has an apparent molecular weight, according to established by gel electrophoresis of approximately 48-50 Dalton.