MXPA00007124A - Antibodies against human il-12 - Google Patents

Abstract

The present invention relates to p75 heterodimer specific anti-human IL-12 antibodies that are characterized by a higher potency and greater efficacy in neutralizing human IL-12 bioactivity than known heterodimer specific IL-12 monoclonal antibodies. The heterodimer specific antibodies recognize one or more epitopes of the human IL-12 p75 heterodimer, but do not bind to the p40 subunit alone. The heterodimer specific IL-12 antibodies neutralize rhesus monkey IL-12 bioactivity with a potency similar to their potency for neutralizing human IL-12 bioactivity making them useful IL-12 antagonists.

Description

ANTIBODIES AGAINST HUMAN IL-12 Description of the Invention This invention relates generally to antibodies to IL-12 and, more specifically, to monoclonal and polyclonal antibodies to anti-human IL-12. Interleukin-12 (IL-12), formerly known as the maturation factor of cytotoxic lymphocytes or stimulating factor of natural killer cells, is a 75-kDa heterodimeric cytokine (p75), composed of subunits 40- kDa (p40) and 35-kDa (p35) linked with disulfide. Subunits p40 and p35 are polypeptides containing 306 amino acid residues and 197 amino acid residues, respectively (Gubler U., et al., Proc.Nat.Acid.Sci.USA, Vol.88, 4143-4147 (1991)). The p75 heterodimer is the biologically active form of IL-12 (Gubler, U., et al., 1991, Proc. Nati, Acad. Sci. USA, 88: 4143; Wolf, SF, et al., 1991, J. Immunol. ., 146: 3074). The p75 heterodimer of IL-12 activates and promotes immune responses mediated by cells against foreign antigens, through the stimulation of helper cell production REF.121323 (collaborators) Thl, stimulating natural killer cells (NK) and activated T cells, improving the lytic activity of cells NK / LAK and stimulating the production of IFN-? leaving at rest and activating the T and NK cells. It has been shown that the p40 subunit of IL-12 is produced in greater quantity than the p35 subunit and is found both in the monomeric form and in the diurnal form (Podlaski, F.J., et al., 1992, Arch. Biochem. Biophys., 294: 230; D'Andrea, A., et al., 1992, J. Exp. Med., 176: 13087). The p40 homodimer of IL-12 is a potent antagonist of IL-12 (Ling, P., et al., 1995, J. Immunol., 154: 116, Gillessen, S. et al., 1995, Eur. J. Immunol., 25: 200). In contrast to the p40 subunit, the p35 subunit of IL-12 has no known biological activity and the p35 protein has only been found in association with the p40 subunit as part of the p75 heterodimer of IL-12. Accordingly, there are two important types of epitopes presented by human IL-12: (1) the epitopes presented by the p40 subunit; and (2) the epitopes presented by the three-dimensional conformation of the p75 heterodimer of IL-12. Accordingly, we designate antibodies that recognize the epitopes present in the p75 heterodimeric protein of IL-12, but that do not recognize the epitopes present in the p40 subunit protein of IL-12, called "heterodimer-specific" antibodies. . It has been discovered that the known antibodies of IL-12 are not optimally effective in the substantial neutralization of the bioactivity of IL-12. Antibodies to IL-12 that reacts immunologically with the p40 subunit do not optimally block the bioactivity of human IL-12. For example, the use of antibodies that react with the epitopes presented by the p40 subunit is particularly problematic, because production of the p75 heterodimer of IL-12 has been shown to result in inactive p40 subunits in excess of the bioactive p75 heterodimer. (Podlaski, FJ, 1992, Arch.Biochem, Biophys, 294: 230, D'Andrea, A. and collaborators, 1992, J. Exp. Med., 176: 1387). Consequently, p40 antibodies are not as effective as antibodies specific for heterodimers in reducing the deleterious effects of IL-12, since the p40 subunit alone is not bioactive and p40 antibodies tend to bind p40 subunits inactive more than with those subunits that are part of a bioactive p75 heterodimer.

Even known antibodies that react only with the p75 heterodimer do not effectively neutralize the bioactivity of IL-12. For example, an antibody specific to the p75 heterodimer of IL-12 that has been previously identified as 20C2 (Chizzonite Y collaborators, Cytokine, 6: A82a (1994) and D'Andrea et al., J. Exp. Mied., Vol. 176, 1387-1398 (1992)), can not substantially block the production of IFN-? and the proliferation of PHA-activated lymphoblasts stimulated by human IL-12. Antibodies specific to heterodimers are needed that more effectively neutralize the bioactivity of IL-12, with the purpose of reducing the harmful effects of IL-12. It is known that the highest levels of IL-12 in serum or tissue are related to the development and progression of autoimmune disorders. Thus, IL-12 antibodies are useful antagonists for controlling diseases with pathologies mediated by immunological mechanisms, in particular, those diseases associated with the activity of aberrant Thl-type helper cells. Examples of the aforementioned autoimmune disorders include: multiple sclerosis, inflammatory bowel disease (IBD), which includes Crohn's disease and ulcerative colitis, rheumatoid arthritis and autoimmune diabetes mellitus. Other disease conditions that have been shown to benefit from the administration of IL-12 antibodies include: transplant / graft versus host disease and septic shock. In accordance with this invention, it has been discovered that IL-12 antibodies obtained from a mammal that has deficiencies in the gene encoding the p35 subunit and / or the gene encoding the p40 subunit, substantially neutralize the bioactivity of IL -12. In accordance with this invention, for the first time, antibodies that substantially neutralize the bioactivity of human IL-12 are produced by using the methods described herein. Unlike other antibodies specific to the p75 heterodimer of IL-12, antibodies specific to the heterodimers of the present invention neutralize at least 90% of the bioactivity of human IL-12. Likewise, antibodies specific for the p75 heterodimer of IL-12 of the present invention cross-react with the IL-12 specific antibodies of the rhesus monkey. The IL-12 antibodies specific to the p75 heterodimer described herein are effective therapeutic agents for use in blocking the bioactivity of IL-12, for the purpose of treating conditions mediated by undesired, stimulated immune responses. for IL-12. The IL-12 antibodies specific to the highly neutralizing heterodimers described herein are particularly useful inhibitors of the proliferation of human PHA-activated lymphoblasts stimulated by human IL-12 and the production of IFN-γ and PHA-activated human lymphoblasts. Fig. 1 is a graph depicting the capture of human IL-12 labeled with 125 I performed by the antibodies contained in the supernatants of the hybridomas HIL-12F3-5F2 (hereinafter referred to as "5F2"), HIL-12F3 -16F2 (in the present called "16F2"), HIL-12F3-16G2 (referred to in this document as "16C-2"), HIL-12F3-20E11 (hereinafter referred to as "20E11") and HIL-12F1-17E2 (referred to herein as "17E2") (unfilled bars). The presence of the human p40 subunit of unlabeled human IL-12 during the immunoprecipitation reaction (filled bars) did not block the capture of human IL-12 labeled with 125I by monoclonal antibodies 5F2, 16F2, 16G2 and 20E11, which shows that these antibodies do not have a high affinity with the p40 subunit of IL-12 alone.

Fig. 2 shows the isoelectric focusing patterns of anti-human IL-12 monoclonal antibodies specific for the p75 heterodimer 20C2, 16G2, 16F2, 20E11 and 5F2. As indicated in Fig. 2, the oncological antibodies 20C2, 20E11 and 5F2 are i = unique immunoglobulins. Monoclonal antibodies 16G2 and 16F2 appear identical by isoelectric focusing, although both differ from 20C2, 20E11 and 5F2. Fig. 3 is a graph showing the inhibition of the proliferation of PHA-activated human lymphoblasts stimulated by natural human IL-12 by the monoclonal antibodies of IL-12 specific for the p75 heterodimer. 20C2 (+), 16G2 (?) 16F2 (O), 20E11 (+) and 5F2 (A). The inhibition of the proliferation of activated human PHA lymphoblasts stimulated by natural human IL-12 was determined with respect to the level of 0.25 ng / ml of proliferation of human PHA-activated lymphoblasts stimulated by human IL-12, in the absence of IL-12 antibodies, which in Fig. 3 is represented as a horizontal dotted line at 9940 cpm, and the proliferation reference levels of PHA-activated human 1-infoblasts, ie, in the absence of both antibodies of IL-12 as of IL-12, which in Fig. 3 are represented as a horizontal dotted line at 1480 cpm. As indicated in Fig. 3, monoclonal antibodies to IL-12, 16G2 (?), 16F2 (O), 20E (+) and 5F2 (A) inhibit 0.25 ng / ml of PHA lymphoblast proliferation Activated stimulated by human IL-12, at least 90%. In contrast, as shown by Fiq. 3, the already known 20C2 (+) antibody does not substantially inhibit the proliferation of PHA-activated human lymphoblasts stimulated by IL-12. Fig. 4 is a graph showing inhibition of the proliferation of PHA-activated human lymphoblasts stimulated by rhesus monkey IL-12, by monoclonal antibodies to IL-12 specific to the P75 heterodimer 16G2 (?), 16F2 (0), 20E1K +) and 5F2 (A) of the present invention, compared to the already known 20C2 (+) antibody. The level of proliferation of lymphoblasts in the presence of 0.5 ng / ml of IL-12 of the rhesus monkey and in the absence of antibodies of IL-12 is represented by the horizontal dotted line found at the upper end of the graph. The reference level of lymphoblast proliferation, that is, both in the absence of IL-12 and in the absence of IL-12 antibodies, is represented by a horizontal dotted line drawn at the lower end of the graph . As depicted in FIG. 4, the antibodies of the present invention are potent inhibitors of the proliferation of PHA-activated lymphoblasts, stimulated by the IL-12 of the rhesus monkey, as opposed to the 20C2 (+) antibody which has an effect inhibitory effect on lymphoblast proliferation stimulated by IL-12 of the rhesus monkey. Fig. 5 is a graph showing the inhibition of IFN-α production by the monoclonal antibodies specific for the heterodimer p75, 16F2 (O), 16G2 (H), 20EII (A), 5F2 (•) and 20C2 (*). As seen in Fig. 5, the antibodies 16F2 (O), 16G2 (B), 20E11 (A) and 5F2 (•) inhibit 0.25 ng / ml of the production of IFN-? stimulated by human IL-12 by at least 90%. The dashed horizontal line drawn at the lower end of the graph represents the production of the IFN-? of reference, in the absence of IL-12. In contrast, as indicated in Fig. 5, the monoclonal antibody 20C2 (*) is unable to inhibit 0.25 ng / ml of IFN-α production. stimulated by IL-12 in more than 65%. Fig. 6 is a nucleotide sequence encoding a portion of the heavy chain variable region of the p75 heterodimer-specific antibody 16G2 and the deduced amino acid sequence of this nucleotide sequence.

Fig. 7 is a nucleotide sequence that encodes a portion of the heavy chain variable region of the p75 heterodimer-specific antibody 20E11 and the deduced amino acid sequence of this nucleotide sequence. In accordance with the present invention, it has been found that when IL-12 antibodies are produced from mammals exhibiting a deficiency in the gene encoding the p35 subunit of IL-12 and / or in the gene encoding the p40 subunit of IL-12, IL-12 antibodies are obtained that selectively react immunologically with epitopes of the p75 heterodimer of IL-12 and that are identified by their ability to react immunologically selectively with the heterodimer p75 of the IL-12 huama, but that do not react with the p40 subunit alone. In contrast to the known p75 antibodies of IL-12, antibodies that substantially neutralize the bioactivity of human IL-12, that is, neutralize at least about 90% of the bioactivity of human IL-12, they are produced through the methods described here. In addition, antibodies specific to the p75 heterodimer of IL-12 of the present invention, cross-react with IL-12 of the rhesus monkey.

The IL-12 antibodies described herein neutralize at least 90% of the bioactivity of human IL-12, by inhibiting about 90% and at least the proliferation of human PHA-activated lymphoblasts induced by IL-12, in concentrations that reach an approximate value of 0.5 μg / ml, and / or the inhibition of approximately 90% of the production of IFN-? stimulated by IL-12, by the PHA-activated human lymphoblasts, at concentrations of at least 0.5 μg / ml. It has also been shown that the antibodies described herein specifically inhibit the proliferation of activated human PHA lymphoblasts induced by IL-12, but not those induced by IL-2. The PHA-activated lymphoblasts are prepared as follows. Separate peripheral blood mononuclear cells (PBMC) from the Peripheral Blood Mononuclear Cells (Gately et al., J. Nati. Cancer Inst., 69: 1245 (1982)) and are stimulated with 0.1% PHA-P (Difco Labs., Detroit, MI). After three days, the cultures are divided 1: 1 with a fresh medium and 50U / ml of recombinant human IL-2, as described in Gately, MK, Chizzonite, R. and Presky, DH, "Measurement of human and mouse interleukin 12", Current Protocols in Immunology, vol. 1. J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., John Wiley & Sons, Inc., New York, 1995, pgs. 6.16.1-6.16.15. PHA-activated lymphoblasts are used after an additional one-day incubation period. In accordance with the present invention, antibodies of IL-12 are identified by their ability to selectively bind to the epitope presented by the p75 heterodimer, although they do not react immunologically with any epitope presented by the p40 subunit. This selectivity is defined by the fact that the IL-12 antibodies of this invention will react, at a certain minimum concentration, with an epitope presented exclusively by a given amount of the p75 heterodimer, although they will not react at that concentration with an epitope presented. by the p40 subunit of that given amount of this p75 heterodimer. Thus, the antibodies of this invention have a higher affinity for an epitope exclusively presented by the? 75 heterodimer than by any epitope presented by the p40 subunit. It is possible to employ any conventional analysis to identify the selective binding of the antibodies with the p75 heterodimer. Generally, in such an assay, the antibodies are incubated with the p75 heterodimer of human IL-12, in order to determine whether the antibodies bind with the p75 heterodimer. As well, the antibodies are incubated with the p75 heterodimer of human IL-12, in the presence and absence of the p40 subunit, in order to determine whether the presence of the p40 subunit blocks the capture or binding of the p75 heterodimer antibodies. For example, competitive immunoprecipitation assays (see Example 7 herein) can be used to demonstrate that the antibodies described herein immunologically react selectively with the p75 heterodimer of human IL-12, although they are not immunologically reactive with the p40 subunit alone.

In accordance with the present invention, the IL-12 antibodies described herein are produced by the use of "knock-out" mammals. The knock-out mammals are deficient in the gene encoding the p35 subunit and / or in the gene encoding the p40 subunit of IL-12 and, therefore, do not express the p75 heterodimer of IL-12. When immunized with the p75 heterodimer of IL-12, the knock-out mammal deficient in the p35 subunit of IL-12 and / or in the p40 subunit of IL-12 recognizes the p-75 heterodimer of IL-12. 12 as foreign and produces antibodies for it. Preferably, the knock-out mammal is a mouse. In accordance with the present invention, knock-out mammals are produced by methods that have been described in the art. Knock-out mammals can be produced by conventional means, for example, by mutation of the gene encoding the p35 subunit of IL-12 and / or the p40 subunit of IL-12. For example, mice carrying a mutation in the p35 subunit gene of IL-12 can be produced as described by Mattner, F. et al., Eur. J. Immunol., 26: 1553-1559 (1996). Mice carrying a mutation in the p40 subunit gene of IL-12 can be produced as described by Magram, J. et al., Immunity, 4: 471-481 (1996). In accordance with the present invention, monoclonal and polyclonal antibodies that immunologically react selectively with the p75 heterodimer of human IL-12 are produced from the activated cells of the aforementioned knock-out mammals, by any means conventional that is known in the art. In general, antibodies are produced through: (a) immunization of a knock-out mammal deficient in a gene encoding the p35 subunit and / or the p40 subunit with the human p75 heterodimer, to produce antibodies; (b) obtaining antibodies from the immunized mammal; and (c) the selection of the antibodies according to their ability to selectively bind with the epitope presented by the p75 heterodimer, in order to obtain the selective binding antibodies. The monoclonal antibodies of IL-12 of the present invention that selectively immunologically react with the p75 heterodimer of human IL-12, are normally produced through a method that includes the following steps: (1) immunizing a knock mammal -out, as for example, a mouse deficient in the gene encoding the p35 subunit of IL-12 and / or the p40 subunit of IL-12, with the p75 heterodimer of human IL-12; (2) selecting immunized cells from the knock-out mammal that have been activated to express antibodies against IL-12, such as, for example, splenocytes or lymph node cells; (3) fusing the harvested cells with the myeloma cells to form the hybridoma cells; (4) selecting the hybridoma cells that secrete antibodies that recognize human IL-12, for example, by testing a medium conditioned with the hybridoma to detect the presence of anti-human IL-12 antibodies, for example, by the use of ELISA or immunoprecipitation analysis, which use labeled or unlabeled human IL-12; and (5) determining whether the antibodies are specific to the p75 heterodimer, demonstrating that the antibodies react immunologically with an epitope of the p75 heterodimer of IL-12, but that they are not immunologically reactive with any epitope of the p40 subunit, by incubation of antibodies with the p75 heterodimer of human IL-12, to determine if the antibodies bind with the p75 heterodimer, and the subsequent incubation of the antibodies with the p75 heterodimer of human IL-12, in the presence and absence of the subunit p40, for the purpose of determining whether the presence of the p40 subunit blocks the capture or binding of the p75 heterodimer antibodies. For example, competitive immunoprecipitation analyzes can be used (see Example 7 herein) to demonstrate that the antibodies described herein immunologically react selectively with the p75 heterodimer of human IL-12, but that they are not immunologically reactive with the p40 subunit alone. The method for producing the IL-12 monoclonal antibodies specific to the p75 heterodimer of the present invention may also comprise the step of determining the ability of the IL-12 antibodies specific for the heterodimers to inhibit the bioactivity of IL. -12, both human and rhesus monkey, in any in vitro or in vivo analysis system to establish the bioactivity of IL-12, such as, for example, the analyzes to determine the proliferation stimulated by IL-12 of the Activated lymphocytes, production stimulated by IL-12 of IFN-? or the increase stimulated by IL-12 of cytolytic activity. The anti-human IL-12 antibodies of the present invention can be separated to a substantially pure form, by standard methods known in the art, such as, for example, ammonium sulfate precipitation, affinity chromatography or the chromatography with ion exchange. Variations in the method for obtaining the antibodies of the present invention also fall within the scope of the present invention. Methods known in the art can be used, such as, for example, Western blot, competitive immunoprecipitation analyzes or cross-block binding assays to determine if the antibodies are specific to the p75 heterodimer. In addition to the mice, mammals such as rats and rabbits deficient in the p35 subunit gene of IL-12 and / or in the p40 subunit gene of IL-12 with the p75 heterodimer of the IL-12, for the purpose of producing the antibodies described herein. The deficiency or mutation in the p35 subunit gene of IL-12 and / or in the p40 subunit gene of IL-12 can be any deficiency or mutation that results in a lack of expression of the pl / 5 heterodimer of IL-12. Likewise, it is possible to use any conventional method to obtain mammalian cells carrying a mutation in the p35 subunit gene of IL-12 and / or in the p40 subunit gene of IL-12, which results in a phenotype deficient in p75 of IL-12. In accordance with the present invention, activated mammalian cells expressing antibodies to the p75 heterodimer of human IL-12 can be obtained by immunization of a mouse or other mammal with recombinant IL-12 or natural human IL-12. Natural human IL-12 and recombinant human IL-12 can be prepared by any conventional technique known in the art, such as, for example, the techniques provided in the examples herein. Suitable cell lines of myelomas, i.e., fusion counterparts for use in the production of hybridomas secreting the IL-12 antibodies of the present invention, include myeloma cell lines that are well known in the art. , such as, for example, cell lines SP2 / 0 and NS / O. The myeloma cells of the mouse SP2 / 0 are preferred. Preferably, the fusion counterpart of the myeloma and the mammalian cell activated against the p75 heterodimer of IL-12 are derived from the same species. Hybridoma cells producing the antibodies of the present invention can be selected and isolated by any of the methods known in the art. Preferably, the myeloma cells and the lymphocytes activated against the p75 heterodimer of IL-12 are cultured together, in media containing a selection agent capable of killing the myeloma cells, but not the lymphocytes. Hybridomas are formed from myeloma cells that fuse with lymphocytes activated against the p75 heterodimer of 1L-12. The aforementioned hybridoma cells are capable of developing in the media containing the selection agent because the DNA of the lymphocytes provides the melanoma cell line with the necessary gene encoding an enzyme which prevents the toxic effects of the selection agent, allowing an alternative metabolic pathway to replace the blocked metabolic pathway by the selection agent. Every unfused lymphocyte dies, because it does not transform and has finite and short life periods. According to the present invention, a selection agent suitable for use in the selection of the hybridoma cells is aminopterin. A medium that is preferred for the culture of the hybridoma cells is the Iscove Modified Dulbecco's Medium (IMDM) supplemented with 10% FBS (Hyclone), 100 Units / ml of penicillin G (Iscove's Modified Dulbecco's Medium).

(Bio Whittaker), 100 μg / ml streptomycin (Bio Whittaker), 250 μg / ml Fungizone (Bio Whittaker), 2 mM glutamine (Bio Whittaker), 100 μg / ml gentamicin sulfate (Bio Whittaker), 50 μM 2-mercaptoethanol (BioRad), 100 μM hypoxanthine (Sigma), 400 nM aminopterin (Sigma), 16 μM thymidine (Sigma) and 2.5% P388D1 supernatant (produced as described by Nordan, RP and collaborators, J. Immunsl, 15139: 813 (1987)). The potency of the IL-12 antibodies of the present invention is determined with respect to the concentration of IL-12 antibodies, at which 50% of the maximum inhibition of the bioactivity of IL-12 occurs. , as measured by the proliferation analyzes of human lymphoblasts stimulated by IL-12 or IFN-α production. The anti-human IL-12 antibodies of the present invention exhibit a higher potency than the IL-12 antibodies specific for the previously characterized heterodimers. Also, the anti-human antibodies of the present invention exhibit a higher efficiency, as calculated by the degree of maximal inhibition of lymphocyte proliferation stimulated by IL-12 or IFN-α production, than IL antibodies. -12 specific to the het-erodimers previously characterized.

The potency and efficacy of the antibodies described herein can be determined by any conventional assay known in the art, such as, for example, lymphoblast proliferation assays induced by the IL-12 or IFN-β synthesis analyzes In accordance with the present invention, any method known in the art can be employed for the purpose of determining the inhibition of lymphoblast proliferation stimulated by IL-12 by IL-12 antibodies. In general, human lymphocytes can be activated by a number of methods, including treatment with mitogenic lectins, for example, phytohaemagglutinin A (PHA) or other activating agents, alone or in combination, such as the cytokines, the fobol esters and the ionophores, the antibodies directed against the cell surface molecules or any other method that leads to the activation of the lymphocytes. The activated lymphocytes are then incubated with and without IL-12, in the presence or absence of the antibodies and the lymphocyte proliferation index is measured by determining the DNA synthesis index, calculating the incorporation of 3H-thymidine into the DNA, by means of the counting the number of cells present after several treatment periods or any other method that can be used to observe the cell proliferation index. Inhibition of proliferation is determined by comparing lymphocyte proliferation at a defined concentration of IL-12, in the presence or absence of various concentrations of anti-IL-12 antibodies. In a conventional lymphocyte proliferation assay, the inhibition of the proliferation of human PHA-activated lymphoblasts stimulated by human IL-12 is determined with respect to the proliferation levels of human PHA-activated lymphoblasts stimulated by human IL-12 without any addition of antibodies and the reference levels of the proliferation of activated human lymphoblasts, that is, proliferation in the absence of both IL-12 and antibodies. In general, proliferation levels stimulated by IL-12 yield approximately 10,000-80,000 cpm in our standard human lymphocyte proliferation assay, while reference levels of proliferation yield around 5,000-20,000 cpm. As a consequence of the inherent variability between batches of stimulated PHA-activated human lymphoblasts, only those assays in which the proliferation ratio is greater than the reference proliferation (ie, the stimulation index) was equal to 3 were considered valid. or reached a value greater than 3, for the measurement of proliferation stimulated with the'IL-12. According to the present invention, any conventional method for determining the inhibition of IFN-α production can be employed. for the antibodies of IL-12. For example, activated human lymphocytes, prepared as described herein or activated human peripheral blood mononuclear cells (PBMCs), prepared by treatment of whole blood or isolated PBMCs with mitogenic agents. which include: lectins, cytokines, fobol esters, ionophores or antibodies directed against cell surface molecules, alone or in combination or by any other method that leads to the production of activated human PBMC, are incubated with or without IL-12 and a variety of other agents, for example, IL-2 and / or IL-1β, in the absence and in the presence of the antibodies. The production of IFN-? it is then determined, for example, by taking a sample of the culture medium and determining the concentration of IFN-α. by ELISA or any other method that can quantitatively calculate IFN-Y. The inhibition of IFN-α production is determined by comparing the production of IFN-? at a defined concentration of IL-12, in the absence and in the presence of various concentrations of anti-IL-12 antibodies. In a conventional assay of the synthesis of IFN-α, the inhibition of IFIT-β is determined. with respect to the production of the IF-? stimulated by IL-12 and reference levels of IFN-α production, ie the synthesis of IFN-? in the presence or absence of IL-12. In general, the production levels of IFN-y stimulated by IL-12 are approximately 7-220 ng / ml, where the reference production levels yield around 1-3 ng / ml. The antibodies of the present neutralize the bioactivity of IL-12 of the rhesus monkey with a potency similar to its potency to inhibit the bioactivity of the Human IL-12, which makes them useful antagonists of IL-12 for in vivo studies in the rhesus monkey. The increased potency and efficacy of these anti-human IL-12 antibodies and their cross-reactivity with the IL-12 of the rhesus monkey makes them excellent candidates for designating effective IL-12 antagonists for use in humans. In particular, the present invention provides four antibodies, 5F2, 16F2, 16G2 and 20E11 for the p75 heterodimer of human IL-12. The corresponding cell lines of the hybridoma producing these antibodies have been deposited on December 11 under the conditions of the Budapest Treaty in the American Collection of Crop Types under accession numbers ATCC HB-12446, HB-12447, HB-12449 and HB-12448, respectively. However, the present invention is not limited to these four antibodies. Any of the antibodies having the characteristics described herein is within the scope of the present invention. Fig. 6 provides the sequence of nucleotides encoding a portion of the heavy chain variable region of the p75 heterodimer-specific antibody 16G2 and the deduced amino acid sequence of is the nucleotide sequence. The nucleotide sequence encoding a portion of the heavy chain variable region of the p75 heterodimer-specific antibody 20E11 and the deduced amino acid sequence of this nucleotide sequence is provided in Fig. 7. Those skilled in the art will recommend that they can be practiced conservative amino acid changes in the constant regions of the IL-12 antibodies specific for the heterodimers herein, without significantly affecting the binding affinity / specificity of the antigens. It is presumed that IL-12 antibodies specific for heterodimers containing amino acid changes in the regions of variable structures or in the complementary determining regions will have a greater effect on the binding affinity / specificity of the antigens. The IL-12 antibodies of the present invention may be whole antibodies, including the two full-length heavy chains and the two full-length light chains. Alternatively, antibodies to IL-12 can be constructs such as single-chain antibodies or "mini" antibodies that retain binding activity with one or more enitopos of the p75 heterodimer of IL-12. Said constructions can be prepared by methods that are known in the art, such as, for example, PCR-mediated cloning and the assembly of single-chain antibodies for expression in E. coli. (as described in "Antibody Engineering, The practical approach series, J.McCafferty, HRn Hoogenboom, and DJ Chiswell, editors, Oxford University Press, 1996.) In this type of construction of the variable portions of the light and heavy chains of One molecule of the antibody is amplified with PCR from cDNA.The resulting amplicons are then assembled, for example, in the second PCR step, through a linker DNA encoding a flexible protein linker that is composed of the amino acids GLY and This linker allows variable portions of the light and heavy chains to fold so that the antigen agglutination gap is regenerated and the antigen binds with similarities commonly comparable to those of the dimeric progenitor immunoglobulin molecule. Full length The anti-human IL-12 antibodies described herein can be humanized to form antibodies possessing the same, or substantially the same affinity for the heterodimer p-75 of IL-12 as anti-human mammalian IL-12 antibodies, but substantially not immunogenic in humans. For example, a humanized IL-12 antibody in accordance with the present invention can include heavy and light chain structural regions of human antibodies. Preferably, the amino acid sequences of framework regions of humanized antibodies are from about 60% to 95% identical to the donor structural regions. Humanized antibodies can be produced by genetic recombination techniques that are well known in the art. Methods for producing humanized immunoglobulins are described, for example, in the U.S. Patent. No. 5,530,101. The IL-12 antibodies of the present invention are useful antagonists for the control of diseases with pathologies mediated by immunological mechanisms, in particular, the diseases associated with a higher bioactivity of IL-12 which results in the activity of the helper type Thl aberrant cell. In accordance with the present invention, antibodies of IL-12 are used to treat autoimmune disorders of humans or other mammals, such as, for example: multiple sclerosis, rheumatoid arthritis, autoimmune diabetes mellitus and intestinal disease inflammatory (IBD, from Inflammatory Bowel Disease), which includes Crohn's disease and ulcerative colitis. The antibodies described herein may also be used to treat other disease conditions that have been shown to benefit from the administration of IL-12 antibodies and include, for example: transplant / graft versus transplant disease. host and septic shock. Those skilled in the art will be able to determine the dose ranges for the administration of IL-12 antibodies herein, without undue experimentation. In general, the appropriate dosages are sufficient to produce the desired effect, ie, they neutralize at least 90% of the bioactivity of IL-12. However, the dosage should not be so significant as to cause adverse side effects, such as inconvenient cross-reactions, anaphylactic reactions and the like. In general, the dosage will vary according to the age, condition, sex and severity of the patient's illness, contraindications if any, immune tolerance and other such variables, which the individual physician should to adapt. Antibodies to IL-12 can be administered parenterally, by injection or by gradual perfusion, over a period of time. They can be administered intravenously, intramuscularly or subcutaneously. Preparations for parenteral administration include sterile aqueous and non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are: propylene glycol, polyethylene glycol, vegetable oils such as, for example, olive oil and injectable orcanic esters, such as ethyl oleate. Aqueous carriers include: water, alcoholic / aqueous solutions, emulsions or suspensions, including buffered media and saline. Parenteral vehicles include: sodium chloride solution, Ringers dextrose, dextrose and sodium chloride, fixed or Ringer's lactate oils. Intravenous vehicles include: fluid and nutrient restorers, electrolyte restorers, such as those based on Ringer's dextrose and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like. See, in general, Reimington's Pharmaceutical Science, 16th Ed., Mack Eds., 1980. Preferred dosages of the IL-12 antibodies of the present invention range in a range of approximately 0.1 mg / kg to 10 mg / kg, two to three times per week. However, the dosage and the dosage plan for the administration of the IL-12 antibodies herein may vary depending on the individual to be treated, the antibody administered and the variables that have been previously disclosed.

In accordance with the present invention, IL-12 antibodies can be administered alone or in combination with other therapeutically active agents. The following examples described below are provided so that the invention described herein can be fully understood. These examples are provided for illustrative purposes and should not be construed as limiting this invention in any way.

EXAMPLE 1 Preparation of Natural Human IL-12 Blood is drawn from normal voluntary donors into syringes containing preservative-free heparin (Sicpa, St. Louis, MO, USA) to provide a final concentration of ~ 5 heparin units / ml blood . A volume of heparinized blood was diluted in 9 volumes of a medium consisting of a 1: 1 mixture of RPMI 1640 and Dulbecco's modified Eagle medium, supplemented with 0.1 mM non-essential amino acids, 60 μg / ml HCl arginine, 10 mM HEPES buffer, 2 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin (all marketed by GIBCO BRL, Grand Island, NY, USA), 50 μM 2-mercaptoethanol (Fisher Scientific, Fair Lawn, NJ, USA) and 1 mg / ml dextrose (Fisher). To this mixture was incorporated interferon-? human, 20 U / ml, (PeproTech, Inc., Rocky Hill, NJ, USA) and Pansorbine cells (formalinized Staphylococcus aureus, Cowan strain; Calbiochem, San Diego, CA, USA) at a final dilution of 1/4000. (Before being used in cultures, Pansorbine cells were washed twice with the Dulbecco phosphate buffered saline solution (GIBCO BRL) and reconstituted to the same volume that is supplied from the factory.) The resulting cell suspension was divided in aliquots, in 162 cm2 cell culture flasks (Costar, Cambridge, Y-7, USA), at a rate of 80 ml / bottle and the bottles were incubated horizontally at 37 ° C, in a humidified atmosphere of 5% C02 / 95% air, for 24 hours. Fluids from the culture supernatant were then harvested by centrifugation and sterilized by filtration through a 0.22μ filter (Costar). The IL-12 heterodimer plus the p40 of IL-12 were purified from the culture supernatants through immunoaffinity chromatography, using a protein G sepharose column (PGS). 4AI, as described below, for the purification of rhesus IL-12, with the exception that the elution buffer contained 0.01% gelatin (Sigma) to minimize protein loss due to non-specific adsorption towards the surfaces. The eluate was dialyzed for 4 to 6 hours, against 100-200 volumes of Dulbecco phosphate buffered saline and then overnight, against the same volume of RPMI 1640 containing 100 μg / ml gentamicin. . The dialysed eluates were sterilized by passage through a 0.22μ filter and then subjected to an ELISA analysis, in order to determine the heterodimer content of IL-12 and p40 of IL-12 ( Gately, MK, Chizzonite, R. and Presky, DH, "Measurement of Human and Mouse Interleukin 12", Current Protocols Immunology, Vol 1. JE Coligan, AM Kruisbeek, DH Margulies, EM Shevach and Strober, eds., John Wiley &Sons, Inc., New York, 1995, pp. 6.16.1-6.16.15) and to determine the bioactivity of IL-12 (In general, the weight ratio of p40 of IL-12: heterodimer of IL-12, as calculated by ELISA, was approximately 5: 1.

EXAMPLE 2 Production of Recombinant Human IL-12 Recombinant human IL-12 was prepared, characterized and generated as presented in U.S. Patent No. 5.53.657 and the content of which is herein incorporated by reference.

EXAMPLE 3 Generation of rhesus monkey IL-12 The cDNA sequences of p35 and p40 sulbunites were prepared for rhesus monkey IL-12 (F.Villinger v., J. Immunol., 155: 3946-3954 (1995 )) for expression in negative CHO-dhfr cells on two separate plasmids, using conventional procedures (Current protocols in molecular biology, F. Pusubel, ed., Wiley and Sons, Inc., New York (1993)). The clones were obtained from a non-amplified population of cells and their production of IL-12 was observed by an ELISA specific for IL-12. An optimal production clone was selected and adapted to growth in a medium without CHO serum (Sigma). The cells were subsequently developed in spinner cultures for the purposes of protein production. The IL-12 of the rhesus monkey was purified from the supernatants, by antibody affinity chromatography. The affinity column was produced by cross-linking 10 mg of p40 mAb 2-4A1 of anti-human IL-12 (Chizzonite et al., J. Immunol., 147: 1548-1556 (1991)) with Protein G Sepharose ( Pharmacia Biotech), using 10 mM dimethyl pimelimidate (Pierce, Rockford, IL, USA), at a density of 1 mg mAb / ml gel (Stem and Podlaski, Tech. In Protein Chem. IV, Acad. Press, New York , 353-360 (1993)). The serum free CHO supernatant containing the rhesus IL-12 was filtered through a 0.2 μm filter and loaded directly onto the 10 ml column 2-41 PGS, previously equilibrated in PBS, pH 7.2. The circulation speed was 1 ml / min. The column was washed with 10 volumes of PBS and eluted with 0.1 M Glycine-HCL, 0.15 M NaCl, pH 3.0. The eluate was immediately neutralized with 3M Tris-HCl, pH 9. The affinity column was able to bind ~ 2mgs of the rhesus IL-12 / cycle, including excess p40 monomer, as determined by Bradford and SDS-PAGE . The other pollutants were at trace levels. To further concentrate and purify rhesus IL-12, the eluate containing IL-12 was dialyzed against 20 mM sodium phosphate pH 7 and loaded onto an S-Sepharose column, conditioned with the same solution as buffer. The circulation speed was 1 ml / min. All the protein binds. The column was washed with 10 volumes of phosphate buffer and then eluted with phosphate buffer containing 0.3M NaCl. The eluted set was subjected to an analysis to detect endotoxin, using the LrL equipment of Biowittaker and it was detected that it was <10 EU / mg of protein. Western blot analysis, using the mAb 2-4A1 as the detection reagent, indicated the heterodimer of rhesus IL-12 at ~ 80 kDa, as well as an apparent surplus of p40 monomer at 40 kDa. The SDS-PAGE colored with coomassie blue indicates an additional prominent protein of equal intensity to the p80 heterodimer at ~ 70 kDa. Both the 80 and 70 kDa proteins are reduced to their monomeric forms after treatment with 2-mercaptoethanol, although the last protein band does not react with mAb 2-4A1.

EXAMPLE 4 Preparation, Characterization and Urination of Hybridoma Antibodies Mice that had a mutation in the p35 subunit gene of IL-12 in the Balb / c inheritance were produced, as described in Mattner, F. et al. , Eur. J. Immunol., 26: 1553-1559 (1996). Mice deficient in p35 of IL-12 were immunized intraperitoneally with 5 μg of purified recombinant human IL-12 in Freund's complete adjuvant. The mice received 3 subsequent intraperitoneal booster injections of 5 μg of human IL-12 in incomplete Freund's adjuvant over a period of 2.5 months. Final injections of 5μg of human IL-12 in PBS (50 μg i.p. and 25 μg i.v.) were given at three and two days prior to splenectomy, followed by an i.p. of 50 μg of human IL-12 in PBS, one day before splenectomy. The splenocytes of these mice were harvested and fused with the SP2 / 0 cells of the mouse myeloma, in a ratio of 1: 1, using 50% w / v of polyethylene glycol 1500 (Boehringer Mannheim) according to the Herzenberg method. , in Selected Methods in Cellullar Immunology, ed. B. Mishell and S. Shiigi, W. H. Freeman and Co., New York, 1980, p. 351-372. The fused cells were plated at a density of 60,000 total cells / well in 96-well cluster plates, in IMDM supplemented with 10% FBS (Hycione), 100 Units / ml penicillin G (Biowhittaker), 100 μg / ml streptomycin (Biowhittaker), 250 μg / ml Fungizone (Biowhittaker), 2 mM glutamine (Biowittaker), 100 μg / ml gentamicin sulfate (Biowittaker), 50 mM 2-mercaptoethanol (BioRad) ), 100 mM hypoxanthine (Sigma), 400 μM aminopterin (Sigma), 16 mM thymidine (Siq a) and 2.5% P388D1 supernatant (produced as described by Nordan, RP et al., J. Immunol.

Vol.139: 813 (1987) Hybridoma supernatants were subjected to an analysis to detect specific anti-human IL-12 antibodies, by immunoprecipitation of the Human IL-12 labeled with 125I as described below. Hybridoma cell lines secreting the anti-human IL-12 antibodies were cloned by limiting dilution. The antibodies were purified from the oils by sequential treatment with caprylic acid and ammonium sulfate, as described above.

(Reik, L. et al, J. Imol Methods, 100: 123-130 (1987)).

EXAMPLE 5 Preparation of human IL-12 labeled with 125 I Recombinant human IL-12 was radiolabelled to a specific activity of about 2200 Ci / mmol, using a modification of the procedure with Iodogen (Pierce Chemical Co.), previously described in Chizzonite et al., J. Immunol., 147: 1548-1556 (1991) and Chizzonite et al., J. Immunol., 148: 3117-3124 (1992), incorporated herein by reference. Iodogen was dissolved in chloroform and 0.05 mg was dried in a 12 x 15 mm borosilocate glass tube. For radiolabelling, 1.0 mCi Na [125I], (Amersham, Chicago, 111., USA) was incorporated into a tube coated with Iodogen, which contained 0.05 ml of Tris-iodination buffer (25 mM Tris-HCl). , pH 7.5, 0.4 M NaCl and lmM EDTA and incubated for 6 minutes at room temperature The activated 125I solution was transferred to a tube containing 0.1 ml of IL-12 (31.5 μg) in Tris-iodination buffer and the reaction was incubated for 6 minutes at room temperature. At the end of the incubation, 0.05 ml of Iodogen stop buffer (10 mg / ml tyrosine, 10% glycerol in Dulbecco's PBS, pH 7.40) was added and allowed to react for 5 minutes. Subsequently the mixture was diluted with 1% BSA (w / v), in 1.0 ml of Tris-iodination buffer and applied to a desalting column Bio-Gel P10DG (BioRad Laboratories (BRL)) for chromatography. The column was eluted with 1% BSA (w / v) in Tris-iodination buffer and the fractions (lml) containing the peak amounts of labeled protein were combined and diluted to lxlO8 cpm / ml with 1% BSA ( w / v) in Tris-iodization buffer. The precipitable radioactivity of the TCA (10% final TCA concentration) generally exceeded 95% of the total radioactivity. The radio-specific activity of recombinant human IL-12 was usually around 2200 Ci / mmol.

EXAMPLE 6 Immunoprecipitation analysis of human IL-12 labeled with 12SI Nunc Maxisorp 96-well detachable plates were coated with a purified antibody of rabbit affinity against mouse IgG (Cappel, Durham, NC, USA), by incubation for 18 hours, added with 100 pl / cavity of 5 μg / ml of rabbit anti-mouse IgG, in a carbonate coating buffer (15mM Na2C03 / NaHCO3 1035mM), pH 9.6. The coated plates were washed with PBS / 0.05% Tween-20/0, 01% Thimerosol and then blocked by incubation with 200ml of 1% (w / v) of BSA / PBS / O.01% Thimerosol, for 4 hours. hours, at 37 ° C. The supernatants of the hybridoma (75pA) were incorporated into the cavities coated with IgG anti-mouse and incubated for 3 hours at 22 ° C. The cavities were washed 3 times with 300 ml of PBS / O, 05% Tween-20/0, 01% Thimerosol and then 100,000 cpm of 125 I-labeled human IL-12 were incorporated in each of the cavities, in 100 ml of antibody dilution buffer (PBS / 1% BSP 0 w / v) / NaCl / 0.5% 0.05M Tween-20 / 0.01% Thimerosol). After 18 hours at 4 ° C, the wells were washed 3 times with 200 thousand PBS / 0.05% Tween-20/0, 01% Thimerosol. Then, the cavities were separated and the amount of radioactivity bound to the cavities was determined, using a gamma counter. In some experiments, after incubation of the hybridoma supernatants in the cavities coated with rabbit anti-mouse IgG, 100 ml of conditioned supernatant of the COS cells transfected with the p40 of human IL-12, prepared as described above, were incubated. described earlier (Gubler et al., Proc. Nati, Acad. Sci. 88: 4143-4147 (19-01)), in the cavities, for one hour, at 37 ° C before adding human IL-12 labeled with 125I to determine whether captured anti-human IL-12 antibodies bound to the p40 subunit of human IL-12.

EXAMPLE 7 Identification of Monoclonal Anti-human IL-12 Antibodies A 96-cavity immunoprecipitation assay was employed to identify the hybridomas secreting anti-human IL-12 antibodies. Hybridoma supernatants were incubated in the absence and in the presence of 100 μl of COS cell supernatant, which contained the p40 subunit of IL-12, as described above. Human 125I-labeled IL-12 (100,000 cpm / well) was added and the amount of 125I-labeled human TL-12 captured on the wells was determined. Fig. 1 indicates that the antibodies contained in the supernatants of the hybridomas 5F2, 16F2, 16G2, 20E11 and 17E2 captured human IL-12 labeled with 125I. Also, the presence of the p40 subunit of unlabeled human IL-12 during the immunoprecipitation reaction did not block the capture of 125I-labeled human IL-12 by the 5F2, 16F2, 16G2 and 20Ell antibodies, demonstrating that these antibodies do not have a high affinity for the p40 subunit of IL-12 alone. In contrast, the presence of the p40 subunit of unlabeled human IL-12 during the immunoprecipitation reaction completely blocked the capture of human IL-12 labeled with 125I by 17E2, which shows that 17E2 recognized the p40 subunit of human IL-12.

EXAMPLE 8 Analytical isoelectric focus of anti-human IL-12 monoclonal antibodies The analytical isoelectric approach was carried out by using an Ampholine PAG plate from Pharmacia Biotech, pH 3.5-9.5 (code No. 80). -1124-80, Uppsala, Sweden). The isoelectric focus was performed in accordance with the factory instructions, using phosphoric acid electrode solutions ÍM sodium hydroxide IN. The gel was loaded with 5 samples, each of which contained a single immunoglobulin, ie, 20E11, 5F2, 20C2, 16G2 and 16F2. The standards were those of the pH 3-10 Calibration Equipment for the isoelectric focus of Pharmacia Biotech (code No. 17-0471-01). The conditions of execution of the cycle were 1000 volts, 10 watts, 2.5 hours, 4 ° C. The gel was stained with silver, using the Silver Coloring Kit of Pharmacia Biotech Plusone for proteins (Code No. 17-1150-01) in accordance with the factory instructions. Figure 2 shows the isoelectric focusing patterns of the anti-human IL-12 monoclonal antibodies 20C2, 16G2, 16F2, 20E11 and 5F2.

EXAMPLE 9 Isoelectric Focusing Patterns of Anti-human IL-12 Monoclonal Antibodies As depicted in Fig. 2, monoclonal antibodies 20C2 (Chizzoni et al., Citckine, 6: A82a (1994)), 20E11 and 5F2 they are serum immunoglobulins. Monoclonal antibodies 16G2 and 16F2 appear identical by isoelectric focusing, although both differ from 20C2,20E11 and 5F2. The pl of these antibodies is in the range of a pH 5-6.

EXAMPLE 10 Generation of PHA-activated fibroblasts On day 4, the PHA-activated peripheral blood mononuclear cells (PBMC) were used to determine both the proliferation induced by natural human IL-12 and the proliferation induced by IL-12 of the rhesus monkey. The PBMCs were separated (Gately and Colaboradores, J.

Nati Cancer Inst., 69: 1245 (1982)) and stimulated with 0.1% PHA-P (Difco Labs., Detroit, MI, USA). After three days, the cultures were divided into a ratio of 1: 1 with fresh medium and 50 U of recombinant / ml of human IL-12, as described (Gately, M.K., Chizzonite, R. and Presky, D.H., Measurement of human and mouse interleukin 12, Current Protocols in Immunology, vol.I., J.E. Coligan, A.M. Kruisbeek, D.H.

Margulies, E.M. Shevach and W. Strober, eds., John Wiley & Sons, Inc., New York, 1995, pgs. 6.16.1-6.16.15). The supplemented medium used for cell culture was the one described above for the production of natural human IL-12, with 5% human AB serum (Irvine Scientific, Santa Ana, Ca, USES) . EXAMPLE 11 Analysis of Lymphocyte Proliferation The effects of various anti-human IL-12 monoclonal antibodies on the proliferation of human PHA-activated lymphoblasts, stimulated with IL-2 and IL-12, were determined by a method based on MK Gately et al. (Gately, MK, Chizzonite, R. and Presky, DH, Measurement of human and mouse interleukin 12, Current Protocols in Immunology, vol I., JE Coligan, AM Kruisbeek, DH Margulies, EM Shevach and W. Strober, eds., John Wiley &Sons, Inc., New York, 1995, pp. 6.16.1-6.16.15). On day 4, the PHA-activated lymphoblasts, prepared as described above, were harvested, washed and resuspended in a supplemented medium at a rate of 4 x 10 5 cells / ml and incubated in 96-well plates (2 x 10 4 cells). / cavity) with the purified monoclonal anti-human IL-12 antibody and the relevant cytokine, i.e., human or monkey IL-12. Aliquots of 25 μl of natural human IL-12 in a ratio of 1 ng / ml or of II-12 of monkey were mixed in a ratio of 2 ng / ml, with aliquots of 25 μl of various dilutions of monoclonal antibodies of the Anti-human IL-12 (mAbs). The final antibody concentration in the cavities varied between 0.0005 μg / ml and 0.5 μg / ml. An identical, separate set of cavities containing the various anti-human IL-12 mAbs and recombinant IL-12 was prepared in order to determine the effects of anti-human IL-12 mAbs on stimulated proliferation. with IL-2, as a measure of inhibitory specificity. A conventional dose response curve varying from 250 pg or 500 pg per human or monkey IL-12 cavity, respectively, to 0 pg without added antibodies was also included for the purpose of determining IL response capacity. -12. Plates containing mixtures of cytokines and antibodies were incubated for 30 minutes at 37 ° C and then aliquots of 50 μl of the cell suspension were added to the cavities. Culture plates were maintained at 37 ° C in a humidified atmosphere of 5% C02 in air, for 48 hours before pulsing 3H-thymidine. Fifty μl of 10 μCi / ml of 3 H-thymidine (diluted in media supplemented with 5% FCS, instead of 5% human AB serum) were added to each well. After incubation for an additional period of 6 hours, at 37 ° C, the contents of the cavities were harvested on glass fiber filters, through a cell harvester and the incorporation of 3H-thymidine in the cell was measured. Cellular DNA, by using a liquid scintillation counter. The values indicated in Figs. 3 and 4 are the averages of the tripled cavities.

EXAMPLE 12 Inhibition of proliferation of PHA-astivated lymphoblasts stimulated with cytokines, by anti-human IL-12 monoclonal antibodies. The proliferation of PHA-activated human lymphoblasts stimulated with 0.25 ng / ml IL.12 was inhibited, in a dose-dependent manner, by the 5F2, 16F2, 16G2, and 20E11 antibodies (Fig. 3). The potencies of these anti-human antibodies - defined as the concentration that produces 50% of the maximum inhibition (IC50) of 0.25 ng / ml of the proliferation stimulated by IL-12 - are: 0.03 μg / ml for 5F2; 0.01 μg / ml for 16F2; 0.01 μg / ml for 16G2 and 0.01 μg / ml for 20E11. The maximum (9440 cpm) and reference (1480 cpm) levels of the proliferation of lymphoblasts are represented by the horizontal dotted lines found at the upper and lower ends of the graphs, respectively. As indicated in FIG. 3, antibodies 5F2, 16F2, 16G2 and 20E11 were able to inhibit the proliferation of PHA-activated lymphoblasts stimulated by human IL-12 by at least 90%. In contrast, also as indicated in Fig. 3, the anti-human antibody 20C2, specific for p75 of IL-12, which has been previously identified (Chizzonite et al., Cytokine, 6: A82a (1994)) is incapable. of substantially inhibiting the bioactivity of human IL-12.

In addition, as shown in Fig. 4, antibodies 5F2, 16F2, 16G2 and 20E11 potently inhibited the proliferation of human PHA-activated lymphoblasts stimulated with 0.5 ng / ml of specific antibodies to IL-12 of the rhesus monkey , with an IC50 similar to that observed with proliferation stimulated by human IL-12. In contrast, 20C2 only exhibited a minimal inhibitory effect on the proliferation stimulated by IL-12 of the rhesus monkey. Therefore, the antibodies 5F2, 16F2, 16G2 and 20E11 appear to have good cross-reactivity with respect to the IL-12 of the rhesus monkey, while the cross-reactivity of the 20C2 is much less. None of these monoclonal antibodies inhibited the proliferation induced by IL-2, which shows that its effect on proliferation stimulated by IL-12 was specific for IL-12 and not due to a generalized inhibition of cell proliferation.

EXAMPLE 13 Analysis of the synthesis of interferon-? The synthesis of interferon-? (IFN-?) By using the PHA-activated human lymphoblasts of day 4, produced as described above. The medium used was a 1: 1 mixture of RPMI 1640 and Dulbecco's modified Eagle's medium, supplemented as previously described for the preparation of natural human IL-12 and also containing heat-inactivated fetal bovine serum. (56 ° C, 30 min.) At 5% (Hyclone, Logan, UT, USA) instead of the human AB serum. One ml cultures were placed in duplicate in the cavities of 24-well tissue culture plates (Costar). In each cavity, 5 x 105 PHA-activated lymphoblasts, 0.25 ng / ml of purified natural human IL-12, 20 units / ml of recombinant human IL-2, 1 ng / ml of recombinant human IL-1β (provided by Dr. R. Chizzonite, Hoffmann-La Roche) and the indicated concentrations of anti-human IL.12 antibodies. Initially, all reagents except lymphoblasts were added to the cavities and incubated at 37 ° C for 30 minutes, followed by the addition of the lymphoblasts. Subsequently cultures were incubated for 24 hours, at 37 ° C in a humidified atmosphere of 5% C02 in air. At the end of this period, the fluids of the culture supernatant were harvested by centrifugation and subjected to a test to determine their IFN-α content by the use of an ELISA. The amount of IF? -? produced in the cultures that contained the lymphoblasts with IL-2 + IL-1 but did not contain IL-12, was always less than 15% and, normally,, less than 5% than that produced in cultures containing 0.25 ng / ml of IL-12, in addition to IL-2 + IL-1. The ELISA to measure the IF? -? human used antibodies of the IF? -? monoclonal antibodies from Endogen (Woburn, MA). Plates? Unc EIA (Fisher) were coated overnight at 4 ° C, with 100 μl / well of 1 μl / ml IF? -? antihuman (Endogen # M-700A) in a coating buffer (α2C03 0, 015M +? aHC03 0, 035M in distilled water, pH 9, 6). The next morning, the coating buffer was rapidly removed from the cavities and the cavities were blocked by the addition of 200 μl / cavity of Dulbecco phosphate buffered saline solution (D-PBS); Fisher), which contained 1% bovine serum albumin (Sigma). After incubation for 1 hour at room temperature, the plates were washed with D-PBS, which contained 0.05% Tween 20 (Sigma) and 100 μl aliquots of IFN-α. recombinant human (Endogen) or culture supernatants, diluted in the assay buffer (D-PBS + 0.5% bovine serum albumin + 0.05% Tween 20), were incorporated into the cavities. Subsequently, the plates were incubated for 2 hours at room temperature, with stirring. After this, the plates were again washed and each well received 100 μl of 300 ng / ml IF? -? biotinylated anti-human (Endogen # M-701-B) in the test buffer. The plates were incubated for 1 hour at 37 ° C and subsequently washed. Aliquots of 100 μl streptavidin-peroxidase (Sigma) diluted 1: 1000 in the test buffer in each well were then added and the plates were incubated for 30 minutes at 37 ° C. The plates were re-washed and then allowed to develop, by the addition of 100 μl aliquots of a 1: 1 mixture of TMB Peroxidase Substrate and Peroxidase B Solution (Kirkegaard &Perry Laboratories, Gaithersburg, MD , USES) . The reaction was stopped after 312 minutes, through the incorporation of 50 μl / well of H3PO4 μM and the absorbance was read at 450 nm with the subtraction of the reference at 650 nm.

EXAMPLE 14 Inhibition of interferon-α production stimulated with cytokines by monoclonal anti-human IL-12 antibodies The production of IFN-α was inhibited. by the PHA-activated human lymphoblasts, stimulated with 0.25 ng / ml of human IL-12, in a dose-dependent manner, by the antibodies 5F2, 16F2, 16G2 and 20E11 (Fig. 5). The potencies of these anti-human antibodies, defined as the concentration that produces 50% of the maximum inhibition (IC50) 0.25ng / ml of IFN-α production stimulated by human IL-12, are: 0.02 μg / ml for 5F2; 0.02 μg / ml for 16F2; 0.01 μg / ml for 16G2 and 0.02 μg / ml for 20E11. These anti-human IL-12 antibodies specific for heterodimers were able to inhibit more than 90% of IFN-α production. stimulated by IL-12 when used at 0.5 μg / ml. In contrast, p75-specific anti-human antibody 20C2 of IL-12 p75 (Chizzonite et al., Cytokine,: A82a (1994)) is less potent and incapable of inhibiting the production of IFN-α. stimulated by IL-12 more than by 65%, at concentrations less than or equal to 0.5 μg / ml.

EXAMPLE 15 Analysis of the sequence of the genes encoding the variable region of the heavy chains of the antibodies present in the hybridoma cell lines producing anti-human IL-12 antibodies Total RNA was extracted from the hybridoma cells , using the Ultraspec RNA separation system and following the factory protocol (Biotecx, Houston, TX, USA). The cDNA of the first filament was synthesized from μg of the total RNA and the oligo-DT primers, in a volume of 20 μl. A 4 μl aliquot of the cDNA reaction mixture was used as a template for PCR amplification of the variable region of heavy chains of mouse IgG, using primers that were designed according to the sequence information of structure 1 and 4, as reported by Dattamajumdar et al. (AK Dattamajumdar et al., Immunogenetics 43: 141-151 (1996)). A 30 cycle PCR reaction was carried out, using an annealing temperature of 50 ° C. The entire PCR reaction was extracted with phenol, precipitated with ethanol and run on an agarose gel with low melting temperature (* low-melt) to separate the amplicon.The DNA fragment was excised from the gel, 70 ° C and 5 μl were reamplified in a 30-cycle PCR reaction to generate more material.The reamplified amplicon was gel purified and sequenced, using a Sanger sequencing method, based on fluorescence, with an automatic sequencer Applied Biosystems Incorporated.

EXAMPLE 16 Nucleotide and deduced amino acid sequences of the variable region of heavy chains of monoclonal anti-human IL-12 antibodies The nucleotide sequences of a portion of the variable region of the immunoglobulin heavy chain gene encompassing the structural region (FR, from the English Framework Region) 1, the complementary determining region (CDR, from the English Compl ementary Determining Region) 1, FR2, CDR2, FR3, CDR3 and FR4 of the IL-12 antibodies produced by the lines HIL-12F3-16G2 and HIL-12F3-20E11 hybridoma cell lines and the amino acid sequences deduced therefrom are shown in FIG. 6 and in Fig. 7, respectively. The CDR sequences are underlined. The comparison of the available sequence information indicated that the heavy chains of the antibodies produced by the hybridomas HIL-12F3-16G2 and HIL-12F3-20E11 exhibit a 94% homology at the DNA level and a similarity of 93% in the level of amino acids.

LIST OF SEQUENCES (I) GENERAL INFORMATION: (i) APPLICANT: (A) Name: F. Hoffman-La Ro AG (B) Street: Grenzacherstrasse (C) City: Basle (D) State: BS (E) Country: Switzerland (F) Zip Code: CH-4002 (G) Facsimile: 061-6881395 (H) Telex: 962292/965542 hlr c (ii) TITLE OF THE INVENTION: ANTIBODIES AGAINST THE HUMAN 12 (iii) NUMBER OF SEQUENCES: 4 (iv) ) COMPUTER READING FORM: (A) TYPE OF MEDIUM: diskette (B) COMPUTER: IBM, compatible with PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.25 (USA) (2) INFORMATION FOR ID SEQ NO: l: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 321 base pairs (B) TYPE: nucleic acid (C) FILAMENTS: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: rat (G) TYPE CELL: Hybridoma (H) CELL LINE: HIL-12F3-16G2 (IX) CHARACTERISTIC: ( A) NAME / KEY: CDS (B) LOCATION: 1.321 (xi) DESCRIPTION OF THE SEQUENCE: NO. SEC ID CTG GAG GCA TCA GGA CCT AGC CTC GTG AAA CCT TCT CAG ACT 48 CTG TCC Leu Glu Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln Thr Leu Ser 1 5 10 fifteen CTC ACC TGT TCT GTC ACT GGC GAC TCC ATC ACC AGT GGT TAC 96 TGG AAC Leu Thr Cys Ser Val Thr Gly Asp Ser lie Thr Ser Gly Tyr Trp Asn 20 25 30 TGG ATC CGG AAA TTC CCA GGG AAT AAA TTT GAG TAC ATG GGA 144 TTC ATA Trp lie Arg Lys Phe Pro Gly Asn Lys Phe Glu Tyr Met Gly Phe He 35 40 45 AGT TAT AGT GGT AGC ACT TAC AAT AAT CCA TCT CTC AAA AAT 192 CGA GTC Ser Tyr Ser Gly Ser Thr Tyr Asn Asn Pro Ser Leu Lys Asn Arg Val 50 55 60 TCC ATC ACT CGA GAC ACA TCC AAT AAC CAG TAC TAC CTG CAG 240 TTG AGT Ser He Thr Arg Asp Thr Ser Asn Asn Gln Tyr Tyr Leu Gln Leu Ser 65 70 75 80 TCT GTG ACT ACT GAG GAC TCA GCC ACA TAT TAC TGT GCA AGA 288 TCT TCG Ser Val Thr Thr Glu Asp Ser Wing Thr Tyr Tyr Cys Wing Arg Ser Ser 85 90 95 GAT GCT TTG GAC TAC TGG GGC GCA GGG ACC ACG 321 Asp Ala Leu Asp Tyr Trp Gly Ala Gly Thr Thr 100 105 (2) INFORMATION FOR NO. SEC ID DO NOT. 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 107 amino acids (B) TYPE: amino acid (D) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: NO. SEC ID : 2 Leu Glu Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln Thr Leu Ser 1 5 10 15 Leu Thr Cys Ser Val Thr Gly Asp Ser He Thr Ser Gly Tyr Trp Asn 20 25 30 Trp He Arg Lys Phe Pro Gly Asn Lys Phe Glu Tyr Met Gly Phe He 35 40 45 Ser Tyr Ser Gly Ser Thr Tyr Asn Asn Pro Ser Leu Lys Asn Arg Val Ser He Thr Arg Asp Thr Ser Asn Asn Gln Tyr Tyr Leu Gln Leu Ser 65 70 75 80 Ser Val Thr Thr Glu Asp Ser Wing Thr Tyr Tyr Cys Wing Arg Ser Ser 85 90 95 sp Wing Leu Asp Tyr Trp Gly Wing Gly Thr Thr 100 105 (2) INFORMATION FOR NO. SEC ID DO NOT. 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 308 base pairs (D) TYPE: nucleic acid (E) FILAMENTS: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (B) ORGANISM: rat (G) TYPE CELL: Hybridoma (H) CELL LINE: HIL-12F3-20E11 (ix) CHARACTERISTIC: (A) NAME / KEY : CDS (B) LOCATION: 1.306 (xi) DESCRIPTION OF THE SEQUENCE: NO. SEC ID: 3 GAG GCA TCA GGA CCT AGC CTC GTG AAA CCT TCT CAG ACT CTG 4í CC CTC Glu Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln Thr Leu Ser Leu 1 10 15 ACC TGT TCT GTC ACT GGC GAC TCC ATC ACC AGT GGT TAC TGG 96 AAC TGG Thr Cys Ser Val Thr Gly Asp Ser He Thr Ser Gly Tyr Trp Asn Trp 20 25 30 ATC CGG AAA TTC CCA GAT AAT ACA CTT GAG TAC ATG GGA TAC 144 ATA AGT He Arg Lys Phe Pro Asp Asn Thr Leu Glu Tyr Met Gly Tyr He Ser 35 40 45 TAC AGT GGT AGT ACT TAC TAC AAT CCA TCT CTC AGA AGT CGA 192 ATC TCC Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Arg Ser Arg He Ser 50 55 60 ATC ACT CGA GAC ACA TCC AAG AAC CAG TAC TCC ATG CAG TTG 240 AAT TCT He Thr Arg Asp Thr Ser Lys Asn Gln Tyr Ser Met Gln Leu Asn Ser 65 70 75 80 GTG ACT ACT GAG GAC ACA GCC ACA TAT TAC TGT GCA AGA TCC 2í TCG GAT Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Ser Ser Asp 85 90 95 GCT ATG GAC TAC TGG GGC GC 308 Wing Met Asp Tyr Trp Gly 100 (2) INFORMATION FOR NO, SEC ID. DO NOT. 4: (i) CHARACTERISTICS OF THE SEQUENCE: (C) LENGTH: 102 amino acids (D) TYPE: amino acid (D) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: NO. SEC ID Glu Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln Thr Leu Ser Leu 1 5 10 15 Thr Cys Ser Val Thr Gly Asp Ser He Thr Ser Gly Tyr Trp Asn Trp 20 25 30 He Arg Lys Phe Pro Asp Asn Thr Leu Glu Tyr Met Gly Tyr He Ser 35 40 45 Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Arg Ser Arg He Ser 50 55 60 He Thr Arg Asp Thr Ser Lys Asn Gln Tyr Ser Met Gln Leu Asn Ser 65 70 75 80 Val Thr Thr Glu Asp Thr Wing Thr Tyr Tyr Cys Wing Arg Ser Ser Asp 85 90 95 Wing Met Asp Tyr Trp Glu 100 It is noted that in relation to this date, the best known method for the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers.

Claims (16)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:
1. l.A monoclonal antibody to human IL-12 which is composed of a p35 subunit and a p40 subunit, wherein said monoclonal antibody: (a) reacts immunologically with an epitope presented by the p75 heterodimer of human IL-12, but is not immunologically reactive with respect to any epitope presented by said p40 subunit; and (b) neutralizes at least 90% of the bioactivity of human IL-12.
2. 2. The antibody according to claim 1, wherein the antibody neutralizes at least 90% of the bioactivity of human IL-12 by inhibiting the proliferation of lymphoblasts stimulated by IL-12, PHA-activated where the concentration of this antibody is 0.5μg / ml and the concentration of human IL-12 is 0.25μg / ml.
3. 3. The antibody according to claim 1, wherein the antibody neutralizes at least 90% of the bioactivity of human IL-12 by inhibiting the proliferation of IL-12 stimulated by IFN-α. where the concentration of this antibody is 0.5μg / ml and the The concentration of human IL-12 is 0.25μg / ml.
4. 4. The antibody according to any one of claims 1 to 3, wherein the antibody cross-reacts with the IL-12 of the rhesus monkey.
5. 5. The antibody according to any one of claims 1 to 4, wherein the antibody is produced from a mouse-cell line.
6. 6. The antibody according to any one of claims 1 to 5, wherein the antibody is a monoclonal antibody.
7. 7. The antibody according to any one of claims 1 to 6, wherein the antibody is produced by a hybridoma having the designation number of ATCC HB-12446, HB-12447, HB-12448, or HB-12449.
8. 8. The antibody according to claims 1-7, wherein the antibody is humanized.
9. 9. A hybridoma that produces an antibody as claimed in any of claims 1 to 8.
10. 10. A pharmaceutical composition comprising at least one antibody as claimed in any of claims 1 to 8.
11. 11. A method for producing an antibody that immunologically reacts selectively with the human IL-12p75 heterodimer which is composed of a p35 subunit and a p40 subunit comprising the steps of: (a) immunizing a mammal that is deficient in a gene which encodes said p35 subunit or said p40 subunit with the human IL-12p75 heterodimer to produce antibodies; (b) obtaining antibodies from the immunized mammal; tracing said antibodies with respect to their ability to selectively bind to an epitope presented by the p75 heterodimer to obtain said antibody that is selectively ligated.
12. 12. A method for producing a monoclonal antibody that immunologically reacts selectively with the human IL-12p75 heterodimer which is composed of a p35 subunit and a p40 subunit, comprising the steps of: (a) immunizing a mammal that has a deficiency of a gene encoding said p35 subunit or said p40 subunit with the human IL-12p75 heterodimer to produce antibodies; (b) harvesting cells that produce antibodies from the immunized mammal; (c) forming a hybridoma that produces a monoclonal antibody from said cells and obtaining said monoclonal antibody; (d) tracing said monoclonal antibody produced by said hybridoma with respect to the ability to selectively bind to an epitope presented by the p75 heterodimer to obtain said monoclonal antibody that is selectively linked.
13. 13. The method according to claim 12, wherein the antibodies produced by the hybridoma are further screened and selected with respect to their ability to cross-react with the IL-12 of the rhesus monkey.
14. 14. The antibody as claimed in any of claims 1 to 8 provided that it is produced by a process as claimed in any of claims 11 to 13 or a process comprising the process as claimed in any of claims 11 to the 13th
15. 15. The antibody as claimed in any of claims 1 to 8 as a therapeutically active agent.
16. 16. The use of any of the antibodies claimed in any of claims 1 to 9 for the preparation of a medicament for the control of diseases with pathologies that are mediated by immune mechanisms, particularly, diseases associated with bioactivity IL-12 increased that results in aberrant activity of Thl-type collaborating cells.