ES2365712T3 - ANTIBODIES AGAINST NIK, ITS PREPARATION AND USE. - Google Patents

Abstract

An antibody or an antigen-binding fragment thereof that specifically detects the endogenously phosphorylated NF-kappaB-inducing kinase, NIK, which is characterized in that said antibody or said antigen-binding fragment is capable of specifically binding to a portion of the amino acid sequence that appears in SEQ ID NO: 5 which comprises a phosphorylated threonine at amino acid position 559.

Description

Field of the Invention

The present invention relates to immunoregulatory molecules. More specifically, the present invention relates to antibodies or antibody fragments capable of specifically binding to the NF-B (NIK) / MAP3K14 inducing kinase, or to a specific portion thereof, and their use, for example for regulate a biochemical activity of NIK and / or to allow the detection of NIK or a specific portion thereof.

Background of the invention

There is no satisfactory treatment or treatment for numerous lethal and / or very debilitating diseases associated with a deregulated activity of NF-B molecules, including malignant diseases and diseases associated with pathological immune responses, such as autoimmune, allergic, inflammatory diseases. and related to transplants.

NF-NB family molecules are complexes of eukaryotic transcription factors critical for the regulation of immune responses, cell growth and survival (Ghosh, S. et al., 1998, Annu. Rev. Immunol. , 16: 225-260) which are inducibly activated by almost all members of the TNF / NGF receptor family. NF-B molecules are normally sequestered in the cytoplasmic compartment by physical association with a family of anchrine-rich cytoplasmic inhibitors called IB, which includes IB and related proteins (Baldwin, AS, Jr., 1996 , Annu. Rev. Immunol., 14: 649683). In response to various stimuli, including cytokines, mitogens and certain viral gene products, the IB is rapidly phosphorylated at Ser32 and Ser36, and is ubiquitinated and subsequently degraded by the 26S proteasome. This allows the released NF-seB to be translocated to the nucleus and participate in the transactivation of target genes (Mercury, F. and Manning, AM, 1999, Curr. Opin. Cell Biol., 11: 226-262; Pahl, HL , 1999, Oncogene, 18: 6853-6866). Recent molecular cloning studies have identified a multi-subunit IB kinase complex (IKK) that mediates phosphorylation induced by IB signals, an NF-B inhibitor. The IKK complex is composed of two catalytic subunits, IKK and IKK, and a regulatory subunit, IKK (NEMO). The catalytic activity of IKK and IKK can be activated by a multitude of different NF-B inducers, including inflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin (IL) -1, the receptor of T cells (TCR) and costimulatory protein T cells, CD28 (Karin, M. and Ben-Neriah, Y., 2000, Annu. Rev. Immunol., 18: 621-663).

The NF-B inducing kinase (NIK) / MAP3K14 (WIPO Publication No. WO 97 / 37016A1 of the present inventors) is critical for the activation of NF-B. For example, it has been shown that NIK overexpression leads to strong activation of NF-B (published in Wallach, D. et al., 2002, Arthritis Res., 4, Suppl. 3: S189-196), and It has been shown that the expression of catalytically inactive NIK mutants leads to effective inhibition of NF-B activation in response to a variety of known NF-B activators, such as LMP1, TNF receptor (TNFR) - 1, TNFR-2, RANK, human Toll receptor, CD3 / CD28, IL-1 receptor (IL-1R), Tax protein of human T-cell lymphotropic virus (HTLV) -1, and lipopolysaccharide (LPS) (Malinin , NL et al., 1997, Nature, 385: 540-544; Sylla, BS et al., 1998, Proc. Natl. Acad. Sci. USA, 95: 10106-10111; Damay, BG et al., 1999, J. Biol. Chem., 274: 7724-7731; Lin, X. et al., 1999, Immunity, 10: 271-280; Geleziunas, R. et al., 1998, Mol. Cell. Biol., 18: 5157-5165). The directed disruption of the NIK gene (Yin, L. et al., 2001, Science, 291: 2165-2165) and the study of the breed of mice with "alinfoplasia" (aly) that carry a point mutation of a natural missense Gly885Arg in NIK (Shinkura, R. et al., 1999, Nat. Genet., 22: 74-77) revealed that NIK plays an essential role in the development of lymphoid organs. Both aly / aly mice and mice with inactivated NIK genes manifest a systemic absence of lymph nodes and Peyer's plaques, disorganized splenic and thymic architectures, and immunodeficiency whose most elastic characteristics are low levels of serum immunoglobulins and a lack of graft rejection ( Shinkura, R. et al., 1999, Nat. Genet., 22: 74-77). These anomalies seem to reflect aberrant signaling by a variety of receptors. The development of deficiencies in NIK-mutant mice resembles what appears in mice deficient in the LT- receptor (LT-R), which suggests that NIK also participates in signaling by this particular receptor. It has been shown that an impaired proliferative capacity of B cells in aly / aly mice correlates with a poor response of these cells to the LPS and the CD40 ligand (CD40L; Garceau, N. et al., 2000, J. Exp. Med., 191: 381-386), and the presence of an excessive amount of B1 cells in the peritoneal cavity of mice can be attributed to defects in the peritoneal cell housing in the intestine-associated lymphatic tissue system (GALT) as a consequence of poor signaling of chemokine receptors in secondary lymphoid tissue (Fagarasan, S. et al., 2000, J. Exp. Med., 191: 1477-1486).

An important and general role for NIK in signaling cytokine receptors has been demonstrated recently in studies conducted by Wallach et al. which have demonstrated, using a two-hybrid system, that the  chain of the IL-2 receptor, or “common  chain,” which is a signaling component of the receptors for IL-2, IL-4, IL-7, IL-9, IL-13, IL-15 and IL-21, is specifically associated with NIK (WO 03/087380). It has been found that overexpression of the common  chain enhances the activation of NF-B mediated by NIK, and after stimulation with IL-2 or IL-15, it has been found that NIK and signalosome components bind to the common  chain. Therefore, these results indicate the involvement of NIK in signaling through the wide variety of cytokine receptors that comprise the common  chain as a signaling subunit.

Apart from these and other contributions to the regulation of the development and function of the immune system, NIK is also involved in the regulation of various non-immunological functions. The aly / aly mice (but not the mice with inactivated NIK genes) show poor development of the mammary glands (Miyawaki, S. et al., 1994, Eur. J. Immunol., 24: 429-434). In addition, in vitro studies have implicated NIK in signaling that leads to the differentiation of skeletal muscle cells (Canicio, J. et al., 2001, J. Biol. Chem., 276: 2022820233) and survival and differentiation of neurons (Foehr, ED et al., 2000, J. Biol. Chem., 275: 3402134024).

Consistent with the suggested role of NIK as a mediator in the activation of NF-B, fibroblasts derived from aly / aly mice and mice with inactivated NIK genes do not activate NF-B in response to LT- activation R. In addition, overregulation by LT-regR of VCAM-1, which occurs through the activation of NF-B, is abnormal in mouse embryonic fibroblasts aly / aly (MEF; Matsumoto, M. et al., 1999 , J. Immunol, 163: 1584-1591). Poor phosphorylation of IB in the response of aly / aly B lymphocytes to the CD40 coupling has also been noted. In contrast, in dendritic cells of these mice, CD40-induced phosphorylation of IB is normal (Garceau, N. et al., 2000, J. Exp. Med., 191: 381-386). Aly / aly peritoneal cells are also unable to respond to SLC chemokine with increased NF-B activity (Fagarasan, S. et al., 2000, J. Exp. Med., 191: 14771486).

Evaluation of the pattern of NF-B species expressed in lymphoid organs of aly / aly mice indicates that, apart from their role in regulating the complex or NF-B complexes formed by ReI proteins (A + p50 ) and IB, the NIK also participates in the control of the expression / activation of other NF-B species. Most notably, aly / aly lymphocytes are deficient in p52, a species of NF-B that is formed specifically in mature B lymphocytes through proteolytic processing of an inactive precursor, p100 (NFB2), which suggests a deficiency in the conversion of p100 to p52 (Yamada, T. et al., 2000, J. Immunol., 165: 804812). Indeed, it has been shown that NIK participates in site-specific phosphorylation of p100. Both lead directly to the phosphorylation of IKK which, in turn, phosphorylates to p100. This phosphorylation acts as a molecular trigger for the ubiquitination and active processing of p100 to form p52. It has been found that this p100 processing activity disappears with the aly mutation (Xiao, G. et al., 2001, Mol. Cell, 7: 401-409; Senftleben, U. et al., 2001. Science, 293: 1495-1499).

In view of the structural homology of NIK with MAP3K, some attempts have been made to explore the involvement of NIK in the ERK, JNK and p38 cascades, the other three major protein kinase cascades that involve MAP3K (Akiba, H. et al., 1998, J. Biol. Chem., 273: 13353-13358). It has been shown that NIK is involved in the ERK cascade in PC12 pheochromocytoma cells (Foehr, E.D. et al., 2000, J. Biol. Chem., 275: 34021-34024). Evidence has also been presented that, in certain cells, NIK can participate in signaling for phosphorylation of Jun, the current target below the JNK cascade, regardless of this particular cascade (Akiba, H. et al., 1998 , J. Biol. Chem., 273: 13353-13358; Natoli, G. et al., 1997, J. Biol. Chem., 272: 26079-26082). Overall, these findings indicate that NIK in effect acts as a mediator of NF-FB activation but may also have other functions, and that it exerts these functions in a specific manner of cells and receptors.

Similar to other MAP3K, NIK can be activated as a result of phosphorylation of the "activation loop" within the NIK molecule. Indeed, the mutation of a phosphorylation site within this loop (Thr559) prevents the activation of NF-B after the overexpression of NIK (Lin, X. et al., 1999, Immunity, 10: 271-280). In addition, NIK activity appears to be regulated through the ability of the upstream and downstream regions of their kinase motif to bind to each other (Lin et al., Molec. Cell Biol. (18), 10: 5899-5907 , 1998). The C-terminal region of NIK downstream of its kinase moiety has proven to be able to bind directly to IKK (Regnier,

C.H.

et al., 1997, Cell, 90: 373-383), as well as p100 (Xiao, G. and Sun, SC, 2000, J. Biol. Chem., 275: 21081-21085) and TRAF2 (Malinin, NL et al., 1997, Nature, 385: 540-544). These interactions appear to be necessary for the function of NIK in NF-B signaling. The N-terminal region of the NIK contains a negative regulatory domain (NRD) that is composed of a basic motif (BR) and a repeated proline motif (PRR) (Xiao, G. et al., 2001, Mol. Cell , 7: 401-409). Apparently, the N-terminal NRD interacts with the C-terminal region of NIK in cis, thereby inhibiting the binding of NIK to its substrate (IKK and p100). Ectopically expressed NIK appears to spontaneously form oligomers in which these junctions of the N-terminal to C-terminal regions in each NIK molecule appear to be disrupted, and show a higher level of constitutive activity (Lin, X. et al. , 1999, Immunity, 10: 271-280). It is very likely that the binding of the C-terminal region of NIK to the adapter protein associated with the TRAF2 TNF receptor, as well as to other TRAFs (Malinin, N.L. et al., 1997, Nature, 385: 540-544; Rothe,

M.

et al., 1994, Cell, 78: 681-692; Takeuchi, M. et al., 1996, J. Biol. Chem., 271: 19935-19942) participate in the activation of NF-B by NIK.

Evidence has been presented that NIK, through the union of its C-terminal region to IKK, can activate the IKK complex. It has been shown that it is able to phosphorylate Ser176 in the IKK activation loop, and thereby activate this molecule (Ling, L. et al., 1998, Proc. Natl Acad. Sci. USA, 95: 3792-3797 ). Consistent with this mode of action, studies on the mechanisms responsible for the deficiency in the activation of NF-B by LT-R in MEF of aly / aly have indicated that the NIK mutation eliminates the activation of the IKK signalosome and the consequent phosphorylation of IB (Matsushima, A. et al., 2001, J. Exp. Med., 193: 631-636). The ability of NIK to bind p100 directly through its C-terminal region and phosphorylate it suggests that p100 acts as a direct substrate of NIK (Xiao, G. and Sun, SC, 2000, J. Biol. Chem., 375: 21081-21085). However, a recent study suggests that NIK mediates phosphorylation of p100 indirectly, through phosphorylation and, therefore, the activation of IKK, which in turn phosphorylates p100 (Senftleben, U. et al. , 2001, Science, 293: 1495-1499).

Therefore, the activation of NF-B molecules by NIK represents a critical control point for the regulation of NF-B activity.

As described above, a deregulated NF-B activity is associated with the pathogenesis of various important human diseases, such as numerous malignant diseases and diseases associated with pathological immune responses (published in Yamamoto and Gaynor, 2001, J. Clin. Invest. ., 107: 135-142). For example, activation of the NF-B pathway is very involved in the pathogenesis of chronic inflammatory diseases, such as asthma, and rheumatoid arthritis (Tak and Firestein, 2001, J. Clin. Invest., 107: 7 -11; Karin, M. and Ben-Neriah, Y., 2000, Annu. Rev. Immunol., 18: 621-663), and inflammatory bowel disease. In addition, an altered regulation of NF-pareceB seems to be involved in the pathogenesis of other diseases, such as arteriosclerosis (Collins and Cybulsky, 2001, J. Clin. Invest., 107: 255-264; Leonard, WJ et al. , 1995, Immunol. Rev., 148: 97-114) and Alzheimer's disease (Mattson and Camandola, 2001, J. Clin. Invest., 107: 247-254; Lin, X. et al., 1999, Immunity , 10: 271-280), in which the inflammatory response is at least partially involved.

Several lines of evidence indicate that NF--B activation of cytokine genes is an important contributor to the pathogenesis of asthma, which is characterized by the infiltration of inflammatory cells and the deregulation of many cytokines and chemokines in the lung (Ling, L. et al., 1998, Proc. Natl. Acad. Sci. USA, 95: 3792-3797). Cytokines, such as TNF, that activate NF-estánB are in high amounts in the synovial fluid of patients with rheumatoid arthritis and contribute to the chronic inflammatory changes and synovial hyperplasia seen in the joints of these patients (Malinin, NL et al., 1997, Nature, 385: 540-544). The administration of antibodies directed against TNF or a truncated TNF receptor that binds to TNF greatly improves the symptoms of patients with rheumatoid arthritis.

The increase in the production of proinflammatory cytokines by lymphocytes and macrophages has also been implicated in the pathogenesis of inflammatory bowel diseases, including Crohn's disease and ulcerative colitis (Matsumoto, M. et al., 1999, J. Immunol., 163: 1584-1591). NF-FB activation is observed in specimens of mucosal biopsies of patients with active Crohn's disease and ulcerative colitis. The treatment of patients suffering from inflammatory bowel diseases with steroids decreases NF-B activity in biopsy specimens and reduces clinical symptoms. These results indicate that stimulation of the NF-B pathway is involved in the enhanced inflammatory response associated with these diseases.

Atherosclerosis is activated by numerous attacks on the endothelium and smooth muscle of the wall of damaged vessels (Matsushima et al., 2001). A large number of growth factors, cytokines and chemokines released from endothelial cells, smooth muscle, macrophages and lymphocytes are involved in this chronic inflammatory and fibroproliferative process (Matsushima, A. et al., 2001, J. Exp. Med., 193: 631-636). The regulation by NF-B of genes involved in the inflammatory response and in the control of cell proliferation is already understood to play an important role in the onset and progression of atherosclerosis.

As described above, it has been shown that abnormalities in the regulation of the NF-B pathway are implicated in the pathogenesis of Alzheimer's disease. For example, immunoreactivity to NF-B has been found predominantly and around the types of early neuritic plaques in Alzheimer's disease, while mature plaque types show a very reduced NF-actividadB activity (Mercury, F .and Manning, AM, 1999, Curr. Opin. Cell Biol., 11: 226-232). Therefore, the activation of NF-seB occurs at the onset of the formation of neuritic plaques and neuronal apoptosis during the early stages of Alzheimer's disease. Therefore, these data indicate that activation of the NF-B pathway plays a role in a number of diseases that have an inflammatory component involved in their pathogenesis.

In addition to the role in the pathogenesis of diseases associated with deregulated immune responses, hyperactivation or constitutive activation of the NF-B pathway has also been implicated in the pathogenesis of various human cancers. An abnormally high and / or constitutive activation of the NF-B pathway is frequently observed in a variety of human malignancies, including leukemias, lymphomas and solid tumors (Miyawaki, S. et al., 1994, Eur. J. Immunol ., 24: 249-234). These abnormalities produce abnormally high and / or high constitutive levels of NF-B in the nucleus of a variety of tumors, including breast, ovarian, prostate and colon cancers. Most of these changes are probably due to alterations in regulatory proteins that activate signaling pathways that lead to activation of the NF--B pathway. However, mutations that inactivate IB proteins, in addition to amplification and rearrangement of genes encoding members of the NF-B family, can produce the increased nuclear levels of NF-B that are observed in some tumors. (Emmerich et al., Blood, November 1, 94 (9): 3129-3134, 1999).

Since, as described above, NIK is critical for the activation of NF-B, a potentially potent strategy to regulate the activation of NF-B and, therefore, to treat diseases associated with an NF-actividad activity Deregulated B, involves identifying antibodies capable of specifically binding to NIK or a specific portion thereof and thereby preventing or inhibiting the activation of NF--B by NIK.

Prior art strategies have failed to provide antibodies capable of binding to phosphorylated NIK in amino acid T559. An antibody of this type, which was supposedly specific for phosphorylated NIK in T559, was acquired by the inventors last year (2002) in Santa Cruz (SC12957R in Nacalai Tesque News [online], vol. 12, 2001). However, the antibody did not function properly when the present inventors used it and also bound to an NIK mutant that cannot phosphorylate on T559. This year, Santa Cruz removed it from its catalog. EP-A2 1,201,765 describes antibodies against a different kinase called kinase that interacts with Nck (NIK) / MAP4k4.

At present, a method for detecting different molecular species of NIK, comprising non-phosphorylated and active phosphorylated species, employs overexpression in epitope-labeled NIK cells and an anti-marker antibody. This method of detecting phosphorylated NIK has many limitations; One of them is that the method cannot be used to specifically detect endogenously activated NIK.

Therefore, the need (and its obtaining) of antibodies capable of binding to endogenous phosphorylated NIK is widely recognized.

Summary of the invention

The invention relates to a polyclonal, monoclonal, chimeric, humanized, human or anti-anti-idiotypic antibody,

or to its fragments, which is capable of specifically binding to an amino acid sequence that appears in SEQ ID NO: 5, or to a portion thereof, in which the amino acid sequence, or a portion thereof, comprises a phosphorylated threonine at amino acid position 559 of SEQ ID NO: 5.

In one embodiment of the invention, the antibody fragment is selected from the group consisting of a single chain Fv, a Fab, a Fab ', an F (ab') 2, and a CDR, which is capable of specifically binding to the amino acid sequence that appears in SEQ ID NO: 5, or a portion thereof, such as that which appears in SEQ ID NO: 6 and 3, in which the amino acid sequence, or a portion thereof, comprises a threonine phosphorylated at amino acid position 559 of SEQ ID NO: 5.

In another embodiment, the invention provides a polyclonal or monoclonal IgG antibody, such as antibodies generated by hybridoma NIK-P4 30.12, deposited in the CNCM as No. I-3095.

Also described is a polyclonal, monoclonal, chimeric, humanized, human or anti-anti-idiotypic antibody, or its fragments, which is capable of specifically binding to NIK or its mutein, its functional derivative, its active fraction, its circularly permuted derivative or its salt, the antibody being prepared by immunizing a mammal with an amino acid sequence, or a portion of the amino acid sequence that appears in SEQ ID NO: 5, preferably the amino acid portion of SEQ ID NO: 3.

In a preferred embodiment of the invention, the antibody of the invention is capable of regulating a biochemical activity of a NIK molecule and / or specifically detecting phosphorylated NIK, or a specific portion thereof, for example by an analysis of Western blot, ELISA or immunoprecipitation.

In another aspect, the invention provides monoclonal antibodies generated by hybridoma NIK-P4 30.12, deposited in the CNCM as No. I-3095.

The invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and, as an active ingredient, a polyclonal, monoclonal, chimeric, humanized, human or anti-anti-idiotypic antibody,

or its fragments, which is capable of specifically binding to the amino acid sequence that appears in SEQ ID NO: 5, or to a portion thereof, in which the amino acid sequence, or a portion thereof, comprises a phosphorylated threonine at amino acid position 559 of SEQ ID NO: 5. More specifically, the pharmaceutical composition comprises an antibody or a fragment of an antibody that is also capable of regulating a biochemical activity of a NIK molecule, for example, inhibiting the signaling of CD40 and / or CD70.

In another aspect, the invention relates to a method for regulating a biochemical activity of NIK, said method comprising contacting the NIK molecule with a polyclonal, monoclonal, chimeric, humanized, human or anti-anti-idiotypic antibody, or its fragments, which is capable of specifically binding to the amino acid sequence that appears in SEQ ID NO: 5, or to a portion thereof, in which the amino acid sequence, or a portion thereof, comprises a phosphorylated threonine at the position of the amino acid 559 of SEQ ID NO: 5, thereby regulating a biochemical activity of a NIK molecule. More specifically, said contacting of the NIK molecule with said preparation can be accomplished by administering said preparation to an individual.

In one embodiment, the invention relates to a composition of matter comprising a substrate, such as an affinity chromatography matrix, covalently linked to a peptide with the amino acid sequence that appears in SEQ ID NO: 5, or to a portion of this, for example SEQ ID NO: 3, in which the amino acid sequence, or a portion thereof, comprises a phosphorylated threonine at amino acid position 559, to selectively capture the antibody or antibody fragment that is capable of specifically binding to the target antigen.

In another embodiment, the invention relates to a method of treating a disease caused or aggravated by the activity of NIK, which comprises the administration of an antibody that is capable of specifically binding to the amino acid sequence that appears in SEQ ID. NO: 5. or to a portion thereof, in which the amino acid sequence, or a portion thereof, comprises a phosphorylated threonine at amino acid position 559 of SEQ ID NO: 5, to an individual in need thereof.

In another embodiment, the invention relates to a method for the purification of a NIK-binding protein, which comprises contacting a sample, such as body fluids, cell extracts and DNA expression banks, containing the NIK samples and the NIK-binding protein, with an antibody of the invention, co-immunoprecipitate NIK and NIK-binding protein, wash the produced immune complex, and recover the NIK-binding protein from the immune complex using a competing peptide derived from NIK.

In another embodiment, the invention provides the use of an antibody according to any one of claims 1 to 12, for the development of an ELISA.

In another embodiment, the invention provides the use of an antibody according to the invention, for the immunopurification of NIK or its mutein, its functional derivative, its active fraction, its circularly permuted derivative or its salt.

In addition, in one embodiment, the invention indicates the use of an antibody or a fragment of an antibody that is capable of specifically binding to the amino acid sequence that appears in SEQ ID NO: 5, or a portion thereof, in which the amino acid sequence, or a portion thereof, comprises a phosphorylated threonine at amino acid position 559 of SEQ ID NO: 5, for the manufacture of a medicament for the treatment of a disease caused or aggravated by NIK activity .

In a preferred embodiment of the invention, the disease is associated with pathological immune responses, such as an autoimmune, allergic, inflammatory and transplant-related disease, and preferably asthma, rheumatoid arthritis, inflammatory bowel disease, atherosclerosis and Alzheimer's disease.

In another preferred embodiment of the invention, the disease is a malignant disease.

In addition, the invention indicates the use of an antibody or a fragment of an antibody of the invention in a method for identifying a ligand that is capable of inducing NIK-mediated activation of NIK-mediated in a cell. In one embodiment of the invention, the method comprises the step of introducing the antibody or antibody fragment into a cell, incubating the cell with individual ligands, controlling the activation of NF-yB and preferably the activation of NF-aB at through the canonical pathway, for example by controlling the degradation of IBa, and selecting the ligand that has affected the activation of NF-medianteB by specific blocking of NIK by the antibody. In a preferred embodiment, the cell in the method for identifying the ligand is of lymphoblast type, such as Ramos, BJAB and Jurkat cells.

In addition, the invention provides a method for preparing a monoclonal antibody, which comprises culturing a cloned hybridoma comprising a splenic cell from a mammal immunized with an amino acid sequence comprising SEQ ID NO: 6, or a portion thereof, in the that the amino acid sequence, or a portion thereof, comprises a phosphorylated threonine at amino acid position 559 of SEQ ID NO: 5, and a homogenous or heterogeneous lymphoid cell in a liquid medium or mammalian abdomen, to allow the hybridoma produce and accumulate the monoclonal antibody.

The invention is defined by the claims.

Brief description of the drawings

The invention is described herein, by way of example only, with reference to the accompanying drawings. With specific reference to the drawings in detail, it is emphasized that the details shown are only examples and are offered only for an illustrative analysis of the preferred embodiments of the present invention, and are presented to provide what is believed to be the more useful and easily understandable description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show the structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, and the description together with the drawings will make it apparent to those skilled in the art how various forms of the invention can be realized in practice. In the drawings:

Figure 1 shows a diagram representing the NIK, T559 domains, the N-terminal region, the kinase region, and the NIK C-terminal domain (405-420), etc.

Figure 2 shows a sequence representing the amino acid sequence of human NIK (SEQ ID NO: 5). The NIK-derived peptide used for immunization, SEQ ID NO: 3, is underlined.

Figure 3 shows the amino acid sequence of the NIK activation loop (SEQ ID NO: 4).

Figure 4 shows an immunoprecipitation (IP) with different monoclonal antibodies prepared using the phosphorylated peptide as it appears in SEQ ID NO: 3. The cells were transfected with pCS3MTNIK (WT-NIK), or with the vector encoding the non-phosphorylated version of the protein, pCS3MTNIKT559A (NIK-). Transfected cells were collected and lysed in 2 ml of 1% NP-40 buffer. 100 ul of this lysate were used for each IP. IP was performed with G protein spheres adsorbed with the indicated antibodies. A Western blot detection and a positive control for IP were performed with the anti-myc antibody.

Figure 5 shows the differential binding of the antibody prepared with the peptide of SEQ ID NO: 3 to the phosphorylated peptide derived from the activation loop by ELISA. The control and the phosphorylated peptide derived from the activation loop peptide (SEQ ID NO: 3) were coated on microtiter plates at a concentration of 10 µg / ml. The undiluted hybridoma culture supernatant from clone NK-P4 30.12 was added for 1 hour at 37 ° C. Anti-mouse HRP was used as secondary antibody at a 1: 10K dilution and OD 405 was measured using an ABTS substrate.

Figure 6 shows the effect of NIK-P4 30.12 on the degradation of IB induced by CD70, CD40 or TNF. Ramos cells were transfected with IgG (negative control) or NIK-P4 30.12 antibody (-pNIK). Transfected cells were not treated or treated with CD70, CD40 and TNF for 20 min, 30 min and 20 min, respectively. After treatment with the ligand, the cells were lysed and subjected to Western blot analysis probed with an anti-IB antibody.

Figure 7 shows the effect of NIK-P4 30.12 with respect to a different antibody generated against an NIK peptide also located in the kinase domain but outside the activation loop, on the I40 degradation induced by CD40. The cells were transfected with IgG (negative control), NIK-P4 30.12 antibody (p4) or a different antibody generated against an NIK peptide also located in the kinase domain but outside the activation loop (SEQ ID NO: 7, 81) . Transfected cells were not treated (-cd40) or treated with CD40 (+ cd40) for 30 min. After treatment with the ligand, the cells were lysed and subjected to Western blot analysis probed with an anti-IB antibody.

Description of preferred embodiments

The present invention relates to an antibody or a fragment of an antibody that is capable of specifically binding to the NF-B (NIK) / MAP3K14 inducing kinase (SEQ ID NO: 5) phosphorylated in the T559 moiety, or a specific portion of it, and its use. Specifically, the antibody of the present invention can be used to regulate a biochemical activity of NIK, and to specifically detect phosphorylated NIK in the T559 moiety,

or a specific portion of it. By virtue of allowing said regulation and said detection, the antibody can be used, respectively, to regulate the activation of NF-B and, therefore, to treat a disease associated with a deregulated NF-B activity, and to characterize aspects of normal / pathological processes / biological / biochemical states that involve NIK, or a specific portion thereof.

There is no satisfactory treatment or treatment for numerous lethal and / or very debilitating diseases associated with a deregulated NF-B activity. These diseases include malignant diseases and diseases associated with pathological immune responses, such as autoimmune, allergic, inflammatory and transplant-related diseases. Since NIK is a critical activator of NF-B, a potentially potent strategy to treat a disease associated with deregulated NF-implicaB activity involves identifying antibodies capable of specifically binding to phosphorylated NIK, or a specific portion of this, and use said antibodies to regulate therapeutically the NF-B activity. By virtue of allowing the specific detection of phosphorylated NIK, or a specific portion thereof, these antibodies also allow the characterization of aspects of normal / pathological processes / biological / biochemical states that involve phosphorylated NIK, or a specific portion of is.

While the present invention was being implemented, an antibody was unexpectedly generated that was capable of specifically binding or binding optimally specifically to NIK (SEQ ID NO: 5) phosphorylated at T559, or to a specific portion of this, such as the portion that appears in SEQ ID NO: 3. The ability of the antibody of the present invention to specifically bind phosphorylated NIK in T559, or a portion thereof, such as the portion that appears in SEQ ID NO: 3, is exclusive in relation to all antibodies in the art previous.

Therefore, by clear contrast with the prior art antibodies, the antibody of the present invention can be used to optimally regulate a biochemical activity, such as a kinase activity of NIK, or a specific portion thereof, and to optimally detect the NIK phosphorylated in T559, or a specific portion thereof. The antibody of the invention can also be used to co-immunoprecipitate and isolate regulatory factors that bind to phosphorylated NIK in T559.

Those skilled in the art will appreciate that an antibody, such as that of the present invention, can be treated using conventional methods, for example as described hereinbelow, to generate one or more types of antibody fragments having binding characteristics. to the antigen fundamentally identical to those of the untreated antibody.

Therefore, according to one aspect of the present invention, there is provided an antibody or an antibody fragment that is capable of specifically binding to the phosphorylated NIK in T559, or a portion thereof, such as the amino acid sequence that appears in SEQ ID NO: 3.

The term "a portion of a peptide" is defined as at least one tripeptide within the activation loop, in which one of the amino acids is phosphorylated threonine and the amino acids adjacent to the N-terminal and the C-terminal are glycine and glutamic acid. , respectively, that is, GT (p) E.

The portions of the phosphorylated activation loop (SEQ ID NO: 4) having as a penultimate amino acid the phosphorylated T599 are hereinafter referred to as "target antigen". T599 is the threonine residue at position T599 in the complete NIK protein (SEQ ID NO: 5) and is also the threonine residue that is conserved in the portions derived from it, for example, threonine at position 11 is SEQ ID NO : 3, threonine at position 26 in SEQ ID NO: 4, and threonine at position 26 in SEQ ID NO: 6.

As described hereinbelow, the antibody can be used to optimally regulate the biochemical activity of the NF-B (NIK) / MAP3K14 inducing kinase, such as a kinase activity, and therefore can be used to optimally regulate the NF-B activation, and therefore can be used to optimally treat a disease associated with deregulation of NF-B activity. In addition, the antibody can be used exclusively to detect the region of the phosphorylated activation loop of NIK, or any of several specific portions thereof, and can be used to optimally detect any of several specific portions of the region of the loop of NIK activation. As such, the antibody can be used to optimally characterize aspects of normal / pathological processes / biological / biochemical states that involve phosphorylated NIK in T559, or a specific portion thereof.

As used herein, the term "treat", when referring to a disease, indicates preventing the onset of a disease, alleviating a disease, mitigating or eliminating the symptoms of a disease, slowing or reversing the progress of a disease. disease, or cure a disease.

As used herein, the term "antibody" refers to an intact or substantially complete antibody molecule.

As used herein, the term "antibody fragment" refers to a molecule that comprises a portion of an antibody capable of specifically binding an antigen, an antigenic determinant or an epitope.

As described in the examples section below, the amino acid sequence shown in SEQ ID NO: 3, 4 and 6 represents amino acid residues 549-560, 534-566 and 534-560 of human NIK, respectively, and are located in the human NIK activation loop.

Preferably, the antibody of the invention is capable of binding to the target antigen with maximum affinity. The antibodies of the invention, for example, antibodies that have the ability to bind to the target antigen according to the invention, are capable of specifically and efficiently detecting phosphorylated NIK in T559 by Western blot analysis.

The antibodies of the invention, for example, antibodies that have the ability to bind to the target antigen according to the invention, are capable of specifically and efficiently detecting phosphorylated NIK in T559 by ELISA.

The antibodies of the invention, for example, antibodies that have binding capacity to the target antigen according to the invention, are capable of specifically and efficiently immunoprecipitating phosphorylated NIK in T559.

In a specific embodiment, polyclonal and monoclonal antibodies specific for phosphorylated NIK were generated, using immunizing peptide 549-560 (SEQ ID NO: 3) with the amino acid sequence SLLTGDYIPGT (p) E, in which T (p) is phosphorylated threonine and is located in the penultimate amino acid of the sequence.

In addition, other peptides derived from the activation loop can be used with the amino acid sequence DFGHAVCLQPDGLGKSLLTGDYIPGT (p) E (SEQ ID NO: 6), or a portion thereof, as an immunizing peptide, with the proviso that the phosphorylated T599 is located in the penultimate amino acid of the sequence.

Alternatively, a cysteine, a lysine or any other amino acid can be chemically linked to the N-terminal end of the immunizing peptide.

The term "specifically binds" according to the invention refers to antibodies that can bind to phosphorylated NIK in T559 but, by contrast, have low binding capacity or do not bind to non-phosphorylated NIK. The differential binding of the antibody of the invention can be assayed by different assays, for example by ELISA, Western blot analysis and immunoprecipitation.

To generate a monoclonal antibody according to the invention different races of immunized mice can be used, preferably the SJL breed. In addition, different hematopoietic organs of the mouse immunized for fusion can be used to generate the hybridoma, for example spleen and / or lymph nodes.

The antibodies prepared according to the invention are capable of recognizing the complete NIK protein, or its fragments, in a phosphorylated state.

In another embodiment, it has been shown that the antibodies according to the invention can inhibit the degradation of IB and the consequent activation of NF-B induced by CD40 or CD70 but not by the TNF ligand. These results demonstrate that the antibody of the invention can specifically inhibit the activation of NF-B.

Therefore, the antibody according to the invention can be used for the therapy in diseases in which the signaling of CD70 and CD40 is involved in the pathogenesis of the disease and / or in diseases in which TNF / NGF family ligands induce the activation of NF-B mediated by NIK through the canonical route. The parameters indicative of the canonical activation pathway are, for example, the degradation of IB, the phosphorylation of IB and the translocation of p65.

In view of the experimental findings, the antibody of the invention can be used in a method to identify a ligand capable of inducing NIK-mediated activation of NIK-mediated in a cell. In one embodiment of the invention, said method comprises the step of introducing the antibody, or a fragment of the antibody according to the invention, or an irrelevant IgG as a control, in a cell, incubating the cell with individual ligands, controlling the indicator parameters of the NF-B activation, preferably by controlling the indicator parameters of canonical pathway activation, such as IB degradation, IB phosphorylation and p65 translocation, and selecting the ligand by which the NF-B activation has been specifically affected by blocking NIK by the antibody or its fragment.

Inhibition of NIK activation observed with the antibody of the invention, for example, NIK-P4 30.12, does not occur with other NIK-specific antibodies capable of binding to the kinase domains that exclude the region of the activation loop of NIK

In general, an antibody of the present invention capable of binding to the target antigen with maximum affinity will allow optimal infraregulation of a biochemical activity mediated or associated with the target antigen. Similarly, the present antibody capable of specifically binding to the target antigen with maximum affinity in general will allow detection of the target antigen with optimal sensitivity.

By virtue of the ability described above of the antibody to specifically bind to a target antigen of the present invention, in particular by virtue of the exclusive ability of the antibody to bind specifically to the target antigen of the present invention located in a functional region. of NIK, the antibody can be used to underregulate a biochemical activity of NIK. In particular, by virtue of the exclusive ability described above of the antibody to specifically bind to a target antigen of the present invention located in the region of the NIK activation loop, the antibody can be used to optimally regulate NIK activity. It will be appreciated that, since this kinase activity is crucial to activate the nuclear factor NFB, as described above, an antibody of the present invention capable of optimally underregulating said kinase activity can be used to optimally underregulate NF- activity. B.

NF-B proteins are a family of proteins that share a 300 amino acid domain, the "Rel homology domain." The Rel homology domain mediates DNA binding, dimerization and nuclear transport of NF-B proteins. In addition to the Rel homology domain, some members of the NF-B family also contain a transactivation domain (for example, c-Rel, RelB, and p65). Members p50 and p52 of NF-B occur after activation by lysis of the inactive precursors p105 and p100, respectively. P50 and p52c have DNA binding dimerization properties but do not have strong transactivation domains. It is the differential generation of these proteins, their ability to heterodimerize with different family members, and the interaction of these proteins with different components of the transcription apparatus that contributes to the various activation effects of the NF--B pathway. NIK does not affect all NF-B species but only specific NF-B species, for example, induces the degradation of p100 and the generation of p52c, in response to specific inducers, for example lymphotoxins. Therefore, modulation of the NIK, for example using the antibodies of the invention, is likely to affect specific aspects of the pathological implications of NF-FB activation. This is an advantage since the general non-specific inhibition of NF-B is dangerous, for example because it can cause extensive apoptosis in the liver.

As used herein, the term "under-regulate", when related to a biochemical activity, refers to preventing, reducing or inhibiting said biochemical activity.

When used to inhibit a biochemical activity of NIK, the antibody is preferably capable of specifically binding with a maximum number of target antigens within a portion of NIK associated with said biochemical activity. In particular, when used to inhibit NIK kinase activity, the antibody is preferably capable of specifically binding to a maximum number of target antigens located within the amino acid sequences of the NIK activation loop region that appear in SEQ ID NO: 3 and 4. Although an antibody of the present invention capable of specifically binding only a target antigen located within a functional region of the NIK, such as the region of the activation loop, can be used to inhibit effectively, a biochemical function of NIK, such as the kinase activity, associated with said functional region, an antibody capable of specifically binding with a maximum number of target antigens located within said functional region will more effectively underregulate kinase activity. , by virtue of interfering with a maximum number of functional epitopes within the functional region.

Alternatively, to inhibit the kinase activity of NIK, the antibody may be advantageously capable of specifically binding to a maximum number of target antigens located within the amino acid sequences that appear in SEQ ID NO: 3, 4 and 6.

By virtue of the ability described above of the antibody to specifically bind to a target antigen of the present invention, the antibody can be used to specifically detect a phosphorylated NIK in T559 with optimal sensitivity, relative to prior art methods. , and can be used exclusively to specifically detect a phosphorylated NIK in T559 and / or a target antigen of the present invention.

The antibody can be used primarily for any application that benefits from a reagent capable of binding to the target antigen of the present invention with optimal affinity. These applications include, for example, affinity purification and, therefore, the identification and characterization of specific NIK ligands.

Therefore, the antibody of the invention can be used for immunopurification of NIK, or a portion thereof. In a preferred embodiment, an antibody of the invention can be used in the capture step for the purification of NIK, or a portion thereof.

As described and illustrated in the examples section below, an antibody of the present invention capable of specifically binding with a phosphorylated polypeptide comprising the amino acid sequence that appears in SEQ ID NO: 3, 4, 5 and 6 can be used to specifically detect, according to the indications in the section, the amino acid sequences of the peptides and phosphorylated proteins that appear in SEQ ID NO: 3, 4, 5 and 6, respectively.

Preferably, an antibody of the present invention capable of specifically binding to a target antigen of the present invention is derived from serum of animals immunized with said target antigen (hereinafter referred to as "antiserum"). As described and illustrated in the examples section below, an antiserum of the present invention capable of specifically binding the amino acid sequence shown in SEQ ID NO: 3 can be generated by immunizing a mammal with the antigen target according to the protocol indicated in the section. Other indications for generating the preparation by immunization of a mammal are provided hereinbelow.

Indications for obtaining a polypeptide, such as the target antigen, are given below.

Depending on the application and purpose, the preparation can be advantageously employed in the form of an unpurified antiserum, or it can be purified in various ways before use. For example, the preparation can be used in the form of: (i) a purified preparation of an antibody with a specific isotype or set of isotypes; (ii) a purified preparation of an antibody or an antibody fragment that is capable of specifically binding phosphorylated peptides specifically with the amino acid sequence shown in SEQ ID NO: 3, 4, 5 and / or 6; (iii) a purified preparation of an antibody or a fragment of an antibody that is capable of binding with a desired affinity to a target antigen of the present invention.

A preparation of the present invention in the form of an unpurified antiserum may be convenient and satisfactory for use in various applications. For example, as described and illustrated in the examples section below, an unpurified antiserum of the present invention, in particular an unpurified antiserum generated by immunization with the amino acid sequence shown in SEQ ID NO: 3, can be used to effectively detect a phosphorylated NIK by ELISA analysis. In general, for applications that benefit from a preparation of the present invention capable of binding to a target antigen of the present invention with a range of affinities / specificities, an unpurified antibody of the present invention, such as an antiserum of The present invention. This unpurified preparation may often be suitable for a given application, since the heterogeneity of the polyclonal antibody or a mixture of antibody fragments contained therein often includes one or more antibodies or antibody fragments that have an affinity / specificity of suitable binding for the target antigen.

Alternatively, an antibody or a fragment of a purified antibody of the present invention can be advantageously employed in applications, such as those involving administration of the antibody or antibody fragment to an individual, and in which detection is desirable. of the target antigen with optimal sensitivity.

As used herein, the term "individual" refers to a human being.

For example, an IgG antibody preparation of the present invention can be advantageously purified from an antiserum of the present invention using an affinity purification of G protein, preferably through an immunoprecipitation of G protein. As described and an antiserum of the present invention derived from an animal immunized with the phosphorylated peptide with the amino acid sequence shown in SEQ ID NO: 3 and purified by an immunoprecipitation with G protein according to the invention is illustrated in the examples section below. The protocol indicated in the section can be used to detect with optimal sensitivity, by Western blot analysis, immunoprecipitation and ELISA, phosphorylated polypeptides with the amino acid sequence that appears in SEQ ID NO: 3, 4, 5 and 6 .

An antibody or a fragment of a purified antibody of the present invention capable of specifically binding to the target antigen can be advantageously used to regulate, with optimal specificity, a biochemical activity of NIK associated with the target antigen, and to detect the antigen target with optimal specificity. In particular, an antibody or a fragment of a purified antibody of the present invention capable of specifically binding to a target antigen of the present invention comprised within the amino acid sequence that appears in SEQ ID NO: 3, 4, 5 and 6 can be used advantageously to regulate the kinase activity of NIK with optimal specificity. In general, for applications that benefit from optimum reproducibility, standardization or precision, an antibody or a fragment of a purified antibody of the present invention capable of specifically binding to the target antigen will, in general, be optimal in relation to an unpurified preparation of the present invention.

Purification of the antibody or antibody fragment capable of specifically binding to the target antigen can be achieved, for example, by purifying a preparation of the present invention, such as an unpurified antiserum of the present invention, by affinity chromatography using an affinity chromatography using substrate covalently bound to the target antigen. This target antigen bound to the substrate can be used, according to the conventional affinity chromatography methodology, to selectively capture the antibody or antibody fragment capable of specifically binding to the target antigen.

The substrate is preferably an affinity chromatography matrix. An affinity chromatography matrix, being a substrate optimized to perform affinity chromatography, can be advantageously used to achieve optimal affinity purification.

Substrates having various structural and chemical characteristics can be used to perform purification.

Preferably, the substrate comprises a carbohydrate or its derivative. Preferably, the carbohydrate is agarose, sepharose or cellulose.

Preferably, the substrate is a sphere, a resin, or a plastic surface.

Normally substrates, such as spheres, resins, or plastic surfaces comprising carbohydrates, such as agarose, Sepharose or cellulose, are used to practice affinity chromatography in the art.

Extensive indications for practicing affinity chromatography are provided in the literature of the art, such as that employed by said substrate (for example, refers to Wilchek, M. and Chaiken, I., 2000, Methods Mol. Biol., 147 : 1-6; Jack, GW, Immunoaffinity chromatography, Mol. Biotechnol., 1, 59-86; Narayanan, SR, 1994, Journal of Chromatography, A 658: 237-258; Nisnevitch, M. and Firer, MA, 2001 , J. Biochem. Biophys. Methods, 49: 467-480; Janson, JC and Kristiansen, T. in: "Packings and Stationary Phases in Chromatography Techniques" (ed. Unger, KK), p. 747 (Marcel Dekker, New York, 1990); Clonis, YD in: "HPLC of Macromolecules: A Practical Approach",

p. 157 (IRL Press, Oxford, 1989); Nilsson, J. et al., 1997, Protein Expr. Purif., 11: 1-16).

Alternatively, a preparation of the present invention can be purified using a variety of conventional protein purification techniques, such as ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, chromatography with concanavalin A, chromatoenfoque and differential solubilization.

An antibody or a fragment of an antibody of the present invention capable of specifically binding to a target antigen of the present invention with a desired affinity can be advantageously used to achieve a desired level of regulation of a biochemical activity of NIK associated with the target antigen, and can be used advantageously to detect the target antigen with a desired sensitivity. In particular, an antibody or a fragment of an antibody of the present invention capable of specifically binding with maximum affinity to a target antigen of the present invention comprised in the kinase region of the NIK can be used advantageously to optimally underregulate the NIK kinase activity. Similarly, an antibody or a fragment of an antibody of the present invention capable of specifically binding to the target antigen with maximum affinity can be advantageously used to detect the target antigen with optimal sensitivity.

Purification of the antibody or antibody fragment capable of binding to the target antigen with a desired affinity from a preparation of the present invention, such as an unpurified antiserum of the present invention, can be achieved, for example, by purification with an affinity chromatography of an unpurified (or more preferably purified with G protein) antibody of the present invention, using the target antigen as an affinity ligand, and by selective elution of an antibody or a fragment of an antibody bound to a substrate under controlled stringency conditions (for example, under conditions of controlled pH and / or salt concentration). In particular, an antibody or a fragment of an antibody of the present invention capable of binding to the target antigen with maximum affinity can be conveniently obtained by elution under conditions of maximum and effective stringency (for example, under conditions of minimum or maximum pH and / o maximum effective salt concentration). Generally, an antibody or an antibody fragment can bind to a cognate antigen attached to a substrate under conditions of physiological pH and saline concentration, and said antibody or antibody fragment can generally be eluted from the substrate by lowering the pH to 2.5 or lower, or increasing the pH to 11 or higher.

Those skilled in the art will appreciate that an antibody or a fragment of an antibody having an affinity characterized by a dissociation constant of up to 10-12 for a cognate antigen can be obtained using standard techniques.

As described hereinbefore, the preparation may advantageously comprise an antibody or a fragment of an antibody bound to any of several types of detectable molecules.

A preparation of the present invention comprising an antibody or a fragment of an antibody bound to a detectable molecule can be used to detect the target antigen specifically bound by the antibody.

or by the antibody fragment.

The preparation may comprise an antibody or a fragment of an antibody bound to any of numerous types of detectable molecules, depending on the application and purpose.

For example, depending on the application and the purpose, the detectable molecule may advantageously be a fluorophore, an enzyme, a light emitting molecule, or a radioisotope.

Preferably, the detectable molecule is an enzyme.

An enzyme can be used advantageously to allow detection of the target antigen by any of several enzyme-based detection methods. Examples of such methods include an enzyme-linked immunoabsorbent assay (ELISA; for example, to detect the target antigen in a solution), an enzyme-linked chemiluminescent assay (for example, to detect the complex in a mixture of proteins so separated electrophoretic), and an enzyme-linked histochemical assay (for example, to detect the complex in a fixed tissue).

As described and illustrated in the examples section below, a preparation of the present invention comprising an antibody bound to an enzyme can be used to effectively detect NIK by Western blot analysis or an ELISA.

Numerous types of enzymes can be used to detect the target antigen, depending on the application and the purpose.

Examples of suitable enzymes include horseradish peroxity (HPR), gala-galactosidase, and alkaline phosphatase (AP).

Extensive indications for practicing enzyme-based molecular detection methods are provided in the literature of the technique (for example, refers to Khatkhatay, MI and Desai, M., 1999, J. Immunoassay, 20: 151-183; Wisdom, GB, 1994, Methods Mol. Biol., 32: 433-440; Ishikawa, E. et al., 1983, J. Immunoassay, 4: 209327; Oellerich, M., 1980, J. Clin. Chem. Clin. Biochem ., 18: 197-208; Schurrs, AH and van Weemen, BK, 1980, J.

Immunoassay, 1: 22-249.

A preparation of the present invention comprising an antibody or an antibody fragment bound to a fluorophore can be advantageously employed to detect the target antigen by any of numerous fluorescence-based molecular detection methods. Depending on the application and purpose, such methods include fluorescence activated flow cytometry (FACS; for example, to characterize the expression or presentation of the target antigen in a suspended cell population), fluorescence confocal microscopy (for example, to detect the molecule in a living or dead tissue or cell in three dimensions), fluorescence in situ hybridization (FISH), fluorescence resonance energy transfer (FRET; for example, to detect a specific intermolecular association involving the target antigen), fluorescence histochemistry (for example, to detect the molecule in a fixed histological sample) and the like.

Various types of fluorophores can be used, depending on the application and purpose, to detect the target antigen.

Examples of suitable fluorophores include phytoerythrin, fluorescein isothiocyanate (FITC), Cy-chromium, rhodamine, green fluorescent protein (GFP), blue fluorescent protein (BFP), Texas red and the like.

Extensive indications regarding fluorophore selection, methods for attaching fluorophores to various types of molecules, such as an antibody or an antibody fragment of the present invention, and methods for using said immunoconjugates are provided in the literature of the art. fluorescent to detect molecules (for example, refers to Richard P. Haughland, "Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals 1992-1994", 5th edition, Molecular Probes, Inc. (1994); U.S. Patent No. 6,037. 137 of Oncoimmunin Inc .; Hermanson, "Bioconjugate Techniques", Academic Press, New York, NY (1995); Kay, M. et al., 1995, Biochemistry, 34: 293; Stubbs et al., 1996, Biochemistry, 35 : 937; Gakamsky, D. et al., "Evaluating Receptor Stoichiometry by Fluorescence Resonance Energy Transfer", in "Receptors: A Practical Approach", 2nd ed., Stanford,

C. and Horton, R. (eds.), Oxford University Press, United Kingdom (2001); U.S. Patent No. 6,350,466 to Targesome, Inc.).

Examples of suitable light emitting molecules include luminol.

Examples of suitable radioisotopes include [125] iodine, [35] sulfur, [3] hydrogen, [32] phosphorus, etc.

The detectable molecule may be bound to the antibody or to the fragment of an antibody in various ways, depending on the application and purpose, and the nature of the molecules involved. Extensive indications are provided in the literature of the art for binding a detectable molecule to an antibody or a fragment of an antibody (for example, it refers to "Using Antibodies: A Laboratory Manual", Ed. Harlow, David Lane (eds.) Cold Spring Harbor Laboratory Press (1999); also refers to the extensive indications provided by The American Chemical Society, for example at http://www.chemistry.org/portal/Chemistry). Those skilled in the art, such as chemists, possess the knowledge required to properly practice such chemical synthesis techniques.

Accordingly, a preparation of the present invention comprising an antibody or a fragment of an antibody bound to a detectable molecule can be used to efficiently and exclusively detect the target antigen primarily in any context.

Depending on the application and the purpose, the preparation may advantageously be a preparation of any of various types of antibody fragments.

The antibody fragment is preferably a single chain Fv (scFv), or more preferably a Fab, Fab ’, F (ab’) 2 or CDR.

An antibody fragment has the advantage of being smaller than the antibody of origin from which it is derived but maintains substantially identical binding specificity to the target antigen, or binding specificity and binding affinity, than the antibody of origin. Therefore, an antibody fragment, by virtue of being smaller than the antibody of origin, will generally have a better biodistribution and diffusion properties (for example, systemically in vivo, or in isolated tissues) than the antibody of origin. . An antibody fragment that substantially lacks an Fc region, such as a single stranded Fv, a Fab ', a Fab, an F (ab') 2 or a CDR, is advantageous for applications involving exposure of the preparation to a molecule capable of specifically binding to said Fc region and in which said binding is not desirable. Generally this may involve an unwanted binding of an Fc region exposed to a cognate Fc receptor, or a component of the Fc binding complement (eg, the Clq complement component, present in the serum). Fc receptors occur on the surface of numerous immunological cell types, including professional APCs, such as dendritic cells; B lymphocytes; and granulocytes, such as neutrophils, basophils, eosinophils, monocytes, macrophages and mast cells. Therefore, the absence of an Fc region of the antibody fragment may be particularly advantageous in preventing unwanted activation of Fc-mediated immune cells or a complement cascade mediated by a complement component, in particular when the preparation is administered in vivo. To an individual.

An F (ab ’) 2 is a fragment of an antibody molecule that contains a divalent antigen binding portion of a molecule of an antibody.

An F (ab ’) 2 preparation of the present invention can be conveniently obtained using conventional methods of the art, treating an antibody preparation of the present invention, such as an antiserum of the present invention, with the enzyme pepsin. The resulting F (ab ’) 2 product is a 5S particle.

An Fab or Fab ’is a fragment of an antibody molecule that contains a monovalent antigen-binding portion of an antibody.

The CDR can be generated for example as described in EP0585939, or as described by Stranberg et al. (Protein Eng., January 2001, 14 (1): 67-74). The CDR according to the invention can be a modified CDR that has an enhanced effect on the modulation of the NIK. An example of methods of modifying active peptides is described in Sawa et al., 1999 (J. Med. Chem., 42, 3289-3299).

A Fab 'preparation of the present invention can be conveniently obtained using conventional methods in the art, treating an antibody preparation of the present invention, such as an antiserum of the present invention, with the enzyme pepsin, followed by the reduction of the F (ab ') 2 resulting. This reduction can be done using a thiol reducing agent, and optionally using a blocking group for the sulfhydryl groups that result from the breakage of the disulfide bonds. This treatment generates two monovalent Fab ’3.5S fragments and Fc fragment.

A Fab preparation can be conveniently obtained using conventional methods of the art, treating an antibody preparation of the present invention, such as an antiserum of the present invention, with the papain enzyme to produce the intact light chain and a portion of the heavy chain composed of the variable domains and CH1.

Extensive indications for generating an antibody fragment by enzymatic treatment of an antibody are provided in the literature of the art (eg, refers to Goldenberg, US Pat. Nos.

4,036,945 and 4,331,647; Porter, R.R., 1959, Biochem. J., 73: 119-126).

A single chain Fv (also referred to in the art as "scFv") is a single chain molecule that includes the variable region of the light chain and the variable region of the heavy chain linked through a suitable polypeptide linker.

A preparation of F (ab ’) 2, Fab’, Fab, or single chain Fv, or CDR of the present invention can be obtained using recombinant techniques.

Preferably, a recombinant antibody fragment is obtained by isolating mRNA from B lymphocytes from animals immunized with the target antigen, generating cDNA from the mRNA by RT-PCR, and using the cDNA to construct a phage display bank of antibody fragments. B lymphocytes can be conveniently isolated from the spleen or, alternatively, from the blood, bone marrow or lymph nodes of the immunized animal.

Recombinant phages that exhibit an antibody fragment that possesses a desirable target antigen binding property can be selected from the bank by sequential enrichment of phages having said binding property from a large excess of unbound clones. This selection can be achieved using any of several techniques, including immunoadsorption on an immobilized target antigen; immunoadsorption using a specific elution; using a biotinylated antigen; affinity purification in columns; or direct immunoadsorption on cells. Upon selection, phages that exhibit non-specific antibody fragments can be removed by washing, and bound phages carrying scFv that exhibit the desired target antigen binding property are eluted and amplified by infection in E. coli. When a recombinant phage having an antibody fragment having a desired target antigen binding property has been isolated, the polynucleotide sequence encoding the variable regions of the antibody fragment can be recovered from the phage display packet and cloned into a Recombinant prokaryotic or eukaryotic expression vector using conventional methodology (eg, refers to "Current Protocols in Molecular Cloning", Ausubel et al. (eds.), Greene Publishing and Wiley Interscience, New York, NY (1989); Sambrook et al., infra and associated references). This prokaryotic expression vector can be used to produce the recombinant antibody fragment purified in E. coli (eg, referred to Studier et al., 1990, Methods in Enzymol., 185: 60-89). This eukaryotic expression vector can be used to genetically transform a eukaryotic cell for expression of the recombinant antibody fragment.

Extensive indications are provided in the literature for obtaining and using a phage display bank of antibody fragments from B-lymphocyte mRNA (for example, it is referred to Hoogenboom et al., 1998, Immunotechnology, 4: 1- 20; Kand et al., 1991, Proc. Natl. Acad. Sci. USA, 88: 4363; Barbas et al., 1991, Proc. Natl. Acad. Sci. USA, 88: 7978; Garrard et al., 1991 , Biotechnology, 9: 1373-1377; Hoogenboom et al., 1991, Nucleic Acids Res., 19: 4133-4137; Sharon et al., 2000, Combinational Chemistry and High Throughput Screening, 3: 185-196; US Pat. No. 5,698,426, 5,658,727, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658 .727, 5,733,743 and 5,969,108; PCT publications WO 92/01047, WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982 and WO 95 / 20401; Brinkman et al., 1995, J. Immunol. Methods, 182: 41-50; Ames et al., 1995, J. Immunol. Methods, 184: 177-186 ; Kettleborough et al., 1994, Eur. J. Immunol., 24: 952-958; Persic et al., 1997, Gene, 187: 9-18; and Burton et al., 1994, Advances in Immunology, 57: 191-280; Pluckthun, in: "The Pharmacology of Monoclonal Antibodies", vol. 113, Rosenburg and Moors (eds.), Springer-Verlag, New York, pp. 269-315 (1994); Hoogenboom et al., 1998, Immunotechnology, 4: 1-20).

It will be appreciated that the methodology described above can be used to obtain a preparation of monoclonal antibody fragments of the present invention having essentially any desired affinity and / or specificity to the target antigen. This preparation can be used in various applications that benefit from a reagent capable of binding to the target antigen with said defined target antigen binding characteristics.

The present invention provides a method for preparing a monoclonal antibody, which comprises culturing a cloned hybridoma comprising a splenic cell from a mammal immunized with an amino acid sequence comprising SEQ ID NO: 6, or a portion thereof, for example the which appears in SEQ ID NO: 3, in which the amino acid sequence, or a portion thereof, comprises a phosphorylated threonine at amino acid position 559 of SEQ ID NO: 5, and a homogenous or heterogeneous lymphoid cell in a medium mammalian fluid or abdomen, to allow the hybridoma to produce and accumulate the monoclonal antibody.

An example of a hybridoma that can be used for the preparation of monoclonal antibodies is the hybridoma clone NIK-P4 30.12, deposited in the CNCM as No. I-3095.

Since a Fab 'has a structure fundamentally similar to a Fab, a preparation of the present invention comprising a Fab' can be used fundamentally interchangeably with another comprising a Fab, wherein said Fab 'and Fab fundamentally comprise the same variable regions of heavy and light chain. As will normally be the case, for applications that benefit from a preparation of the present invention comprising an antibody fragment capable of binding to the target antigen with maximum affinity, an F (ab ') 2 preparation of the present invention may be better that a Fab, Fab 'or scFv preparation of the present invention, due to the divalent binding of an F (ab') 2 to the target antigen in relation to the monovalent binding of said monovalent antibody fragment.

As mentioned hereinabove, depending on the application and purpose, the preparation of antibodies or antibody fragments may originate from any of several mammalian species.

A preparation of antibodies or antibody fragments of the present invention that originates from a desired species can be obtained from the serum of the animal of said species immunized with the target antigen.

Preferably, the antibody or antibody fragment originates in mice.

Said antibody may be monovalent, bivalent or multivalent, and may or may not have crosslinking activity. The antibodies used according to the present invention may be polyclonal antibodies, such as antibodies produced in rabbits, or monoclonal antibodies.

Preferably, the invention relates to the use of an antibody preparation selected from the group consisting of polyclonal, monoclonal, chimeric, humanized, human or anti-anti-idiotypic antibodies, or fragments thereof. Preferably, the antibody or antibody fragment originates in mice.

The term "monoclonal antibody" (mAb) is intended to include monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, antibodies against anti-idiotypic antibodies (anti-anti-Id antibody) that can be labeled in soluble or bound form, as well as their fragments, provided by any known technique, such as enzymatic dissociation, peptide synthesis or recombinant techniques.

A monoclonal antibody contains a substantially homogeneous population of antigen specific antibodies, said population containing substantially similar epitope binding sites. MAbs can be obtained by methods known to those skilled in the art. See, for example, Kohler and Milstein, Nature, 256: 495-497 (1975).

A monoclonal antibody is said to be "capable of binding" to a molecule if it is capable of reacting specifically with the molecule to thereby bind the molecule to the antibody. The term "epitope" refers to the portion of any molecule capable of binding an antibody, which can also be recognized by said antibody. Epitopes or "antigenic determinants" usually consist of clusters of molecules with chemically active surface, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics, as well as specific loading characteristics.

An "antigen" is a molecule or a portion of a molecule that is capable of being bound by an antibody, said antigen also being able to induce an animal to produce an antibody capable of binding to an epitope of that antigen. An antigen may have one or more of an epitope. The specific reaction mentioned above indicates that the antigen will react, in a very selective manner, with an epitope on its corresponding antibody and not with the multitude of other antibodies that can be evoked by other antigens (document 4,376,110; Ausubel et al., eds., Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory (1988); and Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience NY (1992-1996)). Such antibodies may be of any class of immunoglobulins, including IgG, IgM.

An anti-idiotypic (anti-Id) antibody is an antibody that recognizes exclusive determinants generally associated with the antigen binding site of an antibody. An Id antibody can be prepared by immunizing an animal of the same species and genetic type (eg, a mouse breed) as a source of the mAb with which the anti-Id is being prepared.

The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody against these idiotypic determinants (the anti-Id antibody). See, for example, U.S. Patent No. 4,699,880.

The anti-Id antibody can also be used as an "immunogen" to induce an immune response in another animal, producing the so-called anti-anti-Id antibody. The anti-anti-Id may be epitopically identical to the original mAb, which induced the anti-Id. Therefore, using antibodies against the idiotypic determinants of a mAb, it is possible to identify other clones that express antibodies with identical specificity.

Accordingly, mAbs generated against NIK fragments can be used to induce anti-Id antibodies in suitable animals, such as BALB / c mice. Spleen cells from said immunized mice are used to produce anti-Id hybridomas that secrete anti-Id mAbs. In addition, anti-Id mAbs can be coupled to a vehicle, such as barnacle hemocyanin (KLH) and used to immunize other BALB / c mice. The serum of these mice will contain anti-anti-Id antibodies that have the binding properties of the original mAb specific for an epitope or NIK fragment.

Therefore, anti-Id mAbs have their own idiotypic epitopes, or "idiotope", structurally similar to the epitope being evaluated.

A preparation of the present invention of an antibody or a human or humanized antibody fragment may be preferable for applications that involve administration of the preparation to an individual. For example, the antibody or the human or humanized antibody fragment in general tends to be optimally immunologically tolerated and, therefore, will show an optimal half-life in vivo in a human being and, thereby, will show optimum efficacy. Further indications are given below regarding the production and use of human or humanized antibodies.

Antibodies, including antibody fragments, useful in the present invention can be used to quantitatively or qualitatively detect NIK or its mutein, its functional derivative, its active fraction, its circularly permuted derivative, its salt, or a portion thereof. , in a sample, or to detect the presence of cells expressing NIK or its mutein, its functional derivative, its active fraction, its circularly permuted derivative, its salt, or a portion thereof. This can be achieved with immunofluorescence techniques that employ a fluorescently labeled antibody together with fluorescence microscopy, flow cytometry or fluorometric detection.

The antibodies (or their fragments) of the present invention can be used histologically, as in immunofluorescence or immunoelectronic microscopy, for in situ detection of NIK, or portions thereof, of the present invention. In situ detection can be performed by removing a histological specimen from a patient, and providing the labeled antibody of the present invention to said specimen. The antibody (or a fragment) is preferably provided by applying or coating the labeled antibody (or a fragment) on a biological sample. By using this procedure it is possible to determine not only the presence of NIK, or portions of it, but also its distribution over the studied tissue. Using the present invention, those skilled in the art will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified to achieve such detection in situ.

The biological sample can be coupled to a solid phase carrier or carrier, such as nitrocellulose or other solid carrier or carrier, that is capable of immobilizing soluble cells, cell particles or proteins. The carrier or carrier can then be washed with suitable buffers, followed by a treatment with a labeled antibody according to the present invention, as indicated above. The solid phase carrier or carrier can then be washed with the buffer a second time to remove unbound antibody. The amount of marker attached to said vehicle or solid support can then be detected by conventional means.

A "solid phase support", "solid phase vehicle", "solid support", "solid vehicle", "support" or "vehicle" is any support or vehicle capable of binding antigens or antibodies. Well-known supports or vehicles include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabros and magnetite. The nature of the vehicle may be soluble to some extent or insoluble for the purposes of the present invention. The support material may have almost any possible structural configuration, with the proviso that the coupled molecule is capable of binding to an antigen or an antibody. Therefore, the configuration of the support or the vehicle can be spherical, such as a sphere, cylindrical, such as the inner surface of a test tube or the outer surface of a rod. Alternatively, the surface can be flat, such as a sheet, a test strip, etc. Preferred supports or vehicles include polystyrene spheres. Those skilled in the art will know many other suitable vehicles for binding antibodies or antigens, or will be able to determine them using routine experimentation.

The binding activity of a given set of antibodies of the invention, as indicated above, can be determined according to well known methods. Those skilled in the art will be able to determine the optimal operating and test conditions for each determination using routine experimentation.

Other steps, such as washing, removal, agitation, filtration and the like, can be added to the tests as usual or necessary for the specific situation.

One of the ways in which an antibody according to the present invention can be labeled is by binding it to an enzyme and using it in an enzyme immunoassay (EIA). This enzyme, in turn, when later exposed to an appropriate substrate, will react with the substrate in such a way that it produces a chemical moiety that can be detected, for example, by spectrophotometric, fluorometric or visual means. Enzymes that can be used to detectably label the antibody include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase , glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholine esterase. Detection can be carried out by colorimetric methods that employ a chromogenic substrate for the enzyme. Detection can also be performed by visual comparison of the degree of enzymatic reaction of a substrate by comparison with similarly prepared standards.

Detection can be performed using any of a variety of different immunoassays. For example, by radiolabeling antibodies or antibody fragments it is possible to detect R-PTPase using a radioimmunoassay (RIA). A good description of RIA can be found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T.S. et al., North Holland Publishing Company, NY (1978), making specific reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard,

T.

The radioactive isotope can be detected by means, such as the use of a g-counter or scintillation counter or by autoradiography.

It is also possible to label an antibody according to the present invention with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of an appropriate wavelength, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent marker compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emitting metals, such as 152E or others of the lanthanide series. These metals can bind to the antibody using metal chelating groups, such as diethylenetriaminepentaacetic acid (ETPA).

The antibody can also be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescently labeled antibody is then determined by detecting the presence of the luminescence that arises during the development of a chemical reaction. Examples of particularly useful chemiluminescent marker compounds are luminol, isoluminol, thermic acridinium ester, imidazole, acridinium salt and oxalate ester.

Similarly, a bioluminescent compound can be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems, in which a catalytic protein increases the effectiveness of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. The bioluminescent compounds important for labeling purposes are luciferin, luciferase and aequorin.

An antibody preparation of the present invention can be adapted for use in an immunometric assay, also known as a "two-site" or "sandwich" assay. In a typical immunometric assay, an amount of an unlabeled antibody (or a fragment of an antibody) binds to a solid carrier or carrier, and an amount of detectably labeled soluble antibody is added to allow detection and / or quantification of the ternary complex formed between the solid phase antibody, the antigen and the labeled antibody.

Typically and preferably, immunometric assays include "direct" assays in which the antibody bound to the solid phase first contacts the sample being tested to extract the antigen from the sample by forming a complex of solid phase binary antigen-antibody. After an appropriate incubation period, the solid carrier or carrier is washed to remove the residue from the fluid sample, including the unreacted antigen, if any, and then contacted with the solution containing an unknown amount of labeled antibody. (which acts as an "indicator molecule"). After a second incubation period to allow the labeled antibody to form a complex with the antigen bound to the vehicle

or solid support through the unlabeled antibody, the solid carrier or carrier is washed a second time to remove the unreacted labeled antibody.

In another type of "sandwich" assay, which may also be useful with the antigens of the present invention, so-called "simultaneous" and "inverse" assays are used. A simultaneous assay involves a single incubation step, since the antibody bound to the solid carrier or carrier and the labeled antibody are both added to the sample being tested at the same time. After completion of the incubation, the solid carrier or carrier is washed to remove the residue from the fluid sample and the labeled antibody that has not complexed. The presence of the labeled antibody associated with the carrier or solid support is then determined, as would be done in a conventional "direct" sandwich assay.

In the "reverse" assay, the discontinuous addition, first of a solution of the labeled antibody to the fluid sample, is used, followed by the addition of the unlabeled antibody bound to a solid carrier or carrier after a suitable incubation period. After a second incubation, the solid phase is washed in a conventional manner to remove the residue from the sample being tested and the unreacted labeled antibody solution. The labeled antibody associated with a solid carrier or carrier is then determined as in the "simultaneous" and "direct" assays.

The preparation can be used per se or can be formulated as an active ingredient in a pharmaceutical composition.

Therefore, according to the present invention, a pharmaceutical composition comprising a pharmaceutically acceptable carrier and, as active ingredient, the antibody or antibody fragment of the present invention is provided.

The methods for formulating the antibody or antibody fragment of the present invention as an active ingredient in a pharmaceutical composition, and the methods for taking advantage of said pharmaceutical composition are described herein below.

As described above, by virtue of its ability to specifically bind a region, such as a functional region, of the NIK, the antibody or the antibody fragment of the present invention can be used to regulate a biochemical activity of a NIK molecule associated with said functional region.

Therefore, according to another aspect of the present invention, a method for regulating a biochemical activity of an NIK molecule is provided. The method is performed by contacting the NIK molecule with an antibody or an antibody fragment of the present invention.

Preferably, the method is used to regulate the biochemical activity of a human NIK molecule.

As described above, since the antibody or antibody fragment of the present invention is capable of specifically binding to phosphorylated NIK in such a way as to underregulate NIK kinase activity, and since such activity is necessary for activation of NF-B, as detailed in detail in the section "Field and background of the invention" above, the method can be used to optimally regulate NF-B activity.

Therefore, by virtue of allowing the optimal underregulation of NF-B activity, the method can be used to optimally treat in an individual a disease associated with a deregulated NF-B activity, in particular excessive or NF-B activity or constitutive

When the method according to this aspect of the present invention is used to treat the disease in the individual, contact of the NIK molecule with the antibody or with the antibody fragment can be advantageously performed by administering the antibody or the antibody fragment to An individual.

Preferably, administration of the antibody or antibody fragment is accomplished by administering the pharmaceutical composition of the present invention, which comprises the antibody or antibody fragment of the present invention as an active ingredient.

The antibody or antibody fragment is preferably administered to achieve a sufficient level of antibody fragment bound to the target antigen to achieve a desired regulation of biochemical activity.

Those skilled in the art, for example a physician, more preferably a physician specialized in the disease, have the experience required to determine a suitable therapeutic protocol, including a suitable route of administration, and an adequate dosage of the antibody or antibody fragment, to effectively treat the disease according to the indications of the present invention.

NIK is normally an intracellular molecule and, as such, to optimally contact the antibody or antibody fragment with the NIK molecule in a cell, such as a cell characterized by a deregulated NF--B activity, the The method is preferably practiced in such a way that contact of the antibody or antibody fragment with the NIK molecule within the cell is facilitated.

This intracellular contact can be facilitated in various ways, depending on the application and purpose.

For example, said intracellular contact can be made by contacting the cell with the antibody or antibody fragment in association with a lipid-based vehicle capable of facilitating the penetration of the antibody or antibody fragment into the cell.

Alternatively, said intracellular contact can be made by genetically transforming the cell with an expression vector capable of expressing the antibody fragment of the present invention intracellularly.

Suitable types of lipid vehicles to facilitate the entry of the antibody or antibody fragment into the cell include liposomes and immunoliposomes. Immunoliposomes, by virtue of allowing specific transport of the cell type of molecules, can be advantageously used to selectively transport the antibody or antibody fragment to cells affected by a disease, wherein said cells express distinctive surface antigens. , such as markers of inflammation in cells that show a pathological immune response (e.g., CD25 or CD69 in activated T lymphocytes), or tumor associated antigens in malignant cells (e.g., HER-2 in adenocarcinoma cells, MAGE- 1 in melanoma cells, and the like).

In the literature of the art, broad indications are provided for using said lipid-based vehicles for intracellular transport of therapeutic molecules, such as an antibody or a fragment of an antibody of the present invention, to diseased cells (for example, referred a Abra, RM et al., 2002, J. Liposome Res., 12: 1-3; Park, JW, 2002, Breast Cancer Res., 4 (3): 95-99; Bendas, G., 2001, BioDrugs , 15: 215-224; Maruyama, K., 2000, Biol. Pharm. Bull., 23: 791-799; Hong, K. et al., 1999, Ann. NY Acad. Sci., 886: 293-296 ; Margalit, R., 1995, Crit. Rev. Ther. Drug Carrier Syst., 12: 233-261; Storm, G. and Crommelin, DJ, 1997, Hybridoma, 16: 119-125; Park, JW et al. , 1997, Adv. Pharmacol., 40: 399-435).

In the art the production of a recombinant antibody fragment, such as the recombinant antibody fragment of the present invention, is usually performed intracellularly. A recombinant antibody fragment expressed intracellularly may be referred to as "intrabody" in the art. In the literature of the art, broad indications are provided for using intracellular expression of a recombinant antibody fragment capable of specifically binding with a biomolecule to regulate a biochemical activity, such as an enzymatic activity, of the biomolecule in a cell (by example, refers to Mhashilkar, AM et al., 2002, Gene Ther., 9: 307-319; Arafat, W. et al., 2000, Cancer Gene Ther., 7: 1250-1256; Cohen, PA et al. ., 1998, Oncogene, 17: 2445-2456; Hassanzadeh, Gh. G. et al., 1998, FEBS Lett., 437: 81-86; Richardson, JH et al., 1998, Gene Ther., 5: 635 -644; for general indications to express a recombinant antibody fragment in a cell, for example, refer to der Maur, AA et al., 2002, J. Biol. Chem., 277: 45075-45085; Zhu, Q . et al., 1999, J. Immunol. Methods, 231: 207-222; Wirtz, P. and Steipe, B., 1999, Protein Sci., 8: 2245-2250; Ohage, E. and Steipe, B. , 1999, J. Mol. Biol., 291: 1119-1128).

The genetic transformation of a mammalian cell with an expression vector can be performed using any of several commonly used methods of the art, such as stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. Extensive indications for practicing such methods are provided in the literature of the technique (for example, refer to Sambrook et al., Infra and associated references; Chang et al., In: "Somatic Gene Therapy", CRC Press, Ann Arbor, MI (1995); Vega et al., In: "Gene Targeting", CRC Press, Ann Arbor, MI (1995); Vectors, A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston, MA (1988); Gilboa et al., 1986, Biotechniques, 4: 504-512; for vectors that involve the central nervous system, refer, for example, to US Patent 4,866,042; for positive-negative selection methods to induce homologous recombination it refers, for example, to US patents 5,464,764 and 5,487,992).

Further indications are given hereinbelow with respect to the production and use of expression vectors to express an antibody fragment of the present invention in a cell.

As described hereinbefore, the method according to this aspect of the present invention can be used to optimally treat a disease associated with a deregulated NF-B activity. NFB activity in general is involved in the activation of a very wide range of immunological responses, including those triggered by lymphocyte antigen receptors, lymphocyte costimulatory receptors, TNF receptors, interleukin receptors, LMP1, RANK, the human Toll receptor, and lipopolysaccharide (LPS). Since these receptors are involved in mediating a very wide range of types of immunological responses, the method according to this aspect of the present invention can be used to treat numerous diseases whose pathogenesis is associated with said immunological responses. These diseases include autoimmune diseases, inflammatory diseases, transplant-related diseases, and allergic diseases. For example, it has been shown that NF-B is involved in the pathogenesis of various examples of such diseases, including allergic diseases, such as asthma, autoimmune diseases, such as rheumatoid arthritis, inflammatory diseases, such as inflammatory bowel disease, atherosclerosis, and Alzheimer's disease, and transplant-related diseases, such as graft rejection. In addition, it has been shown that deregulated NF-B signaling is associated with various malignant diseases.

As such, the method according to this aspect of the present invention can be used to effectively treat diseases, such as autoimmune, inflammatory, transplant-related, allergic and malignant diseases.

Specific examples of diseases that can be treated according to this aspect of the present invention are listed hereinafter.

As described hereinbefore, the target antigen, which is a polypeptide, can be obtained in various ways.

Preferably, the target antigen is obtained by a conventional chemical synthesis methodology.

Alternatively, the target antigen can be obtained by proteolytic disruption of NIK expressed in nature, or it can be obtained through conventional recombinant techniques using in vitro expression systems (for example, it is referred to Sambrook et al., Infra and associated references ).

The target antigen can be chemically synthesized using, for example, conventional solid phase techniques. These techniques include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, and classical solution synthesis. Solid phase polypeptide synthesis procedures are well known in the art (eg, referred to Stewart et al., In "Solid Phase Peptide Synthesis", 2nd ed., Pierce Chemical Company (1984)).

A synthetic polypeptide can be purified by a preparative high resolution liquid chromatography procedure, as described in Creighton, T. (Proteins, structures and molecular principles, WH Freeman and Co., NY (1983)), and its amino acid sequence It can be confirmed by conventional amino acid sequencing procedures.

As described hereinbefore, the preparation is preferably obtained by immunizing a mammal with the target antigen.

The generation of the preparation in vivo can be carried out advantageously by repeated injection of the target antigen into a mammal in the presence of an adjuvant according to a program that reinforces the production of antibodies in the serum. In cases where the target antigen is too small to produce a suitable immunogenic response (referred to in the art as "hapten"), the hapten can be coupled to an antigenically neutral vehicle, such as a barnacle hemocyanin vehicle (KLH) or of serum albumin (for example, bovine serum albumin (BSA)) (for example, refers to US Patent Nos. 5,189,178 and 5,239,078). The coupling of the hapten with the vehicle can be performed using various methods well known in the art. For example, a direct coupling to the amino groups can be performed, optionally followed by a reduction of the imino bond formed. Alternatively, the vehicle can be coupled using condensing agents, such as dicyclohexylcarbodiimide or other carbodiimide dehydrating agents. Connector compounds can also be used to perform the coupling; at Pierce Chemical Company, Rockford, IL, homobifunctional and heterobifunctional connectors are available. The resulting immunogenic complex can then be injected into suitable mammalian subjects, such as mice, rabbits and the like. Following the in vivo generation of an antibody, its serum titration in the host mammal can be easily measured using immunoassay procedures that are well known in the art.

As described hereinbefore, the preparation may advantageously comprise an antibody or a fragment of a humanized antibody.

Chimeric antibodies and methods for their production are known in the art (Cabilly et al., European patent application 125023 (published November 14, 1984); Taniguchi et al., European patent application 171496 (published 19 February, 1985); Morrison et al., European Patent Application 173494 (published March 5, 1986); Neuberger et al., PCT application WO 8601533 (published March 13, 1986); Kudo et al., application European Patent 184187 (published June 11, 1986); Robinson et al., International Patent Application No. WO 8702671 (published May 7, 1987); Riechmann et al., and Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, supra.

"Human antibodies" are molecules that contain the variable and constant region of human immunoglobulin. Fully human antibodies are particularly suitable for therapeutic use, since anti-idiotypic immunogenicity will be significantly reduced or, ideally, absent. One method for the preparation of fully human antibodies is the "humanization" of the humoral immune system of mice, that is, the production of mouse breeds capable of producing human Ig (xenorratons) by the introduction of human immunoglobulin (Ig) loci in mice in which endogenous Ig genes have been inactivated. Ig loci are extremely complex in terms of their physical structure and the redisposition and gene expression processes required to produce, ultimately, a broad immune response. Antibody diversity is mainly generated by combinatorial redisposition between the different V, D and J genes present in the Ig loci. These loci also contain intercalated regulatory elements, which control antibody expression, allelic exclusion, class switching and affinity maturation. The introduction of non-redisposed human Ig transgenes in mice has shown that the mouse recombination machinery is compatible with human genes. In addition, hybridomas that secrete antigen-specific hu-mAbs of various isotypes can be obtained by immunizing xenorratons with antigens.

Fully human antibodies and methods for their production are known in the art (Méndez et al. (1997); Buggeman et al. (1991); Tomizuka et al. (2000); WO 98/24893).

As used herein, the term "muteins" refers to NIK analogs, in which one or more of the amino acid residues of the NIK components that appear in nature is replaced by different amino acid residues, or are Deleted, or one or more amino acid residues is added to the original NIK sequence, without significantly changing the activity of the resulting products compared to the original NIK. These muteins are prepared by known site-directed synthesis and / or mutagenesis techniques, or by any other known technique suitable for this.

Muteins according to the present invention include proteins encoded by a nucleic acid, such as DNA or RNA, which hybridizes with DNA or RNA, which encodes an NIK, according to the present invention, under stringent conditions. The term "stringent conditions" refers to hybridization and subsequent washing conditions, which those skilled in the art conventionally call "stringent." See Ausubel et al., Current Protocols in Molecular Biology, supra, Interscience, N.Y., §§6.3 and 6.4 (1987, 1992), and Sambrook et al. (Sambrook, J.C., Fritsch, E.F. and Maniatis, T. (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

Without limitation, examples of stringent conditions include washing conditions at 12 ° C-20 ° C below the calculated Tm of the hybrid under study, for example in 2 x SSC and 0.5% SDS for 5 minutes, 2 x SSC and 0.1% SDS for 15 minutes; 0.1 x SSC and 0.5% SDS at 37 ° C for 30-60 minutes, and then 0.1 x SSC and 0.5% SDS at 68 ° C for 30-60 minutes. Those skilled in the art will understand that stringency conditions also depend on the length of the DNA sequences, oligonucleotide probes (such as 10-40 bases) or mixed oligonucleotide probes. If mixed probes are used, it is preferable to use tetramethylammonium chloride (TMAC) instead of SSC. See Ausubel, supra.

Any mutein of this type preferably has an amino acid sequence sufficiently duplicative of that of the NIK to have a substantially similar activity, or even better, than that of the NIK.

In a preferred embodiment, any mutein of this type has at least 40% coincidence or homology with the NIK amino acid sequence. More preferably, it has at least 50%, at least 60%, at least 70%, at least 80%, or most preferably at least 90% coincidence or homology with it.

The coincidence reflects the relationship between two or more polypeptide sequences, or two or more polynucleotide sequences, determined by comparing the sequences. In general, the coincidence refers to an exact correspondence of nucleotide to nucleotide, or amino acid to amino acid, of the two polynucleotide sequences or the two polypeptide sequences, respectively, along the length of the sequences being compared.

For sequences in which there is no exact match, a "match percentage" can be determined. In general, the two sequences that are being compared are aligned to achieve maximum correlation between the sequences. This may include inserting "gaps" in one or both sequences to enhance the degree of alignment. The percentage of coincidence can be determined along the full length of each of the sequences being compared (the so-called global alignment), which is particularly suitable for sequences with an equal or very similar length, or at shorter defined lengths (the so-called local alignment), which is more suitable for sequences with different lengths.

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The methods for comparing the coincidence and homology of two or more sequences are well known in the art. Thus, for example, the programs available in the Wisconsin Sequence Analysis Package, version 9.1 (Devereux, J. et al., 1984, Nucleic Acids Res., January 11, 1984, 12 (1 Pt 1): 387- 395), for example the BESTFIT and GAP programs, to determine the percentage of coincidence between two polynucleotides, and the percentage of coincidence and the percentage of homology between two polypeptide sequences. BESTFIT uses Smith and Waterman's “local homology” algorithm (J. Theor. Biol., 1981, July 21, 91 (2): 379-380, and J. Mol. Biol., March 25, 1981, 147 (1): 195-197, 1981), and find the best individual region of similarity between two sequences. Other programs for determining the match and / or similarity between sequences are also known in the art, for example the BLAST family of programs (Altschul, SF et al., 1990, J. Mol. Biol., October 5, 1990, 215 (3): 403-410; Proc. Natl. Acad. Sci. USA, July 1990, 87 (14): 5509-5513; Altschul SF et al., Nucleic Acids Res., September 1, 1991, 25 (17): 3389-3402, accessible through the NCBI homepage at www.ncbi.nlm.nih.gov) and FASTA (Pearson, WR, Methods Enzymol., 1990, 183: 63-98; Pearson, J Mol. Biol., February 13, 1998, 276 (1): 71-84).

The NIK muteins that can be used according to the present invention, or the nucleic acids encoding them, include a finite set of substantially corresponding sequences such as peptides or substitution polynucleotides that can be routinely obtained by those skilled in the art without unnecessary experimentation. , based on the indications and teachings presented herein.

Preferred changes for the muteins according to the present invention are those known as "conservative" substitutions. NIK conservative amino acid substitutions may include synonymous amino acids within a group that has sufficiently similar physicochemical properties such that the substitution between group members will retain the biological function of the molecule. It is clear that amino acid insertions and deletions can also be made in the sequences defined above without altering their function, in particular if the insertions or deletions involve only a few amino acids, for example less than thirty, and preferably less than ten, and not eliminate or displace amino acids that are critical for a functional conformation, for example cysteine residues. Proteins and muteins produced by said deletions and / or insertions are within the scope of the present invention.

Preferably, the synonymous amino acid groups are those defined in Table A. More preferably, the synonymous amino acid groups are those defined in Table B, and most preferably, the synonymous amino acid groups are those defined in table C.

TABLE A: Preferred group of synonyms amino acids

Amino acid

 Synonym group

Be

Be, Thr, Gly, Asn

Arg

Arg, Gln, Lys, Glu, His

Leu

Ile, Phe, Tyr, Met, Val, Leu

Pro

Gly, Wing, Thr, Pro

Thr

Pro, Be, Wing, Gly, His, Gln, Thr

To

Gly, Thr, Pro, Wing

Val

Met, Tyr, Phe, Ile, Leu, Val

Gly

Wing, Thr, Pro, Be, Gly

Ile

Met, Tyr, Phe, Val, Leu, Ile

Phe

Trp, Met, Tyr, Ile, Val, Leu, Phe

Tyr

Trp, Met, Phe, Ile, Val, Leu, Tyr

Cys

 Be, Thr, Cys

His

Glu, Lys, Gln, Thr, Arg, His

Gln

Glu, Lys, Asn, His, Thr, Arg, Gln

Asn

Gln, Asp, Being, Asn

Lys

Glu, Gln, His, Arg, Lys

Asp

Glu, Asn, Asp

Glu

Asp, Lys, Asn, Gln, His, Arg, Glu

Met

Phe, Ile, Val, Leu, Met

Trp

 Trp

TABLE B: Most preferred group of synonyms amino acids

Amino acid

 Synonym group

Be

Be

Arg

His, Lys, Arg

Leu

Leu, Ile, Phe, Met

Pro

Wing, Pro

Thr

 Thr

To

Pro, wing

Val

Val, Met, Ile

Gly

 Gly

Ile

Ile, Met, Phe, Val, Leu

Phe

Met, Tyr, Ile, Leu, Phe

Tyr

Phe, Tyr

Cys

Cys, Being

His

His, Gln, Arg

Gln

Glu, Gln, His

Asn

Asp, Asn

Lys

Lys, Arg

Asp

Asp, Asn

Glu

Glu, Gln

Met

Met, Phe, Ile, Val, Leu

Trp

 Trp

TABLE C: Most preferred group of synonyms amino acids

Amino acid

 Synonym group

Be

Be

Arg

Arg

Leu

Leu, Ile, Met

Pro

Pro

Thr

 Thr

To

To

Val

Val

Gly

 Gly

Ile

Ile, Met, Leu

Phe

Phe

Tyr

Tyr

Cys

Cys, Being

His

 His

Gln

Gln

Asn

Asn

Lys

Lys

Asp

Asp

Glu

Glu

Met

Met, Ile, Leu

Trp

 Trp

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Examples of production of amino acid substitutions in proteins that can be used to obtain NIK muteins, for use in the present invention, include any step of known methods, such as those presented in US Patents 4,959,314, 4,588,585 and 4,737,462, by Mark et al .; 5,116,943, from Koths et al .; 4,965,195, from Namen et al .; 4,879,111, from Chong et al .; and 5,017,691, by Lee et al .; and the substituted lysine proteins presented in US Patent No. 4,904,584 (Shaw et al.).

"Functional derivatives," as used herein, include NIK derivatives and their muteins that can be prepared from functional groups that appear as side chains on the moieties, or are additions to N- or C groups. -terminals, by means known in the art, and are included in the invention on the condition that they remain pharmaceutically acceptable, that is, that they do not destroy the activity of the protein that is substantially similar to the activity of NIK and that they do not confer toxic properties to the compositions that contain them.

An "active fraction" according to the present invention may be, for example, a fragment of NIK. The term "fragment" refers to any subset of the molecule, that is, a shorter peptide that maintains the desired biological activity. Fragments can be easily prepared by removing amino acids from any end of the NIK molecule, and testing the resulting fragment for its activity. Proteases are known that remove one amino acid each time from the N-terminal or C-terminal of a polypeptide and, therefore, the determination of fragments that retain the desired biological activity involves only routine experimentation.

As active fractions of NIK, its muteins and fused proteins, the present invention further includes any fragment or precursor of the polypeptide chain of the protein molecule alone or together with associated molecules or moieties attached thereto, for example sugar moieties or phosphate, or aggregates of the protein molecule or of the sugar moieties themselves, with the proviso that said fraction has an activity substantially similar to NIK.

The term "salts" herein refers to salts of the carboxyl groups and acid addition salts of amino groups of the NIK molecule or its analogs. The salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts and the like, and salts with organic bases such as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like. Acid addition salts include, for example, salts with mineral acids, such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids, such as, for example, acetic acid or oxalic acid. Of course, any of these salts must maintain the biological activity of NIK.

The term "circularly permuted", as used herein, refers to a linear molecule in which the terminals have joined, directly or through a connector, to produce a circular molecule, and then the circular molecule is opened in another site to produce a new linear molecule with terminals different from the terminals of the original molecule. Circular permutations include molecules whose structure is equivalent to a molecule that has been circularized and then opened. Therefore, a circularly permuted molecule can be synthesized de novo as a linear molecule and never undergo a circularization and opening stage. The concrete circular permutation of a molecule is indicated in parentheses that contain the amino acid residues between which the peptide bond is removed. Circularly permuted molecules, which may include DNA, RNA and proteins, are single-stranded molecules, which have their normal terminals fused, often through a connector, and contain new terminals in another position. See Goldenberg et al., J. Mol. Biol., 165: 407-413 (1983), and Pan et al., Gene, 125: 111-114 (1993).

Circular permutation is functionally equivalent to taking a linear chain molecule, fusing the ends to form a circular molecule, and then cutting the circular molecule at a different site to form a new linear chain molecule with different terminals. Therefore, circular permutation has the effect of essentially conserving the sequence and the coincidence of the amino acids of a protein while generating new terminals at different sites.

Antibodies or humanized antibody fragments are genetically modified antibodies or chimeric antibody fragments that have (preferably minimal) portions that are derived from non-human antibodies. Humanized antibodies include antibodies in which the regions determining the complementarity of a human antibody (receptor antibody) are replaced by residues of a region determining the complementarity of a non-human species (donor antibody), such as a mouse, a rat

or a rabbit, which has the desired functionality. In some cases, the residues of the Fv framework regions of the human antibody are replaced by the corresponding non-human residues. Humanized antibodies may also comprise moieties that are not found in the recipient antibody or in the sequences of the region determining the complementarity or framework imported. In general, the humanized antibody will comprise substantially all of at least one, and generally two, variable domains, in which all or substantially all complementarity determining regions correspond to those of a non-human antibody, and all or substantially all of the framework regions correspond to those of a relevant human consensus sequence. Humanized antibodies also optimally contain at least a portion of a constant region of an antibody, such as an Fc region, generally derived from a human antibody (see, for example, Jones et al., 1986, Nature, 321: 522-525 ; Riechmann et al., 1988, Nature, 332: 323-329; and Presta, 1992, Curr. Op. Struct. Biol, 2: 593-596). Methods for humanizing antibodies or non-human antibody fragments are well known in the art. In general, a humanized antibody has one or more amino acid residues introduced from a source that is not human. These non-human amino acid residues are often referred to as imported residues, which are generally obtained from an imported variable domain. Humanization can be performed primarily as described (see, for example, Jones et al., 1986, Nature, 321: 522-525; Riechmann et al., 1988, Nature, 332: 323-327; Verhoeyen et al., 1988, Science, 239: 1534-1536; US Patent No. 4,816,567), replacing the determining regions of human complementarity with the corresponding determining regions of rodent complementarity. Accordingly, said humanized antibodies are chimeric antibodies, in which substantially less of an intact human variable domain has been replaced by the corresponding sequence of a non-human species. In practice, humanized antibodies may generally be human antibodies in which some of the residues of the complementarity determining region and some framework residues may be substituted by residues from similar sites in rodent antibodies. Antibodies or human antibody fragments can also be produced using various techniques known in the art, including phage display banks (see, for example, Hoogenboom and Winter, 1991, J. Mol. Biol., 227: 381; Marks et al., 1991, J. Mol. Biol, 222: 581; Cole et al., "Monoclonal Antibodies and Cancer Therapy", Alan R. Liss, p. 77 (1985); Boerner et al., 1991, J. Immunol., 147: 86-95). Humanized antibodies can also be prepared by introducing sequences encoding human immunoglobulin loci in transgenic animals, for example, in mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. After antigen exposure, a production of human antibodies is observed in these animals that closely resembles that observed in humans in all aspects, including gene redisposition, chain assembly and antibody repertoire. Extensive indications for practicing such an approach are provided in the literature of the art (for example, it refers to US Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425 and 5,661,016; Marks et al., 1992, Bio / Technology, 10: 779-783; Lonberg et al., 1994, Nature, 368: 856-859; Morrison, 1994, Nature, 368: 812-813; Fishwild et al., 1996 , Nature Biotechnology, 14: 845-851; Neuberger, 1996, Nature Biotechnology, 14: 826; Lonberg and Huszar, 1995, Intern. Rev. Immunol., 13: 65-93).

As described hereinbefore, an antibody fragment of the present invention can be advantageously expressed intracellularly using an expression vector.

A preferred approach to genetically modifying a cell of an individual with an expression vector is to use a viral vector. Viral vectors offer several advantages, including greater transformation and transport efficiency targeting specific cell types and propagation therein. Viral vectors can also be modified with specific receptors or ligands to alter target specificity through specific cell receptors, such as cancer cell receptors. This directed transport capability can be used to direct the expression of an antibody or an antibody fragment of the present invention in a cell characterized by a deregulated NF-actividadB activity.

Retroviral vectors represent a class of vectors suitable for use with the present invention. Defective retroviruses are commonly used as vectors to genetically modify mammalian cells, such as epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes and bone marrow cells, to express recombinant proteins. Portions of the retroviral genome can be removed to make the retrovirus replication defective, and retroviruses with defective replication can then be encapsidated into virions, which can be used to infect target cells through the use of an auxiliary virus using conventional techniques. Extensive indications are provided in the literature of the art to generate a retroviral vector capable of expressing a recombinant protein in a mammalian cell (for example, it is referred to Miller, AD, 1990, Blood, 76: 271; Sambrook et al., infra and associated references).

Another suitable expression vector may be an adenoviral vector. Adenoviral vectors are gene transfer vectors that have been thoroughly studied and are commonly used. The key advantages of adenoviral vectors include a relatively high transduction efficiency in dividing and quiescent cells, a natural tropism towards a wide range of epithelial tissues, and the easy production of high titres. Adenoviral DNA is transported to the nucleus but does not integrate into it. Therefore, the risk of mutagenesis with adenoviral vectors is minimized. Extensive indications for producing and exploiting adenoviral vectors for treating diseases are provided in the literature of the art (for example, refer to Russel, WC, 2000, J. Gen. Virol., 81: 57-63; for indications regarding The use of adenoviral vectors for cancer treatment refers, for example, to Seth et al., Adenoviral vectors for cancer gene therapy, in: P. Seth (ed.), Adenoviruses: Basic biology to Gene Therapy, Landes, Austin , TX, pp. 103-120 (1999)).

A specific example of a suitable adenoviral vector is the Ad-TK adenovirus derived vector. This vector expresses a herpesvirus thymidine kinase (TK) gene for positive or negative selection and includes an expression module for the desired recombinant sequences. This vector can be used to infect cells that have an adenoviral receptor that includes most cancers of epithelial origin (Sandmair et al., 2000, Hum. Gene. Ther., 11: 2197-2205).

A suitable viral expression vector may also be a chimeric adenoviral / retroviral vector that combines retroviral and adenoviral components, and which has been shown to be more effective than traditional expression vectors. Extensive indications for producing and exploiting such vectors are provided in the literature of the art (eg, refers to Pan et al., 2002, Cancer Letters, 184: 179-188).

The expression vector can be administered in various ways. If viral vectors are used, the procedure can take advantage of their target specificity and, therefore, it is not necessary for such vectors to be administered locally at an anatomical site affected by the disease. However, local administration can provide faster and more effective treatment. The administration of viral vectors can also be performed, for example by intravenous or subcutaneous injection to the individual. After injection, viral vectors will be circulating until they recognize host cells with the appropriate target specificity for their infection.

As described hereinbefore, the present invention provides a pharmaceutical composition comprising the antibody or antibody fragment of the present invention as an active ingredient.

As used herein, the term "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components, such as physiologically acceptable carriers and excipients. The objective of a pharmaceutical composition is to facilitate the administration of the active ingredients to an organism.

Here, the term "active ingredients" refers to the antibody or antibody fragment of the present invention that produces the biological effect.

Hereinafter, the terms "physiologically acceptable vehicle" and "pharmaceutically acceptable vehicle", which may be interchangeable, refer to a vehicle or diluent that does not cause significant irritation in an organism and that does not repeal biological activity and properties of the active ingredients administered. Adjuvants are included in these expressions.

Here, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for drug formulation and administration can be found in “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition.

Suitable routes of administration of the pharmaceutical composition may include, for example, the oral, rectal, transmucosal route, especially transnasal, intestinal or parenteral administration, including intramuscular, subcutaneous and intramedullary injection, as well as intrathecal, direct intraventricular injection. , intravenous, intraperitoneal, intranasal or intraocular.

Alternatively, the pharmaceutical composition can be administered in a local rather than systemic manner, for example by injecting the pharmaceutical composition directly into a region of a tissue of the individual.

The pharmaceutical compositions of the present invention can be manufactured by processes well known in the art, for example, by mixing, dissolving, granulating, dragee, levigation, emulsion, encapsulation, entrapment or lyophilization processes.

Thus, pharmaceutical compositions for use according to the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliary substances, which facilitate the processing of active ingredients in preparations that can be used pharmaceutically. The appropriate formulation depends on the route of administration chosen.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers, such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. These penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can be formulated easily by combining the active ingredients with pharmaceutically acceptable carriers well known in the art. These vehicles allow formulating the pharmaceutical composition as tablets, pills, dragees, capsules, liquids, gels, syrups, thick suspensions, suspensions and the like, for oral ingestion by the patient. Pharmacological preparations for oral use can be prepared using a solid excipient, optionally crushing the resulting mixture, and processing the granule mixture, after adding suitable auxiliary substances if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose; and / or physiologically acceptable polymers, such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents, such as cross-linked polyvinylpyrrolidone, agar or alginic acid, or salts thereof, such as sodium alginate, may be added.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and solvents or mixtures of suitable organic solvents. Dyes or pigments may be added to the tablets or to the coatings of the dragees for identification or to characterize the different dose combinations of active ingredients.

Pharmaceutical compositions that can be used orally include hard capsules made of gelatin, as well as sealed soft capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The hard capsules may contain the active ingredients mixed with a filler, such as lactose, binders, such as starches, lubricants, such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition stabilizers can be added. All formulations for oral administration must be in dosages suitable for the route of administration chosen.

For oral administration, the compositions may take the form of tablets or tablets formulated in the conventional manner.

For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently transported in the form of an aerosol spray presentation from a pressurized container or a nebulizer with the aid of a suitable propellant, for example, dichlorodifluoromethane , trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve for administering a dosed amount. Capsules and cartridges, for example gelatin, for use in a dispenser can be formulated to contain a powder mixture of the active ingredients and a suitable powder base, such as lactose or starch.

The pharmaceutical composition described herein can be formulated for parenteral administration, for example, by a bolus injection or a continuous infusion. Formulations for injection may be presented in unit dosage form, for example in ampoules or in multi-dose containers, optionally with an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating agents, such as suspending, stabilizing and / or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. In addition, suspensions of the active ingredients can be prepared as appropriate water or oil-based injection suspensions. Suitable lipophilic carriers or solvents include fatty oils, such as sesame oil, or esters of synthetic fatty acids, such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the active ingredients to allow the preparation of highly concentrated solutions.

As an alternative, the active ingredients may be in powder form for constitution with a suitable vehicle, for example a solution with a sterile pyrogen-free aqueous base, before use.

The pharmaceutical composition of the present invention can also be formulated in rectal compositions, such as suppositories or retention enemas, using, for example, bases for conventional suppositories, such as cocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in the context of the present invention include compositions in which the active ingredients are contained in an amount effective to achieve the proposed objective. More specifically, a therapeutically effective amount means an amount of active ingredients (the antibody or antibody fragment of the present invention) capable of preventing, alleviating or improving the symptoms of a disease, or prolonging the survival of the individual who is is trying.

The determination of a therapeutically effective amount is within the skill of those skilled in the art, especially in light of the detailed description provided herein.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titration. This information can be used to more accurately determine useful doses in humans.

The toxicity and therapeutic efficacy of the active ingredients described herein can be determined by conventional pharmaceutical procedures in vitro, in cell cultures or in experimental animals. Data obtained from these in vitro and cell culture assays and in animal studies can be used to formulate a dosage range for use in humans. The dosage may vary depending on the dosage form used and the route of administration used. The exact formulation, route of administration and dosage can be chosen by each physician in view of the patient's disorder (for example, refer to Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", chap. 1 , p. 1).

The dosage amount and interval can be adjusted individually to provide plasma or brain levels of the active ingredients that are sufficient to exert a desired therapeutic effect (minimum effective concentration, EMF). EMF will vary with each preparation, but can be estimated. from in vitro data. The dosages necessary to achieve EMF will depend on the individual characteristics and the route of administration. Screening assays can be used to determine plasma concentrations.

Depending on the severity and the degree of response of the disorder to be treated, the dosage may be a single administration or a plurality of administrations, lasting the course of treatment from several days to several weeks or until cure or cure is achieved. decreased disease status.

The amount of a composition to be administered depends, of course, on the individual being treated, the severity of the disease, the route of administration, the judgment of the attending physician, etc.

The compositions of the present invention, if desired, may be presented in a container or dispensing device, such as an FDA-approved kit, which may contain one or more unit dosage forms containing the active ingredients. The package may comprise, for example, a sheet of metal or plastic, such as a blister pack. The package or dispensing device may be accompanied by instructions for administration. The container or dispensing device may also be accompanied by a note associated with the container in a manner prescribed by a government agency that regulates the manufacture, use or sale of pharmaceutical products, which note reflects the approval by the agency of the form of compositions or human or veterinary administration. This note, for example, may be a label with U.S. approval. Food and Drug Administration for prescription drugs, or an insert of the approved product. Compositions comprising an antibody or an antibody fragment of the invention formulated in a compatible pharmaceutical carrier can also be prepared, placed in a suitable container, and labeled for the treatment of an indicated disorder, as detailed above.

As described hereinbefore, the present invention can be used to treat a disease associated with a pathological immune response.

Examples of such diseases include diseases associated with type I hypersensitivity (immediate or IgE-mediated), diseases associated with type II hypersensitivity (antibody-mediated), diseases associated with type IV hypersensitivity (T-cell mediated) , diseases associated with delayed type hypersensitivity (DTH), autoimmune diseases, and diseases associated with graft transplantation.

Other examples of diseases associated with hypersensitivity include, for example, diseases associated with type III hypersensitivity (mediated by the immune complex), diseases associated with inflammation, diseases associated with an infection, and diseases associated with idiopathic hypersensitivity.

Examples of type I hypersensitivity include allergic diseases, such as asthma, skin rashes, hives, pollen allergy, dust mite allergy, poison allergy, cosmetic allergy, latex allergy, chemical allergy, drug allergy, insect sting allergy, animal dander allergy, stinging plant allergy, poison ivy allergy, and food allergies.

Examples of type II hypersensitivity include rheumatoid diseases, rheumatoid autoimmune diseases, rheumatoid arthritis (Krenn, V. et al., 2000, Histol. Histopathol, 15: 791), spondylitis, ankylosing spondylitis (Jan Voswinkel et al., 2001, Arthritis Res. 3: 189), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Erikson, J. et al., 1998, Immunol. Res., 17:49), sclerosis, systemic sclerosis (Renaudineau, Y. et al ., 1999, Clin. Diagn. Lab. Immunol., 6: 156; Chan, OT et al., 1999, Immunol. Rev., 169: 107), glandular diseases, autoimmune glandular diseases, autoimmune pancreatic diseases, diabetes, diabetes Type I (Zimmet, P., 1996, Diabetes Res. Clin. Pract., 34, Suppl .: S125), thyroid diseases, anthoimmune thyroid diseases, Graves disease (Orgiazzi, J., 2000, Endocrinol. Metab. Clin North Am., 29: 339), thyroiditis, autoimmune thyroiditis esp Ontaneous (Braley-Mullen, H. and Yu, S., 2000, J. Immunol., 165 (12): 7262), Hashimoto's thyroiditis (Toyoda, N. et al., Nippon Rinsho, 1999, 57 (8) : 1810), myxedema, idiopathic myxedema (Mitsuma, T., Nippon Rinsho, 1999 58 (9): 1759), autoimmune reproductive diseases, ovarian diseases, ovarian autoimmunity (Garza, KM et al., J. Reprod. Immunol., 1998, 37 (2): 87), autoimmune anti-sperm infertility (Diekman, AB et al., Am. J. Reprod. Immunol., 2000, 43 (3): 134), repeated fetal loss (Tincani, A . et al., Lupus, 1998, 7, Suppl. 2: S107-109), neurodegenerative diseases, neurological diseases, neurological autoimmune diseases, multiple sclerosis (Cross, AH et al., J. Neuroimmunol., 2001, 112 (1 -2): 1), Alzheimer's disease (Oron, L. et al.,

J. Neural Transm. Suppl., 1997, 49:77), myasthenia gravis (Infante, AJ and Kraig, E., Int. Rev. Immunol., 1999, 18 (12): 83), motor neuropathies (Kornberg, AJ, J. Clin. Neurosci., 2000, 7 (3): 191), Guillain-Barre syndrome, neuropathies and autoimmune neuropathies (Kusunoki, S., Am. J. Med., Sci., 2000, 319 (4): 234), diseases Myasthenics, Lambert-Eaton Myasthenic Syndrome (Takamori, M., Am. J. Med. Sci., 2000, 319 (4): 204), paraneoplastic neurological diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy, non-rigid person syndrome paraneoplastic, cerebellar atrophies, progressive cerebellar atrophies, encephalitis, Rasmussen encephalitis, amyotrophic lateral sclerosis, Sideham's chorea, Gilles de la Tourette's syndrome, polyocrinopathies, autoimmune polyocrinopathies (Antoine, JC and Honnorat, J., Rev. Neurol , 2000, 156 (1) .23), neuropathies, dysimmunological neuropathies (Nobile-Orazio, E. et al., Electroencephalogr. Clin. Neurophys iol. Suppl., 1999, 50: 419), neuromiotonia, acquired neuromiotonia, congenital multiple arthrogryposis (Vincent, A. et al., Ann. N.Y. Acad. Sci., 1998, 841: 482), cardiovascular diseases, autoimmune cardiovascular diseases, atherosclerosis (Matsuura, E. et al., Lupus, 1998, 7, Suppl. 2: S135), myocardial infarction (Vaarala, O., Lupus , 1998, 7, Suppl. 2: S132), thrombosis (Tincani, A. et al., Lupus, 1998, 7, Suppl. 2: S107109), granulomatosis, Wegener granulomatosis, arteritis, Takayasu arteritis and Kawasaki syndrome (Praprotnik, S. et al., Wein Klin Wochenschr, 2000, 112 (15-16): 660), autoimmune disease anti-factor VIII (Lacroix-Desmazes, S. et al., Semin. Thromb. Hemost., 2000 , 26 (2): 157), vasculitis, necrotizing small vessel vasculitis, microscopic polyangiitis, Churg and Strauss syndrome, glomerulonephritis, pauciimmunological focal necrotizing glomerulonephritis, crescentic glomerulonephritis (Noel, LH, Ann. Med. Interne (Paris), 2000 , 151 (3): 178), antiphospholipid syndrome (Flamholz, R. et al., J. Clin. Apheresis, 1999, 14 (4): 171), heart failure, antibodies to adrenoceptor agonist-in heart failure (Wallukat G. et al, Am J. Cardiol, 1999, 83 (12A):... 75H), thrombocytopenic purpura (Moccia, F., Ann. Ital. Med. Int., 1999, 14 (2): 114), hemolytic anemia, autoimmune hemolytic anemia (Efremov, DG et al., Leuk. Lymphoma, 1998, 28 (3-4): 285), gastrointestinal diseases, autoimmune diseases of the gastrointestinal tract, intestinal diseases, chronic inflammatory bowel disease (García Herola, A. et al., Gastroenterol. Hepatol., January 2000, 23 (1): 16), celiac disease (Landau, YE and Shoenfeld, Y., Harefuah, January 2000, 138 (2): 122), autoimmune diseases of the musculature, myositis, autoimmune myositis, Sjogren's syndrome (Feist, E. et al., Int. Arch. Allergy Immunol., 2000, 123 (1 ): 92), autoimmune smooth muscle disease (Zauli, D. et al., Biomed. Pharmacother., 1999, 53 (5-6): 234), liver diseases, autoimmune liver diseases, autoimmune hepatitis (Manns, MP, J. Hepatol., 2000, 33 (2): 326) and primary biliary cirrhosis (Strassburg, CP et al., Eur. J.

Gastroenterol Hepatol., 1999, 11 (6): 595).

Examples of type III hypersensitivity include diseases mediated by immune effector cells, such as, for example, neutrophils or macrophages activated, for example, by immune complexes through Fc receptors, such as, for example, Fc receptors.

Examples of type IV hypersensitivity include rheumatoid diseases, rheumatoid arthritis (Tisch, R. and McDevitt, HO, Proc. Natl. Acad. Sci. USA, 1994, 91 (2): 437), systemic diseases, systemic autoimmune diseases, Systemic lupus erythematosus (Datta, SK, Lupus, 1998, 7 (9): 591), glandular diseases, glandular autoimmune diseases, pancreatic diseases, pancreatic autoimmune diseases, type I diabetes (Castano, L. and Eisenbarth, GS, Ann. Rev. Immunol., 8: 647), thyroid diseases, autoimmune thyroid diseases, Graves disease (Sakata, S. et al., Mol. Cell Endocrinol., 1993, 92 (1): 77), ovarian diseases (Garza, KM et al., J. Reprod. Immunol., 1998, 37 (2): 87), prostatitis, autoimmune prostatitis (Alexander, RB et al., Urology, 1997, 50 (6): 893), polyglandular syndrome, syndrome autoimmune polyglandular syndrome, autoimmune polyglandular syndrome type I (Hara, T. et al. , Blood, 1991, 77 (5): 1127), neurological diseases, autoimmune neurological diseases, multiple sclerosis, neuritis, optic neuritis (Soderstrom, M. et al., J. Neurol. Neurosurg Pshychiatry, 1994, 57 (5): 544), myasthenia gravis (Oshima, M. et al., Eur. J. Immunol., 1990, 20 (12): 2563), rigid person syndrome (Hiemstra, HS et al., Proc. Natl. Acad. Sci. USA, 2001, 98 (7): 3988), cardiovascular diseases, cardiac autoimmunity in Chagas disease (Cunha-Neto, E. et al., J. Clin. Invest. , 1996, 98 (8): 1709), autoimmune thrombocytopenic purpura (Semple, JW et al., Blood, 1996, 87 (10): 4245), T-helper antilinecyte autoimmunity (Caporossi, AP et al., Viral Immunol., 1998, 11 (1): 9), hemolytic anemia (Sallah, S. et al., Ann. Hematol., 1997, 74 (3): 139), liver diseases, autoimmune liver diseases, hepatitis, chronic active hepatitis (Franco , A. et al., Clin. Immunol. Immunopathol., 1990, 54 (3): 382), biliary cirrhosis, primary biliary cirrhosis (Jones, DE, Clin. Sci. (Colch), 1996, 91 (5): 551), nephritic diseases, autoimmune nephritic diseases, nephritis, interstitial nephritis (Kelly, CJ , J. Am. Soc. Nephrol., 1990, 1 (2): 140), connective tissue diseases, ear diseases, autoimmune connective tissue diseases, autoimmune ear disease (Yoo, T.J. et al., Cell Immunol., 1994, 157 (1): 249), inner ear disease (Gloddek, B. et al., Ann. NY Acad. Sci., 1997, 830: 266), skin diseases , skin diseases, dermal diseases, bullous skin diseases, vulgar pemphigus, bullous pemphigoid, and foliaceous pemphigus.

Examples of diseases associated with delayed type hypersensitivity include contact dermatitis and drug rash.

Examples of autoimmune diseases include cardiovascular diseases, rheumatoid diseases, gladular diseases, gastrointestinal diseases, skin diseases, liver diseases, neurological diseases, muscle diseases, nephritic diseases, reproductive-related diseases, connective tissue diseases and systemic diseases.

Examples of autoimmune cardiovascular diseases include atherosclerosis (Matsuura, E. et al., Lupus, 1998, 7, supplement 2: S135), myocardial infarction (Vaarala, O., Lupus, 1998, 7, supplement 2: S132 ), thrombosis (Tincani, A. et al., Lupus, 1998, 7, Suppl. 2: S107-109), Wegener granulomatosis, Takayasu arteritis, Kawasaki syndrome (Praprotnik, S. et al., Wein Klin Wochenschr , 2000, 112 (15-16): 660), anti-factor VIII autoimmune disease (Lacroix-Desmazes, S. et al., Semin. Thromb. Hemost., 2000, 26 (2): 157), vessel vasculitis small necrotizing, microscopic polyangiitis, Churg and Strauss syndrome, pauciimmunologic and crescent focal necrotizing glomerulonephritis (Noel, LH, Ann. Med. Interne (Paris), 2000, 151 (3): 178), antiphospholipid syndrome (Flamholz, R. et to the.,

J. Clin. Apheresis, 1999, 14 (4): 171), antibody-induced heart failure (Wallukat, G. et al., Am. J. Cardiol., 1999, 83 (12A): 75H), thrombocytopenic purpura (Moccia, F. , Ann. Ital. Med. Int., 1999, 14 (2): 114; Semple,

J.W. et al., Blood, 1996, 87 (10): 4245), autoimmune hemolytic anemia (Efremov, DG et al., Leuk. Lymphoma, 1998, 28 (3-4): 285; Sallah, S. et al., Ann. Hematol., 1997, 74 (3): 139), cardiac autoimmunity in Chagas disease (Cunha-Neto, E. et al., J. Clin. Invest., 1996, 98 (8): 1709), and auxiliary T antilinfocitos autoimmunity (Caporossi, AP et al., Viral Immunol., 1998, 11 (1): 9).

Examples of autoimmune rheumatoid diseases include rheumatoid arthritis (Krenn, V. et al., 2000, Histol. Histopathol., 15 (3): 791; Tisch, R., McDevitt, HO, 1994, Proc. Natl. Acad. Sci. USA, 91 (2): 437) and ankylosing spondylitis (Jan Voswinkel et al., 2001, Arthritis Res., 3 (3): 189).

Examples of autoimmune glandular diseases include autoimmune diseases of the pancreas, type I diabetes (Castano, L. and Eisenbarth, GS, 1990, Ann. Rev. Immunol., 8: 647; Zimmet, P., Diabetes Res. Clin. Pract. ., 1996, 34, Suppl .: S125), anthoimmune thyroid diseases, Graves disease (Orgiazzi, J., Endocrinol. Metab. Clin. North Am., 2000, 29: 339; Sakata, S. et al., Mol Cell Endocrinol., 1993, 92 (1): 77), spontaneous autoimmune thyroiditis (Braley-Mullen, H. and Yu, S., J. Immunol., 2000, 165 (12): 7262), Hashimoto's thyroiditis (Toyoda, N. et al., Nippon Rinsho, 1999, 57 (8): 1810), idiopathic myxedema (Mitsuma, T., Nippon Rinsho, 1999, 58 (9): 1759), ovarian autoimmunity (Garza, KM et al., J. Reprod. Immunol., 1998, 37 (2): 87), autoimmune anti-sperm infertility (Diekman, AB et al., Am. J. Reprod. Immunol., 2000, 43 (3): 134), autoimmune prostatitis (Alexander, RB et al., Urology, 1997, 50 (6): 893) and polyglandul syndrome Autoimmune type I (Hara T. et al., Blood, 1991, 77 (5): 1127).

Examples of autoimmune gastrointestinal diseases include chronic inflammatory bowel disease (García Herola, A. et al., Gastroenterol. Hepatol., January 2000, 23 (1): 16), celiac disease (Landau,

Y.E. and Shoenfeld, Y., Harefuah, 2000, 138 (2): 122), colitis, ileitis and Crohn's disease.

Examples of autoimmune skin diseases include autoimmune bullous skin diseases, such as pemphigus vulgaris, pemphigus bullous and pemphigus foliaceus.

Examples of autoimmune liver diseases include hepatitis, chronic autoimmune active hepatitis (Franco, A. et al., Clin. Immunol. Immunopathol., 1990, 54 (3): 382), primary biliary cirrhosis (Jones, DE, Clin. Sci (Colch), 1996, 91 (5): 551; Strassburg, CP et al., Eur. J. Gastroenterol. Hepatol., 1999, 11 (6): 595) and autoimmune hepatitis (Manns, MP, J. Hepatol ., 2000, 33 (2): 326).

Examples of autoimmune neurological diseases include multiple sclerosis (Cross, AH et al., J. Neuroimmunol., 2001, 112 (1-2): 1), Alzheimer's disease (Oron, L. et al., J. Neural Transm Suppl., 1997, 49:77), myasthenia gravis (Infante, AJ and Kraig, E., Int. Rev. Immunol., 1999, 18 (1-2): 83; Oshima, M. et al., Eur J. Immunol., 1990, 20: 2563), neuropathies, motor neuropathies (Kornberg, AJ, J. Clin. Neurosci., 2000, 7 (3): 191), Guillain-Barre syndrome, and autoimmune neuropathies (Kusunoki , S., Am. J. Med., Sci., 2000, 319 (4): 234), myasthenia, myasthenic Lambert-Eaton syndrome (Takamori, M., Am. J. Med. Sci., 2000, 319 (4): 204), paraneoplastic neurological diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy, and rigid person syndrome (Hiemstra, HS et al., Proc. Natl. Acad. Sci. USA, 2001, 98 (7): 3988 ), non-paraneoplastic rigid person syndrome, progressive cerebellar atrophies, encephalitis, Rasmuss encephalitis in, amyotrophic lateral sclerosis, Sideham's chorea, Gilles de la Tourette syndrome, and autoimmune polyocrinopathies (Antoine,

J.C. and Honnorat, J., Rev. Neurol (Paris), 2000, 156 (1) .23), dysimmunological neuropathies (Nobile-Orazio, E. et al., Electroencephalogr. Clin. Neurophysiol. Suppl., 1999, 50: 419 ), acquired neuromiotonia, congenital multiple arthrogryposis (Vincent, A. et al., Ann. NY Acad. Sci., 1998, 841: 482), neuritis, optic neuritis (Soderstrom, M. et al., J. Neurol. Neurosurg Psychiatry, 1994, 57 (5): 544) and neurodegenerative diseases.

Examples of autoimmune muscle diseases include myositis, autoimmune myositis and primary Sjogren's syndrome (Feist, E. et al., Int. Arch. Allergy Immunol., 2000, 123 (1): 92) and autoimmune smooth muscle disease (Zauli , D. et al., Biomed. Pharmacother., 1999, 53 (5-6): 234)

Examples of autoimmune nephritic diseases include nephritis and autoimmune interstitial nephritis (Kelly, C.J., J. Am. Soc. Nephrol., 1990, 1 (2): 140).

Examples of autoimmune diseases related to reproduction include repeated fetal loss (Tincani, A. et al., Lupus, 1998, 7, Suppl. 2: S107-109).

Examples of autoimmune connective tissue diseases include ear diseases, autoimmune ear diseases (Yoo, TJ et al., Cell Immunol., 1994, 157 (1): 249), and autoimmune diseases of the inner ear (Gloddek, B. et al., Ann. NY Acad. Sci., 1997, 830: 266).

Examples of autoimmune systemic diseases include systemic lupus erythematosus (Erikson, J. et al., 1998, Immunol. Res., 17 (1-2): 49) and systemic sclerosis (Renaudineau, Y. et al., Clin. Diagn Lab. Immunol., 1999, 6: 156; Chan, OT et al., Immunol. Rev., 1999, 169: 107).

Examples of infectious diseases include chronic infectious diseases, subacute infectious diseases, acute infectious diseases, viral diseases, bacterial diseases, protozoan diseases, parasitic diseases, fungal diseases, mycoplasmic diseases and prion diseases.

Examples of transplant-related diseases include graft rejection, chronic graft rejection, subacute graft rejection, hyperacute graft rejection, acute graft rejection and graft versus host disease.

Examples of grafts include gene grafts, allografts, xenografts, cell grafts, tissue grafts, organ grafts and grafts of appendages.

Examples of cell grafts include pluripotential cell grafts, progenitor cell grafts, hematopoietic cell grafts, embryonic cell grafts and nerve cell grafts.

Examples of tissue grafts include skin grafts, bone grafts, nerve grafts, bowel grafts, corneal grafts, cartilage grafts, heart tissue grafts, heart valve grafts, dental grafts, hair follicle grafts and grafts of muscle

Examples of organ grafts include kidney grafts, heart grafts, skin grafts, liver grafts, pancreatic grafts, lung grafts and bowel grafts.

Examples of appendix grafts include arm grafts, leg grafts, hand grafts, foot grafts, finger grafts, toe grafts and sex organ grafts.

Examples of inflammatory diseases include injuries, neurodegenerative diseases, ulcers, inflammation associated with prosthetic implants, menstruation, septic shock, anaphylactic shock, toxic shock syndrome, cachexia, necrosis, gangrene, musculoskeletal inflammation and idiopathic inflammation.

Examples of prosthetic implants include breast implants, silicone implants, dental implants, penile implants, cardiac implants, artificial joints, bone fracture repair devices, bone replacement implants, drug delivery implants, catheters, pacemakers. , respiratory tubes and stenosis implants.

Examples of ulcers include skin ulcers, pressure ulcers, gastric ulcers, peptic ulcers, canker sores, nasopharyngeal ulcers, esophageal ulcers, duodenal ulcers, ulcerative colitis and gastrointestinal ulcers.

Examples of injuries include abrasions, bruises, cuts, puncture injuries, lacerations, impact injuries, concussions, bruises, thermal burns, freezing, chemical burns, sunburn, drying, radiation burns, radioactivity burns, smoke inhalation, torn muscles, muscle pulls, torn tendons, tendon pulls, ligament tears, torn ligaments, hyperextension, torn cartilage, bone fractures, pinched nerves and gunshot wounds.

Examples of musculoskeletal inflammations include muscle inflammations, myositis, tendon inflammations, tendinitis, ligament inflammations, cartilage inflammation, joint inflammations, synovial inflammations, carpal tunnel syndrome and bone inflammations.

As used herein, the term "approximately" refers to 10%.

Other advantages of the present invention will be apparent to those skilled in the art after studying the following examples. In addition, each of the various embodiments and aspects of the present invention, as claimed in the section of the claims that follows, finds experimental support in the following examples.

Examples

Refer to the following examples, which, together with the above descriptions, illustrate the invention in a non-limiting manner.

In general, the nomenclature used herein and the laboratory procedures employed in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. These techniques are fully explained in the bibliography. See, for example, "Molecular Cloning: A Laboratory Manual", Sambrook et al., 1989; "Current Protocols in Molecular Biology", volumes I-III, Ausubel, R.M., ed., 1994; Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland, 1989; Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York, 1988; Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds.), "Genome Analysis: A Laboratory Manual Series", vol. 1-4, Cold Spring Harbor Laboratory Press, New York, 1998; the methodologies indicated in US Patents 4,666,828, 4,683,202, 4,801,531, 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", volumes I-III, Cellis, J.E., ed., 1994; "Current Protocols in Immunology", volumes I-III, Coligan, J.E., ed., 1994; Stites et al, eds., "Basic and Clinical Immunology", 8th edition, Appleton & Lange, Norwalk, CT, 1994; Mishell and Shiigi, eds., "Selected Methods in Cellular Immunology", W.H. Freeman and Co., New York, 1980; The available immunoassays are thoroughly described in the patent and scientific literature, see, for example, US Patents No. 3,791,932, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, 3,879 .262, 3,901,654, 3,935,074, 3,984,533, 3,993,345, 4,034,074, 4,098,876, 4,879,219, 5,011,774 and 5,281,521; "Oligonucleotide Synthesis", Gait, M.J., ed., 1984; "Nucleic Acid Hybridization", Hames, B.D. and Higgins, S.J., eds., 1985; "Transcription and Translation", Hames, B.D. and Higgins, S.J., eds., 1984; "Animal Cell Culture", Freshney, R.I., ed., 1986; "Immobilized Cells and Enzymes", IRL Press, 1986; "A Practical Guide to Molecular Cloning," Perbal, B., 1984, and "Methods in Enzymology," vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA, 1990; Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual", CSHL Press, 1996.

Other general references are provided throughout this document. It is believed that the procedures contained therein are well known in the art and are provided for the convenience of the reader.

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those usually understood by those skilled in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein may be employed in the practice or test of the present invention, suitable methods and materials are described below.

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Example 1: Generation of antibodies capable of binding to phosphorylated NIK in amino acid T559

As described above, there is no satisfactory treatment or treatment available for numerous diseases associated with a deregulated NF-B activity, including malignant diseases and diseases associated with pathological immune responses, such as autoimmune, allergic, inflammatory and related diseases. transplants Since NIK is a critical activator of NF-B, a potentially potent strategy to treat such diseases involves identifying antibodies capable of specifically binding to NIK, and thereby preventing or inhibiting the activation of NF-B by NIK. These antibodies will also be useful as detection reagents, allowing the characterization of normal and pathological aspects of biochemical processes that involve NIK. However, the prior art has not been able to provide adequate or optimally suitable antibodies for said purposes. It has been indicated that phosphorylation of the amino acid T559 located in the NIK activation loop is important for its activity. No suitable antibody with such specificity has been indicated.

Therefore, a candidate antibody for NIK inhibition is one that recognizes phosphorylated NIK in the amino acid residue T559 or its fragments.

To generate polyclonal antibodies specific for phosphorylated NIK, each of the overlapping sets of peptides derived from the NIK activation loop shown in Table 1 (all contain phosphotreonin at position 559 (Figure 1)) were used to immunize groups of rabbits.

Table 1. Activation loop and peptides used for immunization

Name of the peptide *

Amino acid sequence

553-566

CGDYIPGT (p) ETHMAPE

553-562

KGDYIPGT (p) ETH

549-560

CSLLTGDYIPGT (p) E

534-566 Activation loop

DFGHAVCLQPDGLGKSLLTGDYIPGT (p) ETHMAPE

*

 the numbers correspond to the amino acid coordinates of the peptide within the NIK sequence.

*

 Note that in peptide 549-560 and 553-566 a cysteine has been added to the N-terminal end, and that in peptide 553-562 a lysine has been added at the N-terminal end.

It was observed by ELISA (example 3), using microtiter plates coated with phosphopeptides and non-phosphorylated peptides, that the phosphorylated peptide that appears in SEQ ID NO: 3, of the three peptides derived from the activation loop tested, in which threonine Phosphorylated is the penultimate amino acid, it was the only effective peptide to produce antibodies specific for the phosphorylated version of the peptide.

In view of the results obtained with polyclonal antibodies, monoclonal antibodies were prepared using the phosphorylated peptide indicated in SEQ ID NO: 3 (549-560) for the immunization of Balb / C mice. Three fusions were performed using spleen and lymph node of immunized Balb / C mice, although the resulting antibodies were not specific or had a low titration (assayed by ELISA).

When SJL mice immunized for fusions were used instead of Balb / C mice, approximately 24 clones were generated that produced very specific antibodies to the NIK T (p) 559 peptide (assayed by ELISA or Western blot analysis).

Example 2: Control of antibody specificity by immunoprecipitation

Other experiments were performed to check if antibodies prepared against phosphorylated peptide 549-560 were able to recognize the complete NIK protein, and if they were able to differentiate the phosphorylation state of the protein. The experimental scenario used included overexpression of myc-labeled NIK (example 11) or a myc-labeled NIK mutant that has threonine at position 559 replaced by alanine in 293T cells. After NIK overexpression, the cells were lysed, and mutant NIK or NIK, unable to be phosphorylated, were immunoprecipitated (IP) with the monoclonal antibodies of clones 869, 815, 521, 254, 91, 62, 32, 30 or 3, prepared with phosphorylated 549-560 peptide. Immunoprecipitated NIK was detected with a Western blot analysis using an anti-myc antibody.

More specifically, for overexpression of the NIK 3 x 106 293T cells were seeded on a 15 cm plate, and the cells were transiently transfected with pCS3MTNIK or pCS3MTNIKT559A. Twenty-four hours later the cells were harvested and lysed in 2 ml of 1% NP-40 buffer. 100 ul of the lysate was used for each IP. IP was performed by contacting the cell lysate with 30 ul of G protein spheres adsorbed with the respective antibodies for 4 hours at 4 ° C. Western blot analysis and a positive control for IP were performed with the anti-myc antibody.

The anti-myc antibody detected both phosphorylated and non-phosphorylated forms of NIK immunoprecipitated with the above antibodies. The results in Figure 4 demonstrate that all antibodies tested were capable of immunoprecipitating NIK. It was found that clones 869, 815, 521, 254, 91, 62, 32, 30 and 3 were specific for the phosphorylated form of NIK.

Therefore, the results indicate that, as a result of the procedure used, it was possible to generate antibodies specific for the phosphorylated NIK species.

Example 3: Control of the specificity of the NIK-P4 30.12 antibody by ELISA

The non-phosphorylated control peptide or phosphorylated type 549-560, at a concentration of 10 ug / ml, was used to coat wells of a microtiter plate. The supernatant from an undiluted hybridoma culture of clone NIK-P4 30.12 was charged for 1 hour at 37 ° C to each of the coated wells. Anti-mouse HRP (Jackson) was used at a dilution of 1: 10K as a secondary antibody, and OD 405 was measured using an ABTS substrate.

The results observed (Figure 5) demonstrate that NIK-P4 30.12 antibodies recognize only the phosphorylated form of the peptide by ELISA.

Example 4: Demonstration of the inhibitory activity of the NIK-P4 30.12 antibody in signaling through CD40 and CD70

TNF CD70, CD40 and TNF receptor ligands induce degradation of IB and, consequently, induce activation of NF-B. To verify whether NIK-P4 30.12 is capable of inhibiting the activity of NIK, the effect of NIK-P4 30.12 antibody on degradation induced by an I ligaB ligand was tested,

Approximately 0.5 x 10 6 Ramos cells were cultured in 6-well plates and transfected with NIK-P4 30.12 (-pNIK) or a non-relevant control IgG (see protein transfection of Example 12), and after transfection they were induced with a 1: 4 dilution of the medium containing CD70 or CD40 ligand (see the preparation of the medium containing ligand in example 13) for 20 min and 30 min, respectively, or with TNF used at a concentration of 50 ng / ml for 20 min, or remained untreated. After induction with the ligand, the cells were lysed in 45 µl of 1% NP40 buffer + 15 µl of sample loading buffer, and 30 µl of the lysate was loaded in a 10% SDS-PAGE and underwent Western blot analysis, using the anti-IB antibody (obtained from Transduction Laboratories) for the detection of IB.

It was observed (Figure 6) that in untreated (control) cells, IB was present in cells transfected with IgG or with anti-pNIK. However, in all ligand-treated cells, the IB degraded, indicating the activation of NF-B. The degradation of IB and, consequently, the activation of NF-B does not occur in cells transfected with NIK-P4 30.12 antibodies, except when the ligand used is TNF. This result indicates that the phosphorylation of NIK in the T559 moiety is important for the activation of NF-porB by CD70 and CD40 and, therefore, the NIK-P4 30.12 antibody can be used for therapy in diseases in which CD70 signaling and CD40 is involved in the pathogenesis of the disease.

Example 5: The anti-phosphoThr559-NIK antibody blocks the degradation of CD40-induced IB

The following experiment was performed to confirm that the inhibition of NIK activation observed with NIK-P4

30.12 is not produced with antibodies generated against other domains in the kinase.

Approximately 0.5 x 106 BJAB cells were transfected with non-specific IgG, with specific antibody against the N-terminal region of the NIK kinase domain (specific for residues 405-420) (81), or with NIK-P4 30.12. All transfected cells were treated with CD40L, as in the previous example, for 30 min, or remained untreated. After treatment, the cells were lysed in 1% NP40 buffer, and IBa in the cell was monitored by Western blot analysis as in the previous example.

It was observed (Figure 7) that CD40 induces the degradation of IBa in cells treated with CD40. No inhibition of IBa degradation was observed using very effective antibodies against a region of the kinase domain (81) different from the region used to develop the NIK-P4 30.12 antibody, and the only antibody that inhibited the degradation of IBa It was NIK-P4 30.12. This result indicates that NIK-P4 30.12 antibodies generated against phosphorylated T559 in the NIK activation loop inhibit the degradation of IB and, therefore, inhibit the activation of NF-porB by a treatment with CD40L.

The results of examples 4 and 5 demonstrate that NIK does not participate in the activation of the canonical pathway by TNF in lymphoblast cells, such as BJAB and Ramos. However, the results demonstrate that the function of NIK is crucial for the activation of the canonical pathway by other ligands, such as, for example, CD40L and CD70. Therefore, the results also demonstrate that NIK-P4 30.12 antibodies can be used to identify ligands that induce NIK-mediated activation of the canonical pathway.

Example 6: Preparation of a polyclonal antibody

Rabbits were immunized with the peptides indicated in SEQ ID NO: 1, 2 or 3, for the generation of specific polyclonal antibodies.

The first immunization was performed with 100 µg of KLH peptide that was dissolved in 1: 1 complete Freund's adjuvant and injected subcutaneously. A second immunization was performed three weeks later with the same amount of peptide-KLH and injected intramuscularly three weeks later with incomplete Freund's adjuvant. These two immunizations were followed by a booster with the same amount of KLH peptide dissolved in PBS and administered subcutaneously with an interval of three weeks.

Example 7: Preparation of monoclonal antibodies

Monoclonal antibodies were prepared as described in Eshzar, Z., in: "Hybridoma Technology in the Biosciences and Medicine", chapter 1, Springer, T.A. (eds.), Timothy, A., Plenum Publishing Corp., New York, 1985, using the spleen of SJL positive mice exposed to the peptide of SEQ ID NO: 7, 11 and 12 of the Ny C-terminal kinase for fusion of spleen and lymph node.

Hybridoma culture supernatants were assayed by Western blot analysis to detect myc labeled NIK. 1.5 x 106 293-T cells were seeded on 10 cm plates and transfected 24 hours later with pCS3MTNIK. Twenty-four hours later the transfected cells were collected and lysed in 1 ml of 1% NP40 lysis buffer. 40 µl of the lysate was loaded per lane with cell lysate transfected with pcDNA3 as control (C). Western blots were probed with the respective hybridoma culture supernatant.

Example 8: Immunoprecipitation with immune serum

For immunoprecipitation of the recombinant NIK fusion protein from the transfectant protein lysate using immune serum, 1.5 µl of the immune serum was mixed with 25 µl of spheres conjugated with G protein and 50 µl of lysate, and the Volume was completed up to 750 µl with lysis buffer. The mixture was incubated at 4 ° C for 2 hours. After incubation, the spheres were washed three times with lysis buffer, boiled in 30 µl of sample buffer, and microcentrifuged at 15,000 rpm for 2 minutes. The supernatant (immunoprecipitate) was collected and analyzed for the presence of human NIK by 10% SDS-PAGE.

Immunoprecipitation with the hybridoma supernatant

A 239-T lysate expressing the myc-labeled NIK protein was used as a substrate to test the immunoprecipitation ability of anti-NIK monoclonal antibodies. 50 µl of G protein spheres were mixed with 500 ul of hybridoma culture supernatant and incubated at approximately 22 ° C for 1 hour. The spheres were washed with 1% NP-40 lysis buffer three times and used for immunoprecipitation. Immunoprecipitation (IP) was performed for 2 hours at +4 ° C. After IP, the spheres were washed three times and boiled with 50 µl of Laemli buffer and 25 µl of supernatant was loaded onto a 10% SDS-PAGE.

Example 9: Western blot analysis

For a transfer multi-selection Biorad in 20-well slots, a 180 µl aliquot of lysate from 293-T cells transfected with PCS3MTNIK was analyzed by a preparative SDS-PAGE gel. An anti-myc antibody was used as a positive control at a 1: 1,000 dilution.

Example 10: ELISA

The lysate from cells transfected with pcHis-NIK was diluted 25 times in assay buffer, and 50 µl / well was used to coat wells of an ELISA plate. The detection of the peptide coupled to BSA or coated NIK was performed using a 1: 100 dilution of anti-NIK immune serum, and the assay was revealed using a sheep anti-mouse antibody conjugated to HRP, with ABTS as an enzyme substrate. The OD405 of the samples was determined after a reaction time of 20 minutes.

Example 11: Production of recombinant human NIK

For the expression of recombinant human NIK condensed with an affinity marker myc (myc-NIK) or polyhistidine (His-NIK), 3 x 106 or 1.5 x 106 293-T cells, respectively, were transfected with the vector PCS3MTNIK expression encoding the myc-labeled NIK (the NIK nucleotide sequence of WO 9737016) or pcHis-NIK encoding His-labeled NIK, respectively. The cells were transfected by the calcium phosphate method [Ca3 (PO4) 2] in a 10 cm diameter culture plate using 20 µg or 10 µg of DNA from the expression vector. Twenty-four hours after transfection, cells transfected with PCS3MTINIK or pcHis-NIK were collected, sedimented and lysed in 1.5 ml or 1 ml, respectively, of 1% NP-40 protein lysis buffer.

Example 12: Transfection of the antibody to the cell

To test the effect of the antibodies inside the cells, the antibody was introduced into the cells using the Pierce Pro-Ject reagent using the following protocol: 2 x 105 cells were seeded in a 6-well plate and cultured overnight . 10 µl of Pro-JectTM reagent (dissolved in 250 µl of methanol was used

or chloroform) and evaporated under a laminar flow hood for 2 hours. 5-10 µg of protein, the Ab-FITC control or the monoclonal antibody tested in 50-100 µl were diluted.

The dried Pro-JectTM film was hydrated with 50-100 µl of the diluted protein solution by pipetting up and down approximately 3 to 5 times and incubating at room temperature for 5 minutes and vortexing for a few seconds at a rate Low to moderate The final volume of the ProJectTM reagent / protein mixture was brought to 1,000 µl with serum free medium.

The medium of the cells to be tested is removed by centrifugation, washed once with serum-free medium and the Pro-JectTM reagent / protein mixture was transferred directly to the cells.

Example 13: Preparation of medium containing CD70 or CD40 used for induction

293T cells were transfected with a molecule of a CD40 / CD70 construct with leucine zipper marked with Flag (Walczak et al., 1999; Fanslow et al., 1994). In this molecule, the ligand is already multimerized by virtue of the leucine zipper condensed to it. DNA encoding the flag-labeled leucine zipper ligand was cloned into the pcDNA mammalian expression vector (Invitrogen) and expressed transiently in HEK-293 cells. The supernatant of these cells was collected three days after the transfection, sterilized by filtration and used.

The citation or identification of any bibliographic reference in this application should not be considered an acknowledgment that said bibliographic reference is available as prior art for the present invention.

The hybridoma clone NIK-P4 30.12 was deposited in the Collection Nationale de Culture de Microorganismes (CNCM), Institut Pasteur, Paris, under the Budapest Treaty with deposit number I-3095. Hybridomas were registered at the CNCM on October 2, 2003.

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<110> Yeda Research and Development Co. Ltd. WALLACH, David RAMAKRISHNAN, Paraneswaran

<120> Antibodies against NIK, their preparation and use

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Claims (28)

1. An antibody or an antigen-binding fragment thereof that specifically detects the endogenously phosphorylated NF-kappaB-inducing kinase, NIK, which is characterized in that said antibody or said antigen-binding fragment is capable of binding so specific to a portion of the amino acid sequence that appears in SEQ ID NO: 5 which comprises a phosphorylated threonine at amino acid position 559. 2. The antibody or antibody fragments of claim 1, wherein said antibody is a polyclonal, monoclonal, chimeric, humanized, human or anti-anti-idiotypic antibody. 3. The antibody or antibody fragments of claim 1 or 2, wherein the portion of SEQ ID NO: 5 comprises:

(to)

 SEQ ID NO: 6; or

(b)

 SEQ ID NO: 3.

4. The antibody or antibody fragments of any one of claims 1 to 3, wherein said antibody is an IgG antibody. 5. The antibody or antibody fragments of any one of claims 1 to 4, wherein said antibody fragment is selected from the group consisting of a single chain Fv, a Fab, a Fab ', an F (ab' ) 2 and a CDR. 6. The antibody or antibody fragment of any one of claims 1 to 5, wherein said antibody or antibody fragment is also capable of regulating a biochemical activity of an NIK molecule. 7. The antibody or antibody fragment of any one of claims 1 to 6 capable of specifically detecting phosphorylated NIK by:

(to)

 a Western blot analysis;

(b)

 ELISA; or

(C)

 immunoprecipitation

8.- A monoclonal antibody generated by the hybridoma NIK-P4 30.12 deposited in the CNCM as nº I-3095, or the fragments of this antigen binding antibody. 9.- A hybridoma clone deposited in the CNCM as nº I-3095. 10. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and, as an active ingredient, the antibody or antibody fragments of any one of claims 1 to 8. 11. A pharmaceutical composition comprising the antibody or antibody fragments of any one of claims 1 to 8, and a pharmaceutically acceptable carrier for treating autoimmune, allergic, inflammatory, infectious and transplant-related diseases. 12. The use of the antibody or antibody fragments of any one of claims 1 to 8, and a pharmaceutically acceptable carrier for the preparation of a pharmaceutical composition for treating autoimmune, allergic, inflammatory, infectious and transplant-related diseases. 13. The pharmaceutical composition of claim 11 for the treatment of asthma, rheumatoid arthritis, inflammatory bowel disease, atherosclerosis and Alzheimer's disease. 14. The use of claim 12, wherein the disease is selected from asthma, rheumatoid arthritis, inflammatory bowel disease, atherosclerosis and Alzheimer's disease. 15.- A composition of matter comprising a substrate covalently bound to a peptide with the amino acid sequence that appears in SEQ ID NO: 3, in which the amino acid sequence comprises a phosphorylated threonine in the position corresponding to the position of the amino acid 559 of SEQ ID NO: 5, to selectively capture the antibody or fragment of this antibody according to any one of claims 1 to 8, which is capable of specifically binding to the target antigen. 16. The composition of matter of claim 15, wherein said substrate is an affinity chromatography matrix. 17. The composition of matter of claim 15, wherein said substrate comprises a carbohydrate or a derivative of said carbohydrate. 18. The composition of matter of claim 17, wherein said carbohydrate is selected from the group consisting of agarose, Sepharose and cellulose. 19. The composition of matter of claim 15, wherein said substrate is selected from the group consisting of a sphere, a resin or a plastic surface. 20. A method for preparing the antibody of claim 8 comprising culturing the hybridoma clone according to claim 9 to allow the hybridoma to produce and accumulate the antibody according to claim 8. 21. An in vitro method for identifying a ligand capable of inducing NIK-mediated activation of NIK-B in a cell, comprising introducing the antibody or fragment of this antibody according to any one of claims 1 to 8 into a cell, incubate the cell with individual ligands, control the activation of NF-B, and select the ligand that has affected the activation of NF-medianteB by specifically blocking NIK activity by said antibody or antibody fragment. 22. The method of claim 21, wherein the activation of NF-B is determined by controlling the degradation of IBalfa. 23. The method of claims 21 or 22, wherein the cells are of the lymphoblast type. 24. The method of claims 21 or 22, wherein the cells are selected from Ramos, BJAB and Jurkat cells. 25. A method for the purification of a NIK-binding protein, which comprises contacting a sample containing NIK and the NIK-binding protein with an antibody or a fragment of this antibody according to any one of claims 1 to 8, coinmunoprecipitate the NIK and the NIK binding protein, wash the immune complex produced, and recover the NIK binding protein from the immune complex using a competing peptide derived from NIK. 26. The method of claim 25, wherein the sample is selected from body fluids, cell extracts, and DNA expression banks. 27. The use of the antibody or antibody fragment of any one of claims 1 to 8 for the development of an ELISA. 28.- The use of the antibody or antibody fragment of any one of claims 1 to 8 for the immunological purification of NIK or its mutein, its functional derivative, its active fraction, its circularly permuted derivative or its salt.