Title of Invention	AN ISOLATED MAMMALIAN ANTI 1L-12 ANTIBODY AND A COMPOSITION COMPRISING THE SAME
Abstract	The present invention provides isolated human, primate, rodent, mammalian, chimeric, humanized and/or CDR-grafted anti-IL-12 antibodies, immunoglobulins, cleavage products and other specified portions and variants thereof, as well as anti-IL-12 antibody compositions, encoding or complementary nucleic acids, vectors, host cells, compositions, formulations, devices, transgenic animals, transgenic plants, and methods of making and using thereof, as described and enabled herein, in combination with what is known in the art. The present invention also provides at least one isolated anti-IL-12 antibody as described herein. An antibody according to the present invention includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule, such as but not limited to at least one complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof, that can be incorporated into an antibody of the present invention. An antibody of the invention can include or be derived from any mammal, such as but not limited to a human, a mouse, a rabbit, a rat, a rodent, a primate, or any combination thereof, and the like.

Title of Invention

AN ISOLATED MAMMALIAN ANTI 1L-12 ANTIBODY AND A COMPOSITION COMPRISING THE SAME

Abstract

The present invention provides isolated human, primate, rodent, mammalian, chimeric, humanized and/or CDR-grafted anti-IL-12 antibodies, immunoglobulins, cleavage products and other specified portions and variants thereof, as well as anti-IL-12 antibody compositions, encoding or complementary nucleic acids, vectors, host cells, compositions, formulations, devices, transgenic animals, transgenic plants, and methods of making and using thereof, as described and enabled herein, in combination with what is known in the art. The present invention also provides at least one isolated anti-IL-12 antibody as described herein. An antibody according to the present invention includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule, such as but not limited to at least one complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof, that can be incorporated into an antibody of the present invention. An antibody of the invention can include or be derived from any mammal, such as but not limited to a human, a mouse, a rabbit, a rat, a rodent, a primate, or any combination thereof, and the like.

Full Text	BACKGROUND OF THE INVENTION FIELD OF THE INVENTION This application is based in part on and claims priority to, U.S. Provisional 60/223,358 filed August 7. 2000 and 60/236.827 filed September 29, 2000. each of which is entirely incorporated herein by reference. The present invention relates to antibodies, including specified portions or variants, specific for at least one Interleukin-12 (IL-12) protein or fragment thereof, as well as nucleic acids encoding such anti-IL-I2 antibodies, complementary nucleic acids, vectors, host cells, and methods of making and using thereof, including therapeutic formulations, administration and devices. RELATED ART Interleukin-12 (IL-12) is a heterodimeric cytokine consisting of glycosylated polypeptide chains of 35 and 40 kD which are disulfide bonded. The cytokine is synthesized and secreted by antigen presenting cells including dendritic cells, monocytes, macrophages, B cells, Langerhans cells and keratinocytes as well as natural killer (NK) cells. IL-12 mediates a variety of biological processes and has been referred to as NK cell stimulatory factor (NKSF), T-cell stimulating factor, cytotoxic T-lymphocyte maturation factor and EBV-transformed B-cell line factor (Curfs, J.H.A.J., et al., Clinical Microbiology Reviews, 10:742-780 (1997)). Interieukin-12 can bind to the IL-12 receptor expressed on the plasma membrane of cells (e.g., T cells, NK cell), thereby altering (e.g., initiating, preventing) biological processes. For example, the binding of IL-12 to the. IL-12 receptor can stimulate the proliferation of pre- activated T cells and NK cells, enhance the cytolytic activity of cytotoxic..T cells (CTL), NK cells and LAK (lymphokine activated killer) cells, induce production of gamma interferon (IFN GAMMA) by T cells and NK cells and induce differentiation of naive ThO cells into Thl cells that produce IFN GAMMA and IL-2 (Trinchieri, G., Annual Review of Immunology, 13:251- 276(1995)). In particular, IL-12 is vital for the generation: of cytolytic cells (e.g., NK, CTL) and for mounting a cellular immune response (e.g., a Thl cell mediated immune response). Thus, IL-12 is critically important in the generation and regulation of both protective immunity (e.g., eradication of infections) and pathological immune responses (e.g., autoimmunity) (Hendrzak, J.A. and Brunda, M.J., Laboratory Investigation, 72:619-637 (1995)). Accordingly, an immune response (e.g., protective, or pathogenic) can be enhanced, suppressed or prevented by manipulation of the biological activity of IL-12 in vivo, for example, by means of an antibody. Non-human mammalian, chimeric, polyclonal (e.g.. anti-sera) and/or monoclonal antibodies (Mabs) and fragments (e.g., proteolytic digestion or fusion protein products thereof) are potential therapeutic agents that are being investigated in some cases to attempt to treat certain diseases. However, such antibodies or fragments can elicit an immune response when administered to humans. Such an immune response can result in an immune complex- mediated clearance of the antibodies or fragments from the circulation, and make repeated administration unsuitable for therapy, thereby reducing the therapeutic benefit to the patient and limiting the readministration of the antibody or fragment. For example, repeated administration of antibodies or fragments comprising non-human portions can lead to serum sickness and/or anaphalaxis. In order to avoid these and other problems, a number of approaches have been taken to reduce the immunogenicity of such antibodies and portions thereof, including chimerization and humanization, as well known in the art. These and other approaches, however, still can result in antibodies or fragments having some immunogenicity, low affinity, low avidity, or with problems in cell culture, scale up, production, and/or low yields. Thus, such antibodies or fragments can be less than ideally suited for manufacture or use as therapeutic proteins. Accordingly, there is a need to provide anti-IL-12: antibodies or fragments that overcome one more of these problems, as well as improvements over known antibodies or fragments thereof. SUMMARY OF THE INVENTION The present invention provides isolated human, primate, rodent, mammalian, chimeric, humanized and/or CDR-grafted anti-IL-12 antibodies, immunoglobulins, cleavage products and other specified portions and variants thereof, as well as anti-IL-12 antibody compositions, encoding or complementary nucleic acids, vectors, host cells, compositions, formulations, devices, transgenic animals, transgenic plants, and methods of making and using thereof, as described and enabled herein, in combination with what is known in the art. The present invention also provides at least one isolated anti-IL-12 antibody as described herein. An antibody according to the present invention includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule, such as but not limited to at least one complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof, that can be incorporated into an antibody of the present invention. An antibody of the invention can include or be derived from any mammal, such as but not limited to a human, a mouse, a rabbit, a rat, a rodent, a primate, or any combination thereof, and the like. MISSING AT THE TIME OF PUBLICATION The present invention provides. in one aspect. isolated nucleic acid molecules comprising, complementary, or hybridizing to, a polynucleotide encoding at least one IL-12 anti-idiotype antibody, comprising at least one specified sequence, domain, portion or variant thereof. The present invention further provides recombinant vectors comprising said IL-12 anti-idiotype antibody encoding nucleic acid molecules, host cells containing such nucleic acids and/or recombinant vectors, as we'll as methods of making and/or using such anti- idiotype antiobody nucleic acids, vectors and/or host cells. The present invention also provides at least one method for expressing at least one anti-IL-12 antibody, or IL-12 anti-idiotype antibody, in a host cell, comprising culturing a host cell as described herein under conditions wherein at least one anti-IL-12 antibody is expressed in detectable and/or recoverable amounts. The present invention also provides at least one composition comprising (a) an isolated anti-IL-12 antibody encoding nucleic acid and/or antibody as described herein; and (b) a suitable carrier or diluent. The carrier or diluent can optionally be pharmaceutically acceptable, according to known carriers or diluents. The composition can optionally further comprise at least one further compound, protein or composition. The present invention further provides at least one anti-IL-12 antibody method or composition, for administering a therapeutically effective amount to modulate or treat at least one IL-12 related condition in a cell, tissue, organ, animator patient and/or, prior to, subsequent to, or during a related condition, as described herein. The present invention also provides at least one composition, device and/or method of delivery of a therapeutically or prophylactically effective amount of at least one anti-IL-12 antibody, according to the present invention. The present invention further provides at least one anti-IL-12 antibody method or composition, for diagnosing at least one IL-12 related condition in a cell; tissue, organ, animal or patient and/or, prior to, subsequent to, or during a related condition, as described herein. The present invention also provides at least one composition, device and/or method of delivery for diagnosing of at least one anti-IL-12 antibody, according to the present invention. DESCRIPTION OF THE FIGURES Figures 1A and 1B are graphs showing concentration-dependent binding of human anti-IL-12 mAbs to immobilized human IL-12. Anti-IL-12 antibodies were serially diluted in 1% BSA/PBS and incubated on rhIL-12 coated plates for 1 hour at 37°C. Plates were washed twice with 0.02% Tween 20 (polyoxyethylene(20) sorbitan monolaurate), 0.15M saline and then probed with horse radish peroxidase (HRP) labeled goat anti-human IgG kappa specific antibody for 1 hour at room temperature. Plates were again washed, developed with o- phenylenediaminc (OPD) substrate and the optical density (OD) of each well was measured at 490 nm. Figure 2: Lanes from left to right in Figures A and B contain human IL-12, human IL- 12 p40, murine IL-12. and prestained molecular weight markers. Figure 2A shows bands stained from total protein. The primary bands in each lane are human IL-12 (75 kd), p40 human IL-12 (40 kd). and murine IL-12 (75 kd). Figure 2B shows a western blot prepared from a gel identical to that shown in Figure 2A. Blot was reacted with C340 followed by HRP labeled goat anti-human IgG and specifically detected human IL-12 (monomer and multimers) and human IL-12 p40 only. A control blot (not shown) reacted with HRP labeled goat anti- human IgG did not display any bands. Figure 3: Reverse transcription-PCR analysis of IFNg gene expression in human PBL's treated with IL-2, IL-12, IL-2+IL-12 with and without anti-IL-12 antibody C340, 8.6.2, isotype control antibody. Total RNA was reverse transcribed, amplified by PCR using gene- specific primers. The level of b-actin mRNA in each sample was also determined which served as a control for mRNA integrity and content. Figure 4 is a histogram showing that human anti-IL-12 mAb (C340) inhibits; production of interferon-y (IFNg) by monocyte depleted CD3+ peripheral blood mononuclear cells (PBMC) stimulated with IL-2 plus IL-12. PBMC were cultured for five hours in control media (no added cytokines), media supplemented with IL-12 (0.1 ng/ml) plus IL-2 (50 IU/ml) (IL-12/IL-2), control media containing mAb C340 (10 ug/ml) and IL-12/IL-2 media containing mAb C340 (10 m/ml). Intracellular IFNg was measured by two color immunostaining with CD3-PE and IFNg-FITC. Data are shown for one donor. Figure 5 is a graph showing dose-dependent inhibition of IFNg secretion by IL-2 plus IL-12 stimulated peripheral blood lymphocytes with two different lots of a human anti-IL-12 mAb (C340). Human PBL (8 x 106/ml) were cultured for 24 hours with 10 U/ml IL-2, IL-2 plus 400 pg/ml IL-12, or IL-2 plus IL-12 and mAb C340 as indicated. The culture supematents were removed and assayed for IFNg by EIA. Figure 6 is a histogram showing dose-dependent inhibition of IL-12 plus IL-2 induced LAK cell cytotoxicity by a human anti-IL-12 mAb (C340). LAK effector cells (human PBL, 8 x 106/ml) were cultured for 24 hours with IL-12 (400 pg/ml) plus IL-2 (10 U/ml) and mAb C340 (5000 ng/ml or 50 ng/ml as indicated). The LAK, effector cells were washed and cultured with 5lCr labeled Raji target cells for four hours at an effector to target (E:T) ration of 80:1, and the quantity of 5lCr released into the media upon Raji cell lysis was measured. Results are expressed as the mean of three normal donors standard error. EL-12 positive control (IL-12) is effector cells incubated with IL-12 and without antibody. Background (BKGD) is effector cells incubated without IL-12 or antibody. Figures 7A and 7B are histograms showing that IL-I2 plus lL-2-induced expression of CD95 on CD3+ peripheral blood mononuclear cells is inhibited by human anti-IL-12 mAb (C340). PBMC were cultured for 72 hours in media containing 0.1 ng/ml IL-12 and a suboptimal dose of IL-2 (50 IU/ml) in the presence or absence of mAb C340 (10 mg/ml). CD95 expression was measured flow cytometry of cells stained with anti-CD95-FITC. Gating was performed using two-color analysis (CD3 or CD56-PE vs. CD95-FITC) and forward vs. orthogonal light scatter. Figure 8 is a graph showing that recombinant human anti-human IL-12 antibodies (rC340) bind to immobilized IL-12 in a manner that is indistinguishable from purified mAb C340. The concentration of rC340 in the supernatants of three rC340-producing recombinant cell lines was determined, and the supernatants were evaluated for IL-12 binding in an ELISA. Plates were coated with 2 u,g/ml human IL-12 and incubated with purified mAb C340 from the original hybridoma (standard) or the supernatants of recombinant cell lines. IL-12-bound antibody was detected using alkaline phosphatase-conjugated goat anti-human IgG(heavy chain + light chain). Figures 9A - 9C are graphs showing growth kinetics and the quantity of antibody secreted by three independently-derived rC340-producing recombinant cell subclones (Figure 9 A, subclone C379B; Figure 9B, subclone C381A; Figure 9C. subclone C389A). Recombinant cells were seeded into T75 flasks at a starting density of 2 x 10s cells/ml in standard media. At various times, cells were resuspended and the number of live cells and the quantity (mg/ml) of rC340 in the media were determined. DESCRIPTION OF THE INVENTION The present invention provides isolated, recombinant and/or synthetic anti-IL- 12 human, primate, rodent, mammalian, chimeric, humanized or CDR-grafted, antibodies and IL-12 anti-idiotype antibodies thereto, as well as compositions and encoding nucleic acid molecules comprising at least one polynucleotide encoding at least one anti-IL-12 antibody or anti-idiotype antibody. The present invention further includes, but is not limited to, methods of making and using such nucleic acids and antibodies and anti-idiotype antibodies, including diagnostic and therapeutic compositions, methods and devices. As used herein, an "anti-Interleukin-12 antibody," "anti-IL-12 antibody," "anti-IL-12 antibody portion," or "anti-IL-12 antibody fragment" and/or "anti-IL-12 antibody variant" and the like include any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule, such as but not limited to at least one complementarity determinng region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof, or at least one portion of an IL-12 receptor or binding protein, which can be incorporated into an antibody of the present invention. Such antibody optionally further affects a specific ligand. such as but not limited to where such antibody modulates, decreases, increases, antagonizes, angonizes. mitigates, aleviates. blocks, inhibits, abrogates and/or interferes with at least one IL-12 activity or binding, or with IL-12 receptor activity or binding, in vitro, in situ and/or in vivo. As a non-limiting example, a suitable anti-IL-12 antibody, specified portion or variant of the present invention can bind at least one IL-12, or specified portions, variants or domains thereof. A suitable anti-IL-12 antibody, specified portion, or variant can also optionally affect at least one of IL-12 activity or function, such as but not limited to, RNA, DNA or protein synthesis, IL-12 release. IL-12 receptor signaling, membrane IL-12 cleavage, IL-12 activity, IL-12 production and/or synthesis. The term "antibody "is further intended to encompass antibodies, digestion fragments, specified portions and variants thereof, including antibody mimetics or comprising portions of antibodies that mimic the structure and/or function of an anitbody or specified fragment or portion thereof, including single chain antibodies and fragments thereof. Functional fragments include antigen-binding fragments that bind to a mammalian IL-12. For example, antibody fragments capable of binding to IL-12 or portions thereof, including, but not limited to Fab (e.g., by papain digestion), Fab' (e.g., by pepsin digestion and partial reduction) and F(ab'), (e.g., by pepsin digestion), facb (e.g., by plasmin digestion), pFc' (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsin digestion, partial reduction and reaggregation), Fv or scFv (e.g., by molecular biology techniques) fragments, are encompassed by the invention (see, e.g., Colligan, Immunology, supra). Such fragments can be produced by enzymatic cleavage, synthetic or recombinant techniques, as known in the art and/or as described herein, antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a combination gene encoding a F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CH, domain and/or hinge region of the heavy chain. The various portions of antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. As used herein, the term "human antibody" refers to an antibody in which substantially svery part of the protein (e.g., CDR, framework, CL, CH domains (e.g., CH1. CH2, CH3), hinge, (VL, VH)) is substantially non-immunogenic in humans, with only minor sequence changes or /ariations. Similarly, antibodies designated primate (monkey, babboon, chimpanzee, etc.), odent (mouse, rat, rabbit, guinea pid, hamster, and the like) and other mammals designate such species, sub-genus, genus, sub-family, family specific antibodies. Further, chimeric antibodies include any combination of the above. Such changes or variations optionally and preferably retain or reduce the immunogenicity in humans or other species relative to non- modified antibodies. Thus, a human antibody is distinct from a chimeric or humanized antibody. It is pointed out that a human antibody can be produced by a non-human animal or prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and/or light chain) genes. Further, when a human antibody is a single chain antibody, it can comprise a linker peptide that is not found in native human antibodies. For example, an Fv can comprise a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain. Such linker peptides are considered to be of human origin. Bispecific, heterospecific, heteroconjugate or similar antibodies can also be used that are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for at least one IL-12 protein, the other one is for any other antigen. Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature 305:537 (1983)). Because of the random, assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibo'dy molecules, of which only one has the correct bispecific structure. The purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed, e.g., in WO 93/08829, US Patent Nos, 6210668, 6193967, 6132992, 6106833, 6060285, 6037453, 6010902,5989530, 5959084, 5959083,5932448, 5833985, 5821333, 5807706, 5643759, 5601819,5582996, 5496549, 4676980, WO 91/00360, WO 92/00373, EP 03089, Traunecker et at, EMBO J. 10:3655 (1991), Suresh et al., Methods in Enzymology 121:210 (1986), each entirely incorporated herein by reference. Anti-IL-12 antibodies (also termed IL-12 antibodies) useful in the methods and compositions of the present invention can optionally be characterized by high affinity binding to IL-12 and optionally and preferably having low toxicity. In particular, an antibody, specified fragment or variant of the invention, where the individual components, such as the variable region, constant region and framework, individually and/or collectively, optionally and preferably possess low immunogenicity, is useful in the present invention. The antibodies that can be used in the invention are optionally characterized by their ability to treat patients for extended periods with measurable alleviation of symptoms and low and/or acceptable toxicity. Low or acceptable immunogenicity and/or high affinity, as well as other suitable properties, can contribute to the therapeutic results achieved. "Low immunogenicity" is defined herein as raising significant HAHA. HACA or HAMA responses in less than about 75%, or preferably less than about 50% of the patients treated and/or raising low litres in the patient treated (less than about 300. preferably less than about 100 measured with a double antigen enzyme immunoassay) (sec. e.g., Elliott et al., Lancet 344:1125-1127 (1994), entirely incorporated herein by reference). Utility The isolated nucleic acids of the present invention can be used for production of at least one anti-IL-12 antibody or specified variant thereof, which can be used to measure or effect in an cell, tissue, organ or animal (including mammals and humans), to diagnose, monitor, modulate, treat, alleviate, help prevent the incidence of, or reduce the symptoms of, at least one IL-12 condition, selected from, but not limited to, at least one of an immune disorder or disease, a cardiovascular disorder or disease, an infectious, malignant, and/or neurologic disorder or disease, or other known\|or specified IL-12 related condition. Such a method can comprise administering an effective amount of a composition or a pharmaceutical composition comprising at least one anti-IL-12 antibody to a ceil, tissue, organ, animal or patient in need of such modulation, treatment, alleviation, prevention, or reduction in symptoms, effects or mechanisms. The effective amount can comprise an amount of about 0.001 to 500 mg/kg per single (e.g., bolus), multiple or continuous administration, or to achieve a serum concentration of 0.01-5000 mg/ml serum concentration per single, multiple or continuous adminstration, or any effective range or value therein, as done and determined using known methods, as described herein or known in the relevant arts. Citations All publications or patents cited herein are entirely incorporated herein by reference as they show the state of the art at the time of the present invention and/or to provide description and enablement of the present invention. Publications refer to any scientific or patent publications, or any other information available in any media format, including all recorded, electronic or printed formats. The following references are entirely incorporated herein by reference: Ausubel, et al., ed.. Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, NY (1989); Harlow and Lane, antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001), Colligan et al., Current Protocols in Protein Science. John Wiley & Sons, NY, NY, (1997-2001). Antibodies of the Present Invention At least one anti-IL-12 antibody of the present invention can be optionally produced by a cell line, a mixed cell line, an immortalized cell or clonal population of immortalized cells, as well known in the art. See. e.g., Ausubel. et al., ed.. Current Protocols in Molecular Biology, John Wiley &. Sons. Inc., NY. NY (1987-2001): Sambrook. et al.. Molecular Cloning: A Laboratory Manual. 2nd Edition. Cold Spring Harbor. NY (1989); Harlow and Lane, antibodies, a Laboratory Manual. Cold Spring Harbor. NY (1989); Colligan, et al., eds., Current Protocols in Immunology. John Wiley &. Sons. Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science. John Wiley & Sons, NY. NY, (1997-2001), each entirely incorporated herein by reference. Human antibodies that are specific for human IL-12 proteins or fragments thereof can be raised against an appropriate immunogenic antigen, such as isolated and/or IL-12 protein or a portion thereof (including synthetic molecules, such as synthetic peptides). Other specific or general mammalian antibodies can be similarly raised. Preparation of immunogenic antigens, and monoclonal antibody production can be performed using any suitable technique. In one approach, a hybridoma is produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as. but not limited to, Sp2/0, Sp2/0-AGl4, NSO, NS1, NS2, AE-1, L.5, >243, P3X63Ag8.653. Sp2 SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U937, MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI. K-562, COS, RATI, NIH 3T3, HL-60, MLA 144. KAMAIWA, NEURO 2A, or the like, or heteromylomas, fusion products thereof, or any cell or fusion cell derived therefrom, or any other suitable cell line as known in the art See, e.g., www.atcc.org, www.lifetech.com., and the like, with antibody producing cells, such as, but not limited to, isolated or cloned spleen, peripheral blood, lymph, tonsil, or other immune or B cell containing cells, or any other cells expressing heavy or light chain constant or variable or framework or CDR sequences, either as endogenous or heterologous nucleic acid, as recombinant or endogenous, viral, bacterial, algal, prokaryotic, amphibian, insect, reptilian, fish, mammalian, rodent, equine, ovine, goat, sheep, primate, eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triple stranded, hybridized, and the like or any combination thereof. See, e.g., Ausubel, supra, and Colligan, Immunology, supra, chapter 2, entirely incorporated herein by reference. Antibody producing cells can also be obtained from the peripheral blood or, preferably the spleen or lymph nodes, of humans or other suitable animals that have been immunized with the antigen of interest. Any other suitable host cell can also be used for expressing heterologous or endogenous nucleic acid encoding an antibody, specified fragment or variant thereof, of the present invention. The fused cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other suitable known methods, and cloned by limiting dilution or cell sorting, or other known methods. Cells which produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA). Other suitable methods of producing or isolating antibodies of the requisite specificity can be used, including, but not limited to, methods that select recombinant antibody from a peptide or protein library e.g., but not limited to, a bacteriophage, ribosome, oligonucleotide, RNA, cDNA, or the like, display library; e.g., as available from Cambridge antibody Technologies, Cambridgeshire. UK; MorphoSys, Martinsreid/Planegg, DE; Biovation, Aberdeen, Scotland, UK: Biolnvent, Lund, Sweden; Dyax Corp., Enzon, Affymax/Biosite; Xoma, Berkeley, CA; Ixsys. See. e.g., EP 368,684, PCT/GB91/01134; PCT/GB92/01755; PCT/GB92/002240; PCT.GB92.00883; PCT/GB93/00605; US 08/350260(5/12/94); PCT/GB94/01422; PCT/GB94/02662; PCT/GB97/01835: (CAT/MRC); WO90/14443; WO90/14424; WO90/14430; PCT/US94/1234; W092/18619; WO96/07754; (Scripps); EP 614 989 (MorphoSys); WO95/16027 (Biolnvent); WO88/06630; WO90/3809 (Dyax); US 4,704,692 (Enzon); PCT/US91/02989(Affymax); WO89/06283; EP371 998; EP 550400; (Xoma); EP 229 046; PCI7US91.07149 (Ixsys); or stochastically generated peptides or proteins - US 5723323,5763192, 5S14476, 5817483, 5S24514, 5976862, WO 86/05803, EP 590 689 (Ixsys, now Applied Molecular Evolution (AME), each entirely incorporated herein by reference) or that rely upon immunization of transgenic animals (e.g., SCID mice, Nguyen et al., Microbiol. Immunol. 41:901-907 (1997); Sandhu et al., Crit. Rev. Biotechnol. 16:95-118 (1996); Eren et al., Immunol. 93:154-161 (1998), each entirely incorporated by reference as well as related patents and application) that are capable of producing a repertoire of human antibodies, as known in the art and/or as described herein. Such techniques, include, but are not limited to, ribosome display (Hanes et al., Proc. Natl. Acad. Sci. USA, 94:4937-4942 (May 1997); Hanes et al., Proc. Natl. Acad. Sci. USA, 95:14130-14135 (Nov: 1998)); single cell antibody producing technologies (e.g., selected lymphocyte antibody method ("SLAM") (US pat No. 5,627,052, Wen et al., J. Immunol. 17:887-892 (1987); Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-7848 (1996)); gel microdroplet and flow cytometry (Powell et al., Biotechnol. 8:333-337 (1990); One Cell Systems, Cambridge, MA; Gray et al., J. Imm. Meth. 182:155-163 (1995); Kenny et al., Bio/Technol. 13:787-790 (1995)); B-cell selection (Steenbakkers et al., Molec. Biol. Reports 19:125-134 (1994); Jonak et al., Progress Biotech, Vol. 5, In Vitro Immunization in Hybridoma Technology, Borrebaeck, ed., Elsevier Science Publishers B.V., Amsterdam, Netherlands (1988)). Methods for engineering or humanizing non-human or human antibodies can also be used and are well known in the an. Generally, a humanized or engineered antibody has one or more amino acid residues from a source which is non-human, e.g., but not limited to mouse, rat, rabbit, non-human primate or other mammal. These human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable, constant or other domain of a known human sequence. Known human fg sequences are disclosed, e.g., www.ncbi.nlm.nih.gov/entrez/query.fcgl: www.atcc.org/phage/hdb.html; www. sciquest.com/; www.abcam.com/; www.antibodyresource.com/onlinecomp.html; www.public.iastate.edu/~pedro/rcsearch-tools.html; www.mgcn.uni- heidelberg.de/SD/IT/lT.html; www.whfreeman.com/immunology/CH05/kuby05.htm. www.library.thinkquest.org/12429/Immune/Antibody.html; www.hhmi.org/grants/lecrures/l996/vlab/; www.path.cam.ac.uk/~mrc7/mikeimages.html; www.antibodyresource.com/; mcb.harvard.edu/BioLinks/Immunolog.html.www.immunologylink.com/; pathbox.wustl.edu/~hcenter/index.html; www.biotech.ufl.edu/~hcl/; www.pebio.com/pa/340913/340913.html; www.naI.usda.gov/awic/pubs/antibody/; www.m.ehimoe-u.ac.jp/~yasuhito/Elisa.html; www.biodesign.com/table.asp; www.icnet.uk/axp/facs/davtes/links.html; www.biotech.ufl.edu/~fccl/protocol.html; www.isac- net.org/sites_geo.htmI; aximtl .imt.uni-marburg.de/~rek/AEPStart.html; baserv.uci.kun.nl/~jraats/linksl .html; www.recab.uni-hd.de/immuno.bme.nwu.edu/; www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html; www.ibt.unam.mx/vir/V_mice.html; imgt.cnusc.fr 8104/; www.biochem.ucl.ac.uk/~martin/abs/index.html; antibody.bath.ac.uk/; abgen.cvm.tamu.edu/lab/wwwabgen.html; www.uni2h.ch/~honegger/AHOseminar/Slide01 .html; www.cryst.bbk.ac.uk/~ubcg07s/; www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm; www.path.cam.ac.uk/-mrc7/humanisation/TAHHP.html: www.ibt.unarn.rnx/vir/structure/stat_aim.html; www.biosci.missouri.edu/smithgp/index.html; www.cryst.bioc.cam. ac.uk/~fmolina/Web-pages/Pept/sportech.html; www.jerini.de/fr_products.htm; www.patents.ibm.com/ibm.html.Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Dept. Health (1983), each entirely incorporated herein by reference. Such imported sequences can be used to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic, as known in the art. Generally part or all of the non-human or human CDR sequences are maintained while the non-human sequences of the variable and constant regions are replaced with human or other amino acids, antibodies can also optionally be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be optionally prepared by a process of analysis of the parental sequences and vanous conceptual humanized products using three- dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding. Humanization or engineering of antibodies of the present invention can be performed using any known method, such as but not limited to those described in, Winter (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)), Sims et al., J. Immunol. 151: 2296 (1993); Chothiaand Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), US patent Nos: 5723323, 5976862, 5824514, 5817483, 5814476, 5763192, 5723323, 5,766886, 5714352, 6204023, 6180370, 5693762, 5530101, 5585089, 5225539; 4816567, PCT/: US98/16280, US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP 229246, each entirely incorporated herein by reference, included references cited therein. The anri-IL-12 antibody can also be optionally generated by immonization of a transgenic animal (e.g., mouse, rat, hamster, non-human primate, and the like) capable of producing a repertoire of human antibodies, as described herein and/or as known in the art. Cells that produce a human anri-IL-12 antibody can be isolated from such animals and immortalized using suitable methods, such as the methods described herein. Transgenic mice that can produce a repertoire of human antibodies that bind to human antigens can be produced by known methods (e.g., but not limited to, U.S. Pat. Nos: 5,770,428, 5,569,825, 5,545,806, 5,625,126, 5,625,825, 5,633,425, 5,661,016 and 5,789,650 issued to Lonberg et al.; Jakobovits et al. WO 98/50433, Jakobovits et al. WO 98/24893, Lonberg et al WO 98/24884, Lonberg et al. WO 97/13852, Lonberg et al. WO 94/25585, Kucherlapate et aL WO 96/34096, Kucherlapate et al. EP 0463 151 B1, Kucherlapate et al. EP 0710 719 Al, Surani et al. US. Pat. No. 5,545,807, Bruggemann et al. WO 90/04036, Bruggemann et al. EP 0438 474 B1, Lonberg et al. EP 0.814 259 A2, Lonberg et al. GB 2 272 440 A, Lonberg et al. Nature 368:856-859 (1994), Taylor et al., Int. Immunol. 6(4)579-591 (1994), Green et al, Nature Genetics 7:13-21 (1994), Mendez et al., Nature Genetics 15:146-156 (1997), Taylor et al.. Nucleic Acids Research 20(23):6287-6295 (1992), Tuaillon et al., Proc Natl Acad Sci USA 90(8)3720-3724 (1993), Lonberg et al., Int Rev Immunol 13(l):65-93 (1995) and Fishwald et at., Nat Biotechnol 14(7):845-851 (1996), which are each entirely incorporated herein by reference). Generally, these mice comprise at least one transgene comprising DNA from at least one human immunoglobulin locus that is functionally rearranged, or which can undergo functional rearrangement. The endogenous immunoglobulin loci in such mice can be disrupted or deleted to eliminate the capacity of the animal to produce antibodies encoded by endogenous genes. Screening antibodies for specific binding to similar proteins or fragments can be conveniently achieved using peptide display libraries. This method involves the screening of large collections of peptides for individual members having the desired function or structure, antibody screening of peptide display libraries is well known in the art. The displayed peptide sequences can be from 3 to 5000 or more amino acids in length, frequently from 5-100 amino acids long, and often from about 8 to 25 amino acids long. In addition to direct chemical synthetic methods for generating peptide libraries, several recombinant DNA methods have been described. One type involves the display of a peptide sequence on the surface of a bacteriophage or cell. Each bacteriophage or cell contains the nucleotide sequence encoding the particular displayed peptide sequence. Such methods are described in PCT Patent Publication Nos. 91/17271,91/18980,91/19818, and 93/08278. Other systems for generating libraries of peptides have aspects of both in vitro chemical synthesis and recombinant methods. See, PCT Patent Publication Nos. 92/05258, 92/14843, and 96/19256. See also, U.S. Patent Nos. 5,658,754; and 5,643,768. Peptide display libraries, vector, and screening kits are commercially available from such suppliers as Invitrogen (Carlsbad, CA), and Cambridge Antibody Technologies (Cambridgeshire, UK). See, e.g., U.S. Pat. Nos. 4704692,4939666,4946778, 5260203, 5455030,5518889, 5534621, 5656730, 5763733, 5767260,5856456, assigned to Enzon; 5223409,5403484,5571698,5837500, assigned to Dyax, 5427908,5580717, assigned to Affymax; 5885793, assigned to Cambridge antibody Technologies; 5750373, assigned to Genentech, 5618920, 5595898, 5576195, 5698435, 5693493,5698417, assigned to Xoma, Colligan, supra; Ausubel, supra; or Sambrook, supra, each of the above patents and publications entirely incorporated herein by reference. Antibodies of the present invention can also be prepared using at least one anti-IL-12 antibody encoding nucleic acid to provide transgenic animals or mammals, such as goats, cows, horses, sheep, and the like, that produce such antibodies in their milk. Such animals can be provided using known methods. See, e.g., but not limited to, US patent nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; 5,304,489, and the like, each of which is entirely incorporated herein by reference. Antibodies of the present invention can additionally be prepared using at least one anti-IL-12 antibody encoding nucleic acid to provide transgenic plants and cultured plant cells (e.g., but not limited to tobacco and maize) that produce such antibodies, specified portions or variants in the plant parts or in cells cultured therefrom. As a non-limiting example, transgenic tobacco leaves expressing recombinant proteins have been successfully used to provide large amounts of recombinant proteins, e.g., using an inducible promoter. See, e.g., Cramer et al., Curr. Top. Microbol. Immunol. 240:95-118 (1999) and references cited therein. Also, transgenic maize have been used to express mammalian proteins at commercial production levels, with biological activities equivalent to those produced in other recombinant systems or purified from natural sources. See. e.g., Hood et al.. Adv. Exp. Med. Biol. 464:127-147 (1999) and references cited therein, antibodies have also been produced in large amounts from transgenic plant seeds including antibody fragments, such as single chain antibodies (scFv's), including tobacco seeds and potato tubers. See, e.g., Conrad et al., Plant Mol. Biol. 38:101- 109 (1998) and reference cited therein. Thus, antibodies of the present invention can also be produced using transgenic plants, according to know methods. See also, e.g., Fischer et al., Biotechnol. Appl. Biochem. 30:99-108 (Oct., 1999), Ma et al.. Trends Biotechnol. 13:522-7 (1995); Ma et al., Plant Physiol. 109:341-6 (1995); Whitelam et al., Biochem. Soc. Trans. 22:940-944 (1994); and references cited therein. See, also generally for plant expression of antibodies, but not limited to, Each of the above references is entirely incorporated herein by reference. The antibodies of the invention can bind human IL-12 with a wide range of affinities (KD). In a preferred embodiment, at least one human mAb of the present invention can optionally bind human IL-12 with high affinity. For example, a human mAb can bind human IL-12 with a KD equal to or less than about 10"7 M, such as but not limited to, 0.1-9.9 (or any range or value therein) X 10-7 10-8, l0-9,10-10, 10-11, 10-12, 10-13 or any range or value therein. The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method. (See, for example, Berzofsky, et al., "Antibody-Antigen Interactions," In Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York, NY (1984); Kuby, Janis Immunology, W. H. Freeman and Company: New York, NY (1992); and methods described herein). The measured affinity of a particular antibody-antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH). Thus, measurements of affinity and other antigen-binding parameters (e.g., KD, Ka, Kd) are preferably made with standardized solutions of antibody and antigen, and a standardized buffer, such as the buffer described herein. Nucleic Acid Molecules Using the information provided herein, such as the nucleotide sequences encoding at least 70-100% of the contiguous amino acids of at least one of SEQ ID NOS:l, 2, 3,4, 5, 6,7, 8, specified fragments, variants or consensus sequences thereof, or a deposited vector comprising ar least one of these sequences, a nucleic acid molecule of the present invention encoding at least one anti-IL-12 antibody can be obtained using methods descnbed herein or as known in the an. Nucleic acid molecules of the present invention can be in the form of RNA, such as mRNA, hnRNA. tRNA or any other form, or in the form of DNA, including, but not limited to, cDNA and genomic DNA obtained by cloning or produced synthetically, or any combinations thereof. The DNA can be triple-stranded, double-stranded or single-stranded, or any combination thereof. Any portion, of at least one strand of the DNA or RNA can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also referred to as the an ti-sense strand. Isolated nucleic acid molecules of the present invention can include nucleic acid molecules comprising an open reading frame (ORF), optionally with one or more introns, e.g., but not limited to, at least one specified portion of at least one CDR, as CDR1, CDR2 and/or CDR3 of at least one heavy chain (e.g.. SEQ ID NOS: 1 -3) or light chain (e.g., SEQ ID NOS: 4-6); nucleic acid molecules comprising the coding sequence for an anti-IL-12 antibody or variable region (e.g., SEQ ID NOS:,7.8): and nucleic acid imolecules which comprise a nucleotide sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode at least one anti-IL-12 antibody as described herein and/or as known in the art. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate such degenerate nucleic acid variants that code for specific anti-IL-12 antibodies of the present invention. See, e.g., Ausubel, et aL, supra, and such nucleic acid variants are included in the present invention. Non-limiting examples of isolated nucleic acid molecules of the present inveniton include SEQ ID NOS: 10- 15, corresponding to non-limiting examples of a nucleic acid encoding, respectively, HC CDR1, HC CDR2, HC CDR3, LC CDR1. LC CDR2, LC CDR3, HC variable region and LC variable region. In another aspect, the invention provides isolated nucleic acid molecules encoding a(n) anti-IL-12 antibody having an amino acid sequence as encoded by the nucleic acid contained in the plasmid deposited as designated clone names_______________________________and ATCC Deposit Nos.________________________________________, respectively, deposited on______________________________. As indicated herein, nucleic acid molecules of the present invention which comprise a nucleic acid encoding an anti-IL-12 antibody can include, but are not limited to, those encoding the amino acid sequence of an antibody fragment, by itself; the coding sequence for the entire antibody or a portion thereof: the coding sequence for an antibody, fragment or portion, as well as additional sequences, such as the coding sequence of at least one signal leader or fusion peptide, with or without the aforementioned additional coding sequences, such as at least one mtron. together with additional, non-coding sequences, including but not limited to. non-codmc 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription. mRNA processing, including splicing and polyadenylation signals (for example - ribosome binding and stability of mRNA). an additional coding sequence that codes for additional amino acids, such as those that provide additional functionalities. Thus, the sequence encoding an antibody can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused antibody comprising an antibody fragment or portion. Polynucleotides Which Selectively Hybridize to a Polynucleotide as Described Herein The present invention provides isolated nucleic acids that hybridize under selective hybridization conditions to a polynucleotide disclosed herein. Thus, the polynucleotides of this embodiment can be used for isolating, detecting, and/or quantifying nucleic acids comprising such polynucleotides. For example, polynucleotides of the present invention can be used to identify, isolate, or amplify partial or full-length clones in a deposited library. In some embodiments, the polynucleotides are genomic or cDNA sequences isolated, or otherwise complementary to, a cDNA from a human or mammalian nucleic acid library. Preferably, the cDNA library comprises at least 80% full-length sequences, preferably at least 85% or 90% full-length sequences, and more preferably at least 95% full-length sequences. The cDNA libraries can be normalized to increase the representation of rare sequences. Low or moderate stringency hybridization conditions are typically, but not exclusively, employed with sequences having a reduced sequence identity relative to complementary sequences. Moderate and high stringency conditions can optionally be employed for sequences of greater identity. Low stringency conditions allow selective hybridization of sequences having about 70% sequence identity and can be employed to identify onhologous or paralogous sequences. Optionally, polynucleotides of this invention will encode at least a portion of an antibody encoded by the polynucleotides described herein. The polynucleotides of this invention embrace nucleic acid sequences that can be employed for selective hybridization to a polynucleotide encoding an antibody of the present invention. Sec, e.g., Ausubel, supra; Colligan, supra, each entirely incorporated herein by reference. Construction of Nucleic Acids The isolated nucleic acids of the present invention can be made using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, or combinations thereof, as well- known in the art. The nucleic acids can conveniently comprise sequences in addition to a polynucleotide of the present invention. For example, a multi-cloning site comprising one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in isolation of the polynucleotide. Also, translatable sequences can be inserted to aid in the isolation of the translated polynucleotide of the present invention. For example, a hexa-histidine marker sequence provides a convenient means to purify the proteins of the present invention. The nucleic acid of the present invention - excluding the coding sequence - is optionally a vector, adapter, or linker for cloning and/or expression of a polynucleotide of the present invention. Additional sequences can be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell. Use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra; or Sambrook, supra) Recombinant Methods for Constructing Nucleic Acids The isolated nucleic acid compositions of this invention, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methodologies known to those of skill in the art. In some embodiments, oligonucleotide probes that selectively hybridize, under stringent conditions, to the polynucleotides of the present invention are used to identify the desired sequence in a cDNA or genomic DNA library. The isolation of RNA, and construction of cDNA and genomic libraries, is well known to those of ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook, supra) Nucleic Acid Screening and Isolation Methods A cDNA or genomic library can be screened using a probe based upon the sequence of a polynucleotide of the present invention, such as those disclosed herein. Probes can be used to hybridize.with genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms. Those of skill in the art will appreciate that various degrees of stringency of hybridization can be employed in the assay; and either the hybridization of the wash medium can be stringent. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for duplex formation to occur. The degree of stringency can be controlled by one or more of temperature, ionic strength, pH and the presence of a partially denaturing solvent such as formamide. For example, the stringency of hybridization is conveniently varied by changing the polarity of the reactant solution through, for example, manipulation of the concentration of formamide within the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding will vary in accordance with the stringency of the hybridization medium and/or wash medium. The degree of complementarity will optimally be 100%, or 70-100%, or any range or value therein. However, it should be understood that minor sequence variations in the probes and primers can be compensated for by reducing the stringency of the hybridization and/or wash medium. Methods of amplification of RNA or DNA arc well known in the art and can be used according to the present invention without undue experimentation, based on the teaching and guidance presented herein. Known methods of DNA or RNA amplification include, but are not limited to, polymerase chain reaction (PCR) and related amplification processes (see, e.g., U.S. Patent Nos. 4,683,195. 4,683,202. 4.S00.159. 4,965,188, to Mullis, et al.; 4,795.699 and 4,921,794 to Tabor, et al; 5.142,033 to Innis: 5.122,464 to Wilson, et al.; 5,091,310 to Innis; 5,066,584 to Gyllensten, et al; 4.889,818 to Gelfand, et al; 4,994,370 to Silver, et al; 4,766,067 to Biswas; 4,656,134 to Ringold) and RNA mediated amplification that uses anti-sense RNA to the target sequence as a template for double-stranded DNA synthesis (U.S. Patent No. 5,130,238 to Malek, et al, with the tradename NASBA), the entire contents of which references are incorporated herein by reference. (See, e.g., Ausubel, supra; or Sambrook, supra.) For instance, polymerase chain reaction (PCR) technology can be used to amplify the sequences of polynucleotides of the present invention and related genes directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods can also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to direct persons of skill through in vitro amplification methods are found in Berger, supra, Sambrook, supra, and Ausubel, supra, as well as Mullis, et al., U.S. Patent No. 4,683,202 (1987); and Innis, et al., PCR Protocols A Guide to Methods and Applications, Eds., Academic Press Inc., San Diego, CA (1990). Commercially available kits for genomic PCR amplification are known in the art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech). Additionally, e.g., the T4 gene 32 protein (Boehringer Mannheim) can be used to improve yield of long PCR products. Synthetic Methods for Constructing Nucleic Acids The isolated nucleic acids of the present invention can also be prepared by direct chemical synthesis by known methods (see, e.g., Ausubel, et al., supra). Chemical synthesis generally produces a single-stranded oligonucleotide, which can be converted into double- stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill in the art will recognize that while chemical synthesis of DNA can be limited to sequences of about 100 or more bases, longer sequences can be obtained by the ligation of shorter sequences. Recombinant Expression Cassettes The present invention further provides recombinant expression cassettes comprising a nucleic acid of the present invention. A nucleic acid sequence of the present invention, for example a cDNA or a genomic sequence encoding an antibody of the present invention, can be used to construct a recombinant expression cassette that can be introduced into at leas: one desired host cell. A recombinant expression cassette will typically comprise a polynucleotide of the present invention operably linked to transcriptional initiation reyulatory sequences that will direct the transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the present invention. In some embodiments, isolated nucleic acids that serve as promoter, enhancer, or other elements can be introduced in the appropriate position (upstream, downstream or in intron) of a non-heterologous form of a polynucleotide of the present invention so as to up or down regulate expression of a polynucleotide of the present invention. For example, endogenous promoters can be altered in vivo or in vitro by mutation, deletion and/or substitution. Vectors And Host Cells The present invention also relates to vectors that include isolated nucleic acid molecules of the present invention, host cells that are genetically engineered with the recombinant vectors, and the production of at least one anti-IL-12 antibody by recombinant techniques, as is well known in the art. See. e.g., Sambrook, et al., supra; Ausubel, et al., supra, each entirely incorporated herein by reference. The polynucleotides can optionally be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid: If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells. The DNA insert should be operanvely linked to an appropriate promoter. The expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (e.g., UAA, UGA or UAG) appropriately positioned at the end of the mRNA to be translated, with UAA and UAG preferred for mammalian or eukaryotic cell expression. Expression vectors will preferably but optionally include at least one selectable marker. Such markers include, e.g., but not limited to, methotrexate (MTX), dihydrofolate reductase (DHFR. US Pat.Nos. 4,399.216; 4,634,665; 4.656,134; 4,956,288; 5.149,636; 5,179,017), ampicillin, neomycin (G418), mycophenolic acid, or glutamme synthetase (GS, US Pat.Nos. 5.122,464; 5.770,359; 5,827,739) resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria or prokaryotics (the above patents are entirely incorporated hereby by reference). Appropnate culture mediums and conditions for the above-described host cells are known in the an. Suitable vectors will be readily apparent to the skilled artisan. Introduction of a vector construct into a host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection. canonic lipid-medtated transfection, electroporation. transduction, infection or other known methods. Such methods arc described in the art. such as Sambrook. supra. Chapters 1-4 and 16-18; Ausubel. supra. Chapters 1,9, 13. 15, 16. At least one antibody of the present invention can be expressed in a modified form, such as a fusion protein, and can include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, can be added to the N-termmus of an antibody to improve stability and persistence in the host cell, during purification, or dunng subsequent handling and storage. Also, peptide moieties can be added to an antibody of the present invention to facilitate purification. Such regions can be removed prior to final preparation of an antibody or at least one fragment thereof. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18. Those of ordinary skill in the art are knowledgeable in the numerous expression systems available for expression of a nucleic acid encoding a protein of the present invention. Alternatively, nucleic acids of the present invention can be expressed in a host cell by turning on (by manipulation) in a host cell that contains endogenous DNA encoding an antibody of the present invention. Such methods are well known in the art, e.g., as described in US patent Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, entirely incorporated herein by reference. Illustrative of cell cultures useful for the production of the antibodies, specified portions or variants thereof, are mammalian cells. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions or bioreactors Can also be used. A number of suitable host cell lines capable of expressing intact glycosylated proteins have been developed in the art, and include the COS-1 (e.g.. ATCC CRL 1650), COS-7 (e.g., ATCC CRL- 1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610) and BSC-l (e.g., ATCC CRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2A)-Agl4, 293 cells, HeLa cells and the like, which are readily available from, for example, American Type Culture Collection. Manassas, Va (www.atcc.org). Preferred host cells include cells of lymphoid origin such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC Accession Number CRL-1580) and SP2/0-Agl4 cells (ATCC Accession Number CRL-1851). In a particularly preferred embodiment, the recombinant cell is a P3X63Ab8.653 or a SP2/0-Agl4 cell. Expression vectors for these cells can include one or more of the following expression control sequences, such as. but not limited to an origin of replication: a promoter (e.g., late or early SV40 promoters, the CMV promoter (US Pat.Nos. 5.168.062; 5.385.839). an HSV tk promoter, a pgk (phosphoglyccratc kinase) promoter, an EF-l alpha promoter (US Pat.No. 5,266.491). at least one human immunoglobulin promoter, an enhancer, and/or processing information sites, such as nbosome binding sites. RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences. See, e.g., Ausubel et al.. supra; Sambrook, et al., supra. Other celts useful for production of nucleic acids or proteins of the present invention are known and/or available, for instance, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (www.atcc.org) or other known or commercial sources. When eukaryotic host cells are employed, polyadenlyation or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript can also be included. An example of a splicing sequence is the VPl intron from SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)). Additionally, gene sequences to control replication in the host cell can be incorporated into the vector, as known in the art Purification of an antibody An anti-IL-12 antibody can be recovered and purified from recombinant cell cultures by well-known methods including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography ("HPLC") can also be employed for purification. See, e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons. NY, NY. (1997-2001), e.g., Chapters 1, 4. 6. 8. 9, 10, each entirely incorporated herein by reference. Antibodies of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the antibody of the present invention can be glycosylated or can be non-glycosylated, with glycosylated preferred. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, Protein Science, supra. Chapters 12-14, all entirely incorporated herein by reference. Anti-IL-12 Antibodies The isolated antibodies of the present invention comprise an antibody encoded by any one of the polynucleotides of the present invention as discussed more fully herein, or any isolated or prepared antibody. Preferably, the human antibody or antigen-binding fragment binds human IL-12 and, thereby partially or substantially neutralizes at least one biological activity of the protein. An antibody, or specified portion or variant thereof, that partially or preferably substantially neutralizes at least one biological activity of at least one IL-12 protein or fragment can bind the protein or fragment and thereby inhibit activitys mediated through the binding of EL- 12 to the IL-12 receptor or through other IL-12-dependent or mediated mechanisms. As used herein, the term "neutralizing antibody" refers to an antibody that can inhibit an IL-12-dependent activity by about 20-120%, preferably by at least about 10,20, 30,40, 50,55, 60,65,70, 75, 80, 85,90,91, 92,93, 94, 95,96, 97,98,99, 100% or more depending on the assay. The capacity of an anti-IL-12 antibody to inhibit an IL-12-dependent activity is preferably assessed by at least one suitable IL-12 protein or receptor assay, as described herein and/or as known in the art. A human antibody of the invention can be of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and can comprise a kappa or lambda light chain. In one embodiment, the human antibody comprises an IgG heavy chain or defined fragment, for example, at least one of isotypes, IgGl, IgG2, IgG3 or IgG4. Antibodies of this type can be prepared by employing a transgenic mouse or other trangenic non-human mammal comprising at least one human light chain (e.g., IgG, IgA and IgM (e.g., yl, y2, y3, y4) transgenes as described herein and/or as known in the art. In another embodiment, the anti-human IL-12 human antibody comprises an IgG I heavy chain and a IgGl light chain. At least one antibody of the invention binds at least one specified epitope specific to at least one IL-12 protein, subunit, fragment, portion or any combination thereof. The at least one epitope can comprise at least one antibody binding region that comprises at least one portion of said protein, which epitope is preferably comprised of at least one extracellular, soluble, hydrophillic, external or cytoplasmic portion of said protein. The at least one specified epitope can comprise any combination of at least one amino acid sequence of atieast 1-3 amino acids to the entire specified portion of contiguous amino acids of the SEQ ED NO:9. Generally, the human antibody or antigen-binding fragment of the present invention will comprise an antigen-binding region that comprises at least one human complementarity determining region (CDR1, CDR2 and CDR3) or variant of at least one heavy chain variable region and at least one human complementarity determining region (CDR1, CDR2 and CDR3) or variant of at least one light chain variable region. As a non-limiting example, the antibody or antigen-binding portion or variant can comprise at least one of the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:3, and/or a light chain CDR3 having the amino acid sequence of SEQ ID NO:6. In a particular embodiment, the antibody or antigen-binding fragment can have an antigen-binding region that comprises at least a portion of at least one heavy chain CDR (i.e.. CDR1, CDR2 and/or CDR3) having the amino acid sequence of the corresponding CDRs 1. 2 and/or 3 (e.g.. SEQ ID NOS: 1. 2, and/or 3). In another particular embodiment, the antibody or antigen-binding portion or variant can have an antigen-binding region that comprises at least a portion of at least one light chain CDR (i.e., CDR1. CDR2 and/or CDR3) having the amino acid sequence of the corresponding CDRs 1, 2 and/or 3 (e.g., SEQ ID NOS: 4. 5, and/or 6). In a preferred embodiment the three heavy chain CDRs and the three light chain CDRs of the anitbody or antigen-binding fragment have the amino acid sequence of the corresponding CDR of at least one of mAb 12B75, C340, or any others as described herein. Such antibodies can be prepared by chemically joining together the various portions (e.g., CDRs, framework) of the antibody using conventional techniques, by preparing and expressing a (i.e., one or more) nucleic acid molecule that encodes the antibody using conventional techniques of recombinant DNA technology or by using any other suitable method. The anti-IL-12 antibody can comprise at least one of a heavy or light chain variable region having a defined amino acid sequence. For example, in a preferred embodiment, the anti-EL-12 antibody comprises at least one of at least one heavy chain variable region, optionally having the amino acid sequence of SEQ ID NO:7 and/or at least one light chain variable region, optionally havingithe amino acid sequence of SEQ ID NO:8.' antibodies that bind to human IL-12 and that comprise a defined heavy or light chain variable region can be prepared using suitable methods, such as phage display (Katsube, Y., et al, Int J Mol. Med, l(5):863-868 (1998)) or methods that employ transgenic animals, as known in the art and/or as described herein. For example, a transgenic mouse, comprising a functionally rearranged human immunoglobulin heavy chain transgene and a transgene comprising DNA from a human immunoglobulin light chain locus that can undergo functional rearrangement, can be immunized with human IL-12 or a fragment thereof to elicit the production of antibodies. If desired, the antibody producing cells can be isolated and hybridomas or other immortalized antibody-producing cells can be prepared as described herein and/or as known in the art. Alternatively, the antibody, specified portion or variant can be expressed using the encoding nucleic acid or portion thereof in a suitable host cell. The invention also relates to antibodies, antigen-binding fragments, immunoglobulin chains and CDRs comprising amino acids in a sequence that is substantially the same as an amino acid sequence described herein. Preferably, such ;antibodies or antigen-binding fragments and antibodies comprising such chains or CDRs can bind human IL-12 with high affinity (e.g., KD less than or equal to about 10-9 M). Amino acid sequences that are substantially the same as the sequences described herein include sequences comprising conservative amino acid substitutions, as well as amino acid deletions and/or insertions. A conservative amino acid substitution refers to the replacement of a first amino acid by a second amino acid that has chemical and/or physical properties (e.g, charge, structure, polarity, hydrophobicity/ hydrophilicity) that are similar to those of the first amino acid. Conservative substitutions include replacement of one amino: acid by another within the following groups: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y). K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine (I), proline ( (M), cysteine (C) and glycine (G); F, W and Y; C, S and T. Amino Acid Codes The amino acids that make up anti-IL-12 antibodies of the present invention are often abbreviated. The amino acid designations can be indicatedby designating the amino acid by its single letter code, its three letter code; name, or three nucleotide codon(s) as is well understood in the art (see Alberts, B., et al., Molecular Biology of The Cell, Third Ed., Garland Publishing, lnc.,New York, 1994): An anti-IL-12 antibody of the present invention can include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation, as specified herein. Of course, the number of amino acid substitutions a skilled artisan would make depends on many factors, including those described above. Generally speaking, the number of amino acid substitutions, insertions or deletions for any given anti-IL-12 Ig-derived protein, fragment or variant will not be more than 40, 30, 20, 19, 18. 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, such as 1-30 or any range or value therein, as specified herein. Amino acids in an anti-IL-12 antibody of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as, but not limited to at least one IL-12 neutralizing activity. Sites that are critical for antibody binding can also be identified by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992) and de Vos, et al., Science 255:306-312 (1992)). Anti-IL-12 antibodies of the present invention can include, but are not limited to, at least one portion, sequence or combination selected from 5 to all of the contiguous amino acids of at least one of SEQ ID NOS: 1, 2,3, 4, 5, 6. IL-12 antibodies or specified portions or variants of the present invention can include, but are not limited to, at least one portion, sequence or combination selected from at least 3-5 contiguous amino acids of SEQ ID NO:l, 5-17 contiguous amino acids of SEQ ID NO:2,5-10 contiguous amino acids of SEQ ID NO:3, 5-11 contiguous amino acids of SEQ ID NO:4, 5-7 contiguous amino acids of SEQ ID NO:5; 5-9 contiguous amino acids of SEQ ID NO:6; Leu21, Lys76, Met83, Ser85 of SEQ ID NO:7. A(n) anti-IL-12 antibody can further optionally comprise a polypeptide of at least one of 70-100% of 5, 17, 10, 11, 7, 9, 119, or 108 contiguous amino acids of at least one of SEQ IDNOS:l,2,3,4,5,6,7or8. "In one embodiment, the amino acid sequence of an immunoglobulin chain, or portion thereof (e.g., variable region, CDR) has about 70-100% identity (e.g., 70, 71, 72, 73,. 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100 or any range or value therein) to the amino acid sequence of the corresponding chain of at least one of SEQ ID NOS:7, 8. For example, the amino acid sequence of a light chain variable region can be compared with the sequence of SEQ ID NO: 8, or the amino acid sequence of a heavy chain CDR3 can be compared with SEQ ID NO:3. Preferably, 70-100% amino acid identity (i.e., 90, 91, 92, 93. 94. 95. 96, 97, 98, 99, 100 or any range or value therein) is determined using a suitable computer algorithm, as known in the art. Exemplary heavy chain and light chain variable regions sequences are provided in SEQ ID NOS: 7 and 8. The antibodies of the present invention, or specified variants thereof, can comprise any number of contiguous amino acid residues from an antibody of the present invention, wherein that number is selected from the group of integers consisting of from 10- 100% of the number of contiguous residues in an anti-IL-12 antibody. Optionally, this subsequence of contiguous amino acids is at least about 10. 20, 30, 40, 50. 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160. 170, 180, 190,200,210, 220, 230,240,250 or more amino acids in length, or any range or value therein. Further, the number of such subsequences can be any integer selected from the group consisting of from 1 to 20, such as at least 2, 3,4, or 5. As those of skill will appreciate, the present invention includes at least one biologically active antibody of the present invention- Biologically active antibodies have a specific activity at least 20%, 30%, or 40%, and preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95%-1000% of that of the native (non-synthetic), endogenous or related and known antibody. Methods of assaying and quantifying measures of enzymatic activity and substrate specificity, are well known to those of skill in the art. In another aspect, the invention relates to human antibodies and antigen-binding fragments, as described herein, which are modified by the covalent attachment of an organic moiety. Such modification can produce an antibody or antigen-binding fragment with improved pharmacokinetic properties (e.g., increased in vivo serum half-life). The organic moiety can be a linear or branched hydrophilic polymeric group, fatty acid group, or fatty acid ester group. In particular embodiments, the hydrophilic polymeric group can have a molecular weight of about 800 to about 120,000 Daltons and can be a polyalkane glycol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, and the fatty acid or fatty acid ester group can comprise from about eight to about forty carbon atoms. The modified antibodies and antigen-binding fragments of the invention can comprise one or more organic moieties that are covalently bonded, directly or indirectly, to the antibody. Each organic moiety that is bonded to an antibody or antigen-binding fragment of the invention can independently be a hydrophilic polymeric group, a fatty acid group or a fatty acid ester group. As used herein, the term "fatty acid" encompasses mono-carboxylic acids and di-carboxylic acids. A "hydrophilic polymeric group," as the term is used herein, refers to an organic polymer that is more soluble in water than in octane. For example, polylysine is more soluble in water than in octane. Thus, an antibody modified by the covalent attachment of polylysine is encompassed by the invention. Hydrophilic polymers suitable for modifvinu antibodies of the invention can be linear or branched and inciude. for example, polyalkanc glycols (e.g.. PEG, monomethoxy-polycthylene glycol (mPEG). PPG and the like), carbohydrates (e.g., dextran. cellulose, oligosaccharides, polysaccharides and the like), polymers of hydrophilic amino acids (e.g., polylysine. polyarginine, polyaspartate and the like), polyalkane oxides (e.g., polyethylene oxide. polypropylene oxide and the like) and polyvinyl pyrolidone. Preferably, the hydrophilic polymer that modifies the antibody of the invention has a molecular weight of about 800 to about 150,000 Daltons as a separate molecular entity. For example PEG5000 and PEG20000 wherein the subscript is the average molecular weight of the polymer in Daltons, can be used. The hydrophilic polymeric group can be substituted with one to about six alkyl, fany acid or fatty acid ester groups. Hydrophilic polymers that are substituted with a fatty acid or fatty acid ester group can be prepared by employing suitable methods. For example, a polymer comprising an amine group can be coupled to a carboxylate of the fatty acid or fatty acid ester, and an activated carboxylate (e.g., activated with N, N-carbonyl diimidazole) on a fatty acid or fatty acid ester can be coupled to a hydroxyl group on a polymer. Fatty acids and fatty acid.esters suitable for modifying antibodies of the invention can be saturated or can contain one or more units of unsaruration. Fatty acids that are suitable for modifying antibodies of the invention include, for example, n-dodecanoate (C,,, laurate), n- tetradecanoate (Cl4, myristate), n-octadecanoate (C18, stearate), n-eicosanoate (CM, arachidatc) , n-docosanoate (C22, behenate), n-triacontanoatc (C30), n-tetracontanoate (C40), cis-?9- octadecanoate (C18, oleate), all cis-?5,8,l 1,14-eicosatetraenoate (C20, arachidonate), octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like. Suitable fatty acid esters include mono-esters of dicarboxylic acids that comprise a linear or branched lower alkyl group. The lower alkyl group can comprise from one to about twelve, preferably one to about six, carbon atoms. The modified human antibodies and antigen-binding fragments can be prepared using suitable methods, such as by reaction with one or more modifying agents. A "modifying agent" as the term is used herein, refers to a suitable organic group (e.g., hydrophilic polymer, a fatty acid, a fatty acid ester) that comprises an activating group. An "activating group" is a chemical moiety or functional group that can, under appropriate conditions, react with a second chemical group thereby forming a covalent bond between the modifying agon and the second chemical group. For example, amine-reactive activating groups include electrophilic groups such as tosylate, mesylate, halo (chloro, bromo. fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfides, 5-thioI-2-nitrobenzoic acid thiol (TNB- thiol), and the like. An aldehyde functional group can be coupled to amine- or hydrazide- containing molecules, and an azide group can react with a trivalent phosphorous group to form phosphoramidate or phosphorimide linkages. Suitable methods to introduce activating groups into molecules are known in the art (see for example. Herrnanson, G. T., Bioconjugate Techniques. Academic Press: San Diego, CA (1996)). An activating group can be bonded directly to the organic group (e.g., hydrophilic polymer, fatty acid, fatty acid ester), or through a linker moiety, for example a divalent C1-C12 group wherein one or more carbon atoms can be replaced by a heteroatom such as oxygen, nitrogen or sulfur. Suitable linker moieties include, for example, tetraethylene glycol, -(CH2)3-, -NH-(CH2)6-NH-, -(CH2)2-NH- and -CHv-O-CH2- CH2-0-CH2-CH2-O-CH-NH-. Modifying agents that comprise a linker moiety can be produced, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc- ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylate. The Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine that can be coupled to another carboxylate as described, or can be reacted with maleic anhydride and the resulting product cyclized to produce an activated maleimido derivative of the fatty acid. (See, for example, Thompson, et al., WO 92/16221 the entire teachings of which are incorporated herein by reference.) The modified antibodies of the invention can be produced by reacting a human antibody or antigen-binding fragment with a modifying agent. For example','the organic moieties can be bonded to the antibody in a non-site specific manner by employing an amine- reactive modifying agent, for example, an NHS ester of PEG. Modified human antibodies or antigen-binding fragments can also be prepared by reducing disulfide bonds (e.g., intra-chain disulfide bonds) of an antibody or antigen-binding fragment. The reduced antibody or antigen- binding fragment can then be reacted with a thiol-reactive modifying agfent to produce the modified antibody of the invention. Modified human antibodies and antigen-binding fragments comprising an organic moiety that is bonded to specific sites of an antibody of the present invention can be prepared using suitable methods, such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci. 6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1): 59-68 (1996); Capellas et al., Biotechnol. Bioeng, 56(4):456-463 (1997)), and the methods described in Herrnanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, CA (1996). ANTI-IDIOTYPE ANTIBODIES TO ANTI-IL-12 IG DERIVED PROTEIN COMPOSITIONS In addition to monoclonal or chimeric anti-lL-12 antibodies, the present invention is also directed to an anti-idiotypic (anti-Id) antibody specific for such antibodies of the invention. An anti-Id antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding region of another antibody. The anti-Id can be prepared by immunizing an animal of the same species and genetic type (e.g. mouse strain) as the source of the Id antibody with the antibody or a CDR containing region thereof The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody and produce an anti-Id antibody. The anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody. ANTI-IL-12 IG DERIVED PROTEIN COMPOSITIONS The present invention also provides at least one anti-IL-12 antibody composition comprising at least one, at least two, at least three, at least four, at least five, at least six or more anti-IL-12 antibodies thereof, as described herein and/or as known in the art that are provided in a non-naturally occurring composition, mixture or form. Such compositions comprise non-naturally occurring compositions comprising at least one or two full length, C- and/or N-terminally deleted variants, domains, fragments, or specified variants, of the anti-EL- 12 antibody amino acid sequence selected from the group consisting of 70-100% of the contiguous amino acids of SEQ ID NOS:l, 2,3,4, 5, 6, 7 or 8, or specified fragments, domains or variants thereof. Preferred anti-IL-12 derived protein, fragment or variant compositions include at least one or two full length, fragments, domains or variants as at least one CDR containing portions of the anti-IL-12 antibody sequence of 70-100% of SEQ ID NOS: 1,2, 3, 4, 5, 6, or specified fragments, domains or variants thereof. Further preferred compositions comprise 40-99% of at least one of 70-100% of SEQ ID NOS: 1,2,3,4.5,6, orspecified fragments, domains or variants thereof. Such composition percentages are by weight, volume, concentration, molarity, or molality as liquid or dry solutions, mixtures, suspension, emulsions or colloids, as known in the art or as described herein. Anti-IL-12 annbody compositions of the present invention can further comprise at least one of any suitable and effective amount of a composition or pharmaceutical composition comprising at least one anti-IL-12 antibody to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy, optionally further comprising at least one selected from at least one TNF antagonist (e.g., but not limited to a TNF antibody or fragment, a soluble TNF receptor or fragment, fusion proteins thereof, or a small molecule TNF antagonist), an antirheumatic (e.g., methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), a muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anethetic. a neuromuscular blocker, an antimicrobial (e.g., aminoglycoside, an antifungal, an antiparasitic, an antiviral, a carbapenem, cephalosporin, a flurorquinolone, a macrolide, a penicillin, a sulfonamide, a tetracycline, another antimicrobial), an antipsoriatic, a corticosteriod, an anabolic steroid, a diabetes related agent, a mineral, a nutritional, a thyroid agent, a vitamin, a calcium related hormone, an antidiarrheal, an antitussive, an antiemetic, an antiulcer, a laxative, an anticoagulant, an erythropieitin (e.g., epoetin alpha), a filgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), an immunization, an immunoglobulin, an immunosuppressive (e.g., basilixirriab, cyclosporine, daclizumab), a growth hormone, a hormone replacement drug, an estrogen receptor modulator, a mydriatic, a cycloplegic, an alkylating agent, an antimetabolite, a mitotic inhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, an antipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an asthma medication, a beta agonist, an inhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn, an epinephrine or analog, dornase alpha (Pulmozyme), a cytokine or a cytokine antagonist. Non-limiting examples of such cytokines include, but are not limted to, any of IL-1 to IL-23. Suitable dosages are well known in the art. See, e.g., Wells et al.„eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, CT (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda. CA (2000), each of which references are entirely incorporated herein by reference. Such anti-cancer or anti-infectives can also include toxin molecules that are associated, bound, co-formulated or co-administered with at least one antibody of the present invention.- The toxin can optionally act to selectively kill the pathologic cell or tissue. The pathologic cell can be a cancer or other cell. Such toxins can be, but are not limited to, purified or recombinant toxin or toxin fragment comprising at least one functional cytotoxic domain of toxin, e.g., selected from at least one of ricin. diphtheria toxin, a venom toxin, or a bacterial toxin. The term toxin also includes both endotoxins and exotoxins produced by any naturally occurring, mutant or recombinant bacteria or S'iruses which may cause any pathological condition in humans and other mammals, including toxin shock, which can result in death. Such toxins may include, but are not limited, to, enterotoxigenic E. coli heat-labile enterotoxin (LT), heat-stable enterotoxin (ST), Shigella cytotoxin, Aeromonas enterotoxins, toxic shock syndrome toxin-1 (TSST-1), Staphylococcal enterotoxin A (SEA), B (SEB), or C (SEC), Streptococcal enterotoxins and the like. Such bacteria include, but are not limited to, strains of a species of enterotoxigenic E. coli (ETEC), emerohemorrhagic E. coli (e.g., strains of serotype 0157:H7), Staphylococcus species (e.g., Staphylococcus aureus. Staphylococcus pyogenes), Shigella species (e.g., Shigella dysenteriae. Shigella flexneri, Shigella boydii, and Shigella sonnei), Salmonella species (e.g., Salmonella typhi. Salmonella cholera-suis. Salmonella enteritidis), Clostridium species (e.g., Clostridium perfringens, Clostridium dificile, Clostridium botulinum), Camphlobacter species (e.g., Camphlobacter jejuni, Camphlobacter fetus), Heliobacter species, (e.g., Heliobacter pylori), Aeromonas species (eg., Aeromonas sobria, Aeromonas hydrophila, Aeromonas caviae), Pleisomonas shigelloides, Yersina enterocolitica, Vibrios species (e.g., Vibrios cholerae. Vibrios parahemolyticus), Klebsiella species, Pseudomonas aeruginosa, and Streptococci. See, e.g., Stein, ed., INTERNAL MEDICINE, 3rd ed., pp 1-13, Little, Brown and Co., Boston, (1990); Evans etal, eds., Bacterial Infections of Humans: Epidemiology and Control, 2d. Ed.', pp 239-254, Plenum Medical Book Co., New York (1'991); Mandell et al, Principles and Practice of Infectious Diseases, 3d. Ed., Churchill Livingstone, New York (1990); Berkow et al, eds., The Merck Manual, 16th edition, Merck and Co., Rahway, N.J., 1992; Wood et al, FEMS Microbiology Immunology, 76:121-134 (1991s); Marracket al, Science, 248:705-711 (1990), the contents of which references are incorporated entirely herein by reference. Anti-IL-12 antibody compounds, compositions or combinations of the present invention can further comprise at least one of any suitable auxiliary, such as, but not: limited to, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like. Pharmaceutically acceptable auxiliaries are preferred. Non-limiting examples of, and methods of preparing such sterile solutions arc well known in the art. such as, but limited to, Gennaro. Ed., Remington 's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, PA) 1990. Pharmaceutically acceptable carriers can be routinely selected that are suitable for the mode of administration, solubility and/or stability of the anti-IL-12 antibody, fragment or variant composition as well known in the art or as described herein. Pharmaceutical excipients and additives useful in the present composition include but are not limited to proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid/antibody components, which1 can also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine,,phenylalanine, aspartame, and the like. One preferred amino acid is glycine. Carbohydrate excipients suitable for use in the invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, .starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol,,xylitol sorbitol {glucitol), myoinositol and the like. Preferred carbohydrate excipients for use in the present invention are mannitol, trehalose, and raffinose. Anti-IL-12 antibody compositions can also include a buffer or a pH adjusting agent; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers. Preferred buffers for use in the present compositions are organic acid salts such as citrate. Additionally, anti-IL-12 antibody compositions of the invention can include polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins. such as 2-hydroxypropyl-p-cyclodextrin), polyethylene glycols, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "TWEEN 20" and 'TWEEN 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA). These and additional known pharmaceutical excipients and/or additives suitable for use in the anti-IL-12 antibody, portion or variant compositions according to the invention are known in the art, e.g., as listed in "Remington: The Science & Practice of Pharmacy", 19* ed., Williams & Williams, (1995), and in the "Physician's Desk Reference", 52nd ed., Medical Economics, Montvale, NJ (1998), the disclosures of which are entirely incorporated herein by reference. Preferrred carrier or excipient materials are carbohydrates (e.g., saccharides and alditols) and buffers (e.g., citrate).or polymeric agents. Formulations As noted above, the invention provides for stable formulations, which is preferably a phosphate buffer with saline or a chosen salt, as well as preserved solutions and formulations containing a preservative as well as multi-use preserved formulations suitable for pharmaceutical or veterinary use,,comprising at least one anti-IL-12 antibody in a pharmaceutically acceptable formulation. Preserved formulations contain at least one known preservative or optionally selected from the group consisting of at least one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkom'um chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof in an aqueous diluent Any suitable concentration or mixture can be used as known in the art, such as 0.001-5%, or any range or value therein, such as, but not limited to 0.001, 0.003,0.005,0.009,0.01, 0.02, 0.03,0.05, 0.09,0.1, 0.2,0.3, 0.4., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0,1.1,1.2,1.3,1.4,1.5, pt.6, 1.7, 1.8, 1.9.2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3,4.5, 4.6,4.7, 4.8,4.9, or any range or value therein. Non-limiting examples include, no preservative, 0.1-2% m-cresol (e.g., 0.2, 0.3. 0.4, 0.5,0.9, 1.0%), 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1., 1.5, 1.9, 2.0, 2.5%),; 0.001-0.5% thimerosal (e.g., 0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05,0.075, 0.09, 0.1,0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like. As noted above, the invention provides an article of manufacture, comprising packaging material and at least one vial comprising a solution of at least one anti-IL-12 antibody with the prescribed buffers and/or preservatives, optionally in an aqueous diluent, wherein said packaging material comprises a label that indicates that such solution can be held over a period of 1. 2, 3, 4. 5. 6. 9, 12. 18. 20. 24, 30, 36. 40, 48, 54, 60. 66, 72 hours or greater. The invention further comprises an article of manufacture, comprising packaging material, a first viai comprising lyophilized at least one anti-IL-12 antibody, and a second viai comprising an aqueous diluent of prescribed buffer or preservative, wherein said packaging material comprises a label that instructs a patient to reconstitute the at least one anti-IL-12 antibody in the aqueous diluent to form a solution that can be held over a period of twenty-four hours or greater. The at least one anti-IL-12antibody used in accordance with the present invention can be produced by recombinant means, including from mammalian cell or transgenic preparations, or can be purified from other biological sources, as described herein or as known in the art. The range of at least one anti-IL-12 antibody in the product of the present invention includes amounts yielding upon reconstitution, if in a wet/dry system, concentrations from about 1.0 ug/ml to about 1000 mg/nil, although lower and higher concentrations are operable and are dependent on the intended delivery vehicle, e.g., solution formulations will differ from transdermal patch, pulmonary, transmucosal, or osmotic or micro pump methods. Preferably, the aqueous diluent optionally further comprises a pharmaceutically acceptable preservative. Preferred preservatives include those selected from the group consisting of phenol, m-cresoi, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof The concentration of preservative used in the formulation is a concentration sufficient to yield an anti-microbial effect. Such concentrations are dependent on the preservative selected and are readily determined by the skilled artisan. Other excipients, e.g. isotonicity agents; buffers, antioxidants, preservative enhancers, can be optionally and preferably added to the diluent. An isotonicity agent, such as glycerin, is commonly used at known concentrations. A physiologically tolerated buffer is preferably added to provide improved pH control. The formulations can cover a wide range of pHs, such as from about pH 4 to about pH 10, and preferred ranges from about pH 5 to about pH 9, and a most preferred range of about 6.0 to about 8.0. Preferably the formulations of the present invention have pH between about 6.8 and about 7.8. Preferred buffers include phosphate buffers, most preferably sodium phosphate, particularly phosphate buffered saline (PBS). Other additives, such as a pharmaceutically acceptable solubilizers like Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block copolymers), and PEG (polyethylene glycol) or non-ionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or 188, Pluronic® polyls, other block co-polymers, and chelators such as EDTA and EGTA can optionally be added to the formulations or compositions to reduce aggregation. These additives are particularly useful if a pump or plastic container is used to administer the formulation. The presence of pharmaceutically acceptable surfactant mitigates the propensity for the protein to aggregate. The formulations of the present invention can be prepared by a process which comprises mixing at least one anti-IL-12 antibody and a preservative selected from the group consisting of phenol, m-cresol, p-cresbl o-cfesol, chlorpcresol, benzyl alcohol, alkylparaben, (methyl, ethyl, propyl, butyl and the like), benzalkoniurn chloride, benzethonium chloride, sodium dehydroacetate and thimeirosal or mixtures thereof in an aqueous diluent. Mixing the at least one anti-IL-12 antibody and preservative in an aqueous diluent is carried out using conventional dissolution and mixing procedures. To prepare a suitable formulation, for example, a measured amount of at least one anti-IL-12 antibody in buffered solution is combined with the desired preservative in a buffered solution in quantities sufficient to provide the protein and preservative at the desired concentrations. Variations of this process would be recognized by one of ordinary skill in the art. For example, the order the components are added, whether additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that can be optimized for the concentration and means of administration used. The claimed formulations can be provided to patients as clear solutions or as dual vials comprising a vial of lypphilized at least one anti-IL-12 antibody that is reconstituted with a second vial containing water, a preservative and/or excipients, preferably a phosphate buffer and/or saline and a chosen salt, in an aqueous diluent. Either a single solution vial or dual vial requiring reconstitution can be reused multiple times and can suffice for a single or multiple cycles of patient treatment and thus can provide a more conver/xent treatment regimen than currently available. The present claimed articles of manufacture are useful for administration over a period of immediately to twenty-four hours or greater., Accordingly, the presently claimed articles of manufacture offer significant advantages to the patient. Formulations of the invention can optionally be safely stored at temperatures of from about 2 to about 40°C and retain the biologically activity of the protein for extended periods of time, thus, allowing a package label indicating that the solution can be held and/or used over a period of 6, 12,18, 24, 36,48, 72, or 96 hours or greater. If preserved diluent is used, such label can include use up to 1-12 months, one-half, one and a half, and/or two years. The solutions of at least one anti-IL-12 antibody in the invention can be prepared by a process that comprises mixing at least one antibody in an aqueous diluent. Mixing is carried out using conventional dissolution and mixing procedures. To prepare a suitable diluent, for example, a measured amount of at least one antibody in water or buffer is combined in quantities sufficient to provide the protein and optionally a preservative or buffer at the desired concentrations. Variations of this process would be recognized by one of ordinary skill in the art. For example, the order the components are added, whether additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that can be optimized for the concentration and means of administration used. The claimed products can be provided to patients as clear solutions or as dual vials comprising a vial of lyophilized at least one anti-IL-12 antibody that is reconstituted with a second vial containing the aqueous diluent. Either a single solution vial or dual vial requiring reconstitution can be reused multiple times and can suffice for a single or multiple cycles of patient treatment and thus provides a more convenient treatment regimen than currently available. The claimed products can be provided indirectly to patients by providing to pharmacies, clinics, or other such institutions and facilities, clear solutions or dual vials comprising a vial of lyophilized at least one anti-IL-12 antibody that is reconstituted with a second vial containing the aqueous diluent. The clear solution in this case can be up to one liter or even larger in size, providing a large reservoir from which smaller portions of the at least one antibody solution can be retrieved one or multiple times for transfer into smaller vials and provided by the pharmacy or clinic to their customers and/or patients. Recognized devices comprising these single vial systems include those pen- injector devices for delivery of a solution such as BD Pens, BD Autojector®, Humaject®' NovoPen®, B-D®Pen, AutoPen®, and OptiPen®, GenotropinPen®; Genotronorm Pen®, Humatro Pen®, Reco-Pen®, Roferon Pen®, Biojector®, iject®, J-tip Needle-Free Injector®, Intraject®, Medi-Ject®, e.g., as made or developed by Becton Dickensen (Franklin Lakes, NJ, www.bectondickenson.com), Disetronic (Burgdorf, Switzerland, www.disetronic.com; Bioject, Portland, Oregon (www.bioject.com); National Medical Products , Weston Medical (Peterborough, UK, www.weston-medical.com), Medi-Ject Corp (Minneapolis, MN, www.mediject.com). Recognized devices comprising a dual vial system include those pen- injector systems for reconstituting a jyophilized drug in a cartridge for delivery of the reconstituted solution such as the HumatroPen®. The products presently claimed include packaging material. The packaging material provides, in addition to the information required by the regulatory agencies, the conditions under which the product can be used. The packaging material of the present invention provides instructions to the patient to reconstitute the at least one anti-IL-12 antibody in the aqueous diluent to form a solution and to use the solution over a period of 2-24 hours or greater for the two viai, wet/dry, product. For the single vial, solution product, the label indicates that such solution can be used over a period of 2-24 hours or greater. The presently claimed products are useful for human pharmaceutical product use. The formulations of the present invention can be prepared by a process that comprises mixing at least one anti-IL-12 antibody and a selected buffer, preferably a phosphate buffer containing saline or a chosen salt. Mixing the at least one antibody and buffer in an aqueous diluent is carried out using conventional dissolution and mixing procedures. To prepare a suitable formulation, for example, a measured amount of at least one antibody in water or buffer is combined with the desired buffering agent in water in quantities sufficient to provide the protein and buffer at the desired concentrations. Variations of this process would be recognized by one of ordinary skill in the art. For example, the order the components are added, whether additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that can be optimized for the concentration and means of administration used. The claimed stable or preserved formulations can be provided to patients as clear solutions or as dual vials comprising a vial of lyophilized at least one anti-IL-12 antibody that is reconstituted with a second viai containing a preservative or buffer and excipients in an aqueous diluent. Either a single solution vial or dual vial requiring reconstitution can be' reused multiple times and can suffice for a single or multiple cycles of patient treatment and thus provides a more convenient treatment regimen than currently available. At least one anti-IL-12 antibody in either the stable or preserved formulations or solutions described herein, can be administered to a patient in accordance with the present invention via a variety of delivery methods including SC or IM injection; transdermal, pulmonary, transmucosal, implant, osmotic pump, cartridge, micro pump, or other means appreciated by the skilled artisan, as well-known in the art. Therapeutic Applications The present invention also provides a method for modulating or treating at least one immune related disease, in a cell, tissue, organ, animal, or patient including, but not limited to, at least one of rheumatoid arthritis, juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, psoriatic arthritis, ankylosing spondilitis, gastric ulcer, seronegative arthropathies, osteoarthritis, inflammatory bowel disease, ulcerative colitis, systemic lupus erythematosis, antiphospholipid syndrome, iridocyclitis/uveitis/optic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis/wegener's granulomatosis, sarcoidosis, orchitis/vasectomy reversal procedures, allergic/atopic diseases, asthma, allergic rhinitis, eczema, allergic contact dermatitis, allergic conjunctivitis, hypersensitivity pneumonitis, transplants, organ transplant rejection, graft-versus-host disease, systemic inflammatory response syndrome, sepsis syndrome, gram positive sepsis, gram negative sepsis, culture negative sepsis, fungal sepsis, neutropenic fever, urosepsis, meningococcemia. trauma/hemorrhage, bums, ionizing radiation exposure, acute pancreatitis, adult respiratory distress syndrome, rheumatoid arthritis, alcohol-induced hepatitis, chronic inflammatory pathologies, sarcoidosis. Crohn's pathology, sickle cell anemia, diabetes, nephrosis, atopic diseases, hypersensitity reactions, allergic rhinitis, hay fever, perennial rhinitis, conjunctivitis, endometriosis, asthma, urticaria, systemic anaphalaxis, dermatitis, pernicious anemia, hemolytic disesease, thrombocytopenia, graft rejection of any organ or tissue, kidney translplant rejection, heart transplant rejection, liver transplant rejection, pancreas transplant rejection, lung transplant rejection, bone marrow transplant (BMT) rejection, skin allograft rejection, cartilage transplant rejection, bone graft rejection, small bowel transplant rejection, fetal thymus implant rejection, parathyroid transplant rejection, xenograft rejection of any organ or tissue, allograft rejection, anti-receptor hypersensitivity reactions, Graves disease, Raynoud's disease, type B insulin-resistant diabetes, asthma, myasthenia gravis, antibody- meditated cytotoxicity, type HI hypersensitivity reactions, systemic lupus erythematosus, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes syndrome), polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin changes syndrome, antiphospholipid syndrome, pemphigus, scleroderma, mixed connective tissue disease, idiopathic Addison's disease, diabetes mellitus, chronic active hepatitis, primary billiary cirrhosis, vitiligo, vasculitis, post-MI cardiotomy syndrome, type IV hypersensitivity , contact dermatitis, hypersensitivity pneumonitis, allograft rejection, granulomas due to intracellular organisms, drug sensitivity, metabolic/idiopatfuc, Wilson's disease, hemachromatosis, alpha-1-antitrypsin deficiency, diabetic retinopathy, hashimoto's thyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axis evaluation, primary biliary cirrhosis, thyroiditis, encephalomyelitis, cachexia, cystic fibrosis, neonatal chronic lung disease, chronic obstructive pulmonary disease (COPD), familial hematophagocytic lymphohistiocytosis, dermatologic conditions, psoriasis, alopecia, nephrotic syndrome, nephritis, glomerular nephritis, acute renal failure, hemodialysis, uremia, toxicity, preeclampsia, okt3 therapy, anti-cd3 therapy, cytokine therapy, chemotherapy, radiation therapy (e.g., including but not limited toasthenia, anemia, cachexia, and the like), chronic salicylate intoxication, and the like. See, e.g., the Merck Manual, 12th-17th Editions, Merck & Company, Rahway, NJ (1972, 1977, 1982, 1987, 1992, 1999), Pharmacotherapy Handbook, Wells et al., eds., Second Edition, Appleton and Lange, Stamford, Conn. (1998, 2000), each entirely incorporated by reference. The present invention also provides a method for modulating or treating at least one cardiovascular disease in a cell, tissue, organ, animal, or patient, including, but not limited to, MISSING AT THE TIME OF PUBLICATION MISSING AT THE TIME OF PUBLICATION concurrently, and/or after, at least one selected from at least one TNF antagonist (e.g., but not limited to a TNF antibody or fragment, a soluble TNF receptor or fragment, fusion proteins thereof, or a small molecule TNF antagonist), an antirheumatic (e.g., methotrexate, auranofm, aurothioglucose, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide. sulfasalzine). a muscle relaxant, a narcotic, a non-steroid anti- inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anethetic, a neuromuscular blocker, an antimicrobial (e.g., aminoglycoside, an antifungal, an antiparasitic, an antiviral, a carbapenem, cephalosporin, a flurorquinolone, a macrolide, a penicillin, a sulfonamide, a tetracycline, another antimicrobial), an antipsoriatic, a corticosteriod, an anabolic steroid, a diabetes related agent, a mineral, a nutritional, a thyroid agent, a vitamin, a calcium related hormone, an antidiarrheal. an antitussive, an antiemetic, an antiulcer, a laxative, an anticoagulant, an erythropieitin (e.g., epoetin alpha), a filgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), an immunization, an immunoglobulin, an immunosuppressive (e.g., basiliximab, cyclpsporine, daclizumab), a growth hormone, a hormone replacement drug, an estrogen receptor modulator, a mydriatic, a cycloplegic, an alkylating agent, an antimetabolite, a mitotic inhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, an antipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an asthma medication, a beta agonist, an inhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn, an epinephrine or analog, domase alpha (Pulmozyme), a cytokine or a cytokine antagonistm. Suitable dosages are well known in the art. See, e.g., Wells et al., eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, CT (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, CA (2000), each of which references are entirely incorporated herein by reference. TNF antagonists suitable for compositions, combination therapy; co-administration, devices and/or methods of the present invention (further comprising at least one anti body, specified portion and variant thereof, of the present invention), include, but are not limited to, anti-TNF antibodies, antigen-binding fragments thereof, and receptor molecules which bind specifically to TNF; compounds which prevent and/or inhibit TNF synthesis, TNF release or its action on target cells, such as thalidomide, tenidap, phosphodiesterase inhibitors (e.g, pentoxifylline and rolipram), A2b adenosine receptor agonists and A2b adenosine receptor enhancers; compounds which prevent and/or inhibit TNF receptor signalling, such as mitogen activated protein (MAP) kinase inhibitors; compounds which block and/or inhibit membrane TNF cleavage, such as metalloproteinase inhibitors; compounds which block and/or inhibit TNF activity, such as angiotensin converting enzyme (ACE) inhibitors (e.g., captopril); and compounds which block and/or inhibit TNF production and/or synthesis, such as MAP kinase inhibitors. As used herein, a "tumor necrosis factor antibody," "TNF antibody," "TNF antibody," or fragment and the like decreases, blocks, inhibits, abrogates or interferes with TNF activity in vitro, in situ and/or preferably in vivo. For example, a suitable TNF human antibody of the present invention can bind TNFa and includes anti-TNF antibodies, antigen- binding fragments thereof, and specified mutants or domains thereof that bind specifically to TNFa. A suitable TNF anttibody or fragment can also decrease block, abrogate, interfere, prevent and/or inhibit TNF RNA, DNA or protein synthesis, TNF release, TNF receptor signaling, membrane TNF cleavage, TNF activity, TNF production and/or synthesis. Chimeric antibody cA2 consists of the antigen binding variable region of the high- affinity neutralizing mouse anti-human TNFa IgGl antibody, designated A2, and the constant . regions of a human IgGI, kappa immunoglobulin. The human IgGl Fc region improves allogeneic antibody effector function, increases the circulating serum half-life and decreases the immunogenicity of the antibody. The avidity and epitope specificity of the chimeric antibody cA2 is derived from the variable region of the murine antibody A2. In a particular embodiment, a preferred source for nucleic acids encoding the variable region of the murine antibody A2 is the A2 hybridoma cell line. Chimeric A2 (cA2) neutralizes the cytotoxic effect of both natural and recombinant human TNFa in a dose dependent manner. From binding assays of chimeric antibody cA2 and recombinant human TNFa, the affinity constant of chimieric antibody cA2 was calculated to be 1.04x1010M-1. Preferred methods for determining monoclonal antibody specificity and affinity by competitive inhibition can be found! in Harlow, et al., antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988; Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, New York, (1992-2000); Kozbor et al, Immunol. Today. 4:12-19 (1983); Ausubel etal, eds. Current Protocols in Molecular Biology, Wiley Interscience, New York (1987-2000); and Muller, Meth. Enzymol., 92:589-601 (1983), which references are entirely incorporated herein by reference. In a particular embodiment, murine monoclonal antibody A2 is produced by a cell line designated c134A. Chimeric antibody cA2 is produced by a cell line designated cl68A. Additional examples of monoclonal anti-TNF antibodies that can be used in the present invention are described in the art (see, e.g., U.S. Patent No. 5,231,024; Moller, A. et al., Cytokine 2(3):162-169 (1990); U.S. Application No. 07/943,852 (filed September 11, 1992); Rathjen et al, Intemational!Publicatioh No. WO 91/02078 (published February 21, 1991); Rubin et al, EPO Patent Publication No. 0 218 868 (published April 22, 1987); Yone et al., EPO Patent Publication No. 0 288 088 (October 26. 1988); Liang, et al., Biochem. Biophys. Res. Comm. 137:847-854 (1986); Meager, et al., Hybridoma 6:305-311 (1987); Fendly et al., Hybridoma 6:359-369 (19S7); Bringman, etal., Hybridoma 6:489-507 (1987); and Hirai, et al., J. Immunol. Meth. 96:57-62 (1987), which references are entirely incorporated herein by reference). TNF Receptor Molecules Preferred TNF receptor molecules useful in the present invention are those that bind TNF with high affinity (see, e.g., Feldmann et at., International Publication No. WO 92/07076 (published April 30, 1992); Schall et al., Celt 67:361-370 (1990); and Loetscher et al., Cell 67:351-359 (1990), which references are entirely incorporated herein by reference) and optionally possess low immunogenicity. In particular, the 55 kDa (p55 TNF-R) and the 75 kDa (p75 TNF-R) TNF cell surface-receptors are useful in the present invention. Truncated forms of these receptors, comprising the extracellular domains (ECD) of the receptors or functional portions thereof (see, e.g., Corcoran et at., Eur. J. Biochem. 223:831-840 (1994)), are also useful in the present invention. Truncated forms of the TNF receptors, comprising the ECD, have been detected in urine and serum as 30 kDa and 40 kDa TNF inhibitory binding proteins (Engelmann, H. etal., J. Biol. Chem. 265:1531-1536(1990)). TNF receptor multimeric molecules and TNF immunoreceptor fusion molecules, and derivatives and fragments or portions thereof, are additional examples of TNF receptor molecules which are useful in the methods and compositions of the present invention. The TNF receptor molecules which can be used in the invention are characterized by their ability to treat patients for extended periods with good to excellent alleviation of symptoms and low toxicity. Low immunogenicity and/or high affinity, as well as other undefined properties, can contribute to the therapeutic results achieved. TNF receptor multimeric molecules useful in the present invention comprise all or a functional portion of the ECD of two or more TNF receptors linked via one or more polypeptide linkers or other nonpeptide linkers, such as polyethylene glycol (PEG). The multimeric molecules can further comprise a signal peptide of a secreted protein to direct expression of the multimeric molecule. These multimeric molecules and methods for their production have been described in U.S. Application No. 08/437,533 (filed May 9, 1995), the content of which is entirely incorporated herein by reference. TNF immunoreceptor fusion molecules useful in the methods and compositions of the present invention comprise at least one portion of one or more immunoglobulin molecules and all or a functional portion of one or more TNF receptors. These immunoreceptor fusion molecules can be assembled as monomers, or hetero- or homo-multimers. The immunoreceptor fusion molecules can also be monovalent or multivalent. An example of such a TNF immunoreceptor fusion molecule is TNF receptor/IgG fusion protein. TNF immunoreceptor fusion molecules and methods for their production have been described in the an (Lesslauer et al.. Eur. J. Immunol. 27:2883-2886 (199.1); Ashkenazi et al., Proc. Natl. Acad. Sci. USA 55:10535-10539 (1991); Peppel etal.,J. Exp. Med. 774:1483-1489 (1991); Kolls et al., Proc. Natl. Acad. Sci. USA 97:215-219 (1994); Butler et al.. Cytokine 6(6):616- 623 (1994); Baker et al., Eur. J. Immunol. 24:2040-2048 (1994); Beutler et al., U.S. Patent No. 5,447,851; and U.S. Application No,08/442,133 (filed May 16, 1995), each of which references are entirely incorporated herein by reference). Methods for producing immunoreceptor fusion molecules can also be found in Capon et al., U.S. Patent No. 5,116,964; Capon et al., U.S. Patent No, 5,225,538; and Capon et al., Nature 337:525-531 (1989), which references are entirely incorporated herein by reference. A functional equivalent, derivative, fragment or region of TNF receptor molecule refers to the portion of the TNF receptor molecule, or the portion of the TNF receptor molecule sequence which encodes TNF receptor molecule, that is of sufficient size and sequences to functionally resemble TNF receptor molecules that can be used in the present invention (e.g., bind TNF with high affinity and possess low immunogenicity). A functional equivalent of TNF receptor molecule also includes,modified TNF receptor molecules that functionally resemble TNF receptor molecules thatcan be used in the present invention (e.g., bind TNF with high affinity and possess low immunogenicity). For example, a functional equivalent of TNF receptor molecule can contain a"SILENT" codon or one or more amino acid substitutions, deletions or additions (e.g., substitution of one acidic amino acid for another acidic amino acid; or substitution of one codon encoding the same or different hydrophobic amino acid for another codon encoding a hydrophobic amino acid). See Ausubel et al.. Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience, New York (1987-2000). Cytokines include any known cytokine. See, e.g., www.CopewithCytokines.com. Cytokine antagonists include, but are not limited to, any antibody, fragment or mimetic, any soluble receptor, fragment or mimetic, any small molecule antagonist, or any combination thereof. Therapeutic Treatments. Any method of the present invention can comprise a method for treating a IL-12 mediated disorder, comprising administering an effective amount of a composition or pharmaceutical composition comprising at least one anti-IL-12 antibody to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy. Such a method can optionally further comprise co-administration or combination therapy for treating such immune diseases, wherein the administering of said at least one anti-IL-12 antibody, specified portion or variant thereof, further comprises administering, before concurrently, and/or after, at least one selected from at least one of at least one selected from at least one TNF antagonist (e.g., but not limited to a TNF antibody or fragment, a soluble TNF receptor or fragment, fusion proteins thereof, or a small molecule TNF antagonist), an antirheumatic (e.g., methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), a muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anethetic, a neuromuscular blocker, an antimicrobial (e.g., aminoglycoside, an antifungal, an antiparasitic, an antiviral, a carbapenem, cephalosporin, a flurorquinolone, a macrolide, a penicillin, a sulfonamide, a tetracycline, another antimicrobial), an antipsoriatic, a corticosteriod, an anabolic steroid, a diabetes related agent, a mineral, a nutritional, a thyroid agent, a vitamin, a calcium related hormone, an antidiarr heal, an antitussive, an antiemetic, an antiulcer, a laxative, an anticoagulant, an erythropieitin (e.g., epoetin alpha), a filgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), an immunization, an immunoglobulin, an immunosuppressive (e.g., basiliximab, cyclosporine, daclizumab), a growth hormone, a hormone replacement drug, an estrogen receptor modulator, a mydriatic, a cycloplegic, an alkylating agent, an antimetabolite, a mitotic inhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, an antipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an asthma medication, a beta agonist, an inhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn, an epinephrine or analog, domase alpha (Pulmozyme), a cytokine or a cytokine antagonist Typically, treatment of pathologic conditions is effected by administering an effective amount or dosage of at least one anti-IL-12 antibody composition that total, on average, a range from at least about 0.01 to 500 milligrams of at least one anti-IL- 12antibody per kilogram of patient per dose, and preferably from at least about 0.1 to 100 milligrams antibody /kilogram of patient per single or multiple administration, depending upon the specific activity of contained in the composition. Alternatively, the effective serum concentration can comprise 0.1-5000 ug/ml serum concentration per single or multiple adminstration. Suitable dosages are known to medical practitioners and will, of course, depend upon the particular disease state, specific activity of the composition being administered, and the particular patient undergoing treatment. In sortie instances, to achieve the desired therapeutic amount, it can be necessary to provide for repeated administration, i.e., repeated individual administrations of a particular monitored or metered dose, where the individual administrations are repeated until the desired daily dose or effect is achieved. Preferred doses can optionally include 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8,0.9,1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19,20, 21, 22,23, 24, 25, 26,27, 28,29,30,31,32, 33, 34, 35, 36, 37, 38, 39. 40, 41, 42, 43, 44, 45. 46. 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 62. 63, 64, 65. 66, 67, 68, 6.9, 70, 71, 72, 73, 74, 75, 76. 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89. 90,91, 92, 93, 94, 95, 96, 97, 98, 99 and/or 100-500 mg/kg/administration, or any range, value or fraction thereof, or to achieve a serum concentration of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9, 2.0. 2.5, 2.9. 3.0, 3.5, 3.9, 4.0,4.5, 4.9, 5.0. 5.5. 5.9. 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9,9.0,9.5.9.9, 10. 10.5, 10.9, 11, 11.5, 11.9.20. 12.5. 12.9! 13.0, 13.5, 13.9, 14.0, 14.5,4.9, 5.0, 5.5., 5.9. 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 12, 12.5, 12.9. 13.0, 13.5. 13.9, 14, 14.5, 15, 15.5, 15.9. 16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9, 19, 19.5, 19.9, 20, 20.5,20.9, 21,22,23, 24, 25,26, 27, 28, 29, 30, 35,40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 96, 100,200,300,400, 500, 600,700, 800, 900, 1000,1500, 2000, 2500, 3000, 3500,4000,4500, and/or 5000,ug/ml serum concentration per single or multiple administration, or any range, value or fraction thereof. Alternatively, the dosage: administered can vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; age, health, and weight of the recipient;; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired. Usually a dosage of active ingredient can be about 0.1 to LOO milligrams per kilogram of body weight. Ordinarily 0.1 to 50, and preferably 0.1 to 10 milligrams per kilogram per administration or in sustained release form is effective to obtain desired results. As a non-limiting example, treatment of humans or animals can be provided as a one- time or periodic dosage of at least one antibody of the present invention 0.1 to 100 mg/kg, such as 0.5,0.9,1.0, l.l, 1.5, 2, 3,4, 5, 6, 7, 8,9, 10, 11,12, 13, 14, 15,16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26,27, 28, 29, 30,40,45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17,18, 19, 20, 21, 22,23, 24,25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively or additionally, at least one of week 1,2, 3, 4, 5, 6, 7, 8, 9, 10,11,12, 13, 14,15, 16,17, 18, 19, 20,21, 22, 23,24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42,43, 44, 45, 46, 47, 48,49, 50, 51, or 52, or alternatively or additionally, at least one of 1,2, 3, 4, 5, 6„ 7, 8, 9, 10,11, 12,13, 14, 15, 16, 17, 18, 19, or 20 years, or any combination thereof, using single, infusion or repeated doses. Dosage forms (composition) suitable for internal administration generally contain from about 0.1 milligram to about 500 milligrams of active ingredient per unit or container. In these pharmaceutical compositions the active ingredient will ordinarily be present in an amount of about 0.5-99.999% by! weight based on the total weight of the composition. For parenteral administration, the antibody can be formulated as a solution, suspension, emulsion or lyophilized powder in association, or separately provided, widi a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 1-10% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils can also be used. The vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by known or suitable techniques. Suitable pharmaceutical carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field. Alternative Administration Many known and developed modes of can be used according to the present invention for administering pharmaceutically effective amounts of at least one anti-IL-12 antibody according to the present invention. While pulmonary administration is used in the following description, other modes of administration can be used according to the present invention with suitable results. EL-12 antibodies of the present invention can be. delivered in a carrier, as a solution, emulsion, colloid, or suspension, or as a dry powder, using any of a variety of devices and methods suitable for administration by inhalation or other modes described here within or known in the art. Parenteral Formulations and Administration Formulations for parenteral administration can contain as common excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. Aqueous or oily suspensions for injection can be prepared by using an appropriate emulsifier of humidifier and a suspending agent, according to known methods. Agents for injection can be a non-toxic, non-orally administrable diluting agent such as aquous solution or a sterile injectable solution or suspension in a solvent As the usable vehicle or solvent, water, Ringer's solution, isotonic saline, etc. are allowed; as an ordinary solvent, or suspending solvent, sterile involatile oil can be used. For these purposes, any kind of involatile oil and fatty acid can be used, including natural or synthetic or semisynthetic fatty oils or fatty acids; natural or synthetic or semisynthtetic mono- or di- or tri- glycerides. Parental administration is known in the art and includes, but is not limited to, conventional means of injections, a gas pressured needle-less injection device as described in U.S. Pat. No. 5,851,198, and a laser perforator device as described in U.S. Pat. No. 5,839,446 entirely incorporated herein by reference. Alternative Delivery The invention further relates to the administration of at least one anti-IL-12 antibody by parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous. intracavitary, intracelial. intracelebellar, intracerebroventricular. intracolic. intracervical, intragastric, intrahepatic, intramyocardial, intraosteal. intrapelvic. intrapericardiac. intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarcnal. intraretinal, lintraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal means. At least one anti-IL-12 antibody composition can ibe prepared for use for parenteral (subcutaneous, intramuscular or intravenous) or any other administration particularly in the form of liquid solutions or suspensions; for use in vaginal or rectal administration particularly in semisolid forms such as, but not limited to, creams and suppositories: for buccal, or sublingual administration such as, but not limited to, in the form of tablets or capsules; or intranasally such as, but not limited to, the form of powders, nasal drops or aerosols or certain agents; or transdermally such as not limited to a gel, ointment, lotion, suspension or patch delivery system with chemical enhancers such as dimethyl sulfoxide to either modify the skin structure or to increase the drug concentration in the transdermal patch (Junginger, et: al. In "Drug Permeation Enhancement"; Hsieh, D. S., Eds., pp. 59-90 (Marcel Dekker, Inc. New York 1994, entirely incorporated herein by reference), or with oxidizing agents that enable the application of formulations containing proteins and peptides onto the skin (WO 98/53847), or applications of electric fields to create transient transport pathways such as electroporation, or to increase the mobility of charged drugs through the skin such as iontophoresis, or application of ultrasound such as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) (the above publications and patents being entirely incorporated herein by reference). Pulmonary/Nasal Administration For pulmonary administration, preferably at least one anti-IL-12 antibody composition is delivered in a particle size effective for reaching the lower airways of the lung or sinuses. According to the invention, at least one anti-IL-12 antibody can be delivered by any of a variety of inhalation or nasal devices known in the art for administration of a therapeutic agent by inhalation. These devices capable of depositing aerosolized formulations in the sinus cavity or alveoli of a patient include metered dose inhalers, nebulizers, dry powder generators, sprayers, and the like. Other devices suitable for directing the pulmonary or nasal administration of antibodies are also known in the art. All such devices can use of formulations suitable for the administration for the dispensing of antibody in an aerosol. Such aerosols can be comprised of either solutions (both aqueous and non aqueous) or solid particles. Metered dose inhalers like the Ventolin® metered dose inhaler, typically use a propellent gas and require actuation during inspiration (See, e.g., WO 94/16970, WO 98/35888). Dry powder inhalers like Turbuhaler™ (Astra), Rotahaler® (Glaxo), Diskus® (Glaxo), Spiros™ inhaler (Dura), devices marketed by Inhale Therapeutics, and the Spinhaler* powder inhaler (Fisons), use breath-actuation of a mixed powder (US 4668218 Astra, EP 237507 Astra. WO 97/25086 Glaxo. WO 94/08552 Dura. US 5458135 Inhale, WO 94/06498 Fisons. entirely incorporated herein by reference). Nebulizers like AERx™ Aradigm, the Ultravent® nebulizer (Mallinckrodt), and the Acom II® nebulizer (Marquest Medical Products) (US 5404871 Aradigm. WO 97/22376). the above references entirely incorporated herein by reference, produce aerosols from solutions, while metered dose inhalers, dry powder inhalers, etc. generate small particle aerosols. These specific examples of commercially available inhalation devices are intended to be a representative of specific devices suitable for the practice of this invention, and are not intended as limiting the scope of the invention. Preferably, a composition comprising at least one anti-IL-12 antibody is delivered by a dry powder inhaler or a sprayer. There are a several desirable features of an inhalation device for administering at least one antibody of the present invention. For example, delivery by the inhalation device is advantageously reliable, reproducible, and accurate. The inhalation device can optionally deliver small dry particles, e.g. less than about 10 µm, preferably about 1-5 um, for good respirability. Administration of IL-12 antibody Compositions as a Spray A spray including IL-12 antibody composition protein can be produced by forcing a suspension or solution of at least one anti-IL-12 antibody through a nozzle under pressure. The nozzle size and configuration, the applied pressure, and the liquid feed rate can be chosen to achieve the desired output and particle size. An electrospray can be produced, for example, by an electric field in connection with a capillary or nozzle feed. Advantageously, particles of at least one anti-IL-12 antibody composition protein delivered by a sprayer have a particle size less than about 10 urn, preferably in the range of about 1 urn to about 5 µm, and most preferably about 2 urn to about 3 µm. Formulations of at least one anti-IL-12 antibody composition protein suitable for use with a sprayer typically include antibody composition protein in an aqueous solution at a concentration of about 0.1 mg to about 100 mg of at least one anti-IL-12 antibody composition protein per ml of solution or mg/gm, or any range or value therein, e.g., but not lrnited to, .1, .2., .3, .4, .5, .6, .7, .8, .9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/ml or mg/gm. The formulation can include agents such as an excipient, a, buffer, an isotonicity agent, a preservative, a surfactant, and, preferably, zinc. The formulation can also include an excipient or agent for stabilization of the antibody composition protein, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulk proteins useful in formulating antibody composition proteins include albumin, protamine, or the like. Typical carbohydrates useful in formulating antibody composition proteins include sucrose, mannitol, lactose, trehalose, glucose, or the like. The antibody composition protein formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation of the antibody composition protein caused by atomization of the solution in forming an aerosol. Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range between 0.001 and 14% by weight of the formulation. Especially preferred surfactants for purposes of this invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, or the like. Additional agents known in the art for formulation of a protein such as IL-12 antibodies, or specified portions or variants, can also be included in the formulation. Administration of IL-12 antibody compositions by a Nebulizer antibody composition protein can be administered by a nebulizer, such as jet nebulizer or an ultrasonic nebulizer. Typically, in a jet nebulizer, a compressed air source is used to create a high-velocity air jet through an orifice. As the gas expands beyond the nozzle, a low- pressure region is created, which draws a solution of antibody composition protein through a capillary tube connected to a liquid reservoir. The liquidstream from the capillary tube is sheared into unstable filaments and droplets as it exits the tube, creating the aerosol. A range of configurations, flow rates, and baffle types can be employed to achieve the desired performance characteristics from a given jet nebulizer. In an ultrasonic nebulizer, high- frequency electrical energy is used to create vibrational, mechanical energy,typically employing a piezoelectric transducer. This energy is transmitted to the formulation of antibody composition protein either directly or through a coupling fluid, creating an aerosol including the antibody composition protein. Advantageously, particles of antibody composition protein delivered by a nebulizer have a particle size less than about 10 µm, preferably in the range of about 1 µm to about 5 µm, and.most preferably about 2 µm to about 3 µm. Formulations of at least one anti-IL-12 antibody suitable for use with a nebulizer, either jet or ultrasonic, typically include a concentration of about 0.1 mg to about 100 mg of at least one anti-IL-12 antibody protein per ml of solution. The formulation can include agents such as an excipient, a buffer, an isptonicity agent, a preservative, a surfactant, and, preferably, zinc. The formulation can also include an excipient or agent for stabilization of the at least one anti-IL-12 antibody composition protein, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulk proteins useful in formulating at least one anti-IL-12 antibody composition proteins include albumin, protamine, or the like. Typical carbohydrates useful in formulating at least one anti-IL-12 antibody include sucrose, mannitol, lactose, trehalose, glucose, or the like. The at least one anti-IL-12 antibody formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation of the at least one anti-IL- 12 antibody caused by atomization of the solution in forming an aerosol. Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbital fatty acid esters. Amounts will generally range between 0.001 and 4% by weight of the formulation. Especially preferred surfactants for purposes of this invention are polyoxyethylene sorbitan mono-oleate, polysorbate 80. polysorbate 20, or the like. Additional agents known in the art for formulation of a protein such as antibody protein can also be included in the formulation. Administration of IL-12 antibody compositions By A Metered Dose Inhaler In a metered dose inhaler (MDI), a propellant, at least one anti-IL-12 antibody, and any excipients or other additives are contained in a canister as a mixture including a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol, preferably containing particles in the size range of less than about 10 µm, preferably about 1 µm to about 5 µm, and most preferably about 2 µm to about 3 µm. The desired aerosol particle size can be obtained by employing a formulation of antibody composition protein produced by various methods known to those of skill in the art, including jet-milling, spray drying, critical point condensation, or the like. Preferred metered dose inhalers include those manufactured by 3M or Glaxo and employing a hydrofluorocarbon propellant. Formulations of at least one anti-IL-12 antibody for use with a metered-dose inhaler device will generally include a finely divided powder containing at least one anti-IL-12 antibody as a suspension in a non-aqueous medium, for example, suspended in a propellant with the aid of a surfactant. The propellant can be any conventional material employed for this purpose, such as chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromeuiane, dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a(hyoVofluroalkane-l34a), HFA-227 (hydrofluroalkane-227), or the like. Preferably the propellant is a hydrofluorocarbon. The surfactant can be chosen to stabilize the at least one anti-IL-12 antibody as a suspension in the propellant, to protect the active agent against chemical degradation, and the like. Suitable surfactants include sorbitan trioleate, soya lecithin, oleic acid, or the like. In some cases solution aerosols are preferred using solvents such as ethanol. Additional agents known in the art for formulation of a protein such as protein can also be included in the formulation. One of ordinary skill in the art will recognize that the methods of the current invention can be achieved by pulmonary administration of at least one anti-IL-12 antibody compositions via devices not described herein. Oral Formulations and Administration Formulations for oral rely on the co-administration of adjuvants (e.g.. resorcinols and nonionic surfactants such as polyoxyethylenc oleyl ether land n-hexadecylpolyethyiene ether) to increase artificially the permeability of the intestinal walls, as well as the co-administration of enzymatic inhibitors (e.g., pancreatic trypsin inhibitors, diisopropylfluorophosphate (DFF) and trasylol) to inhibit enzymatic degradation. The active constituent compound of the solid- type dosage form for oral administration can be mixed with at least one additive, including sucrose, lactose, cellulose, mannitol. trehalose, raffinose, maltitol, dextran, starches, agar, arginates, chitins. chitosans. pectins, gum tragacanth. gum arabic, gelatin, collagen, casein, albumin, synthetic or semisynthetic polymer, and glyceride. These dosage forms can also contain other type(s) of additives, e.g.. inactive diluting agent, lubricant such as magnesium stearate, paraben, preserving agent such as sorbic acid, ascorbic acid, .alpha.-tocopherol, antioxidant such as cysteine, disintegrator, binder, thickener, buffering agent, sweetening agent, flavoring agent, perfuming agent, etc. Tablets and pills can be further processed into enteric-coated preparations. The liquid preparations for oral administration include emulsion, syrup, elixir, suspension and solution preparations allowable for medical use. These preparations can contain inactive diluting agents ordinarily used in said field, e.g., water. Liposomes have also been described as drug delivery systems for insulin and heparin (U.S. Pat. No. 4,239,754). More recently, microspheres of artificial polymers of mixed amino acids (proteinoids) have been used to deliver pharmaceuticals (U.S. Pat. No. 4,925,673). Furthermore, carrier compounds described in U.S. Pat. No. 5,879,681 and U.S. Pat. No. 5,5,871,753 are used to deliver biologically active agents orally are known in the art. Mucosal Formulations and Administration For absorption through mucosal surfaces, compositions and methods of administering at least one anti-EL-12 antibody include an emulsion comprising a plurality of submicron particles, a mucoadhesive macromolecule, a bioactive peptide, and an aqueous continuous phase, which promotes absorption through mucosal surfaces by achieving mucoadhesion of the emulsion particles (U.S. Pat. Nos. 5,514,670). Mucous surfaces suitable for application of the emulsions of the present invention can include corneal, conjunctival, buccal, sublingual, nasaL vaginal, pulmonary, stomachic, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration, e.g. suppositories, can contain as excipients, for example, polyalkyleneglycols, vaseline, cocoa butter, and the like. Formulations for intranasal administration can be solid and contain as excipients, for example, lactose or can be aqueous or oily solutions of nasal drops. For buccal administration excipients include sugars, calcium stearate, magnesium stearate, pregelinatined starch, and the like (U.S. Pat. Nos. 5,849,695). Transdermal Formulations and Administration For transdermal administration, the at least one anti-IL-12 antibody is encapsulated in a delivery device such as a liposome or polymeric nanoparticles, microparticle, microcapsule, or microspheres (referred to collectively as microparticles unless otherwise stated). A number of suitable devices are known, including microparticles made of synthetic polymers such as polyhydroxy acids such as polylactic acid, polyglycolic acid and copolymers thereof, polyorthoesters. polyanhydrides, and polyphosphazenes. andnatural polymers such as collagen, polyamino acids, albumin and other proteins, alginate and other polysaccharides, and combinations thereof (U.S. Pat. Nos. 5,814,599). Prolonged Administration and Formulations It can be sometimes desirable to deliver the compounds of the present invention to the subject over prolonged periods of time, for example, for periods of one week to one year from a single administration. Various slow release, depot or implant dosage forms can be utilized. For example, a dosage form can contain a pharmaceutically acceptable non-toxic salt of the compounds that has a low degree of solubility in body fluids, for example, (a) an acid addition salt with a polybasic acid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or di-sulfonic acids, polygalacturonic acid, and the like; (b) a salt with a polyvalent metal cation such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and the like, or with an organic cation formed from e.g.,N,N'-dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of (a) and (b) e.g. a zinc tannate salt. Additionally, the ' compounds of the present invention or, preferably, a relatively insoluble salt such as those just described, can be formulated in a gel, for example, an aluminum monostearate gel with, e.g. sesame oil, suitable for injection. Particularly preferred salts are zinc salts, zinc tannate salts, pamoate salts, and the like. Another type of slow release depot formulation for injection would contain the compound or salt dispersed for encapsulated in a slow degrading, non-toxic, non- antigenic polymer such as a polylactic acid/polyglycolic acid polymer for example as described in U.S. Pat. No. 3,773,919. The compounds or, preferably, relatively insoluble salts such as those described above can also be formulated in cholesterol matrix silastic pellets, particularly for use in animals. Additional slow release, depot or implant formulations, e.g. gas or liquid liposomes are known in the literature (U.S. Pat. Nos. 5,770,222 and "Sustained and Controlled Release Drug Delivery Systems", J. R. Robinson ed., Marcel Dekker, Inc., N.Y., 1978). Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided byway of illustration and are not intended as limiting. MISSING AT THE TIME OF PUBLICATION The vector pC4 is used for the expression of IL-12 antibody. Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146). The plasmid contains the mouse DHFR gene under control of the S V40 early promoter. Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (e.g., alpha minus MEM. Life Technologies, Gaithersburg, MD) supplemented with the. chemotherapeutic agent methotrexate. The amplification of the DHFR genes in cells resistant to methotrexate (MTX) has been well documented (see, e.g., F. W. Alt, etal., J. Biol. Chem. 253:1357-1370 (1978); J. L. Hamlin and C. Ma, Biochem. et Biophys. Acta 1097:107-143 (1990); and M. J. Page and ML A. Sydenham, Biotechnology 9:64-68 (1991)). Cells grown in increasing concentrations of MTX develop resistance to the drug by overproducing the target enzyme, DHFR, as a result of amplification of the DHFR gene. If a second gene is linked to the DHFR gene, it is usually co-amplified and over-expressed. It is known in the art that this approach can be used to develop cell lines carrying more than 1,000 copies of the amplified gene(s). Subsequently, when the methotrexate is withdrawn, cell lines are obtained that contain the amplified gene integrated into one or more chromosome(s) ofthe host cell. Plasmid pC4 contains for expressing the gene of interest the strong promoter ofthe long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985)) plus a fragment isolated .from the enhancer ofthe immediate early gene of human cytomegalovirus (CMV) (Boshart, etal., Cell 41:521-530 (1985)). • Downstream ofthe promoter are BamHI, Xbal, and Asp718 restriction enzyme cleavage sites that allow integration of the genes. Behind these cloning sites the plasmid contains the 3' intron and polyadenylation site of the rat preproinsulin gene. Other high efficiency promoters can also be used for the expression, e.g., the human b-actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HTV and HTLVI". Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express the IL-12 in a regulated way in mammalian cells (M. Gossen, and H. Bujard, Proc. Natl. Acad. Sci. USA 89: 5547-5551 (1992)). For the polyadenylation of the mRNA other signals, e.g., from the human growth hormone or globin genes can be used as well. Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methotrexate. The plasmid pC4 is digested with restriction enzymes and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The vector is then isolated from a 1% agarose gel. The DNA sequence encoding the complete IL-12 antibody is used, e.g., as presented in SEQ ID NOS:INSERT MAB AA SEQ ID I, and INSERT MAB AA SEQ ID N02, corresponding to HCand LC variable regions, of a.IL-12 antibody of the present invention, according to known method steps. Isolated nucleic acid encoding a suitable human constant region (i.e., HC and LC regions) is also used in this construct (e.g.. as provided in vector p 1351: INSERT ATCC ACCESSION NUMBER AND ADDITIONAL HC/LCplasmids) The isolated variable and constant region encoding DNA and the dephosphorylated vector are then ligated with T4 DNAligase. E. coli HB101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC4 using, for instance, restriction enzyme analysis. Chinese hamster ovary (CHO) cells lacking an active DHFR gene are used for transfection. 5 g of the expression plasmid pC4 is cotrahsfected with 0.5 g of the plasmid pSV2-neo using lipofectin. The plasmid pSV2neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 g /ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 g/mlG418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrationso'f methotrexate are then transferred to new 6-well plates containing even higher concentrations/of methotrexate (1 mM, 2 mM, 5 mM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained that grow at a concentration of 100 - 200 mM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reverse phase HPLC analysis. Example 2: Generation of High Affinity Human IgG Monoclonal Antibodies Reactive With Human IL-12 Using Transgenic Mice Summary Transgenic mice have been used that contain human heavy and light chain immunoglobulin genes to generate high affinity, completely human, monoclonal antibodies that can be used therapeutically to inhibit the action of IL-12 for the treatment of one or more EL-12-mediated disease. (CBA/J x C57/BL6/J) F2 hybrid mice containing human variable and constant region antibody transgenes for both heavy and light chains are immunized with human recombinant IL-12 (Taylor et al., Intl. Immunol. 6:5J79-591 (1993); Lonberg, el: al., Nature 368:856-859 (1994); Neuberger, M., Nature Biotech. 14:826(1996); Fishwild, et al., Nature Biotechnology 14:845-851 (1996)). Several fusions yielded one or more panels of completely human IL-12 reactive IgG monoclonal antibodies. The completely human anti-EL- 12 antibodies are further characterized. All are IgGl . Such antibodies are found to have affinity constants somewhere between lxl09and 9xl012. The unexpectedly high affinities of these fully human monoclonal antibodies make them suitable candidates for therapeutic applications in IL-12 related diseases, pathologies or disorders. Abbreviations BSA - bovine serum albumin C02 - carbon dioxide DMSO - dimethyl sulfoxide EIA - enzyme immunoassay FBS - fetal bovine serum H202 - hydrogen peroxide HRP - horseradish peroxidaseID - interadermal Ig - immunoglobulin IL-12 - interleukin-12 DP - intraperitoneal IV - intravenous Mab - monoclonal antibody OD - optical density OPD - o-Phenylenediamine dihydrochloride PEG - polyethylene glycol PSA - penicillin, streptomycin, amphotericin RT - room temperature SQ - subcutaneous v/v - volume per volume w/v - weight per volume Materials and Methods Animals Transgenic mice that can express human antibodies are known in the art (and are commecially available (e.g., from GenPharm International, San Jose, CA; Abgenix, Freemont, CA, and others) that express human immunoglobulins but not mouse IgM or Ig . For example, such transgenic mice contain human sequence transgenes that undergo V(D)Jjoining, heavy-chain class switching, and somatic mutation to generate a repertoire of human sequence immunoglobulins (Lonberg, et al., Nature 368:856-859 (1994)). The light chain transgenecan be derived, e.g., in part from a yeast artificial chromosome clone that includes nearly half of the germline human V region. In addition, the heavy-chain transgene can encode both human u and human 1 (Fishwild, et al., Nature Biotechnology 14:845-851 (1996)) and/or 3 constant regions. Mice derived from appropriate genotopic lineages can be used in the immunization and fusion processes to generate fully human monoclonal antibodies to EL-12. Immunization One or more immunization schedules can be used to generate the anti-IL-12 human hybridomas. The first several fusions can be performed after the following exemplary immunization protocol, but other similar known protocols can be used. Several 14-20 week old female and/or surgically castrated transgenic male mice are immunized IP and/or ID with 1-1000 µg of recombinant human IL-12 emulsified with an equal volume of TITERMAX or complete Freund's adjuvant in a final volume of 100-400µL (e.g., 200). Each mouse can also optionally receive 1 -10 jo.g in 100 pX physiological saline at each of 2 SQ sites. The mice can then be immunized 1-7,5-12, 10-18, 17-25 and/or 21-34 days later IP (1-400 ug) and SQ (1- 400 ug x 2) with IL-12 emulsified with an equal volume of TITERMAX or incomplete Freund's adjuvant. Mice can be bled 12-25 and 25-40 days later by retro-orbital puncture without anti-coagulant. The blood is then allowed to clot at RT for one hour and the serum is collected and titered using an IL-12 EIA assay according to known methods. Fusions are performed when repeated injections do not cause titers to lincrease. At that time, the mice can be given a final IV booster injection of 1-400 ug IL-12 diluted in 100 p.L physiological saline. Three days later, the mice can be euthanized by cervical dislocation and the spleens removed aseptically and immersed in 10 mL of cold phosphate buffered saline (PBS) containing 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B (PSA). The splenocytes are harvested by sterilely perfusing the spleen with PSA-PBS. The cells are washed once in cold PSA-PBS, counted using Trypan blue dye exclusion and resuspended in RPMI1640 media containing 25 mM Hepes. j Cell Fusion Fusion can be carried out at a 1:1 to 1:10 ratio of murine myeloma cells to viable spleen cells according to known methods, e.g., as known in the art As a non-limiting example, spleen cells and myeloma cells can be pelleted together. The pellet can then be slowly resuspended, over 30 seconds, in 1 mL of 50% (w/v) PEG/PBS Solution (PEG molecular weight 1.450, Sigma) at 37 C. The fusion can then be stopped by slowly adding 10.5 mL of RPMI 1640 medium containing 25 mM Hepes (37 C) over 1 minute. The fused cells are centrifuged for 5 minutes at 500-1500 rpm. The cells are then resuspended in HAT medium (RPMI 1640 medium containing 25 mM Hepes, 10% Fetal Clone I serum (Hyclone), 1 mM sodium pyruvate, 4 mM L-glutamine, 10 ug/mL gentamicin, 2.5% Origen culturing supplement (Fisher), 10% 653-conditioned RPMI 1640/Hepes media, 50 uM 2-mercaptoethanol. 100 uM hypoxanthine, 0.4 uM aminppterin, and 16 uM thymidine) and then plated at 200 u.L/well in fifteen 96-well flat bottom tissue culture plates. The plates are then placed in a humidified 37 C incubator containing 5% C02 and 95% air for 7-10 days. Detection of Human IgG Anti-IL-12 Antibodies in Mouse Serum Solid phase EIA's can be used to screen mouse sera for human IgG antibodies specific for human IL-I2. Briefly, plates can be coated with IL-12 at 2 µg/mL in PBS overnight. After washing in 0.15M saline containing 0.02% (v/v) Tween 20, the wells can be blocked with 1% (w/v) BSA in PBS. 200 uL/well for 1 hour at RT. Plates are used immediately or frozen at -20 C for future use. Mouse serum dilutions are incubated on the IL-12 coated plates at 50 uL/well at RT for 1 hour. The plates are washed and then probed with 50 uL/well HRP-labeled goat anti-human IgG, Fc specific diluted 1:30,000 in 1% BSA-PBS for i hour at RT. The plates can again be washed and 100 uL/well of the citrate-phosphate substrate solution (0.1M citric acid and 0.2M sodium phosphate, 0.01% H2O2 and 1 mg/mL OPD) is added for 15 minutes at RT. Stop solution (4N sulfuric acid) is then added at 25 µL/well and the OD's are read at 490 nm via an automated plate spectrophotometer. Detection of Completely Human Immunoglobulins in Hybridoma Supernates Growth positive hybridomas secreting fully human immunoglobulins can be detected using a suitable EIA. Briefly, 96 well pop-out plates (VWR, 610744) can be coated with 10 µg/mL goat anti-human IgG Fc in sodium carbonate buffer overnight at 4 C. The plates are washed and blocked with 1% BSA-PBS for one hour at 37°C and used immediately or frozen at -20 C. Undiluted hybridoma supernatants are incubated on the plates for one hour at 37°C. The plates are washed and probed with HRP labeled goat anti-human kappa diluted 1:10,000 in 1% BSA-PBS for one hour at 37°C. The plates are then incubated with substrate solution as described above. Determination of Fully Human Anti-IL-12 Reactivity Hybridomas, as above, can be simultaneously assayed for reactivity to EL-12 using a suitable RIA or other assay. For example, supernatants are incubated on goat anti-human IgG Fc plates as above, washed and then probed with radiolabeled IL-12 with appropriate counts per well for 1 hour at RT. The wells are washed twice with PBS and bound radiolabled EL-12 is quantitated using a suitable counter. Human IgGl anti-IL-12 secreting hybridomas can be expanded in cell culture and serially subcloned by limiting dilution. The resulting clonal populations can be expanded and cryopreserved in freezing medium (95% FBS, 5% DMSO) and stored in liquid nitrogen. Isotyping Isotype determination of the antibodies can be accomplished using an EIA in a format similar to that used to screen the mouse immune sera for specific titers. IL-12 can be coated on 96- well plates as described above and purified antibody at 2 µg/mL can be incubated on the plate for one hour at RT. The plate is washed and probed with HRP labeled goat anti-human IgG, or HRP labeled goat anti-human IgG3 diluted at 1:4000 in 1% BSA-PBS for one hour at RT. The plate is again washed and incubated, with substrate solution as described above. Binding Kinetics of Human Anti-Human IL-12 Antibodies With Human IL-12 Binding characteristics for antibodies can be suitably assessed using an IL-12 capture EIA and BIAcore technology, for example. Graded concentrations of purified human IL-12 antibodies can be assessed for binding to EIA plates coated with 2 µg. mL of IL-12 in assays as described above. The OD's can be then presented as semi-log plots showing relative binding efficiencies. Quantitative binding constants can be obtained, e.g., as follows, or by any other known suitable method. A BIAcore CM-5 (carboxymethyl) chip is placed in a BIAcore 2000 unit HBS buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v P20 surfactant, pH 7.4) is flowed over a flow cell of the chip at 5 µL/minute until a stable baseline is obtained. A solution (100 pX) of 15 mg of EDG (N-ethyl-N'-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride) in 200 µL water is added to 100 µL of a solution of 2.3 mg of NHS (N-hydroxysuccinimide) in 200 µL water. Forty (40) µL;of the resulting solution is injected onto the chip. Six µL of a solution of human IL-12 (15 µg/mL in 10 mM sodium acetate, pH 4.8) is injected onto the chip, resulting in an increase of ca. 500 RU. The buffer is changed to TBS/Ca/Mg/BSA running buffer (20 mM Tris, 0.15 M sodium chloride. 2 mM calcium chloride, 2 mM magnesium acetate, 0.5% Triton X-100,25 µg/mL BSA, pH 7.4) and flowed over the chip overnight to equilibrate it and to hydrolyze or cap any unreacted succinimide esters. Antibodies are dissolved in the running buffer at 33.33,16.67, 8.33, and 4.17 nM. The flow rate is adjusted to 30 µL/min and the instrument temperature to 25 C. Two flow cells are used for the kinetic runs, one on which IL-12 had been immobilized (sample) and a second, underivatized flow cell (blank). 120 µL of each antibody concentratiorris injected over the flow cells at 30 µL/min (association phase) followed by an uninterrupted 360 seconds of buffer flow (dissociation phase). The surface of the chip is regenerated (interleukin-12 /antibody complex dissociated) by two sequential injections of 30 µL each of 2 M guanidine thiocyanate. Analysis of the data is done using BIA evaluation 3.0 or CLAMP 2.0, as known in the art For each antibody concentration the blank sensogram is subtracted from the sample sensogram. A global fit is done for both dissociation (kd. sec-1) and association (k2, mol-1 sec-1) and the dissociation constant (KD, mol) calculated (kd/k2). Where the antibody affinity is high enough that the RUs of antibody captured are >100, additional dilutions of the antibody are run. Results and Discussion Generation of Anti-Human IL-12 Monoclonal Antibodies Several fusions are performed and each fusion is seeded in 15 plates (1440 wells/fusion) that yield several dozen antibodies specific for human IL-12. Of these, some are found to consist of a combination of human and mouse Ig chains. The remaining hybridomas secret anti-IL-12 antibodies consisting solely of human heavy and light chains. Of the human hybridomas all are expected to be IgGI. Binding Kinetics of Human Anti-Human IL-12 Antibodies ELISA analysis confirms that purified antibody from most or all of these hybridomas bind IL-12 in a concentration-dependent manner. Figures 1-2 show the results of the relative binding efficiency of these antibodies. In this case, the avidity of the antibody for its cognate antigen (epitope) is measured. It should be noted that binding IL-12 directly to the EI A plate can cause denaturation of the protein and the apparent binding affinities cannot be reflective of binding to undenatured protein. Fifty percent binding is found over a range of concentrations. Quantitative binding constants are obtained using BIAcore analysis of the human antibodies and reveals that several of the human monoclonal antibodies are very high affinity with KD in the range of 1x10-9 to 7xl0-12. Conclusions Several fusions are performed utilizing splenocytes from hybrid mice containing human variable and constant region antibody transgenes that are immunized with human EL- 12. A set of several completely human IL-12 reactive IgG monoclonal antibodies of the IgGI isotype are generated. The completely human anti-IL-12 antibodies are further characterized. Several of generated antibodies have affinity constants between lxl 09 and 9xl012. The unexpectedly high affinities of these fully human monoclonal antibodies make them suitable for therapeutic applications in IL-12-dependent diseases, pathologies or related conditions. Example 3: C340 is a Neutralizing Human Monoclonal Antibody The bioactivity of IL-12 was shown to be neutralized by C340 in a variety of IL-12 dependent activity assays. Since IL-12 enhances IFN GAMMA production by NK cells and T lymphocytes, the effect of C340 antibody on the upregulation of IFN GAMMA mRNA and the effect of C340 on the production of IFN GAMMA protein was examined (Trinchieri, G., Current Opinion in Immunology, 9:17-23 (1997), Morris, S.C., et al., Journal of Immunology, 152:1047-1056 (1994)). The ability of C340 to neutralize IL-12 driven induction of lymphokine activated killer (LAK) cell activity was also investigated in these studies (Kutza, J. and Murasko, D.M., Mechanisms of Ageing and Development, 90:209-222 (1996), Stem, A.S., et al., Proceedings of the National Academy of Sciences of the U.S.A., 87:6808-6812 (1990)). Lastly, the effect of C340 on IL-12-mediated upregulation of CD95 cell surface expression on T and NK cells was tested (Medvedev, A.E., et ai.. Cytokine. 9:394- 404(1997)). Inhibition of IFN gamma mRNA Transcnption To determine whether C340 inhibits IL-12. IL-2 induced IFN GAMMA gene transcription in human PBL. a reverse transcription-PCR assay was performed. Specific primers for b-actin (a control for mRNA integrity and content) and IFN GAMMA were used to amplify the cDNA obtained from stimulated human PBL. Figure 3 shows C340 down regulates IFN GAMMA mRNA in IL-12/IL-2 activated (2 hour) PBMC. Inhibition of Intracellular IFN GAMMA as Measured by Flow Cytometry In response to various signals and as a measure of activation, T cells and NK cells can be induced to secrete cytokines. More specifically, PBL treated with IL-2 and IL-12 initiate substantial synthesis of IFN gamma within 4-8 hours after stimulation. This production can be detected in the cytoplasm of Brefeldin-A treated PBL by flow cytometry. Figure 4 demonstrates a 60% reduction in IFN GAMMA production in such cultures when C340 IL-12 was added in conjunction with IL-12 for five hours. Inhibition of IL-12 induced ,IFN GAMMA Secretion Figure 5 clearly shows that two different lots of C340 inhibited the secretion of EFN GAMMA by peripheral blood lymphocytes in a dose-dependent fashion. Four hundred picograms of IL-12 were premixed with varying amounts of C340 and then added to IL-2 stimulated cultures of PBL's. When IFN GAMMA was measured by EIA after an 18-24 hour incubation, markedly diminished amounts of EFN GAMMA were detected with as little as 1 ?g/mL of C340 antibody. Inhibition of IL-12 Induced LAK Cell Cytotoxicity Raji cells, an IL-12 sensitive Burkitt lymphoma derived cell line; is an NK cell resistant. LAK cell sensitive cell line. Raji cells, in triplicate, were cultured for four hours with LAK cells which had been activated with 400 pg/mL IL-12 and 10 U/mL IL-2 in the presence or absence of the human monoclonal antibody C340 (5000 ng/mL or 50 ng/mL). Figure 6 shows the results from three normal, healthy donors. IL-12 + IL-2 activation of effector cells resulted in an increasing cytotoxic activity over that of cells activated with IL-2 alone. The C340 antibody inhibited this IL-12 dependent effect. The magnitude of inhibition was related to antibody concentration, with the highest concentration tested reducing cytotoxicity to background levels. Inhibition of CD95 Upregulation Reports have described IL-12-induced upregulation. of CD95 on the surface of highly purified CD56+ PBL. As can be seen in Figure 7A and 7B, distributional flow cytometric analysis revealed that CD95 expression was significantly upregulated on CD3+ T cells and CD56+ NIC cells after treatment with IL-12 plus fL-2 for 72 hours. Concomitant anti-IL-12 treatment inhibited CD95 expression in both CD3+ and CD56+ populations. CD3+ cells were inhibited by -50% (Figure 7A). whereas CD56+ cells were inhibited by -85% (Figure 7B), as evidenced by a diminished MFI index (percent greater then unstimulated control). Example 4: Gene cloning and characterization Genomic DNA fragments containing either the C340 heavy chain gene or the C340 light chain were cloned and purified. Genomic DNA purified from C340 hybridoma cells was partially digested with Sau3A restriction enzyme and size-selected by centrifugal fractionation through a 10-40% sucrose gradient. DNA fragments in the size range of 15-23 kb were cloned into the bacteriophage vector, EMBL3, [commercially available ?] and packaged into phage particles. Several packaging reactions resulted in a library of 1 million bacteriophage clones. Approximately 600,000 clones from the library were screened by plaque hybridization using 32P-labeled genomic DNA fragments that contained either human IgGl heavy chain constant region sequences or human kappa light chain constant region sequences as probe. Thirteen heavy chain and nine light chain clones were detected. Of these, three heavy chain clones and four light chain clones were purified by two more rounds of screening. One of the heavy chain clones and two of the light chain clones were shown to contain the 5' and 3' ends of the coding sequences by PCR analysis of bacteriophage DNA. The DNA insert in heavy chain (HC) clone H4 was 16 kb in size and includes 3.6 kb of 5' flanking and at least 2 kb of 3' flanking sequence. The DNA insert in light chain (LC) clone LCI was 15 kb in size and included 4.4 kb of 5' flanking and 6.0 kb of 3' flanking sequence. The complete inserts were removed from the bacteriophage vector as Sall fragments and cloned between the Xhol and Sall sites of plasmid expression vector pl351, which provided a gpt selectable marker gene. Because there was an internal Sall site in the heavy chain variable region coding sequence, two Sail fragments had to be transferred from bacteriophage H4 to the p1351 expression vector. The resulting heavy and light chain expression plasmids were1 termed p1560 and pl558, respectively. The orientations of the heavy and light chain genes in these two plasmids relative to the p1351 vector sequences were determined using restriction enzyme analysis and PCR, respectively. In both cases, the orientations were such that the 5' end of the Ab gene fragment was proximal to the 3' end of the gpt gene. Both strands of the coding regions of the cloned genes were sequenced. The sequences of plasmids pl560 and pl558 are presented in Figures 11A-11K and Figures 13A-13J, respectively. Example 5: Preparation of recombinant cell lines Heavy chain plasmid pl560 was linearized by digestion with Pvul restriction enzyme and light chain plasmid p1558 was linearized using Sall restriction enzyme. p3X63Ag8.653 (653) and SP2/0-Agl4 (SP2/0) cells were separately transfected with the premixed linearized plasmids by electroporation and cells cultured and transfectants selected using mycophenolic acid as described (Knight, et aL Molecular Immunology 30:1443 (1993)). Cell supernatants From mycophenolic acid-resistant colonies were assayed approximately two weeks later for human IgG (i.e., recombinant C340 (rC340)). For this, cell supernatants were incubated on 96-well ELISA plates that were coated with goat antibodies specific for the Fc portion of human IgG. Human IgG which bound to the coated plate was detected using alkaline phosphatase-conjugated goat anti-human IgG (heavy chain + light chain) antibody and alkaline phosphatase substrates as described (Knight, et al., Molecular Immunology 30:1443 (1993)). Cells of the higher producing clones were transferred to 24-well culture dishes in standard media and expanded (IMDM, 5% FBS, 2 mM glutamine, mycophenolic acid selection mix). The amount of antibody produced (i.e., secreated into the media of spent cultures) was carefully quantified by ELISA using purified C340 mAb as the standard. Selected clones were then expanded in T75 flasks and the production of human IgG by these clones was quantified by ELISA. Based on these values, six independent 653 transfectants and three independent SP2/0 transfectants were subcloned (by seeding an average of one cell per well in 96 well plates), the quantity of antibody produced by the subclones was determined by assaying (ELISA) supernatants from individual subclone colonies. Three subclones, '653 transfectant 19-20 (C379B) and the SP2/0 transfectants 84-81 (C381A) and 22-56 (C389A), were selected for further analysis. Assay for rC340 antigen binding. Prior to subcloning selected cell lines as described above, cell supernatants from three parental lines (653 transfectants clone 2 and clone 18 and SP2/0 transfectant clone 1) were used to test the antigen binding characteristics of rC340. The concentrations of rC340 in the three cell supernatant samples were first determined by ELISA. Titrating amounts of the supernatant samples, or purified C340 positive control, were then incubated in 96-well plates coated with 2 µg/ml of human IL-12. Bound mAb was then detected with alkaline phosphatase-conjugated goat anti-human IgG (heavy chain + light chain) antibody and the appropriate alkaline phosphatase substrates. As shown in Figure 8, rC340 bound specifically to human IL-12 in a manner indistinguishable from the original C340 mAb. Characterization of selected cell lines. Growth curve analyses were performed on C379B, C381A, andC3S9A by seeding T75 flasks with a starting cell density of 2 X 105 cells/ml in standard media or SFM-5 serum-free media and then monitoring cell number and rC340 concentration on a daily basis until the cultures were spent. The results of cultures in standard media are shown in Figures 9A - 9C. Maximal C340 mAb production levels for C379B. C381A, and C389A were 135 µg/ml, 150 4g/ml, and 110 µg/ml, respectively. Attempts to adapt C379B cells to SFM-5 media were not successful. C381A cells produced the same amount of rC340 in SFM-5 media as in standard media, whereas C389A cells produced only half as much rC340 in SFM-5 media as in standard media. The stability of rC340 mAb production over time for the three subclones was assessed by culturing cells in 24-well dishes with standard media or standard media without nycophenolic acid selection for varying periods of time. Lines C379B and C381A were »bserved to stably produce rC340, in the presence or absence of selection for a period of 30 lays (the maximum time tested) and 75 days, respectively. Line C389A was unstable and after 13 days of culture produced just 20% as much antibody as at the beginning of the study. It will be clear that the invention can be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims. We Claim: 1. An isolated mammalian anti-lL-12 antibody, comprising at least one of (i) all of the heavy chain complementarity determining regions (CDR) amino acid sequences of SEQ ID NOS: 1,2, and 3; (ii) all of the light chain CDR amino acids sequences of SEQ ID NOS: 4, 5 and 6 and (iii) at least one variable region comprising SEQ ID NO:7 or 8. 2. The anti-IL-12 antibody as claimed in claim 1, wherein said antibody binds IL-12 with an affinity of at least one selected from at least 10-9 M, at least 10-10 M, at least 10-11 M, or at least 1012 M. 3. The anti-IL-12 antibody as claimed in claim 1, wherein said antibody substantially neutralizes at least one activity of at least one IL-12 protein. 4. An isolated nucleic acid molecule encoding at least one isolated mammalian anti-IL-12 antibody having at least one variable region comprising SEQ ID NO:7 or 8. 5. An isolated nucleic acid vector comprising the isolated nucleic acid molecule as claimed in claim 4. 6. A method for producing an anti-IL-12 antibody, comprising translating the nucleic acid molecule as claimed in claim 4 under conditions in vitro, such that the IL-12 antibody is expressed in detectable or recoverable amounts. 7. An anti-idiotype antibody or fragment that specifically binds an isolated mammalian anti-IL-12 antibody having at least one variable region comprising SEQ ID NO:7 or 8. 8. A medical device, comprising the isolated mammalian anti-IL-12 antibody having at least, one variable region comprising SEQ ID NO:7 or 8, wherein said device is suitable to contacting or administering said anti-IL-12 antibody by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraostel, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal. 9. A method for producing an isolated mammalian anti-IL-12 antibody having at least one variable region comprising SEQ ID NO:7 or 8, comprising providing a host cell or transgenic non- human animal or transgenic plant or plant cell capable of expressing in recoverable amounts said antibody. 10. An anti-IL-12 antibody produced by the method as claimed in claim 9. 11. The anti-IL-12 antibody as claimed in claim 1, wherein said antibody binds IL-12 with an affinity of at least one selected from at least 10-9-M, at least 10-10 M, at least 10-11 M, or at least 10-12M. 12. The anti-IL-12 antibody as claimed in claim 13, wherein said antibody substantially neutralizes at least one activity of at least one IL-12 protein. 13. As isolated nucleic acid molecule encoding an isolated mammalian anti-IL-12 antibody having at least one of (i) all of the heavy chain CDR amino acid sequences of SEQ ID NOS: 1,2, and 3; (ii) all of the light chain CDR amino acids sequences of SEQ ID NOS: 4, 5 and 6. 14. An isolated nucleic acid vector comprising the isolated nucleic acid molecule as claimed in claim 13. 15. A method for producing an anti-lL-12 antibody, comprising translating the nucleic acid molecule as claimed in claim 16 under conditions in vitro, such that the anti-lL-12 antibody is expressed in detectable or recoverable amounts. 16. An anti-idiotype antibody or fragment that specifically binds an isolated mammalian anti-lL-12 antibody having at least one of (i) all of the heavy chain CDR amino acid sequences of SEQ ID NOS: 1,2, and 3; and (ii) all of the light chain CDR amino acids sequences of SEQ ID NOS: 4, 5 and 6. 17. A medical device, comprising an isolated mammalian anti-IL-12 antibody having at least one of (i) all of the heavy chain CDR amino acid sequences of SEQ) ID NOS: 1, 2 and 3; and (ii) all of the light chain CDR amino acids sequences of SEQ ID NOS: 4, 5, and 6, wherein said device is suitable to contacting or administering said anti-IL-12 antibody by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal. 18. A method for producing an isolated mammalian anti-IL-12 antibody having at least one of (i) all of the heavy chain CDR amino acid sequences of SEQ ID NOS: 1, 2, and 3; and (ii) all of the light chain CDR amino acids sequences of SEQ ID NOS: 4, 5, and 6, comprising providing a host cell or transgenic non-human animal or transgenic plant or plant cell capable of expressing in recoverable amounts said antibody. 19. An anti-IL-12 antibody produced by the method as claimed in claim 18. 20. An isolated mammalian anti-IL-12 antibody, comprising at least one heavy chain or light chain CDR having the amino acid sequence of at least one of SEQ ID NOS: 1, 2, 3, 4, 5 and 6. 21. The anti-IL-12 antibody as claimed in claim 20, wherein said antibody binds IL-12 with an affinity of at least one selected from at least 10-9 M, at least 10-10 M, at least 10-11 M, or at least 10-12 M. 22. The anti-IL-12 antibody as claimed in claim 20, wherein said antibody substantially neutralizes at least one activity of at least one IL-12 protein. 23. An isolated nucleic acid molecule encoding at least one isolated mammalian anti-IL-12 antibody having at least one heavy chain or light chain CDR having the amino acid sequence of at least one of SEQ ID NOS: 1, 2, 3, 4, 5 and 6. 24. An isolated nucleic acid vector comprising the isolated nucleic acid molecule as claimed in claim 23. 25. A method for producing an anti-IL-12 antibody, comprising translating the nucleic acid molecule as claimed in claim 23 under conditions in vitro, such that the anti-IL-12 antibody is expressed in detectable or recoverable amounts. 26. An anti-idiotype antibody or fragment that specifically binds an isolated mammalian anti-IL-12 antibody having at least one heavy chain or light chain CDR having the amino acid sequence of at least one of SEQ ID NOS: 1, 2, 3, 4, 5 and 6. 27. A medical device, comprising an isolated mammalian anti-IL-12 antibody having at least one heavy chain or light chain CDR having the amino acid sequence of at least one of SEQ ID NOS: 1, 2, 3, 4, 5 and 6, wherein said device is suitable to contacting or administering said at least one anti-IL-12 antibody by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal. 28. A method for producing an isolated mammalian anti-IL-12 antibody having at least one heavy chain or light chain CDR having the amino acid sequence of at least one of SEQ ID NOS; 1, 2, 3, 4, 5 and 6, comprising providing a host cell or transgenic non- human animal or transgenic plant or plant cell capable of expressing in recoverable amounts said antibody. 29. An anti-IL-12 antibody produced by the method as claimed in claim 28. 30. An isolated mammalian anti-IL-12 antibody that binds to the same region of a IL-12 protein as an antibody comprising at least one heavy chain or light chain CDR having the amino acid sequence of at least one of SEQ ID NOS: 1, 2, 3, 4, 5, and 6. 31. The anti-IL-12 antibody as claimed in claim 30, wherein said antibody binds IL-12 with an affinity of at least one selected from at least 10"9 M, at least 10-10 M, at least 10-11 M, or at least 10-12 M. 32. The anti-IL-12 antibody as claimed in claim 30, wherein said antibody substantially neutralizes at least one activity of at least one IL-12 protein. 33. An isolated nucleic acid molecule encoding an isolated mammalian anti-IL-12 antibody that binds to the same region of an IL-12 protein as an antibody comprising at least one heavy chain or light chain CDR having the amino acid sequence of at least one of SEQ ID NOS: 1,2, 3,4, 5, and 6. 34. An isolated nucleic acid vector comprising the isolated nucleic acid molecule as claimed in claim 33. 35. A method for producing at. least one anti-IL-12 antibody, comprising translating the nucleic acid molecule as claimed in claim 33 under conditions in vitro, such that the anti-IL-12 antibody is expressed in detectable or recoverable amounts. 36. An anti-idiotype antibody or fragment that specifically binds an isolated mammalian anti-IL-12 antibody that binds to the same region of an IL-12 protein as an antibody comprising at least one heavy chain or light chain CDR having the amino acid sequence of at least one of SEQ ID NOS; 1, 2, 3, 4, 5 and 6. 37. A medical device, comprising an isolated mammalian anti-IL-12 antibody that binds to the same region of an IL-12 protein as an antibody comprising at least one heavy chain or light chain CDR having the amino acid sequence of at least one of SEQ ID NOS: 1, 2, 3, 4, 5, 6, wherein said device is suitable for contacting or administering said anti-IL-12 antibody by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal. 38. A method for producing an isolated mammalian anti-IL-12 antibody that binds to the same region of an IL-12 protein as an antibody comprising at least one heavy chain or light chain CDR having the amino acid sequence of at least one of SEQ ID NOS: 1, 2, 3, 4, 5, and 6, comprising a host cell or transgenic non-human animal or transgenic plant or plant cell capable of expressing in recoverable amounts said antibody. 39. An anti-IL-12 antibody produced by the method as claimed in claim 38. 40. An isolated mammalian anti-IL-12 antibody, comprising a heavy chain variable region amino acid sequence of SEQ ID NO: 7 and a light chain variable region amino acid sequence of SEQ ID NO: 8. 41. The anti-IL-12 antibody as claimed in claim 40, wherein said antibody binds IL-12 with an affinity of at least one selected from at least 10-9 M, at least 10-10 M, at least 10-11M, or at least 10-12 M. 42. The anti-IL-12 antibody as claimed in claim 40, wherein said antibody substantially neutralizes at least one activity of at least one IL-12 protein. 43. An isolated nucleic acid molecule encoding at least one isolated mammalian anti-IL-12 antibody comprising a heavy chain variable region amino acid sequence of SEQ ID NO: 7 and a light chain variable region amino acid sequence of SEQ ID NO: 8. 44. An isolated nucleic acid vector comprising the isolated nucleic acid molecule as claimed in claim 43. 45. A method for producing an anti-IL-12 antibody, comprising translating the nucleic acid molecule as claimed in claim 44 under conditions in vitro, such that the 1L-12 antibody is expressed in detectable or recoverable amounts. 46. An anti-idiotype antibody or fragment that specifically binds an isolated mammalian anti-IL-12 antibody comprising a heavy chain variable region amino acid sequence of SEQ ID NO: 7 and a light chain variable region amino acid sequence of SEQ ID NO: 8. 47. A medical device, comprising an isolated mammalian anti-IL-12 antibody comprising a heavy chain variable region amino acid sequence of SEQ ID NO:7 and a light chain variable region amino acid sequence of SEQ ID NO: 8, wherein said device is suitable to contacting or administering said anti-IL-12 antibody by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal. 48. A method for producing an isolated mammalian anti-IL-12 antibody comprising a heavy chain variable region amino acid sequence of SEQ ID NO.: 7 and a light chain variable region amino acid sequence of SEQ ID NO: 8, comprising providing a host cell or transgenic non-human animal or transgenic plant or plant cell capable of expressing in recoverable amounts said antibody. 49. An anti-IL-12 antibody produced by the method as claimed in claim 48. The present invention provides isolated human, primate, rodent, mammalian, chimeric, humanized and/or CDR-grafted anti-IL-12 antibodies, immunoglobulins, cleavage products and other specified portions and variants thereof, as well as anti-IL-12 antibody compositions, encoding or complementary nucleic acids, vectors, host cells, compositions, formulations, devices, transgenic animals, transgenic plants, and methods of making and using thereof, as described and enabled herein, in combination with what is known in the art. The present invention also provides at least one isolated anti-IL-12 antibody as described herein. An antibody according to the present invention includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule, such as but not limited to at least one complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof, that can be incorporated into an antibody of the present invention. An antibody of the invention can include or be derived from any mammal, such as but not limited to a human, a mouse, a rabbit, a rat, a rodent, a primate, or any combination thereof, and the like.

Full Text

BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
This application is based in part on and claims priority to, U.S. Provisional 60/223,358
filed August 7. 2000 and 60/236.827 filed September 29, 2000. each of which is entirely
incorporated herein by reference.
The present invention relates to antibodies, including specified portions or variants,
specific for at least one Interleukin-12 (IL-12) protein or fragment thereof, as well as nucleic
acids encoding such anti-IL-I2 antibodies, complementary nucleic acids, vectors, host cells, and
methods of making and using thereof, including therapeutic formulations, administration and
devices.
RELATED ART
Interleukin-12 (IL-12) is a heterodimeric cytokine consisting of glycosylated polypeptide
chains of 35 and 40 kD which are disulfide bonded. The cytokine is synthesized and secreted by
antigen presenting cells including dendritic cells, monocytes, macrophages, B cells, Langerhans
cells and keratinocytes as well as natural killer (NK) cells. IL-12 mediates a variety of
biological processes and has been referred to as NK cell stimulatory factor (NKSF), T-cell
stimulating factor, cytotoxic T-lymphocyte maturation factor and EBV-transformed B-cell line
factor (Curfs, J.H.A.J., et al., Clinical Microbiology Reviews, 10:742-780 (1997)).
Interieukin-12 can bind to the IL-12 receptor expressed on the plasma membrane of cells
(e.g., T cells, NK cell), thereby altering (e.g., initiating, preventing) biological processes. For
example, the binding of IL-12 to the. IL-12 receptor can stimulate the proliferation of pre-
activated T cells and NK cells, enhance the cytolytic activity of cytotoxic..T cells (CTL), NK
cells and LAK (lymphokine activated killer) cells, induce production of gamma interferon (IFN
GAMMA) by T cells and NK cells and induce differentiation of naive ThO cells into Thl cells
that produce IFN GAMMA and IL-2 (Trinchieri, G., Annual Review of Immunology, 13:251-
276(1995)). In particular, IL-12 is vital for the generation: of cytolytic cells (e.g., NK, CTL)
and for mounting a cellular immune response (e.g., a Thl cell mediated immune response).
Thus, IL-12 is critically important in the generation and regulation of both protective immunity
(e.g., eradication of infections) and pathological immune responses (e.g., autoimmunity)
(Hendrzak, J.A. and Brunda, M.J., Laboratory Investigation, 72:619-637 (1995)). Accordingly,
an immune response (e.g., protective, or pathogenic) can be enhanced, suppressed or prevented by
manipulation of the biological activity of IL-12 in vivo, for example, by means of an antibody.
Non-human mammalian, chimeric, polyclonal (e.g.. anti-sera) and/or monoclonal
antibodies (Mabs) and fragments (e.g., proteolytic digestion or fusion protein products thereof)
are potential therapeutic agents that are being investigated in some cases to attempt to treat
certain diseases. However, such antibodies or fragments can elicit an immune response when
administered to humans. Such an immune response can result in an immune complex-
mediated clearance of the antibodies or fragments from the circulation, and make repeated
administration unsuitable for therapy, thereby reducing the therapeutic benefit to the patient
and limiting the readministration of the antibody or fragment. For example, repeated
administration of antibodies or fragments comprising non-human portions can lead to serum
sickness and/or anaphalaxis. In order to avoid these and other problems, a number of
approaches have been taken to reduce the immunogenicity of such antibodies and portions
thereof, including chimerization and humanization, as well known in the art. These and other
approaches, however, still can result in antibodies or fragments having some immunogenicity,
low affinity, low avidity, or with problems in cell culture, scale up, production, and/or low
yields. Thus, such antibodies or fragments can be less than ideally suited for manufacture or
use as therapeutic proteins.
Accordingly, there is a need to provide anti-IL-12: antibodies or fragments that
overcome one more of these problems, as well as improvements over known antibodies or
fragments thereof.
SUMMARY OF THE INVENTION
The present invention provides isolated human, primate, rodent, mammalian, chimeric,
humanized and/or CDR-grafted anti-IL-12 antibodies, immunoglobulins, cleavage products
and other specified portions and variants thereof, as well as anti-IL-12 antibody compositions,
encoding or complementary nucleic acids, vectors, host cells, compositions, formulations,
devices, transgenic animals, transgenic plants, and methods of making and using thereof, as
described and enabled herein, in combination with what is known in the art.
The present invention also provides at least one isolated anti-IL-12 antibody as
described herein. An antibody according to the present invention includes any protein or
peptide containing molecule that comprises at least a portion of an immunoglobulin molecule,
such as but not limited to at least one complementarity determining region (CDR) of a heavy
or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region,
a heavy chain or light chain constant region, a framework region, or any portion thereof, that
can be incorporated into an antibody of the present invention. An antibody of the invention
can include or be derived from any mammal, such as but not limited to a human, a mouse, a
rabbit, a rat, a rodent, a primate, or any combination thereof, and the like.
MISSING AT THE TIME OF PUBLICATION
The present invention provides. in one aspect. isolated nucleic acid molecules
comprising, complementary, or hybridizing to, a polynucleotide encoding at least one IL-12
anti-idiotype antibody, comprising at least one specified sequence, domain, portion or variant
thereof. The present invention further provides recombinant vectors comprising said IL-12
anti-idiotype antibody encoding nucleic acid molecules, host cells containing such nucleic
acids and/or recombinant vectors, as we'll as methods of making and/or using such anti-
idiotype antiobody nucleic acids, vectors and/or host cells.
The present invention also provides at least one method for expressing at least one
anti-IL-12 antibody, or IL-12 anti-idiotype antibody, in a host cell, comprising culturing a host
cell as described herein under conditions wherein at least one anti-IL-12 antibody is expressed
in detectable and/or recoverable amounts.
The present invention also provides at least one composition comprising (a) an
isolated anti-IL-12 antibody encoding nucleic acid and/or antibody as described herein; and (b)
a suitable carrier or diluent. The carrier or diluent can optionally be pharmaceutically
acceptable, according to known carriers or diluents. The composition can optionally further
comprise at least one further compound, protein or composition.
The present invention further provides at least one anti-IL-12 antibody method or
composition, for administering a therapeutically effective amount to modulate or treat at least
one IL-12 related condition in a cell, tissue, organ, animator patient and/or, prior to,
subsequent to, or during a related condition, as described herein.
The present invention also provides at least one composition, device and/or method of
delivery of a therapeutically or prophylactically effective amount of at least one anti-IL-12
antibody, according to the present invention.
The present invention further provides at least one anti-IL-12 antibody method or
composition, for diagnosing at least one IL-12 related condition in a cell; tissue, organ, animal
or patient and/or, prior to, subsequent to, or during a related condition, as described herein.
The present invention also provides at least one composition, device and/or method of
delivery for diagnosing of at least one anti-IL-12 antibody, according to the present invention.
DESCRIPTION OF THE FIGURES
Figures 1A and 1B are graphs showing concentration-dependent binding of human
anti-IL-12 mAbs to immobilized human IL-12. Anti-IL-12 antibodies were serially diluted in
1% BSA/PBS and incubated on rhIL-12 coated plates for 1 hour at 37°C. Plates were washed
twice with 0.02% Tween 20 (polyoxyethylene(20) sorbitan monolaurate), 0.15M saline and
then probed with horse radish peroxidase (HRP) labeled goat anti-human IgG kappa specific
antibody for 1 hour at room temperature. Plates were again washed, developed with o-
phenylenediaminc (OPD) substrate and the optical density (OD) of each well was measured at
490 nm.
Figure 2: Lanes from left to right in Figures A and B contain human IL-12, human IL-
12 p40, murine IL-12. and prestained molecular weight markers. Figure 2A shows bands
stained from total protein. The primary bands in each lane are human IL-12 (75 kd), p40
human IL-12 (40 kd). and murine IL-12 (75 kd). Figure 2B shows a western blot prepared
from a gel identical to that shown in Figure 2A. Blot was reacted with C340 followed by HRP
labeled goat anti-human IgG and specifically detected human IL-12 (monomer and multimers)
and human IL-12 p40 only. A control blot (not shown) reacted with HRP labeled goat anti-
human IgG did not display any bands.
Figure 3: Reverse transcription-PCR analysis of IFNg gene expression in human
PBL's treated with IL-2, IL-12, IL-2+IL-12 with and without anti-IL-12 antibody C340, 8.6.2,
isotype control antibody. Total RNA was reverse transcribed, amplified by PCR using gene-
specific primers. The level of b-actin mRNA in each sample was also determined which
served as a control for mRNA integrity and content.
Figure 4 is a histogram showing that human anti-IL-12 mAb (C340) inhibits;
production of interferon-y (IFNg) by monocyte depleted CD3+ peripheral blood mononuclear
cells (PBMC) stimulated with IL-2 plus IL-12. PBMC were cultured for five hours in control
media (no added cytokines), media supplemented with IL-12 (0.1 ng/ml) plus IL-2 (50 IU/ml)
(IL-12/IL-2), control media containing mAb C340 (10 ug/ml) and IL-12/IL-2 media
containing mAb C340 (10 m/ml). Intracellular IFNg was measured by two color
immunostaining with CD3-PE and IFNg-FITC. Data are shown for one donor.
Figure 5 is a graph showing dose-dependent inhibition of IFNg secretion by IL-2 plus
IL-12 stimulated peripheral blood lymphocytes with two different lots of a human anti-IL-12
mAb (C340). Human PBL (8 x 106/ml) were cultured for 24 hours with 10 U/ml IL-2, IL-2
plus 400 pg/ml IL-12, or IL-2 plus IL-12 and mAb C340 as indicated. The culture
supematents were removed and assayed for IFNg by EIA.
Figure 6 is a histogram showing dose-dependent inhibition of IL-12 plus IL-2 induced
LAK cell cytotoxicity by a human anti-IL-12 mAb (C340). LAK effector cells (human PBL, 8
x 106/ml) were cultured for 24 hours with IL-12 (400 pg/ml) plus IL-2 (10 U/ml) and mAb
C340 (5000 ng/ml or 50 ng/ml as indicated). The LAK, effector cells were washed and
cultured with 5lCr labeled Raji target cells for four hours at an effector to target (E:T) ration
of 80:1, and the quantity of 5lCr released into the media upon Raji cell lysis was measured.
Results are expressed as the mean of three normal donors standard error. EL-12 positive control
(IL-12) is effector cells incubated with IL-12 and without antibody. Background (BKGD) is
effector cells incubated without IL-12 or antibody.
Figures 7A and 7B are histograms showing that IL-I2 plus lL-2-induced expression of
CD95 on CD3+ peripheral blood mononuclear cells is inhibited by human anti-IL-12 mAb
(C340). PBMC were cultured for 72 hours in media containing 0.1 ng/ml IL-12 and a
suboptimal dose of IL-2 (50 IU/ml) in the presence or absence of mAb C340 (10 mg/ml).
CD95 expression was measured flow cytometry of cells stained with anti-CD95-FITC. Gating
was performed using two-color analysis (CD3 or CD56-PE vs. CD95-FITC) and forward vs.
orthogonal light scatter.
Figure 8 is a graph showing that recombinant human anti-human IL-12 antibodies
(rC340) bind to immobilized IL-12 in a manner that is indistinguishable from purified mAb
C340. The concentration of rC340 in the supernatants of three rC340-producing recombinant
cell lines was determined, and the supernatants were evaluated for IL-12 binding in an ELISA.
Plates were coated with 2 u,g/ml human IL-12 and incubated with purified mAb C340 from the
original hybridoma (standard) or the supernatants of recombinant cell lines. IL-12-bound
antibody was detected using alkaline phosphatase-conjugated goat anti-human IgG(heavy
chain + light chain).
Figures 9A - 9C are graphs showing growth kinetics and the quantity of antibody
secreted by three independently-derived rC340-producing recombinant cell subclones (Figure
9 A, subclone C379B; Figure 9B, subclone C381A; Figure 9C. subclone C389A).
Recombinant cells were seeded into T75 flasks at a starting density of 2 x 10s cells/ml in
standard media. At various times, cells were resuspended and the number of live cells and the
quantity (mg/ml) of rC340 in the media were determined.
DESCRIPTION OF THE INVENTION
The present invention provides isolated, recombinant and/or synthetic anti-IL-
12 human, primate, rodent, mammalian, chimeric, humanized or CDR-grafted, antibodies and
IL-12 anti-idiotype antibodies thereto, as well as compositions and encoding nucleic acid
molecules comprising at least one polynucleotide encoding at least one anti-IL-12 antibody or
anti-idiotype antibody. The present invention further includes, but is not limited to, methods
of making and using such nucleic acids and antibodies and anti-idiotype antibodies, including
diagnostic and therapeutic compositions, methods and devices.
As used herein, an "anti-Interleukin-12 antibody," "anti-IL-12 antibody," "anti-IL-12
antibody portion," or "anti-IL-12 antibody fragment" and/or "anti-IL-12 antibody variant" and
the like include any protein or peptide containing molecule that comprises at least a portion of
an immunoglobulin molecule, such as but not limited to at least one complementarity
determinng region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy
chain or light chain variable region, a heavy chain or light chain constant region, a framework
region, or any portion thereof, or at least one portion of an IL-12 receptor or binding protein,
which can be incorporated into an antibody of the present invention. Such antibody optionally
further affects a specific ligand. such as but not limited to where such antibody modulates,
decreases, increases, antagonizes, angonizes. mitigates, aleviates. blocks, inhibits, abrogates
and/or interferes with at least one IL-12 activity or binding, or with IL-12 receptor activity or
binding, in vitro, in situ and/or in vivo. As a non-limiting example, a suitable anti-IL-12
antibody, specified portion or variant of the present invention can bind at least one IL-12, or
specified portions, variants or domains thereof. A suitable anti-IL-12 antibody, specified
portion, or variant can also optionally affect at least one of IL-12 activity or function, such as
but not limited to, RNA, DNA or protein synthesis, IL-12 release. IL-12 receptor signaling,
membrane IL-12 cleavage, IL-12 activity, IL-12 production and/or synthesis. The term
"antibody "is further intended to encompass antibodies, digestion fragments, specified portions
and variants thereof, including antibody mimetics or comprising portions of antibodies that
mimic the structure and/or function of an anitbody or specified fragment or portion thereof,
including single chain antibodies and fragments thereof. Functional fragments include
antigen-binding fragments that bind to a mammalian IL-12. For example, antibody fragments
capable of binding to IL-12 or portions thereof, including, but not limited to Fab (e.g., by
papain digestion), Fab' (e.g., by pepsin digestion and partial reduction) and F(ab'), (e.g., by
pepsin digestion), facb (e.g., by plasmin digestion), pFc' (e.g., by pepsin or plasmin digestion),
Fd (e.g., by pepsin digestion, partial reduction and reaggregation), Fv or scFv (e.g., by
molecular biology techniques) fragments, are encompassed by the invention (see, e.g.,
Colligan, Immunology, supra).
Such fragments can be produced by enzymatic cleavage, synthetic or recombinant
techniques, as known in the art and/or as described herein, antibodies can also be produced in a
variety of truncated forms using antibody genes in which one or more stop codons have been
introduced upstream of the natural stop site. For example, a combination gene encoding a
F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CH,
domain and/or hinge region of the heavy chain. The various portions of antibodies can be
joined together chemically by conventional techniques, or can be prepared as a contiguous
protein using genetic engineering techniques.
As used herein, the term "human antibody" refers to an antibody in which substantially
svery part of the protein (e.g., CDR, framework, CL, CH domains (e.g., CH1. CH2, CH3), hinge,
(VL, VH)) is substantially non-immunogenic in humans, with only minor sequence changes or
/ariations. Similarly, antibodies designated primate (monkey, babboon, chimpanzee, etc.),
odent (mouse, rat, rabbit, guinea pid, hamster, and the like) and other mammals designate
such species, sub-genus, genus, sub-family, family specific antibodies. Further, chimeric
antibodies include any combination of the above. Such changes or variations optionally and
preferably retain or reduce the immunogenicity in humans or other species relative to non-
modified antibodies. Thus, a human antibody is distinct from a chimeric or humanized
antibody. It is pointed out that a human antibody can be produced by a non-human animal or
prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human
immunoglobulin (e.g., heavy chain and/or light chain) genes. Further, when a human antibody
is a single chain antibody, it can comprise a linker peptide that is not found in native human
antibodies. For example, an Fv can comprise a linker peptide, such as two to about eight
glycine or other amino acid residues, which connects the variable region of the heavy chain
and the variable region of the light chain. Such linker peptides are considered to be of human
origin.
Bispecific, heterospecific, heteroconjugate or similar antibodies can also be used that
are monoclonal, preferably human or humanized, antibodies that have binding specificities for
at least two different antigens. In the present case, one of the binding specificities is for at least
one IL-12 protein, the other one is for any other antigen. Methods for making bispecific
antibodies are known in the art. Traditionally, the recombinant production of bispecific
antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs,
where the two heavy chains have different specificities (Milstein and Cuello, Nature 305:537
(1983)). Because of the random, assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different antibo'dy molecules, of
which only one has the correct bispecific structure. The purification of the correct molecule,
which is usually done by affinity chromatography steps, is rather cumbersome, and the product
yields are low. Similar procedures are disclosed, e.g., in WO 93/08829, US Patent Nos,
6210668, 6193967, 6132992, 6106833, 6060285, 6037453, 6010902,5989530, 5959084,
5959083,5932448, 5833985, 5821333, 5807706, 5643759, 5601819,5582996, 5496549,
4676980, WO 91/00360, WO 92/00373, EP 03089, Traunecker et at, EMBO J. 10:3655
(1991), Suresh et al., Methods in Enzymology 121:210 (1986), each entirely incorporated
herein by reference.
Anti-IL-12 antibodies (also termed IL-12 antibodies) useful in the methods and
compositions of the present invention can optionally be characterized by high affinity binding
to IL-12 and optionally and preferably having low toxicity. In particular, an antibody,
specified fragment or variant of the invention, where the individual components, such as the
variable region, constant region and framework, individually and/or collectively, optionally
and preferably possess low immunogenicity, is useful in the present invention. The antibodies
that can be used in the invention are optionally characterized by their ability to treat patients
for extended periods with measurable alleviation of symptoms and low and/or acceptable
toxicity. Low or acceptable immunogenicity and/or high affinity, as well as other suitable
properties, can contribute to the therapeutic results achieved. "Low immunogenicity" is
defined herein as raising significant HAHA. HACA or HAMA responses in less than about
75%, or preferably less than about 50% of the patients treated and/or raising low litres in the
patient treated (less than about 300. preferably less than about 100 measured with a double
antigen enzyme immunoassay) (sec. e.g., Elliott et al., Lancet 344:1125-1127 (1994), entirely
incorporated herein by reference).
Utility
The isolated nucleic acids of the present invention can be used for production of at least
one anti-IL-12 antibody or specified variant thereof, which can be used to measure or effect in
an cell, tissue, organ or animal (including mammals and humans), to diagnose, monitor,
modulate, treat, alleviate, help prevent the incidence of, or reduce the symptoms of, at least
one IL-12 condition, selected from, but not limited to, at least one of an immune disorder or
disease, a cardiovascular disorder or disease, an infectious, malignant, and/or neurologic
disorder or disease, or other known|or specified IL-12 related condition.
Such a method can comprise administering an effective amount of a composition or a
pharmaceutical composition comprising at least one anti-IL-12 antibody to a ceil, tissue,
organ, animal or patient in need of such modulation, treatment, alleviation, prevention, or
reduction in symptoms, effects or mechanisms. The effective amount can comprise an amount
of about 0.001 to 500 mg/kg per single (e.g., bolus), multiple or continuous administration, or
to achieve a serum concentration of 0.01-5000 mg/ml serum concentration per single, multiple
or continuous adminstration, or any effective range or value therein, as done and determined
using known methods, as described herein or known in the relevant arts.
Citations
All publications or patents cited herein are entirely incorporated herein by reference as
they show the state of the art at the time of the present invention and/or to provide description
and enablement of the present invention. Publications refer to any scientific or patent
publications, or any other information available in any media format, including all recorded,
electronic or printed formats. The following references are entirely incorporated herein by
reference: Ausubel, et al., ed.. Current Protocols in Molecular Biology, John Wiley & Sons,
Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd
Edition, Cold Spring Harbor, NY (1989); Harlow and Lane, antibodies, a Laboratory Manual,
Cold Spring Harbor, NY (1989); Colligan, et al., eds., Current Protocols in Immunology, John
Wiley & Sons, Inc., NY (1994-2001), Colligan et al., Current Protocols in Protein Science.
John Wiley & Sons, NY, NY, (1997-2001).
Antibodies of the Present Invention
At least one anti-IL-12 antibody of the present invention can be optionally produced
by a cell line, a mixed cell line, an immortalized cell or clonal population of immortalized
cells, as well known in the art. See. e.g., Ausubel. et al., ed.. Current Protocols in Molecular
Biology, John Wiley &. Sons. Inc., NY. NY (1987-2001): Sambrook. et al.. Molecular Cloning:
A Laboratory Manual. 2nd Edition. Cold Spring Harbor. NY (1989); Harlow and Lane,
antibodies, a Laboratory Manual. Cold Spring Harbor. NY (1989); Colligan, et al., eds.,
Current Protocols in Immunology. John Wiley &. Sons. Inc., NY (1994-2001); Colligan et al.,
Current Protocols in Protein Science. John Wiley & Sons, NY. NY, (1997-2001), each entirely
incorporated herein by reference.
Human antibodies that are specific for human IL-12 proteins or fragments thereof can
be raised against an appropriate immunogenic antigen, such as isolated and/or IL-12 protein or
a portion thereof (including synthetic molecules, such as synthetic peptides). Other specific or
general mammalian antibodies can be similarly raised. Preparation of immunogenic antigens,
and monoclonal antibody production can be performed using any suitable technique.
In one approach, a hybridoma is produced by fusing a suitable immortal cell line (e.g.,
a myeloma cell line such as. but not limited to, Sp2/0, Sp2/0-AGl4, NSO, NS1, NS2, AE-1,
L.5, >243, P3X63Ag8.653. Sp2 SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U937, MLA 144, ACT IV,
MOLT4, DA-1, JURKAT, WEHI. K-562, COS, RATI, NIH 3T3, HL-60, MLA 144.
KAMAIWA, NEURO 2A, or the like, or heteromylomas, fusion products thereof, or any cell
or fusion cell derived therefrom, or any other suitable cell line as known in the art See, e.g.,
www.atcc.org, www.lifetech.com., and the like, with antibody producing cells, such as, but not
limited to, isolated or cloned spleen, peripheral blood, lymph, tonsil, or other immune or B cell
containing cells, or any other cells expressing heavy or light chain constant or variable or
framework or CDR sequences, either as endogenous or heterologous nucleic acid, as
recombinant or endogenous, viral, bacterial, algal, prokaryotic, amphibian, insect, reptilian,
fish, mammalian, rodent, equine, ovine, goat, sheep, primate, eukaryotic, genomic DNA,
cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA,
tRNA, single, double or triple stranded, hybridized, and the like or any combination thereof.
See, e.g., Ausubel, supra, and Colligan, Immunology, supra, chapter 2, entirely incorporated
herein by reference.
Antibody producing cells can also be obtained from the peripheral blood or, preferably
the spleen or lymph nodes, of humans or other suitable animals that have been immunized with
the antigen of interest. Any other suitable host cell can also be used for expressing
heterologous or endogenous nucleic acid encoding an antibody, specified fragment or variant
thereof, of the present invention. The fused cells (hybridomas) or recombinant cells can be
isolated using selective culture conditions or other suitable known methods, and cloned by
limiting dilution or cell sorting, or other known methods. Cells which produce antibodies with
the desired specificity can be selected by a suitable assay (e.g., ELISA).
Other suitable methods of producing or isolating antibodies of the requisite specificity
can be used, including, but not limited to, methods that select recombinant antibody from a
peptide or protein library e.g., but not limited to, a bacteriophage, ribosome, oligonucleotide,
RNA, cDNA, or the like, display library; e.g., as available from Cambridge antibody
Technologies, Cambridgeshire. UK; MorphoSys, Martinsreid/Planegg, DE; Biovation,
Aberdeen, Scotland, UK: Biolnvent, Lund, Sweden; Dyax Corp., Enzon, Affymax/Biosite;
Xoma, Berkeley, CA; Ixsys. See. e.g., EP 368,684, PCT/GB91/01134; PCT/GB92/01755;
PCT/GB92/002240; PCT.GB92.00883; PCT/GB93/00605; US 08/350260(5/12/94);
PCT/GB94/01422; PCT/GB94/02662; PCT/GB97/01835: (CAT/MRC); WO90/14443;
WO90/14424; WO90/14430; PCT/US94/1234; W092/18619; WO96/07754; (Scripps); EP 614
989 (MorphoSys); WO95/16027 (Biolnvent); WO88/06630; WO90/3809 (Dyax); US
4,704,692 (Enzon); PCT/US91/02989(Affymax); WO89/06283; EP371 998; EP 550400;
(Xoma); EP 229 046; PCI7US91.07149 (Ixsys); or stochastically generated peptides or
proteins - US 5723323,5763192, 5S14476, 5817483, 5S24514, 5976862, WO 86/05803, EP
590 689 (Ixsys, now Applied Molecular Evolution (AME), each entirely incorporated herein
by reference) or that rely upon immunization of transgenic animals (e.g., SCID mice, Nguyen
et al., Microbiol. Immunol. 41:901-907 (1997); Sandhu et al., Crit. Rev. Biotechnol. 16:95-118
(1996); Eren et al., Immunol. 93:154-161 (1998), each entirely incorporated by reference as
well as related patents and application) that are capable of producing a repertoire of human
antibodies, as known in the art and/or as described herein. Such techniques, include, but are
not limited to, ribosome display (Hanes et al., Proc. Natl. Acad. Sci. USA, 94:4937-4942 (May
1997); Hanes et al., Proc. Natl. Acad. Sci. USA, 95:14130-14135 (Nov: 1998)); single cell
antibody producing technologies (e.g., selected lymphocyte antibody method ("SLAM") (US
pat No. 5,627,052, Wen et al., J. Immunol. 17:887-892 (1987); Babcook et al., Proc. Natl.
Acad. Sci. USA 93:7843-7848 (1996)); gel microdroplet and flow cytometry (Powell et al.,
Biotechnol. 8:333-337 (1990); One Cell Systems, Cambridge, MA; Gray et al., J. Imm. Meth.
182:155-163 (1995); Kenny et al., Bio/Technol. 13:787-790 (1995)); B-cell selection
(Steenbakkers et al., Molec. Biol. Reports 19:125-134 (1994); Jonak et al., Progress Biotech,
Vol. 5, In Vitro Immunization in Hybridoma Technology, Borrebaeck, ed., Elsevier Science
Publishers B.V., Amsterdam, Netherlands (1988)).
Methods for engineering or humanizing non-human or human antibodies can also be
used and are well known in the an. Generally, a humanized or engineered antibody has one or
more amino acid residues from a source which is non-human, e.g., but not limited to mouse,
rat, rabbit, non-human primate or other mammal. These human amino acid residues are often
referred to as "import" residues, which are typically taken from an "import" variable, constant
or other domain of a known human sequence. Known human fg sequences are disclosed, e.g.,
www.ncbi.nlm.nih.gov/entrez/query.fcgl: www.atcc.org/phage/hdb.html; www. sciquest.com/;
www.abcam.com/; www.antibodyresource.com/onlinecomp.html;
www.public.iastate.edu/~pedro/rcsearch-tools.html; www.mgcn.uni-
heidelberg.de/SD/IT/lT.html; www.whfreeman.com/immunology/CH05/kuby05.htm.
www.library.thinkquest.org/12429/Immune/Antibody.html;
www.hhmi.org/grants/lecrures/l996/vlab/; www.path.cam.ac.uk/~mrc7/mikeimages.html;
www.antibodyresource.com/;
mcb.harvard.edu/BioLinks/Immunolog.html.www.immunologylink.com/;
pathbox.wustl.edu/~hcenter/index.html; www.biotech.ufl.edu/~hcl/;
www.pebio.com/pa/340913/340913.html; www.naI.usda.gov/awic/pubs/antibody/;
www.m.ehimoe-u.ac.jp/~yasuhito/Elisa.html; www.biodesign.com/table.asp;
www.icnet.uk/axp/facs/davtes/links.html; www.biotech.ufl.edu/~fccl/protocol.html; www.isac-
net.org/sites_geo.htmI; aximtl .imt.uni-marburg.de/~rek/AEPStart.html;
baserv.uci.kun.nl/~jraats/linksl .html; www.recab.uni-hd.de/immuno.bme.nwu.edu/;
www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html; www.ibt.unam.mx/vir/V_mice.html;
imgt.cnusc.fr 8104/; www.biochem.ucl.ac.uk/~martin/abs/index.html; antibody.bath.ac.uk/;
abgen.cvm.tamu.edu/lab/wwwabgen.html;
www.uni2h.ch/~honegger/AHOseminar/Slide01 .html; www.cryst.bbk.ac.uk/~ubcg07s/;
www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm;
www.path.cam.ac.uk/-mrc7/humanisation/TAHHP.html:
www.ibt.unarn.rnx/vir/structure/stat_aim.html; www.biosci.missouri.edu/smithgp/index.html;
www.cryst.bioc.cam. ac.uk/~fmolina/Web-pages/Pept/sportech.html;
www.jerini.de/fr_products.htm; www.patents.ibm.com/ibm.html.Kabat et al., Sequences of
Proteins of Immunological Interest, U.S. Dept. Health (1983), each entirely incorporated
herein by reference. Such imported sequences can be used to reduce immunogenicity or
reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or
any other suitable characteristic, as known in the art. Generally part or all of the non-human or
human CDR sequences are maintained while the non-human sequences of the variable and
constant regions are replaced with human or other amino acids, antibodies can also optionally
be humanized with retention of high affinity for the antigen and other favorable biological
properties. To achieve this goal, humanized antibodies can be optionally prepared by a process
of analysis of the parental sequences and vanous conceptual humanized products using three-
dimensional models of the parental and humanized sequences. Three-dimensional
immunoglobulin models are commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display probable three-dimensional
conformational structures of selected candidate immunoglobulin sequences. Inspection of
these displays permits analysis of the likely role of the residues in the functioning of the
candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of
the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and
combined from the consensus and import sequences so that the desired antibody characteristic,
such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues
are directly and most substantially involved in influencing antigen binding. Humanization or
engineering of antibodies of the present invention can be performed using any known method,
such as but not limited to those described in, Winter (Jones et al., Nature 321:522 (1986);
Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)), Sims et
al., J. Immunol. 151: 2296 (1993); Chothiaand Lesk, J. Mol. Biol. 196:901 (1987), Carter et
al., Proc. Natl. Acad. Sci. U.S.A 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993),
US patent Nos: 5723323, 5976862, 5824514, 5817483, 5814476, 5763192, 5723323,
5,766886, 5714352, 6204023, 6180370, 5693762, 5530101, 5585089, 5225539; 4816567,
PCT/: US98/16280, US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334,
GB91/01134, GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP 229246, each
entirely incorporated herein by reference, included references cited therein.
The anri-IL-12 antibody can also be optionally generated by immonization of a
transgenic animal (e.g., mouse, rat, hamster, non-human primate, and the like) capable of
producing a repertoire of human antibodies, as described herein and/or as known in the art.
Cells that produce a human anri-IL-12 antibody can be isolated from such animals and
immortalized using suitable methods, such as the methods described herein.
Transgenic mice that can produce a repertoire of human antibodies that bind to human
antigens can be produced by known methods (e.g., but not limited to, U.S. Pat. Nos: 5,770,428,
5,569,825, 5,545,806, 5,625,126, 5,625,825, 5,633,425, 5,661,016 and 5,789,650 issued to
Lonberg et al.; Jakobovits et al. WO 98/50433, Jakobovits et al. WO 98/24893, Lonberg et al
WO 98/24884, Lonberg et al. WO 97/13852, Lonberg et al. WO 94/25585, Kucherlapate et aL
WO 96/34096, Kucherlapate et al. EP 0463 151 B1, Kucherlapate et al. EP 0710 719 Al,
Surani et al. US. Pat. No. 5,545,807, Bruggemann et al. WO 90/04036, Bruggemann et al. EP
0438 474 B1, Lonberg et al. EP 0.814 259 A2, Lonberg et al. GB 2 272 440 A, Lonberg et al.
Nature 368:856-859 (1994), Taylor et al., Int. Immunol. 6(4)579-591 (1994), Green et al,
Nature Genetics 7:13-21 (1994), Mendez et al., Nature Genetics 15:146-156 (1997), Taylor et
al.. Nucleic Acids Research 20(23):6287-6295 (1992), Tuaillon et al., Proc Natl Acad Sci USA
90(8)3720-3724 (1993), Lonberg et al., Int Rev Immunol 13(l):65-93 (1995) and Fishwald et
at., Nat Biotechnol 14(7):845-851 (1996), which are each entirely incorporated herein by
reference). Generally, these mice comprise at least one transgene comprising DNA from at
least one human immunoglobulin locus that is functionally rearranged, or which can undergo
functional rearrangement. The endogenous immunoglobulin loci in such mice can be
disrupted or deleted to eliminate the capacity of the animal to produce antibodies encoded by
endogenous genes.
Screening antibodies for specific binding to similar proteins or fragments can be
conveniently achieved using peptide display libraries. This method involves the screening of
large collections of peptides for individual members having the desired function or structure,
antibody screening of peptide display libraries is well known in the art. The displayed peptide
sequences can be from 3 to 5000 or more amino acids in length, frequently from 5-100 amino
acids long, and often from about 8 to 25 amino acids long. In addition to direct chemical
synthetic methods for generating peptide libraries, several recombinant DNA methods have been
described. One type involves the display of a peptide sequence on the surface of a bacteriophage
or cell. Each bacteriophage or cell contains the nucleotide sequence encoding the particular
displayed peptide sequence. Such methods are described in PCT Patent Publication Nos.
91/17271,91/18980,91/19818, and 93/08278. Other systems for generating libraries of peptides
have aspects of both in vitro chemical synthesis and recombinant methods. See, PCT Patent
Publication Nos. 92/05258, 92/14843, and 96/19256. See also, U.S. Patent Nos. 5,658,754; and
5,643,768. Peptide display libraries, vector, and screening kits are commercially available from
such suppliers as Invitrogen (Carlsbad, CA), and Cambridge Antibody Technologies
(Cambridgeshire, UK). See, e.g., U.S. Pat. Nos. 4704692,4939666,4946778, 5260203,
5455030,5518889, 5534621, 5656730, 5763733, 5767260,5856456, assigned to Enzon;
5223409,5403484,5571698,5837500, assigned to Dyax, 5427908,5580717, assigned to
Affymax; 5885793, assigned to Cambridge antibody Technologies; 5750373, assigned to
Genentech, 5618920, 5595898, 5576195, 5698435, 5693493,5698417, assigned to Xoma,
Colligan, supra; Ausubel, supra; or Sambrook, supra, each of the above patents and publications
entirely incorporated herein by reference.
Antibodies of the present invention can also be prepared using at least one anti-IL-12
antibody encoding nucleic acid to provide transgenic animals or mammals, such as goats,
cows, horses, sheep, and the like, that produce such antibodies in their milk. Such animals can
be provided using known methods. See, e.g., but not limited to, US patent nos. 5,827,690;
5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; 5,304,489, and the like, each of which
is entirely incorporated herein by reference.
Antibodies of the present invention can additionally be prepared using at least one
anti-IL-12 antibody encoding nucleic acid to provide transgenic plants and cultured plant cells
(e.g., but not limited to tobacco and maize) that produce such antibodies, specified portions or
variants in the plant parts or in cells cultured therefrom. As a non-limiting example, transgenic
tobacco leaves expressing recombinant proteins have been successfully used to provide large
amounts of recombinant proteins, e.g., using an inducible promoter. See, e.g., Cramer et al.,
Curr. Top. Microbol. Immunol. 240:95-118 (1999) and references cited therein. Also,
transgenic maize have been used to express mammalian proteins at commercial production
levels, with biological activities equivalent to those produced in other recombinant systems or
purified from natural sources. See. e.g., Hood et al.. Adv. Exp. Med. Biol. 464:127-147 (1999)
and references cited therein, antibodies have also been produced in large amounts from
transgenic plant seeds including antibody fragments, such as single chain antibodies (scFv's),
including tobacco seeds and potato tubers. See, e.g., Conrad et al., Plant Mol. Biol. 38:101-
109 (1998) and reference cited therein. Thus, antibodies of the present invention can also be
produced using transgenic plants, according to know methods. See also, e.g., Fischer et al.,
Biotechnol. Appl. Biochem. 30:99-108 (Oct., 1999), Ma et al.. Trends Biotechnol. 13:522-7
(1995); Ma et al., Plant Physiol. 109:341-6 (1995); Whitelam et al., Biochem. Soc. Trans.
22:940-944 (1994); and references cited therein. See, also generally for plant expression of
antibodies, but not limited to, Each of the above references is entirely incorporated herein by
reference.
The antibodies of the invention can bind human IL-12 with a wide range of affinities
(KD). In a preferred embodiment, at least one human mAb of the present invention can
optionally bind human IL-12 with high affinity. For example, a human mAb can bind human
IL-12 with a KD equal to or less than about 10"7 M, such as but not limited to, 0.1-9.9 (or any
range or value therein) X 10-7 10-8, l0-9,10-10, 10-11, 10-12, 10-13 or any range or value therein.
The affinity or avidity of an antibody for an antigen can be determined experimentally
using any suitable method. (See, for example, Berzofsky, et al., "Antibody-Antigen
Interactions," In Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York, NY
(1984); Kuby, Janis Immunology, W. H. Freeman and Company: New York, NY (1992); and
methods described herein). The measured affinity of a particular antibody-antigen interaction
can vary if measured under different conditions (e.g., salt concentration, pH). Thus,
measurements of affinity and other antigen-binding parameters (e.g., KD, Ka, Kd) are preferably
made with standardized solutions of antibody and antigen, and a standardized buffer, such as
the buffer described herein.
Nucleic Acid Molecules
Using the information provided herein, such as the nucleotide sequences encoding at
least 70-100% of the contiguous amino acids of at least one of SEQ ID NOS:l, 2, 3,4, 5, 6,7,
8, specified fragments, variants or consensus sequences thereof, or a deposited vector
comprising ar least one of these sequences, a nucleic acid molecule of the present invention
encoding at least one anti-IL-12 antibody can be obtained using methods descnbed herein or as
known in the an.
Nucleic acid molecules of the present invention can be in the form of RNA, such as
mRNA, hnRNA. tRNA or any other form, or in the form of DNA, including, but not limited to,
cDNA and genomic DNA obtained by cloning or produced synthetically, or any combinations
thereof. The DNA can be triple-stranded, double-stranded or single-stranded, or any
combination thereof. Any portion, of at least one strand of the DNA or RNA can be the coding
strand, also known as the sense strand, or it can be the non-coding strand, also referred to as
the an ti-sense strand.
Isolated nucleic acid molecules of the present invention can include nucleic acid
molecules comprising an open reading frame (ORF), optionally with one or more introns, e.g.,
but not limited to, at least one specified portion of at least one CDR, as CDR1, CDR2 and/or
CDR3 of at least one heavy chain (e.g.. SEQ ID NOS: 1 -3) or light chain (e.g., SEQ ID NOS:
4-6); nucleic acid molecules comprising the coding sequence for an anti-IL-12 antibody or
variable region (e.g., SEQ ID NOS:,7.8): and nucleic acid imolecules which comprise a
nucleotide sequence substantially different from those described above but which, due to the
degeneracy of the genetic code, still encode at least one anti-IL-12 antibody as described
herein and/or as known in the art. Of course, the genetic code is well known in the art. Thus,
it would be routine for one skilled in the art to generate such degenerate nucleic acid variants
that code for specific anti-IL-12 antibodies of the present invention. See, e.g., Ausubel, et aL,
supra, and such nucleic acid variants are included in the present invention. Non-limiting
examples of isolated nucleic acid molecules of the present inveniton include SEQ ID NOS: 10-
15, corresponding to non-limiting examples of a nucleic acid encoding, respectively, HC
CDR1, HC CDR2, HC CDR3, LC CDR1. LC CDR2, LC CDR3, HC variable region and LC
variable region.
In another aspect, the invention provides isolated nucleic acid molecules encoding a(n)
anti-IL-12 antibody having an amino acid sequence as encoded by the nucleic acid contained
in the plasmid deposited as designated clone names_______________________________and
ATCC Deposit Nos.________________________________________, respectively, deposited
on______________________________.
As indicated herein, nucleic acid molecules of the present invention which comprise a
nucleic acid encoding an anti-IL-12 antibody can include, but are not limited to, those
encoding the amino acid sequence of an antibody fragment, by itself; the coding sequence for
the entire antibody or a portion thereof: the coding sequence for an antibody, fragment or
portion, as well as additional sequences, such as the coding sequence of at least one signal
leader or fusion peptide, with or without the aforementioned additional coding sequences, such
as at least one mtron. together with additional, non-coding sequences, including but not limited
to. non-codmc 5' and 3' sequences, such as the transcribed, non-translated sequences that play
a role in transcription. mRNA processing, including splicing and polyadenylation signals (for
example - ribosome binding and stability of mRNA). an additional coding sequence that codes
for additional amino acids, such as those that provide additional functionalities. Thus, the
sequence encoding an antibody can be fused to a marker sequence, such as a sequence
encoding a peptide that facilitates purification of the fused antibody comprising an antibody
fragment or portion.
Polynucleotides Which Selectively Hybridize to a Polynucleotide as Described Herein
The present invention provides isolated nucleic acids that hybridize under selective
hybridization conditions to a polynucleotide disclosed herein. Thus, the polynucleotides of this
embodiment can be used for isolating, detecting, and/or quantifying nucleic acids comprising
such polynucleotides. For example, polynucleotides of the present invention can be used to
identify, isolate, or amplify partial or full-length clones in a deposited library. In some
embodiments, the polynucleotides are genomic or cDNA sequences isolated, or otherwise
complementary to, a cDNA from a human or mammalian nucleic acid library.
Preferably, the cDNA library comprises at least 80% full-length sequences, preferably at
least 85% or 90% full-length sequences, and more preferably at least 95% full-length sequences.
The cDNA libraries can be normalized to increase the representation of rare sequences. Low or
moderate stringency hybridization conditions are typically, but not exclusively, employed with
sequences having a reduced sequence identity relative to complementary sequences. Moderate
and high stringency conditions can optionally be employed for sequences of greater identity.
Low stringency conditions allow selective hybridization of sequences having about 70%
sequence identity and can be employed to identify onhologous or paralogous sequences.
Optionally, polynucleotides of this invention will encode at least a portion of an antibody
encoded by the polynucleotides described herein. The polynucleotides of this invention embrace
nucleic acid sequences that can be employed for selective hybridization to a polynucleotide
encoding an antibody of the present invention. Sec, e.g., Ausubel, supra; Colligan, supra, each
entirely incorporated herein by reference.
Construction of Nucleic Acids
The isolated nucleic acids of the present invention can be made using (a) recombinant
methods, (b) synthetic techniques, (c) purification techniques, or combinations thereof, as well-
known in the art.
The nucleic acids can conveniently comprise sequences in addition to a polynucleotide of
the present invention. For example, a multi-cloning site comprising one or more endonuclease
restriction sites can be inserted into the nucleic acid to aid in isolation of the polynucleotide.
Also, translatable sequences can be inserted to aid in the isolation of the translated polynucleotide
of the present invention. For example, a hexa-histidine marker sequence provides a convenient
means to purify the proteins of the present invention. The nucleic acid of the present invention -
excluding the coding sequence - is optionally a vector, adapter, or linker for cloning and/or
expression of a polynucleotide of the present invention.
Additional sequences can be added to such cloning and/or expression sequences to
optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or
to improve the introduction of the polynucleotide into a cell. Use of cloning vectors, expression
vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra; or Sambrook,
supra)
Recombinant Methods for Constructing Nucleic Acids
The isolated nucleic acid compositions of this invention, such as RNA, cDNA, genomic
DNA, or any combination thereof, can be obtained from biological sources using any number of
cloning methodologies known to those of skill in the art. In some embodiments, oligonucleotide
probes that selectively hybridize, under stringent conditions, to the polynucleotides of the present
invention are used to identify the desired sequence in a cDNA or genomic DNA library. The
isolation of RNA, and construction of cDNA and genomic libraries, is well known to those of
ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook, supra)
Nucleic Acid Screening and Isolation Methods
A cDNA or genomic library can be screened using a probe based upon the sequence of a
polynucleotide of the present invention, such as those disclosed herein. Probes can be used to
hybridize.with genomic DNA or cDNA sequences to isolate homologous genes in the same or
different organisms. Those of skill in the art will appreciate that various degrees of stringency of
hybridization can be employed in the assay; and either the hybridization of the wash medium can
be stringent. As the conditions for hybridization become more stringent, there must be a greater
degree of complementarity between the probe and the target for duplex formation to occur. The
degree of stringency can be controlled by one or more of temperature, ionic strength, pH and the
presence of a partially denaturing solvent such as formamide. For example, the stringency of
hybridization is conveniently varied by changing the polarity of the reactant solution through, for
example, manipulation of the concentration of formamide within the range of 0% to 50%. The
degree of complementarity (sequence identity) required for detectable binding will vary in
accordance with the stringency of the hybridization medium and/or wash medium. The degree of
complementarity will optimally be 100%, or 70-100%, or any range or value therein. However,
it should be understood that minor sequence variations in the probes and primers can be
compensated for by reducing the stringency of the hybridization and/or wash medium.
Methods of amplification of RNA or DNA arc well known in the art and can be used
according to the present invention without undue experimentation, based on the teaching and
guidance presented herein.
Known methods of DNA or RNA amplification include, but are not limited to,
polymerase chain reaction (PCR) and related amplification processes (see, e.g., U.S. Patent
Nos. 4,683,195. 4,683,202. 4.S00.159. 4,965,188, to Mullis, et al.; 4,795.699 and 4,921,794 to
Tabor, et al; 5.142,033 to Innis: 5.122,464 to Wilson, et al.; 5,091,310 to Innis; 5,066,584 to
Gyllensten, et al; 4.889,818 to Gelfand, et al; 4,994,370 to Silver, et al; 4,766,067 to Biswas;
4,656,134 to Ringold) and RNA mediated amplification that uses anti-sense RNA to the target
sequence as a template for double-stranded DNA synthesis (U.S. Patent No. 5,130,238 to
Malek, et al, with the tradename NASBA), the entire contents of which references are
incorporated herein by reference. (See, e.g., Ausubel, supra; or Sambrook, supra.)
For instance, polymerase chain reaction (PCR) technology can be used to amplify the
sequences of polynucleotides of the present invention and related genes directly from genomic
DNA or cDNA libraries. PCR and other in vitro amplification methods can also be useful, for
example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic
acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic
acid sequencing, or for other purposes. Examples of techniques sufficient to direct persons of
skill through in vitro amplification methods are found in Berger, supra, Sambrook, supra, and
Ausubel, supra, as well as Mullis, et al., U.S. Patent No. 4,683,202 (1987); and Innis, et al., PCR
Protocols A Guide to Methods and Applications, Eds., Academic Press Inc., San Diego, CA
(1990). Commercially available kits for genomic PCR amplification are known in the art. See,
e.g., Advantage-GC Genomic PCR Kit (Clontech). Additionally, e.g., the T4 gene 32 protein
(Boehringer Mannheim) can be used to improve yield of long PCR products.
Synthetic Methods for Constructing Nucleic Acids
The isolated nucleic acids of the present invention can also be prepared by direct
chemical synthesis by known methods (see, e.g., Ausubel, et al., supra). Chemical synthesis
generally produces a single-stranded oligonucleotide, which can be converted into double-
stranded DNA by hybridization with a complementary sequence, or by polymerization with a
DNA polymerase using the single strand as a template. One of skill in the art will recognize that
while chemical synthesis of DNA can be limited to sequences of about 100 or more bases, longer
sequences can be obtained by the ligation of shorter sequences.
Recombinant Expression Cassettes
The present invention further provides recombinant expression cassettes comprising a
nucleic acid of the present invention. A nucleic acid sequence of the present invention, for
example a cDNA or a genomic sequence encoding an antibody of the present invention, can be
used to construct a recombinant expression cassette that can be introduced into at leas: one
desired host cell. A recombinant expression cassette will typically comprise a polynucleotide of
the present invention operably linked to transcriptional initiation reyulatory sequences that will
direct the transcription of the polynucleotide in the intended host cell. Both heterologous and
non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the
nucleic acids of the present invention.
In some embodiments, isolated nucleic acids that serve as promoter, enhancer, or other
elements can be introduced in the appropriate position (upstream, downstream or in intron) of a
non-heterologous form of a polynucleotide of the present invention so as to up or down regulate
expression of a polynucleotide of the present invention. For example, endogenous promoters can
be altered in vivo or in vitro by mutation, deletion and/or substitution.
Vectors And Host Cells
The present invention also relates to vectors that include isolated nucleic acid
molecules of the present invention, host cells that are genetically engineered with the
recombinant vectors, and the production of at least one anti-IL-12 antibody by recombinant
techniques, as is well known in the art. See. e.g., Sambrook, et al., supra; Ausubel, et al.,
supra, each entirely incorporated herein by reference.
The polynucleotides can optionally be joined to a vector containing a selectable
marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate,
such as a calcium phosphate precipitate, or in a complex with a charged lipid: If the vector is a
virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced
into host cells.
The DNA insert should be operanvely linked to an appropriate promoter. The
expression constructs will further contain sites for transcription initiation, termination and, in
the transcribed region, a ribosome binding site for translation. The coding portion of the
mature transcripts expressed by the constructs will preferably include a translation initiating at
the beginning and a termination codon (e.g., UAA, UGA or UAG) appropriately positioned at
the end of the mRNA to be translated, with UAA and UAG preferred for mammalian or
eukaryotic cell expression.
Expression vectors will preferably but optionally include at least one selectable
marker. Such markers include, e.g., but not limited to, methotrexate (MTX), dihydrofolate
reductase (DHFR. US Pat.Nos. 4,399.216; 4,634,665; 4.656,134; 4,956,288; 5.149,636;
5,179,017), ampicillin, neomycin (G418), mycophenolic acid, or glutamme synthetase (GS,
US Pat.Nos. 5.122,464; 5.770,359; 5,827,739) resistance for eukaryotic cell culture, and
tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria or
prokaryotics (the above patents are entirely incorporated hereby by reference). Appropnate
culture mediums and conditions for the above-described host cells are known in the an.
Suitable vectors will be readily apparent to the skilled artisan. Introduction of a vector
construct into a host cell can be effected by calcium phosphate transfection, DEAE-dextran
mediated transfection. canonic lipid-medtated transfection, electroporation. transduction,
infection or other known methods. Such methods arc described in the art. such as Sambrook.
supra. Chapters 1-4 and 16-18; Ausubel. supra. Chapters 1,9, 13. 15, 16.
At least one antibody of the present invention can be expressed in a modified form,
such as a fusion protein, and can include not only secretion signals, but also additional
heterologous functional regions. For instance, a region of additional amino acids, particularly
charged amino acids, can be added to the N-termmus of an antibody to improve stability and
persistence in the host cell, during purification, or dunng subsequent handling and storage.
Also, peptide moieties can be added to an antibody of the present invention to facilitate
purification. Such regions can be removed prior to final preparation of an antibody or at least
one fragment thereof. Such methods are described in many standard laboratory manuals, such
as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17
and 18.
Those of ordinary skill in the art are knowledgeable in the numerous expression systems
available for expression of a nucleic acid encoding a protein of the present invention.
Alternatively, nucleic acids of the present invention can be expressed in a host cell by
turning on (by manipulation) in a host cell that contains endogenous DNA encoding an antibody
of the present invention. Such methods are well known in the art, e.g., as described in US patent
Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, entirely incorporated herein by reference.
Illustrative of cell cultures useful for the production of the antibodies, specified portions
or variants thereof, are mammalian cells. Mammalian cell systems often will be in the form of
monolayers of cells although mammalian cell suspensions or bioreactors Can also be used. A
number of suitable host cell lines capable of expressing intact glycosylated proteins have been
developed in the art, and include the COS-1 (e.g.. ATCC CRL 1650), COS-7 (e.g., ATCC CRL-
1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610) and BSC-l (e.g.,
ATCC CRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2A)-Agl4,
293 cells, HeLa cells and the like, which are readily available from, for example, American
Type Culture Collection. Manassas, Va (www.atcc.org). Preferred host cells include cells of
lymphoid origin such as myeloma and lymphoma cells. Particularly preferred host cells are
P3X63Ag8.653 cells (ATCC Accession Number CRL-1580) and SP2/0-Agl4 cells (ATCC
Accession Number CRL-1851). In a particularly preferred embodiment, the recombinant cell
is a P3X63Ab8.653 or a SP2/0-Agl4 cell.
Expression vectors for these cells can include one or more of the following expression
control sequences, such as. but not limited to an origin of replication: a promoter (e.g., late or
early SV40 promoters, the CMV promoter (US Pat.Nos. 5.168.062; 5.385.839). an HSV tk
promoter, a pgk (phosphoglyccratc kinase) promoter, an EF-l alpha promoter (US Pat.No.
5,266.491). at least one human immunoglobulin promoter, an enhancer, and/or processing
information sites, such as nbosome binding sites. RNA splice sites, polyadenylation sites (e.g., an
SV40 large T Ag poly A addition site), and transcriptional terminator sequences. See, e.g.,
Ausubel et al.. supra; Sambrook, et al., supra. Other celts useful for production of nucleic acids
or proteins of the present invention are known and/or available, for instance, from the American
Type Culture Collection Catalogue of Cell Lines and Hybridomas (www.atcc.org) or other
known or commercial sources.
When eukaryotic host cells are employed, polyadenlyation or transcription terminator
sequences are typically incorporated into the vector. An example of a terminator sequence is the
polyadenlyation sequence from the bovine growth hormone gene. Sequences for accurate
splicing of the transcript can also be included. An example of a splicing sequence is the VPl
intron from SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)). Additionally, gene sequences to
control replication in the host cell can be incorporated into the vector, as known in the art
Purification of an antibody
An anti-IL-12 antibody can be recovered and purified from recombinant cell cultures
by well-known methods including, but not limited to, protein A purification, ammonium
sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction chromatography, affinity
chromatography, hydroxylapatite chromatography and lectin chromatography. High
performance liquid chromatography ("HPLC") can also be employed for purification. See,
e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John
Wiley & Sons. NY, NY. (1997-2001), e.g., Chapters 1, 4. 6. 8. 9, 10, each entirely
incorporated herein by reference.
Antibodies of the present invention include naturally purified products, products of
chemical synthetic procedures, and products produced by recombinant techniques from a
eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells.
Depending upon the host employed in a recombinant production procedure, the antibody of the
present invention can be glycosylated or can be non-glycosylated, with glycosylated preferred.
Such methods are described in many standard laboratory manuals, such as Sambrook, supra,
Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, Protein
Science, supra. Chapters 12-14, all entirely incorporated herein by reference.
Anti-IL-12 Antibodies
The isolated antibodies of the present invention comprise an antibody encoded by any
one of the polynucleotides of the present invention as discussed more fully herein, or any isolated
or prepared antibody. Preferably, the human antibody or antigen-binding fragment binds human
IL-12 and, thereby partially or substantially neutralizes at least one biological activity of the
protein. An antibody, or specified portion or variant thereof, that partially or preferably
substantially neutralizes at least one biological activity of at least one IL-12 protein or fragment
can bind the protein or fragment and thereby inhibit activitys mediated through the binding of EL-
12 to the IL-12 receptor or through other IL-12-dependent or mediated mechanisms. As used
herein, the term "neutralizing antibody" refers to an antibody that can inhibit an IL-12-dependent
activity by about 20-120%, preferably by at least about 10,20, 30,40, 50,55, 60,65,70, 75, 80,
85,90,91, 92,93, 94, 95,96, 97,98,99, 100% or more depending on the assay. The capacity of
an anti-IL-12 antibody to inhibit an IL-12-dependent activity is preferably assessed by at least
one suitable IL-12 protein or receptor assay, as described herein and/or as known in the art. A
human antibody of the invention can be of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype
and can comprise a kappa or lambda light chain. In one embodiment, the human antibody
comprises an IgG heavy chain or defined fragment, for example, at least one of isotypes, IgGl,
IgG2, IgG3 or IgG4. Antibodies of this type can be prepared by employing a transgenic mouse
or other trangenic non-human mammal comprising at least one human light chain (e.g., IgG, IgA
and IgM (e.g., yl, y2, y3, y4) transgenes as described herein and/or as known in the art. In
another embodiment, the anti-human IL-12 human antibody comprises an IgG I heavy chain and
a IgGl light chain.
At least one antibody of the invention binds at least one specified epitope specific to at
least one IL-12 protein, subunit, fragment, portion or any combination thereof. The at least
one epitope can comprise at least one antibody binding region that comprises at least one
portion of said protein, which epitope is preferably comprised of at least one extracellular,
soluble, hydrophillic, external or cytoplasmic portion of said protein. The at least one
specified epitope can comprise any combination of at least one amino acid sequence of atieast
1-3 amino acids to the entire specified portion of contiguous amino acids of the SEQ ED NO:9.
Generally, the human antibody or antigen-binding fragment of the present invention
will comprise an antigen-binding region that comprises at least one human complementarity
determining region (CDR1, CDR2 and CDR3) or variant of at least one heavy chain variable
region and at least one human complementarity determining region (CDR1, CDR2 and CDR3)
or variant of at least one light chain variable region. As a non-limiting example, the antibody
or antigen-binding portion or variant can comprise at least one of the heavy chain CDR3
having the amino acid sequence of SEQ ID NO:3, and/or a light chain CDR3 having the amino
acid sequence of SEQ ID NO:6. In a particular embodiment, the antibody or antigen-binding
fragment can have an antigen-binding region that comprises at least a portion of at least one
heavy chain CDR (i.e.. CDR1, CDR2 and/or CDR3) having the amino acid sequence of the
corresponding CDRs 1. 2 and/or 3 (e.g.. SEQ ID NOS: 1. 2, and/or 3). In another particular
embodiment, the antibody or antigen-binding portion or variant can have an antigen-binding
region that comprises at least a portion of at least one light chain CDR (i.e., CDR1. CDR2
and/or CDR3) having the amino acid sequence of the corresponding CDRs 1, 2 and/or 3 (e.g.,
SEQ ID NOS: 4. 5, and/or 6). In a preferred embodiment the three heavy chain CDRs and the
three light chain CDRs of the anitbody or antigen-binding fragment have the amino acid
sequence of the corresponding CDR of at least one of mAb 12B75, C340, or any others as
described herein. Such antibodies can be prepared by chemically joining together the various
portions (e.g., CDRs, framework) of the antibody using conventional techniques, by preparing
and expressing a (i.e., one or more) nucleic acid molecule that encodes the antibody using
conventional techniques of recombinant DNA technology or by using any other suitable
method.
The anti-IL-12 antibody can comprise at least one of a heavy or light chain variable
region having a defined amino acid sequence. For example, in a preferred embodiment, the
anti-EL-12 antibody comprises at least one of at least one heavy chain variable region,
optionally having the amino acid sequence of SEQ ID NO:7 and/or at least one light chain
variable region, optionally havingithe amino acid sequence of SEQ ID NO:8.' antibodies that
bind to human IL-12 and that comprise a defined heavy or light chain variable region can be
prepared using suitable methods, such as phage display (Katsube, Y., et al, Int J Mol. Med,
l(5):863-868 (1998)) or methods that employ transgenic animals, as known in the art and/or as
described herein. For example, a transgenic mouse, comprising a functionally rearranged
human immunoglobulin heavy chain transgene and a transgene comprising DNA from a
human immunoglobulin light chain locus that can undergo functional rearrangement, can be
immunized with human IL-12 or a fragment thereof to elicit the production of antibodies. If
desired, the antibody producing cells can be isolated and hybridomas or other immortalized
antibody-producing cells can be prepared as described herein and/or as known in the art.
Alternatively, the antibody, specified portion or variant can be expressed using the encoding
nucleic acid or portion thereof in a suitable host cell.
The invention also relates to antibodies, antigen-binding fragments, immunoglobulin
chains and CDRs comprising amino acids in a sequence that is substantially the same as an
amino acid sequence described herein. Preferably, such ;antibodies or antigen-binding
fragments and antibodies comprising such chains or CDRs can bind human IL-12 with high
affinity (e.g., KD less than or equal to about 10-9 M). Amino acid sequences that are
substantially the same as the sequences described herein include sequences comprising
conservative amino acid substitutions, as well as amino acid deletions and/or insertions. A
conservative amino acid substitution refers to the replacement of a first amino acid by a second
amino acid that has chemical and/or physical properties (e.g, charge, structure, polarity,
hydrophobicity/ hydrophilicity) that are similar to those of the first amino acid. Conservative
substitutions include replacement of one amino: acid by another within the following groups:
lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate (E); asparagine (N),
glutamine (Q), serine (S), threonine (T), tyrosine (Y). K, R, H, D and E; alanine (A), valine
(V), leucine (L), isoleucine (I), proline (

(M), cysteine (C) and glycine (G); F, W and Y; C, S and T.
Amino Acid Codes
The amino acids that make up anti-IL-12 antibodies of the present invention are often
abbreviated. The amino acid designations can be indicatedby designating the amino acid by
its single letter code, its three letter code; name, or three nucleotide codon(s) as is well
understood in the art (see Alberts, B., et al., Molecular Biology of The Cell, Third Ed., Garland
Publishing, lnc.,New York, 1994):
An anti-IL-12 antibody of the present invention can include one or more amino acid
substitutions, deletions or additions, either from natural mutations or human manipulation, as
specified herein.
Of course, the number of amino acid substitutions a skilled artisan would make
depends on many factors, including those described above. Generally speaking, the number of
amino acid substitutions, insertions or deletions for any given anti-IL-12 Ig-derived protein,
fragment or variant will not be more than 40, 30, 20, 19, 18. 17, 16, 15, 14, 13, 12, 11, 10, 9, 8,
7, 6, 5, 4, 3, 2, 1, such as 1-30 or any range or value therein, as specified herein.
Amino acids in an anti-IL-12 antibody of the present invention that are essential for
function can be identified by methods known in the art, such as site-directed mutagenesis or
alanine-scanning mutagenesis (e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells,
Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at
every residue in the molecule. The resulting mutant molecules are then tested for biological
activity, such as, but not limited to at least one IL-12 neutralizing activity. Sites that are
critical for antibody binding can also be identified by structural analysis such as
crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith, et al., J. Mol.
Biol. 224:899-904 (1992) and de Vos, et al., Science 255:306-312 (1992)).
Anti-IL-12 antibodies of the present invention can include, but are not limited to, at
least one portion, sequence or combination selected from 5 to all of the contiguous amino acids
of at least one of SEQ ID NOS: 1, 2,3, 4, 5, 6.
IL-12 antibodies or specified portions or variants of the present invention can include,
but are not limited to, at least one portion, sequence or combination selected from at least 3-5
contiguous amino acids of SEQ ID NO:l, 5-17 contiguous amino acids of SEQ ID NO:2,5-10
contiguous amino acids of SEQ ID NO:3, 5-11 contiguous amino acids of SEQ ID NO:4, 5-7
contiguous amino acids of SEQ ID NO:5; 5-9 contiguous amino acids of SEQ ID NO:6;
Leu21, Lys76, Met83, Ser85 of SEQ ID NO:7.
A(n) anti-IL-12 antibody can further optionally comprise a polypeptide of at least one
of 70-100% of 5, 17, 10, 11, 7, 9, 119, or 108 contiguous amino acids of at least one of SEQ
IDNOS:l,2,3,4,5,6,7or8.
"In one embodiment, the amino acid sequence of an immunoglobulin chain, or portion
thereof (e.g., variable region, CDR) has about 70-100% identity (e.g., 70, 71, 72, 73,. 74, 75,
76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100
or any range or value therein) to the amino acid sequence of the corresponding chain of at least
one of SEQ ID NOS:7, 8. For example, the amino acid sequence of a light chain variable
region can be compared with the sequence of SEQ ID NO: 8, or the amino acid sequence of a
heavy chain CDR3 can be compared with SEQ ID NO:3. Preferably, 70-100% amino acid
identity (i.e., 90, 91, 92, 93. 94. 95. 96, 97, 98, 99, 100 or any range or value therein) is
determined using a suitable computer algorithm, as known in the art.
Exemplary heavy chain and light chain variable regions sequences are provided in SEQ
ID NOS: 7 and 8. The antibodies of the present invention, or specified variants thereof, can
comprise any number of contiguous amino acid residues from an antibody of the present
invention, wherein that number is selected from the group of integers consisting of from 10-
100% of the number of contiguous residues in an anti-IL-12 antibody. Optionally, this
subsequence of contiguous amino acids is at least about 10. 20, 30, 40, 50. 60, 70, 80, 90, 100,
110,120, 130, 140, 150, 160. 170, 180, 190,200,210, 220, 230,240,250 or more amino acids in
length, or any range or value therein. Further, the number of such subsequences can be any
integer selected from the group consisting of from 1 to 20, such as at least 2, 3,4, or 5.
As those of skill will appreciate, the present invention includes at least one biologically
active antibody of the present invention- Biologically active antibodies have a specific activity at
least 20%, 30%, or 40%, and preferably at least 50%, 60%, or 70%, and most preferably at least
80%, 90%, or 95%-1000% of that of the native (non-synthetic), endogenous or related and
known antibody. Methods of assaying and quantifying measures of enzymatic activity and
substrate specificity, are well known to those of skill in the art.
In another aspect, the invention relates to human antibodies and antigen-binding
fragments, as described herein, which are modified by the covalent attachment of an organic
moiety. Such modification can produce an antibody or antigen-binding fragment with
improved pharmacokinetic properties (e.g., increased in vivo serum half-life). The organic
moiety can be a linear or branched hydrophilic polymeric group, fatty acid group, or fatty acid
ester group. In particular embodiments, the hydrophilic polymeric group can have a molecular
weight of about 800 to about 120,000 Daltons and can be a polyalkane glycol (e.g.,
polyethylene glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymer, amino acid
polymer or polyvinyl pyrolidone, and the fatty acid or fatty acid ester group can comprise from
about eight to about forty carbon atoms.
The modified antibodies and antigen-binding fragments of the invention can comprise
one or more organic moieties that are covalently bonded, directly or indirectly, to the antibody.
Each organic moiety that is bonded to an antibody or antigen-binding fragment of the
invention can independently be a hydrophilic polymeric group, a fatty acid group or a fatty
acid ester group. As used herein, the term "fatty acid" encompasses mono-carboxylic acids
and di-carboxylic acids. A "hydrophilic polymeric group," as the term is used herein, refers to
an organic polymer that is more soluble in water than in octane. For example, polylysine is
more soluble in water than in octane. Thus, an antibody modified by the covalent attachment
of polylysine is encompassed by the invention. Hydrophilic polymers suitable for modifvinu
antibodies of the invention can be linear or branched and inciude. for example, polyalkanc
glycols (e.g.. PEG, monomethoxy-polycthylene glycol (mPEG). PPG and the like),
carbohydrates (e.g., dextran. cellulose, oligosaccharides, polysaccharides and the like),
polymers of hydrophilic amino acids (e.g., polylysine. polyarginine, polyaspartate and the
like), polyalkane oxides (e.g., polyethylene oxide. polypropylene oxide and the like) and
polyvinyl pyrolidone. Preferably, the hydrophilic polymer that modifies the antibody of the
invention has a molecular weight of about 800 to about 150,000 Daltons as a separate
molecular entity. For example PEG5000 and PEG20000 wherein the subscript is the average
molecular weight of the polymer in Daltons, can be used. The hydrophilic polymeric
group can be substituted with one to about six alkyl, fany acid or fatty acid ester groups.
Hydrophilic polymers that are substituted with a fatty acid or fatty acid ester group can be
prepared by employing suitable methods. For example, a polymer comprising an amine group
can be coupled to a carboxylate of the fatty acid or fatty acid ester, and an activated
carboxylate (e.g., activated with N, N-carbonyl diimidazole) on a fatty acid or fatty acid ester
can be coupled to a hydroxyl group on a polymer.
Fatty acids and fatty acid.esters suitable for modifying antibodies of the invention can
be saturated or can contain one or more units of unsaruration. Fatty acids that are suitable for
modifying antibodies of the invention include, for example, n-dodecanoate (C,,, laurate), n-
tetradecanoate (Cl4, myristate), n-octadecanoate (C18, stearate), n-eicosanoate (CM, arachidatc)
, n-docosanoate (C22, behenate), n-triacontanoatc (C30), n-tetracontanoate (C40), cis-?9-
octadecanoate (C18, oleate), all cis-?5,8,l 1,14-eicosatetraenoate (C20, arachidonate),
octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like.
Suitable fatty acid esters include mono-esters of dicarboxylic acids that comprise a linear or
branched lower alkyl group. The lower alkyl group can comprise from one to about twelve,
preferably one to about six, carbon atoms.
The modified human antibodies and antigen-binding fragments can be prepared using
suitable methods, such as by reaction with one or more modifying agents. A "modifying
agent" as the term is used herein, refers to a suitable organic group (e.g., hydrophilic polymer,
a fatty acid, a fatty acid ester) that comprises an activating group. An "activating group" is a
chemical moiety or functional group that can, under appropriate conditions, react with a
second chemical group thereby forming a covalent bond between the modifying agon and the
second chemical group. For example, amine-reactive activating groups include electrophilic
groups such as tosylate, mesylate, halo (chloro, bromo. fluoro, iodo), N-hydroxysuccinimidyl
esters (NHS), and the like. Activating groups that can react with thiols include, for example,
maleimide, iodoacetyl, acrylolyl, pyridyl disulfides, 5-thioI-2-nitrobenzoic acid thiol (TNB-
thiol), and the like. An aldehyde functional group can be coupled to amine- or hydrazide-
containing molecules, and an azide group can react with a trivalent phosphorous group to form
phosphoramidate or phosphorimide linkages. Suitable methods to introduce activating groups
into molecules are known in the art (see for example. Herrnanson, G. T., Bioconjugate
Techniques. Academic Press: San Diego, CA (1996)). An activating group can be bonded
directly to the organic group (e.g., hydrophilic polymer, fatty acid, fatty acid ester), or through
a linker moiety, for example a divalent C1-C12 group wherein one or more carbon atoms can be
replaced by a heteroatom such as oxygen, nitrogen or sulfur. Suitable linker moieties include,
for example, tetraethylene glycol, -(CH2)3-, -NH-(CH2)6-NH-, -(CH2)2-NH- and -CHv-O-CH2-
CH2-0-CH2-CH2-O-CH-NH-. Modifying agents that comprise a linker moiety can be
produced, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-
ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of l-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (EDC) to form an amide bond between the free amine and
the fatty acid carboxylate. The Boc protecting group can be removed from the product by
treatment with trifluoroacetic acid (TFA) to expose a primary amine that can be coupled to
another carboxylate as described, or can be reacted with maleic anhydride and the resulting
product cyclized to produce an activated maleimido derivative of the fatty acid. (See, for
example, Thompson, et al., WO 92/16221 the entire teachings of which are incorporated
herein by reference.)
The modified antibodies of the invention can be produced by reacting a human
antibody or antigen-binding fragment with a modifying agent. For example','the organic
moieties can be bonded to the antibody in a non-site specific manner by employing an amine-
reactive modifying agent, for example, an NHS ester of PEG. Modified human antibodies or
antigen-binding fragments can also be prepared by reducing disulfide bonds (e.g., intra-chain
disulfide bonds) of an antibody or antigen-binding fragment. The reduced antibody or antigen-
binding fragment can then be reacted with a thiol-reactive modifying agfent to produce the
modified antibody of the invention. Modified human antibodies and antigen-binding
fragments comprising an organic moiety that is bonded to specific sites of an antibody of the
present invention can be prepared using suitable methods, such as reverse proteolysis (Fisch et
al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate Chem., 5:411-417
(1994); Kumaran et al., Protein Sci. 6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1):
59-68 (1996); Capellas et al., Biotechnol. Bioeng, 56(4):456-463 (1997)), and the methods
described in Herrnanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, CA
(1996).
ANTI-IDIOTYPE ANTIBODIES TO ANTI-IL-12 IG DERIVED PROTEIN
COMPOSITIONS
In addition to monoclonal or chimeric anti-lL-12 antibodies, the present
invention is also directed to an anti-idiotypic (anti-Id) antibody specific for such antibodies of
the invention. An anti-Id antibody is an antibody which recognizes unique determinants
generally associated with the antigen-binding region of another antibody. The anti-Id can be
prepared by immunizing an animal of the same species and genetic type (e.g. mouse strain) as
the source of the Id antibody with the antibody or a CDR containing region thereof The
immunized animal will recognize and respond to the idiotypic determinants of the immunizing
antibody and produce an anti-Id antibody. The anti-Id antibody may also be used as an
"immunogen" to induce an immune response in yet another animal, producing a so-called
anti-anti-Id antibody.
ANTI-IL-12 IG DERIVED PROTEIN COMPOSITIONS
The present invention also provides at least one anti-IL-12 antibody composition
comprising at least one, at least two, at least three, at least four, at least five, at least six or
more anti-IL-12 antibodies thereof, as described herein and/or as known in the art that are
provided in a non-naturally occurring composition, mixture or form. Such compositions
comprise non-naturally occurring compositions comprising at least one or two full length, C-
and/or N-terminally deleted variants, domains, fragments, or specified variants, of the anti-EL-
12 antibody amino acid sequence selected from the group consisting of 70-100% of the
contiguous amino acids of SEQ ID NOS:l, 2,3,4, 5, 6, 7 or 8, or specified fragments, domains
or variants thereof. Preferred anti-IL-12 derived protein, fragment or variant compositions
include at least one or two full length, fragments, domains or variants as at least one CDR
containing portions of the anti-IL-12 antibody sequence of 70-100% of SEQ ID NOS: 1,2, 3,
4, 5, 6, or specified fragments, domains or variants thereof. Further preferred compositions
comprise 40-99% of at least one of 70-100% of SEQ ID NOS: 1,2,3,4.5,6, orspecified
fragments, domains or variants thereof. Such composition percentages are by weight, volume,
concentration, molarity, or molality as liquid or dry solutions, mixtures, suspension, emulsions
or colloids, as known in the art or as described herein.
Anti-IL-12 annbody compositions of the present invention can further comprise at
least one of any suitable and effective amount of a composition or pharmaceutical composition
comprising at least one anti-IL-12 antibody to a cell, tissue, organ, animal or patient in need of
such modulation, treatment or therapy, optionally further comprising at least one selected from
at least one TNF antagonist (e.g., but not limited to a TNF antibody or fragment, a soluble
TNF receptor or fragment, fusion proteins thereof, or a small molecule TNF antagonist), an
antirheumatic (e.g., methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, gold
sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), a muscle relaxant,
a narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a
sedative, a local anethetic. a neuromuscular blocker, an antimicrobial (e.g., aminoglycoside, an
antifungal, an antiparasitic, an antiviral, a carbapenem, cephalosporin, a flurorquinolone, a
macrolide, a penicillin, a sulfonamide, a tetracycline, another antimicrobial), an antipsoriatic, a
corticosteriod, an anabolic steroid, a diabetes related agent, a mineral, a nutritional, a thyroid
agent, a vitamin, a calcium related hormone, an antidiarrheal, an antitussive, an antiemetic, an
antiulcer, a laxative, an anticoagulant, an erythropieitin (e.g., epoetin alpha), a filgrastim (e.g.,
G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), an immunization, an
immunoglobulin, an immunosuppressive (e.g., basilixirriab, cyclosporine, daclizumab), a
growth hormone, a hormone replacement drug, an estrogen receptor modulator, a mydriatic, a
cycloplegic, an alkylating agent, an antimetabolite, a mitotic inhibitor, a radiopharmaceutical,
an antidepressant, antimanic agent, an antipsychotic, an anxiolytic, a hypnotic, a
sympathomimetic, a stimulant, donepezil, tacrine, an asthma medication, a beta agonist, an
inhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn, an epinephrine or
analog, dornase alpha (Pulmozyme), a cytokine or a cytokine antagonist. Non-limiting
examples of such cytokines include, but are not limted to, any of IL-1 to IL-23. Suitable
dosages are well known in the art. See, e.g., Wells et al.„eds., Pharmacotherapy Handbook, 2nd
Edition, Appleton and Lange, Stamford, CT (2000); PDR Pharmacopoeia, Tarascon Pocket
Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda. CA (2000), each of
which references are entirely incorporated herein by reference.
Such anti-cancer or anti-infectives can also include toxin molecules that are
associated, bound, co-formulated or co-administered with at least one antibody of the present
invention.- The toxin can optionally act to selectively kill the pathologic cell or tissue. The
pathologic cell can be a cancer or other cell. Such toxins can be, but are not limited to,
purified or recombinant toxin or toxin fragment comprising at least one functional cytotoxic
domain of toxin, e.g., selected from at least one of ricin. diphtheria toxin, a venom toxin, or a
bacterial toxin. The term toxin also includes both endotoxins and exotoxins produced by any
naturally occurring, mutant or recombinant bacteria or S'iruses which may cause any
pathological condition in humans and other mammals, including toxin shock, which can result
in death. Such toxins may include, but are not limited, to, enterotoxigenic E. coli heat-labile
enterotoxin (LT), heat-stable enterotoxin (ST), Shigella cytotoxin, Aeromonas enterotoxins,
toxic shock syndrome toxin-1 (TSST-1), Staphylococcal enterotoxin A (SEA), B (SEB), or C
(SEC), Streptococcal enterotoxins and the like. Such bacteria include, but are not limited to,
strains of a species of enterotoxigenic E. coli (ETEC), emerohemorrhagic E. coli (e.g., strains
of serotype 0157:H7), Staphylococcus species (e.g., Staphylococcus aureus. Staphylococcus
pyogenes), Shigella species (e.g., Shigella dysenteriae. Shigella flexneri, Shigella boydii, and
Shigella sonnei), Salmonella species (e.g., Salmonella typhi. Salmonella cholera-suis.
Salmonella enteritidis), Clostridium species (e.g., Clostridium perfringens, Clostridium
dificile, Clostridium botulinum), Camphlobacter species (e.g., Camphlobacter jejuni,
Camphlobacter fetus), Heliobacter species, (e.g., Heliobacter pylori), Aeromonas species (eg.,
Aeromonas sobria, Aeromonas hydrophila, Aeromonas caviae), Pleisomonas shigelloides,
Yersina enterocolitica, Vibrios species (e.g., Vibrios cholerae. Vibrios parahemolyticus),
Klebsiella species, Pseudomonas aeruginosa, and Streptococci. See, e.g., Stein, ed.,
INTERNAL MEDICINE, 3rd ed., pp 1-13, Little, Brown and Co., Boston, (1990); Evans etal,
eds., Bacterial Infections of Humans: Epidemiology and Control, 2d. Ed.', pp 239-254, Plenum
Medical Book Co., New York (1'991); Mandell et al, Principles and Practice of Infectious
Diseases, 3d. Ed., Churchill Livingstone, New York (1990); Berkow et al, eds., The Merck
Manual, 16th edition, Merck and Co., Rahway, N.J., 1992; Wood et al, FEMS Microbiology
Immunology, 76:121-134 (1991s); Marracket al, Science, 248:705-711 (1990), the contents of
which references are incorporated entirely herein by reference.
Anti-IL-12 antibody compounds, compositions or combinations of the present
invention can further comprise at least one of any suitable auxiliary, such as, but not: limited to,
diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like.
Pharmaceutically acceptable auxiliaries are preferred. Non-limiting examples of, and methods
of preparing such sterile solutions arc well known in the art. such as, but limited to, Gennaro.
Ed., Remington 's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, PA)
1990. Pharmaceutically acceptable carriers can be routinely selected that are suitable for the
mode of administration, solubility and/or stability of the anti-IL-12 antibody, fragment or
variant composition as well known in the art or as described herein.
Pharmaceutical excipients and additives useful in the present composition include but
are not limited to proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars,
including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as
alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers),
which can be present singly or in combination, comprising alone or in combination 1-99.99%
by weight or volume. Exemplary protein excipients include serum albumin such as human
serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like.
Representative amino acid/antibody components, which1 can also function in a buffering
capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid,
cysteine, lysine, leucine, isoleucine, valine, methionine,,phenylalanine, aspartame, and the
like. One preferred amino acid is glycine.
Carbohydrate excipients suitable for use in the invention include, for example,
monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the
like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like;
polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, .starches, and the like;
and alditols, such as mannitol, xylitol, maltitol, lactitol,,xylitol sorbitol {glucitol), myoinositol
and the like. Preferred carbohydrate excipients for use in the present invention are mannitol,
trehalose, and raffinose.
Anti-IL-12 antibody compositions can also include a buffer or a pH adjusting agent;
typically, the buffer is a salt prepared from an organic acid or base. Representative buffers
include organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic
acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine
hydrochloride, or phosphate buffers. Preferred buffers for use in the present compositions are
organic acid salts such as citrate.
Additionally, anti-IL-12 antibody compositions of the invention can include polymeric
excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g.,
cyclodextrins. such as 2-hydroxypropyl-p-cyclodextrin), polyethylene glycols, flavoring
agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g.,
polysorbates such as "TWEEN 20" and 'TWEEN 80"), lipids (e.g., phospholipids, fatty acids),
steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).
These and additional known pharmaceutical excipients and/or additives suitable for
use in the anti-IL-12 antibody, portion or variant compositions according to the invention are
known in the art, e.g., as listed in "Remington: The Science & Practice of Pharmacy", 19* ed.,
Williams & Williams, (1995), and in the "Physician's Desk Reference", 52nd ed., Medical
Economics, Montvale, NJ (1998), the disclosures of which are entirely incorporated herein by
reference. Preferrred carrier or excipient materials are carbohydrates (e.g., saccharides and
alditols) and buffers (e.g., citrate).or polymeric agents.
Formulations
As noted above, the invention provides for stable formulations, which is
preferably a phosphate buffer with saline or a chosen salt, as well as preserved solutions and
formulations containing a preservative as well as multi-use preserved formulations suitable for
pharmaceutical or veterinary use,,comprising at least one anti-IL-12 antibody in a
pharmaceutically acceptable formulation. Preserved formulations contain at least one known
preservative or optionally selected from the group consisting of at least one phenol, m-cresol,
p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol,
formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparaben (methyl,
ethyl, propyl, butyl and the like), benzalkom'um chloride, benzethonium chloride, sodium
dehydroacetate and thimerosal, or mixtures thereof in an aqueous diluent Any suitable
concentration or mixture can be used as known in the art, such as 0.001-5%, or any range or
value therein, such as, but not limited to 0.001, 0.003,0.005,0.009,0.01, 0.02, 0.03,0.05,
0.09,0.1, 0.2,0.3, 0.4., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0,1.1,1.2,1.3,1.4,1.5, pt.6, 1.7, 1.8, 1.9.2.0,
2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3,4.5,
4.6,4.7, 4.8,4.9, or any range or value therein. Non-limiting examples include, no
preservative, 0.1-2% m-cresol (e.g., 0.2, 0.3. 0.4, 0.5,0.9, 1.0%), 0.1-3% benzyl alcohol (e.g.,
0.5, 0.9, 1.1., 1.5, 1.9, 2.0, 2.5%),; 0.001-0.5% thimerosal (e.g., 0.005, 0.01), 0.001-2.0%
phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075,
0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05,0.075, 0.09, 0.1,0.2, 0.3, 0.5, 0.75,
0.9, 1.0%), and the like.
As noted above, the invention provides an article of manufacture, comprising
packaging material and at least one vial comprising a solution of at least one anti-IL-12
antibody with the prescribed buffers and/or preservatives, optionally in an aqueous diluent,
wherein said packaging material comprises a label that indicates that such solution can be held
over a period of 1. 2, 3, 4. 5. 6. 9, 12. 18. 20. 24, 30, 36. 40, 48, 54, 60. 66, 72 hours or greater.
The invention further comprises an article of manufacture, comprising packaging material, a
first viai comprising lyophilized at least one anti-IL-12 antibody, and a second viai comprising
an aqueous diluent of prescribed buffer or preservative, wherein said packaging material
comprises a label that instructs a patient to reconstitute the at least one anti-IL-12 antibody in
the aqueous diluent to form a solution that can be held over a period of twenty-four hours or
greater.
The at least one anti-IL-12antibody used in accordance with the present invention can
be produced by recombinant means, including from mammalian cell or transgenic
preparations, or can be purified from other biological sources, as described herein or as known
in the art.
The range of at least one anti-IL-12 antibody in the product of the present invention
includes amounts yielding upon reconstitution, if in a wet/dry system, concentrations from
about 1.0 ug/ml to about 1000 mg/nil, although lower and higher concentrations are operable
and are dependent on the intended delivery vehicle, e.g., solution formulations will differ from
transdermal patch, pulmonary, transmucosal, or osmotic or micro pump methods.
Preferably, the aqueous diluent optionally further comprises a pharmaceutically
acceptable preservative. Preferred preservatives include those selected from the group
consisting of phenol, m-cresoi, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben
(methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride,
sodium dehydroacetate and thimerosal, or mixtures thereof The concentration of preservative
used in the formulation is a concentration sufficient to yield an anti-microbial effect. Such
concentrations are dependent on the preservative selected and are readily determined by the
skilled artisan.
Other excipients, e.g. isotonicity agents; buffers, antioxidants, preservative enhancers,
can be optionally and preferably added to the diluent. An isotonicity agent, such as glycerin, is
commonly used at known concentrations. A physiologically tolerated buffer is preferably
added to provide improved pH control. The formulations can cover a wide range of pHs, such
as from about pH 4 to about pH 10, and preferred ranges from about pH 5 to about pH 9, and a
most preferred range of about 6.0 to about 8.0. Preferably the formulations of the present
invention have pH between about 6.8 and about 7.8. Preferred buffers include phosphate
buffers, most preferably sodium phosphate, particularly phosphate buffered saline (PBS).
Other additives, such as a pharmaceutically acceptable solubilizers like Tween
20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorbitan
monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic F68
(polyoxyethylene polyoxypropylene block copolymers), and PEG (polyethylene glycol) or
non-ionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or 188, Pluronic® polyls,
other block co-polymers, and chelators such as EDTA and EGTA can optionally be added to
the formulations or compositions to reduce aggregation. These additives are particularly
useful if a pump or plastic container is used to administer the formulation. The presence of
pharmaceutically acceptable surfactant mitigates the propensity for the protein to aggregate.
The formulations of the present invention can be prepared by a process which
comprises mixing at least one anti-IL-12 antibody and a preservative selected from the group
consisting of phenol, m-cresol, p-cresbl o-cfesol, chlorpcresol, benzyl alcohol, alkylparaben,
(methyl, ethyl, propyl, butyl and the like), benzalkoniurn chloride, benzethonium chloride,
sodium dehydroacetate and thimeirosal or mixtures thereof in an aqueous diluent. Mixing the
at least one anti-IL-12 antibody and preservative in an aqueous diluent is carried out using
conventional dissolution and mixing procedures. To prepare a suitable formulation, for
example, a measured amount of at least one anti-IL-12 antibody in buffered solution is
combined with the desired preservative in a buffered solution in quantities sufficient to provide
the protein and preservative at the desired concentrations. Variations of this process would be
recognized by one of ordinary skill in the art. For example, the order the components are
added, whether additional additives are used, the temperature and pH at which the formulation
is prepared, are all factors that can be optimized for the concentration and means of
administration used.
The claimed formulations can be provided to patients as clear solutions or as
dual vials comprising a vial of lypphilized at least one anti-IL-12 antibody that is reconstituted
with a second vial containing water, a preservative and/or excipients, preferably a phosphate
buffer and/or saline and a chosen salt, in an aqueous diluent. Either a single solution vial or
dual vial requiring reconstitution can be reused multiple times and can suffice for a single or
multiple cycles of patient treatment and thus can provide a more conver/xent treatment regimen
than currently available.
The present claimed articles of manufacture are useful for administration over
a period of immediately to twenty-four hours or greater., Accordingly, the presently claimed
articles of manufacture offer significant advantages to the patient. Formulations of the
invention can optionally be safely stored at temperatures of from about 2 to about 40°C and
retain the biologically activity of the protein for extended periods of time, thus, allowing a
package label indicating that the solution can be held and/or used over a period of 6, 12,18,
24, 36,48, 72, or 96 hours or greater. If preserved diluent is used, such label can include use
up to 1-12 months, one-half, one and a half, and/or two years.
The solutions of at least one anti-IL-12 antibody in the invention can be
prepared by a process that comprises mixing at least one antibody in an aqueous diluent.
Mixing is carried out using conventional dissolution and mixing procedures. To prepare a
suitable diluent, for example, a measured amount of at least one antibody in water or buffer is
combined in quantities sufficient to provide the protein and optionally a preservative or buffer
at the desired concentrations. Variations of this process would be recognized by one of
ordinary skill in the art. For example, the order the components are added, whether additional
additives are used, the temperature and pH at which the formulation is prepared, are all factors
that can be optimized for the concentration and means of administration used.
The claimed products can be provided to patients as clear solutions or as dual
vials comprising a vial of lyophilized at least one anti-IL-12 antibody that is reconstituted with
a second vial containing the aqueous diluent. Either a single solution vial or dual vial
requiring reconstitution can be reused multiple times and can suffice for a single or multiple
cycles of patient treatment and thus provides a more convenient treatment regimen than
currently available.
The claimed products can be provided indirectly to patients by providing to
pharmacies, clinics, or other such institutions and facilities, clear solutions or dual vials
comprising a vial of lyophilized at least one anti-IL-12 antibody that is reconstituted with a
second vial containing the aqueous diluent. The clear solution in this case can be up to one
liter or even larger in size, providing a large reservoir from which smaller portions of the at
least one antibody solution can be retrieved one or multiple times for transfer into smaller vials
and provided by the pharmacy or clinic to their customers and/or patients.
Recognized devices comprising these single vial systems include those pen-
injector devices for delivery of a solution such as BD Pens, BD Autojector®, Humaject®'
NovoPen®, B-D®Pen, AutoPen®, and OptiPen®, GenotropinPen®; Genotronorm Pen®, Humatro
Pen®, Reco-Pen®, Roferon Pen®, Biojector®, iject®, J-tip Needle-Free Injector®, Intraject®,
Medi-Ject®, e.g., as made or developed by Becton Dickensen (Franklin Lakes, NJ,
www.bectondickenson.com), Disetronic (Burgdorf, Switzerland, www.disetronic.com; Bioject,
Portland, Oregon (www.bioject.com); National Medical Products , Weston Medical
(Peterborough, UK, www.weston-medical.com), Medi-Ject Corp (Minneapolis, MN,
www.mediject.com). Recognized devices comprising a dual vial system include those pen-
injector systems for reconstituting a jyophilized drug in a cartridge for delivery of the
reconstituted solution such as the HumatroPen®.
The products presently claimed include packaging material. The packaging
material provides, in addition to the information required by the regulatory agencies, the
conditions under which the product can be used. The packaging material of the present
invention provides instructions to the patient to reconstitute the at least one anti-IL-12
antibody in the aqueous diluent to form a solution and to use the solution over a period of 2-24
hours or greater for the two viai, wet/dry, product. For the single vial, solution product, the
label indicates that such solution can be used over a period of 2-24 hours or greater. The
presently claimed products are useful for human pharmaceutical product use.
The formulations of the present invention can be prepared by a process that
comprises mixing at least one anti-IL-12 antibody and a selected buffer, preferably a
phosphate buffer containing saline or a chosen salt. Mixing the at least one antibody and
buffer in an aqueous diluent is carried out using conventional dissolution and mixing
procedures. To prepare a suitable formulation, for example, a measured amount of at least one
antibody in water or buffer is combined with the desired buffering agent in water in quantities
sufficient to provide the protein and buffer at the desired concentrations. Variations of this
process would be recognized by one of ordinary skill in the art. For example, the order the
components are added, whether additional additives are used, the temperature and pH at which
the formulation is prepared, are all factors that can be optimized for the concentration and
means of administration used.
The claimed stable or preserved formulations can be provided to patients as
clear solutions or as dual vials comprising a vial of lyophilized at least one anti-IL-12 antibody
that is reconstituted with a second viai containing a preservative or buffer and excipients in an
aqueous diluent. Either a single solution vial or dual vial requiring reconstitution can be'
reused multiple times and can suffice for a single or multiple cycles of patient treatment and
thus provides a more convenient treatment regimen than currently available.
At least one anti-IL-12 antibody in either the stable or preserved formulations or
solutions described herein, can be administered to a patient in accordance with the present
invention via a variety of delivery methods including SC or IM injection; transdermal,
pulmonary, transmucosal, implant, osmotic pump, cartridge, micro pump, or other means
appreciated by the skilled artisan, as well-known in the art.
Therapeutic Applications
The present invention also provides a method for modulating or treating at least one
immune related disease, in a cell, tissue, organ, animal, or patient including, but not limited to,
at least one of rheumatoid arthritis, juvenile rheumatoid arthritis, systemic onset juvenile
rheumatoid arthritis, psoriatic arthritis, ankylosing spondilitis, gastric ulcer, seronegative
arthropathies, osteoarthritis, inflammatory bowel disease, ulcerative colitis, systemic lupus
erythematosis, antiphospholipid syndrome, iridocyclitis/uveitis/optic neuritis, idiopathic
pulmonary fibrosis, systemic vasculitis/wegener's granulomatosis, sarcoidosis,
orchitis/vasectomy reversal procedures, allergic/atopic diseases, asthma, allergic rhinitis,
eczema, allergic contact dermatitis, allergic conjunctivitis, hypersensitivity pneumonitis,
transplants, organ transplant rejection, graft-versus-host disease, systemic inflammatory
response syndrome, sepsis syndrome, gram positive sepsis, gram negative sepsis, culture
negative sepsis, fungal sepsis, neutropenic fever, urosepsis, meningococcemia.
trauma/hemorrhage, bums, ionizing radiation exposure, acute pancreatitis, adult respiratory
distress syndrome, rheumatoid arthritis, alcohol-induced hepatitis, chronic inflammatory
pathologies, sarcoidosis. Crohn's pathology, sickle cell anemia, diabetes, nephrosis, atopic
diseases, hypersensitity reactions, allergic rhinitis, hay fever, perennial rhinitis, conjunctivitis,
endometriosis, asthma, urticaria, systemic anaphalaxis, dermatitis, pernicious anemia,
hemolytic disesease, thrombocytopenia, graft rejection of any organ or tissue, kidney
translplant rejection, heart transplant rejection, liver transplant rejection, pancreas transplant
rejection, lung transplant rejection, bone marrow transplant (BMT) rejection, skin allograft
rejection, cartilage transplant rejection, bone graft rejection, small bowel transplant rejection,
fetal thymus implant rejection, parathyroid transplant rejection, xenograft rejection of any
organ or tissue, allograft rejection, anti-receptor hypersensitivity reactions, Graves disease,
Raynoud's disease, type B insulin-resistant diabetes, asthma, myasthenia gravis, antibody-
meditated cytotoxicity, type HI hypersensitivity reactions, systemic lupus erythematosus,
POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal
gammopathy, and skin changes syndrome), polyneuropathy, organomegaly, endocrinopathy,
monoclonal gammopathy, skin changes syndrome, antiphospholipid syndrome, pemphigus,
scleroderma, mixed connective tissue disease, idiopathic Addison's disease, diabetes mellitus,
chronic active hepatitis, primary billiary cirrhosis, vitiligo, vasculitis, post-MI cardiotomy
syndrome, type IV hypersensitivity , contact dermatitis, hypersensitivity pneumonitis, allograft
rejection, granulomas due to intracellular organisms, drug sensitivity, metabolic/idiopatfuc,
Wilson's disease, hemachromatosis, alpha-1-antitrypsin deficiency, diabetic retinopathy,
hashimoto's thyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axis evaluation, primary
biliary cirrhosis, thyroiditis, encephalomyelitis, cachexia, cystic fibrosis, neonatal chronic lung
disease, chronic obstructive pulmonary disease (COPD), familial hematophagocytic
lymphohistiocytosis, dermatologic conditions, psoriasis, alopecia, nephrotic syndrome,
nephritis, glomerular nephritis, acute renal failure, hemodialysis, uremia, toxicity,
preeclampsia, okt3 therapy, anti-cd3 therapy, cytokine therapy, chemotherapy, radiation
therapy (e.g., including but not limited toasthenia, anemia, cachexia, and the like), chronic
salicylate intoxication, and the like. See, e.g., the Merck Manual, 12th-17th Editions, Merck
& Company, Rahway, NJ (1972, 1977, 1982, 1987, 1992, 1999), Pharmacotherapy Handbook,
Wells et al., eds., Second Edition, Appleton and Lange, Stamford, Conn. (1998, 2000), each
entirely incorporated by reference.
The present invention also provides a method for modulating or treating at least one
cardiovascular disease in a cell, tissue, organ, animal, or patient, including, but not limited to,
MISSING AT THE TIME OF PUBLICATION
MISSING AT THE TIME OF PUBLICATION
concurrently, and/or after, at least one selected from at least one TNF antagonist (e.g., but not
limited to a TNF antibody or fragment, a soluble TNF receptor or fragment, fusion proteins
thereof, or a small molecule TNF antagonist), an antirheumatic (e.g., methotrexate, auranofm,
aurothioglucose, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine
sulfate, leflunomide. sulfasalzine). a muscle relaxant, a narcotic, a non-steroid anti-
inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anethetic, a
neuromuscular blocker, an antimicrobial (e.g., aminoglycoside, an antifungal, an antiparasitic,
an antiviral, a carbapenem, cephalosporin, a flurorquinolone, a macrolide, a penicillin, a
sulfonamide, a tetracycline, another antimicrobial), an antipsoriatic, a corticosteriod, an
anabolic steroid, a diabetes related agent, a mineral, a nutritional, a thyroid agent, a vitamin, a
calcium related hormone, an antidiarrheal. an antitussive, an antiemetic, an antiulcer, a
laxative, an anticoagulant, an erythropieitin (e.g., epoetin alpha), a filgrastim (e.g., G-CSF,
Neupogen), a sargramostim (GM-CSF, Leukine), an immunization, an immunoglobulin, an
immunosuppressive (e.g., basiliximab, cyclpsporine, daclizumab), a growth hormone, a
hormone replacement drug, an estrogen receptor modulator, a mydriatic, a cycloplegic, an
alkylating agent, an antimetabolite, a mitotic inhibitor, a radiopharmaceutical, an
antidepressant, antimanic agent, an antipsychotic, an anxiolytic, a hypnotic, a
sympathomimetic, a stimulant, donepezil, tacrine, an asthma medication, a beta agonist, an
inhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn, an epinephrine or
analog, domase alpha (Pulmozyme), a cytokine or a cytokine antagonistm. Suitable dosages
are well known in the art. See, e.g., Wells et al., eds., Pharmacotherapy Handbook, 2nd Edition,
Appleton and Lange, Stamford, CT (2000); PDR Pharmacopoeia, Tarascon Pocket
Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, CA (2000), each of
which references are entirely incorporated herein by reference.
TNF antagonists suitable for compositions, combination therapy; co-administration,
devices and/or methods of the present invention (further comprising at least one anti body,
specified portion and variant thereof, of the present invention), include, but are not limited to,
anti-TNF antibodies, antigen-binding fragments thereof, and receptor molecules which bind
specifically to TNF; compounds which prevent and/or inhibit TNF synthesis, TNF release or
its action on target cells, such as thalidomide, tenidap, phosphodiesterase inhibitors (e.g,
pentoxifylline and rolipram), A2b adenosine receptor agonists and A2b adenosine receptor
enhancers; compounds which prevent and/or inhibit TNF receptor signalling, such as mitogen
activated protein (MAP) kinase inhibitors; compounds which block and/or inhibit membrane
TNF cleavage, such as metalloproteinase inhibitors; compounds which block and/or inhibit
TNF activity, such as angiotensin converting enzyme (ACE) inhibitors (e.g., captopril); and
compounds which block and/or inhibit TNF production and/or synthesis, such as MAP kinase
inhibitors.
As used herein, a "tumor necrosis factor antibody," "TNF antibody," "TNF
antibody," or fragment and the like decreases, blocks, inhibits, abrogates or interferes with
TNF activity in vitro, in situ and/or preferably in vivo. For example, a suitable TNF human
antibody of the present invention can bind TNFa and includes anti-TNF antibodies, antigen-
binding fragments thereof, and specified mutants or domains thereof that bind specifically to
TNFa. A suitable TNF anttibody or fragment can also decrease block, abrogate, interfere,
prevent and/or inhibit TNF RNA, DNA or protein synthesis, TNF release, TNF receptor
signaling, membrane TNF cleavage, TNF activity, TNF production and/or synthesis.
Chimeric antibody cA2 consists of the antigen binding variable region of the high-
affinity neutralizing mouse anti-human TNFa IgGl antibody, designated A2, and the constant .
regions of a human IgGI, kappa immunoglobulin. The human IgGl Fc region improves
allogeneic antibody effector function, increases the circulating serum half-life and decreases
the immunogenicity of the antibody. The avidity and epitope specificity of the chimeric
antibody cA2 is derived from the variable region of the murine antibody A2. In a particular
embodiment, a preferred source for nucleic acids encoding the variable region of the murine
antibody A2 is the A2 hybridoma cell line.
Chimeric A2 (cA2) neutralizes the cytotoxic effect of both natural and recombinant
human TNFa in a dose dependent manner. From binding assays of chimeric antibody cA2 and
recombinant human TNFa, the affinity constant of chimieric antibody cA2 was calculated to be
1.04x1010M-1. Preferred methods for determining monoclonal antibody specificity and affinity
by competitive inhibition can be found! in Harlow, et al., antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988; Colligan et al.,
eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience,
New York, (1992-2000); Kozbor et al, Immunol. Today. 4:12-19 (1983); Ausubel etal, eds.
Current Protocols in Molecular Biology, Wiley Interscience, New York (1987-2000); and
Muller, Meth. Enzymol., 92:589-601 (1983), which references are entirely incorporated herein
by reference.
In a particular embodiment, murine monoclonal antibody A2 is produced by a cell line
designated c134A. Chimeric antibody cA2 is produced by a cell line designated cl68A.
Additional examples of monoclonal anti-TNF antibodies that can be used in the
present invention are described in the art (see, e.g., U.S. Patent No. 5,231,024; Moller, A. et
al., Cytokine 2(3):162-169 (1990); U.S. Application No. 07/943,852 (filed September 11,
1992); Rathjen et al, Intemational!Publicatioh No. WO 91/02078 (published February 21,
1991); Rubin et al, EPO Patent Publication No. 0 218 868 (published April 22, 1987); Yone et
al., EPO Patent Publication No. 0 288 088 (October 26. 1988); Liang, et al., Biochem. Biophys.
Res. Comm. 137:847-854 (1986); Meager, et al., Hybridoma 6:305-311 (1987); Fendly et al.,
Hybridoma 6:359-369 (19S7); Bringman, etal., Hybridoma 6:489-507 (1987); and Hirai, et
al., J. Immunol. Meth. 96:57-62 (1987), which references are entirely incorporated herein by
reference).
TNF Receptor Molecules
Preferred TNF receptor molecules useful in the present invention are those that bind
TNF with high affinity (see, e.g., Feldmann et at., International Publication No. WO
92/07076 (published April 30, 1992); Schall et al., Celt 67:361-370 (1990); and Loetscher et
al., Cell 67:351-359 (1990), which references are entirely incorporated herein by reference)
and optionally possess low immunogenicity. In particular, the 55 kDa (p55 TNF-R) and the 75
kDa (p75 TNF-R) TNF cell surface-receptors are useful in the present invention. Truncated
forms of these receptors, comprising the extracellular domains (ECD) of the receptors or
functional portions thereof (see, e.g., Corcoran et at., Eur. J. Biochem. 223:831-840 (1994)),
are also useful in the present invention. Truncated forms of the TNF receptors, comprising the
ECD, have been detected in urine and serum as 30 kDa and 40 kDa TNF inhibitory binding
proteins (Engelmann, H. etal., J. Biol. Chem. 265:1531-1536(1990)). TNF receptor
multimeric molecules and TNF immunoreceptor fusion molecules, and derivatives and
fragments or portions thereof, are additional examples of TNF receptor molecules which are
useful in the methods and compositions of the present invention. The TNF receptor molecules
which can be used in the invention are characterized by their ability to treat patients for
extended periods with good to excellent alleviation of symptoms and low toxicity. Low
immunogenicity and/or high affinity, as well as other undefined properties, can contribute to
the therapeutic results achieved.

TNF receptor multimeric molecules useful in the present invention comprise all or a
functional portion of the ECD of two or more TNF receptors linked via one or more
polypeptide linkers or other nonpeptide linkers, such as polyethylene glycol (PEG). The
multimeric molecules can further comprise a signal peptide of a secreted protein to direct
expression of the multimeric molecule. These multimeric molecules and methods for their
production have been described in U.S. Application No. 08/437,533 (filed May 9, 1995), the
content of which is entirely incorporated herein by reference.
TNF immunoreceptor fusion molecules useful in the methods and compositions of the
present invention comprise at least one portion of one or more immunoglobulin molecules and
all or a functional portion of one or more TNF receptors. These immunoreceptor fusion
molecules can be assembled as monomers, or hetero- or homo-multimers. The
immunoreceptor fusion molecules can also be monovalent or multivalent. An example of such
a TNF immunoreceptor fusion molecule is TNF receptor/IgG fusion protein. TNF
immunoreceptor fusion molecules and methods for their production have been described in the
an (Lesslauer et al.. Eur. J. Immunol. 27:2883-2886 (199.1); Ashkenazi et al., Proc. Natl.
Acad. Sci. USA 55:10535-10539 (1991); Peppel etal.,J. Exp. Med. 774:1483-1489 (1991);
Kolls et al., Proc. Natl. Acad. Sci. USA 97:215-219 (1994); Butler et al.. Cytokine 6(6):616-
623 (1994); Baker et al., Eur. J. Immunol. 24:2040-2048 (1994); Beutler et al., U.S. Patent No.
5,447,851; and U.S. Application No,08/442,133 (filed May 16, 1995), each of which
references are entirely incorporated herein by reference). Methods for producing
immunoreceptor fusion molecules can also be found in Capon et al., U.S. Patent No.
5,116,964; Capon et al., U.S. Patent No, 5,225,538; and Capon et al., Nature 337:525-531
(1989), which references are entirely incorporated herein by reference.
A functional equivalent, derivative, fragment or region of TNF receptor molecule
refers to the portion of the TNF receptor molecule, or the portion of the TNF receptor molecule
sequence which encodes TNF receptor molecule, that is of sufficient size and sequences to
functionally resemble TNF receptor molecules that can be used in the present invention (e.g.,
bind TNF with high affinity and possess low immunogenicity). A functional equivalent of
TNF receptor molecule also includes,modified TNF receptor molecules that functionally
resemble TNF receptor molecules thatcan be used in the present invention (e.g., bind TNF
with high affinity and possess low immunogenicity). For example, a functional equivalent of
TNF receptor molecule can contain a"SILENT" codon or one or more amino acid
substitutions, deletions or additions (e.g., substitution of one acidic amino acid for another
acidic amino acid; or substitution of one codon encoding the same or different hydrophobic
amino acid for another codon encoding a hydrophobic amino acid). See Ausubel et al..
Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience,
New York (1987-2000).
Cytokines include any known cytokine. See, e.g., www.CopewithCytokines.com.
Cytokine antagonists include, but are not limited to, any antibody, fragment or mimetic, any
soluble receptor, fragment or mimetic, any small molecule antagonist, or any combination
thereof.
Therapeutic Treatments. Any method of the present invention can comprise a
method for treating a IL-12 mediated disorder, comprising administering an effective amount
of a composition or pharmaceutical composition comprising at least one anti-IL-12 antibody to
a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy. Such
a method can optionally further comprise co-administration or combination therapy for
treating such immune diseases, wherein the administering of said at least one anti-IL-12
antibody, specified portion or variant thereof, further comprises administering, before
concurrently, and/or after, at least one selected from at least one of at least one selected from at
least one TNF antagonist (e.g., but not limited to a TNF antibody or fragment, a soluble TNF
receptor or fragment, fusion proteins thereof, or a small molecule TNF antagonist), an
antirheumatic (e.g., methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, gold
sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), a muscle relaxant,
a narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a
sedative, a local anethetic, a neuromuscular blocker, an antimicrobial (e.g., aminoglycoside, an
antifungal, an antiparasitic, an antiviral, a carbapenem, cephalosporin, a flurorquinolone, a
macrolide, a penicillin, a sulfonamide, a tetracycline, another antimicrobial), an antipsoriatic, a
corticosteriod, an anabolic steroid, a diabetes related agent, a mineral, a nutritional, a thyroid
agent, a vitamin, a calcium related hormone, an antidiarr heal, an antitussive, an antiemetic, an
antiulcer, a laxative, an anticoagulant, an erythropieitin (e.g., epoetin alpha), a filgrastim (e.g.,
G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), an immunization, an
immunoglobulin, an immunosuppressive (e.g., basiliximab, cyclosporine, daclizumab), a
growth hormone, a hormone replacement drug, an estrogen receptor modulator, a mydriatic, a
cycloplegic, an alkylating agent, an antimetabolite, a mitotic inhibitor, a radiopharmaceutical,
an antidepressant, antimanic agent, an antipsychotic, an anxiolytic, a hypnotic, a
sympathomimetic, a stimulant, donepezil, tacrine, an asthma medication, a beta agonist, an
inhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn, an epinephrine or
analog, domase alpha (Pulmozyme), a cytokine or a cytokine antagonist
Typically, treatment of pathologic conditions is effected by administering an effective
amount or dosage of at least one anti-IL-12 antibody composition that total, on average, a range
from at least about 0.01 to 500 milligrams of at least one anti-IL- 12antibody per kilogram of
patient per dose, and preferably from at least about 0.1 to 100 milligrams antibody /kilogram of
patient per single or multiple administration, depending upon the specific activity of contained in
the composition. Alternatively, the effective serum concentration can comprise 0.1-5000 ug/ml
serum concentration per single or multiple adminstration. Suitable dosages are known to medical
practitioners and will, of course, depend upon the particular disease state, specific activity of the
composition being administered, and the particular patient undergoing treatment. In sortie
instances, to achieve the desired therapeutic amount, it can be necessary to provide for repeated
administration, i.e., repeated individual administrations of a particular monitored or metered
dose, where the individual administrations are repeated until the desired daily dose or effect is
achieved.
Preferred doses can optionally include 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8,0.9,1,2,3,4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19,20, 21, 22,23, 24, 25, 26,27, 28,29,30,31,32,
33, 34, 35, 36, 37, 38, 39. 40, 41, 42, 43, 44, 45. 46. 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
59,60, 62. 63, 64, 65. 66, 67, 68, 6.9, 70, 71, 72, 73, 74, 75, 76. 77, 78, 79, 80, 81, 82, 83, 84, 85,
86, 87, 88. 89. 90,91, 92, 93, 94, 95, 96, 97, 98, 99 and/or 100-500 mg/kg/administration, or any
range, value or fraction thereof, or to achieve a serum concentration of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2,
1.5, 1.9, 2.0. 2.5, 2.9. 3.0, 3.5, 3.9, 4.0,4.5, 4.9, 5.0. 5.5. 5.9. 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5,
8.9,9.0,9.5.9.9, 10. 10.5, 10.9, 11, 11.5, 11.9.20. 12.5. 12.9! 13.0, 13.5, 13.9, 14.0, 14.5,4.9,
5.0, 5.5., 5.9. 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9,
12, 12.5, 12.9. 13.0, 13.5. 13.9, 14, 14.5, 15, 15.5, 15.9. 16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5,
18.9, 19, 19.5, 19.9, 20, 20.5,20.9, 21,22,23, 24, 25,26, 27, 28, 29, 30, 35,40, 45, 50, 55,60,
65, 70, 75, 80, 85, 90, 96, 100,200,300,400, 500, 600,700, 800, 900, 1000,1500, 2000, 2500,
3000, 3500,4000,4500, and/or 5000,ug/ml serum concentration per single or multiple
administration, or any range, value or fraction thereof.
Alternatively, the dosage: administered can vary depending upon known factors, such
as the pharmacodynamic characteristics of the particular agent, and its mode and route of
administration; age, health, and weight of the recipient;; nature and extent of symptoms, kind of
concurrent treatment, frequency of treatment, and the effect desired. Usually a dosage of
active ingredient can be about 0.1 to LOO milligrams per kilogram of body weight. Ordinarily
0.1 to 50, and preferably 0.1 to 10 milligrams per kilogram per administration or in sustained
release form is effective to obtain desired results.
As a non-limiting example, treatment of humans or animals can be provided as a one-
time or periodic dosage of at least one antibody of the present invention 0.1 to 100 mg/kg, such
as 0.5,0.9,1.0, l.l, 1.5, 2, 3,4, 5, 6, 7, 8,9, 10, 11,12, 13, 14, 15,16, 17, 18, 19, 20,21, 22,
23, 24, 25, 26,27, 28, 29, 30,40,45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one
of day 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17,18, 19, 20, 21, 22,23, 24,25, 26,
27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively or additionally, at least
one of week 1,2, 3, 4, 5, 6, 7, 8, 9, 10,11,12, 13, 14,15, 16,17, 18, 19, 20,21, 22, 23,24,25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42,43, 44, 45, 46, 47, 48,49, 50,
51, or 52, or alternatively or additionally, at least one of 1,2, 3, 4, 5, 6„ 7, 8, 9, 10,11, 12,13,
14, 15, 16, 17, 18, 19, or 20 years, or any combination thereof, using single, infusion or
repeated doses.
Dosage forms (composition) suitable for internal administration generally contain
from about 0.1 milligram to about 500 milligrams of active ingredient per unit or container. In
these pharmaceutical compositions the active ingredient will ordinarily be present in an
amount of about 0.5-99.999% by! weight based on the total weight of the composition.
For parenteral administration, the antibody can be formulated as a solution,
suspension, emulsion or lyophilized powder in association, or separately provided, widi a
pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline,
Ringer's solution, dextrose solution, and 1-10% human serum albumin. Liposomes and
nonaqueous vehicles such as fixed oils can also be used. The vehicle or lyophilized powder
can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical
stability (e.g., buffers and preservatives). The formulation is sterilized by known or suitable
techniques.
Suitable pharmaceutical carriers are described in the most recent edition of
Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field.
Alternative Administration
Many known and developed modes of can be used according to the present invention
for administering pharmaceutically effective amounts of at least one anti-IL-12 antibody
according to the present invention. While pulmonary administration is used in the following
description, other modes of administration can be used according to the present invention with
suitable results.
EL-12 antibodies of the present invention can be. delivered in a carrier, as a solution,
emulsion, colloid, or suspension, or as a dry powder, using any of a variety of devices and
methods suitable for administration by inhalation or other modes described here within or
known in the art.
Parenteral Formulations and Administration
Formulations for parenteral administration can contain as common excipients sterile
water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin,
hydrogenated naphthalenes and the like. Aqueous or oily suspensions for injection can be
prepared by using an appropriate emulsifier of humidifier and a suspending agent, according to
known methods. Agents for injection can be a non-toxic, non-orally administrable diluting
agent such as aquous solution or a sterile injectable solution or suspension in a solvent As the
usable vehicle or solvent, water, Ringer's solution, isotonic saline, etc. are allowed; as an
ordinary solvent, or suspending solvent, sterile involatile oil can be used. For these purposes,
any kind of involatile oil and fatty acid can be used, including natural or synthetic or
semisynthetic fatty oils or fatty acids; natural or synthetic or semisynthtetic mono- or di- or tri-
glycerides. Parental administration is known in the art and includes, but is not limited to,
conventional means of injections, a gas pressured needle-less injection device as described in
U.S. Pat. No. 5,851,198, and a laser perforator device as described in U.S. Pat. No. 5,839,446
entirely incorporated herein by reference.
Alternative Delivery
The invention further relates to the administration of at least one anti-IL-12 antibody
by parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial,
intraabdominal, intracapsular, intracartilaginous. intracavitary, intracelial. intracelebellar,
intracerebroventricular. intracolic. intracervical, intragastric, intrahepatic, intramyocardial,
intraosteal. intrapelvic. intrapericardiac. intraperitoneal, intrapleural, intraprostatic,
intrapulmonary, intrarectal, intrarcnal. intraretinal, lintraspinal, intrasynovial, intrathoracic,
intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal
means. At least one anti-IL-12 antibody composition can ibe prepared for use for parenteral
(subcutaneous, intramuscular or intravenous) or any other administration particularly in the
form of liquid solutions or suspensions; for use in vaginal or rectal administration particularly
in semisolid forms such as, but not limited to, creams and suppositories: for buccal, or
sublingual administration such as, but not limited to, in the form of tablets or capsules; or
intranasally such as, but not limited to, the form of powders, nasal drops or aerosols or certain
agents; or transdermally such as not limited to a gel, ointment, lotion, suspension or patch
delivery system with chemical enhancers such as dimethyl sulfoxide to either modify the skin
structure or to increase the drug concentration in the transdermal patch (Junginger, et: al. In
"Drug Permeation Enhancement"; Hsieh, D. S., Eds., pp. 59-90 (Marcel Dekker, Inc. New
York 1994, entirely incorporated herein by reference), or with oxidizing agents that enable the
application of formulations containing proteins and peptides onto the skin (WO 98/53847), or
applications of electric fields to create transient transport pathways such as electroporation, or
to increase the mobility of charged drugs through the skin such as iontophoresis, or application
of ultrasound such as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) (the above
publications and patents being entirely incorporated herein by reference).
Pulmonary/Nasal Administration
For pulmonary administration, preferably at least one anti-IL-12 antibody composition
is delivered in a particle size effective for reaching the lower airways of the lung or sinuses.
According to the invention, at least one anti-IL-12 antibody can be delivered by any of a
variety of inhalation or nasal devices known in the art for administration of a therapeutic agent
by inhalation. These devices capable of depositing aerosolized formulations in the sinus cavity
or alveoli of a patient include metered dose inhalers, nebulizers, dry powder generators,
sprayers, and the like. Other devices suitable for directing the pulmonary or nasal
administration of antibodies are also known in the art. All such devices can use of
formulations suitable for the administration for the dispensing of antibody in an aerosol. Such
aerosols can be comprised of either solutions (both aqueous and non aqueous) or solid
particles. Metered dose inhalers like the Ventolin® metered dose inhaler, typically use a
propellent gas and require actuation during inspiration (See, e.g., WO 94/16970, WO
98/35888). Dry powder inhalers like Turbuhaler™ (Astra), Rotahaler® (Glaxo), Diskus®
(Glaxo), Spiros™ inhaler (Dura), devices marketed by Inhale Therapeutics, and the Spinhaler*
powder inhaler (Fisons), use breath-actuation of a mixed powder (US 4668218 Astra, EP
237507 Astra. WO 97/25086 Glaxo. WO 94/08552 Dura. US 5458135 Inhale, WO 94/06498
Fisons. entirely incorporated herein by reference). Nebulizers like AERx™ Aradigm, the
Ultravent® nebulizer (Mallinckrodt), and the Acom II® nebulizer (Marquest Medical Products)
(US 5404871 Aradigm. WO 97/22376). the above references entirely incorporated herein by
reference, produce aerosols from solutions, while metered dose inhalers, dry powder inhalers,
etc. generate small particle aerosols. These specific examples of commercially available
inhalation devices are intended to be a representative of specific devices suitable for the
practice of this invention, and are not intended as limiting the scope of the invention.
Preferably, a composition comprising at least one anti-IL-12 antibody is delivered by a dry
powder inhaler or a sprayer. There are a several desirable features of an inhalation device for
administering at least one antibody of the present invention. For example, delivery by the
inhalation device is advantageously reliable, reproducible, and accurate. The inhalation device
can optionally deliver small dry particles, e.g. less than about 10 µm, preferably about 1-5 um,
for good respirability.
Administration of IL-12 antibody Compositions as a Spray
A spray including IL-12 antibody composition protein can be produced by forcing a
suspension or solution of at least one anti-IL-12 antibody through a nozzle under pressure.
The nozzle size and configuration, the applied pressure, and the liquid feed rate can be chosen
to achieve the desired output and particle size. An electrospray can be produced, for example,
by an electric field in connection with a capillary or nozzle feed. Advantageously, particles of
at least one anti-IL-12 antibody composition protein delivered by a sprayer have a particle size
less than about 10 urn, preferably in the range of about 1 urn to about 5 µm, and most
preferably about 2 urn to about 3 µm.
Formulations of at least one anti-IL-12 antibody composition protein suitable for use
with a sprayer typically include antibody composition protein in an aqueous solution at a
concentration of about 0.1 mg to about 100 mg of at least one anti-IL-12 antibody composition
protein per ml of solution or mg/gm, or any range or value therein, e.g., but not lrnited to, .1,
.2., .3, .4, .5, .6, .7, .8, .9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/ml or mg/gm. The
formulation can include agents such as an excipient, a, buffer, an isotonicity agent, a
preservative, a surfactant, and, preferably, zinc. The formulation can also include an excipient
or agent for stabilization of the antibody composition protein, such as a buffer, a reducing
agent, a bulk protein, or a carbohydrate. Bulk proteins useful in formulating antibody
composition proteins include albumin, protamine, or the like. Typical carbohydrates useful in
formulating antibody composition proteins include sucrose, mannitol, lactose, trehalose,
glucose, or the like. The antibody composition protein formulation can also include a
surfactant, which can reduce or prevent surface-induced aggregation of the antibody
composition protein caused by atomization of the solution in forming an aerosol. Various
conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and
alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range
between 0.001 and 14% by weight of the formulation. Especially preferred surfactants for
purposes of this invention are polyoxyethylene sorbitan monooleate, polysorbate 80,
polysorbate 20, or the like. Additional agents known in the art for formulation of a protein
such as IL-12 antibodies, or specified portions or variants, can also be included in the
formulation.
Administration of IL-12 antibody compositions by a Nebulizer
antibody composition protein can be administered by a nebulizer, such as jet nebulizer
or an ultrasonic nebulizer. Typically, in a jet nebulizer, a compressed air source is used to
create a high-velocity air jet through an orifice. As the gas expands beyond the nozzle, a low-
pressure region is created, which draws a solution of antibody composition protein through a
capillary tube connected to a liquid reservoir. The liquidstream from the capillary tube is
sheared into unstable filaments and droplets as it exits the tube, creating the aerosol. A range
of configurations, flow rates, and baffle types can be employed to achieve the desired
performance characteristics from a given jet nebulizer. In an ultrasonic nebulizer, high-
frequency electrical energy is used to create vibrational, mechanical energy,typically
employing a piezoelectric transducer. This energy is transmitted to the formulation of
antibody composition protein either directly or through a coupling fluid, creating an aerosol
including the antibody composition protein. Advantageously, particles of antibody
composition protein delivered by a nebulizer have a particle size less than about 10 µm,
preferably in the range of about 1 µm to about 5 µm, and.most preferably about 2 µm to about
3 µm.
Formulations of at least one anti-IL-12 antibody suitable for use with a nebulizer,
either jet or ultrasonic, typically include a concentration of about 0.1 mg to about 100 mg of at
least one anti-IL-12 antibody protein per ml of solution. The formulation can include agents
such as an excipient, a buffer, an isptonicity agent, a preservative, a surfactant, and, preferably,
zinc. The formulation can also include an excipient or agent for stabilization of the at least one
anti-IL-12 antibody composition protein, such as a buffer, a reducing agent, a bulk protein, or a
carbohydrate. Bulk proteins useful in formulating at least one anti-IL-12 antibody
composition proteins include albumin, protamine, or the like. Typical carbohydrates useful in
formulating at least one anti-IL-12 antibody include sucrose, mannitol, lactose, trehalose,
glucose, or the like. The at least one anti-IL-12 antibody formulation can also include a
surfactant, which can reduce or prevent surface-induced aggregation of the at least one anti-IL-
12 antibody caused by atomization of the solution in forming an aerosol. Various conventional
surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and
polyoxyethylene sorbital fatty acid esters. Amounts will generally range between 0.001 and
4% by weight of the formulation. Especially preferred surfactants for purposes of this
invention are polyoxyethylene sorbitan mono-oleate, polysorbate 80. polysorbate 20, or the
like. Additional agents known in the art for formulation of a protein such as antibody protein
can also be included in the formulation.
Administration of IL-12 antibody compositions By A Metered Dose Inhaler
In a metered dose inhaler (MDI), a propellant, at least one anti-IL-12 antibody, and
any excipients or other additives are contained in a canister as a mixture including a liquefied
compressed gas. Actuation of the metering valve releases the mixture as an aerosol, preferably
containing particles in the size range of less than about 10 µm, preferably about 1 µm to about
5 µm, and most preferably about 2 µm to about 3 µm. The desired aerosol particle size can be
obtained by employing a formulation of antibody composition protein produced by various
methods known to those of skill in the art, including jet-milling, spray drying, critical point
condensation, or the like. Preferred metered dose inhalers include those manufactured by 3M
or Glaxo and employing a hydrofluorocarbon propellant.
Formulations of at least one anti-IL-12 antibody for use with a metered-dose inhaler
device will generally include a finely divided powder containing at least one anti-IL-12
antibody as a suspension in a non-aqueous medium, for example, suspended in a propellant
with the aid of a surfactant. The propellant can be any conventional material employed for this
purpose, such as chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a
hydrocarbon, including trichlorofluoromethane, dichlorodifluoromeuiane,
dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a(hyoVofluroalkane-l34a),
HFA-227 (hydrofluroalkane-227), or the like. Preferably the propellant is a
hydrofluorocarbon. The surfactant can be chosen to stabilize the at least one anti-IL-12
antibody as a suspension in the propellant, to protect the active agent against chemical
degradation, and the like. Suitable surfactants include sorbitan trioleate, soya lecithin, oleic
acid, or the like. In some cases solution aerosols are preferred using solvents such as ethanol.
Additional agents known in the art for formulation of a protein such as protein can also be
included in the formulation.
One of ordinary skill in the art will recognize that the methods of the current invention
can be achieved by pulmonary administration of at least one anti-IL-12 antibody compositions
via devices not described herein.
Oral Formulations and Administration
Formulations for oral rely on the co-administration of adjuvants (e.g.. resorcinols and
nonionic surfactants such as polyoxyethylenc oleyl ether land n-hexadecylpolyethyiene ether)
to increase artificially the permeability of the intestinal walls, as well as the co-administration
of enzymatic inhibitors (e.g., pancreatic trypsin inhibitors, diisopropylfluorophosphate (DFF)
and trasylol) to inhibit enzymatic degradation. The active constituent compound of the solid-
type dosage form for oral administration can be mixed with at least one additive, including
sucrose, lactose, cellulose, mannitol. trehalose, raffinose, maltitol, dextran, starches, agar,
arginates, chitins. chitosans. pectins, gum tragacanth. gum arabic, gelatin, collagen, casein,
albumin, synthetic or semisynthetic polymer, and glyceride. These dosage forms can also
contain other type(s) of additives, e.g.. inactive diluting agent, lubricant such as magnesium
stearate, paraben, preserving agent such as sorbic acid, ascorbic acid, .alpha.-tocopherol,
antioxidant such as cysteine, disintegrator, binder, thickener, buffering agent, sweetening
agent, flavoring agent, perfuming agent, etc.
Tablets and pills can be further processed into enteric-coated preparations. The liquid
preparations for oral administration include emulsion, syrup, elixir, suspension and solution
preparations allowable for medical use. These preparations can contain inactive diluting agents
ordinarily used in said field, e.g., water. Liposomes have also been described as drug delivery
systems for insulin and heparin (U.S. Pat. No. 4,239,754). More recently, microspheres of
artificial polymers of mixed amino acids (proteinoids) have been used to deliver
pharmaceuticals (U.S. Pat. No. 4,925,673). Furthermore, carrier compounds described in U.S.
Pat. No. 5,879,681 and U.S. Pat. No. 5,5,871,753 are used to deliver biologically active agents
orally are known in the art.
Mucosal Formulations and Administration
For absorption through mucosal surfaces, compositions and methods of administering
at least one anti-EL-12 antibody include an emulsion comprising a plurality of submicron
particles, a mucoadhesive macromolecule, a bioactive peptide, and an aqueous continuous
phase, which promotes absorption through mucosal surfaces by achieving mucoadhesion of the
emulsion particles (U.S. Pat. Nos. 5,514,670). Mucous surfaces suitable for application of the
emulsions of the present invention can include corneal, conjunctival, buccal, sublingual, nasaL
vaginal, pulmonary, stomachic, intestinal, and rectal routes of administration. Formulations
for vaginal or rectal administration, e.g. suppositories, can contain as excipients, for example,
polyalkyleneglycols, vaseline, cocoa butter, and the like. Formulations for intranasal
administration can be solid and contain as excipients, for example, lactose or can be aqueous
or oily solutions of nasal drops. For buccal administration excipients include sugars, calcium
stearate, magnesium stearate, pregelinatined starch, and the like (U.S. Pat. Nos. 5,849,695).
Transdermal Formulations and Administration
For transdermal administration, the at least one anti-IL-12 antibody is encapsulated in
a delivery device such as a liposome or polymeric nanoparticles, microparticle, microcapsule,
or microspheres (referred to collectively as microparticles unless otherwise stated). A number
of suitable devices are known, including microparticles made of synthetic polymers such as
polyhydroxy acids such as polylactic acid, polyglycolic acid and copolymers thereof,
polyorthoesters. polyanhydrides, and polyphosphazenes. andnatural polymers such as
collagen, polyamino acids, albumin and other proteins, alginate and other polysaccharides, and
combinations thereof (U.S. Pat. Nos. 5,814,599).
Prolonged Administration and Formulations
It can be sometimes desirable to deliver the compounds of the present invention to the
subject over prolonged periods of time, for example, for periods of one week to one year from
a single administration. Various slow release, depot or implant dosage forms can be utilized.
For example, a dosage form can contain a pharmaceutically acceptable non-toxic salt of the
compounds that has a low degree of solubility in body fluids, for example, (a) an acid addition
salt with a polybasic acid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic
acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or di-sulfonic acids,
polygalacturonic acid, and the like; (b) a salt with a polyvalent metal cation such as zinc,
calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and the
like, or with an organic cation formed from e.g.,N,N'-dibenzyl-ethylenediamine or
ethylenediamine; or (c) combinations of (a) and (b) e.g. a zinc tannate salt. Additionally, the '
compounds of the present invention or, preferably, a relatively insoluble salt such as those just
described, can be formulated in a gel, for example, an aluminum monostearate gel with, e.g.
sesame oil, suitable for injection. Particularly preferred salts are zinc salts, zinc tannate salts,
pamoate salts, and the like. Another type of slow release depot formulation for injection would
contain the compound or salt dispersed for encapsulated in a slow degrading, non-toxic, non-
antigenic polymer such as a polylactic acid/polyglycolic acid polymer for example as
described in U.S. Pat. No. 3,773,919. The compounds or, preferably, relatively insoluble salts
such as those described above can also be formulated in cholesterol matrix silastic pellets,
particularly for use in animals. Additional slow release, depot or implant formulations, e.g. gas
or liquid liposomes are known in the literature (U.S. Pat. Nos. 5,770,222 and "Sustained and
Controlled Release Drug Delivery Systems", J. R. Robinson ed., Marcel Dekker, Inc., N.Y.,
1978).
Having generally described the invention, the same will be more readily understood by
reference to the following examples, which are provided byway of illustration and are not
intended as limiting.
MISSING AT THE TIME OF PUBLICATION
The vector pC4 is used for the expression of IL-12 antibody. Plasmid pC4 is a
derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146). The plasmid contains the
mouse DHFR gene under control of the S V40 early promoter. Chinese hamster ovary- or other
cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by
growing the cells in a selective medium (e.g., alpha minus MEM. Life Technologies,
Gaithersburg, MD) supplemented with the. chemotherapeutic agent methotrexate. The
amplification of the DHFR genes in cells resistant to methotrexate (MTX) has been well
documented (see, e.g., F. W. Alt, etal., J. Biol. Chem. 253:1357-1370 (1978); J. L. Hamlin and
C. Ma, Biochem. et Biophys. Acta 1097:107-143 (1990); and M. J. Page and ML A. Sydenham,
Biotechnology 9:64-68 (1991)). Cells grown in increasing concentrations of MTX develop
resistance to the drug by overproducing the target enzyme, DHFR, as a result of amplification
of the DHFR gene. If a second gene is linked to the DHFR gene, it is usually co-amplified and
over-expressed. It is known in the art that this approach can be used to develop cell lines
carrying more than 1,000 copies of the amplified gene(s). Subsequently, when the
methotrexate is withdrawn, cell lines are obtained that contain the amplified gene integrated
into one or more chromosome(s) ofthe host cell.
Plasmid pC4 contains for expressing the gene of interest the strong promoter ofthe
long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol.
5:438-447 (1985)) plus a fragment isolated .from the enhancer ofthe immediate early gene of
human cytomegalovirus (CMV) (Boshart, etal., Cell 41:521-530 (1985)). • Downstream ofthe
promoter are BamHI, Xbal, and Asp718 restriction enzyme cleavage sites that allow
integration of the genes. Behind these cloning sites the plasmid contains the 3' intron and
polyadenylation site of the rat preproinsulin gene. Other high efficiency promoters can also be
used for the expression, e.g., the human b-actin promoter, the SV40 early or late promoters or
the long terminal repeats from other retroviruses, e.g., HTV and HTLVI". Clontech's Tet-Off
and Tet-On gene expression systems and similar systems can be used to express the IL-12 in a
regulated way in mammalian cells (M. Gossen, and H. Bujard, Proc. Natl. Acad. Sci. USA 89:
5547-5551 (1992)). For the polyadenylation of the mRNA other signals, e.g., from the human
growth hormone or globin genes can be used as well. Stable cell lines carrying a gene of
interest integrated into the chromosomes can also be selected upon co-transfection with a
selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one
selectable marker in the beginning, e.g., G418 plus methotrexate.
The plasmid pC4 is digested with restriction enzymes and then dephosphorylated
using calf intestinal phosphatase by procedures known in the art. The vector is then isolated
from a 1% agarose gel.
The DNA sequence encoding the complete IL-12 antibody is used, e.g., as presented in
SEQ ID NOS:INSERT MAB AA SEQ ID I, and INSERT MAB AA SEQ ID N02,
corresponding to HCand LC variable regions, of a.IL-12 antibody of the present invention,
according to known method steps. Isolated nucleic acid encoding a suitable human constant
region (i.e., HC and LC regions) is also used in this construct (e.g.. as provided in vector
p 1351: INSERT ATCC ACCESSION NUMBER AND ADDITIONAL HC/LCplasmids)
The isolated variable and constant region encoding DNA and the dephosphorylated
vector are then ligated with T4 DNAligase. E. coli HB101 or XL-1 Blue cells are then
transformed and bacteria are identified that contain the fragment inserted into plasmid pC4
using, for instance, restriction enzyme analysis.
Chinese hamster ovary (CHO) cells lacking an active DHFR gene are used for
transfection. 5 g of the expression plasmid pC4 is cotrahsfected with 0.5 g of the plasmid
pSV2-neo using lipofectin. The plasmid pSV2neo contains a dominant selectable marker, the
neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics
including G418. The cells are seeded in alpha minus MEM supplemented with 1 g /ml
G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner,
Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1
g/mlG418. After about 10-14 days single clones are trypsinized and then seeded in 6-well
petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM,
200 nM, 400 nM, 800 nM). Clones growing at the highest concentrationso'f methotrexate are
then transferred to new 6-well plates containing even higher concentrations/of methotrexate
(1 mM, 2 mM, 5 mM, 10 mM, 20 mM). The same procedure is repeated until clones are
obtained that grow at a concentration of 100 - 200 mM. Expression of the desired gene
product is analyzed, for instance, by SDS-PAGE and Western blot or by reverse phase HPLC
analysis.
Example 2: Generation of High Affinity Human IgG Monoclonal Antibodies Reactive
With Human IL-12 Using Transgenic Mice
Summary
Transgenic mice have been used that contain human heavy and light chain
immunoglobulin genes to generate high affinity, completely human, monoclonal antibodies
that can be used therapeutically to inhibit the action of IL-12 for the treatment of one or more
EL-12-mediated disease. (CBA/J x C57/BL6/J) F2 hybrid mice containing human variable and
constant region antibody transgenes for both heavy and light chains are immunized with
human recombinant IL-12 (Taylor et al., Intl. Immunol. 6:5J79-591 (1993); Lonberg, el: al.,
Nature 368:856-859 (1994); Neuberger, M., Nature Biotech. 14:826(1996); Fishwild, et al.,
Nature Biotechnology 14:845-851 (1996)). Several fusions yielded one or more panels of
completely human IL-12 reactive IgG monoclonal antibodies. The completely human anti-EL-
12 antibodies are further characterized. All are IgGl . Such antibodies are found to have
affinity constants somewhere between lxl09and 9xl012. The unexpectedly high affinities of
these fully human monoclonal antibodies make them suitable candidates for therapeutic
applications in IL-12 related diseases, pathologies or disorders.
Abbreviations
BSA - bovine serum albumin
C02 - carbon dioxide
DMSO - dimethyl sulfoxide
EIA - enzyme immunoassay
FBS - fetal bovine serum
H202 - hydrogen peroxide
HRP - horseradish peroxidaseID - interadermal
Ig - immunoglobulin
IL-12 - interleukin-12
DP - intraperitoneal
IV - intravenous
Mab - monoclonal antibody
OD - optical density
OPD - o-Phenylenediamine dihydrochloride
PEG - polyethylene glycol
PSA - penicillin, streptomycin, amphotericin
RT - room temperature
SQ - subcutaneous
v/v - volume per volume
w/v - weight per volume
Materials and Methods
Animals
Transgenic mice that can express human antibodies are known in the art (and are
commecially available (e.g., from GenPharm International, San Jose, CA; Abgenix, Freemont,
CA, and others) that express human immunoglobulins but not mouse IgM or Ig . For
example, such transgenic mice contain human sequence transgenes that undergo V(D)Jjoining,
heavy-chain class switching, and somatic mutation to generate a repertoire of human sequence
immunoglobulins (Lonberg, et al., Nature 368:856-859 (1994)). The light chain transgenecan
be derived, e.g., in part from a yeast artificial chromosome clone that includes nearly half of
the germline human V region. In addition, the heavy-chain transgene can encode both
human u and human 1 (Fishwild, et al., Nature Biotechnology 14:845-851 (1996)) and/or 3
constant regions. Mice derived from appropriate genotopic lineages can be used in the
immunization and fusion processes to generate fully human monoclonal antibodies to EL-12.
Immunization
One or more immunization schedules can be used to generate the anti-IL-12 human
hybridomas. The first several fusions can be performed after the following exemplary
immunization protocol, but other similar known protocols can be used. Several 14-20 week
old female and/or surgically castrated transgenic male mice are immunized IP and/or ID with
1-1000 µg of recombinant human IL-12 emulsified with an equal volume of TITERMAX or
complete Freund's adjuvant in a final volume of 100-400µL (e.g., 200). Each mouse can also
optionally receive 1 -10 jo.g in 100 pX physiological saline at each of 2 SQ sites. The mice can
then be immunized 1-7,5-12, 10-18, 17-25 and/or 21-34 days later IP (1-400 ug) and SQ (1-
400 ug x 2) with IL-12 emulsified with an equal volume of TITERMAX or incomplete
Freund's adjuvant. Mice can be bled 12-25 and 25-40 days later by retro-orbital puncture
without anti-coagulant. The blood is then allowed to clot at RT for one hour and the serum is
collected and titered using an IL-12 EIA assay according to known methods. Fusions are
performed when repeated injections do not cause titers to lincrease. At that time, the mice can
be given a final IV booster injection of 1-400 ug IL-12 diluted in 100 p.L physiological saline.
Three days later, the mice can be euthanized by cervical dislocation and the spleens removed
aseptically and immersed in 10 mL of cold phosphate buffered saline (PBS) containing 100
U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B (PSA). The
splenocytes are harvested by sterilely perfusing the spleen with PSA-PBS. The cells are
washed once in cold PSA-PBS, counted using Trypan blue dye exclusion and resuspended in
RPMI1640 media containing 25 mM Hepes.
j
Cell Fusion
Fusion can be carried out at a 1:1 to 1:10 ratio of murine myeloma cells to viable
spleen cells according to known methods, e.g., as known in the art As a non-limiting
example, spleen cells and myeloma cells can be pelleted together. The pellet can then be
slowly resuspended, over 30 seconds, in 1 mL of 50% (w/v) PEG/PBS Solution (PEG
molecular weight 1.450, Sigma) at 37 C. The fusion can then be stopped by slowly adding
10.5 mL of RPMI 1640 medium containing 25 mM Hepes (37 C) over 1 minute. The fused
cells are centrifuged for 5 minutes at 500-1500 rpm. The cells are then resuspended in HAT
medium (RPMI 1640 medium containing 25 mM Hepes, 10% Fetal Clone I serum (Hyclone),
1 mM sodium pyruvate, 4 mM L-glutamine, 10 ug/mL gentamicin, 2.5% Origen culturing
supplement (Fisher), 10% 653-conditioned RPMI 1640/Hepes media, 50 uM
2-mercaptoethanol. 100 uM hypoxanthine, 0.4 uM aminppterin, and 16 uM thymidine) and
then plated at 200 u.L/well in fifteen 96-well flat bottom tissue culture plates. The plates are
then placed in a humidified 37 C incubator containing 5% C02 and 95% air for 7-10 days.
Detection of Human IgG Anti-IL-12 Antibodies in Mouse Serum
Solid phase EIA's can be used to screen mouse sera for human IgG antibodies specific
for human IL-I2. Briefly, plates can be coated with IL-12 at 2 µg/mL in PBS overnight. After
washing in 0.15M saline containing 0.02% (v/v) Tween 20, the wells can be blocked with 1%
(w/v) BSA in PBS. 200 uL/well for 1 hour at RT. Plates are used immediately or frozen at -20
C for future use. Mouse serum dilutions are incubated on the IL-12 coated plates at 50
uL/well at RT for 1 hour. The plates are washed and then probed with 50 uL/well
HRP-labeled goat anti-human IgG, Fc specific diluted 1:30,000 in 1% BSA-PBS for i hour at
RT. The plates can again be washed and 100 uL/well of the citrate-phosphate substrate
solution (0.1M citric acid and 0.2M sodium phosphate, 0.01% H2O2 and 1 mg/mL OPD) is
added for 15 minutes at RT. Stop solution (4N sulfuric acid) is then added at 25 µL/well and
the OD's are read at 490 nm via an automated plate spectrophotometer.
Detection of Completely Human Immunoglobulins in Hybridoma Supernates
Growth positive hybridomas secreting fully human immunoglobulins can be detected
using a suitable EIA. Briefly, 96 well pop-out plates (VWR, 610744) can be coated with 10
µg/mL goat anti-human IgG Fc in sodium carbonate buffer overnight at 4 C. The plates are
washed and blocked with 1% BSA-PBS for one hour at 37°C and used immediately or frozen
at -20 C. Undiluted hybridoma supernatants are incubated on the plates for one hour at 37°C.
The plates are washed and probed with HRP labeled goat anti-human kappa diluted 1:10,000 in
1% BSA-PBS for one hour at 37°C. The plates are then incubated with substrate solution as
described above.
Determination of Fully Human Anti-IL-12 Reactivity
Hybridomas, as above, can be simultaneously assayed for reactivity to EL-12 using a
suitable RIA or other assay. For example, supernatants are incubated on goat anti-human IgG
Fc plates as above, washed and then probed with radiolabeled IL-12 with appropriate counts per
well for 1 hour at RT. The wells are washed twice with PBS and bound radiolabled EL-12 is

quantitated using a suitable counter.
Human IgGl anti-IL-12 secreting hybridomas can be expanded in cell culture and
serially subcloned by limiting dilution. The resulting clonal populations can be expanded and
cryopreserved in freezing medium (95% FBS, 5% DMSO) and stored in liquid nitrogen.
Isotyping
Isotype determination of the antibodies can be accomplished using an EIA in a format
similar to that used to screen the mouse immune sera for specific titers. IL-12 can be coated
on 96- well plates as described above and purified antibody at 2 µg/mL can be incubated on
the plate for one hour at RT. The plate is washed and probed with HRP labeled goat
anti-human IgG, or HRP labeled goat anti-human IgG3 diluted at 1:4000 in 1% BSA-PBS for
one hour at RT. The plate is again washed and incubated, with substrate solution as described
above.
Binding Kinetics of Human Anti-Human IL-12 Antibodies With Human IL-12
Binding characteristics for antibodies can be suitably assessed using an IL-12 capture
EIA and BIAcore technology, for example. Graded concentrations of purified human IL-12
antibodies can be assessed for binding to EIA plates coated with 2 µg. mL of IL-12 in assays as
described above. The OD's can be then presented as semi-log plots showing relative binding
efficiencies.
Quantitative binding constants can be obtained, e.g., as follows, or by any other known
suitable method. A BIAcore CM-5 (carboxymethyl) chip is placed in a BIAcore 2000 unit
HBS buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v P20 surfactant, pH 7.4)
is flowed over a flow cell of the chip at 5 µL/minute until a stable baseline is obtained. A
solution (100 pX) of 15 mg of EDG (N-ethyl-N'-(3-dimethyl-aminopropyl)-carbodiimide
hydrochloride) in 200 µL water is added to 100 µL of a solution of 2.3 mg of NHS
(N-hydroxysuccinimide) in 200 µL water. Forty (40) µL;of the resulting solution is injected
onto the chip. Six µL of a solution of human IL-12 (15 µg/mL in 10 mM sodium acetate, pH
4.8) is injected onto the chip, resulting in an increase of ca. 500 RU. The buffer is changed to
TBS/Ca/Mg/BSA running buffer (20 mM Tris, 0.15 M sodium chloride. 2 mM calcium
chloride, 2 mM magnesium acetate, 0.5% Triton X-100,25 µg/mL BSA, pH 7.4) and flowed
over the chip overnight to equilibrate it and to hydrolyze or cap any unreacted succinimide
esters.
Antibodies are dissolved in the running buffer at 33.33,16.67, 8.33, and 4.17 nM. The
flow rate is adjusted to 30 µL/min and the instrument temperature to 25 C. Two flow cells are
used for the kinetic runs, one on which IL-12 had been immobilized (sample) and a second,
underivatized flow cell (blank). 120 µL of each antibody concentratiorris injected over the
flow cells at 30 µL/min (association phase) followed by an uninterrupted 360 seconds of buffer
flow (dissociation phase). The surface of the chip is regenerated (interleukin-12 /antibody
complex dissociated) by two sequential injections of 30 µL each of 2 M guanidine thiocyanate.
Analysis of the data is done using BIA evaluation 3.0 or CLAMP 2.0, as known in the
art For each antibody concentration the blank sensogram is subtracted from the sample
sensogram. A global fit is done for both dissociation (kd. sec-1) and association (k2, mol-1 sec-1)
and the dissociation constant (KD, mol) calculated (kd/k2). Where the antibody affinity is high
enough that the RUs of antibody captured are >100, additional dilutions of the antibody are
run.
Results and Discussion
Generation of Anti-Human IL-12 Monoclonal Antibodies
Several fusions are performed and each fusion is seeded in 15 plates (1440
wells/fusion) that yield several dozen antibodies specific for human IL-12. Of these, some are
found to consist of a combination of human and mouse Ig chains. The remaining hybridomas
secret anti-IL-12 antibodies consisting solely of human heavy and light chains. Of the human
hybridomas all are expected to be IgGI.
Binding Kinetics of Human Anti-Human IL-12 Antibodies
ELISA analysis confirms that purified antibody from most or all of these hybridomas
bind IL-12 in a concentration-dependent manner. Figures 1-2 show the results of the relative
binding efficiency of these antibodies. In this case, the avidity of the antibody for its cognate
antigen (epitope) is measured. It should be noted that binding IL-12 directly to the EI A plate
can cause denaturation of the protein and the apparent binding affinities cannot be reflective of
binding to undenatured protein. Fifty percent binding is found over a range of concentrations.
Quantitative binding constants are obtained using BIAcore analysis of the human
antibodies and reveals that several of the human monoclonal antibodies are very high affinity
with KD in the range of 1x10-9 to 7xl0-12.
Conclusions
Several fusions are performed utilizing splenocytes from hybrid mice containing
human variable and constant region antibody transgenes that are immunized with human EL-
12. A set of several completely human IL-12 reactive IgG monoclonal antibodies of the
IgGI isotype are generated. The completely human anti-IL-12 antibodies are further
characterized. Several of generated antibodies have affinity constants between lxl 09 and
9xl012. The unexpectedly high affinities of these fully human monoclonal antibodies make
them suitable for therapeutic applications in IL-12-dependent diseases, pathologies or related
conditions.
Example 3: C340 is a Neutralizing Human Monoclonal Antibody
The bioactivity of IL-12 was shown to be neutralized by C340 in a variety of
IL-12 dependent activity assays. Since IL-12 enhances IFN GAMMA production by NK cells
and T lymphocytes, the effect of C340 antibody on the upregulation of IFN GAMMA mRNA
and the effect of C340 on the production of IFN GAMMA protein was examined (Trinchieri,
G., Current Opinion in Immunology, 9:17-23 (1997), Morris, S.C., et al., Journal of
Immunology, 152:1047-1056 (1994)). The ability of C340 to neutralize IL-12 driven
induction of lymphokine activated killer (LAK) cell activity was also investigated in these
studies (Kutza, J. and Murasko, D.M., Mechanisms of Ageing and Development, 90:209-222
(1996), Stem, A.S., et al., Proceedings of the National Academy of Sciences of the U.S.A.,
87:6808-6812 (1990)). Lastly, the effect of C340 on IL-12-mediated upregulation of CD95
cell surface expression on T and NK cells was tested (Medvedev, A.E., et ai.. Cytokine. 9:394-
404(1997)).
Inhibition of IFN gamma mRNA Transcnption
To determine whether C340 inhibits IL-12. IL-2 induced IFN GAMMA gene
transcription in human PBL. a reverse transcription-PCR assay was performed. Specific
primers for b-actin (a control for mRNA integrity and content) and IFN GAMMA were used to
amplify the cDNA obtained from stimulated human PBL. Figure 3 shows C340 down
regulates IFN GAMMA mRNA in IL-12/IL-2 activated (2 hour) PBMC.
Inhibition of Intracellular IFN GAMMA as Measured by Flow Cytometry
In response to various signals and as a measure of activation, T cells and NK cells can
be induced to secrete cytokines. More specifically, PBL treated with IL-2 and IL-12 initiate
substantial synthesis of IFN gamma within 4-8 hours after stimulation. This production can be
detected in the cytoplasm of Brefeldin-A treated PBL by flow cytometry. Figure 4
demonstrates a 60% reduction in IFN GAMMA production in such cultures when C340 IL-12
was added in conjunction with IL-12 for five hours.
Inhibition of IL-12 induced ,IFN GAMMA Secretion
Figure 5 clearly shows that two different lots of C340 inhibited the secretion of EFN
GAMMA by peripheral blood lymphocytes in a dose-dependent fashion. Four hundred
picograms of IL-12 were premixed with varying amounts of C340 and then added to IL-2
stimulated cultures of PBL's. When IFN GAMMA was measured by EIA after an 18-24 hour
incubation, markedly diminished amounts of EFN GAMMA were detected with as little as 1
?g/mL of C340 antibody.
Inhibition of IL-12 Induced LAK Cell Cytotoxicity
Raji cells, an IL-12 sensitive Burkitt lymphoma derived cell line; is an NK cell
resistant. LAK cell sensitive cell line. Raji cells, in triplicate, were cultured for four hours
with LAK cells which had been activated with 400 pg/mL IL-12 and 10 U/mL IL-2 in the
presence or absence of the human monoclonal antibody C340 (5000 ng/mL or 50 ng/mL).
Figure 6 shows the results from three normal, healthy donors. IL-12 + IL-2 activation of
effector cells resulted in an increasing cytotoxic activity over that of cells activated with IL-2
alone. The C340 antibody inhibited this IL-12 dependent effect. The magnitude of inhibition
was related to antibody concentration, with the highest concentration tested reducing
cytotoxicity to background levels.
Inhibition of CD95 Upregulation
Reports have described IL-12-induced upregulation. of CD95 on the surface of highly
purified CD56+ PBL. As can be seen in Figure 7A and 7B, distributional flow cytometric
analysis revealed that CD95 expression was significantly upregulated on CD3+ T cells and
CD56+ NIC cells after treatment with IL-12 plus fL-2 for 72 hours. Concomitant anti-IL-12
treatment inhibited CD95 expression in both CD3+ and CD56+ populations. CD3+ cells were
inhibited by -50% (Figure 7A). whereas CD56+ cells were inhibited by -85% (Figure 7B), as
evidenced by a diminished MFI index (percent greater then unstimulated control).
Example 4: Gene cloning and characterization
Genomic DNA fragments containing either the C340 heavy chain gene or the C340
light chain were cloned and purified. Genomic DNA purified from C340 hybridoma cells was
partially digested with Sau3A restriction enzyme and size-selected by centrifugal fractionation
through a 10-40% sucrose gradient. DNA fragments in the size range of 15-23 kb were cloned
into the bacteriophage vector, EMBL3, [commercially available ?] and packaged into phage
particles. Several packaging reactions resulted in a library of 1 million bacteriophage clones.
Approximately 600,000 clones from the library were screened by plaque hybridization using
32P-labeled genomic DNA fragments that contained either human IgGl heavy chain constant
region sequences or human kappa light chain constant region sequences as probe. Thirteen
heavy chain and nine light chain clones were detected. Of these, three heavy chain clones and
four light chain clones were purified by two more rounds of screening. One of the heavy chain
clones and two of the light chain clones were shown to contain the 5' and 3' ends of the coding
sequences by PCR analysis of bacteriophage DNA. The DNA insert in heavy chain (HC)
clone H4 was 16 kb in size and includes 3.6 kb of 5' flanking and at least 2 kb of 3' flanking
sequence. The DNA insert in light chain (LC) clone LCI was 15 kb in size and included 4.4
kb of 5' flanking and 6.0 kb of 3' flanking sequence. The complete inserts were removed from
the bacteriophage vector as Sall fragments and cloned between the Xhol and Sall sites of
plasmid expression vector pl351, which provided a gpt selectable marker gene. Because there
was an internal Sall site in the heavy chain variable region coding sequence, two Sail
fragments had to be transferred from bacteriophage H4 to the p1351 expression vector. The
resulting heavy and light chain expression plasmids were1 termed p1560 and pl558,
respectively. The orientations of the heavy and light chain genes in these two plasmids
relative to the p1351 vector sequences were determined using restriction enzyme analysis and
PCR, respectively. In both cases, the orientations were such that the 5' end of the Ab gene
fragment was proximal to the 3' end of the gpt gene. Both strands of the coding regions of the
cloned genes were sequenced. The sequences of plasmids pl560 and pl558 are presented in
Figures 11A-11K and Figures 13A-13J, respectively.
Example 5: Preparation of recombinant cell lines
Heavy chain plasmid pl560 was linearized by digestion with Pvul restriction enzyme
and light chain plasmid p1558 was linearized using Sall restriction enzyme. p3X63Ag8.653
(653) and SP2/0-Agl4 (SP2/0) cells were separately transfected with the premixed linearized
plasmids by electroporation and cells cultured and transfectants selected using mycophenolic
acid as described (Knight, et aL Molecular Immunology 30:1443 (1993)). Cell supernatants
From mycophenolic acid-resistant colonies were assayed approximately two weeks later for
human IgG (i.e., recombinant C340 (rC340)). For this, cell supernatants were incubated on
96-well ELISA plates that were coated with goat antibodies specific for the Fc portion of
human IgG. Human IgG which bound to the coated plate was detected using alkaline
phosphatase-conjugated goat anti-human IgG (heavy chain + light chain) antibody and alkaline
phosphatase substrates as described (Knight, et al., Molecular Immunology 30:1443 (1993)).
Cells of the higher producing clones were transferred to 24-well culture dishes in standard
media and expanded (IMDM, 5% FBS, 2 mM glutamine, mycophenolic acid selection mix).
The amount of antibody produced (i.e., secreated into the media of spent cultures) was
carefully quantified by ELISA using purified C340 mAb as the standard. Selected clones were
then expanded in T75 flasks and the production of human IgG by these clones was quantified
by ELISA. Based on these values, six independent 653 transfectants and three independent
SP2/0 transfectants were subcloned (by seeding an average of one cell per well in 96 well
plates), the quantity of antibody produced by the subclones was determined by assaying
(ELISA) supernatants from individual subclone colonies. Three subclones, '653 transfectant
19-20 (C379B) and the SP2/0 transfectants 84-81 (C381A) and 22-56 (C389A), were selected
for further analysis.
Assay for rC340 antigen binding.
Prior to subcloning selected cell lines as described above, cell supernatants from three
parental lines (653 transfectants clone 2 and clone 18 and SP2/0 transfectant clone 1) were
used to test the antigen binding characteristics of rC340. The concentrations of rC340 in the
three cell supernatant samples were first determined by ELISA. Titrating amounts of the
supernatant samples, or purified C340 positive control, were then incubated in 96-well plates
coated with 2 µg/ml of human IL-12. Bound mAb was then detected with alkaline
phosphatase-conjugated goat anti-human IgG (heavy chain + light chain) antibody and the
appropriate alkaline phosphatase substrates. As shown in Figure 8, rC340 bound specifically
to human IL-12 in a manner indistinguishable from the original C340 mAb.
Characterization of selected cell lines.
Growth curve analyses were performed on C379B, C381A, andC3S9A by seeding T75
flasks with a starting cell density of 2 X 105 cells/ml in standard media or SFM-5 serum-free
media and then monitoring cell number and rC340 concentration on a daily basis until the
cultures were spent. The results of cultures in standard media are shown in Figures 9A - 9C.
Maximal C340 mAb production levels for C379B. C381A, and C389A were 135 µg/ml, 150
4g/ml, and 110 µg/ml, respectively. Attempts to adapt C379B cells to SFM-5 media were not
successful. C381A cells produced the same amount of rC340 in SFM-5 media as in standard
media, whereas C389A cells produced only half as much rC340 in SFM-5 media as in standard
media.
The stability of rC340 mAb production over time for the three subclones was assessed
by culturing cells in 24-well dishes with standard media or standard media without
nycophenolic acid selection for varying periods of time. Lines C379B and C381A were
»bserved to stably produce rC340, in the presence or absence of selection for a period of 30
lays (the maximum time tested) and 75 days, respectively. Line C389A was unstable and after
13 days of culture produced just 20% as much antibody as at the beginning of the study.
It will be clear that the invention can be practiced otherwise than as particularly
described in the foregoing description and examples.
Numerous modifications and variations of the present invention are possible in light of
the above teachings and, therefore, are within the scope of the appended claims.
We Claim:
1. An isolated mammalian anti-lL-12 antibody, comprising at least
one of (i) all of the heavy chain complementarity determining
regions (CDR) amino acid sequences of SEQ ID NOS: 1,2, and 3;
(ii) all of the light chain CDR amino acids sequences of SEQ ID
NOS: 4, 5 and 6 and (iii) at least one variable region comprising
SEQ ID NO:7 or 8.
2. The anti-IL-12 antibody as claimed in claim 1, wherein said
antibody binds IL-12 with an affinity of at least one selected from
at least 10-9 M, at least 10-10 M, at least 10-11 M, or at least 1012 M.
3. The anti-IL-12 antibody as claimed in claim 1, wherein said
antibody substantially neutralizes at least one activity of at least
one IL-12 protein.
4. An isolated nucleic acid molecule encoding at least one isolated
mammalian anti-IL-12 antibody having at least one variable region
comprising SEQ ID NO:7 or 8.
5. An isolated nucleic acid vector comprising the isolated nucleic
acid molecule as claimed in claim 4.
6. A method for producing an anti-IL-12 antibody, comprising
translating the nucleic acid molecule as claimed in claim 4 under
conditions in vitro, such that the IL-12 antibody is expressed in
detectable or recoverable amounts.
7. An anti-idiotype antibody or fragment that specifically binds an
isolated mammalian anti-IL-12 antibody having at least one
variable region comprising SEQ ID NO:7 or 8.
8. A medical device, comprising the isolated mammalian anti-IL-12
antibody having at least, one variable region comprising SEQ ID
NO:7 or 8, wherein said device is suitable to contacting or
administering said anti-IL-12 antibody by at least one mode
selected from parenteral, subcutaneous, intramuscular, intravenous,
intrarticular, intrabronchial, intraabdominal, intracapsular,
intracartilaginous, intracavitary, intracelial, intracerebellar,
intracerebroventricular, intracolic, intracervical, intragastric,
intrahepatic, intramyocardial, intraostel, intrapelvic,
intrapericardiac, intraperitoneal, intrapleural, intraprostatic,
intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,
intrasynovial, intrathoracic, intrauterine, intravesical, bolus,
vaginal, rectal, buccal, sublingual, intranasal, and transdermal.
9. A method for producing an isolated mammalian anti-IL-12
antibody having at least one variable region comprising SEQ ID
NO:7 or 8, comprising providing a host cell or transgenic non-
human animal or transgenic plant or plant cell capable of
expressing in recoverable amounts said antibody.
10. An anti-IL-12 antibody produced by the method as claimed in
claim 9.
11. The anti-IL-12 antibody as claimed in claim 1, wherein said
antibody binds IL-12 with an affinity of at least one selected from
at least 10-9-M, at least 10-10 M, at least 10-11 M, or at least 10-12M.
12. The anti-IL-12 antibody as claimed in claim 13, wherein said
antibody substantially neutralizes at least one activity of at least
one IL-12 protein.
13. As isolated nucleic acid molecule encoding an isolated mammalian
anti-IL-12 antibody having at least one of (i) all of the heavy chain
CDR amino acid sequences of SEQ ID NOS: 1,2, and 3; (ii) all of
the light chain CDR amino acids sequences of SEQ ID NOS: 4, 5
and 6.
14. An isolated nucleic acid vector comprising the isolated nucleic
acid molecule as claimed in claim 13.
15. A method for producing an anti-lL-12 antibody, comprising
translating the nucleic acid molecule as claimed in claim 16 under
conditions in vitro, such that the anti-lL-12 antibody is expressed in
detectable or recoverable amounts.
16. An anti-idiotype antibody or fragment that specifically binds an
isolated mammalian anti-lL-12 antibody having at least one of (i)
all of the heavy chain CDR amino acid sequences of SEQ ID NOS:
1,2, and 3; and (ii) all of the light chain CDR amino acids
sequences of SEQ ID NOS: 4, 5 and 6.
17. A medical device, comprising an isolated mammalian anti-IL-12
antibody having at least one of (i) all of the heavy chain CDR
amino acid sequences of SEQ) ID NOS: 1, 2 and 3; and (ii) all of
the light chain CDR amino acids sequences of SEQ ID NOS: 4, 5,
and 6, wherein said device is suitable to contacting or
administering said anti-IL-12 antibody by at least one mode
selected from parenteral, subcutaneous, intramuscular, intravenous,
intrarticular, intrabronchial, intraabdominal, intracapsular,
intracartilaginous, intracavitary, intracelial, intracerebellar,
intracerebroventricular, intracolic, intracervical, intragastric,
intrahepatic, intramyocardial, intraosteal, intrapelvic,
intrapericardiac, intraperitoneal, intrapleural, intraprostatic,
intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,
intrasynovial, intrathoracic, intrauterine, intravesical, bolus,
vaginal, rectal, buccal, sublingual, intranasal, and transdermal.
18. A method for producing an isolated mammalian anti-IL-12
antibody having at least one of (i) all of the heavy chain CDR
amino acid sequences of SEQ ID NOS: 1, 2, and 3; and (ii) all of
the light chain CDR amino acids sequences of SEQ ID NOS: 4, 5,
and 6, comprising providing a host cell or transgenic non-human
animal or transgenic plant or plant cell capable of expressing in
recoverable amounts said antibody.
19. An anti-IL-12 antibody produced by the method as claimed in
claim 18.
20. An isolated mammalian anti-IL-12 antibody, comprising at least
one heavy chain or light chain CDR having the amino acid
sequence of at least one of SEQ ID NOS: 1, 2, 3, 4, 5 and 6.
21. The anti-IL-12 antibody as claimed in claim 20, wherein said
antibody binds IL-12 with an affinity of at least one selected from
at least 10-9 M, at least 10-10 M, at least 10-11 M, or at least 10-12 M.
22. The anti-IL-12 antibody as claimed in claim 20, wherein said
antibody substantially neutralizes at least one activity of at least
one IL-12 protein.
23. An isolated nucleic acid molecule encoding at least one isolated
mammalian anti-IL-12 antibody having at least one heavy chain or
light chain CDR having the amino acid sequence of at least one of
SEQ ID NOS: 1, 2, 3, 4, 5 and 6.
24. An isolated nucleic acid vector comprising the isolated nucleic
acid molecule as claimed in claim 23.
25. A method for producing an anti-IL-12 antibody, comprising
translating the nucleic acid molecule as claimed in claim 23 under
conditions in vitro, such that the anti-IL-12 antibody is expressed in
detectable or recoverable amounts.
26. An anti-idiotype antibody or fragment that specifically binds an
isolated mammalian anti-IL-12 antibody having at least one heavy
chain or light chain CDR having the amino acid sequence of at
least one of SEQ ID NOS: 1, 2, 3, 4, 5 and 6.
27. A medical device, comprising an isolated mammalian anti-IL-12
antibody having at least one heavy chain or light chain CDR
having the amino acid sequence of at least one of SEQ ID NOS: 1,
2, 3, 4, 5 and 6, wherein said device is suitable to contacting or
administering said at least one anti-IL-12 antibody by at least one
mode selected from parenteral, subcutaneous, intramuscular,
intravenous, intrarticular, intrabronchial, intraabdominal,
intracapsular, intracartilaginous, intracavitary, intracelial,
intracerebellar, intracerebroventricular, intracolic, intracervical,
intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,
intrapericardiac, intraperitoneal, intrapleural, intraprostatic,
intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,
intrasynovial, intrathoracic, intrauterine, intravesical, bolus,
vaginal, rectal, buccal, sublingual, intranasal, and transdermal.
28. A method for producing an isolated mammalian anti-IL-12
antibody having at least one heavy chain or light chain CDR
having the amino acid sequence of at least one of SEQ ID NOS; 1,
2, 3, 4, 5 and 6, comprising providing a host cell or transgenic non-
human animal or transgenic plant or plant cell capable of
expressing in recoverable amounts said antibody.
29. An anti-IL-12 antibody produced by the method as claimed in
claim 28.
30. An isolated mammalian anti-IL-12 antibody that binds to the same
region of a IL-12 protein as an antibody comprising at least one
heavy chain or light chain CDR having the amino acid sequence of
at least one of SEQ ID NOS: 1, 2, 3, 4, 5, and 6.
31. The anti-IL-12 antibody as claimed in claim 30, wherein said
antibody binds IL-12 with an affinity of at least one selected from
at least 10"9 M, at least 10-10 M, at least 10-11 M, or at least 10-12 M.
32. The anti-IL-12 antibody as claimed in claim 30, wherein said
antibody substantially neutralizes at least one activity of at least
one IL-12 protein.
33. An isolated nucleic acid molecule encoding an isolated mammalian
anti-IL-12 antibody that binds to the same region of an IL-12
protein as an antibody comprising at least one heavy chain or light
chain CDR having the amino acid sequence of at least one of SEQ
ID NOS: 1,2, 3,4, 5, and 6.
34. An isolated nucleic acid vector comprising the isolated nucleic
acid molecule as claimed in claim 33.
35. A method for producing at. least one anti-IL-12 antibody,
comprising translating the nucleic acid molecule as claimed in
claim 33 under conditions in vitro, such that the anti-IL-12
antibody is expressed in detectable or recoverable amounts.
36. An anti-idiotype antibody or fragment that specifically binds an
isolated mammalian anti-IL-12 antibody that binds to the same
region of an IL-12 protein as an antibody comprising at least one
heavy chain or light chain CDR having the amino acid sequence of
at least one of SEQ ID NOS; 1, 2, 3, 4, 5 and 6.
37. A medical device, comprising an isolated mammalian anti-IL-12
antibody that binds to the same region of an IL-12 protein as an
antibody comprising at least one heavy chain or light chain CDR
having the amino acid sequence of at least one of SEQ ID NOS: 1,
2, 3, 4, 5, 6, wherein said device is suitable for contacting or
administering said anti-IL-12 antibody by at least one mode
selected from parenteral, subcutaneous, intramuscular, intravenous,
intrarticular, intrabronchial, intraabdominal, intracapsular,
intracartilaginous, intracavitary, intracelial, intracerebellar,
intracerebroventricular, intracolic, intracervical, intragastric,
intrahepatic, intramyocardial, intraosteal, intrapelvic,
intrapericardiac, intraperitoneal, intrapleural, intraprostatic,
intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,
intrasynovial, intrathoracic, intrauterine, intravesical, bolus,
vaginal, rectal, buccal, sublingual, intranasal, and transdermal.
38. A method for producing an isolated mammalian anti-IL-12
antibody that binds to the same region of an IL-12 protein as an
antibody comprising at least one heavy chain or light chain CDR
having the amino acid sequence of at least one of SEQ ID NOS: 1,
2, 3, 4, 5, and 6, comprising a host cell or transgenic non-human
animal or transgenic plant or plant cell capable of expressing in
recoverable amounts said antibody.
39. An anti-IL-12 antibody produced by the method as claimed in
claim 38.
40. An isolated mammalian anti-IL-12 antibody, comprising a heavy
chain variable region amino acid sequence of SEQ ID NO: 7 and a
light chain variable region amino acid sequence of SEQ ID NO: 8.
41. The anti-IL-12 antibody as claimed in claim 40, wherein said
antibody binds IL-12 with an affinity of at least one selected from
at least 10-9 M, at least 10-10 M, at least 10-11M, or at least 10-12 M.
42. The anti-IL-12 antibody as claimed in claim 40, wherein said
antibody substantially neutralizes at least one activity of at least
one IL-12 protein.
43. An isolated nucleic acid molecule encoding at least one isolated
mammalian anti-IL-12 antibody comprising a heavy chain variable
region amino acid sequence of SEQ ID NO: 7 and a light chain
variable region amino acid sequence of SEQ ID NO: 8.
44. An isolated nucleic acid vector comprising the isolated nucleic
acid molecule as claimed in claim 43.
45. A method for producing an anti-IL-12 antibody, comprising
translating the nucleic acid molecule as claimed in claim 44 under
conditions in vitro, such that the 1L-12 antibody is expressed in
detectable or recoverable amounts.
46. An anti-idiotype antibody or fragment that specifically binds an
isolated mammalian anti-IL-12 antibody comprising a heavy chain
variable region amino acid sequence of SEQ ID NO: 7 and a light
chain variable region amino acid sequence of SEQ ID NO: 8.
47. A medical device, comprising an isolated mammalian anti-IL-12
antibody comprising a heavy chain variable region amino acid
sequence of SEQ ID NO:7 and a light chain variable region amino
acid sequence of SEQ ID NO: 8, wherein said device is suitable to
contacting or administering said anti-IL-12 antibody by at least one
mode selected from parenteral, subcutaneous, intramuscular,
intravenous, intrarticular, intrabronchial, intraabdominal,
intracapsular, intracartilaginous, intracavitary, intracelial,
intracerebellar, intracerebroventricular, intracolic, intracervical,
intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,
intrapericardiac, intraperitoneal, intrapleural, intraprostatic,
intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,
intrasynovial, intrathoracic, intrauterine, intravesical, bolus,
vaginal, rectal, buccal, sublingual, intranasal, and transdermal.
48. A method for producing an isolated mammalian anti-IL-12
antibody comprising a heavy chain variable region amino acid
sequence of SEQ ID NO.: 7 and a light chain variable region amino
acid sequence of SEQ ID NO: 8, comprising providing a host cell
or transgenic non-human animal or transgenic plant or plant cell
capable of expressing in recoverable amounts said antibody.
49. An anti-IL-12 antibody produced by the method as claimed in
claim 48.

The present invention provides isolated human, primate, rodent, mammalian, chimeric,
humanized and/or CDR-grafted anti-IL-12 antibodies, immunoglobulins, cleavage products
and other specified portions and variants thereof, as well as anti-IL-12 antibody compositions,
encoding or complementary nucleic acids, vectors, host cells, compositions, formulations,
devices, transgenic animals, transgenic plants, and methods of making and using thereof, as
described and enabled herein, in combination with what is known in the art.
The present invention also provides at least one isolated anti-IL-12 antibody as
described herein. An antibody according to the present invention includes any protein or
peptide containing molecule that comprises at least a portion of an immunoglobulin molecule,
such as but not limited to at least one complementarity determining region (CDR) of a heavy
or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region,
a heavy chain or light chain constant region, a framework region, or any portion thereof, that
can be incorporated into an antibody of the present invention. An antibody of the invention
can include or be derived from any mammal, such as but not limited to a human, a mouse, a
rabbit, a rat, a rodent, a primate, or any combination thereof, and the like.

Documents:

150-kolnp-2003-abstract.pdf

150-kolnp-2003-assignment.pdf

150-kolnp-2003-claims.pdf

150-kolnp-2003-correspondence.pdf

150-kolnp-2003-description (complete).pdf

150-kolnp-2003-drawings.pdf

150-kolnp-2003-examination report.pdf

150-kolnp-2003-form 1.pdf

150-kolnp-2003-form 18.pdf

150-kolnp-2003-form 2.pdf

150-kolnp-2003-form 26.pdf

150-kolnp-2003-form 3.pdf

150-kolnp-2003-form 5.pdf

150-KOLNP-2003-FORM-27.pdf

150-kolnp-2003-granted-abstract.pdf

150-kolnp-2003-granted-assignment.pdf

150-kolnp-2003-granted-claims.pdf

150-kolnp-2003-granted-correspondence.pdf

150-kolnp-2003-granted-description (complete).pdf

150-kolnp-2003-granted-drawings.pdf

150-kolnp-2003-granted-examination report.pdf

150-kolnp-2003-granted-form 1.pdf

150-kolnp-2003-granted-form 18.pdf

150-kolnp-2003-granted-form 2.pdf

150-kolnp-2003-granted-form 26.pdf

150-kolnp-2003-granted-form 3.pdf

150-kolnp-2003-granted-form 5.pdf

150-kolnp-2003-granted-reply to examination report.pdf

150-kolnp-2003-granted-sequence listing.pdf

150-kolnp-2003-reply to examination report.pdf

150-kolnp-2003-specification.pdf

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Patent Number

236195

Indian Patent Application Number

150/KOLNP/2003

PG Journal Number

41/2009

Publication Date

09-Oct-2009

Grant Date

07-Oct-2009

Date of Filing

07-Feb-2003

Name of Patentee

CENTOCOR, INC.

Applicant Address

200 GREAT VALLEY PARKWAY, MALVERN, PA

Inventors:

#	Inventor's Name	Inventor's Address
1	DAVID PERITT	407 CONSHOHOCKEN STATE ROAD, BALA CYNWYD, PA 19004
2	JILL GILES KOMAR	31 BLAKELY ROAD, DOWNINGTOWN, PA 19335
3	DAVID M. KNIGHT	2430 WHITEHORSE ROAD, BERWYN, PA 19312
4	BERNARD SCALLON	139 HEMLOCK DRIVE, COLLEGEVILLE, PA 19426
5	DAVID SHEALY	1351 PENNS RIDGE PLACE, DOWNINGTOWN, PA 19335

PCT International Classification Number

C12N 15/13

PCT International Application Number

PCT/US2001/24720

PCT International Filing date

2001-08-07

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/236,827	2000-09-29	U.S.A.
2	60/223,358	2000-08-07	U.S.A.
3	09/920,262	2001-08-01	U.S.A.