UA81100C2 - Isolated monoclonal human antibody that binds to human il-12 - Google Patents

Abstract

The present invention relates to at least one novel anti-IL-12 antibodies, including isolated nucleic acids that encode at least one anti-IL-12 antibody, IL-12, vectors, host cells, transgenic animals or plants, and methods of making and using thereof, including therapeutic compositions, methods and devices.

Description

This application is based in part on, and claims priority to (05 Rgomysiopa! (provisional application) 60/223,358 filed Aug. 7, 2000 and 60/236,827 filed Sep. 29, 2000|, each of which is incorporated herein by reference in its entirety.

The present invention relates to antibodies, including the specified parts or variants, specific to at least one Interleukin-12 (I/-12), protein or its fragment, as well as to nucleic acids encoding antibodies to 1-12, complementary nucleic acids , vectors, host cells and methods of their preparation and use, including therapeutic compositions, applications and devices.

Interleukin-12 (11-12) is a heterodimeric cytokine that consists of glycosylated polypeptide chains with a mass of 35 and 40 kDa, linked by a disulfide bond. The cytokine is synthesized and secreted by antigen-presenting cells, including dendritic cells, monocytes, macrophages, B cells,

Langerhans and keratinocytes, as well as natural killer cells (MK cells). 11-12 mediates a variety of biological processes, and is referred to as a MK-cell stimulation factor (MK5B), a T-cell stimulation factor, a cytotoxic T-lymphocyte maturation factor, and a factor of EVvVU-transformed B-cell lines (Sygiv, 5). N. A. »., ey ai, Siipisa! Mishorioyudu Nemiemuv, 10: 742-780 (1997)|.

Interleukin-12 can bind to receptor 1-12 expressed on the plasma membrane of cells (eg, T cells, MK cells), thus changing (eg, initiating, preventing) biological processes.

For example, the binding of 1-12 to the I-12 receptor can stimulate the proliferation of T cells and MK cells, enhance the cytolytic activity of cytotoxic T cells (STI), HAC cells and MkC cells (killers activated by lymphokine), cause the production of gamma interferon (EM SAMMA) by T- and MK-cells and cause the differentiation of simple TpO cells into Tp1-cells that produce IEM SAMMA and 1--2 |ipspieti, a, Appia! Journal of Ittypoiodu, 13: 251-276 (1995)). In particular, I--12 is vital for the formation of cytolytic cells (eg, MK, ST) and for the generation of a cellular immune response (eg, a cellular response mediated by TP1 cells).

Thus, 1--12 is critically important for the development and regulation of both protective immune (e.g., inhibition of infections) and pathological immune response (e.g., autoimmunity) (Nepaggak, 9. A. apa Vgypaa, M.

U., Garogayugu Ipmeziidayop, 72: 619-637 (1995)). Thus, an immune response (eg, protective and pathogenic) can be enhanced, suppressed or prevented through manipulation of biological activity 1-12 ip mimo, for example via an antibody.

Chimeric, polyclonal (eg, antisera) and/or monoclonal antibodies (Mare) and fragments (eg, protein products of their proteolytic cleavage or fusion) of non-human mammals are potential therapeutic agents that have been studied in some cases in an attempted treatment certain diseases. However, such antibodies or fragments may elicit an immune response when administered to humans. Such an immune response may cause immune complex-mediated release of the antibodies or fragments from the circulation and render re-administration unacceptable for therapy, thereby reducing therapeutic benefit to the patient and limiting re-administration use of antibodies or fragments.

For example, repeated administration of antibodies or fragments that include non-human antibody portions may result in serum sickness and/or anaphylaxis. As is known in the art, in order to prevent these and other problems, many approaches have been tried to reduce the immunogenicity of such antibodies and parts thereof, including chimerization and humanization. These and other approaches, however, may provide antibodies or fragments thereof with some immunogenicity, low affinity, low avidity, or difficulty in cell culture, scale-up, production, and/or low product yield. Thus, such antibodies or fragments may not be ideally suited for production or use as therapeutic proteins.

It follows from this that there is a need both for the introduction of antibodies to 1I-12 or their fragments, which will overcome one of the mentioned problems, and for the improvement of known antibodies or their fragments.

The present invention provides isolated human, primate, rodent, mammalian antibodies, chimeric, humanized and/or SOBV-hybridized antibodies to 1-12, immunoglobulins, cleavage products and other specified parts or variants thereof, as well as compositions of antibodies to II/- 12, encoding or complementary nucleic acids, vectors, host cells, compositions, compositions, devices, transgenic animals, and methods of their preparation and use, as described and practiced herein, in combination with those known in the art.

The present invention also provides at least one isolated antibody to II-12 as described herein.

An antibody according to the present invention includes any protein or peptide containing a molecule that is represented by at least a portion of an immunoglobulin molecule, such as, but not limited to, a complementarity-determining region (CDO) of a heavy or light chain, or a region of binding to its ligand, the region of variability of the heavy or light chain, the region of constancy of the heavy or light chain, the framework region, or a part thereof, which can be included in the antibody of this invention. An antibody of the invention may include or be derived from any mammal, such as, but not limited to, human, mouse, rabbit, rat, rodent, primate, or combinations thereof, and the like.

The present invention provides, in one aspect, isolated nucleic acid molecules, including complementary or hybridized, polynucleotides that encode at least one idiotype of antibodies to II--12, including at least one specified sequence, domain, part or variant thereof. The present invention also provides recombinant vectors comprising said nucleic acid molecules encoding an anti-idiotype antibody 1-12, a host cell containing such nucleic acid and/or vectors, as well as methods for obtaining and/or using such anti-idiotype nucleic acids antibodies, vectors and/or host cells.

The present invention also provides at least one method of expressing at least one antibody to 1-12 or antibody to idiotype 1-12 in a host cell, which comprises culturing the host cell as described herein, provided that at least one antibody to 1- 12 is expressed in amounts sufficient for detection and/or isolation.

The present invention also provides at least one composition comprising (a) an isolated nucleic acid antibody to 1-12 and/or an antibody as shown herein; and (b) a suitable carrier or diluent. The carrier or diluent may be, but need not be, pharmaceutically acceptable, in accordance with known carriers or diluents.

The composition may optionally further include at least one component, protein or composition.

The present invention also provides at least one method or composition of an antibody to I-12 for use in a therapeutically effective amount to modulate or treat at least one condition of a cell, tissue, organ, animal or patient associated with I-12 and/or one that precedes it, follows it, runs over for related reasons, as shown here.

The present invention also provides at least one composition, device and/or method for delivering a therapeutically or prophylactically effective amount of at least one antibody to 1-12, according to the present invention.

The present invention further provides at least one method or composition with antibodies to 1-12 for diagnosing at least one condition of a cell, tissue, organ, animal or patient associated with and/or preceding 1-12, follows him, defecting for related reasons, as shown here.

The present invention also provides at least one composition, device and/or method of delivery for the diagnosis of at least one anti-II-12 antibody according to the present invention.

Figures 1A and 18: graphs showing concentration-dependent binding of monoclonal anti-1I.-12 antibodies to immobilized human I/-12. Anti-II-12 antibodies were sequentially washed 1905

B5BA/RV5 and incubated on plates coated with hPI--12 for one hour at 37"C. Then the plates were washed twice with 0.0295 Tween 20 (polyoxyethylene (20) sorbitan monolaurate), 0.15M saline and then tested with horseradish peroxidase (HPR) with labeled goat anti-human anti-kappa antibodies for 1 hour at room temperature.The plates were then washed again, treated with o-phenylenediamine (OR) substrate, and the optical density (00) of each well was measured at 490 nm.

Figure 2: Left to right bands in Figures A and B correspond to human 1-12, human I//12 p40, mouse 1-12 and molecular weight markers. Figure A shows areas stained with total protein.

The original bands from each lane are human I-12 (75kDa), human p40 1-12 (40kDa), and mouse I/-12 (75kDa). On

Figure 28 shows an image obtained from a gel identical to that shown in Figure 2A. The blot was treated with C340 after HER with labeled goat antibodies specific for human anti-Ido and specifically detected only human I/-12 (monomers and multimers) and human I/-12 p40. Control spots (not shown) treated with HER, labeled with goat antibodies specific for human anti-doe showed no zones at all.

Figure 3: Analysis by RSE reverse transcription of the expression of the IEMu gene in human RV5 treated with I1I--2, I-12, 1--2-41-12 with or without anti-II/-12 antibodies C340, 8.6.2, isotype controls antibodies Total EMA was reverse transcribed, amplified by PCR using gene-specific primers.

The level of β-actin tEMA was also determined in each sample, which served as a control for the integrity and content of TEMA.

Figure 4: is a histogram showing that human anti-II-12 TAB (C340) inhibits the production of interferon-γ (IEMu) by CO3-depleted monocytes from peripheral blood mononuclear cells (PBMCs) stimulated with 1--2-1Й1- 12. PBMCs were cultured for five hours in control media (without the addition of cytokines), media with the addition of 1-12 (0.1 Tng/ml) plus 1-2 (50 IU/ml) (C-12/1-2), control environments with the addition of tAb C340 (10 do/ti) and environments 1--12/I-2, which contained tA C340 (10 do/ti). Intercellular IPM was determined using two-color immunostaining of SOZ-RE and IEMu-RITS. Data are given for one donor.

Figure 5: Diagram showing dose-dependent inhibition of IEM secretion by peripheral blood lymphocytes stimulated with 1-2 plus 1-12 two different groups of human anti-1II-12 tAbBr (C340). Human RVI (in106/ti) were cultured for 24 hours with 10Sh/ti I--2, 1-2 plus 4b0orad/ti 1I--12, or 1-2 plus 1-12 and tA C340 as shown. Culture supernatants were removed and examined on IEM using EA.

Figure 6: Histogram showing the dose-dependent inhibition of cytotoxicity of I AK cells induced

I/-12 plus 1-2 antm-11I-12 tAr (0340) of a person. HAC effector cells (human RVI, 8x106/ti) were cultured for 24 hours with 1-12 (400 rd/ti) plus 1-2 (100/ti) and tAB C340 (5000pad/ti or 50pd/ti as shown). GAC effector cells were washed and cultured with Kai target cells labeled with 51Cg for four hours at an effector to target ratio (E:T) of 80:11, and the amount of 51Cg released into the medium as a result of Kai cell lysis was determined. Results are presented as mean with standard error of three normal donors. I/-12 positive control (1-12) are effector cells incubated with and without 1-12 antibodies. The background (VKOB) is effector cells incubated without 1-12 or antibodies.

Figures 7A and 7B: are histograms demonstrating that 1-12 plus IS-2-induced expression of ХО95 on peripheral blood mononuclear cells COZ3- is inhibited by human anti-I./-12 tA (2340). RVMSO were cultured for 72 hours on a medium with a content of 1-12 0.Tpd/ti and a suboptimal dose of 1-2 (5010/tiI) in the presence or absence of tA C340 (10yd/tiI). CO95 expression was measured by flow cytometry of cells treated with anti-CO95-RITS. Light transmission was performed with the help of two-color analysis (SOZ or СО56-РЕ uv. СО95-РІТС) and direct uz. orthogonal light scattering.

Figure 8: The diagram shows that recombinant human antibodies (gC340) to human 1-12 bind to immobilized 1-12 in a manner that does not differ from purified tAb C340. The concentration of "9340 in the supernatant of three recombinant cell lines that produce hC340 was determined, and the binding of the supernatant to 1-12 in EHI5A was determined.

Plates were coated with 2 μl/ti of 11/-12 human and incubated with purified tABb C340 from the original hybridoma (standard) or supernatants of recombinant cell lines. The II -12-threshold of antibodies was determined using goat antibodies to dO (heavy chain-light chain) of a person, conjugated with alkaline phosphatase.

Figure 9A-9C are graphs showing the kinetics of growth and amount of antibody secreted by three independently derived KS-340 producing recombinant cell subclones (Figure 9A, subclone C3798B;

Fig. 9B, subclone SZ381A; Fig. 9C, subclone 389A). Recombinant cells were planted in flasks 175 with an initial density of 2x10 cells/th in a standard medium. After different time intervals, the cells were resuspended and the number of living cells and the value (n/t) of "C34O" in the medium were determined.

This invention offers isolated recombinant and/or synthetic anti-III-12 of humans, primates, rodents,

mammalian, chimeric, humanized or SOC-hybridized, antibodies and anti-idiotypic 1-12 antibodies thereof, in addition, nucleic acid compositions and encoding molecules that include at least one polynucleotide that encodes at least one anti-II-12 antibody or anti-idiotypic antibody . The present invention also includes, but is not limited to, methods of making and using such nucleic acids, antibodies, and anti-idiotypic antibodies, including diagnostic and therapeutic compositions, methods, and devices.

This description uses the terms "anti-interleukin-12 antibody", "anti-II-12 antibody", "anti-II-12 antibody part", "anti-II-12 antibody fragment" and/or "anti-II variant" -12 antibodies" and the like, include any protein or peptide containing a molecule represented by at least a portion of an immunoglobulin molecule, such as, but not limited to, at least one complementarity-determining region (CDO) of a heavy or light chain or part thereof , which binds the ligand, or the variable region of the heavy or light chain, or the constant region of the heavy or light chain, the framework region, or any part thereof, or at least one part of a receptor 1-12 or a binding protein that can be included in the antibody of this invention. Such an antibody may act on a specific ligand, such as, but not limited to, that such antibody modulates, decreases, enhances, antagonizes, agonizes, inhibits, stimulates, blocks, inhibits, abrogates and/or interferes with at least one action 1-12 , at least with one activity or binding of I/-12 or activity or binding of receptor 1-12, ip migo, ip 5yi and/or ip mimo. As a non-limiting example, an acceptable anti-1II-12 antibody, specified part or variant of the present invention can bind to at least one 1-12, with its specified part, variant or domain. An acceptable anti-1/-12 antibody, said portion or variant may also optionally affect at least one activity or function of 1-12, such as, but not limited to: EMA, OMA or protein synthesis, release

I/-12, signaling of receptor 1-12, cleavage of II--12 membranes, activity of I--12, production and/or synthesis of I/-12. The term "antibody" further describes antibodies as a whole, cleavage fragments, specified parts and variants thereof, including antibody mimetics or those containing parts that mimic the structure and/or function of antibodies or specified fragments or parts thereof, including single-chain antibodies and their fragments. Functional fragments include antigen-binding fragments that bind human IL-12. The present invention includes, for example, an antibody fragment capable of binding to IL-12 or a portion thereof, including, but not limited to: Rab (eg, papain hydrolysis),

EPar' (e.g., hydrolysis by pepsin and partial reduction) and K(ab) (e.g., hydrolysis by pepsin), Tas (e.g., hydrolysis by papain) pEs' (e.g., hydrolysis by pepsin or plasmin) Pa (e.g., hydrolysis pepsin, partial reduction and reaggregation), Em or 5cEm (e.g., molecular biology methods) (see, e.g., Soyiiidap,

Ittypoiodu, zirga).

Such fragments may be obtained by enzymatic cleavage, synthesis or recombination methods as known in the art and/or as described herein. Antibodies can also be produced in various truncated forms using antibody genes in which one or more stop codons are inserted above the natural stop sites. For example, a combination of genes encoding part of the heavy chain

E(ar)"2 can be constructed by including the OMA sequence encoding the CH, domain and/or loop link of the heavy chain. Different parts of antibodies can be joined together by conventional chemical means, or made as a whole protein by genetic engineering methods.

As used herein, the term "human antibody" refers to an antibody in which each portion of the protein (eg, SOE, framework, C, C domains (eg, Cn1!, Cn2g, Cn3Z), loop, (Mi, Mn)) are mostly non-immunogenic for humans, with only minor changes in sequence and variants. Similarly, antibodies are specified as primate (monkeys, baboons, chimpanzees, etc.), rodents (mice, rats, rabbits, guinea pigs, gophers, etc.) and other antibodies specified as specific for mammals, species, subgenus, genus , subfamilies, families. Further, chimeric antibodies include any combination of the aforementioned antibodies. Such changes or variations do not necessarily and preferably preserve or reduce immunogenicity in humans or other species, refer to unmodified antibodies. Therefore, human antibodies are different from chimeric or humanized ones. It has been noted that human antibodies can be produced by other animal, prokaryotic or eukaryotic cells capable of expressing a functionally rearranged human immunoglobulin gene (eg, heavy and/or light chain). Further, when the human antibody is a single-chain antibody, it may contain a linker peptide that is not found in natural human antibodies. For example, Em can contain a linker peptide, such as two to eight glycines or other amino acid residues, which connect different regions of the heavy chain and different regions of the light chain. Such a linker peptide can be recognized as a peptide of human origin.

Bispecific, heterospecific, heteroconjugate or similar antibodies can also be used as monoclonal, preferably human or humanized, antibodies that have specific binding to at least two different antigens. In this case, one of the binding specificities must be at least one to 1-12 protein, and the second must be to another protein. Methods of obtaining bispecific antibodies are known from the state of the art. Traditionally, recombinant methods of obtaining bispecific antibodies are based on the simultaneous expression of two pairs of immunoglobulin heavy chains-light chains, where the two heavy chains have different specificities (Miivieip apa Sieiyo, Maishge 305: 537 (1983)). Due to the random selection of heavy and light chains, these hybridomas (quadroms) potentially produce a mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecules is usually done using affinity chromatography, which is quite complex, resulting in low product yields. Подібні процедури описано, наприклад, (М/О 93/08829, патенти 05 6210668, 6193967, 6132992, 6106833, 6060285, 6037453, 6010902, 5989530, 5959084, 5959083, 5932448, 5833985, 5821333, 5807706, 5643759, 5601819, 5582996, 5496549, 4676980, UMO 91/00360, UMO 92/00373, ER 03089, Tgashpeskeg ega!I., EMVO .). 10: 3655 (1991), Z!,gezp ei ai., Meshioaz ip Epguto!odu 121: 210 (1986), which are incorporated herein by reference in their entirety).

Anti-1II-12 antibodies (also called antibodies to 1-12) useful in the methods and compositions of the present invention may, optionally, be characterized by high binding affinity to 1II/-12 and optionally and preferably, low toxicity . In particular, antibodies, fragments or variants of the invention are suitable for this invention, where the individual components, such as the region of variability, the region of constancy and the framework, individually and/or together, optionally and preferably have low immunogenicity.

Antibodies that may be used in the present invention may optionally be characterized by their ability to treat patients over a long period of time with marked relief of symptoms and low and/or acceptable toxicity. Low or acceptable immunogenicity and/or high affinity, as well as acceptable properties, may account for the therapeutic results obtained. "Low immunogenicity" is defined as exceeding the values of NANA, NASA or NAMA responses by less than 7595, or preferably less than 5095 in patients and/or exceeding low titers in patients (less than 300, preferably less than 100 determined by double antigen enzyme immunoassay) (See, e.g., ЕЙПойк ег ай., Gapsei 344: 1125-1127 (1994), which is incorporated herein by reference in its entirety).

Model

The isolated nucleic amino acids of the present invention can be used to produce at least one antibody to 1-12 or a specified variant thereof, which can be used to detect or act on a cell, tissue, organ or animal (including a mammal and humans), diagnose, monitor, modulate , treating, ameliorating, helping to prevent worsening, reducing symptoms of at least one of II--12-related conditions selected from, but not limited to, at least one immune disorder or disease, cardiovascular disorder or disease, infection, malignancy neoplasms, and/or neurological disorders or diseases, or other known or identified conditions associated with II -12.

Such a method may include administering an effective dose of a composition or pharmaceutical composition comprising at least one anti-1II-12 antibody to a cell, tissue, organ, animal or patient for the purposes of such modulation, treatment, enhancement, prevention or reduction of symptoms, effects or mechanisms.

An effective amount may be from 0.001 to 500 td/ti per unit (eg, bolus), multiple or prolonged administration, or to achieve a serum concentration of 0.01-5000 td/ti per single, multiple or prolonged administration, or any effective range thereof. or values as made and determined using known methods and as described herein or known from similar works.

All publications or patents cited herein are hereby incorporated by reference in their entirety to the extent that they reflect the state of the art at the time of this invention and/or assist in the disclosure and practice of this invention. Publications include any scientific or patent publications, or any which information is available in media, including all recorded, electronic or printed media The following links are incorporated by reference in their entirety:

Iaizibei, eyi ai., ed., Sitepi Rgoioso!v ip MoiIesshag Vioiodu, dopp UMieu 5 Bopv, Ips., MU, MU (1987-2001);

ZatbgookK, ei ai., MoIesshag Siopipd: A Igarogaigu Mapia), 2pa Eayop, Soii brgipd Natbog, MU (1989);

Napomapa I ape, anTtMbodyez, a I arogaiyugu Mapiai, Soia brgipd Nagpog, MU (1989); SoiPidap, ei a!., eaav., Sshtepi

Rgoiyusoiv ip Ittypoiodu, adjunct of UMieu 5 Bopv, Ips., MU (1994-2001); SoiPdap ei ai., Sitepi Rgoiosoi!5 ip Rgoieip

Zsiepse, dhopp UMieu 5 Zopv, MU, MU, (1997-2001).

Antibodies of this invention

At least one anti-1III-12 antibody of the present invention may optionally be produced by a cell line, a mixed cell line, immortalized cells, or a clonal population of immortalized cells, as is known in the art. | See eg.

MMPeu b bopv, Ips., MU, MM (1987-2001); Zatgook, ei ai., MoIesshag Siopipd: A I arogaiyugu Mapiai, 2"Eashtsop,

So brgipd Nagrog, MM (1989); Napom apa and ape; antirodiez, and I aroghaiogu Mapiai!, Soia brgipd Nagbog, MU (1989); SoipPidap, yei a!., edv., Sitepi Rgoiosoi!v ip Ittipoidodu, dopp Ulieu b ope, Ips., MU (1994-2001); SoiYidap ei ai., Sstepi Rgoiosoiv ip Rgoieip 5siepse, dop UMieu 5 Bopz, MU, MU, (1997-2001), all fully incorporated by reference).

Human antibodies specific for human I/-12 protein or fragments thereof may have an advantage over a corresponding immunogenic antigen, such as isolated and/or I/-12 protein or a portion thereof (including synthetic molecules such as synthetic peptides). Other specific or general mammalian antibodies may also be preferred. The production of immunogenic antigens and the production of monoclonal antibodies can be performed according to an acceptable method.

For example, the hybridoma can be obtained by fusion of acceptable immortalized cell lines (eg, myeloma cell lines such as, but not limited to, 5рg2/0, 5р2/0-АС14, MBO, M51, M52, AE-1, I. 5 , »243, R3UHbZAd8. 653, 5ra ZAZ, 5ra2 MAI, 5ra2 551, 5ra 5AB, 0937, MI A 144, AST IM, MOI T4, SA-1, Ch"0OAKAT,

MENI, K-562, CO5, VASHI, MIN 313, NI-60, MHA 144, MAMAIMUA, MESHKO 2A, or similar, or heteromyelomas, their fusion products), or any cells or fusion cells derived from them, or other suitable cell lines are known in the art (see, e.g., m/mlu.aiss.ogo, mule. Pegesp.soti| and the like, to cells producing antibodies, such as, but not limited to, isolated or cloned cells spleen, peripheral blood, lymph, tonsil, or other that contain immune or B cells, or other cells that express a heavy or light chain, constant or variable region or framework or SOC sequence, endogenous as well as heterogeneous nucleic acids, recombinant or endogenous viruses, bacteria, algae, prokaryotes, amphibians, insects, reptiles, fish, mammals, rodents, horses, rams, goats, primates, eukaryotes, genomic OMA, SOMA, OMA, mitochondrial OMA or CMA, chloroplast OMA or KMA, ppPKMA,

SUBJECT, SUBJECT, single-, double-, or triple-stranded, hybridized, and similar or a combination thereof. IDiv. e.g., zirga, apa Soiidap, Ittypo!odu, zirga, sparieg 2, which is fully incorporated by reference|.

Antibody-producing cells can be obtained from peripheral blood or, preferably, the spleen or lymph nodes of a human or other suitable animal that has been immunized with the target antigen.

Another acceptable host cell can also be used to express a heterogeneous or endogenous nucleic acid that encodes an antibody, a specific fragment, or a variant thereof, according to the present invention. Fusion cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other acceptable known methods, and cloned by limited dilution or selection of cells, or by other known methods. Cells that produce antibodies with the desired specificity can be selected by an acceptable analytical method (eg, EGIBZA).

Other acceptable methods of obtaining or isolating antibodies of the desired specificity may be used, including, but not limited to, methods by which recombinant antibodies are selected from a peptide or protein library, e.g. but not limited to, bacteriophage, ribosomal, oligonucleotide, EMMA, SOMA or the like; E.g., if available, from display libraries

Satrpdde apirodu Tesppoiodiez, Satrgpddevzpigtye. SHK; Mogrpozuv, Mapipvgeid/Riaped9, OE; Wiomayop,

Aregdeep, bsoyapa, OK: Vioipmepi, pa, Zmedep; Ouah Sogr., Epgop, Apyutakh/Vioziye; Hota, WegKkeieu, SA;

Ikhzuz See, e.g., ER 368, 684, PCT/5891/01134; PCT/5892/01755; PCT/2B92/002240; PCT/2B92/00883;

PCT/ав93/00605; 05 08/350260 (5/12/94); PCT/а894/01422; PCT/а894/02662; PCT/2897/01835; (SAT/ISS);

M/090/14443; M/090/14424; MO90/14430; PCT/O594/1234; M/092/18619; M/096/07754; (Soviet Union); ER 614989 (Mogrpobuvz); UU095/16027 (Vioipulep); UUO88/06630; M/090/3809 (Ouach); 5 4,704,692 2 (Epgop);

PCT/Y591/02989 (ApPutakh); UMO89/06283; ER 371998; ER 550400; (Hota); ER 229046; PCT, 07149 (Ikhvuv)|; or stochastically generated peptides or proteins - (05 5723323, 5763192, 5814476, 5817483, 5524514, 5976862, MO 86/05803, EP 590689 (Ikhzuz, now Arriea MoiIesshag Emoisiop (AMIE) each of which is incorporated by reference in its entirety) , which are used in the immunization of transgenic animals, E.g., 5SIO tese, Mdyuep ei ai., Misgobryo!. Ittypoi!. 41: 901-907 (1997); Zapany ei ai., St. Aem. Vioiespoi. 16: 95-118 ( 1996); Egep et al., Ittypoi. 93: 154-161 (1998), which are hereby incorporated by reference in their entirety, capable of producing a specific list of human antibodies as known in the art and/or described herein. Such techniques, including but not limited to this, "gyrozote aiizriau" (Napez ei ai., Rgos. May). Asad. 5si. OBA, 94: 4937- 4942 (Mau 1997); Napez eyi aiYi., Rgos. May. Asad. Osi. OBA , 95: 14130-14135 (Mom. 1998))|; methods of antibody production with the help of single cells. For example, the method of obtaining antibodies of selected lymphocytes ("ZIGAM") (05 rai. Mo. 5,627,052, UMep ei ai., 9. Ittipoi. 17: 887-892 (1987); Varsook ei ai. , Rhos.

May Asad Axes BOTH 93: 7843-7848 (1996)); microdrop-gel and flow cytometry (Rouei! ei ai.,

Vioyesnpoi!. 8: 333-337 (1990); Ope Sei! Zuvietv, Satrbigdde, MA; Stgau eyi a)., 9). Etc. Meiy. 182: 155-163 (1995);

Keppu ei ai., Vioiesppoi!. 13: 787-790 (1995)); selection of B-cells |BieeprakKkKeg"z ei ai., Moies. Vioi. Nerohiv 19: 125-134 (1994); dopak ei a!i., Rgodgez55 Vioiyesi, Moi. 5, Ip Migo Ittipigayop ip Nubgidota Tesppoiodu, Voiterayesk, ei ., Eivemieg Zsieepse RybiizNegz V. M., Atviegdat, MemShepapavz (1988)|.

Methods of creating or humanizing non-human and human antibodies that are generally known in the art can also be used. In general, humanized or engineered antibodies have one or more amino acid residues from a non-human source, eg, but not limited to, mouse, rat, rabbit, non-human primate, or other mammals. These human amino acid residues are often called "import" residues, which are usually derived from the "Import" variable, constant, or other domain of known human sequences. The known human Id sequences are given, e.g.,

Hmyllu pores sweat.piy.dom/epigeg/diegu.isdi; cute agss.ogo/rpade/par.niti; mlmlu.vsidevisot/; mlmlmu.arsat.sotu/; mlmly.apiirodugezotsgse.sot/opiipesotr. (ti; mly. ribiis.iaziasge.edi/"reago/gezeagspiooiv. piti; mumlu. tdep.ipipeiideIrego.ae/5O/LTLT.NETi; mulu. m piteetap.sot/ttypoiodu/SNOB/Kkyruob. pit; mul Tyugagu Fipkdneviogo /12429/LIttipe/ANTMbroau. piti; muly. pi ti.ogo/dgapivLesiigev/1996/mar/; mly. ra. sat.as.ik/"tgs7/tpikeiitadev.piti; muly. apirodugezotsgse.sot/; ter.Nagmaga .edi/Vio ipko/typoiodu.piti; mli. ittypoioduiipKosot/; ragtrokhlmyvi.edi/-Nsepieg/Ipaeh. pi ti; mumlu.rioitesp sht eits/-psi/ 0 mumlu.rebio.sot/ra/340913/340913. piti; /- mumlu.pai. izda.dom/ajis/ryrz/antirodu/; muly. gp.epite-y.as.|r/"uaziniu/Eiiva. piti; mm. riodevzidp.sot/arie.azr; mlmlu . spe ik/akhr/asz/damiezlLipkv.piti;. milu. riotesn. y. eyi/Tosi/rgoiosoi.piti;. mly. izaspeiogo/ziev deo.piti; akhitN Itipi-tagrygo.de/"hek/AER Ban. piti; Bazegu.ysi.Kip.pi/igaaivLipke1.piti; mumly.tesabr. ipin-npa.de/ltypo.Yute. pumi. edi/; mulu. tto-sre.sat. as. ikK/ti-dos/ryriis /IMT VO. piti; mila. ri ipat.thx/mig/M tise.piti; etc. spivs. 81047; mili. Viospet. si. as. ik/eta pip/arz/Ipayeh. sweat; aNTproau.rak.as.ik/; ardep.smt.aty.edi Larlyummardep. sweat; mumlu.ypi2n.sp/"popedde/ANOzetipag/5iIideb1. pEti; mumlm.sguviVrK. as.ik/"irs9o75/; mm translation tgo.as.ik/SS/ssayemd/ssayemo. sweat; cute cancer. sat.as.ik/e tgs7/pitapizayop/TANNR. drinks; murmured ri pat. tx/mig/5iisshge/vnai ait. sweat; hmm riossi tivzotsShgi.edi/5tipor/ipayeh. sweat; mlmly.sguvi.rios.sat. as.ik/Utoiipal//ler-radez/Reriuvrotesn.pi ti; had |Egipi el Rgodisiv. sweat; mumble raiepiv.Irt.sot/irt.piti. Kabai ei ai., Zedoepsez oi Rgoieip5 oi Ittipoiodisa! Ipiegewzi, 0. 5. Oeri. Neanith (1983), incorporated by reference in its entirety). These import sequences can be used to decrease immunogenicity or decrease, increase or modify binding, affinity, op-gaie, o-gaie, avidity, specificity, half-life, or other acceptable characteristics, as known in the art. In general, some or all of the human or non-human SOC sequence is conserved while the constant and variable regions are replaced by human or other amino acids. Antibodies can also, optionally, be humanized while maintaining high affinity to the antigen and other desired biological properties. To achieve this goal, humanized antibodies can optionally be derived from analysis of parental sequences and various conceptual humanized products using three-dimensional models of parental and humanized sequences. Three-dimensional models of immunoglobulin are generally available and well known to those skilled in the art.

Computer programs are available that illustrate and display possible three-dimensional conformational structures of selected candidates among immunoglobulin sequences. The study of these maps allows to analyze the possible roles of residues in the functioning of candidate immunoglobulin sequences, eg, the analysis of residues that affect the ability of candidate immunoglobulins to bind to an antigen. In this way, EC residues can be selected and combined from consensus and import sequences in such a way that the desired antibody characteristics, such as increased affinity for the target antigen(s), will be obtained. In general, SOC residues are directly and more significantly involved in influencing antigen binding. Humanization and construction of the antibodies of the present invention can be accomplished by any known method, such as, but not limited to, those described in: (Mi/ipieg (Copez et al., Mashge 321: 522 (1986); Wiesptapp et al! ., Mashge 332: 323 (1988); Metoeuep ei ai., Zsieepse 239: 1534 (1988)), 5its ei ai., 9. Ittipoi. 151: 2296 (1993); Spoipia apa G ez5K, 9. Mo. Visi . 196: 901 (1987), Sapeg eyi a!., Rgos. Maiyi). Asai. si. 0. 5. A. 89: 4285 (1992); Rgevia vy ai., 9U. And so on. 151: 2623 (1993), О5 раїепі Мов : 5723323, 5976862, 5824514, 5817483, 5814476, 5763192, 5723323, 5766886, 5714352, 6204023, 6180370, 5693762, 5530101, 5585089, 5225539, 4816567, РСТ/: О598/16280, 00596/18978, 0591/09630, 0591/05939,

O594/01234, 4889/01334, 891/01134, 1892/01755; M/090/14443, UMO90/14424, M090/14430, ЕР229246, all fully incorporated by reference, including references therein).

Anti-1/-12 antibodies can also be obtained by immunization of transgenic animals (eg, mice, rats,

gophers, primates, but not humans, and the like) capable of producing a variety of human antibodies as described and/or known in the art.

Cells producing anti-II/-12 antibodies can also be isolated from such animals and immortalized by suitable methods, such as those described herein.

Transgenic mice that can produce a spectrum of human antibodies that bind to human antigens can be obtained by known methods, such as, but not limited to, . 5. Paradise. Languages: 5,770,428, 5,569,825, 5,545,806, 5,625,126, 5,625,825, 5,633,425, 5,661,016 apa 5,789,650 issued on Gopreg ei a).;

UaKobomiiv ei ai. UUO 98/50433, ChdaKobomiiv eyi a. UUO 98/24893, I oprego eyi aI. UUO 98/24884, I oprego her a. MO 97/13852, I oprego eyi a. MO 94/25585, Kispeparaye eyi ai. UUO 96/34096, Kispeparaye ei aI. ER 0463151 VI1,

Kispeparaie eyi a. ER 0710719 JSC, Bigapi eyi a. 05. Paradise. Mo. 5,545,807, Vpddetapp ei ai. UU/UO 90/04036,

Vgoddetapp ei ai. ER 0438474 В 1, I opregu ei ai. ER 0814259 A2, I oprego ei aI. OV 2272440 A, I oprego eyi a.

Mayte 368: 856-859 (1994), Tauiog et al., Ipi. And so on. 6 (4) 579-591 (1994), Stgeep vy ai, Mayte Sepeiysv 7: 13- 21 (1994), Mepaee? ei ai., Maishge Sepeiysv 15: 146-156 (1997), ei ai., Misiyeis Asidz Nezeagsi 20 (23): 6287-6295 (1992), Tiayop ei ai., Rgos Mai! Asai 5si OBA 90 (8) 3720-3724 (1993), I oprego ei ai., Ipi Vem Ittipoi 13 (1): 65-93 (1995) apa Rivpmaia ei ai., Mai Vioiesppoi! 14 (7): 845-851 (1996), the entirety of which is incorporated by reference).

In general, these mice include at least one OMA containing a transgene from at least one human immunoglobulin locus that is functionally rearranged or can be functionally rearranged. Endogenous immunoglobulin loci in such mice can be truncated or deleted to suppress the animal's ability to produce antibodies encoded by the endogenous genes.

Screening of antibodies for the specificity of binding to similar proteins or fragments can be easily carried out using display peptide libraries. This method involves screening large collections of peptides to identify those that have the desired function or property. Antibody screening in peptic display libraries is well known in practice. Display peptide sequences can be from 3 to 5000 or more amino acids in length, most commonly 5-100 amino acids in length, and even more commonly 8 to 25 amino acids in length. In addition to the methods of generating peptide libraries by direct chemical synthesis, several methods of recombinant OMA have been described.

One type involves displaying a peptide sequence on the surface of a bacteriophage or cell. Each bacteriophage or cell contains a nucleotide sequence that encodes, in particular, a display peptide sequence. This method is described in PCT Raiepi Rybiisayop Mo5 91/17271, 91/18980, 91/19818, and 93/08278). Other systems for generating peptide libraries have features of both chemical synthesis and recombinant methods. See PCT Raiepi Rybiisayop Mo5. 92/05258, 92/14843, and 96/19256. See also, 0. 5.

Raiepi Mov. 5,658,754; apa 5,643,768)|. Peptide display libraries, vectors, and screening kits are commercially available from suppliers such as: Ppmigodep (Saghizride, CA), apa Satgidde Antirodou Tesppoiodie5 (Satrgiddespige, OK). See, for example, Sh. 5. Rai. Mo5. 4704692, 4939666, 4946778, 5260203, 5455030, 5518889, 5534621, 5656730, 5763733, 5767260, 5856456, issued on Eplop; 5223409, 5403484, 5571698, 5837500, issued in Ouah, 5427908, 5580717, issued in APyut; 5885793, issued on Saturyugidde of the anti-genus

ThesppoIodie5; 5750373, issued on Sepepiesp, 5618920, 5595898, 5576195, 5698435, 5693493, 5698417, issued on Khota, Soiida, Zirga; A!, zibei, zirga; oh zatrgook, zirga. Each of the listed patents and publications is incorporated by reference in its entirety).

Antibodies of the present invention may also be produced using at least one nucleic acid encoding an anti-1Y-12 antibody to generate transgenic animals or mammals such as goats, cows, horses, sheep, and the like, to produce such antibodies in their milk. Such animals can be created using known methods. | See e.g., but not limited to these, О5 raiepi pov. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; 5,304,489, et seq., each of which is incorporated by reference in its entirety).

Antibodies of the present invention can additionally be produced using at least one nucleic acid encoding an anti-1II-12 antibody to generate transgenic plants and cultured plant cells (eg, but not limited to, tobacco, corn) that produce such antibodies, said parts or variants in parts of plants or in cells cultured from them. As a non-limiting example, transgenic tobacco leaves that express recombinant proteins have been successfully used to create a large number of recombinant proteins, eg, using an inducible promoter.

See e.g., Stateg ei ai., Sigg. Torus. Misgoroi And so on. 240: 95-118 (1999) and references cited therein).

Also, transgenic maize has been used to express mammalian proteins in commercial quantities, and with biological properties equivalent to those produced in other recombinant systems or isolated from natural sources. | See e.g., Nosa ei a!., Adu. Ex. Honey. Vio!. 464: 127-147 (1999) and references cited therein), antibodies have also been produced in large quantities from seeds of transgenic plants, including antibody fragments such as single-chain antibody (5SEU5), including tobacco seeds and potato roots. (See, e.g., Sopgai et al., Riapi Moi. Vioi. 38: 101-109 (1998) and references cited therein|. Thus, the antibodies of the present invention can also be produced using transgenes, according to known methods. See . also, for example, Rizspeg ei a!., Vioitesppoi. Arri.

Viospet. 30: 99-108 (Osi., 1999), Ma ei ai., Tgepaz Viotesnpoi. 13: 522-7 (1995); Ma ei ai., Riapi Rpuzio!. 109: 341-6 (1995); M/pieYiat ei ai!., Viospet. boss. Tgapv. 22: 940-944 (1994); and references therein). Also general for expression of antibodies in plants, but not limited to this. Each of the above references is incorporated by reference in its entirety.

Antibodies of the invention can bind human anti-I.-12 with a wide spectrum of affinity (Ko). In a preferred embodiment, at least one human tAr can bind human I/-12 with high affinity.

For example, a human tAb can bind 1-12 with a Co equal to or less than 1077M, as well as, but not limited to, 0.1-9.9 (or other value or range of) X 1077, 1098, 1079, 1079, 1071, 1072, 1073, or some other value or range thereof.

The affinity or avidity of an antibody to an antigen can be determined experimentally using the appropriate method, see eg.

Ittypoiodu, Rush, M.E., Ea., Namep Rgev55: Mem/ MoiKk, MU (1984); Kybu, dapiz Ittypoiodu, MU. N. Egeetap apa

Sotrapu: Mem Moik, Moscow State University (1992); and methods are given here). Values of affinity, individual antibody-antigen interactions may vary depending on different conditions of determination (e.g., salt concentration, pH). Thus, determination of affinity and other antigen binding parameters (eg, Co, Ka, Ka) is preferably performed with standardized solutions of antigen and antibody, a standardized buffer, such as the buffer described herein.

Nucleic acid molecules

Using the information provided herein, such as a nucleotide sequence encoding at least 70-10095 contiguous amino acids of at least one of ZEO IO MOB: 1, 2, 3, 4, 5, 6, 7, 8, specific fragments, variants, or consensus sequences thereof , deposited vectors that contain at least one of these sequences, a nucleic acid molecule of the present invention and at least one anti-1I-12 antibody can be obtained using the methods described herein or known in the art.

Nucleic acid molecules of this invention can be in the form of EMA, such as teMA, ppeMA,

IVEMA or any other forms, or in the form of OMA, including but not limited to SOMA and genomic OMA obtained by cloning or synthetically, or any combination thereof. OMA can be three-chain, two-chain or any combination thereof. Any part of at least one chain of OMA or RMA can be a coding chain, also known as a sense sequence, or can be a non-coding chain, also known as an antisense sequence.

Isolated nucleic acid molecules of the present invention may include nucleic acid molecules that have a single open reading frame (OPE), optionally with one or more introns, e.g., but not limited to, at least one specified portion of at least one ORP, such as SOK1, SOBKO and/or

SOCZ, at least one heavy chain (eg, ZEO IU MOB: 1-3) or light chain (eg, ZEO IU MOB: 4-6); and nucleic acid molecules that contain a nucleotide sequence significantly different from that given, but which, due to the degeneracy of the genetic code, can still encode at least one anti-

I/--12 antibody as shown herein or known in the art. Of course, the genetic code is very well known in practice. Thus, it would be a mere mechanical procedure for one skilled in the art to generate such degenerate nucleic acid variants encoding specific anti-1I-12 antibodies. (See, e.g., A!zibrei, ei a!., zirgai, and such nucleic acid variants are also included in the present invention. Non-limiting examples of isolated nucleic acid molecules of the present invention include ZEO IU MOB: 10-15, and are non-limiting examples of nucleic acid that encodes, respectively, НС СОК1, НС СОК2, НС СО3, 1 С СОМ1.

Го сОок2, 1 С СОМ, NS variable region AND С variable region.

On the other hand, the invention provides isolated nucleic acid molecules that encode a(n) anti-1(--12) antibody having the amino acid sequence encoded by the nucleic acid contained in the plasmid deposited as the specified clone name and ATOS Oerosion Mo5, respectively, deposited in .

As indicated herein, nucleic acid molecules of the present invention that contain nucleic acid encoding anti-I-12 antibodies may include, but are not limited to, such encoding amino acid sequence of the antibody fragment, itself; the coding sequence for a complete antibody is part of it; an antibody coding sequence, fragment or part, as well as additional sequences, as well as the coding sequence of at least one signal leader or fusion peptide, with or without the aforementioned additional coding sequence, such as at least one intron together with an additional, non-coding sequence, including but but not limited to, non-coding 5' and 3' sequences, such as transcribed, untranslated sequences that play a role in transcription, processing of TEMA, including splicing and polyadenylation signals (eg, ribosome binding and TEMA stability); an additional coding sequence encoding additional amino acids, such as those providing additional functional characteristics. Thus, the sequence encoding the antibody can be fused to a marker sequence, such as the sequence encoding a peptide, which facilitates purification of the fused antibody, including fragments or part.

Polynucleotides that selectively hybridize to a polynucleotide described herein

The present invention provides isolated nucleic acids that hybridize under certain hybridization conditions with a nucleotide described herein. Therefore, the polynucleotides of this embodiment can be used to isolate, determine and/or count the amount of nucleic acid that contains such polynucleotides.

For example, the polynucleotides of this invention can be used to identify, select or amplify partial or complete clones in a deposit library. In some embodiments, the polynucleotides are genomic or isolated SOMA sequences, or complementary to SOMA from a human or mammalian nucleic acid library.

Preferably, the SOMA library contains at least 8095 complete sequences, preferably at least 8590 or 9095 full-length sequences, more preferably at least 9595 complete sequences.

SOMA libraries can be normalized to increase the presence of rare sequences. Low or medium stringency hybridization conditions are typical, but not exclusive, and are used for sequences with low similarity to complementary sequences. Moderate or strict conditions may sometimes be applied to sequences with greater similarity.

Low stringency conditions provide selective hybridization of sequences with 7095 sequence identity and can be applied to identify orthologous or paralogous sequences.

Optionally, the polynucleotides of this invention will encode at least a portion of the antibody encoded by the polynucleotides described herein. Polynucleotides of this invention include nucleic acid sequences that can be used for selective hybridization with a polynucleotide encoding an antibody of this invention. (See, e.g., A!izibei, zirga; Soiiyidap, zirga, which are incorporated by reference in their entirety).

Construction of nucleic acids

The isolated nucleic acids of this invention can be prepared using (a) recombinant methods, (b) synthetic methods, (c) purification methods, or combinations thereof, which are known in the art.

Nucleic acids may include sequences in addition to the polynucleotide of the present invention.

For example, a multicloned site that includes one or more endonuclease restriction sites can be inserted into the nucleic acid to facilitate isolation of the polynucleotide.

Also, translatable sequences can be introduced to facilitate isolation of the polynucleotide of the present invention. For example, a hexahistidine marker sequence provides an acceptable method for purifying the proteins of this invention. The nucleic acid of this invention, except for the coding sequence, is not necessarily a vector, adapter or linker for cloning and/or expression of the polynucleotide of this invention.

Additional sequences can be added to such cloning and/or expression sequences to optimize their function during cloning and/or expression, to facilitate isolation of a polynucleotide, or to improve the introduction of a polynucleotide into a cell. The use of cloning vectors, expression vectors, adapters and linkers is well known in the art. (see, for example, Ats,izibei, zirga; og Zatgook, zirga)|.

Recombinant methods of nucleic acid construction

The isolated nucleic acid compositions of the present invention, such as EMMA, SOMA, genomic OMA, or any combination thereof, can be obtained from biological sources using many cloning techniques known to those skilled in the art. In some embodiments, oligonucleotide samples that selectively hybridize, under strict conditions, with the polynucleotides of this invention are used to identify the desired sequence in libraries of OMA or genomic SOMA. Isolation of EMA and construction of SOMA and their genome libraries is well known to a specialist in this field (see, for example, A,izibei, zirga; og Zatgook, zirgai.

Nucleic Acid Screening and Methods for Isolation A cOMA or genomic library can be screened using a sample based on a polynucleotide sequence of the present invention, such as described herein. Samples can be used for hybridization with genomic sequences of OMA and CsOMA to isolate homologous genes from the same or another organism. Those skilled in the art will appreciate the various levels of hybridization stringency that can be used in the analysis; hybridization and washing media can also be harsh. By creating more stringent hybridization conditions, the degree of complementarity between the sample and the target product to create a duplex is increased. The level of rigidity can be controlled by one or more parameters such as temperature, ionic strength, pH, and the presence of a partially denaturing agent such as formamide. For example, the stringency of the hybridization can be easily changed by changing the polarity of the reactant solution through, for example, manipulating the concentration of formamide between 090 and 50905. A level of complementarity (sequence identity) is required so that the binding that is detected varies according to the stringency of the hybridization medium and /or washing medium.

The level of complementarity would be optimally 10095 or 70-10095 or some other value or range within these limits.

However, it should be understood that mirror sequence variations in samples and primers can be compensated for by reducing the stringency of the hybridization medium and/or washing medium.

OMA or EMA amplification methods are well known to those skilled in the art and can be used in accordance with the present invention without undue experimentation, using the teaching aids and guidance contained therein.

Amplification methods for OMA or EMA include, but are not limited to, polymerase chain reaction (PCR) and similar amplification processes (see, e.g., Shyu. 5. Raiepi Mo5. 4,683,195, 4,683,202, 4,800,159, 4,965,188, yuyu MiiYiiv, ei ai. ; 4,795,699 APA 4,921,794 Yu Tarbog, ei A1.; 5,142,033 (Yu IPPs: 5,122,999999, 4,3,311; o 5iMmeg, ei a).; 4,766,067 10 Vibzulaz; 4,656,134 io Kypdoid| and KMA-mediated amplifications, which use antisense EMAs to the target sequence as a template for the synthesis of double-stranded OMA (SHO. 5. Raiepi Mo. 5,130,238 o Majek, ei ai. , under the trade name MAZVAI, the entire content of which is fully incorporated by reference (See, e.g., A!,5ibei, zirga; og zatogook, zirga|. For example, polymerase chain reaction (PCR) technology can be used to amplify the polynucleotide sequences of the present invention and similar genes directly from the genomic OMA or SOMA library who PCR and other ip migo amplification techniques can be useful, for example, in cloning nucleic acid sequences that encode proteins to be expressed, in order to generate nucleic acids for use as samples to determine the presence of a desired teMA in a sample, in sequencing nucleic acids acids, or for other needs. Examples of methods sufficient for persons skilled in the art of ip amplification can be found in

IVegdeg, zirga, zatrgook, zirga, and A!zibei, zirga, as well as in Miiyya5, ei aI., SHYU. 5. Raiepi Mo. 4,683,202 (1987); and Ippiv, ei ai., ROSA Rgoiosoiz A Sshide Yuyu Meiipod" apa Arriisayope, Edz., Asadetis Rgev5 Ips., Zap

Oiedo, SA (1990)). Commercially available kits for genomic RSEK amplifications are known. See e.g.

Admapiade-as Sepotis ROSA Kii (Siopiesp)). Additionally, for example, T4 gene 32 protein (Voephipdeg Mapppeit) can be used to improve the product yield of long PCP products.

Synthetic methods of nucleic acid construction

The isolated nucleic acids of this invention can be obtained by direct chemical synthesis using known methods (see, e.g., A!,zibei, ei a!., zirga). Chemical synthesis generally yields a single-stranded oligonucleotide, which can be converted to a double-stranded OMA by hybridization with a complementary sequence or polymerization by UOMA polymerase using a single-stranded OMA as a template.

Those skilled in the art can see that while the chemical synthesis of OMA can be limited to a sequence of about 100 or more bases, longer sequences can be obtained by combining shorter sequences.

Recombinant expression cassettes

The present invention also provides recombinant expression cassettes that include the nucleic acids of the present invention. Nucleic acid sequences of the present invention, for example, OMA or genomic sequences encoding antibodies of the present invention, can be used to construct recombinant expression cassettes that can be introduced into at least one host cell. A recombinant expression cassette will typically include a polynucleotide of this invention operably linked to regulatory transcription initiation sequences that will direct transcription of the polynucleotide in a specific host cell. Both heterologous and non-heterologous (eg, endogenous) promoters can be used for direct expression of the nucleic acids of this invention.

In some embodiments, the isolated nucleic acid may serve as a promoter, enhancer, or other element may be introduced into an appropriate location (upstream, downstream, or intron) of a non-heterologous form of a polynucleotide of the present invention to increase or decrease expression of the polynucleotide of the present invention.

For example, endogenous promoters can be altered by ip mmo or ip migo mutations, deletions and/or substitutions.

Vectors and host cells

The present invention also relates to vectors that include isolated nucleic acids of the present invention, genetically engineered host cells with recombinant vectors, and production of at least one anti-I-12 antibody by recombinant methods known in the art. See e.g., zatyuogook, ei a!., zirga; A!,izibei, ei a!., zirga, each of which is fully incorporated by reference).

Polynucleotides can optionally be introduced into a vector that contains a selective marker for growing in a cell. Generally, the plasma copper vector is introduced into a precipitate, such as a calcium phosphate precipitate, or in a complex with charged lipids. If the vector is a virus, it can be packaged ip migo using an appropriate packaging cell line and then transferred into the host cell.

The OMA insert must be directly linked to the corresponding promoter. The expression construct will further contain sites for transcription initiation, termination, and, in the transcriptional region, a ribosome binding site for translation (e.g., CAA, OACA, or CAA) respectively located at the end of the translated tcMA, with a predominance of CAA and AC for eukaryotic expression cells

Expression vectors will preferably, but not necessarily, include at least one selectable marker.

Such markers include, for example, but are not limited to, methotrexate (MTX), dihydrofolate reductase (ONEK,

Paradise As. 4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017|, ampicillin, neomycin (5418), mycophenolic acid, glutamate synthetase (5, IO5 Rai Mo5. 5,122,464; 5,770,359; 5,827,739|, resistance to culture of eukaryotic cells, and tetracycline or ampicillin resistance genes for cultivation of BE. soybean and other bacteria or prokaryotes (the above patents are fully incorporated herein by reference.) Suitable culture media and conditions for the host cells described above are well known to those skilled in the art.

Acceptable vectors are obvious to those skilled in the art. The introduction of the vector construct into the host cell can be performed by calcium phosphate transfection, OEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other known methods. Such methods are described in works such as (Zatrbgook, 5irga,

Sparieegz" 1-4 and 16-18; Aisybrei!. zirga, Spariegz: 1, 9, 13, 15, 16).

At least one antibody of the present invention may be expressed in a modified form, such as a fusion protein, and may include not only expression signals, but also an additional heterologous functional region. For example, a region of additional amino acids, particularly charged amino acids, can be added to the M-tissue of an antibody to increase stability and stability in the host cell during purification, use, and storage.

Also, peptide moieties can be added to the antibodies of this invention to facilitate purification. Such regions can be removed before the final preparation of the antibody or at least a fragment thereof. Such methods are described in many standard laboratory manuals, such as, |(Zatogook, zirga, Sparieg5 17.29-17.42 and 18.1-18.74; A!,zibei, zirga, Sparieg» 16, 17 and 181.

Such intermediate skills in operation are very common in many expression systems suitable for expressing nucleic acids that encode the protein of this invention.

Alternatively, the nucleic acids of the present invention can be expressed by inclusion (manipulation) in a host cell containing an endogenous OMA that encodes the antibodies of the present invention. Such a method is well known to those skilled in the art, e.g., as described in U.S. Pat.

An illustrative example of cell cultures useful for the production of antibodies, the specified parts and their variants are mammalian cells. Mammalian cell systems are often in the form of monolayers, although suspensions or bioreactors of mammalian cells are also used. Many host cell lines suitable for expressing intact glycosylated proteins have been developed, including СО5-1 (eg, ATOS SK. 1650), СО5-7 (eg, ATSS SVI. 1651), NEK293, VNK21 (eg, ATSS CA -10), CHO (e.g., ATSS SVI. 1610) and cell lines B5C-1 (e.g., ATS SKI -26), Co5-7 cells, CHO cells, per 52 cells, RZUKhbZA98.653,

ZR2/O-AdiYa4, 293 cells, Nega cells and the like, which are always available, for example in (Ategisap

Toure Siyige SoPesiop, Mapavzza5, Ma (mlum/.aiss.og9)). Preferred host cells include cells of lymphoid origin such as myeloma and lymphoma cells. In particular, the preferred host cells are

РХбЗАОдВ, cells 653 (ATSS Assezviop Mitbreg SKI -1580) and 5P2/0-Adi4 cells (ATSS Assezviop Mitreg

SVI-1851).

Expression vectors of these cells may include one or more expression control sequences, such as, but not limited to the origin of replication: a promoter (eg, late or early 5M40 promoters, SMM promoter (OB Rai. Mo5. 5,168,062; 5,385,839), NZM IC promoter , rac promoter (phosphoglycerate kinase), arpa promoter EB-1 (OB Rai Mo. 5,266,491) |, at least one human immunoglobulin promoter, enhancer, and/or processing information sites, such as ribosome binding sites, KMA splicing sites, polyadenylation, e.g., ZM40 large T Addroi A additional site, transcription terminator sequences. known and/or available.

E.g., with |Ategisap Ture Sshshtge SoPesip SaiaIodie oi Seii! Hypez apa Nuridotav (m/mlu.aiss.oga)| or other known commercial sources.

When eukaryotic host cells are used, polyadenylation or transcription termination sequences are usually introduced into the vector. An example of a terminator sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences for precise splicing of the transcript may also be included. An example of a splicing sequence is the MP1 intron from 5М40 IZrgadie, ей ай.,».

Migo!. 45: 773-781 (1983)). In addition, to control replication in the host cell, genetic sequences can be included in the vector, which is well known in the art.

Purification of antibodies

Antibodies to anti-I/-12 can be obtained from recombinant cell culture by well-known methods, 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, hydroxyapatite chromatography and pectin chromatography. High performance liquid chromatography (HPLC") can also be used (see, e.g.,

Sshitepi Rgoiosoiiv ip Rgoieip bsiepse, dhopp UMiieu 5 ope, MU, MM, (1997-2001), Sparieg5 1, 4,6, 8, 9, 10, each fully incorporated by reference|.

Antibodies of the present invention typically include purified products, products of chemical synthesis procedures, products obtained by recombination from a eukaryotic host, including, for example, cells of yeast, higher plants, insects and mammals. Depending on the host used in the process of recombinant production, the antibodies of this invention can be glycosylated or non-glycosylated, with preference given to glycosylated. Such methods are described in many standard laboratory manuals, such as, (Zatrgook, zirga, Zesiiop5 17.37-17.42; Ivybei, vzyrga, Sparieegz 10, 12, 13, 16, 18 apa 20, Soiidap, Rgoieip

Zsiepse, Zirga, Spariegv 12-14, which are fully incorporated by reference).

Anti-1II -2 antibodies

Isolated antibodies of the present invention include antibodies encoded by any of the polynucleotides of the present invention, as will be discussed in more detail herein, or another isolated or prepared antibody. Preferably, a human antibody or an antigen-binding fragment that binds human II/-12 and thus partially or substantially neutralizes at least one of the biological activities of the protein. An antibody or a specified part or variant thereof, which partially or, preferably, to a large extent, neutralizes at least one biological activity of at least one I/-12 protein or fragment, can bind the protein or fragment, and thus inhibit the actions mediated by by binding of 1-12 to the 1-12 receptor or through other 1-12-dependent or mediated mechanisms. The term "neutralizing antibody" as used herein refers to an antibody that can inhibit I-12 to 20-12095, preferably up to 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 10095, or more, depending on the sample.

The ability of anti-II/-12 antibodies to inhibit II/-12-dependent activity is preferably determined by at least one acceptable protein or receptor 1-12 assay as described herein or known in the art. Human antibodies according to the invention may belong to any class (Ids, IDdDA, I9M, ICHE, to, eis.) or isotype, and may include kappa or lambda light chains. In one embodiment, the human antibody comprises a db heavy chain or a defined fragment, e.g., at least one of the isotypes, (951, Ids2, DdDOZ, or di.

Antibodies of this type can be produced using at least one human light chain transgene (eg, dos, IdDA and I9DM (eg, ut, ug, yZ, y4) as described herein and/or known in the art. In another embodiment , the human anti-II-12 antibody includes a heavy chain of IdO1 and a light chain of IdO1.

At least one antibody of the invention binds at least one specified epitope specific to at least one protein 1-12, subunit, fragment, part or combination thereof. At least one epitope may include at least one antibody binding region that includes at least part of said protein, the epitope of which preferably includes at least one extracellular, soluble or cytoplasmic part of said protein. At least one specified epitope may include any combination of at least one amino acid chain of at least 1-3 amino acids to the entire specified portion of continuous amino acids ZEO IU MO:9.

In general, a human antibody or antigen-binding fragment of the present invention will include an antigen-binding region that includes at least one complementarity-determining region (COB1, SOC2, and SOCZ) or a variant of at least one heavy chain variable region, and at least one detection region complementarity (SOK!I, SOMK2 and SOKZ) or a variant of at least one variable region of the light chain. By way of non-limiting example, the antibody or antigen-binding portion may optionally include at least one heavy chain of the HCZ having the amino acid sequence ZEO IO MO:Z and or one light chain of the HCZ having the amino acid sequence 5EO IU

MO:6. In a particular case, the antibody or antigen-binding fragment may have an antigen-binding region that includes at least a portion of at least one heavy chain of a SOC (eg, SOCI, SOMB2, and/or SOC3) that has an amino acid sequence corresponding to SOC -am 1, 2 and/or Z (e.g., zEO IU

Ministry of Health: 1, 2 and/or 3). In another specific embodiment, the antibody or antigen-binding portion or variant may have an antigen-binding region that includes at least a portion of one SOC light chain (e.g.,

SOM, SOK2 and/or SOKZ) which has an amino acid sequence corresponding to SOBs 1, 2 and/or C (e.g.

ZEO IO MOH: 4. 5. and/or 6). In a preferred embodiment, the three heavy chains of the COP and the three light chains of the SOC of the antibody or antigen-binding fragment have amino acid sequences that correspond to the SON of at least one of tAb 12875, C340, or any other as described herein. Such antibodies can be produced by chemically introducing together with various parts (eg, SOC'5, framework) of antibodies using conventional techniques, by making or expressing (eg, one or more) nucleic acid molecules encoding the antibodies using generally known techniques technologies of recombinant OMA or using any other suitable method.

Anti-1I-12 antibodies can include at least one light or heavy chain of the variable region of a defined amino acid sequence. For example, in a preferred embodiment, anti-I/-12 antibodies include at least one of at least one heavy chain variable region that optionally has the amino acid sequence ZEO IU MO:7 and/or at least one light chain variable region that optionally has has the amino acid sequence 5EO IU MO:8. Antibodies that can bind 1-12 people and include defined variable regions of heavy and light chains can be produced by suitable methods, such as phage display (Kaisire, U., ei a)i., Ipi UMoi. Mea, 1 (5): 863-868 (1998)| or by methods using transgenic animals as known in the art and/or described herein. For example, a transgenic mouse that has a functionally rearranged human immunoglobulin heavy chain transgene and a transgene that contains an OMA from a human immunoglobulin light chain locus that has been functionally rearranged can be immunized with human I/-12 or a fragment thereof to induce antibody production. If desired, antibody-producing cells can be isolated and hybridomas or other immortalized antibody-producing cells can be made, as described herein and/or known in the art.

Alternatively, antibodies may be expressed using the encoding nucleic acids or parts thereof in a suitable host cell.

The invention also relates to antibodies, antigen-binding fragments, immunoglobulin chains and SOVs that contain amino acids in a sequence that is significantly similar to the amino acid sequence described herein. Preferably, such antibodies or antigen-binding fragments and antibodies that include such chains or SOVs can bind 1-12 humans with high affinity (eg, a K of less than or equal to about 10:

ZM). Amino acid sequences that are substantially similar to the sequence described herein include sequences that have conservative amino acid substitutions, as well as deletions and/or amino acid inversions. Substitution of a conservative amino acid refers to the replacement of one amino acid with another that has chemical and/or physical properties (such as charge, structure, polarity, hydrophobicity/hydrophilicity) similar to those of the first amino acid. Conservative substitutions include replacing one acid with another within the following groups: lysine (K), arginine (K) and histidine (H); aspartate (0) and glutamate (E); asparagine (M), glutamine (0), serine (5), threonine (T), tyrosine (XU). K, K, H, O and E; alanine (A), valine (M), leucine (U), isoleucine (I), proline (P), phenylalanine (P), tryptophan (M/), methionine (M), cysteine (C) and glycine (C) ; E, UM and M; C, 5 and T.

Amino acid codes

Amino acids that make up anti-1II-12 antibodies of this invention are usually shortened. Amino acid designations can be denoted by a one-letter amino acid code, a three-letter amino acid code or a trinucleotide codon(s), as is well known from the literature (see Airegiv, V., ei aI., Moiesshag Vioiodu oi Tpe

Seyi, Tpiga Ed., Sapapa Rybiizpipo, Ips., Mem Mogk, 1994): 07777771 TFAbr,///////// |Avrays// |бАСсАС/С/С/С/:///:/: /С:(/4ШСУС- 77777777 no 7 |ivoyensipe,d/ FASAD AOS, AOYUIGSGS/7777777777772С mm 77777777 |Mmeye////////// |Meshoppe// AV С///////// ::OE.S:( AI SGC

R 77777771 Ф|Ryu ////////////////// |Ryuile //// |ССАСоСсосссЙ42222г2ш9 о 77777777 (b |Sishatpe // |SAADSASS//////7// /77/7//:/:U/)?-

А 77777770 |Ayu //////// |Ayuipyne / |АСА Or СоАСоС,Сос,соОсССш9

Anti-1II-12 antibodies of the present invention may include one or more amino acid substitutions, deletions, or insertions, both naturally occurring mutations and human engineered ones, as specified herein.

Of course, many amino acid substitutions can be made by one skilled in the art depending on many factors, including those described above. Generally speaking, the number of amino acid substitutions, insertions and deletions for any given protein, fragment or variant thereof derived from anti-1Y -12 Id cannot be greater than 40, 30, 20, 19, 18.17, 16, 15 .

Amino acids in the anti-II-12 antibodies of the present invention that are essential for function can be determined by methods well known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (eg, A!, Zibrei, Zirga, Sparieev 8, 15; Sippipoiate apa Mu/iei5, Science 244: 1081-1085 (1989). The following procedure results in single alanine mutations in each residue of the molecule. The resulting mutant molecules are then tested for biological activity such as, but not limited to, of at least one neutralizing action of I--12. Sites that are critical for antibody binding can also be revealed by structural analysis, such as crystallization, nuclear magnetic resonance, or photoaffinity labeling (Ztiy, ei ai., U. Moi. Vio! . 224: 899-904 (1992) apa de Mov, eyii ai., Zsiepse 255: 306-312 (1992)).

Anti-I/-12 antibodies of the present invention may include, without limitation, at least one part, a sequence or combination of 5 to all continuous amino acids of at least one of ZEO IU MOB: 1, 2, 3, 4, 5, 6.

I/-12 antibodies or specified parts or variants of the present invention may include, but are not limited to, at least one part, a sequence or combination selected from 3-5 continuous amino acids from ZEO IJU MO: 1, 5-17 continuous amino acids ZEO IJU MO: 2, 5-10 continuous amino acids with 5EO IU MO: 3, 5-11 continuous amino acids with ZEO IO MO: 4, 5-7 continuous amino acids with BEO IU

MO: 5; 5-9 continuous amino acids with ZEO IO MO: 6; I ets21, I uz76, Mei83, 5eg85 with ZEO IU MO: 7.

Anti-1/-12 antibodies can also optionally include a polypeptide of at least 70-10090 of 5, 17, 10, 11, 7, 9, 119, or 108 contiguous amino acids, at least one of ZEO IU MOB: 1,2, 3,4, 5,6, 7 or 8.

In one embodiment, the amino acid sequence of the immunoglobulin chain, or part thereof (eg, variable region, VOC) has about 70-10095 similarity (eg, 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 thereof or value) to the amino acid sequence, the corresponding chain from at least one of the ZEO IYUO MOB: 7, 8.

For example, the amino acid sequence of the variable region of the light chain can be compared to the sequence of ZEO IO MO: 8, or the amino acid sequence of the heavy chain of SOKZ can be compared to 5EO IUO MO: 3. Preferably, 70-10095 amino acid similarities (eg, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100 or any range or value thereof) is determined using a computer algorithm well known in practice.

Exemplary sequences of the variable region of the light and heavy chains are presented in 5EO GO MO5: 7 apa 8. Antibodies of the present invention, or variants thereof, may include any number of contiguous amino acid residues from an antibody of the present invention, where this number is selected from the group of integer values, which are within 10-10095 of the number of continuous residues from anti-1II-12 antibodies.

Optionally, the contiguous amino acid subsequence is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 in length. , 210, 220, 230, 240, 250 or more amino acids, or any interval or value thereof. Also, the number of such subsequences may be any integer selected from the group of 1 to 20, such as at least 2, 3, 4, or 5.

Those skilled in the art will appreciate that the present invention includes at least one biologically active antibody of the present invention. Biologically active antibodies have a specific activity of at least 2095, 3095, or 4095, and preferably at least 5095, 6095, or 7095, and more preferably, at least 80905, 9095, or 9595-100092 of the activity of natural (non-synthetic), endogenous or similar and known antibodies. Methods for determining and calculating enzymatic activity and substrate specificity are well known to those skilled in the art.

In another aspect, the invention relates to human antibodies and antigen binding fragments, as described herein, which are modified by covalent attachment of an organic moiety. Such modification can provide antibodies or antigen-binding fragments with improved pharmacokinetic parameters (such as increased serum half-life). The organic part can be a linear or branched hydrophilic polymer group, a fatty acid group, or a fatty acid ester group.

In a particular embodiment of the present invention, the hydrophilic polymer group can have a molecular weight of from about 800 to about 120,000 Da and can be a polyalkane glycol (such as polyethylene glycol (PES), polypropylene glycol (PESb)), a carbohydrate polymer, an amino acid polymer, or polyvinylpyrrolidone, and a fatty acid group or ester fatty acid group may contain from eight to fourteen carbon atoms.

Modified antibodies and antigen-binding fragments of the invention may contain one or more organic particles that are directly or indirectly covalently linked to the antibody. Each organic particle linked to the antibody or antigen binding fragment of the invention can independently be a hydrophilic polymer group, a fatty acid group or an ester fatty acid group. As used herein, the term "fatty acid" encompasses monocarboxylic and dicarboxylic acids. The term "hydrophilic polymer group" as used herein refers to organic polymers that are more soluble in water than octane.

For example, polylysine is more soluble in water than octane. Thus, antibodies modified by covalent attachment of polylysine are covered by the invention. Hydrophilic polymers suitable for modifying the antibodies of the invention may be linear or branched and include, for example, polyalkane glycols (such as PES, monomethoxy polyethylene glycol (MPES), PPO, and the like), carbohydrates (such as dextrin, cellulose, oligosaccharides, polysaccharides, and the like ), polyalkane oxides (such as polyethylene oxide, polypropylene oxide and the like) and polyvinylpyrrolidone. Preferably, the hydrophilic polymer which modifies the antibody of the invention has a molecular weight of from about 800 to about 150,000 Da, as a single molecular unit.

For example, PESvooo and PESgoooo may be used, where the subscript indicates the average molecular weight of the polymer in daltons. The hydrophilic polymer group can be substituted with one to six alkyl, fatty acid or ester fatty acid groups.

Hydrophilic polymers substituted with fatty acids or fatty acid esters can be prepared by suitable methods. For example, a polymer that includes an amino group can be attached to a fatty acid carboxylate or fatty acid ester, and an activated carboxylate (such as an activated

M,M-carbonyldiimidazole) on the fatty acid or fatty acid ester can be attached to the hydroxyl group on the polymer.

Fatty acids and fatty acid esters suitable for modifying the antibodies of the invention may be saturated or have one or more units of unsaturation. Fatty acids suitable for modifying the antibodies of the invention include, for example, p-dodecanoate (Sig2, laurate), p-tetradecanoate (Sta, myristate), p- octadecanoate (Siv, stearate), p-eicosanoate (Sgo, arachidate) p- docosanoate (Sgg, behenate), p-triacontanoate (Szo), p-tetracontanoate (Slo), cis-lU9-octadecanoate (Steve, oleate), all cis-AB5,8,11,14-eicosatetraenoates (Sgo, arachidonate), octanedioic acid, tetradecandioic acid, actandioic acid, docosandioic acid and the like.

Suitable fatty acid esters include monoesters of dicarboxylic acids having a linear or branched lower alkyl group. The lower alkyl group can have from one to twelve, preferably from one to six, carbon atoms.

Modified human antibodies and antigen-binding fragments can be obtained using suitable methods, such as reaction with one or more modifying agents. The term "modifying agent" is used herein to refer to suitable organic groups (such as a hydrophilic polymer, fatty acid or fatty acid ester) having activating groups. The term "activating group" is a chemical unit or functional group that can, under appropriate conditions, react with a second chemical group, thus forming a covalent bond between the modifying agent and the second chemical group. For example, the amenoreactive activating group includes electrophilic groups such as tosylate,

mesylate, halo (chloro, bromo, fluoro, iodo), M-hydroxysuccinimidyl esters (MH), and the like. Activating groups can react with thiols, including, for example, maleimidine, iodoacetyl, acrololol, pyridine disulfides, 5-thiol-2-nitrobenzoic acid thiol (TMB-thiol), and the like. An aldehyde functional group can be attached to an amine- or hydrazide-containing molecule, and an azide group can react with a trivalent phosphorus group to form a phosphoamidate or phosphoimide bond. Suitable methods of introducing groups into molecules are known in the art (see, for example, Neptapzop, 0. T., Viosopishdaie

Tesppidnez, Asadetis Rgev5: Zap Oiedo, SA (1996)). Activating groups can be linked directly to organic groups (such as hydrophilic polymers, fatty acids and fatty acid esters), or through linker moieties, for example, a divalent C1-C2 group in which 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, -(CHg)z-, /-MH-(CHg)v-MH-, /-(CHg)»-MH- and /-CH2-O-CHno-CHg-0O- СН2-СН2-ОО-СН-МН-.

Modifying agents having linker moieties can be prepared, for example, by reacting mono-Bos-alkyldiamines (such as mono-Bos-ethylenediamine, mono-Bos-diaminohexane) with a fatty acid in the presence of 1-ethyl-3-(3-dimethylaminopropyl) )-carbodiimide (EOS), forming 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 trichloroacetic acid (TEA) to expose the primary amine, which can be attached to another carboxylate, as shown, or react with maleic anhydride, and cyclization of the resulting product to give an activated maleimide fatty acid derivative See, for example,

Tpotrzop, ei aiI., UMO 92/16221, the full content of which is fully incorporated herein by reference.

Modified antibodies of the invention can be obtained by interaction of human antibodies or antigen binding fragment with a modifying agent. For example, an organic moiety can be attached to an antibody in a non-site-specific manner using an aminoreactive modifying agent, e.g.

MH5 ester REb.

Modified human antibodies or antigen-binding fragments can also be obtained by reducing disulfide bonds (such as interchain disulfide bonds) of antibodies or antigen-binding fragments. The reconstituted antibodies or antigen-binding fragments can then be reacted with a thiol-reactive modifying fragment to produce modified antibodies of the invention. Modified human antibodies and/or antigen-binding fragments that contain organic moieties that specifically bind to the antibody sites of the present invention can be prepared using suitable methods such as reverse proteolysis. , 3: 147-153 (1992); Mepep ei ai.,

Viosopishdaye Spet., 5: 411-417 (1994); Kitagap ei ai., Rgoieip 5si. 6 (10): 2233-2241 (1997); NP ei a)., Visaga.

Spet., 24 (1): 59-68 (1996); Sareyaz ei ai., Vioyesppoi. Vioepad., 56 (4): 456-463 (1997)), and by the methods described in |Negtapzop, 0. T. Viosopitsdage Tesppidne5, Asadetis Rge55: zap Oiedo, SA (1996).

Derivative protein compositions of anti-idiotypic anti-i! 2 from antibodies

In addition to monoclonal or chimeric anti-1I-12 antibodies, the present invention is also directed to anti-idiotypic (anti-Ia) antibodies specific for such antibodies of the invention. Anti-Ia antibodies are antibodies that recognize unique determinants usually associated with the antigen-binding region of another antibody.

Anti-Ia can be obtained by immunization of animals of the same species and genetic type (for example, mouse lines) as well as the source of Id-antibodies or its region, which is SOC. Immunized animals will recognize and respond to the idiotypic determinants of the immunizing antibodies and produce anti-I antibodies.

Derived protein compositions of anti-1Y -2 IZ

The present invention also provides at least one anti-I-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-I-12 antibodies thereof, as shown herein and /or known from the state of the art, which are presented in the form of a composition, mixture or in a form that does not occur in nature. Such compositions include at least non-naturally occurring compositions including at least one or two amino acid sequences of a full-length anti-1I-12 antibody, variants, domains, fragments, or said variants lacking the C- and/or M-terminal sequence , selected from the group containing 70-10095 of the complete amino acid sequences with 5EO IU

MOB: 1, 2, 3, 4, 5, 6, 7 or 8 or the specified fragments, domains or their variants. Preferred compositions of derivatives of anti-1II-12 proteins, fragments or variants, include at least one or two full-length, fragment or variant part of the sequence of anti-1II-12 antibodies, containing COP, 70-10095 5EO IYU MOB: 1, 2,

With, 4, 5, 6, is the specified fragment, domain or their variant. Such a composition can have a percentage composition by mass, volume, concentration, polarity or molality, both for a liquid and for a dry solution, a mixture. Suspension, emulsion or colloid as known in the art or described herein.

The anti-II/-12 antibody compositions of the present invention may also include at least one of any suitable and effective amounts of a composition or pharmaceutical composition comprising at least one anti-1-12 antibody in a cell, tissue, organ, animal, or patient according to the need for modulation, treatment or therapy, which optionally includes at least one TME antagonist (such as, but not limited to, a TME antibody or fragment, a soluble TME receptor or a fragment, a fusion protein thereof, or a small molecule TME antagonist ), anti-rheumatic agent (such as methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, gold-sodium thiomalate, hadroxychloroquine sulfate, leflunomide, sulfasalazine), muscle relaxant, narcotic, non-steroidal anti-inflammatory agent (NSAID), analgesic, anesthetic, sedative, local anesthetic, neuromuscular blocker, antimicrobial (such as, aminoglycoside, antifungal, antiparasitic, antiviral, carbapenem, cephalosporin, fluoroquinolone, macrolith, peni cilin, sulfonamide, tetracycline, other antimicrobial), antipsoriatic, corticosteroid, anabolic steroid, diabetic agent, mineral and dietary supplement, thyroid agent, vitamin, calcium-related hormone, antidiarrheal, antitussive, antiulcer, laxative , anticoagulant, erythropoietin (such as epoetin alfa), figrastim (such as, 6-C5E, Meprodep), sagramostim (SM-C5BE,

And crew), immunizer, immunoglobulin, immunosuppressant, (such as basiliximab, cyclosporine, daclizumab), growth hormone, hormone replacement drug, estrogen receptor modulator, mydriatic, cyclopedic, alkylating agent, antimetabolite, mitosis inhibitor, radiopharmaceutical, antidepressant, antimanic agent, antipsychotic, antixiolytic, hypnotic, sympathomimetic, stimulant, donepezil, tacrine, asthma medication, beta agonist, inhaled steroid, leukotriene inhibitor,

methylxanthine, cromolyn, epinephrine or analog, dornase alfa (Rytogute), cytokine or cytokine agonist.

Non-limiting examples of such cytokinins include, but are not limited to, any of I/-1 to 1-23. suitable dosages are well known. See eg.

Arrieyup apa Gapde, biatioga, ST (2000); ROV Rpaptasorovia, Tagazsop Roskei Rnapptasorovia 2000, Oeishke

Eayop, Tagazzop Rybiivepipo, Sota Gipda, SA (2000), each of which is incorporated by reference in its entirety).

Anti-cancer and anti-infective agents may also include toxin molecules that are linked, bound, co-formulated or co-administered with at least one antibody of the present invention.

A toxin may optionally act by selectively killing pathogenic cells or tissues. Pathogenic cells can be cancerous or other cells. Such toxins may be, but are not limited to, purified or recombinant toxins, or a toxin fragment that includes at least one cytotoxic toxin domain, such as, selected from at least one of, ricin, snake (animal) toxin, or bacterial toxin. The term "toxin" also includes both endotoxins and exotoxins produced by any natural, mutant or recombinant bacteria or virus that can cause any pathological condition in humans and other mammals, including toxin shock that can lead to death Such toxins may include, but are not limited to, heat-labile enterotoxin (IT), and heat-stable enterotoxin (57), enterotoxigenic E. soya, Cytotoxin ZPpideIPa, Aegotopav enterotoxins, toxic shock syndrome Iohip-1 (T551-1), staphylococcal enterotoxin A (5EA), epiegoihip B (ZEV), or C (ZES), streptococcal enterotoxins and the like. Such bacteria include, but are not limited to, strains and species of enterotoxigenic E. soya (ETES), enterohemorrhagic EE. soybeans (such as strains of serotype 0157:H7), soybeans (such as

Zarnuosossi5 atsgei5, Ziarpuiosossiz ruodepevz), ZpidePya zresiez (such as, ZpideiPa duzepiegiae, ZpidePa

Pehpegi, Zpideia rowidii, apa ZpidePa zoppei), Zaitopeiia z5resiez5 (such as, ZaItopeiIa iurpi, Zaitopeia spoiegaztsiv, Zaitopei Ia epiegiyaiv), Siozipaisht z5resiez (such as, Siovigidiit regitipdep5, Siozigidisht aiysie, Siovigid Satruez roisige) such as, Satruobasieg ie)pi, Satruorasieg Ttef5), Neijsorasieg zresiev, (such as, Neijsorasieg ruiogi), Aegotopach 5zresiez (such as, Aegotopaz zbra, Aegotopaz puagorpiia, Aegotopav samiae), Riesiotopaz 5PidePoide5, Megzipa epiegosoiiisa, Mirpez (obz. zresieg 9., Mibpoz sNoiegae, Myrgioz raganetoi/uiissiv), Kiervziea zresieeh, Reendotopavz aegidiposa, apa bigeriosossi. |See, such as, 5beip, ed.,

IMTEAMAI MEBISIME, Zga ey., pp. 1-13, GishShe, Vgom/p apa So., Voziyuop, (1990); Emapv ei ai., eadz., Vasiegia!

Ipiesiop5e oi Nytape: Eridetioiyudu apa Sopigoi, 26. Ed., pp. 239-254, Riepyt Meais! VookK So., Mem/ Khoik (1991); Mapavai! eyi a), Riipsiriye5 apa Rgasiise oi Ipgesiysiv Oizeazev5, Za. Ea., SpigspiiY Pmipdviope. Mem Hoik (1990); Vegkom yei a, edv., Te Megok Mapiai, 16i1yp eayop, Megsok apa So., Wapmau, M. u., 1992; Mooa hey ay

EREM5 Misgobioiodu Ittypoiodu, 76: 121-134 (1991); Maytask et al., Biopsy, 248: 705-711 (1990), the contents of these references are incorporated by reference in their entirety).

Anti-II-12 antibody mixtures, compositions or combinations of the present invention may also include at least one of any suitable excipient such as, but not limited to, a diluent, binder, stabilizer, buffers, salts, lyophilic solvents, preservatives , adjuvant, or similar.

Pharmaceutically acceptable excipients are preferred. Non-limiting examples and methods of preparing such sterile solutions are well known, such as, but not limited to, (Seppago, Ed.,

Kepiipdiopye Rpaptaseshis! Zsiepse5, 18p Eaiiop, Musk Rybiizpipd So. (Easiop, RA) 1990). Pharmaceutically acceptable carriers, which may be selected in a conventional manner, are suitable for the route of administration, solubility and/or stability of the anti-1I-12 antibody compositions, fragments or variants, as is well known in the art or described herein.

Pharmaceutical excipients and additives useful in this formulation include, but are not limited to, proteins, peptides, amino acids, lipids, and carbohydrates (such as sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; saccharide derivatives such as alditols, aldonic acids, esterified sugars and the like; polysaccharides and polymers of sugars), which may be present alone or in combination, including alone or in combination 1-99.9995 by weight or volume. Exemplary protein excipients include serum albumin, such as human serum albumin (H5A), recombinant human albumin (HA), gelatin, casein, and the like. A representative amino acid/antibody component that can act with a buffering capacity, including alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like.

The predominant amino acid is glycine.

Hydrocarbon fillers suitable for use in the invention include, for example, monosaccharides such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides such as raffinose, melezitose, maltodextrins, dextrins, starches, and the like, alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), myoinositol, and the like. Preferred carbohydrate fillers for use in the invention are mannitol, trehalose and raffinose.

Anti-I/-12 antibody compositions may also include a buffer or pH-adjusting agent; of course, a buffer is a salt derived from an organic acid or base. Typical representatives of buffers are salts of organic acids, such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, phthalic acid; Tris, tromethamine hydrochloride or phosphate buffer. Preferred buffers for use in these compositions are salts of organic acids, such as citrate.

Also, anti-II-12 antibody compositions of the invention may include polymeric fillers/additives such as polyvinylpyrrolidones, ficols (polymeric sugars), dextrates (such as cyclodextrins, such as 2-hydroxypropyl-(3-cyclodextrin), polyethylene glycols, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (such as polysorbates, e.g., TWIN 20 and TWIN 80), lipids (such as phospholipids, fatty acids), steroids (e.g., cholesterol) and chelating agents agents (eg

EOTA).

These and other known pharmaceutical excipients and/or additives suitable for use in anti-1-12 antibody compositions, parts or variants thereof, according to the invention are well known to those skilled in the art, as described in ", 19 ey., UMMiShate 8 UMiPatv, (1995), apa ip

Ipe "Rpuzisiapye OezK Ketegepse", 5274 ed., Medisa! Esopotis5, Mopimaie, MU (1998), disclosures incorporated in their entirety by reference)|. Preferred carriers or bulking materials are carbohydrates (such as saccharides and alditols) and buffers (eg, citrate) or polymeric agents.

Warehouses

As stated above, the invention provides stable formulations, which are preferably phosphate buffered saline or a solution of a specific salt, just as the preserved solution contains preservatives, and the preserved formulations of general purpose suitable for pharmaceutical or veterinary use, which include at least one anti-II /-12 antibody in a pharmaceutically acceptable composition. The preserved formulations contain at least one known preservative or optionally selected from the group consisting of at least one phenol, t-cresol, p-cresol, o-cresol, chlorocresol, benzene alcohol, phenylmercury nitrile, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride, (e.g., hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and trimerosal, or mixtures thereof in an aqueous solvent. Any suitable concentration or mixture known in the art may be used, such as 0.001-595, or any interval 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, 1.5, 1.4, 1.5, 1.6, 1.7, 1.8. ,2.2, 2.3, 2.4, 2.5, 2.6, 2.1,2.8,2.9, 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 interval or value within it. Non-limiting examples include: preservative free, 0.1-295 t-cresol (eg, 0.2, 0.3, 0.4, 0.5, 0.9, 1.095), 0.1-395 benzyl alcohol (eg, 0.5, 0.9, 1.1, 1.5, 1.9, . 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, 1.095), and the like.

As stated above, the invention provides an article consisting of a packaging material and at least one vial containing a solution of at least one anti-1II-12 antibody, with certain buffers and/or preservatives, optionally in an aqueous solvent, wherein said packaging material has indication that this solution can be stored for 1,2, 3,4,5,6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours or more.

The invention also includes an article consisting of a packaging material, a foot vial with lyophilized at least one antibody, and a second vial with an aqueous solvent with a buffer or preservative, where said packaging material is marked with instructions for the patient how to properly dissolve at least one anti-1II- 12 antibody in an aqueous solvent to obtain a solution that can be stored for twenty-four hours or more.

At least one anti-1I-12 antibody, which is used in accordance with the present invention and can be obtained by the method of recombination, and includes preparations from mammalian cells or transgenic, or can be isolated from biological sources, as described herein or known in the art.

A certain set of at least one anti-1II-12 antibody in a product of the present invention may include a certain amount obtained as a result of rehydration, if in a wet/dry system, the concentrations will be from about 1.0 nd/ti to about 1000 td/ti, although lower or higher concentrations are permissible and dependent on the intended carrier or delivery method, for example, formulations in a solution will differ from a transdermal patch, pulmonary, transmucosal or osmotic or micropump method.

Preferably, the aqueous solvent may optionally include a pharmaceutically acceptable amount of preservative. Preferred preservatives include phenol, t-cresol, p-cresol, o-cresol, chlorocresol, benzene alcohol, alkylparaben (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and trimerozal, or their mixtures. Concentrations of the preservative used in the formulations are concentrations sufficient to achieve an antimicrobial effect. Such concentrations depend on the preservative chosen and can always be determined by an experienced professional.

Other excipients, for example, isotonic agents, buffers, antioxidants, preservative enhancers, may optionally be added to the solvent. An isotonic agent such as glycerin is commonly used in known concentrations. Physiologically tolerant buffer, preferably added to improve pH control. The formulations can have a wide range of pH values, such as from about pH 4 to about pH 10, and preferably from about pH 5 to about pH 9, and more preferably from about pH 6.0 to about pH 8.0. Preferred compositions of this invention have a pH value of about pH 6.8 to about pH 7.8. Preferred buffers include phosphate buffers, preferably sodium phosphate, in particular buffered saline (PB5B).

Other additives such as pharmaceutically acceptable solubilizers such as Twin 20 (polyoxyethylene (20) sorbitan monolaurate), Twin 40 (polyoxyethylene (20) sorbitan monolaurate), Twin 80 (polyoxyethylene (20) sorbitan monolaurate), Rygopis Eb8 (block copolymer polyoxyethylene polaoxypropylene ) and REO (polyethylene glycol) or nonionic surfactants, such as polysorbates 20 or 80, or Roiochateg 184 or 188 Rishgopisyu polyls, other block copolymers and chelators, such as EOTA and ESTA, may optionally be added to formulations or compositions to reduce aggregation. These additives, in particular, are useful if a plastic container and pump are used to introduce the composition. The presence of a pharmaceutically acceptable surfactant reduces the tendency of the protein to aggregate.

The composition according to the present invention can be made in a way that includes mixing at least one anti-III/-12 antibody and a preservative selected from the group: phenol, t-cresol, p-cresol, o-cresol, chlorocresol, benzene alcohol, alkylparaben (methyl , ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and trimerozal, or their mixtures in an aqueous solvent. Mixing of at least one anti-1II-12 antibody and preservative in an aqueous solvent is carried out using conventional dissolution and mixing methods. To make an acceptable composition, for example, a measured amount of at least one anti-III-12 antibody in a buffer solution is combined with a desired preservative in a buffer solution in an amount sufficient to obtain the protein and preservative in the desired concentrations. Variations on this process will be apparent to one skilled in the art. For example, to add components, when using additional additives, you need to create appropriate temperature conditions and pH, as well as optimize all factors for concentration and method of administration.

The claimed composition can be prepared for patients as a ready-made solution or as a two-vial with a vial of lyophilized, at least one, anti-II-12 antibody, which is diluted with a second vial containing water, a preservative and/or filler, preferably a phosphate buffer and or a saline solution and selected salt, in an aqueous solvent. Both the single-solution vial and the two-vial that require dilution can be reused multiple times and can accommodate patients for one or more cycles of treatment, and thus provide a more convenient treatment regimen than currently available.

Existing claimed products are suitable for use for up to twenty-four hours or more.

Accordingly, the currently claimed products provide more benefits for patients. The compositions of the invention can, optionally, be reliably stored at temperatures of from about 27 to about 40°C and retain the biological properties of the protein over an extended period of time, thus allowing the indication on the package label that the solution can be stored and/or used after 6 , 12, 18, 24, 36, 48, 72, or 96 hours or more.If a canned solvent is used, such indication may include a shelf life of up to 1-12 months, six months, one and a half years, and/or two years.

A solution of at least one anti-I/-12 antibody of the invention can be prepared by a method that includes mixing at least one antibody in an aqueous solvent. Mixing is carried out using generally accepted methods of dissolution and mixing. To make a suitable solvent, for example, a measured amount of at least one antibody in water or buffer is combined in an amount sufficient to obtain the protein and, optionally, the preservative or buffer in the desired concentrations. Options for this process are readily apparent to one of ordinary skill in the art. For example, to add components, when using additional additives, you need to create appropriate temperature conditions and pH, as well as optimize all factors for concentration and method of administration.

The claimed product can be prepared for patients as a ready-made solution or as a two-vial with a vial of lyophilized, at least one, anti-II-12 antibody, which is diluted with a second vial that contains water, a preservative and/or filler, preferably a phosphate buffer and or a saline solution and selected salt, in an aqueous solvent. | the single-solution vial and the two-vial kit requiring dilution can be reused many times and can accommodate patients for single or multi-cycle treatment and thus provide a more convenient treatment regimen than currently available.

The claimed product can be manufactured not directly for patients, but through manufacturing for pharmacies, clinics or other similar institutions and establishments, in the form of a ready-made solution or as a two-vial with a vial of lyophilized, at least one, anti-III-12 antibody, which is diluted with a second vial, which contains an aqueous solvent. The finished solution in this case can be up to one liter or larger, from such a large reservoir, smaller portions of the solution of at least one antibody can be obtained once or repeatedly to transfer them to smaller vials and offered by pharmacies or clinics to their customers and/or patients.

Commonly accepted devices, including single-vial systems and peninjector devices for solution delivery, such as VO Rep5, VO Asho/|esiogt, Nita)esib, MomoRepF, V-OFRep, AshoRepF, apa

OriiRepF, SepoigoripRepF), Sepoigopogt RepF),, Nitaigo RepF), Neso-RepF), Voiegop RepF, Vio|esyugF,, i|esif,

UZ-Yayre,, MeeaiIe-Rgee IpiesiogF), Ipiga)esiF, Meai-UesiFf, e.g., how made or developed Vesiop biscepzep

IEhapkiip such, MU, mly. resiopaiiskepzop.soti, Oiseiopis

Roshapa, Ogedop |mlmyli.rio|esi.soti|; Myopia! Meaisa! Rhodisie, MUeviop Meayisa! (Regegtfogotsdy, ShK, mulu meviop-teaiisa!.sot)|, Meai-Uesi Sogr |Mippearoii5, MM, m/mliu. teai)esisot|. Generally acceptable devices of two-vial systems, including peninjector devices for diluting lyophilized drugs in a cartridge for delivery of a diluted solution is such as NitayoRepFf.

The products claimed here include packaging material. The packaging material provides, in addition to the information required by the regulatory authorities, the conditions under which the product may be used.

The packaging material of the present invention instructs patients to dilute at least one anti-1-12 antibody in an aqueous solvent to form a solution, and to use the solution within 2-24 hours or more for two vials of dry/liquid product. For one vial solution, the product may contain an indication that such a solution can be used for 2-24 hours or more. The products disclosed herein are suitable for human pharmaceutical use.

The compositions of the present invention can be prepared by a method that includes mixing at least one anti-I/!-12 antibody with a selected buffer, preferably a phosphate buffer containing a saline solution or a selected salt. Mixing of at least one antibody and buffer in an aqueous solvent is carried out by generally accepted methods of dissolution or mixing. To prepare an acceptable formulation, for example, a measured amount of at least one antibody in water or buffer is combined with a desired buffering agent in water at concentrations sufficient to produce the protein and buffer at the desired concentrations. Variations of this process should be apparent to any person of ordinary skill in the art. For example, to add components, when using additional additives, you need to create appropriate temperature conditions and pH, as well as optimize all factors for concentration and method of administration.

The claimed stable or preserved formulations can be provided to patients as ready-made solutions or as two-vial preparations, with a vial of lyophilized at least one anti-II-12 antibody, which is diluted with a second vial containing a preservative or buffer and a filler in an aqueous solvent. Both the single solution vial and the two-vial preparation requiring dilution can be reused many times and can accommodate patients in single or multi-cycle treatment and thus provide a more convenient treatment regimen than currently available.

At least one anti-1I--12 antibody in both stable and preserved formulations or solutions, as described herein, may be administered to patients in accordance with the present invention by various delivery methods, including 5C or IM injection; transdermal, pulmonary, transmucosal, implant, osmotic pump, cartridge, micro pump, or other method designated by one skilled in the art as well known in the art.

Therapeutic Uses

The present invention also provides a method for modulating or treating at least one immune disease of a cell, tissue, organ, animal or patient, including, but not limited to, at least one of: rheumatoid arthritis, juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis spondylitis, gastric ulcer, seronegative arthropathy, osteoarthritis, inflammatory bowel disease, ulcerative colitis, systemic lupus erythematosus, antiphospholipid syndrome, iridocyclitis/ureitis/ocular neuritis, idiopathic pulmonary fibrosis, systemic vasculitis/Wegner's granulomatosis, sarcoidosis, orchitis/total vasectomy procedures, allergic /atopic diseases, asthma, allergic rhinitis, eczema, allergic contact dermatitis, allergic conjunctivitis, hypersensitive pneumonitis, transplant rejection of transplanted organs, "graft versus host" disease, systemic inflammatory response syndrome, sepsis syndrome, gram-positive sepsis, gram-negative sepsis , culture-negative sepsis, fungal sepsis, neuropenic fever, urosepsis, meningococcemia, trauma/hemorrhage, burns, ionizing radiation exposure, acute pancreatitis, adult respiratory distress syndrome, rheumatoid arthritis, alcoholic hepatitis, chronic inflammatory conditions, sarcoidosis, Crohn's disease, sickle cell - cellular anemia, diabetes, nephrosis, atopic diseases, hypersensitivity reactions, allergic rhinitis, hay fever, protracted rhinitis, conjunctivitis, endometriosis, asthma, urticaria, systemic anaphylaxis, dermatitis, pernicious anemia, hemolytic diseases, thrombocytopenia, orean transplant rejection or tissue rejection, transplanted kidney rejection, transplanted heart rejection, transplanted liver rejection, transplanted pancreas rejection, transplanted lung rejection, transplanted bone marrow (BMT), rejection of skin allograft, rejection of transplanted cartilage, rejected rejection of a transplanted bone, rejection of a transplanted small intestine, rejection of a transplanted embryonic thymus, rejection of a transplanted thyroid gland, rejection of a xenograft of any organ or tissue, rejection of allografts, anti-receptor hypersensitivity reactions, Grave's disease, Raynaud's disease (vibrodisease), insulin-resistant diabetes type B, asthma, malignant myasthenia gravis, antibody-mediated cytotoxicity, hypersensitivity reactions type IA, systemic lupus erythematosus, ROEM5 syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gramopathies, and skin change syndrome), polyneuropathy, organomegaly, endocrinopathy, monoclonal gramopathies, and skin change syndrome, antiphospholipid syndrome, pemphigus, scleroderma, mixed connective tissue disease, idiopathic Addison's disease, diabetes, chronic active hepatitis, primary miliary cirrhosis, vitiligo, vasculitis, post-MI cardiotomy syndrome, hypersensitivity type MI, contact dermatitis, hypersensitivity pneumonitis, allograft rejection, intracellular granuloma, drug sensitivity, metabolic/diopathic, Wilson's disease, hemachromatosis, alpha-1 antitrypsin deficiency, diabetic retinopathy, Hashimoto's thyroiditis, osteoporosis, hypothalamic-pituitary evaluation -adrenal axis, primary miliary cirrhosis, thyroiditis, encephalomyelitis, cachexia, cystic fibrosis, chronic lung disease of newborns, chronic obstructive pulmonary disease (COPD), hereditary hematophagocytic lymphohisticystosis, dermatological conditions, psoriasis, alopecia, nephrotic syndrome, nephritis, glomerular nephritis, acute renal failure, hemodialysis, uremia, toxicity, pre-eclampsia, OCKIZ therapy, anti-CAZ therapy, cytokinin therapy, chemotherapy, radiation therapy (eg, includes but not limited to toasthenia, anemia, cachexia, and the like), chronic salicylate poisoning, and the like. |See, for example, Megok Mapiai, 1211-17 Eaiopv, Megek 5 Sotrapu, Wapmau, MU (1972, 1977, 1982, 1987, 1992, 1999), RpaptpasoiShegaru Napabrook, U/eiIv eyi ai., edv., Zesopa Eaiop , Arriyup apa Gapde, Ziatioga, Sopp. (1998, 2000), all are fully incorporated by settling |.

The present invention also provides a method of modulating or treating at least one cardiovascular disease in a cell, tissue, organ, animal or patient, including but not limited to,

Lost in publication

Lost in publication at the same time and/or after at least one selected from at least one TME antagonist, (eg, but not limited to, a TME antibody or fragment, a soluble TME receptor or fragment, fusion proteins thereof, or TME small molecules antagonist), anti-rheumatic (eg, methotrexate, auronafine, aurothioglucose, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquinone sulfate, sulfasalazine leflunomide) muscle relaxant, narcotic, nonsteroidal anti-inflammatory drugs (NSAIDs), analgesic, anesthetic, sedative drugs, local anesthetic , neuromuscular blocker, antimicrobial (eg, aminoglycoside, antifungal, antiparasitic, antiviral, carbapenem, cephalosporin, fluoroquinone, macrolide, penicillin, sulfonamide, tetracycline, other antimicrobial), antipsoriatic, corticosteroid, anabolic steroid, diabetic agents, minerals, nutritional supplements, typhoid agents, vitamins, calcium-related hormone, antidiarrheal, antitussive, antinausea, pro antiulcer, emollient, anticoagulant, erythropoietin (eg, epoetin alfa), filgrastim (eg, c-C5BE,

Meiprodep), sargamostim (5M-C5BE, leucine), immunizer, immunoglobulin, immunosuppressant (eg, basiliximab, cyclosporine, daclizumab), growth hormone, hormone replacement drug, estrogen receptor modulator, mydriatic, mydriatic, alkylating agent, antimetabolite, mitotic inhibitor, radiopharmaceutical, antidepressant, antimanic agent, antipsychotic, antipsychotic, hypnotic, sympathomimetic, stimulant, donepezil, tacrine, asthma medication, beta antagonist, inhaler medication, leukotriene inhibitor, methylxanthine, cromolyn, epinephrine or analog, dornase alfa (Ryitolute), cytokine or antagonist cytokine. Acceptable dosages are well known to those skilled in the art. See for example, Umeii5 ei ai., eav5.,

RPpapgtasoiShegaru Naparook, 2pa Eayop, Arriyup apa IGapde, biatioga, ST (2000); ROA RNaghtasorovia,

Taghsop RosKei Rpaptasoroyeia 2000, YuOyeiihe Eaiiop, Taghavsop Rybiizpipd, Sota Sipda, SA (2000), each of these references is incorporated by reference in its entirety).

TME antagonists are suitable for compositions, combination therapeutic devices and/or methods of the present invention (also including at least one antibody of the present invention, specified portion, or variant thereof), including, but not limited to, anti-TME antibodies, TME-TME antibodies, antigen - its binding fragments and receptor molecules that specifically bind to TME; compounds that prevent and/or inhibit TME synthesis, TME release, or its effect on target cells, such as thalidomide, tenidap, phosphodiesterase inhibitors (eg, pentoxifylline and rolipram), A2o adenosine receptor antagonist, and A2p adenosine receptor enhancer; compounds that prevent and/or inhibit TME receptor signaling, such as mitogen-activated protein kinase (MAP) inhibitors; compounds that block and/or inhibit membrane cleavage of TME, such as metalloproteinase inhibitors; compounds that block and/or inhibit TME action, such as angiotensin-converting enzyme inhibitors (eg, captopril); compounds that block and/or inhibit the synthesis and/or production of TME, such as MAP kinase inhibitors.

As used herein, "tumor necrosis factor antibody", "TME antibody", "TME antibody" or fragments and the like reduce, block, inhibit, abrogate or interfere with the activity of TME ip migo, ip 5yi and/or ip mimo. For example, suitable human TME antibodies of the present invention can bind TMEs, including anti-TME antibodies, antigen-binding fragments thereof, and specified mutants or domains thereof that specifically bind to TMEo. Suitable TME antibodies or fragments thereof are also capable of reducing blocking, inhibiting, abrogating or interfering with EMA, OMA or TME protein synthesis, TME release, TE receptor signaling, TME membrane cleavage, TME activity, production and/or synthesis

TME.

CCA2 chimeric antibodies consist of the antigen-binding variable region of a high-affinity mouse antibody to TMERa dO | human, designated as A2, and constant region TO I, immunoglobulin kappa. Region ES | to | human gives halogenated antibodies an effector function, increases the half-life of serum circulation and reduces the immunogenicity of antibodies. Avidity and epitope specificity of the chimeric cA2 antibody is derived from the murine A2 antibody variable region. In a separate embodiment, the preferred source of nucleic acids encoding the variable region of murine antibody A2 is a cell line hybridoma Ag.

Chimeric A2 (cCA2) neutralizes the cytotoxic effect of natural and recombinant human TMEoso depending on the dosage. When analyzing the binding of A2 antibodies and recombinant human TMER, the affinity constant was determined to be equal to 1.04x10'7M". Preferred methods of determining the specificity of monoclonal antibodies and affinity by competitive inhibition can be found in (Napokhmu, ei ai., antirodieev: A Igarogayugu Mapiai, Soy Zrgipd Nagbrog IGarogasogu Rgez5, Soy Zrgipd Nagrog, Memzh/ Mogk, 1988;

Soiidap ei a)i., edv., Sitepi Rgoiosoi5 ip Itp2ypoiodu, Steepe Rybiizpipd Avzos. apa Umieu Ipiegssieepse, Mem mok, (1992-2000); Cogbrogeia!., IttypoiY. Todau, 4: 72-79 (1983); A!yvyrei eyi ai., ed5. Sitepi Rgoiyuosoiv5 ip

Moiesshag Vioiodu, U/ieu Ipiegesiepse, Mem HogK (1987-2000); apa Miiieg, May. Egaguto)., 92: 589-601 (1983), these references are incorporated herein by reference in their entirety).

In a separate embodiment, monoclonal mouse A2 antibodies were produced by a cell line designated as c134A. Chimeric cA2 antibodies were produced by a cell line designated as c1684A.

Additional examples of monoclonal anti-TME antibodies that can be used in this example are described in the special literature (see, for example, O.5. Raiepi Mo. 5,231,024; Moipeg, A. eia!., Suyuyukipe 2 (3): 162- 169 (1990); 0О.5.Raiepi Arriisayop Mo.07/943,852 (yey Zerietbreg 11, 1992); Vaciep ei ai., Ipiegpaiopai!

Rybiisayop MO.M/091/02078 (ribriizpei Rergoagu 21,1991); Kybip eyi aI., ERO Raiepi Rybiisayop Mo. 0218868 (ririizpei Argii 22, 1987); ope ei aI.,, ERO Raiepi Rybiisayop Mo. 0288088 (Osioreg 26, 1988) Giapd, ei ai.,

Viospet. Viorrpuz. Vez. Sott. 137:847-854 (1986); Meaodegh, ei ai., Nubgidota 6:305-311 (1987); Repaiu ei ai.,

Nubrgidota 6:359-369 (1957); Viipdtap, ei ai., Nurgidota 6:489-507 (1987); apa Nigai, eyi ai., dU. Ittipoi. May 96:57-62 (1987), these references are hereby incorporated by reference in their entirety.

TME receptor molecules

Preferred TME receptor molecules of this invention are those that bind TME with high affinity | see e.g., ReIdtapp ei ai., IpsTegpayopa! Rybiysayop Mo. UMO 92/07076 (Ririizpea Argii 30, 1992); cheers! hey ay., Seii! 61: 361-370 (1990); apa I oeyivspeg eyi ai., Se 61 : 351-359 (1990), these references are fully incorporated herein by reference), and do not necessarily have low immunogenicity. In particular, 55 KOa (works

TME-K) and 75KOa (p75 TME-K) TME cell surface receptors are useful in the present invention. Truncated forms of these receptors that contain the extracellular domains (ECDs) of these receptors or their functional parts (see, e.g., Sogsogap et al., Eig. U. Biospet. 223: 831-840 (1994)) are also suitable for of this invention. Truncated forms of TME receptors, which contain ESO, have been found in urine and serum and identified as 30kKOa and 40kKOa inhibitory proteins of TME binding (EpdeItapp, N. ei ai., 9. Violyi. S/aet. 265: 1531- 1536 (1990)). Multimeric TME receptor molecules and TME immunoreceptor fusion molecules, and derivatives, fragments or parts thereof, are additional examples of TME receptor molecules that are suitable for the methods and compositions of the present invention. The TME receptor molecules that can be used in the invention are characterized by their ability to treat patients over a long period of time with good to excellent relief of symptoms and low toxicity. Low immunogenicity and/or high affinity, as well as other undefined properties, may contribute to therapeutic outcomes.

Multimeric TME receptor molecules useful for this invention include all or a functional part of ESO or TME receptors with one or more polypeptide linkers or other non-polypeptide linkers, such as polyethylene glycol (PEG). Multimeric molecules can also contain a signal peptide of a secreted protein to direct the expression of the multimeric molecule. These multimeric molecules and methods of their preparation were described in (0.5. Raiepi Arriisayop Mo.08/437,533

Mau 9, 1995), the contents of which are incorporated herein by reference in their entirety).

TME 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 TME receptors. These immunoreceptor fusion molecules can assemble as monomers or hetero- or homo-multimers. Immunoreceptor fusion molecules can also be monovalent or multivalent.

An example of such a TME-immunoreceptor fusion molecule is the TME-heseriog/do fusion protein. Fused molecules

TME-immunoreceptor and methods for their preparation were described in the literature (Gezziaceg ei a!., Eig. U. IC 101.

21: 2883-2886 (1991); AzPKepa?i eyi a!., Rhos. Mai). Asad 5 BOTH 88: 10535-10539 (1991); Rerrei ei a|., 9. E, gr.

Honey. 174: 1483-1489 (1991); Koiih5 ei ai!., Rgos. iMeSh., Isi2siI. All together. S BA 91: 215-219 (1994); Vyshyeg eyia!., SuyukKipe 6(6): 616623 (1994); VaKeg eia!., hey. 9. Ittipoi. 24: 2040-2048 (1994); Vesheg her AI., SHO. 5. Raiepi Mo. 5,447,851; apa |. 5. Arriisayop Mo. 08/442, 133 (Pea Mau 16, 1995), each of which is incorporated herein by reference in its entirety. Methods of obtaining fusions of immunoreceptor molecules can also be found in

ISarop ei a!., ). 5. Raiepi Mo. 5,116,964; Sarop ei ai., ). 5. Raiepi Mo. 5,225,538; apa Sarop vi a!., Mashge 337: 525-531 (1989, this reference is incorporated herein by reference in its entirety).

A functional equivalent, derivative, fragment, or region of a TME receptor molecule refers to a portion of a TME receptor molecule, or a portion of the sequence of a TME receptor molecule that encodes a TME receptor molecule that is of sufficient size and sequence to be functionally similar to TME receptor molecules that may be used in the present invention (eg, bind TME with high affinity and have low immunogenicity). A functional equivalent of a TME receptor molecule also includes modified TME receptor molecules that are functionally similar to the TME receptor molecules that can be used in the present invention (eg, bind TME with high affinity and have low immunogenicity). For example, a functional equivalent of a TME receptor molecule may contain a "silent" codon or one or more amino acid substitutions, deletions or additions (ie. or another codon encoding a hydrophobic amino acid). | See A!, Mzybei ei ai., Sygepi

Rgoiyusoiv ip MoiIesshtsag Vioiodu, Sgeyepe Rybiivzpipd Avzos. apa, U/ieu-Ipiegvsiepse, Mem Hoik (1987-2000)).

Cytokinins include any known cytokinin. See e.g., mmlu.SoremppSmiokKipe5.sot. Cytokinin 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 treatment. Any method of the present invention may include a method of treating a 1-12-mediated disorder, including administering an effective amount of a composition or a pharmaceutical composition comprising at least one anti-II-12 antibody to a cell, tissue, organ, animal, or patient in need of such modulation , treatment or therapy. Such a method may optionally include the simultaneous use or combination therapy for the treatment of such immune diseases, where the use of said at least one anti-1II-12 antibody, said part or variant thereof, also covers the use, before, simultaneously, and/ or after at least one selected from an antagonist (eg, but not limited to, a TME antibody or fragment, a soluble TME receptor or fragment, their fusion proteins, or a small molecule TME antagonist), an antirheumatic drug (e.g. 9d, methotrexate, auranofin , aurothioglucose, azathiopyrine, etanercept, sodium aurothiomalate, hydroxychloroquinone sulfate, lerflunomide, sulfasalazine), muscle relaxant, narcotic, nonsteroidal anti-inflammatory drug, analgesic, anesthetic, sedative, local anesthetic, neuromuscular blocker, antimicrobial (eg, aminoglycoside , antifungal, antiparasitic, antiviral, carbapenem, cephalosporin, fluoroquinolone, macrolide, penicillin, sul fonamide, tetracycline, other antimicrobial), antipsoriatic, corticosteroid, anabolic steroid, diabetes-related agent, mineral, dietary supplement, thyroid agent, vitamin, calcium-related hormone, pritillary, antitussive, antiemetic, antiulcer, laxative, anticoagulant, erythropoietin (eg, epoetin alfa), filgrastim (eg, 5-C5E, Meprodep), sargramostim (M-C5E, leucine), immunizer, immunoglobulin, immunosuppressant (eg, basiliximab, cyclosporine, daclizumab), growth hormone, hormone replacement drug, estrogen receptor modulator, mydriatic agent, cycloplegic, acylating agent , antimetabolite, mitotic inhibitor, radiopharmaceutical, antidepressant, antimanic agent, antipsychotic, tranquilizer, hypnotic, sympathomimetic, stimulant, donepezil, tacrine, asthmatic, beta agonist, inhaled steroid, leukotrene inhibitor, methylxanthine, cromolyn, epinephrine or analog, dornase alfa (Riitogute), cytokinin, or cytokinin antagonist.

Of course, the treatment of the pathological conditions is carried out by the use of an effective amount or dose of at least one anti-I-12 antibody composition that is wholly, on average, or in the range of at least about 0.01 to 500 milligrams of at least one anti-I-12 antibodies / kilogram of the patient per dose, and preferably from at least about 0.1 to 100 milligrams, antibodies / kilogram of the patient for one or multiple use, depending on the specificity of the action contained in the composition. Alternatively, the effective serum concentration can be 0.1-5000dd/t! serum, for single or repeated use.

Acceptable dosages are known to medical practitioners and will, of course, depend on the specificity of the disease state, the specificity of the activity of the composition used, and the specificity of the course of treatment. In some examples, to achieve the desired therapeutic amount, it may be necessary to implement multiple administrations, eg, repeated individual administrations of a controlled or metered dose, where individual administrations are repeated until the desired daily dose or result is achieved.

Preferred may 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, AZ, 44, 45, Ab. 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 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 100-500td/kd/application, or any interval, value or part thereof, or until reaching 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, 12, 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, 9.0,9 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 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, 4 500, and/or 5,000 U/ti serum concentration for a single or multiple use, or any interval, value, or part thereof.

Alternatively, the dosage of use may vary, depending on known factors, such as the specifics of the pharmacodynamics of a specific agent, and its method and route of application; age, health, and weight of the recipient; the nature and duration of symptoms, the type of concomitant treatment, the frequency of use, and the desired result. Typically, the dosage of the active ingredient can range from about 0.1 to 100 milligrams per kilogram of body weight. Typically 0.1-50, and preferably 0.1-10 milligrams per kilogram per application or in a slow release form, is effective for obtaining the desired results.

As a non-limiting example, the treatment of a person or an animal can be carried out as a single or periodic dosage of at least one antibody of the present invention 0.1-100tod/code, such as 0.5, 0.9, 1.0, 1.1, 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 tons per day, at least one of per 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 per 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, AZ, 44 , 45, Ab, 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 a single infusion or repeated and doses.

Dosage forms (compositions) suitable for internal use generally contain from about 0.1 milligrams to about 500 milligrams of active ingredient per unit or container. In these pharmaceutical compositions, the active ingredient is usually present in an amount of about 0.5-99.99995 mass, from the total weight of the composition.

For parenteral administration, the antibody may be in the form of a solution, suspension, emulsion, or lyophilized powder in combination with, or separately administered, an acceptable pharmaceutical parenteral carrier. Examples of such transport are water, saline, Singer's solution, glucose solution, and 1-1095-per human serum albumin. Liposomes and non-aqueous vehicles such as non-volatile oils can also be used. The carrier or lyophilized powder may contain additives that maintain isotonicity (eg, sodium chloride, mannitol) and chemical stability (eg, buffers and preservatives). Formulations are sterilized by known or suitable methods.

Suitable pharmaceutical carriers described in the latest edition, (Ketipdiopye Rpagtaseshiisa! Zsiepsev, A.

Yes, the standard link text in this field.

Alternative Application

Many known and currently being developed methods can be used according to this invention to apply pharmaceutically effective amounts of at least one anti-1II-12 antibody according to this invention. While pulmonary administration has been provided in this description, other methods of administration can be used according to this invention with acceptable results.

Antibodies I--12 of the present invention may be presented 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 inhaler use or by other methods described herein or known in practice.

Parenteral Formulations and Application

Formulations for parenteral use may contain, as a common filler, sterile water or saline solution, polyalkyl glycols, such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalene, and the like. Aqueous or oily suspensions for injections can be prepared using a suitable emulsifier or wetting agent and a suspending agent according to known methods.

Agents for injection may be non-toxic, not orally diluted, such as aqueous solutions or a sterile injectable solution or suspension in a solvent. As a suitable carrier or solvent, water, Ringer's solution, isotonic saline, etc. can be used. permitted; sterile non-volatile oil may be used as the usual carrier or suspension solvent. For these purposes, any kind of non-volatile oil and fatty acid can be used, including natural or synthetic or semi-synthetic fatty oils or fatty acids; natural or synthetic or semi-synthetic mono- or di- or triglycerides. Parenteral administration is known in the art and includes, but is not limited to, a conventional injection device, a needleless device for injecting compressed gas as described in (0.5.Rai.Mo.5,851,198), and a laser perforating device as described in (O.5.Rai.Mo.5,839,446 is fully included here by reference).

Alternative Delivery

In addition, the invention relates to the use of at least one anti-1I-12 antibody parenterally, subcutaneously, intramuscularly, intravenously, intraarticularly, intrabronchially, intraperitoneally, intraarticularly, intracartilaginously. intracavitary, intraceliac, intracerebral, intracerebroventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, with bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal means. At least one formulation of the anti-II-12 antibody may be intended for parenteral (subcutaneous, intramuscular or intravenous) or any other use, especially in the form of liquid solutions or suspensions; for vaginal or rectal use, especially in semi-solid forms such as, but not limited to, creams and suppositories: for buccal or sublingual use, such as, but not limited to, tablet or capsule form; or intranasally, such as, but not limited to, in the form of powders, nasal drops or aerosols or certain agents; or transdermally, such as non-limiting, gel, ointment, lotion, suspension, or patch delivery system with a chemical filler, such as dimethyl sulfoxide, to change the structure of the skin or increase the drug concentration in the transdermal patch |(Chipdipdeg, ей а). n "Opd Repteyeayop

Eppapsetepi"; Nvieei, 0. 5., Ev., pp. 59-90 (Magse! OekKeg, Ips. Mem Mogk 1994, fully incorporated by reference) and, or with oxidizing agents, which enable the use of formulations containing proteins and peptides on the skin (M/098/53847|, or the use of electric fields, to create fast transport pathways, such as electroporation, to increase the mobility of charged drugs through the skin, such as iontophoresis, or the use of ultrasound, such as sonophoresis

IU.5.Rai.Mo5.4,309,989 and 4,767,402| (the aforementioned publications and patents are incorporated herein by reference in their entirety).

Pulmonary/Nasal use

For pulmonary administration, preferably at least one anti-1Y-12 antibody formulation is delivered in a particle size effective to reach the lower airways of the lungs or sinuses. According to the invention, at least one anti-1II-12 antibody can be delivered by any of the various inhaler or nasal devices known in practice for the use of a therapeutic agent by inhalation. These devices capable of depositing aerosolized formulations into the patient's sinuses or alveoli include metered dose inhalers, nebulizers, dry powder generators, nebulizers, and the like. Other devices suitable for use for pulmonary or nasal administration of antibodies are also known in the art. All such devices can use formulations suitable for use in aerosolized antibody delivery. Such aerosols may include either solutions (both aqueous and non-aqueous) or solid particles. Metered inhaler dispensers, like the metered metered inhaler Mepiop5 dispenser, usually use a propellant gas and require actuation by inhaling Idv. e.g., MO 94/16970,

UMO 98/35888)|. Dry powder inhalers similar to Tigripa|egtm (Aviga), Noiapa|ego (Stiaho), OizkyzF (Spaho),

ZrigozFippa|eg (Oiga), devices for sale, Ippaie Tpegareshisv, and ZzrippaiegF powder inhaler (Rizopv) which is actuated by inhalation of mixed powder (05 4668218 Aviga, ER 237507 Aviga, MO 97/25086

Siaho, MO 94/08552 Byga, 5 5458135 Ippaie, UMO 94/06498 GRizop5, fully incorporated herein by reference.

Sprayers like AEkh'""Agadidt, OKgamepiFperitsi2eg (MaiPpsKgoayuO, and sprayer Asogp PF (Medical

Products Magdne5z) (05 5404871 Agadidt, UMO 97/223761|, mentioned above, fully incorporated herein by reference), generate aerosols from solutions, while metered dose aerosols, dry powder inhalers, etc. generate small aerosol particles. These specific examples are commercially available inhalation devices are representative of specific devices suitable for this invention and are not a limitation of this invention of the invention.

Preferably, the composition, which includes at least one anti-1I-12 antibody, is delivered as a dry powder inhaler or nebulizer. These are some desirable features of the inhalation device for the use of at least one antibody of this invention. For example, delivery by an inhalation device is much more profitable, easier to reproduce, and more accurate. The inhalation device may optionally deliver small dry particles, for example less than about 10ct, preferably about 1-5dt, to improve respirability.

Application of antibody compositions 1-12 Aerosol

An aerosol containing a protein composition of the antibody I/-12 can be obtained by spraying under pressure through a nozzle of suspensions or solutions of at least one anti-1I-12 antibody.

Nozzle size and configuration, pressure applied, and liquid feed rate can be selected to achieve the desired yield and particle size. An electroaerosol can be obtained, for example, by an electric field in combination with a capillary or a feeding nozzle. Preferably, the particles of at least one anti-II-12 antibody protein composition, delivered by a nebulizer, have a particle size of less than about 10 units, preferably from about 1 unit to about 5 units, and most preferably from about 1 unit to about 1 unit.

Formulations of at least one protein of the anti-1II-12 antibody composition suitable for use in a nebulizer typically include the protein of the antibody composition in an aqueous solution at a concentration of from about 0.1 to about 100% 0.1 of at least one protein of the anti-1II-12 antibody composition per ti of the solution or td/dt or any interval or value therein eg but not limited 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 100td/ti or td/d. The composition may include agents such as a filler, a buffer, an isotonic agent, a preservative, a surfactant, and, preferably, zinc. The composition may also include a filler or protein stabilization agent of the antibody composition, such as a buffer, reducing agent, additional protein, or carbohydrate. Additional proteins useful in the formulation of protein antibody compositions include albumin, protamine, or the like. Typical carbohydrates useful in the formulation of protein antibody compositions include sucrose, mannitol, lactose, trehalose, glucose, or the like. The formulation of the protein antibody composition may also include a surfactant that can reduce or prevent surface aggregation of the protein antibody composition caused by atomization of the solution in the aerosol formation. Various traditional surfactants can be used, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts may generally range from 0.001-1495 of the weight of the formulation. Particularly preferred surfactants for the purposes of the present invention are polyoxyethylene sorbitan monooleal, polysorbate 80, polysorbate 20, or the like. Additional agents known in the art for protein formulations, such as antibodies 1-12, or specified portions or variants, may also be included in the formulation.

Application of antibody compositions 1-12 using a sprayer

The protein composition of the antibody can be applied as a nebulizer, such as a jet nebulizer or an ultrasonic nebulizer. Of course, in a jet sprayer! a source of compressed air is used to create a high-velocity air jet through the opening. As the gas expands after the nozzle, a low-pressure zone is created that pulls the protein solution of the antibody composition through a capillary tube connected to the liquid reservoir. The liquid flow from the capillary tube breaks up into unstable filaments and droplets at the exit of the tube, creating an aerosol. A number of configurations, leak rates, and baffle types can be used to achieve the desired features from a given jet atomizer. In an ultrasonic atomizer, a piezoelectric transducer is usually used to convert high-frequency electrical energy into vibrational, mechanical energy. This energy is transferred to the protein formulation of the antibody composition either directly or through the coupling liquid, creating an aerosol that includes the protein composition of the antibody.

More preferred is an embodiment in which the protein particles of the nebulizer-delivered antibody composition have a particle size of less than about 1 µm, preferably in the range from about 1 µm to about 5 µm, and most preferably from about 2 µm to about 1 µm.

Formulations of at least one anti-IL-12 antibody suitable for use with either a jet or ultrasonic nebulizer typically have a concentration of from about 0.1 t79 to about 100 t4d of at least one anti-IL-12 antibody protein per unit of solution. The formulation may include such agents as, for example, a filler, a buffer, an isotonic agent, a preservative, a surfactant, and, preferably, zinc. The formulation may also include a filler or stabilizer of at least one protein of the anti-I-12 antibody composition, such as a buffer, reducing agent, additional protein, or carbohydrate. Additional proteins useful in the formulation of at least one protein of the anti-II-12 antibody composition include albumin, protamine, or the like. Typical carbohydrates useful in the formulation of at least one anti-1I--12 antibody include sucrose, mannitol, lactose, trehalose, glucose, or the like. At least one anti-II-12 antibody formulation may also include a surfactant that can reduce or prevent surface aggregation of at least one anti-II-12 antibody caused by atomization of the solution in the aerosol formation.

Various suitable surfactants can be used, such as polyoxyethylene fatty acid esters, alcohols and polyoxyethylene sorbital fatty acid esters. The concentration of the substance is usually from about 0.001 to about 495 by weight of the formulation. Particularly preferred surfactants for the purposes of the present invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, or the like. Additional agents known in the art for protein formulations, such as protein antibodies may also be included in the formulation.

Use of I/-12 Antibody Compositions Metered Inhaler In a metered dose inhaler (MEI), a propellant gas, at least one anti-III-12 antibody, and any of the excipients or other additives contained in the container in the form of a mixture, including liquefied , compressed gas. Actuation of the metering valve releases the mixture in the form of an aerosol, which preferably contains particles of a size of less than about 10 µm, preferably about 1 µm to about 5 µm, and most preferably from about 2 µm to about Zd µm. The desired particle size of the aerosol can be obtained, when using the formulation of the protein composition of the antibody obtained by various methods known in the art, including such as jetting, spray drying, critical point condensation, or the like. Preferable metered dose inhalers include manufactured ZM or siaho and use fluorocarbon as a displacing gas.

Formulations of at least one anti-I-12 antibody for use with a metered dose inhaler device generally include a finely dispersed powder containing at least one anti-I-12 antibody as a suspension in a non-aqueous medium, for example, suspended in a displacer gas using a surfactant. The displacing gas can be any commonly accepted material used for this purpose, such as chlorofluorocarbon, hydrochlorofluorocarbon, hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoromethane, NEA-134a (hydrofluoroalkane -134a), NEA-227 (hydrofluoroalkane -227), or similar.

The best propellant is hydrofluorocarbon. The surfactant may be selected to stabilize at least one anti-I-12 antibody as a suspension in a propellant to protect the active agent from chemical degradation, and the like. A suitable surfactant includes sorbitan trioleate, soy lecithin, oleic acid, or the like. In some cases, solution aerosols, which are preferred, use solvents such as ethanol.

Additional agents known in the art for protein formulations, such as protein may also be included in the formulation.

Anyone skilled in the art will understand that the methods of this invention may include pulmonary administration of at least one anti-II-12 antibody formulation using devices not described herein.

Oral Formulation and Application

Oral formulations depend on the simultaneous use of adjuvants (eg, resorcinols and nonionic surfactants, such as polyoxyethylene oleyl ether and p-hexadecylpolyethylene ether) to artificially increase the permeability of the intestinal wall, as well as enzymatic inhibitors (eg, pancreatic trypsin inhibitors , diisopropylfluorophosphate (ORE) and triazolyl), to prevent enzymatic degradation. The basis of the active mixture for the solid dosage form for oral use can be obtained with at least one additive from the listed sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starches, agar, arginates, chitins, chitosans, pectins, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymer, and glyceride. These dosage forms may also contain other type(s) of additives, e.g., an inert diluting agent, a lubricant such as magnesium stearate, a paraben, a preservative such as sorbic acid, ascorbic acid, alpha-tocopherol, an antioxidant such as cysteine, disintegrant, binder, thickener, buffering agent, sweetening agent, flavoring agent, flavoring agent, etc.

Tablets and pills may also have an enteric coating. Liquid preparations for oral use include emulsion, syrup, elixir, suspension and solution suitable for medical use. These preparations may contain inert diluents commonly used in the art, e.g., water. Liposomes are also found as drug delivery systems for insulin and heparin (0.5.Rai.Mo.4,239,754)|. Artificial polymer microspheres of the mixture were also used to deliver pharmaceutical preparations of amino acids (proteinoids), (Sh. 5. Rai. Mo. 4,925,673)|. In addition, the carrier mixtures described in (Yu. 5. Rai. Mo. 5,879,681 and Sh. 5. Rai. Mo. 5,5,871,753| which were used for the delivery of biologically active agents for internal administration are known in practice. -- ------stopped here-------

Mucosal formulation and application

For absorption through mucosal surfaces, compositions and methods of application of at least one anti-II-12 antibody include an emulsion comprising a large number of submicron particles, macromolecules adhesive to the mucosa, bioactive peptides, and an aqueous continuous phase that performs absorption through mucosal surfaces by achieving microadhesion of emulsion particles (0.5.Rai.Mo. 5,514,6701I.

Mucous membranes suitable for application of the emulsions of this invention may include corneal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, gastric, intestinal, and rectal routes of administration. Formulations for vaginal or rectal use, such as suppositories, may contain excipients such as polyalkene glycols, petrolatum, cocoa butter, and the like. Formulations for intranasal use can be solid and contain, as fillers, for example, lactose, or can be aqueous or oily solutions of nasal drops.

Fillers for buccal use include sugars, calcium stearate, magnesium stearate, pregelatinized starch, and the like (Sh.5.Rai.Mo. 5,849,6951I.

Transdermal formulations and applications

For transdermal administration, at least one anti-1II-12 antibody encapsulated in delivery vehicles such as liposomes or polymeric nanoparticles, microparticles, macrocapsules, or microspheres (usually all called microparticles, although sometimes distinguished), there are a number of known delivery vehicles, including synthetic polymeric microparticles made, for example, from polyhydroxy acids, such as polylactic acid, polyglycolic acid and their copolymers, polyorthoesters, polyanhydrides, and polyphosphazenes, and natural polymers, such as collagen, polyamino acids, albumin and other proteins, alginates and other polysaccharides, and their combinations (0.5.Rai.Mo. 5,814,599).

Long-term use and formulation

Sometimes it may be desirable to deliver the compounds of this invention to the subject over a long period of time, for example, from one week to one year from a single application. Various dosage forms of slow release, depot or implant can be used.

For example, the dosage form may contain a pharmaceutically acceptable non-toxic salt of compounds that have a low degree of solubility in biological fluids, for example (a) an acid salt with a polybasic acid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, rathoic acid , alginic acid, polyglutamic acid, naphthalene mono- or disulfonic acids, polygalacturonic acid, and the like; (B) a salt with a multivalent 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., M,M'-dibenzyl- ethylenediamine or ethylenediamine; or (c) combinations of (a) and (b) such as zinc tannate salt. Additionally, the compounds of this 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, for example, sesame oil, suitable for injection. Particularly preferred salts are zinc salts, zinc tannate salts, ratoaige salts, and the like. Another type of slow-release depot formulation for injection could contain a mixture or salt dispersed for encapsulation in a slowly degradable, non-toxic, non-antigenic, polymer, such as a polylactic acid/polyglycolic acid polymer, for example as described in (Sh.5.Rai .Mo.3,773,919|. Mixtures or, preferably, relatively insoluble salts such as those described above may also be formulated into silastic pills with a cholesterol matrix, particularly for use in animals. Additionally, slow-release, depot or implant formulations, such as gas or liquid liposomes known in the literature (0. 5.

Paradise As. 5,770,222 apa "Zyviaipei apa SopigoPei NeiIease YuOgpid Oeiiimegu buzietv", U. A. Vobipzop ed., Magse!

Oekkegt, Ips., M.U., 19781.

The same as set forth in the description of the invention may be made clear from the following examples, which are given by way of illustration and are not intended to be limiting.

Lost in publication

Vector pC4 is used for expression of antibodies 1-12. Plasmid rs4 is a derivative of plasmid rem2-apt (ATSS Aseszvsiop Mo.37146). The plasmid contains the mouse ONERK gene under the control of the earlier 5M40 promoter. Ovary or other Chinese hamster cells deficient in dihydrosulfolate activity that are transfected with these plasmids can be selected by growing the cells on a selective medium

For example, airpa tipiz MEM, Gite Tesppoiodie5, cSypegzrigd, MOI supplemented with the chemotherapeutic agent methotrexate. Amplification of ONEK genes in cells resistant to methotret (MTX) is well described | see, e.g.

R. MU. АЙ,, ей а)., 9. Vioi. Snet 253: 1357-1370 (1978); 9. M. Natiip apa S. Ma, Viospet. her Viorpuv. Asia 1097: 107-143 (1990); apa M. u. Rade apa M. A. budepnat, Vioyesppoiodo 9: 64-68 (1991)). Cells grown at increased concentrations of MTX develop drug resistance by overproducing the target enzyme,

OSNERK, as a result of amplification of the ONEK gene. If the second gene is connected to the ONEK gene, it is usually coamplified and overexpressed. It is known in practice that this approach can be used to obtain cell lines that carry more than 1,000 copies of the amplified gene(s). Subsequently, when the methotrexate is removed, cell lines are obtained that contain the amplified gene linked to one or more chromosomes/(s) of the host cell.

Plasmid Res. expression of the target gene contains the strong promoter of the long terminal repeat (LTC) of the Rous sarcoma virus (Schiep, et al., MoiIes. SeiI. BioI. 5: 438-447 (1985) | plus a fragment isolated from the enhancer of the human cytomegalovirus immediate early gene ( SMU) |Vozpaigi, ei a!., Seii! 41: 521-530 (1985)|.

Laterally located downstream of the promoter are Watni, Hwai, and Azr718 restriction sites by lytic enzymes that allow gene integration. After cloning of the main sites, the plasmid contains the C intron and the polyadenylation site of the rat preproinsulin gene. Other highly efficient promoters for expression may also be used, such as the human β-actin promoter, early or late 5M40 promoters, or the long terminal repeat from another retrovirus, such as HIV and NTIMI, Siopiesp5 Tei-OyY and Tag-Op gene expression systems and the like systems can be used for expression of regulated type 1-12 in mammalian cells M. Sbozzep, apa N. Vshaga, Rgos. May Asai. zZsi OBA 89: 5547-5551 (1992)). Other signals can also be used for tkMA polyadenylation, for example, from human growth hormone or a novel gene. Stable cell lines that carry the target gene integrated into the chromosome can also be selected by cotransfection with a selectable marker such as ori, 5418 or hygromycin. The use of more than one marker early on, such as 5418 with methotrexate, offers significant advantages.

Plasmid pC4 is cleaved by restriction enzymes and then dephosphorylated using calf intestinal phosphatase, a commonly known method. The vector is then separated from the 195 agarose gel.

The OMA sequence encoding the complete antibody 1-12 is used, for example, ZEO IU MOB: IM5EKT

MAV AA 5EO IO 1, and IM5ECT MAV AA 5EO IO MO2, according to the variable regions of NS and I C antibodies

I/-12 of this invention, by appropriate known methods. Nucleic acid isolations encoding a suitable human constant region (eg, NS and IS regions) are also used for this construct (eg, as in vector p 1351: IMBENT ATSS ASSEBBIOM MOMVEA AMO AOOITIOMAG NSL C plasmid).

The isolated OMAs encoding the variable and constant regions and the dephosphorylated vector are then ligated with T4 OMA ligase. Cells of E. soya HB 101 or XI-1 Vie are then transformed and identify bacteria that contain a fragment inserted into plasmin pC4 using, for example, restriction enzyme analysis.

For transfection, Chinese hamster ovary cells (CHO), devoid of the active ONEK gene, are used. 594 pC4 expression plasmins are cotransfected with 0.59 reM2peo plasmids using lipofescin. Plasmid reM2peo contains a dominant selectable marker, the peo gene from Tp5 encodes an enzyme that leads to resistance to a group of antibiotics including 0418. After 2 days, cells are trypsinized and plated on hybridoma cloning plates (sgeipeg, begptapa) in alpha minus MEM with the addition of 10, 25 , or 5bpd/ti of methotrexate with Ttd/ti 5418. after about 10-14 days, individual clones are trypsinized and sown in b-well Petri dishes or 10ti! flasks using different concentrations of methotrexate 50 pM, 100 pM, 200 pM, 400 pM, 800 pM). Clones growing with the highest methotrexate concentrations were then transferred to new b-well plates with even higher methotrexate contents (1tM, 2tM, 5tM, 10tM, 20tM). The same procedure was repeated until clones were obtained that grew at a concentration of 100-200 tM. The expression of the desired gene product was studied, for example, using 505-

RACE and Uueviet Brioi using reverse-phase NRI C-analysis.

Example 2: Obtaining, with the help of transgenic mice, high-affinity monoclonal antibodies (human dO reacting with human I-12

A concise statement

To obtain high-affinity, fully human, monoclonal antibodies that can be used therapeutically to inhibit the action of I/-12 for the treatment of one or more diseases mediated by I--12, transgenic mice were used that had genes for light and heavy chains of human immunoglobulin. Hybrid mice (SBA/) x C57/8VI6/U) E, which had transgenes of the variable and constant regions of human heavy and light chains, were immunized with recombinant human 1-12

ITaupog ei ai., Ipi). Ittypo)y. 6: 579-591 (1993); And oprego, ei a!., Maishge 368: 856-859 (1994); Meiybregoetg, M., Mayte

Vioitesn. 14: 826 (1996); Rizpmd, ei ai!., Journal of Biochemistry 14: 845-851 (1996)). Several fusions yielded one or more panels of fully human II/-12 reactive dO monoclonal antibodies. Fully human anti-I-12 antibodies will be described below. All of them are dO. Such antibodies have been determined to have an affinity constant somewhere between 1x109 and 9x1012, the unexpectedly high affinities of these fully human monoclonal antibodies making them suitable candidates for therapeutic applications in diseases, pathologies or disorders related to 1-12.

Abbreviation

VZA - bovine serum albumin

СО2 - carbon dioxide

OM5O - dimethylsulfoxide

EIA - enzyme immunoassay

EB5 - embryonic bovine serum

Hg02-hydrogen peroxide

NRR - horseradish peroxidase

IO - intradermal

Id - immunoglobulin

I/-12 - interleukin-12

IR - intraperitoneal

IM - intravenous

Mab - monoclonal antibodies

OO - optical density

OPO-o - phenylenediamine dichloride

PE - polyethylene glycol

RBZA - penicillin, streptomycin, amphotericin

VT - room temperature 5O - subcutaneous m/m - volume per volume m/m - weight per volume

Materials and methods

Animals

Well known from the literature are transgenic mice capable of expressing human antibodies that express human immunoglobulins, but not murine IdM or Id, they are also commercially available In eg., from sepRPagt Ipiegpaiiopai, Zap duose, CA; Ardepikh, Egeetopi, SA, and others). For example, such transgenic mice have a human transgene sequence that has been subjected to M(0)U insertion, a heavy chain class switch, and somatic mutations to produce a list of immunoglobulins with human sequence

IHopbego, ei ai., Mayte 368: 856-859 (1994)). A light chain transgene can be obtained, for example, in part from a clone of an artificial yeast chromosome, which includes approximately half of the M region of human gamete lines. Additionally, a heavy chain transgene can encode both and and | (Rizpm/ya, ei ai., Maishge

Biosciences 14: 845-851 (1996). and/or human C constant regions. Mice derived from the appropriate genotypic lines can be used in immunization and fusion processes to produce human monoclonal antibodies to 1-12.

Immunization

One or more immunization regimens can be used to produce a human anti-1I/-12 hybridoma, the first few fusions can be performed after the following exemplary immunization protocol, but other known similar protocols can also be used. Several 14-20-week-old female and/or surgically castrated transgenic male mice were immunized with IR and/or IO of 1-1000n9 recombinant human 1-12 emulsified with an equal volume of TITEEMAH or complete Freund's adjuvant with a final volume of 100-400 (e.g., 200). Each mouse may also optionally receive 1-10c9 in 100% saline at each of the two 50 sites. Mice can then be immunized 1-7, 5-12, 10-18, 17-25 and/or 21-34 days after IR (1-400d9) and 5O (1-400ddhg2) emulsified 1-12 with equal vol. by TITEKMACH or Freund's incomplete adjuvant. Mice can be bled on days 17-25 and 25-40 after retro-orbital puncture without anticoagulant. The blood can then be allowed to settle under CT for one hour, and the serum collected and titrated with 1-12 EIA sample according to known methods. Fusions are performed when repeated injections do not lead to an increase in the titer. At this time, an enhanced injection of 1-400 rn49 11-12 of the physiological solution dissolved in you can be transferred to the final MI.

Three days later, the mice can be euthanized by cervical dislocation and the spleen is aseptically removed and placed in 10 µl of cold phosphate buffer solution (PB5) containing 100 µl/µl. penicillin, 100dd/ti. streptomycin, and 0.25nd4/ti. amphotericin B (RBZA). Spleen cells were harvested by sterile perfusion of the spleen with RBA-RV5. Cells were washed once in cold RBA-RVB5, counted by exclusion using Trypan blue dye, and resuspended in medium

ERMI 1640, which contained 25 tons of stainless steel.

Fusion of cells

Fusion was performed at a ratio of 1:11 to 1:10 mouse myeloma cells to viable spleen cells, according to known methods, that is, methods known from the literature. As a non-limiting example, spleen cells and myeloma cells can be pelleted together. The pellets can then be slowly resuspended, in 30 seconds, in tt. 5095 (m/u) of RES/RVZ solution (molecular weight RES 1.450, 5idta) and 37"C. The fusion can then be stopped by slowly adding 10.5 tp!. medium KERMI 1640 containing 25M Nerez (37"C) over 1 minutes The merged cells are centrifuged for 5 minutes at 500-1500 rpm. the cells are then resuspended in NAT medium (ERMI 1640, which contains 25 tM Nerevz, 1095 serum of Embryonic Clone I (Nusiope), 1 tM sodium pyruvate, 4 tM I--glutamine, 1Ud/ti. gentamicin, 2.590 culture supplement Ogidep (Rizpeg), 1095 653 -standardized ERMI 1640/Nerev environment,

BOM of 2-mercaptoethanol, 100µM hypoxanthine, 0.4dM aminopterin, and 16dM thymidine) and then placed in g00sh lmei!! in fifteen 96-well flat-bottom tissue culture plates. The plates were then placed in a humidified incubator at 37"C with 595 CO" and 9595 air for 7-10 days.

Determination of anti-1II-12 human antibodies in mouse serum

For the screening of mouse sera for Ido antibodies specific to human I--12, a solid-phase EIA"5 can be used. Briefly, the plates can be coated with II/-12 in the amount of 2nd/ti. in RV5 overnight. After washing in 0.15M saline solution containing 0.02905 (m//) Tween 20, wells may be blocked with 1905 (m/u) VBA in РВ5. 200 µl/well for 1 hour at CT. Plates may be used immediately or frozen at -207"C for later use . Diluted mouse serum is incubated on coverslips

I/-12 discs with 50 /well at CT during one time. Plates are washed and tested 50di/well

NAR-labeled goat dos to humans, Es specifically diluted 1:30,000 in 1906 VBA-RV5 for 1 hour at CT. Plates can be washed again by adding 100 cells/well of citrate-phosphate substrate solution (0.1 M citric acid and 0.2 M sodium phosphate, 0.019565 Ng2O» apa 1 tad/t/. ORO) for 15 minutes at CT. Then a stop solution (4N sulfuric acid) is added in the amount of 25 µl/well and 005 is measured at 490pt on an automated plate spectrophotometer.

Determination of fully human immunoglobulins in their hybridoma supernatants

The determination of growth-positive hybrids of fully human immunoglobulins can be determined by suitable EIA. Briefly, 96-well plates with protrusions (MU, 610744) can be covered with 1 Orna/ti. goat to Es to human in sodium carbonate buffer overnight at 4"C. Plates are washed and blocked 195

VZA-RV5 for 1 hour at 37"C and can be used immediately or frozen at -20"C.

Undiluted hybridoma supernatants are incubated in plates for one hour at 37°C. Plates are washed and tested with HER-labeled goat dos kappa to human diluted 1:10,000 in 1956 VBA-RV5 for one hour at 37°C. The plates were then incubated as described above.

Determination of the reactivity of fully human Anti-1Y -12

Hybridomas such as those described above may be simultaneously tested for reactivity to 1-12 using a KIA or other test. For example, supernatants are incubated with goat DCs to human plates as indicated above, washed and then tested with radiolabeled 1-12 with a countermeasure per well for one hour at CT. The wells were washed twice with RV5 and the bound radiolabeled 1-12 was counted with an appropriate counter.

Hybridomas that secreted anti-II-12 Go! human can be developed in cell culture and serially subcloned in limiting dilution. The resulting clonal populations can be expanded and cryopreserved in a freezing environment (9595 ЕВ5, 5956 ОМ5О) and preserved in liquid nitrogen.

Isotyping

Antibody isotype determination can be performed using EIA in a manner similar to that used to screen mouse immune sera for specific titers. I--12 can be applied to 96-well plates as described above and purified antibodies 2dd/ti. can be incubated on plates for one hour under CT. Plates are washed and tested by goats and humans

HER with labeled goat IdOZ to humans diluted 1:4000 in 195 VBA-RV5Z for one hour at

ET. The plate is washed again and incubated with the substrate solution as shown above.

Binding kinetics of human antibodies to 1-12 human from 1-12 human

Antibody binding characteristics can be reasonably assessed using, for example, 1-12 capture of EIA and ViAsog esppoIod. Gradual concentrations of purified antibodies up to 1-12 people can be tested for binding on EIIA plates coated with 2rdad/ti. antibodies I-12 according to the method described above.

Optical density can be represented as a semi-logarithmic plot that reflects the relative efficiency of binding.

A quantitative binding constant can be obtained, for example, as shown below, or by any other suitable method. The ViAsoge CM-5 (carboxymethyl) chip is placed in ViAsoge 2000. HB5 buffer (0.01M

NEREZ, 0.15 M Mass, ZtM EOTA, 0.005965 m/m P2O surfactant, pH 7.4) is passed through the flow of chip cells at a rate of bni/min until the initial level is reached. A solution (10041) of 5td ES (M-ethyl-M'-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride) in 200 ml of water is added to 100! of a solution of 2.3 ml of MH (M-hydroxysuccinimide) in 200 ml of water. Forty (40) shi! of the obtained solution is injected onto the chip. Sixth solution

I--12 of a person (15dd/ti. in 10tM sodium acetate, pH 4.8) is injected on the chip, which leads to an increase in sa. 500 KO. The buffer is replaced with TV5/Ca/Ma/B5A flow buffer (20tM Tris, 0.15M sodium chloride, 27M calcium chloride, 27M magnesium acetate, 0.595 Tgtyop X-100, 25na/t!. VZA, pH 7.4) and flows through the chip overnight to equilibrate it and hydrolyze or block any unreacted succinimide esters.

Antibodies are diluted in flow buffer at 33.33, 16.67, 8.33 and 4.17 pM. The flow rate is adjusted to ZOdi/min, and the temperature of the device to 25"С. Two flow cells are used for kinetic studies, one of which was immobilized I/-12 (sample), and the other was pure. At 12041 of each concentration, the antibody was injected with a cell flow of 30/min (association phase), which was performed with a continuous flow of Zb0Osec buffer (dissociation phase).The surface of the chip was regenerated (dissociated Interleukin-12/antibody complex) by two consecutive applications of Zbdi 2M guanidine thiocyanate.

Data analysis was performed using VIA 3.0 or SI AMP 2.0 evaluation, as known in the art.

For each antibody concentration, pure sensograms were subtracted from sample sensograms. Global alignments were performed for both dissociations (Ka zes") and associations (Ka to!" ves") and the dissociation constant (Ko, toi) (Ka/ka) was determined. Where the affinity of the antibodies was so high that the captured KO of antibodies were »100, additional dilutions of antibodies were carried out.

Results and discussion

Production of 1-12 human monoclonal antibodies

Several fusions were performed and each fusion was seeded in 15 plates (1440 wells/fusion), yielding several dozen antibodies to 1-12 individuals. Of these, some were found to have human and mouse Id chains.

All other hybridoma secretions of anti-1I--12 antibodies contained only human heavy and light chains. All human hybridomas were supposed to be Id.

Binding kinetics of antibodies against 1-12 people

Analysis of EHIZBA confirmed that the purified antibodies of the majority of these hybrids bind I/-12 in a concentration-dependent manner. Figures 1-2 demonstrate the results of the relative binding efficiency of these antibodies. In this case, the avidity of antibodies to their related antigen (epitope) was measured. It should be noted that binding of 1//-12 directly to EIA plates may be caused by denaturation of the protein, and such binding affinities may not reflect binding of non-denatured protein. 1595 binding was observed in a range of concentrations.

The numerical binding constant of the human antibodies was obtained using the ViAsoge assay and showed that some human monoclonal antibodies had very high affinity with K values ranging from 1x10 to 7x10-12,

Conclusion

Some fusions were performed using spleen cells from hybrid mice that had been immunized with II. - 12 people who had transgenes of variable and constant regions of antibodies. A set of monoclonal antibodies reactive (TO) to fully human 1-12 antibodies of the isotype (91. Fully human anti-1II-12 human antibodies will be described below. Some of the obtained antibodies had affinity constants from 1x109 apa to 9x1012. Unexpectedly high affinities of these completely of human monoclonal antibodies make them suitable for therapeutic use in diseases, pathologies or similar conditions associated with 1I--12.

Example 3: C340 neutralizes human monoclonal antibodies

It has been shown that the biological activity of 1-12 can be neutralized in various tests related to the activity of 1-12. Since I--12 enhances the production of IEM SAMMA by MK-cells and T-lymphocytes, the effect of C340 antibodies on the up-regulation of IEM SAMMA and the effect of C340 antibodies on the production of the IEM SAMMA protein were studied. -23 (1997), Mogtiv, 5. S, vy ai., doshpai! oi Ittypoiodu, 152: 1047-1056 (1994)). The ability of C340 to neutralize I--12-directed induction of killer cells activated by lymphokine (GAK) was also investigated in these works |Kiiga, U. apa Mygazko, 0. M.,

Mespapivtv oi Adeipd apa OyemeIortepi, 90: 209-222 (1996), 5ieet, A. 5., eyi a)., Rgoseedipdz oi She Mayopai!

Asadetu oi bZsiepse5 oi ilipe u. 5. A., 87: 6308-6812 (1990)). In conclusion, the effect of C340 on the up-regulation of cell surface expression of CO95, mediated by II, was tested on T- and MK- cells. -12.

IMedmedem, A.E., ейі аі!., Suiokipe, 9: 394404 (1997)|.

Inhibition of transcription THEME IEM datta

A reverse transcription PCR analysis was performed to determine whether C340 inhibits 11-12 transcription of the IEM SAMMA gene induced by 1--2 in RVI. a person Specific p-actin primers (control of tEMA integrity and content) and SAMMA IEM were used to amplify SOMA obtained from stimulated human RVI. Figure C shows that C340 negatively regulates IEM SAMMA tEMA in RVMO activated 1--12/1I -2 (2 hours).

Inhibition of intracellular IEM by SAMMA was determined by flow cytometry

In response to various signals and as an indicator of activation, T- and MK-cells can be induced to secrete cytokines. In more detail, RVI., processed 1-2 and 1-12 initiate significant production of IEM SAMMA during 4-8 hours after stimulation. Production in the cytoplasm can be determined by flow cytometry of RVI treated with VheteIdip-A. Figure 4 shows the 6095 decrease in IEM SAMMA production in cultures where C340 II -12 was added in combination with 1-12 for five hours.

Inhibition of IEM SAMMA secretion induced by 1-12

Figure 5 clearly shows that two different samples of C340 inhibit the secretion of IEM SAMMA by peripheral blood lymphocytes in a dose-dependent manner. 400 picograms were added to various amounts of C340 and then added to the RVI culture stimulated 1-12. when IEM SAMMA was determined using EIA after 18-24 hours of incubation, a significant decrease in the number of IEM SAMMA with C340 antibody content of less than 1 ?d/ti was found.

Inhibition of cytotoxicity of AK-cells induced by 1-12

Kai cells), a cell line sensitive to 1-12, derived from Burkitt's lymphoma, which are resistant MK cells. Cell lines are sensitive to HAC cells. Kaia cells, in triplet. It was cultured for four hours with HAC cells, which were activated 400 rda/ti. 1-12 and 10SHO/t/. 1-2 in the presence or absence of human monoclonal antibodies C340 (5000pad/ti. og 50pd/t/). Figure 6 shows the results from those normal healthy donors. Activation of 1-12--II-2 together, effector cells led to an increase in cytotoxic activity more than activation of 1I--2 alone. C340 antibodies inhibited this 1I--12-dependent effect. The power of enhancement depended on the concentration of antibodies, at the highest concentration cytotoxicity returned to background values.

Inhibition of regulation of C0U5 stimulation

The report describes the up-regulation of CO95 on the surface of highly purified CO56-RVI. As can be seen in Figures 7A and 7B, distributive flow cytometric analysis revealed that the expression of CO95 was significantly stimulated on COZ T cells and apa CO56-MK cells after their treatment 1-12 plus 1-2 within 72 hours. Co-treatment with anti-1Й-12 inhibited the expression of СОО95 both in SOZ- and in СО5b populations.

COX cells were inhibited by 5095, while CO56 - was inhibited by 8595 (Figure 7B), as confirmed by the MPI index (percentage greater than unstimulated control).

Example 4: Gene cloning and identification

Genomic fragments of OMA containing the C340 heavy chain or C340 light chain gene were cloned and purified. The OMA genome purified from a hybrid of C340 cells was partially treated with the restriction enzyme zaizA and separated by size by centrifugation in a 10-4095 sucrose gradient. OMA fragments with sizes in the range of 15-23 Kr were cloned into the bacteriophage vector, EMVI 3, (commercially available 7) and packaged into bacteriophage particles. Some 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 OMA fragments that included either the human IDS1 heavy chain constant region sequence or the human Carr light chain constant region sequence as a template.

Thirteen heavy chain and nine light chain clones were found. Of these, three heavy chain clones and two light chain clones were isolated by an additional two rounds of screening. By means of RSC analysis of bacteriophage OMA, it was shown that one of the clones of the heavy chain and two clones of the light chain contained the 5' and 3' ends of the coding sequence. The OMA insert in the HA clone of the heavy chain (HC) was 16 Kb in size and included 3.6 Kb of the 5'-terminal and at least 2 Kb of the 3'-terminal sequence. The OMA insertion in the NA light chain clone (I C) was 15 Kb in size and included 4.4 Kb of the 5'-end and at least 6.OKb of the 3'-end sequence. The inserts were completely removed from the bacteriophage vector as ZaiII fragments and cloned between the sites of Khpoi! and Zai! expression vector of plasmid p1351, which has a selective marker gene ori. Because they were external ZI sites in the sequence encoding the variable region of the heavy chain, the two

ZaiII fragments had to be transferred from bacteriophage NA to the expression vector ri351. The resulting heavy chain and light chain expression plasmids were named rib5b60O and rib558, respectively. The orientation of heavy and light chain genes in these two plasmids relative to the sequence of the ri351 vector was determined by restriction enzyme analysis and RCE, respectively. In both cases, the orientation was such that the 5-end of the AB gene was close to the 3'-end of the dri gene. Both directions of the coding regions of the cloned genes were sequenced. The sequences of plasmids ri560 and riI558 are presented in Figures 11A-11K and Figures 13A-13, respectively.

Example 5: Production of recombinant cell lines

Plasmid ri560 of the heavy chain was linearized by treatment with the restriction enzyme Ruci and plasmid ri558 was linearized by treatment with the restriction enzyme Za. rahb3zA9d8. 653 (653) and 5P2/0-Adia (5P2/O0) cells were separately transfected by adding linearized plasmids by electroporation, cells were cultured, and transfectants were selected using mycophenolic acid as described in

IKpidni, ей аЙ. Moiiesshag Ittypoiodu 30: 1443 (1993)). Cell supernatants of X colonies resistant to mycophenolic acid were tested for two weeks against human (e.g., recombinant C340 (pi5340)). For this, cell supernatants were incubated in 96-well EHIBA plates, which were coated with goat antibodies specific for E part of human Db. The human Db bound to the coated plates was determined using alkaline phosphatase conjugated with goat anti-human antibodies (heavy chain-light chain) and alkaline phosphatase substrate as described by IKpidni, ei a!., Moiesshag

Ittipoiiodu 30: 1443 (1993)). Cells from highly producing clones were transferred to 24-well culture dishes in standard medium and grown (selective mixture of IMOM, 595 EB5, 2tM glutamine, mycophenolic acid).

The amount of antibodies produced (eg, secreted into the medium of spent cultures) was accurately counted using EHIZA, using C340 tAbB as a standard. The selected clones were then expanded in 175 flasks and the human production of these clones was counted using EGIS.

Based on these values, six independent 653 transfectants and three independent 5P2/0 transfectants were subcloned (by plating an average of one cell per well in 96b-well plates), the number of antibodies produced by the subclones was calculated by EHI5A analysis of supernatants from individual subclones colonies. Three subclones were selected: transfectant 653 19-20 (С3798) and transfectant ЗР2/О 84-81 (С3814А) and 22-56 (СЗ89А), for further analysis.

Analysis of hS340 antigen binding

Before subcloning the selected cell lines as described above, cell supernatants from three parental lines (transfectant 653 clone 2 and clone 18 and transfectant 5P2/0 clone 1) were used for the hC340 binding test. First, the concentrations of C340 in three supernatant samples were determined using EGIS. Titrated amounts of supernatant samples, or purified C340 control, were then incubated in 96-well plates coated with 2 na/t! I/-12 humans. TAr binding was then detected using alkaline phosphatase conjugated with anti-human antibodies (heavy chain and light chain) and the corresponding alkaline phosphatase substrate. As shown in Figure 8, "2340 specifically binds to human 1-12 in the same way as the original C340 tAb.

Identification of selected cell lines

Growth curve analysis was performed on C3798, CZ814A, and CZ89A, by seeding in 175 flasks with an initial cell density of 2X105 cells/t!I in standard medium or serum-free ZEM-5 medium and subsequent daily monitoring of cell number and /Cb340 concentration until cell exhaustion.

The results of cultures on standard medium are presented in Figures 9A-96.

The maximum production levels of C340 tA for C379B, C381A, and SZ89A were 135na/ti, 150rna/ti, and 110dd/ti, respectively. Attempts to adapt C379B cells to 5EM-5 medium were unsuccessful. Cells

C381A produced the same amount of "C340" in the ZEM-5 medium as in the standard medium.

The stability of production of 27340 tA over time for the three subclones was evaluated by cultivation in 24-well plates with standard medium or with standard medium without mycophenolic acid selection at different time periods. Lines C379B and C381A showed stable production of /gC340 in the presence and absence of the selector for 30 days (maximum testing time) and 75 days, respectively.The SZ89A line was unstable and after 43 days of culture produced only a maximum of 2095 antibodies from the start of the study.

It becomes clear that the invention can be implemented in a different way than that partially described in the given description and examples.

Numerous modifications and variants of this invention are possible in the light of the above, as well as within the limits of the given formula.

. LIST OF SEQUENCES 'IN: Shealy, David: Knight, David; Skellon, Bernie; Gotes-Komar, Gilya; Perritt, David "izhe ANTI 12 ANTIBODIES, COMPOSITIONS, METHODS AND USES "1305 SEMO45 "1650" 15 "170" Rhea et 70 "10 1 "B "10 RET "Tiz" Noscho variere "Ac 1

Ti Tuk Yar Ty (U 1 E "5" "i" Ii "I" RET "132 Moto vareov "A

Pe Meeeg Rto UZ Avr bat zr Tse ge Tut Cheh Ro Meh Rre sny 1 5 ni 15 su "210" 3 I "ih yuYu "Zm RET "23" Noshsche zariyene "Ak 3

Dec Av to Su Siv Su Tuk Riv Ayar BV 1st 5 16 "oh "11" her "17" REG "21325 Bosho jam "oh 4

At Ayiv beh Spa ZIU Pe bek Zag tr Gay Av i 5 yuyu "10" 5 "I 7 "rii" VVE "213" Nopo zaristce "dO 5

Av Aza Beh Zeh Tse ip 5eh 1 5 "10 6 "I" 9 - "122" RET "2132 Noto vapivva "A" 6

Siv Siyu Tuh Ap De Kuk Rgo Tut TE 1 5 oh 7 "from Neo "12" VRET "from Noto vyvisvu "hell" 7

Sv Uvi siv ey Uvi Sp et Su Ada On Ua! Tuya Buv Ro ZU S

H 5 10 15

Bek Gay Was Pe bet Sub uz Su Zeg Su Tut Beh Rode Te TV Tuh

CI

Teria U Ter Ua Ate siv Me Rgo SPU uz Su Gen Ayar Ter Te 33 40 45

Ou Ce Mas bst Rgo Uvi Avr Zeh Ar Pe Ahu Tukh Beh Rio et Ve 50 55 50 siv slu in Ua! TE Mei Beh Uvi Ayar eats Zeh Tse Tk TOK Av Tu 55 70 7 80

Soch Zi Ter Asp Beg ii Guz Ada et Ar Tv AV Me tuyu Tukh Suk 55 59 95

Av At Ahe Atu Rio pu biv Su Tut Re der Te Tour LU Sv STU 100 105 th

Tvsisi uv is Mi God Veg 115 "MO" 8 "ih 108 "ie РЖ "3" Noto zariepe "oh

Avr ti biv Me Tvt bir bek ho bek dec pep Tower Aza bek in SI1uU 1 a 160 i8 dAvr Akta Uuai TAZH I1e Tk Su AkYa Vin Beh Zir Ziu yiiye Chek yeh Thx» 20 28 zo

Bema vla Ter tuh biv zip buye Rho Zi" puv Aza Rko Buv beh Pvy her "0 i5

TUuh viv Aiv bek bek iss siv bek so1u Uaii Ro Zek goes Vb Veh 01u va 55 I-1

Bek SI1U Bak biu TI dDlr Re Tk Gay Tk iZa bah bah Pec beep Bo 65 70 75 na

Фио дар о рРВе Аза Тих Тук Тук бул сип бип Тук Ап и1и6 Тут РуОо Тух 85 20 Св

So de piu pip Tu TVh Puye this biz y3Ze Guv Ah 109 105 "2102 a "i" 303 "12" RET "3". Nosho zariyeni «oh In the entrance of two objects ko Uuai Aza Ts Rtgo Agro Echo biu Me BBe rho Suv Pe ii 5 What i8

BKiv Niv beh Zip AB by gez Aka Aza wai Bek dvy Mes Pe ip Muv 2 25 30

Aa Ah bij Tak ope) ziy ne tTuk Ro Suv TVZH Beh biz zZiz iTe Dar

ZE 35 45

Niv biz der i1e Tps buz Avr o Buv Tak Beh Tzhh Ua 91 Aza Suv bech vo 25 BO ho ey 41 beb o oylk was Vvp osi bat was Bey dvo bek Ah biz Tv. 70 75 80

Zek Rpe iLe TBE Avposiu Beh Su Pec Aiia Beh Ake was Tak Bek Wde va 98 55

Mes Men o diva Bey Sub Yineh Zezh bek Iife TUkK ich ber rev Buv mezh Tuk a 105 115 biv Chvi si vBe puz Tk Meb Avp Aiya Buv ped bez Meb Avrorho Puv 115 120 125 vga bid I1e Ve peo dyar biv Avi Mes yinmi Aza Uai That gift 3 this

Zhao i35 134

Mei siv va Bey dvp ore Av Beh Siy Ta Mai ko ila Buv dec bej 145 150 iii58 160

Betsa biy What ho dr o Rve Tuh Muv Tnj Buv Her Muv pey Sun Ti Bei yi55 29 75 yiv Niya deya Red dku IT1e Aku dia uai lpzh T1ie Avno yku uni Meb bee zab iv 185 tTuk ivu dey Aia Bek yZe Thr sich Bey Buv tsuv Avrouai Tuk chai Chai 155 250 898 biz ieva AvroTkrotuh Ro Avrodiz vho Ziu biv mes UuUaii Mai pep Tu 210 215 Neyai spuya vyv TVt bks vich ich Avrosiu T1z tTrh tTcroTat ei vro bip bek aa av 235 29 bej Yaii Mai iv) 01U beh p1u Buv Those Pe TK i1e Failure Ua) pub I:

245,850,255

Zme 01u Avr odiz 01iu 515 Tuh ZhBh Sub niv puvstu 01uU siy uai yiv 285 265 270

Bek Viv Beh Pe) ev rey Be) niv Ppuye puz bi dia» Ziu hiv tTzhv Beh 275 4385 285

So dnr her paz was Avroziv was her ko Mus Av in Jacques Ve ikh 235 285 305

Aka Suv sish Aza tsuv dev Tut Beh Siu Aku Re TNH Sue Ter Tr rey zov zlo ziy za tTvh tah iIZe bau TVzhJ o Abr obez tre Rya bek Chai Buv beh Beh Aka BiuU z25 Za z dec bek der rko bip 0iUu Uvi TVh Suv piu Via Aza lu yvi bek ia 340 za5 zo

Z dk wai vu B3iu Avr oda pu uch tTuk biz tTuh Vezh uzi s Mi Sun 355 360 z55 sii biz dar Beh Aza Suv Bo dia Aza 1 biz bek bey ro T1e Pich 170 375 380 chai Me chai ABr dia Chai Niv Buv Pec buye Tukh b) lad Tukh residential complex bek 385 394 395 oo

Bah Be Na 1128 DK Avoroiie ti1e Buv RE Avrorho rt Ppuv dvy yiv 05 410 415 «ii vch bu RO ek Buya Av) bek Aho ip uai z Uvu bej Thr b zha zhe 430

Tuk Rgo Avr otu Tkr Beh Tnh Bho yid Beh Tu RBa bak ey Tpzh Ve «35 «40 448

Suv in 10 Uvi biz z tsu Zeg Bu Aha Zyy was Puv Avo Jo chai 455 455 chei vne TI Av tsuv Tph Bek Atia TBh Zaz her was hu due dvvoAiz bek "v ato "78 ad yiye Veh Uai Aho Aia ziv Avr o Ahoa Tuk Tut Beh Beh beh Thr Beh 1 "85 439 2955 tkr'Aia Beh Maii Rko Sub vah 50o0 "0: yuYu "i" i" mih DNA "1" KNolo zarikaya "Ae 1 vevivizsia Sas 15 "IN 1 "1" 2135 DNA

"13" "Noto varietya "nah Li rova sa anar pzzhvanae vsvianasi ssetovannv s with "210 15 "211" 30 "2122 DNA "215" Noscho zarievh "shyu" kevnssovte pase s Nyrias Ko "re 13 "I" 33 "o" DNA "213" Nato wabieye "ah 3 power vevssaris rarivtsvre performances; ss 33 "Zh y "4 21 "312" DNA "from /Nomyu varikoye "ayu Matesvisoh asavokse 18 "Zh 15 "aBim 27 "22" DNA "132: Noto zvrishiv "this 5 saksadsvia yasavsivysoi 21 25 . take -О341 2.0 ес: ЛЯ --02 5340 n- o А | --C2333 x "a sya" 0883 x y

E zv. more: - 331

From ke F --0330 from V Sha CHI: --0329 0.5. with B on 00 OT nya 10.000 .000 0190 0010 v.O0

Code of micro ml of purified mats

Fig. A oi 4 and -- SB

SAME zo --- sv fri and mon a 05 Sat dol -ee:

OR 0о500 00050 00005

Antibody concentration (μg/ml)

Fig. 18

GU g

E 8 : 2 E i g h a a what t 5 - t SHE

Uk same: i

Ж хх а 8 E Е ч я З - Ге щ

M o

Se oh re! appeared ze zha

KK Kt 5 Shnny

TC Z Shshchiy Y

Ukr uve o: 00 tova oEatv BUSHNY

ELECT or KV yavy ty i shly lu, NN 4

In kN! Chi ik sawdust ZDA p i i NN z o i V i UT vkros lesynh ti i supy B ser fl vit nn Y ;

Fig, 2A 5 E gZ i

J. Yu

« 2 and 4 E g ki - E ii

ВХ is вх т ш Х х ж Х Х Х xx y y y 5

ZE g. Z c - V z 5

KUN and about Ya

ON

PO p h k "ey soda vane nu a M EN

TEKY st VN sum equ ne ekt o z SHY and m

Ben and

Sha yk and zd ya: kn - yaYah ad «ra u

Sht tu st KI M

Op en

TOK, Av

PC ket ikh hi zhh NI ovo pi a sa I

B hi SHE blya ev nar AV:

Fig. 28

Donor 141 Ponor 1457 pev OVA o PO VN OK Ks V doby i zve ni o MK A San i en an a i KB Vede pane v NA Vash i NN s

In the University of Econ at I. Kontrol

GIVEN o KRU SO VC KV EN I 2. chx 3. A pvh put to VN sia OD i. certain

In OM, I am not yet V

PO Ah PEN VN at 5. PAN. 126340 vna r A ov 6. PAS a o Va i o SY tk m ia o ov (isotype control for SZ340 sen nya VZya p t. oon tvo o pny KN NUK already Mi, E - sha in Zh. SONATA women in isotype control for 8.8.2) and ich ti P ! mon KI o r KE KI shcha NE

Fig. From

With Fr. - x

E. And what in vo

In 8 gYa gZ 70

X in the same

Zh tv h eE 1 h shcha 8 zv ko 20 r vo o ih same v pan MY

MALI PAT -2 -C340

Characteristics of cultures. .

Fig. 4

"Vka. : i ZO So u - ag 3064 kA u, In zksoy CDnE

E zaze zov Bek chne ef zh y ny ov ly jo yah oyu vv as du» cho vh zh chik oyu zhi with his same where and in

In ; From the city, from the city of R

Wow. :

Ey ; sh ay navan i i

Antibody concentration (pgmp)

Fig. 5

5000 Ng

SHABO -chns/mya - "0

B

Ge i x z 30 e - o zy

B. ZY 1035. about Her Kk "And Dy

Fig. 6

In d

Bu 2 sho ve i a 1 | IA t: Shizh oStimo 340 AV

I from xx et: 25 8 o

EK oo. 55 chess 7 55

I am THEIR

Ge g 5

ZE 9

Characteristics

Fig. 7A

B

5 w

E zo. ha in

E E 375

Not in B.

Did you sleep well? up to 25 9 pieces from SZAb and A

It is there

EE with more

IN

Ex at 50

EV x e Ex

VE Characteristics

Fig. 78

And -- cleansing; (5340 va --5B5Z clone 2 078 --653 clone 18 a -su- BROYU "clone Z ii a56 025 ao uperta : ! tp taz tal 102 KTeV maxima 1340

Fig. 8 sha | 160 dreams of KliTINIMA c379v 140 micrograms/ml of oil 126 2 zo

It is food

With | 80 5. okcho ; b

Contact 01238567 8 o 01119134

Dniv Y

Fig. FOR "Thesis. Zo and 140 and Ido. And 420

With : 100

B zomoz b E m y okho t okho : nt ; 1234887 8 8z01119544

days

Fig. 98

ZDO ment tot tttttn zvo. Kpitin/ml

SZ8ZA zia0 007 μg/mi 1. Bkidrya-i2a

And that one is -8. r 5 1010 a E a 50 y i I v oo nt - homo y t 12345579 101112153154

days

Fig. 90

Claims (6)

1. An isolated human monoclonal antibody that binds to I/-12 having a heavy chain variable region containing 5EO IU MO: 7 and a light chain variable region containing 5EO IU MO: 8. 2. An isolated nucleic acid that encodes an isolated human monoclonal antibody that binds to IL-12 having a heavy chain variable region containing HEO IO MO: 7 and a light chain variable region containing cBEO IU MO: 8 . 3. A prokaryotic or eukaryotic host cell containing the isolated nucleic acid according to claim 2. 4. A pharmaceutical composition that contains an isolated human monoclonal antibody that binds to IL-12, which has a heavy chain variable region containing ZEO IU MO: 7 and a light chain variable region containing ZEO IUO MO: 8, and at least one pharmaceutically acceptable carrier or diluent. 5. A method of diagnosing conditions related to 1-12, which includes contacting a composition that contains an effective amount of an isolated human monoclonal antibody that binds to IL-12, which has a heavy chain variable region containing ZEO IU MO: 7, and the variable region of the light chain containing the GEO IO MO: 8, with the material under study. 6. A method of treating conditions associated with II-12, comprising administering to a patient in need thereof a composition comprising an effective amount of an isolated human monoclonal antibody that binds to II-12 having a heavy chain variable region , containing 5EO IC MO: 7, and the variable region of the light chain containing HEO IU MO: 8.