EA040272B1 - TREATMENT OF PSORIASIS WITH ANTIBODIES TO IL-12 AND/OR IL-23 WITH INCREASING THE INTERVAL BETWEEN DOSING - Google Patents

Description

Scope of the invention

The present invention relates to methods for treating an IL-12/23 related disease with an antibody that binds to human IL-12 and/or human IL-23 proteins using specific administration regimens. In particular, it relates to the determination of an increasing dose interval (or maintenance dose) for subcutaneous administration of an anti-IL-12/23p40 antibody and specific pharmaceutical formulations of the antibody, such as ustekinumab, that are safe and effective for administration to patients with an IL-related disease. -12/23.

Prerequisites for the creation of the invention

Interleukin (IL)-12 is a secreted heterodimeric cytokine consisting of 2 disulfide-linked glycosylated protein subunits, designated p35 and p40 according to approximate molecular weights. IL-12 is produced primarily by antigen-presenting cells and stimulates cell-mediated immunity by binding to a double-stranded receptor complex that is expressed on the surface of T cells or natural killer (NK) cells. The beta-1 chain of the IL-12 receptor (IL-12Rβ1) binds to the p40 subunit of IL-12, providing the primary interaction between IL-12 and its receptor. However, it is the binding of IL-12p35 to the second strand of the receptor, IL-12Rβ2, that excites an intracellular signal (eg, STAT4 phosphorylation) and activation of the receptor-bearing cell (Presky et al., 1996). It is believed that IL-12 signaling, which occurs simultaneously with antigen presentation, causes T-cell differentiation to the T-helper 1 (Th1) phenotype, characterized by the production of gamma interferon (IFNy) (Trinchieri, 2003). Th1 cells are believed to stimulate immunity to certain intracellular pathogens, generate isotypes of complement-fixing antibodies, and participate in tumor immune surveillance. Thus, IL-12 is considered to be an important component of the host's immune defense mechanisms.

It has been found that the p40 protein subunit of IL-12 can also combine with a separate protein subunit, designated p19, to form a new cytokine, IL-23 (Oppman et al, 2000). IL-23 signaling is also carried out through a double-stranded receptor complex. Since the p40 subunit is shared by IL-12 and IL-23, therefore the Pffl-12R.pi chain is also shared by IL-12 and IL-23. However, it is the binding of IL-23p19 to the second component of the IL-23 receptor complex, Hn-23R, that excites a specific intracellular IL-23 signal (eg, STAT3 phosphorylation) and subsequent production of IL-17T cells (Parham et al., 2002; Aggarwal et al. 2003). Studies have shown that the biological functions of IL-23 differ from those of IL-12, despite the structural similarity between the two cytokines (Langrish et al., 2005) .

Abnormal regulation of IL-12 and Th1 cell populations has been associated with many immune-mediated diseases, as neutralization of IL-12 by antibodies is effective in treating animal models of psoriasis, multiple sclerosis (MS), rheumatoid arthritis, inflammatory bowel disease, insulin-dependent diabetes mellitus (1st type) and uveitis (Leonard et al., 1995; Hong et al., 1999; Malfait et al., 1998; Davidson et al., 1998). However, since these studies targeted the common p40 subunit, neutralization of both IL-12 and IL-23 occurred in vivo. Therefore, it was not clear which of the two cytokines: IL-12 or IL-23 mediated the disease, and whether it is necessary to inhibit both cytokines in order to achieve suppression of the disease. Additional studies in mice deficient in IL-23p19 or with neutralization of IL-2 3 specific antibodies confirmed that inhibition of IL-23 can provide a positive result equivalent to anti-IL-12p40 strategies (Cua et al., 2003, Murphy et al., 2003 , Benson et al., 2004). Thus, there is evidence for a role for IL-12 and IL-23 in immune-mediated disease.

Psoriasis is a chronic immune system-mediated skin disorder with significant comorbidities such as psoriatic arthritis (PsA), depression, cardiovascular disease, hypertension, obesity, diabetes mellitus, metabolic syndrome, and Crohn's disease. Plaque psoriasis is the most common form of the disease and presents as well-circumscribed erythematous lesions covered with silvery-white scales. The plaques are itchy, painful, and often disfiguring, with a significant proportion of psoriasis patients having plaques on the hands/nails, face, feet, and genitals. Thus, psoriasis can become a physical and psychosocial burden that goes beyond physical dermatological symptoms and interferes with daily activities. For example, psoriasis negatively affects family, marital, social and work relationships, is associated with an increased incidence of depression and an increase in suicidal tendencies.

Histological characterization of psoriatic lesions reveals a thickened epidermis resulting from impaired proliferation and differentiation of keratocytes, as well as infiltration through the dermis and co-localization of CD3+ T lymphocytes and dendritic cells. Although the etiology of psoriasis has not been fully defined, gene and protein analyzes have shown that IL-12, IL-23 and their successors are overexpressed in psoriatic lesions, and some may correlate with the severity of psoriasis. Some therapies used to treat psoriasis

- 1 040272 modulate the levels of IL-12 and IL-23, which presumably contributes to their effectiveness. Th1 and Th17 cells can produce effector cytokines that induce the production of vasodilators, chemoattractants, and expression of adhesion molecules on endothelial cells, which in turn promote monocyte and neutrophil recruitment, T cell infiltration, neovascularization, and keratocyte activation and hyperplasia. Activated keratocytes can produce chemoattractant factors that stimulate the targeted migration of neutrophils, monocytes, T cells, and dendritic cells, thereby initiating an inflammatory cycle and hyperproliferation of keratocytes.

Three Phase 3 clinical trials of the anti-IL-12/23 antibody, ustekinumab, have been published in the treatment of moderate to severe plaque psoriasis. Ustekinumab, given by subcutaneous injection at weeks 0 and 4, and then once every 12 weeks, showed a rapid and sustained clinical response as measured by psoriasis surface area index and severity, an approved measure of efficacy in psoriasis. A phase 3 study compared ustekinumab with etanercept, a tumor necrosis factor (TNF) antagonist, and observed that in patients with moderate to severe psoriasis, ustekinumab was more effective than etanercept over a 12-week period. In two Phase 3 clinical trials, Phoenix I and Phoenix II, the half-life of ustekinumab was approximately 3 weeks. The frequency of immune response against ustekinumab was in the range of 3-5%. In addition, reported adverse events were relatively mild, with most events including susceptibility to mild infections such as nasopharyngitis and upper respiratory tract infections. During 12 weeks of therapy, the infection rate in patients treated with ustekinumab was no higher than the infection rate in patients treated with placebo; the incidence of infection did not increase with the introduction of higher doses of ustekinumab, compared with the introduction of low doses. In addition, the rates of serious infections, cardiovascular events, injection site reactions, and malignancies were low. Taken together, clinical observations of the use of ustekinumab for the treatment of psoriasis confirmed its first-in-class status and confirmed the fundamental role of IL-12 and/or IL-23 in the pathogenesis of psoriasis.

Statement of the Invention

In a first aspect, the invention relates to a method for treating an IL-12/23 related disease in a patient, comprising subcutaneously administering to the patient an anti-IL-12 and/or anti-IL-23 antibody, such as an anti-IL-12/23p40 (IL-12/ 23p40), wherein the anti-IL-12/23p40 antibody is administered at the initial dose, at a subsequent dose 4 weeks later, and at a dosing interval of once every 12 weeks, with this interval (maintenance interval) being increased 28 weeks after the initial dose. The extended dosing interval may be a set interval or vary based on when the patient experiences a relapse of the disease after discontinuation or an increase in the interval of administration of a therapeutic antibody, for example in psoriasis based on a change in the Global Psoriasis Score (PGA) score and/or the Psoriasis Spread and Severity Index (PASI). In one embodiment, the interval between dosing after 28 weeks is increased from once every 12 weeks to once every 16, 20 or 24 weeks at doses of 45 or 90 mg.

In one embodiment, the IL-12/23 associated disease is selected from the group consisting of psoriasis, psoriatic arthritis, lupus, diabetes mellitus, Crohn's disease, ulcerative colitis and other inflammatory bowel diseases, sarcoidosis, ankylosing spondylitis (AS), and axial spondyloarthritis (nrAxSpA). In a preferred embodiment, the disease associated with IL-12/23 is psoriasis. In another embodiment, the IL-12/23 associated disease is psoriatic arthritis.

The invention also relates to a method for the treatment of psoriasis in a patient, comprising subcutaneous administration of an antibody to IL-12/23p40 - ustekinumab (Stelara®) to the patient, wherein ustekinumab is administered at the initial dose, 4 weeks after the initial dose, with an interval between dosing once every 12 weeks to 28 weeks after initial dose, then administered once every 16, 20, or 24 weeks.

In addition, the composition used in the method of the present invention contains a pharmaceutical composition containing an antibody to IL-12/23p40 in an amount of from about 1.0 μg/ml to about 1000 mg/ml, specifically, a dose of 45 or 90 mg. In a preferred embodiment, the anti-IL12/23p40 antibody is ustekinumab (Stelara®). In another embodiment, the pharmaceutical composition contains an isolated anti-IL-12/IL-23p40 antibody that binds to a peptide chain containing residues 1-88 of SEQ ID NO: 9; from about 0.27 to about 0.80 mg L histidine per milliliter of pharmaceutical composition; about 0.69 to about 2.1 mg of L-histidine monohydrochloride monohydrate per milliliter of pharmaceutical composition; from about 0.02 to about 0.06 mg of polysorbate-80 per milliliter of pharmaceutical composition; and about 65 to about 87 mg of sucrose per milliliter of pharmaceutical composition; wherein the diluent is water in the standard state.

In another aspect of the invention, the pharmaceutical composition comprises an isolated anti-IL12/IL-23p40 antibody that has (i) the amino acid sequences of the heavy chain CDRs of SEQ ID NO: 1, SEQ

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ID NO: 2 and SEQ ID NO: 3 and (ii) the amino acid sequences of the light chain CDRs of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; about 0.27 to about 0.80 mg of L-histidine per milliliter of pharmaceutical composition; about 0.69 to about 2.1 mg of L-histidine monohydrochloride monohydrate per milliliter of pharmaceutical composition; from about 0.02 to about 0.06 mg of polysorbate-80 per milliliter of pharmaceutical composition; and about 65 to about 87 mg of sucrose per milliliter of pharmaceutical composition; wherein the diluent is water in the standard state.

Another aspect of the method of the present invention comprises administering a pharmaceutical composition comprising an isolated anti-IL-12/IL-23p40 antibody having the heavy chain variable region amino acid sequence of SEQ ID NO: 7 and the light chain variable region amino acid sequence of SEQ ID NO: 8; about 0.27 to about 0.80 mg of L-histidine per milliliter of pharmaceutical composition; about 0.69 to about 2.1 mg of L-histidine monohydrochloride monohydrate per milliliter of pharmaceutical composition; from about 0.02 to about 0.06 mg of polysorbate-80 per milliliter of pharmaceutical composition; and about 65 to about 87 mg of sucrose per milliliter of pharmaceutical composition; wherein the diluent is water in the standard state.

Another aspect of the method is the administration of a pharmaceutical composition comprising an isolated anti-IL-12/IL-23p40 antibody that has (i) the amino acid sequences of the heavy chain CDRs of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and ( ii) the amino acid sequences of the light chain CDRs of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; about 0.53 mg of L-histidine per milliliter of pharmaceutical composition; about 1.37 mg of L-histidine monohydrochloride monohydrate per milliliter of pharmaceutical composition; about 0.04 mg of polysorbate-80 per milliliter of pharmaceutical composition and about 76 mg of sucrose per milliliter of pharmaceutical composition; wherein the diluent is water in the standard state.

An additional aspect of the method is the administration of a pharmaceutical composition comprising an isolated anti-IL-12/IL-23p40 antibody having the heavy chain variable region amino acid sequence of SEQ ID NO: 7 and the light chain variable region amino acid sequence of SEQ ID NO: 8, wherein the isolated antibody binds to a peptide chain containing residues 1-88 with the sequence of SEQ ID NO: 9; about 0.53 mg of L-histidine per milliliter of pharmaceutical composition; about 1.37 mg of L-histidine monohydrochloride monohydrate per milliliter of pharmaceutical composition; about 0.04 mg of polysorbate-80 per milliliter of pharmaceutical composition and about 76 mg of sucrose per milliliter of pharmaceutical composition; wherein the diluent is water in the standard state.

In another aspect, the method comprises administering a pharmaceutical composition comprising a binding compound that competes for binding to the antibodies described above, optionally at residues 1-88 of SEQ ID NO: 9; about 0.27 to about 0.80 mg of L-histidine per milliliter of pharmaceutical composition; about 0.69 to about 2.1 mg of L-histidine monohydrochloride monohydrate per milliliter of pharmaceutical composition; from about 0.02 to about 0.06 mg of polysorbate-80 per milliliter of pharmaceutical composition and from about 65 to about 87 mg of sucrose per milliliter of pharmaceutical composition; wherein the diluent is water in the standard state.

Brief description of graphic materials

In FIG. 1 shows the molecular structure of the bound IL-12/p40 Fab complex, in ribbon representation.

In FIG. 2 shows the binding site (epitope) of mAb p40 present on the surface of the molecules, in surface and ribbon views. Domain D1 and Fv are shown outside the structure of the complex for clarity. The molecular surface of the D1 domain from p40 is shown. The Fv portion of the Fab fragment is shown as ribbons. Left panel: bottom view of the antibody binding site, i.e. epitope. Middle panel: ~90° angle view from left panel view. Right panel: Band representation of epitope remnants.

In FIG. 3 shows the result of an ELISA evaluation of the binding of an anti-IL-12p40 antibody to various p40 single mutants.

In FIG. 4 shows the relative binding affinity of the mAb for p40 with various p40 mutants.

In FIG. Figure 5 shows the design of ustekinumab, a phase 3b study in a randomized, double-blind, multicentre active-controlled study with a 4-week screening period, an open-label run-in period from week 0 to week 28, a double-blind treatment period from week 28 to week 104, post-treatment period to week 116, and follow-up for reliability by telephone or study center visit at week 124.

In FIG. 6 shows the percentage of subjects who achieved clearance (0) or minimal (1) on the PGA scale at visits from week 28 to week 112 in study CNTO1275PSO3009.

In FIG. 7 shows the percentage of subjects who achieved a PASI response of 75 at visits from week 28 to week 112 in study CNTO1275PSO3009.

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In FIG. 8 shows clearance (0) PGA responses over time from week 28 to week 112.

In FIG. 9 shows the percentage of subjects who achieved a 90 PASI response at visits from week 28 to week 112 in study CNTO1275PSO3009.

Detailed Description of the Preferred Embodiments

In the context of this document, a method for the treatment of psoriasis includes the introduction of isolated, recombinant and/or synthetic human antibodies to IL-12, IL-23 and IL-12/23p40, the use of diagnostic and therapeutic compositions, methods and devices.

As used herein, the terms anti-IL-12 antibody, anti-IL-23 antibody, anti-IL-12/23p40 antibody, anti-IL-12/23p40 antibody, antibody portion or fragment and/or antibody variant, and the like. include any molecule containing a protein or peptide that contains at least a portion of an immunoglobulin molecule, such as, without limitation, at least one complementarity determining (CDR) heavy or light chain region, or a ligand binding portion, a heavy chain variable region or a light chain, a heavy chain or light chain constant region, a framework region, or any portion thereof, or at least one portion of an IL-12 and/or IL23 receptor or binding protein that can be inserted into an antibody of the present invention. Optionally, such an antibody further acts on a specific ligand, for example, without limitation, such an antibody can modulate, decrease, increase, antagonize, agonist, decrease, attenuate, block, inhibit, eliminate and/or interfere with at least one IL activity or binding. -12/23, or activity or binding of the IL-12/23 receptor in vitro, in situ and/or in vivo. As a non-limiting example, a suitable anti-IL-12/23p40 antibody, defined portion or variant of the present invention may bind to at least one IL-12/23 molecule or defined portions, variants or domains thereof. A suitable anti-IL-12/23p40 antibody, a particular portion or variant thereof may also optionally affect at least one activity or function of IL-12/2 3 , for example, without limitation, RNA, DNA or protein synthesis, isolation of IL-12 /23, IL-12/23 receptor signal transduction, membrane cleavage of IL-12/23, IL-12/23 activity, production and/or synthesis of IL-12/2 3.

The term antibody is also intended to cover antibodies, their cleavage fragments, their specific parts and variants, including antibodies that mimic or contain parts of antibodies that mimic the structure and/or function of an antibody or a specific fragment or part thereof, including single-chain antibodies and their fragments. . Functional fragments include antigen-binding fragments that bind to mammalian IL-12/23. For example, the present invention encompasses antibody fragments capable of binding to IL-12/23, or portions thereof, including, but not limited to, Fab fragments (eg, after papain digestion), Fab' (eg, after pepsin digestion and partial reduction), and F (ab') ₂ (eg after pepsin digestion), Facb (eg after plasmin digestion), pFc' (eg after pepsin or plasmin digestion), Fd (eg after pepsin digestion, partial reduction and reaggregation), Fv or scFv (eg, molecular biology techniques) (see, eg, Colligan, Immunology, cited).

Such fragments can be produced by enzymatic cleavage, synthesis or recombination methods known in the art and/or described herein. Antibodies can also be produced in various truncated forms using antibody genes in which one or more stop codons have been inserted upstream of the natural termination site. For example, it is possible to create a combined gene encoding part of the F(ab') ₂ heavy chain that includes DNA sequences encoding the C _H 1 domain and/or the heavy chain hinge region. The different parts of the antibodies can be chemically combined by conventional methods or obtained as a single protein by genetic engineering.

As used herein, the term human antibody refers to an antibody in which essentially every portion of the protein (e.g., CDR, framework, C _L , CH domains (e.g., _CH 1, CH2, C _H 3 ), hinge, V _L domains, VH) is essentially non-immunogenic in humans, with only minor alterations or sequence variations. The human antibody can also be an antibody that is derived from or closely matches human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by germline immunoglobulin sequences (eg, mutations introduced by in vitro random or site-specific mutagenesis, or in vivo somatic mutation). Often this means that the human antibody is essentially non-immunogenic in humans. Human antibodies are classified into groups based on the similarity of their amino acid sequences. Thus, using a sequence similarity search, an antibody with a similar linear sequence can be selected as a template for generating a human antibody. Similarly, antibodies of a certain primate (monkey, baboon, chimpanzee, etc.), rodent (mouse, rat, rabbit, guinea pig, hamster, etc.) and other mammals are determined by the characteristics of antibodies of this species, subgenus, genus, subfamily and family . Additionally, chimeric antibodies may include any combination as described above. Such changes or variations optionally and preferably keep

- 4 040272 or weaken the immunogenicity in humans or other species relative to unmodified antibodies. Thus, a human antibody is distinct from a chimeric or humanized antibody.

It should be noted that a human antibody can be produced by an animal (excluding humans), or by a prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin genes (eg, heavy chain and/or light chain). Additionally, if the human antibody is a single chain antibody, it may contain a linker peptide that is not present in native human antibodies. For example, the Fv may contain a linker peptide, such as two to about eight glycine or other amino acid residues, that connects the heavy chain variable region and the light chain variable region. Such linker peptides are believed to be of human origin.

It is also possible to use bispecific, heterospecific, heteroconjugate or the like antibodies which are monoclonal, preferably human or humanized antibodies having binding specificities for at least two different antigens. In this case, one of the binding specificities is for at least one IL-12/23 protein and the other is for any other antigen. Methods for producing bispecific antibodies are known to those skilled in the art. Typically, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain/light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature 305:537 (1983)). Due to the random distribution of heavy and light chains of immunoglobulins, these hybridomas (quadromas) form a mixture of 10 different possible antibody molecules, with only one of them having the correct bispecific structure. Purification of the correct molecule, which is usually performed by affinity chromatography, is a rather laborious process with a low product yield. Аналогичные процедуры описаны, например, в WO 93/08829, патентах США № 6210668, 6193967, 6132992, 6106833, 6060285, 6037453, 6010902, 5989530, 5959084, 5959083, 5932448, 5833985, 5821333, 5807706, 5643759, 5601819, 5582996, 5496549 , 4676980, WO 91/00360, WO 92/00373, EP 03089, Traunecker et al., EMBO J. 10: 3655 (1991), Suresh et al., Methods in Enzymology 121: 210 (1986), each of which incorporated herein by reference in its entirety.

The anti-IL-12/23p40 antibodies (also referred to as anti-IL-12/23p40 antibodies) (or anti-IL23 antibodies) used in the methods and compositions of the present invention may optionally have high binding affinity for IL-12/23p40 (or IL -23), and optionally and preferably have low toxicity. In particular, an antibody, a specific fragment or variant of the invention can be used in the present invention, wherein the individual components such as the variable region, constant region and framework, alone and/or together, optionally and preferably have low immunogenicity. Antibodies that can be used in the present invention are not necessarily characterized by their ability to provide a therapeutic effect on patients for an extended period, with measurable symptomatic relief and low and/or acceptable toxicity. Low or tolerable immunogenicity and/or high affinity, as well as other acceptable properties, may help achieve therapeutic results. Low immunogenicity, as used herein, refers to inducing significant HANA, HACA, or HAMA antibody responses in less than about 75%, or preferably less than about 50% of treated patients, and/or inducing low titers in treated patients (less than about 300 , preferably less than about 100 as measured by dual antigen enzyme immunoassay) (see Elliott et al., Lancet 344: 1125-1127 (1994), which is incorporated herein by reference in its entirety). The term low immunogenicity can also be defined as the occurrence of titrated anti-IL-12 antibody levels in patients treated with anti-IL-12 antibody, occurring in less than 25% of treated patients, preferably in less than 10% of treated patients, with recommended dose during the recommended course of therapy during the treatment period.

Beneficial features

The isolated nucleic acids of the present invention can be used to produce at least one anti-IL-12/23p40 (or anti-IL-23) antibody, or a specific variant thereof, which can be used to determine the effect in a cell, tissue, organ, or animal ( including mammals and humans), to diagnose, monitor, modulate, treat, ameliorate, prevent the onset of, or reduce symptoms of at least one IL12/23 associated condition selected from, without limitation, at least one of an immune disorder or disease, a cardiovascular disorder or disease, an infectious, malignant and/or neurological disorder or disease, or other known or defined condition associated with IL-12/23.

Such a method may comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one anti-IL-12/23p40 (or anti-IL-23) antibody to a cell, tissue, organ, body of an animal or patient in need of such modulation, treatment, amelioration, prevention or reduction of symptoms, effects or mechanisms. An effective amount may comprise an amount of from about 0.001 to 500 mg/kg for single (eg, bolus), multiple or continuous administration, or to achieve a serum concentration of 0.015000 μg/ml for single, multiple or continuous administration, or be any effective range or value, as established and determined using the known methods described herein or known to specialists in the relevant fields.

Links

All publications or patents cited herein, whether specifically indicated or not, are incorporated herein by reference in their entirety as they indicate the state of the art at the time of the present invention and/or provide description and necessary information for the present invention. Publications include any scientific or patent publications, or any information available in any medium, including all recording, electronic and printed formats. The following sources are incorporated herein by reference in their entirety: Ausubel, et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: a Laboratory Manual, ^2nd Edition, Cold Spring Harbor, NY (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001).

Antibodies of the present invention.

Production and generation

The at least one anti-IL-12/23p40 (or anti-IL-23) antibody used in the method of the present invention may optionally be produced in a cell line, mixed cell line, immortal cell, or immortal cell clonal population, as is well known to those skilled in the art. this area. See, for example, Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: a Laboratory Manual, ^2nd Edition, Cold Spring Harbor, NY (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (19972001), each of which is incorporated herein by reference in its entirety.

A preferred anti-IL-12/23p40 antibody is ustekinumab (Stelara®), which has the heavy chain variable region amino acid sequence of SEQ ID NO: 7 and the light chain variable region amino acid sequence of SEQ ID NO: 8, and also has the heavy chain CDR amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; and the amino acid sequences of the light chain CDRs of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. A preferred anti-IL-23 antibody (which binds specifically to IL-23 but not IL-12) is guselcumab (also called CNTO1959, which contains the variable region sequences of SEQ ID NOS: 106 and 116, described in US patent No. 7935344, the contents of which are hereby incorporated by reference in its entirety) and other antibodies described in US patent No. 7935344.

Human antibodies specific for human IL-12/23p40 or IL-23 proteins or fragments thereof can be generated in response to a suitable immunogenic antigen, such as isolated IL12/23p40 protein, IL-23 protein and/or parts thereof (including synthetic molecules such as synthetic peptides). Other specific or general mammalian antibodies can be obtained in a similar manner. The preparation of immunogenic antigens and production of the monoclonal antibody can be performed by any suitable method.

In one approach, a hybridoma is produced by fusing an acceptable immortal cell line (e.g., a myeloma cell line such as, but not limited to, Sp2/0, Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, L243, P3X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U937, MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMALWA, NEURO 2A, etc., or heteromyelomas, their fusion products, or any cells or fusion cells derived from them, or any other acceptable cell line known in the art) (see, for example, www.atcc .org, www.lifetech.com., etc.), with antibody-producing cells such as, but not limited to, isolated or cloned spleen, peripheral blood, lymph, tonsil, or other immune cells, or cells with B -cells, or any other cell expressing constant or variable or framework sequences or heavy or light chain CDRs, either as an endogenous or heterologous nuc leic acid, in the form of recombinant or endogenous genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single-, double- or triple-stranded, hybridized, etc. or any combination thereof, derived from viruses, bacteria, algae, prokaryotes, amphibians, insects, reptiles, fish, mammals, rodents, horses, sheep, goats, rams, primates, eukaryotes. See, for example, Ausubel, cited and Colligan, Immunology, cited, Chapter 2, incorporated herein by reference in its entirety.

Antibody-producing cells can also be obtained from the peripheral blood, or preferably from the spleen or lymph nodes of a human or other acceptable animal that

- 6 040272 were immunized with the antigen of interest. Any other suitable host cell may also be used to express a heterologous or endogenous nucleic acid encoding an antibody, defined fragment, or variant thereof of the present invention. Fusion cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other known suitable methods, and cloned by limiting dilution, or cell sorting, or other known methods. Cells producing antibodies with the desired specificity can be selected using a suitable assay (eg, ELISA).

Other suitable methods for producing or isolating antibodies of the desired specificity may be used, including, without limitation, methods for selecting a recombinant antibody from a peptide or protein library (e.g., without limitation, display libraries of bacteriophages, ribosomes, oligonucleotides, RNA, cDNA, etc.; e.g. available from Cambridge antibody Technologies, Cambridgeshire, UK; MorphoSys, Martinsride/Planegg, Germany; Biovation, Aberdeen, Scotland, UK; Biolnvent, Lund, Sweden; Dyax Corp., Enzon, Affymax/Biosite; Xoma, Berkeley, CA , USA, Ixsys See e.g. /350260 (12.05.94); PCT/GB94/01422; PCT/GB 94/02662; PCT/GB 97/01835; (CAT/MRC); WO 90/14443; WO 90/14424; WO 90/14430; PCT /US 94/1234; WO 92/18619; WO 96/07754; (Scripps); WO 96/13583, WO 97/08320 (MorphoSys); WO 95/16027 (Biolnvent); WO 88/06630; WO90/3809 ( Dyax); US 4,704,692 (Enzon); PC T/US91/02989 (Affymax); WO89/06283; EP 371998; EP550400; (Xoma); EP 229046; PCT/US 91/07149 (Ixsys); or stochastically derived peptides or proteins - US 5723323, 5763192, 5814476, 5817483, 5824514, 5976862, WO 86/05803, EP 590689 (Ixsys, currently Applied Molecular Evolution (AME), all incorporated herein by reference)), or methods based on immunization of transgenic animals (e.g. SCID mice, see Nguyen et al., Microbiol. Immunol. 41: 901-907 (1997); Sandhu et al., Crit. Rev. Biotechnol. 16: 95- 118 (1996); Eren et al., Immunol. 93: 154-161 (1998, each publication is incorporated herein by reference in its entirety, as are related patents and applications), which are capable of producing a set of human antibodies known to those skilled in the art. this area and/or described in this document. Such methods include, without limitation, ribosome display (Hanes et al., Proc. Natl. Acad. Sci. USA, 94: 4937-4942 (May 1997); Hanes et al., Proc. Natl. Acad. Sci. USA, 95: 14130-14135 (Nov. 1998)); technology for the production of antibodies from a single cell (for example, the method for obtaining antibodies from selected lymphocytes (SLAM) (US patent No. 5,627,052, Wen et al., J. Immunol. 17: 887892 (1987); Babcook et al., Proc. Natl. Acad Sci. USA 93: 7843-7848 (1996)); microdroplet in gel and flow cytometry (Powell et al., Biotechnol. 8: 333-337 (1990); One Cell Systems, Cambridge, MA, USA; Gray et al., J. Imm. Meth. 182: 155-163 (1995); Kenny et al., Bio/Technol. 13: 787-790 (1995)); selection of B cells (Steenbakkers et al., Molec. Biol Reports 19: 125-134 (1994) Jonak et al., Progress Biotech, Vol 5, In Vitro Immunization in Hybridoma Technology, Borrebaeck, ed., Elsevier Science Publishers B.V., Amsterdam, Netherlands (1988)).

Methods well known to those skilled in the art for constructing or humanizing non-human or human antibodies can also be used. As such, a humanized or engineered antibody has one or more amino acid residues from a non-human source, such as, but not limited to, mice, rats, rabbits, primates (excluding humans), or other mammals. These non-human amino acid residues are replaced by residues that are often referred to as imported residues because they are usually taken from imported variable, constant or other domains of known human sequence.

For descriptions of known human Ig sequences, see, for example, the websites: www.ncbi.nlm.nih.gov/entrez/query.fcgi;

www.ncbi.nih.gov/igblast; www.atcc.org/phage/hdb.html; www.mrccpe. cam.ac.uk/ALIGNMENTS.php; www.kabatdatabase. com/top.html; ftp.ncbi.nih. gov/repository/kabat; www.sciquest. com;

www. abeam.com; www. antibody resource. com/onlinecomp.html;

www.public. lastate.edu/-pedro/research_tools.html;

www.whfreeman.com/immunology/CH05/kuby05.htm;

- 7 040272 www.hhmi. org/grants/lectures/1996/vlab;

www.path. cam.ac.uk/~mrc7/mikeimages.html;

mcb. harvard.edu/BioLinks/Immunology.html;

www. immunologylink. com; pathbox.wustl.edu/~hcenter/index.html;

www.appliedbiosysterns. com; www.nal.usda. gov/awic/pubs/antibody;

www.m.ehime-u.ac.jp/~yasuhito/Elisa.html; www.biodesign.com com;

www.cancerresearchuk.org; www.biotech.ufl.edu; www.isac-net.org;

baseserv.uci. kun.nl/~jraats/links1.html; www. recab.unihd. de/immuno.bme.nwu.edu; www.mrc-cpe. cam.ac.uk;

www.ibt.unam.mx/vir/V_mice.html; http://www.bioinf.org.uk/abs;

antibody.bath.ac.uk; www.unizh.ch www.cryst.bbk.ac.uk/~ubcg07s;

www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.html;

www.path. cam.ac.uk/~mrc7/human!sation/TAHHP.html;

www.ibt.unam.mx/vir/structure/stat_aim.html;

www.biosci.missouri.edu/smithgp/index.html; www.jerini.de cm.

See also Rabat et al., Sequences of Proteins of Immunological

Interest, U.S. Dept. Health (1983), all incorporated herein by reference in their entirety.

As known to those skilled in the art, such imported sequences can be used to reduce immunogenicity, or to reduce, enhance or modify binding, affinity, association rate, dissociation rate, avidity, specificity, half-life, or any other suitable characteristic. Generally, residues in the CDR region are directly and to the greatest extent involved in influencing antigen binding. Accordingly, some or all of the non-human or human CDR sequences are retained, and the non-human variable and constant region sequences can be replaced with human or other amino acids.

Antibodies can also optionally be humanized, or human antibodies can be engineered to retain high antigen affinity and other favorable biological properties. To achieve this goal, humanized (or human) antibodies may optionally be generated from the analysis of the original sequences and various conceptual humanized products using three-dimensional models of the original and humanized sequences. Three-dimensional immunoglobulin models are widely available and known to those skilled in the art. There are computer programs that illustrate and display possible three-dimensional conformational structures of selected variants of immunoglobulin sequences. Viewing these images allows one to analyze the likely role of the residues in the functioning of the immunoglobulin sequence variant, that is, to analyze the residues that affect the ability of the immunoglobulin variant to bind to its antigen. Thus, from exemplary matched and imported sequences, framework region (FR) residues can be selected and combined so as to obtain the desired antibody characteristic, eg, increased affinity for the target antigen(s).

In addition, the human anti-IL-12/23p40 (or anti-IL-23) antibody used in the method of the present invention may comprise a human germline light chain backbone. In specific embodiments, the germline light chain sequence is selected from human VK sequences, including, but not limited to, A1, A10, A11, A14, A17, A18, A19, A2, A20, A23, A26, A27, A3, A30, A5, A7, B2, B3, L1, L10, L11, L12, L14, L15, L16, L18, L19, L2, L20, L22, L23, L24, L25, L4/18a, L5, L6, L8, L9, O1, O11, O12, O14, O18, O2, O4 and O8. In certain embodiments, this human germline light chain framework is selected from V1-11, V1-13, V1-16, V1-17, V118, V1-19, V1-2, V1-20, V1-22, V1-3 , V1-4, V1-5, V1-7, V1-9, V2-1, V2-11, V2-13, V2-14, V2-15, V2-17, V2-19, V2-6, V2 -7, V2-8, V3-2, V3-3, V3-4, V4-1, V4-2, V4-3, V4-4, V4-6, V5-1, V5-2, V5-4 and V5-6.

In other embodiments, the human anti-IL-12/23p40 (or anti-IL-23) antibody used in the method of the present invention may comprise a human germline heavy chain backbone. In specific embodiments, this human germline heavy chain framework is selected from VH1-18, VH1-2, VH1-24, VH1-3, VH1-45, VH1-46, VH1-58, VH1-69, VH1-8, VH2 -26, VH2-5, VH2-70, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-35 , VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VH3-7, VH3-72, VH3-73, VH3-74, VH3-9, VH4 -28, VH4-31, VH4-34, VH4-39, VH4-4, VH4-59, VH4-61, VH5-51, VH6-1 and VH7-81.

In specific embodiments, the light chain variable region and/or variable volume

- 8 040272 The heavy chain region contains a framework region or at least part of a framework region (eg containing 2 or 3 sub-regions such as FR2 and FR3). In some embodiments, at least FRL1, FRL2, FRL3 or FRL4 is fully human. In other embodiments, at least FRH1, FRH2, FRH3, or FRH4 is fully human. In some embodiments, at least FRL1, FRL2, FRL3, or FRL4 is a germline (e.g., human germline) sequence or contains typical human matching sequences for a given framework region (widely available from known human Ig sequence sources described above ). In other embodiments, at least FRH1, FRH2, FRH3, or FRH4 is a germline (eg, human germline) sequence or contains typical human matching sequences for a given framework. In preferred embodiments, the framework is a fully human framework.

The humanization or construction of antibodies of the present invention can be performed using any known method, such as, without limitation, the methods described in: Winter (Jones et al., Nature 321: 522 (1986); Riechmann et al., Nature 332: 323 (1988); Verhoeyen et al., Science 239: 1534 (1988)), Sims et al., J. Immunol. 151:2296 (1993); Chothia and Lesk, J. Mol. Biol. 196: 901 (1987), Carter et al., Proc. Natl. Acad. sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol. 151: 2623 (1993), патентах США №: 5723323, 5976862, 5824514, 5817483, 5814476, 5763192, 5723323, 5,766886, 5714352, 6204023, 6180370, 5693762, 5530101, 5585089, 5225539; 4816567, PCT: US 98/16280, US 96/18978, US 91/09630, US 91/05939, US 94/01234, GB 89/01334, GB 91/01134, GB 92/01755; WO 90/14443, WO 90/14424, WO 90/14430, EP 229246 are each incorporated herein by reference in their entirety, including references cited therein.

In certain embodiments, the antibody contains an altered (eg, mutated) Fc region. For example, in some embodiments, the Fc region has been altered to reduce or enhance the effector functions of the antibody. In some embodiments, the Fc region is an isotype selected from IgM, IgA, IgG, IgE, or another isotype. Alternatively or additionally, a combination of amino acid modifications with one or more additional amino acid modifications that alter C1q binding and/or function of complement-dependent cytotoxicity in the Fc region of the IL-12 binding molecule can be used. Of particular interest may be such an initial polypeptide that binds to C1q and exhibits complement-dependent cytotoxicity (CDC). Polypeptides with initial C1q binding activity, optionally additionally having the ability to mediate CDC, can be modified so that one or both of these activities are enhanced. Amino acid modifications that alter C1q and/or modify its complement dependent cytotoxicity function are described, for example, in WO 0042072, which is incorporated herein by reference.

As described above, it is possible to construct the Fc region of a human anti-IL-12/23p40 (or anti-IL-23) antibody of the present invention with an altered effector function, for example, by modifying C1q binding and/or FcyR binding and thus changing complement-dependent cytotoxicity (CDC) activity; and/or antibody-dependent cell-mediated cytotoxicity (ADCC) activity. Effector functions are responsible for activating or decreasing biological activity (eg, in a subject). Examples of effector functions include, without limitation: binding to C1q; CDC; binding to the Fc receptor; ADCC; phagocytosis; downregulation of cell surface receptors (eg, B cell receptor; BCR), etc. Such effector functions may require an Fc region to be combined with a binding domain (eg, an antibody variable domain) and can be assessed by various assays (eg, Fc binding assay, ADCC assay, CDC assay, etc.).

For example, it is possible to generate a variant Fc region of a human anti-IL-12/23p40 (or anti-IL-23) antibody with improved C1q binding and improved FcyRIII binding (eg, having both increased ADCC activity and increased CDC activity). Alternatively, if it is desired to reduce or eliminate this effector function, a variant Fc region with reduced CDC activity and/or reduced ADCC activity can be designed. In other embodiments, it is possible to increase only one of these activities and optionally also decrease the other activity (eg, generate a variant Fc region with increased ADCC activity but reduced CDC activity, and vice versa).

Fc mutations can also be introduced into constructs to alter their interaction with the neonatal Fc receptor (FcRn) and improve their pharmacokinetic properties. A collection of human Fc variants with improved binding to FcRn has been described (Shields et al., (2001). High resolution mapping of the binding site on human IgG1 for FcyRI, FcyRII, FcyRIII, and FcRn and design of IgG1 variants with improved binding to the FcyR, J Biol Chem 276: 6591-6604).

Another type of amino acid substitution serves to change the glycosylation pattern of the Fc region of a human anti-IL-12/23p40 (or anti-IL-23) antibody. Glycosylation of the Fc region is usually

- 9 040272 either N-linked or O-linked. N-linked glycosylation refers to the attachment of a carbohydrate unit to the side chain of an asparagine residue. O-linked glycosylation refers to the addition of one of the sugars: N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5hydroxylysine can also be used.

Recognizable sequences for enzymatic attachment of a carbohydrate moiety to asparagine side chain peptide sequences are asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. Thus, the presence of any of these peptide sequences in a polypeptide creates a potential glycosylation site.

The glycosylation pattern can be altered, for example, by removing one or more glycosylation sites found in the polypeptide and/or adding one or more glycosylation sites that are not present in the polypeptide. The addition of glycosylation sites to the Fc region of a human anti-IL-12/23p40 (or anti-IL-23) antibody is conveniently accomplished by changing the amino acid sequence to contain one or more of the tripeptide sequences described above (for N-linked glycosylation sites). An exemplary glycosylation variant has an Asn 297 amino acid residue substitution in the heavy chain. The change can also be made by adding or replacing one or more of the serine or threonine residues in the sequence of the original polypeptide (for O-linked glycosylation sites). In addition, substitution of Asn 297 for Ala may lead to the removal of one of the glycosylation sites.

In some embodiments, a human anti-IL-12/23p40 (or anti-IL-23) antibody of the present invention is expressed in cells where beta-(1,4)-Nacetylglucosaminyltransferase III (GnT III) is expressed such that GnT III attaches GlcNAc to human antibody to IL-12. Methods for producing antibodies in this way are presented in WO/9954342, WO/03011878, patent publication 20030003097A1 and Umana et al., Nature Biotechnology, 17: 176-180, Feb. 1999; all of which are specifically incorporated herein by reference in their entirety.

An anti-IL-12/23p40 (or anti-IL-23) antibody can also optionally be generated by immunizing a transgenic animal (e.g., mouse, rat, hamster, primate (excluding humans), etc.) capable of producing a repertoire of human antibodies, such as described herein and/or as known to those skilled in the art. Cells that produce human anti-IL12/23p40 (or anti-IL-23) antibodies can be isolated from such animals and immortalized using suitable methods such as those described herein.

Transgenic mice that can produce a repertoire of human antibodies that bind to human antigens can be generated by known methods (e.g., without limitation, described in US Pat. al., issued by Jakobovits et al WO 98/50433, Jakobovits et al WO 98/24893, Lonberg et al WO 98/24884, Lonberg et al WO 97/13852, Lonberg et al WO 94/25585, Kucherlapate et al WO 96/34096, Kucherlapate et al EP 0463151 B1, Kucherlapate et al EP 0710719 Al, Surani et al US Pat. Lonberg et al EP 0814259 A2, Lonberg et al GB 2 272 440 A, in Lonberg et al Nature 368: 856-859 (1994), Taylor et al, Int Immunol 6(4): 579-591 (1994), Green et al, Nature Genetics 7: 13-21 (1994), Mendez et al., Nature Genetics 15: 146-156 (1997), Taylor et al., Nucleic Acids Research 20(23): 6287- 6295 (1992), Tuaillon et al., Proc Natl Acad Sci USA 90(8): 3720-3724 (1993), Lonberg et al., Int Rev Immunol 13(1): 65-93 (1995) and Fishwald et al., Nat Biotechnol 14(7 ): 845-851 (1996), each of which is incorporated herein by reference in its entirety). As such, such mice contain at least one transgene-containing DNA from at least one human immunoglobulin locus that is functionally rearranged, or that may be functionally rearranged. The endogenous immunoglobulin locus in such mice can be disrupted or deleted to thereby deprive the animal of the ability to produce antibodies encoded by the endogenous genes.

Screening of antibodies for binding specificity to similar proteins or fragments is conveniently performed using peptide display libraries. This method involves screening large sets of peptides to identify individual peptides that have a desired function or structure. Screening for antibodies in peptide display libraries is well known to those skilled in the art. Displayed peptide sequences can be 3 to 5000 or more amino acids in length, often 5 to 100 amino acids in length, and often about 8 to 25 amino acids in length. In addition to methods for obtaining peptide libraries by direct chemical synthesis, several methods have been described with recombinant DNA. One such method involves displaying a peptide sequence on the surface of a bacteriophage or cell. Each bacteriophage or cell contains a nucleotide sequence encoding a specific display peptide sequence. Such methods are described in PCT Patent Publications Nos. 91/17271, 91/18980, 91/19818 and 93/08278.

Other systems for generating peptide libraries have aspects of both in vitro chemical synthesis methods and recombination methods. See PCT Patent Publications Nos. 92/05258, 92/14843 and 96/19256. See also US Pat. Nos. 5,658,754 and 5,643,768. Peptide display libraries, vectors, and screening kits are commercially available from manufacturers such as Invitrogen (Carlsbad, CA, USA) and Cambridge antibody Technologies (Cambridgeshire, UK). See, for example, US Pat. Nos. 4,704,692; 4,939,666; 4,946,778; 5223409, 5403484, 5571698, 5837500 issued by Dyax; 5427908, 5580717 issued by Affymax; 5885793 issued by Cambridge antibody Technologies; 5750373 issued by Genentech, 5618920, 5595898, 5576195, 5698435, 5693493, 5698417 issued by Xoma, Colligan, mentioned; Ausubel mentioned; or Sambrook as mentioned, each of these patents and publications is incorporated herein by reference in its entirety.

The antibodies used in the method of the present invention can also be generated using at least one nucleic acid encoding an anti-IL-12/23p40 (or anti-IL23) antibody to generate transgenic animals or mammals such as goats, cows, horses, sheep, rabbits, etc., which produce such antibodies in their milk. Such animals can be created using known methods. See, for example, without limitation, US patent No. 5827690; 5849992; 4873316; 5849992; 5994616; 5565362; 5304489 and the like, each of which is incorporated herein by reference in its entirety.

Antibodies used in the method of the present invention can additionally be produced using at least one nucleic acid encoding an antibody to IL-12/23p40 (or to IL-23) to create transgenic plants and plant cell cultures (for example, without limitation , tobacco and maize) that produce such antibodies, certain parts or variants thereof in plant organs or in cell cultures from them. As a non-limiting example, transgenic tobacco leaves expressing recombinant proteins have been successfully used to generate large amounts of recombinant proteins, for example using an inducible promoter. See, for example, Cramer et al., Curr. top. microbiol. Immunol. 240: 95-118 (1999) and references cited in this publication. Also, transgenic maize has been used to express mammalian proteins at industrial production levels, with biological activity equivalent to proteins produced in other recombination systems or purified from natural sources. See, for example, Hood et al., Adv. Exp. Med. Biol. 464: 127-147 (1999) and references cited in this publication. Antibodies, including antibody fragments such as single chain antibodies (scFv), have also been produced in large quantities from transgenic plant seeds, including tobacco seeds and potato tubers. See, for example, Conrad et al., Plant Mol. Biol. 38: 101-109 (1998) and references cited in this publication. Thus, the antibodies of the present invention can also be produced using transgenic plants according to known methods. See also, for example, Fischer et al., Biotechnol. Appl. Biochem. 30: 99-108 (Oct., 1999), Ma et al., Trends Biotechnol. 13:522-7 (1995); Ma et al., Plant Physiol. 109:341-6 (1995); Whitelam et al., Biochem. soc. Trans. 22:940-944 (1994); and references cited in those publications. Each of the above references is incorporated herein by reference in its entirety.

The antibodies used in the method of the present invention can bind to human IL-12/23p40 (or IL-23) in a wide range of affinity (KD). In a preferred embodiment, the human mAb can optionally bind human IL-12/23p40 (or IL-23) with high affinity. For example, a human mAb can bind human IL-12/23p40 (or IL-23) with a KD equal to or less than about 10 ^-7 M, such as, but not limited to, 0.1-9.9 (or any interval, or with any value in it) X 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 , 10 ^-11 , 10 ^-12 , 10 ^-13 or in any interval or with any value in it.

The affinity or avidity of an antibody for an antigen can be determined experimentally by any suitable method. (See, for example, Berzofsky, et al., Antibody-Antigen Interactions, in Fundamental Immunology, Paul, WE, Ed., Raven Press: New York, NY (1984); Kuby, Janis Immunology, WH Freeman and Company: New York, NY (1992); as well as the methods described herein). The measured affinity of a particular antibody-antigen interaction may vary depending on the measurement under different conditions (eg, salt concentration, pH). Thus, measurements of affinity and other antigen binding parameters (eg, KD, K _a , Kd) are preferably performed in standardized solutions of antibody and antigen, and in a standardized buffer such as the buffer described herein.

Nucleic acid molecules

Using the information provided herein, for example, nucleotide sequences encoding at least 70-100% of consecutive amino acids in at least one of the variable regions or CDRs of the light or heavy chain, with the sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, among other sequences described herein, certain fragments, variants or typical match sequences thereof, or a deposited vector containing at least one of these sequences, a nucleic acid molecule of the present invention can be obtained , encoding at least one anti-IL-12 antibody using the methods described herein or known to those skilled in the art.

The nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, hnRNA, tRNA, or any other form, or in the form of DNA, including, without limitation, cDNA and genomic DNA obtained by cloning, by synthesis, or any combination thereof. The DNA can be three-stranded, double-stranded, single-stranded, or a combination. Any portion of at least one strand of DNA or RNA may be a coding strand, also known as a forward strand, or a non-coding strand, also referred to as a reverse strand.

The isolated nucleic acid molecules used in the method of the present invention may include nucleic acid molecules containing an open reading frame (ORF), optionally with one or more introns, for example, without limitation, for at least one specific portion of at least one CDR such as CDR1, CDR2 and/or CDR3 of at least one heavy chain (eg SEQ ID NO: 1-3) or light chain (eg SEQ ID NO: 4-6); nucleic acid molecules containing a coding sequence for an anti-IL-12/23p40 antibody or variable region (eg, light and heavy chain variable regions of SEQ ID NOs: 7 and 8); and nucleic acid molecules that contain a nucleotide sequence substantially different from the nucleotide sequences described above, but which, due to the degeneracy of the genetic code, encodes at least one antibody to IL-12/23p40 (or to IL-23) , as described herein and/or known to those skilled in the art. Of course, the genetic code is well known to those skilled in the art. Therefore, it would be standard procedure for one skilled in the art to generate such degenerate nucleic acid variants encoding specific anti-IL-12/23p40 (or anti-IL-23) antibodies used in the method of the present invention. See, for example, Ausubel, et al., cited. Such nucleic acid variants are included in the present invention. Non-limiting examples of isolated nucleic acid molecules include nucleic acids encoding HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3, respectively.

As described herein, nucleic acid molecules that contain a nucleic acid encoding an antibody to IL-12/23p40 (or to IL-23) may include, without limitation, one that separately encodes the amino acid sequence of the antibody fragment; the coding sequence for the whole antibody or part thereof; a coding sequence for an antibody, fragment or portion, as well as additional sequences, such as the coding sequence for at least one signal leader or fusion peptide with or without the aforementioned additional coding sequences, such as at least one intron, together with additional non-coding sequences including, without limitation, non-coding 5' and 3' sequences such as transcribed non-translated sequences that are involved in transcription, mRNA processing, including splicing and polyadenylation signals (eg, ribosome binding and mRNA stability); an additional coding sequence that codes for additional amino acids, such as amino acids that provide additional functionality. Thus, an antibody coding sequence can be fused to a marker sequence, such as a peptide coding sequence, to facilitate purification of the fused antibody containing an antibody fragment or portion.

Selective hybridization of polynucleotides with a polynucleotide as described herein

The method of the present invention utilizes isolated nucleic acids which, under selective hybridization conditions, form the fusion polynucleotide described herein. Thus, the polynucleotides of the present embodiment can be used to isolate, detect and/or quantify nucleic acids containing such polynucleotides. For example, the polynucleotides of the present invention can be used to identify, isolate, or amplify partial or full-length clones in a deposited library. In some embodiments, the polynucleotides are genomic DNA or cDNA sequences isolated from or otherwise complementary to cDNA from a human or mammalian nucleic acid library.

The cDNA library preferably contains at least 80% full length sequences, more preferably at least 85 or 90% full length sequences, and most preferably at least 95% full length sequences. cDNA libraries can be normalized to increase the representation of rare sequences. For sequences with little identity to complementary sequences, hybridization is usually, but not exclusively, carried out under conditions of low or moderate stringency. For sequences with greater identity, medium and high stringency conditions are optionally applied. Low stringency conditions allow selective hybridization of sequences with a level of identity of approximately 70% and can be used to identify orthologous or paralogous sequences.

- 12 040272

Optionally, the polynucleotides will encode at least a portion of an antibody. Polynucleotides encompass nucleotide sequences that can be used for selective hybridization with a polynucleotide encoding an antibody of the present invention. See, for example,

Ausubel mentioned; Colligan mentioned, each publication is hereby incorporated by reference in its entirety.

Construction of nucleic acids

As is well known to those skilled in the art, isolated nucleic acids can be obtained by (a) recombination techniques, (b) synthetic techniques, (c) purification techniques, and/or (d) combinations thereof.

Nucleic acids may conveniently contain sequences in addition to the polynucleotide of the present invention. For example, a multiple cloning site containing one or more restriction endonuclease sites can be inserted into the nucleic acid to facilitate isolation of the polynucleotide. In addition, translatable sequences may be inserted to facilitate isolation of the translated polynucleotide of the present invention. For example, a convenient means of purifying the proteins of the present invention is the introduction of a hexahistidine marker sequence. The nucleic acid of the present invention, with the exception of the coding sequence, may optionally be a vector, adapter or linker for cloning and/or expression of the polynucleotide of the present invention.

Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in the isolation of the polynucleotide, or to improve the introduction of the polynucleotide into the cell. The use of cloning vectors, expression vectors, adapters and linkers is well known to those skilled in the art. (See, for example, Ausubel, cited; or Sambrook, cited).

Recombinant methods for constructing nucleic acids

Isolated compositions of nucleic acids, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methods known to those skilled in the art. In some embodiments, oligonucleotide probes that selectively hybridize under stringent conditions to the polynucleotides of the present invention are used to identify a desired sequence in a cDNA or genomic DNA library. RNA isolation and construction of cDNA libraries and genomic libraries is well known to those skilled in the art. (See, for example, Ausubel, cited; or Sambrook, cited).

Methods for Screening and Isolation of Nucleic Acids

Screening of a cDNA or genomic DNA library can be performed with a probe based on a polynucleotide sequence used in the method of the present invention, such as those described herein. Probes can be used to hybridize to genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms. Those skilled in the art will appreciate that varying degrees of stringency in hybridization can be used for analysis; and that either hybridization or wash medium can be stringent. As the hybridization conditions become more stringent, the degree of complementarity between probe and target required to form a duplex increases. The stringency of the conditions can be controlled by one or more of the following: temperature, ionic strength, pH, and the presence of a partially denaturing solvent such as formamide. For example, the stringency of the hybridization conditions is usually changed by changing the polarity of the reagent solution, for example, by changing the concentration of formamide in the range from 0 to 50%. The degree of complementarity (sequence identity) required for detectable binding varies according to the stringency of the hybridization medium and/or wash medium. The optimal degree of complementarity is 100%, or 70 to 100%, or any range or value therein. However, it should be understood that small sequence variations in probes and primers can be compensated for by reducing the stringency of the hybridization medium and/or wash medium.

Methods for amplifying RNA or DNA are well known to those skilled in the art and can be used in accordance with the present invention without undue experimentation, based on the instructions and recommendations provided herein.

Known methods for amplifying DNA or RNA include, but are not limited to, polymerase chain reaction (PCR) and related amplification processes (see, for example, US Pat. issued by Tabor, et al; 5142033 issued by Innis; 5122464 issued by Wilson, et al.; 5091310 issued by Innis; 5066584 issued by Gyllensten, et al; 4889818 issued by Gelfand, et al; 4994370 issued by Silver, et al; 4,766,067 to Biswas; 4,656,134 to Ringold), and RNA-mediated amplification that uses reverse RNA to a target sequence as a template for double-stranded DNA synthesis (U.S. Patent No. 5,130,238 to Malek, et al, tradenamed NASBA), the entire contents of all of these references are incorporated herein by reference. (See, for example, Ausubel mentioned; or

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Sambrook, mentioned).

For example, polymerase chain reaction (PCR) technology can be used to amplify the polynucleotide sequences used in the method of the present invention and their associated genes directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods can also be used, for example, to clone nucleotide sequences encoding proteins to be expressed in order to prepare nucleic acid probes to detect the presence of the desired mRNA in samples, nucleic acid sequencing, or other purposes. Examples of methods sufficient to guide those skilled in the art of in vitro amplification techniques can be found in Berger, cited, Sambrook, cited, and Ausubel, cited, as well as Mullis, et al., US Pat. No. 4,683,202 (1987); and Innis, et al., PCR Protocols: a Guide to Methods and Applications, Eds., Academic Press Inc., San Diego, CA (1990). Commercially available kits for amplifying a genomic PCR sequence are known to those skilled in the art. See, for example, the Advantage-GC Genomic PCR Kit (Clontech). In addition, it is possible to use, for example, protein 32 of the T4 gene (Boehringer Mannheim) to increase the yield of the reaction during PCR of long fragments.

Synthetic Methods for Designing Nucleic Acids The isolated nucleic acids used in the method of the present invention can also be prepared by direct chemical synthesis using known methods (see, for example, Ausubel, et al., cited). Chemical synthesis typically produces a single stranded oligonucleotide that can be converted to double stranded DNA by hybridization with a complementary sequence or polymerization with a DNA polymerase and a single strand as a template. Those skilled in the art will recognize that DNA chemistry can be limited to sequences of 100 bases or more, but short sequences can be ligated to produce longer sequences.

Recombinant Expression Cassettes

The present invention uses recombinant expression cassettes containing a nucleic acid. A nucleotide sequence, such as a cDNA or genomic DNA sequence encoding an antibody of the present invention, can be used to construct a recombinant expression cassette that can be introduced into at least one desired host cell. The recombinant expression cassette typically contains a polynucleotide operably linked to transcriptional initiation regulatory sequences that direct translation of the polynucleotide in its intended host cell. Both heterologous and non-heterologous (ie, endogenous) promoters can be used to direct the expression of nucleic acids.

In some embodiments, isolated nucleic acids that serve as a promoter, enhancer, or other elements can be inserted at the appropriate position (upstream, downstream, or intron) of a non-heterologous form of a polynucleotide of the present invention in such a manner as to promote or repress expression of the polynucleotide. For example, endogenous promoters can be changed in vivo or in vitro by mutation, deletion and/or substitution.

Vectors and host cells

The present invention also relates to vectors that include isolated nucleic acid molecules, to host cells that are genetically engineered with recombinant vectors, and to the production of at least one anti-IL-12/23p40 (or anti-IL-23) antibody by recombinant methods that are well known to those skilled in the art. See, for example, Sambrook et al. mentioned; Ausubel, et al., mentioned; each publication is incorporated herein by reference in its entirety.

The polynucleotides may optionally be coupled to a vector containing a selectable marker for propagation in a host cell. Typically, the plasmid vector is incorporated into a precipitate, such as a calcium phosphate precipitate, or complexed with a charged lipid. If a virus is used as the vector, it can be packaged in vitro using a suitable packaging cell line and then introduced into host cells.

The DNA insert must be operably linked to a suitable promoter. Expression constructs additionally contain sites for transcription initiation and termination, and in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs preferably contains a translation initiation at the beginning and a termination codon (e.g., UAA, UGA, or UAG) appropriately located at the end of the translated mRNA, with UAA and UAG being preferred for expression in mammalian or eukaryotic cells.

Expression vectors preferably, but not necessarily, include at least one selectable marker. Such markers include, for example, without limitation, methotrexate (MTX), dihydrofolate reductase (DHFR) resistance genes, US Pat. Nos. 4,399,216; 4,634,665; 4,656,134; (US Pat. Nos. 5,122,464; 5,770,359; 5,827,739) for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culture in E. coli and other bacteria or proca.

- 14 040272 riots (the above patents are incorporated herein by reference in their entirety). Suitable growth media and conditions for the above host cells are known to those skilled in the art. Acceptable vectors are, of course, known to those skilled in the art. Introduction of the vector construct into the host cell may be by transfection with calcium phosphate, DEAE-dextran, cationic lipids, electroporation, transduction, infection, or other known methods. Such techniques are described in the art, for example, in Sambrook, cited, chapters 1-4 and 16-18; Ausubel, mentioned, chapters 1, 9, 13, 15, 16.

At least one antibody used in the method of the present invention may be expressed in a modified form, such as a fusion protein, and may include not only secretory signals but also additional heterologous functional regions. For example, a region of additional amino acids, especially charged amino acids, can be added to the N-terminus of an antibody to enhance the stability and persistence of the antibody in the host cell and during purification or subsequent handling and storage. In addition, peptide units can be added to the antibody of the present invention to facilitate purification. Such regions can be removed prior to preparation of the finished antibody, or at least one fragment thereof. Such methods are described in numerous standard laboratory manuals, for example Sambrook, cited, chapters 17.29-17.42 and 18.1-18.74; Ausubel, mentioned, chapters 16, 17 and 18.

Those skilled in the art are aware of a variety of expression systems available for expressing a nucleic acid encoding a protein used in the method of the present invention. Alternatively, nucleic acids can be expressed in a host cell by running (by a procedure) in a host cell that contains endogenous DNA encoding the antibody. Such methods are well known to those skilled in the art, such as those described in US Pat.

Mammalian cells are an example of cell cultures used to produce antibodies, certain parts or variants thereof. Mammalian cell systems are often used as cell monolayers, however, mammalian cell suspensions or bioreactors can also be used. Several suitable host cell lines have been developed in the art that are capable of expressing intact glycosylated proteins, in particular the cell lines COS-1 (e.g. ATCC CRL 1650), COS-7 (e.g. ATCC CRL-1651), HEK293, BHK21 (e.g. , ATCC CRL-10), CHO (eg ATCC CRL 1610) and BSC-1 (eg ATCC CRL-26), Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653 cells, SP2/0-Ag14 cells , 293, HeLa cells, and the like, for example, from the American Type Culture Collection, Manassas, VA, USA (www.atcc.org). Preferred host cells include cells of lymphoid origin such as myeloma and lymphoma cells. More preferred are P3X63Ag8.653 host cells (ATCC catalog number CRL-1580) and SP2/0-Ag14 cells (ATCC catalog number CRL-1851). In a particularly preferred embodiment, the recombinant cell is a P3X63Ab8.653 or SP2/0-Ag14 cell line.

Expression vectors for such cells may include one or more of the following expression control sequences such as, but not limited to, an origin of replication; promoter (e.g., SV40 late or early promoters, CMV promoter (US Pat. No. 5,168,062; 5,385,839), HSV tk promoter, pgk (phosphoglycerate kinase) promoter, EF-1-alpha promoter (US Pat. No. 5,266,491), at least one human immunoglobulin, enhancer and/or processing information sites such as ribosome binding sites, RNA splicing sites, polyadenylation sites (e.g. poly-A attachment site of large T-Ag SV40), and transcription terminator sequences See, for example, Ausubel et al. ., cited Sambrook et al., cited Other known and/or commercially available cells can be used to produce nucleic acids or proteins of the present invention, such as the American Type Culture Collection Catalog of Cell Lines and Hybridomas (www.atcc.org ), or from other known or commercial sources.

When eukaryotic host cells are used, polyadenylation or transcription termination sequences are usually inserted into the vector. For example, a polyadenylation sequence from the bovine growth hormone gene can be used as a termination sequence. It is also possible to add sequences for accurate splicing of the transcript. An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al., J. Virol. 45: 773-781 (1983)). In addition, gene sequences for controlling replication in the host cell, known to those skilled in the art, can be added to the vector.

Antibody Purification

An anti-IL-12/23p40 (or anti-IL-23) antibody can be isolated from recombinant cell cultures and purified by well known methods including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. High performance liquid chromatography (HPLC) can also be used for purification. See, for example, Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), e.g., chapters 1, 4, 6, 8, 9 , 10, each publication is incorporated herein by reference in its entirety.

Antibodies useful in the method of the present invention include naturally purified products, chemical synthesis products, and products obtained by recombinant technologies from eukaryotic host cells, including, for example, cells from yeast, higher plants, insects, and mammals. Depending on the host used in the recombinant production method, the antibody may be glycosylated or may be non-glycosylated, with a glycosylated antibody being preferred. Such methods are described in numerous standard laboratory manuals, for example, Sambrook, cited, sections 17.37-17.42; Ausubel, cited, chapters 10, 12, 13, 16, 18, and 20; Colligan, Protein Science, cited, chapters 12-14, all publications are incorporated herein by reference in their entirety.

Antibodies to IL-12/IL-23r40

An anti-IL-12/23p40 (or IL-23) antibody of the present invention includes any protein or peptide containing a molecule that contains at least a portion of an immunoglobulin molecule, such as, without limitation, at least one ligand-binding site (LBP ), such as, without limitation, a complementarity determining region (CDR) of a heavy or light chain, or a ligand-binding region, a heavy or light chain variable region, a framework region (e.g., FR1, FR2, FR3, FR4, or a fragment thereof, further optionally containing at least one substitution, insertion or deletion), heavy or light chain constant region (for example, containing at least one _CH 1, hinge 1, hinge 2, hinge 3, hinge 4, CH2 or CH3, or a fragment thereof, additionally optionally containing at least one substitution, insertion or deletion), or any part thereof that can be inserted into an antibody. The antibody may include or be derived from any mammal, such as, but not limited to, a human, mouse, rabbit, rat, rodent, primate, or any combination thereof, and the like.

The isolated antibodies used in the method of the present invention comprise the amino acid sequences of the antibodies described herein encoded by any suitable polynucleotide, or any isolated or derived antibody. Preferably, the human antibody or antigen-binding fragment binds to human IL-12/23 or IL-23 and thus partially or significantly neutralizes at least one biological activity of that protein. An antibody, or a defined portion or variant thereof, that partially or significantly neutralizes at least one biological activity of at least one IL-12 or IL-23 protein or fragment, can bind the protein or fragment and thus inhibit activities mediated by binding of IL-12 or IL-23 to the IL-12 or IL-23 receptor, or to other IL-12 dependent or mediated mechanisms. In the context of this document, the term "neutralizing antibody" refers to an antibody that can inhibit IL-12 or IL-23 dependent activity by about 20-120%, preferably at least about 10, 20, 30, 40, 50, 55 , 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or more, depending on the method of analysis. The ability of an anti-IL-12/23p40 antibody to inhibit IL-12/23 dependent activity is preferably assessed by at least one acceptable assay for the IL-12/23 protein or its receptor, as described herein and/or as known to those skilled in the art. this area. The human antibody may be any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype, and may contain a kappa or lambda light chain. In one embodiment, the human antibody contains a heavy chain or a specific fragment of IgG, for example, at least one of the IgG1, IgG2, IgG3, or IgG4 isotypes (eg, γ1, γ2, γ3, γ4). Antibodies of this type can be generated using a transgenic mouse or other transgenic mammal (excluding humans) containing transgenes of at least one human light chain (e.g., IgG, IgA, and IgM) as described herein and/or as known to those skilled in the art. areas. In another embodiment, the human anti-human IL-12/23p40 (or anti-IL-23) antibody comprises an IgG1 heavy chain and an IgG1 light chain.

The antibody binds at least one defined epitope specific to at least one IL-12/23 protein, subunit, fragment, portion, or any combination thereof. At least one epitope may contain at least one antibody binding region that contains at least one portion of the protein, and this epitope preferably contains at least one extracellular, soluble, hydrophilic, extrinsic, or cytoplasmic portion of the protein. At least one defined epitope may comprise any combination of at least one amino acid sequence of at least 1-3 amino acids and up to the full defined portion of consecutive amino acids of SEQ ID NO: 9, e.g., amino acid residues 15, 17 -21, 23, 40-43, 45-47, 54-56 and 58-62.

As such, a human antibody or antigen-binding fragment comprises an antigen-binding region that contains at least one human complementarity-determining region (CDR1, CDR2, and CDR3) or a variant of at least one heavy chain variable region and at least one complementarity-determining region. human (CDR1, CDR2 and

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CDR3), or a variant of at least one light chain variable region. CDR sequences can be derived from human germline sequences, or they can closely resemble germline sequences. For example, a CDR from a synthetic library derived from the original non-human CDRs can be used. These CDRs can be generated by inserting conservative substitutions from the original non-human sequence. As a non-limiting example, an antibody or antigen-binding moiety or variant may comprise at least one heavy chain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and/or a light chain CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-6. In a particular embodiment, an antibody or antigen-binding fragment may have an antigen-binding region that contains at least a portion of at least one heavy chain CDR (i.e., CDR1, CDR2, and/or CDR3) having an amino acid sequence corresponding to CDR 1, 2 and/or 3 (for example, SEQ ID NO: 1, 2 and/or 3). In another specific embodiment, an antibody or antigen-binding portion or variant may have an antigen-binding region that contains at least a portion of at least one light chain CDR (i.e., CDR1, CDR2, and/or CDR3) having an amino acid sequence corresponding to CDR 1, 2 and/or 3.

Such antibodies can be produced by chemically linking different parts (e.g., CDRs, scaffolds) of an antibody using conventional methods, preparing and expressing a nucleic acid molecule (i.e., one or more) that encodes an antibody using conventional methods of recombinant DNA technology, or using another suitable method.

An anti-IL-12/2 3p40 (or IL-23) antibody may contain at least one of the heavy or light chain variable regions having a specific amino acid sequence. For example, in a preferred embodiment, the anti-IL-12/23p40 antibody comprises at least one heavy chain variable region optionally having the amino acid sequence of SEQ ID NO: 7 and/or at least one light chain variable region optionally having the amino acid sequence of SEQ ID NO: 8. Antibodies that bind to human IL12/23 and that contain a defined heavy or light chain variable region can be obtained by suitable methods such as phage display (Katsube Y., et al., Int J Mol. Med., 1(5): 863868 (1998)), or methods that use transgenic animals known to those skilled in the art and/or described herein. For example, a transgenic mouse containing an operably rearranged human immunoglobulin heavy chain transgene and a transgene containing DNA from a human immunoglobulin light chain locus that can be functionally rearranged can be immunized with human IL-12/23 or a fragment thereof to produce antibodies. If desired, antibody-producing cells can be isolated and hybridomas or other immortal antibody-producing cells can be prepared as described herein and/or as known to those skilled in the art. Alternatively, an antibody, defined moiety, or variant can be expressed in a suitable host cell with an encoding nucleic acid, or a portion thereof.

The invention also relates to antibodies, antigen-binding fragments, immunoglobulin chains, and CDRs containing amino acids in a sequence substantially identical to the amino acid sequence of an antibody described herein. Preferably, such antibodies or antigen-binding fragments, and antibodies containing such CDR chains or regions, can bind to human IL-12/23 with high affinity (eg, with a KD less than or equal to about 10 ^-9 M). Amino acid sequences substantially the same as those described herein include those containing conservative amino acid substitutions as well as amino acid deletions and/or insertions. A conservative amino acid substitution refers to the replacement of the first amino acid with a second amino acid whose physical and/or chemical properties (eg, charge, structure, polarity, hydrophobicity/hydrophilicity) are similar to those of the first amino acid. Conservative substitutions include, without limitation, substitution of one amino acid for another within the following groups: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), series (S), threonine (T), tyrosine (Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C), and glycine (G); F, W and Y; C, S and T.

Amino acid codes

The amino acids that make up the anti-IL-12/23p40 (or IL-23) antibodies of the present invention are often abbreviated. Amino acid names can be specified using a one-letter amino acid code, a three-letter code, a name, or a three-nucleotide codon(s), as is well known to those skilled in the art (see Alberts B., et al., Molecular Biology of The Cell, Third Ed ., Garland Publishing, Inc., New York, 1994):

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SINGLE LETTER code THREE LETTER code NAME CODON(S) OF THREE NUCLEOTIDES A Ala Alanine GCA, GCC, GCG, GCU C Cys Cysteine UGO, UGU Aspartic D asp GAC, GAU acid Glutamine E Glu GAA, GAG acid F Phe Phenylalanine UUC, UUU G gly Glycine GGA, GGC, GGG, GGU H His Histidine САС, CAU I He Isoleucine AUA, AUG, AUU TO Lys Lysine LAA, AAG L Leu Leucine UUA, UUG, CUA, CUC, CUG, CUU M Met Methionine AUG N Asn Asparagine LAS, AAU P Pro Proline CCA, CCC, CCG, CCU Q gin Glutamine SLA, SAG — AGA, AGG, GGA, CGC, R Arg Arginine GGG, CGU — AGG, AGU, UGA, UCC, S Ser Serene UGG, UCU T Thr Threonine ACA, ACC, ACG, ACU V Vai Valine GUA, GUC, GUG, GUU W trp tryptophan UGG Y Tyr Tyrosine UAC, UAU

An anti-IL-12/23p40 (or anti-IL-23) antibody used in the method of the present invention may include one or more amino acid substitutions, deletions or additions due to natural mutations or human actions described herein.

The number of amino acid substitutions that a skilled artisan can make depends on many factors, including those described above. Generally speaking, the number of amino acid substitutions, insertions or deletions for any given anti-IL-12/23p40 (or anti-IL-23) antibody, fragment or variant will be no more than 40, 30, 20, 19, 18, 17, 16, 15 , 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, such as 1-30, or any interval or value therein, as specified herein.

Amino acids in an anti-IL-12/23p40 (or anti-IL-23) antibody that are essential for its function can be identified by methods known to those skilled in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g., Ausubel, cited, chapters 8, 15; Cunningham and Wells, Science 244: 1081-1085 (1989)). The last procedure involves the addition of alanine point mutations at each residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as, without limitation, at least one IL-12 or IL-23 neutralizing activity. Sites critical for antibody binding can also be identified by structure analysis, for example by crystallization, nuclear magnetic resonance, or photoaffinity labeling (Smith, et al., J. Mol. Biol. 224: 899-904 (1992) and de Vos, et al. al., Science 255: 306-312 (1992)). Anti-IL-12/23p40 antibody residues involved in IL-12 binding were identified based on the co-crystalline structure of the anti-IL-12/23p40 antibody and IL12p40 antigen. They are shown in Table 5 below.

Anti-IL-12/23p40 antibodies may include, without limitation, at least one part, sequence, or combination selected from 5 to all consecutive amino acids from at least one of SEQ ID NOs: 1, 2, 3, 4 ,5, 6.

Anti-IL-12/23p40 antibodies or specific portions or variants may include, without limitation, at least one portion, sequence or combination selected from at least 3-5 consecutive amino acids from SEQ ID NO: 1, 5-17 consecutive amino acids from SEQ ID NO: 2, 510, consecutive amino acids from SEQ ID NO: 3, 5-11, consecutive amino acids from SEQ ID NO: 4, 5-7, consecutive amino acids from SEQ ID NO: 5, 5-9, consecutive amino acids from SEQ ID NO: 6, Leu21, Lys76, Met83, Ser85 from SEQ ID NO: 7.

The anti-IL-12/23p40 antibody may optionally further comprise a polypeptide from

- 18 040272 at least one of 70-100% of 5, 17, 10, 11, 7, 9, 119 or 108 consecutive amino acids in at least one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. In one embodiment, the amino acid sequence of an immunoglobulin chain or portion thereof (e.g., variable region, CDR) has about 70-100% identity (e.g., 70, 71, 72, 73, 74, 75, 76, 77 , 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or any interval, or a value therein) with the amino acid sequence of the corresponding chain of at least one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. For example, the amino acid sequence of the light chain variable region can be compared to the sequences of SEQ ID NO: 4, 5, 6, or 8, or the heavy chain CDR3 amino acid sequence can be compared to the sequences of SEQ ID NO: 1, 2, 3, or 7. Preferably, 70-100% amino acid identity (e.g., 90, 91, 92, 93 , 94, 95, 96, 97, 98, 99, 100 or any interval or value therein) is determined using an acceptable computer algorithm known to those skilled in the art.

Identity, as known to those skilled in the art, is the relationship between two or more polypeptide sequences, or two or more polynucleotide sequences, determined by comparing these sequences. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by matching strings of such sequences. Identity and similarity can be easily calculated by known methods including, without limitation, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., Siam J. Applied Math., 48: 1073 (1988). In addition, percent identity can be derived from amino acid and nucleotide sequence mappings generated with the default settings of the AlignX component in Vector NTI Suite 8.0 (Informax, Frederick, MD, USA).

Preferred identity methods are designed to find the best match between test sequences. Methods for determining identity and similarity are systematized in publicly available computer programs. Preferred computer software methods for determining identity and similarity between two sequences include, without limitation, the GCG software package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul , S. F. et al., J. Molec Biol 215: 403-410 (1990)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894: Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990) The well-known Smith Waterman algorithm can also be used to determine identity.

Preferred parameters for comparing polypeptide sequences include the following:

(1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48:443-453 (1970). Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sc., USA. 89:10915-10919 (1992).

Gap Penalty: 12

Gap Length Penalty: 4.

The program used with these parameters is publicly available as the gap program from Genetics Computer Group, Madison, Wisconsin, USA. The above parameters are the default parameters for peptide sequence comparisons (with no end gap penalty).

Preferred parameters for comparing polynucleotides include the following:

(1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48:443-453 (1970).

Comparison matrix: matches=+10, mismatch=0.

Gap Penalty: 50

Gap Length Penalty: 3.

Available as a gap program from Genetics Computer Group, Madison, Wisconsin, USA. The specified parameters are the default parameters for nucleotide sequence comparisons.

As an example, a polynucleotide sequence may be identical to another sequence, ie 100% identical, or may include up to a certain integer number of nucleotide changes compared to a reference sequence. Such changes are selected from the group consisting of deletion, substitution, including transition and transversion, or insertion of at least one nucleotide, and the change may take place at the 5' or 3' end positions of the reference nucleotide sequence, or anywhere between these terminal positions, and may either be scattered singly among the nucleotides in the reference sequence, or assembled into one or more consecutive groups within the reference sequence. Number

- 19 040272 nucleotide changes are determined by multiplying the total number of nucleotides in the sequence by a number giving the corresponding percent identity (divided by 100) and then subtracting this result from the total number of nucleotides in the sequence, or

n. sub.n. ltorsim. x. sub .n - (x.sub .my), where n.sub.n is the number of nucleotide changes, x.sub.n is the total number of nucleotides in the sequence and y is, for example, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, etc., and any non-integer result of multiplying x.sub.n by y is rounded down to the nearest integer number before subtracting it from x.sub.n.

Changes in the polynucleotide sequence encoding the polypeptide of SEQ ID NO: 7 may result in a senseless, missense, or frameshift mutation in that coding sequence, and thereby a change in the polypeptide encoded by the polynucleotide following such changes. Similarly, the polypeptide sequence may be identical to the reference sequence of SEQ ID NO: 7, i.e., 100% identical, or may include up to a certain integer number of amino acid changes compared to the reference sequence such that the percent identity is less than 100%. Such changes are selected from the group consisting of deletion, substitution, including conservative and non-conservative substitution, or insertion of at least one amino acid, and the changes may take place at positions at the amino or carboxyl terminus of the reference polypeptide sequence, or anywhere in between these terminal positions. , and may either be scattered singly among the amino acids in the reference sequence, or assembled into one or more consecutive groups within the reference sequence. The number of amino acid changes for a given % identity is determined by multiplying the total number of amino acids in SEQ ID NO: 7 by the number giving the corresponding percent identity (divided by 100) and then subtracting this result from the total number of amino acids in SEQ ID NO: 7, or

n.sub.a.ltorsim.x.sub.a -(x.sub.a.y), where n.sub.a is the number of amino acid changes, x.sub.a is the total number of amino acids in SEQ ID NO: 7 , and y is, for example, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, etc., and where any non-integer result of multiplying x.sub.a by y is rounded down to nearest integer before subtracting it from x.sub.a.

Examples of heavy and light chain variable region sequences and portions thereof are shown in SEQ ID NOs: 1-8. The antibodies of the present invention, or specific variants thereof, may contain any number of consecutive amino acid residues from an antibody of the present invention, which number is selected from the group of integers in the range of 10-100% of the number of consecutive residues in an anti-IL-12 antibody. Optionally, the length of a given subsequence of consecutive amino acids is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 , 210, 220, 230, 240, 250 or more amino acids, or any range or value therein. In addition, the number of such subsequences may be any integer selected from the group consisting of 1 to 20, such as at least 2, 3, 4, or 5.

As will be determined by experts, the present invention includes at least one biologically active antibody of the present invention. Biologically active antibodies have a specific activity of at least 20%, 30% or 40%, preferably at least 50%, 60% or 70% and most preferably at least 80%, 90% or 95-100% or more (including, without limitation up to up to a 10-fold increase in specific activity) from the activity of a native (non-synthetic), endogenous or related and known antibody. Methods for qualitative and quantitative analysis of 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 a modification allows the creation of an antibody or antigen-binding fragment with improved pharmacokinetic properties (eg, increased in vivo serum half-life). As an organic link, a linear or branched hydrophilic polymeric group, a fatty acid group or a fatty acid ester group can be used. In some embodiments, the hydrophilic polymeric group may have a molecular weight of from about 800 to about 120,000 Da and be a polyalkane glycol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), a carbohydrate polymer, an amino acid polymer, or polyvinylpyrrolidone, and a fatty acid group or a the fatty acid ester may contain from about eight to about forty carbon atoms.

Modified antibodies and antigen-binding fragments may contain one or more organic units that are covalently linked, directly or indirectly, to the antibody. Each organic unit associated with an antibody or antigen-binding fragment of the present invention may independently be a hydrophilic polymeric group, a fatty acid group, or a fatty acid ester group. In the context of this document, the term fatty acid encompasses monobasic and dibasic carboxylic acids. In the context of this document, the term hydrophilic polymer group means an organic polymer having better solubility in water than in octane. For example, polylysine is more soluble in water than in octane. Thus, an antibody modified by covalently attaching a polylysine is within the scope of the present invention. Hydrophilic polymers suitable for modifying the antibodies of the present invention may be linear or branched, and include, for example, polyalkane glycols (e.g., PEG, monomethoxypolyethylene glycol (mPEG), PPG, etc.), carbohydrates (e.g., dextran, cellulose, oligosaccharides , polysaccharides, and the like), hydrophilic amino acid polymers (eg, polylysine, polyarginine, polyaspartate, and the like), polyalkane oxides (eg, polyethylene oxide, polypropylene oxide, and the like), and polyvinylpyrrolidone. The hydrophilic antibody-modifying polymer of the present invention preferably has a molecular weight of from about 800 to about 150,000 Da as a single molecular moiety. For example, PEG ₅₀₀₀ and PEG ₂₀₀₀ where the subscript indicates the average molecular weight of the polymer in daltons. The hydrophilic polymeric group may have one to about six alkyl, fatty acid, or fatty acid ester substituents. Hydrophilic polymers substituted with a fatty acid or fatty acid ester group can be prepared using suitable methods. For example, an amino group-containing polymer can be coupled to a fatty acid or fatty acid ester carboxylate, and an activated carboxylate (eg, activated with N,N-carbonyldiimidazole) on a fatty acid or fatty acid ester can be coupled to the hydroxyl group of the polymer.

Fatty acids and fatty acid esters suitable for modifying the antibodies of the present invention may be saturated or may contain one or more unsaturated linkages. Fatty acids suitable for modifying antibodies of the present invention include, for example, n-dodecanoate (C12, laurate), n-tetradecanoate (C ₁₄ , myristate), n-octadecanoate (C 18 , stearate), neicosanoate (C ₂₀ , arachidate) , n-docosanoate (C ₂₂ , behenate), n-triacontanoate (Cs ₀ ), n-tetracontanoate (C ₄₀ ), cis-A9-octadecanoate (C ₁₈ , oleate), completely cis-A5,8,11,14- eicosatetraenoate (C ₂₀ , arachidonate), octanedicarboxylic acid, tetradecanedicarboxylic acid, octadecanedicarboxylic acid, docosandicarboxylic acid, and the like. Acceptable fatty acid esters include monoesters of dicarboxylic acids containing a linear or branched lower alkyl group. The lower alkyl group may contain from one to about twelve, preferably from one to about six carbon atoms.

Modified human antibodies and antigen-binding fragments can be obtained using an acceptable method, for example, by reaction with one or more modifying agents. In the context of this document, the term modifying agent means a suitable organic group (eg, hydrophilic polymer, fatty acid, fatty acid ester) that contains an activating group. An activating group' means a chemical unit or functional group which, under suitable conditions, can react with a second chemical group to form a covalent bond between the modifying agent and the second chemical group. For example, amine-reactive activating groups include electrophilic groups such as tosylate, mesylate, halogen (chlorine, bromine, fluorine, iodine), N-hydroxysuccinimidyl esters (NHS), and the like. Activating groups capable of reacting with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfides, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehyde functional group can combine with amine or hydrazide containing molecules, and an azide group can react with a trivalent phosphorus group to form phosphoramidate or phosphorimide bonds. Acceptable methods for introducing activating groups into molecules are known to those skilled in the art (see, for example, Hermanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, CA (1996)). The activating group may be linked to an organic group (e.g. hydrophilic polymer, fatty acid, fatty acid ester) directly or via a linker unit, e.g. a divalent C1-C12 group in which one or more carbon atoms may be replaced by a heteroatom such as oxygen, nitrogen or sulfur. Suitable linker units include, for example, tetraethylene glycol, -(CH ₂ ) ₃ -, -NH-(CH2) ₆ -NH-, -(CH ₂ ) ₂ -NH- and -CH ₂ -O-CH ₂ -CH ₂ - OCH2-CH2-O-CH-NH-. Modifying agents which contain a link can be prepared, for example, by reacting a mono-Boc-alkyldiamine (eg, mono-Boc-ethylenediamine, mono-Bocdiaminohexane) with a fatty acid in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylate. The Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to open a primary amine, which can be combined with another carboxylate as described above, or it can be reacted with maleic anhydride to loop the resulting product to give an activated maleimide fatty acid derivative . (See, for example, Thompson, et al., WO 92/16221, the contents of which are incorporated herein by reference in their entirety).

Modified antibodies can be produced by reacting a human antibody or antigen-binding fragment with a modifying agent. For example, organic units can be linked to the antibody in a non-site specific manner with an amine-reactive modifying agent, such as an NHS ester and PEG. Modified human antibodies or antigen binding

- 21 040272 fragments can also be obtained by reduction of disulfide bonds (eg, intrachain disulfide bonds) of the antibody or antigen-binding fragment. The reconstituted antibody or antigen-binding fragment can then be reacted with a thiol-reactive modifying agent to produce a modified antibody of the present invention.

Modified human antibodies and antigen-binding fragments containing an organic unit that is associated with certain areas of the antibody of the present invention, can be obtained using suitable methods such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3: 147-153 (1992 ); Werlen et al., Bioconjugate Chem., 5: 411-417 (1994); Kumaran et al., Protein Sci. 6(10): 2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1): 59-68 (1996); Capellas et al., Biotechnol. Bioeng., 56(4): 456-463 (1997)), as well as the methods described in Hermanson, G. T., Bioconjugate Techniques , Academic Press: San Diego, CA (1996).

The method of the present invention also employs an anti-IL-12/23p40 antibody composition comprising at least one, at least two, at least three, at least four, at least five, at least six or more anti-IL-12/23p40 antibodies. IL-12/23p40 as described herein and/or known to those skilled in the art, presented as a non-naturally occurring composition, mixture or form. Such compositions are non-naturally occurring compositions containing at least one or two full-length variants, C- and/or N-terminal deletion variants, domains, fragments, or certain amino acid sequence variants of an anti-IL-12/23p40 antibody selected from a group consisting of 70-100% consecutive amino acids from SEQ ID NO: 7 or 8, or their defined fragments, domains or variants. Preferred anti-IL-12/23p40 antibody compositions comprise at least one or two full-length variants, fragments, domains, or variant portions of regions containing at least one CDR or LBP of the anti-IL-12/23p40 antibody sequence described herein, for example, 70-100% of the sequences of SEQ ID NO: 1-6, 7 or 8, or certain fragments, domains or variants thereof. Further preferred compositions contain, for example, 40-99% of at least one of 70-100% of SEQ ID NOs: 1-6, 7 or 8 and the like, or certain fragments, domains or variants thereof. Percentages of a similar composition are determined by weight, volume, concentration, molarity or molality in liquid or dry solutions, mixtures, suspensions, emulsions, particles, powder or colloids, as is known to those skilled in the art or described herein.

Antibody compositions containing additional therapeutically active substances

The antibody compositions used in the method of the present invention may optionally additionally contain an effective amount of at least one compound or protein selected from at least one drug (drug) against infection, drug for the cardiovascular system (CVS), drug for the central nervous systems (CNS), drugs for the autonomic nervous system (ANS), drugs for the respiratory tract, drugs for the gastrointestinal tract (GIT), hormonal drugs, drugs for fluid or electrolyte balance, hematological drugs, antitumor drugs, immunomodulatory drugs, drugs for eyes, ears or nose, topical medicine, nutritional medicine, etc. Such drugs are well known to those skilled in the art, including formulations, indications, dosages, and administration for each drug presented herein (see, for example, Nursing 2001 Handbook of Drugs, ^21st edition, Springhouse Corp., Springhouse, PA, 2001 ; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle River, NJ; Pharmacotherapy Handbook, Wells et al., ed., Appleton & Lange, Stamford, CT, each publication incorporated herein by reference in its entirety).

An example of a drug that can be combined with antibodies for the method of the present invention is an anti-infective drug, which can be at least one selected from amoebicides, or at least one of antiprotozoal, anthelmintic, antifungal, antimalarial, antituberculosis agents, or at least at least one of antileprosy, aminoglycosides, penicillins, cephalosporins, tetracyclines, sulfonamides, fluoroquinolones, antivirals, macrolide anti-infectives and other anti-infectives. The hormonal drug may be at least one selected from corticosteroids, androgens, or at least one of anabolic steroids, estrogens, or at least one progestin, gonadotropin, antidiabetic drug, or at least one of glucagon, thyroid hormone, antagonist thyroid hormone, pituitary hormone and parathyroid hormone-like drugs. The at least one cephalosporin may be at least one selected from cefaclor, cefadroxil, cefazolin sodium, cefdinir, cefepime hydrochloride, cefixime, cefmetazole sodium, cefonicide sodium, cefoperazone sodium, cefotaxime sodium, cefotetan disodium, cefoxitin sodium, cefpodoxime proxetil, cefprozil , ceftazidime, ceftibutene, ceftizoxime sodium, ceftriaxone sodium, cefuroxime axetil, cefuroxime sodium, cephalexin hydrochloride, cephalexin monohydrate, cephradine, and loracarbef.

The at least one corticosteroid may be at least one selected from betamethasone, betamethasone acetate or betamethasone sodium phosphate, betamethasone sodium phosphate, cortisone

- 22 040272 ацетата, дексаметазона, дексаметазона ацетата, дексаметазона натрия фосфата, флудрокортизона ацетата, гидрокортизона, гидрокортизона ацетата, гидрокортизона ципионата, гидрокортизона натрия фосфата, гидрокортизона натрия сукцината, метилпреднизолона, метилпреднизолона ацетата, метилпреднизолона натрия сукцината, преднизолона, преднизолона ацетата, преднизолона натрия фосфата , prednisolone tebutate, prednisone, triamcinolone, triamcinolone acetonide and triamcinolone diacetate. The at least one androgen or anabolic steroid may be at least one selected from danazol, fluoxymesterone, methyltestosterone, nandrolone decanoate, nandrolone phenpropionate, testosterone, testosterone cypionate, testosterone enanthate, testosterone propionate, and testosterone in a transdermal system.

The at least one immunosuppressant may be at least one selected from azathioprine, basiliximab, cyclosporine, daclizumab, lymphocyte immunoglobulin, muromonab CD3, mycophenolate mofetil, mycophenolate mofetil hydrochloride, sirolimus, and tacrolimus.

The at least one topical anti-infective agent may be at least one selected from acyclovir, amphotericin B, azelaic acid cream, bacitracin, butoconazole nitrate, clindamycin phosphate, clotrimazole, econazole nitrate, erythromycin, gentamicin sulfate, ketoconazole, mafenide acetate, metronidazole (topical), miconazole nitrate, mupirocin, naftifine hydrochloride, neomycin sulfate, nitrofurazone, nystatin, silver sulfadiazine, terbinafine hydrochloride, terconazole, tetracycline hydrochloride, thioconazole and tolnaftate. The at least one anti-scabies drug or pediculicide may be at least one selected from crotamiton, lindane, permethrin and pyrethrins. The at least one topical corticosteroid may be at least one selected from betamethasone dipropionate, betamethasone valerate, clobetasol propionate, desonide, deoxymethasone, dexamethasone, dexamethasone sodium phosphate, diflorazone diacetate, fluocinolone acetonide, fluocinonide, flurandrenolide, fluticasone propionate, halcyonide , hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone valerate, mometasone furoate, and triamcinolone acetonide. (See, for example, pp. 1098-1136 in Nursing 2001 Drug Handbook.)

Anti-IL-12/23p40 (or IL-23) antibody compositions may further comprise at least one of any acceptable and effective amounts of a composition or pharmaceutical composition comprising at least one anti-IL-12/23p40 (or IL-23) antibody. 23) that is brought into contact with or introduced into a cell, tissue, organ, animal, or patient in need of such modulation, treatment, or therapy, further optionally containing at least one agent selected from at least one FIO antagonist (e.g., without limitation, a chemical or protein antagonist of PIO, a monoclonal or polyclonal antibody to PIO or a fragment, a soluble PIO receptor (e.g., p55, p70, or p85) or a fragment, a fusion polypeptide thereof, or a small molecule PIO antagonist, such as a PIO binding protein I or II (TBP -1 or TBP-II), nerlimonmab, infliximab, eternacept, CDP-571, CDP-870, afelimomab, lenercept, etc.), antirheumatic drugs (for example, methotrexate, au rananofine, aurothioglucose, azathioprine, etanercept, sodium gold thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), immunization, immunoglobulin, immunosuppressant (eg, basiliximab, cyclosporine, daclizumab), cytokine, or cytokine antagonist. Non-limiting examples of such cytokines include, without limitation, any of IL-1 to IL-23, etc. (eg, IL-1, IL-2, etc.). Acceptable dosages are well known to those skilled in the art. See, for example, Wells et al., eds., Pharmacotherapy Handbook, ^2nd Edition, Appleton and Lange, Stamford, CT (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, CA (2000), each publication is incorporated herein by reference in its entirety.

Compounds, compositions or combinations of antibodies to IL-12/23p40 used in the method of the present invention may additionally contain at least one of any suitable excipients, such as, but not limited to, diluent, binder, stabilizer, buffers, salts, lipophilic diluents, preservative, adjuvant, and the like. Pharmaceutically acceptable excipients are preferred. Non-limiting examples of such sterile solutions and methods for their preparation are well known to those skilled in the art, for example, without limitation, are described in Gennaro, Ed., Remington's Pharmaceutical Sciences, ^18th Edition, Mack Publishing Co. (Easton, PA) 1990. Pharmaceutically acceptable carriers may be selected in a conventional manner based on suitability for the route of administration, solubility, and/or stability of a composition containing an anti-IL-12/23p40 antibody, fragment, or variant, as is well known to those skilled in the art, or described in this document.

Pharmaceutical excipients and additives used in the present composition include, without limitation, proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; sugar derivatives such as alditols, aldonic acids, esterified sugars, and the like; and polysaccharides or polymers of Sugars), which may be present alone or in combination, constituting alone or in combination 1-99.99% by weight or by volume. Examples of protein excipients include serum albumin such as human

- 23 040272 serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, etc.

Typical amino acid/antibody components that can also buffer include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. One preferred amino acid is glycine.

Carbohydrate excipients suitable for use in the present invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides such as raffinose, melicitose, maltodextrins, dextrans, starches, and the like; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucite), myoinositol, and the like. Preferred carbohydrate excipients for use in the present invention are mannitol, trehalose and raffinose.

Anti-IL-12/23p40 (or IL-23) antibody compositions may also include a buffer or pH adjusting agent; typically, the buffer is a salt prepared from an organic or base salt. Typical buffers include salts of organic acids such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid or phthalic acid; buffers Tris, tromethamine hydrochloride or phosphate. Preferred buffers for use in the present compositions are organic acid salts such as citrate.

Additionally, anti-IL-12/23p40 (or anti-IL-23) antibody compositions may include polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (polymeric sugar), dextrates (e.g. cyclodextrins such as 2-hydroxypropyl-p-cyclodextrin), polyethylene glycols, flavors, antimicrobials, sweeteners, antioxidants, antistatic agents, surfactants (eg polysorbates such as TWEEN-20 and TWEEN-80), lipids (eg phospholipids, fatty acids), steroids (eg cholesterol) and chelating agents (eg EDTA).

These and additional known pharmaceutical excipients and/or additives suitable for use in anti-IL-12/23p40 (or anti-IL-23) antibody compositions, parts or variants according to the present invention are known to those skilled in the art, for example, listed in Remington: The Science & Practice of Pharmacy, ^19th ed., Williams & Williams, (1995), and in Physician's Desk Reference, ^52nd ed., Medical Economics, Montvale, NJ (1998), the contents of which are incorporated herein in their entirety. by reference. Preferred carrier materials or excipients are carbohydrates (eg saccharides and alditols) and buffers (eg citrate) or polymeric agents. An example of a carrier molecule is mucopolysaccharide, hyaluronic acid, which can be used for intra-articular administration.

Lineups

As indicated above, the present invention provides stable formulations that preferably contain a phosphate buffer with saline or a selected salt, as well as canned solutions and formulations containing a preservative, as well as canned multiple use formulations suitable for pharmaceutical or veterinary use, containing at least at least one anti-IL-12/23p40 (or IL-23) antibody in a pharmaceutically acceptable formulation. Preserved formulations contain at least one known preservative, or optionally selected from the group consisting of at least one phenol, m-cresol, p-cresol, ocresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (eg, hexahydrate), alkyl paraben (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof in an aqueous diluent. As known to those skilled in the art, any suitable concentration or mixture can be used, such as 0.001-5%, or any range or value therein, such as, without limitation, 0.001, 0.003, 0.005, 0.009, 0.01, 0, 02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2, 2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any interval, or the value in it. Non-limiting examples include no preservatives, 0.1-2% m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), 0.1- 3% benzyl alcohol (e.g. 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g. 0.005, 0 .01%), 0.001-2.0% phenol (e.g. 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkyl paraben (s) (for example, 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0, 2, 0.3, 0.5, 0.75, 0.9, 1.0%), etc.

As noted above, the invention uses an industrial product containing packaging material and at least one vial containing a solution of at least one antibody to IL12/23p40 (or IL-23) with prescribed buffers and/or preservatives, optionally in an aqueous diluent , and the specified packaging material contains a label indicating that such a solution can be stored for a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours or longer. The invention additionally uses an industrial product containing packaging material, the first vial containing a lyophilized antibody to IL-12/23p40 (or to IL-23), and the second

- 24 040272 vial containing an aqueous diluent consisting of a prescribed buffer or preservative, said packaging material containing a label with instructions to the patient on how to dilute an antibody to IL-12/23p40 (or IL-23) in an aqueous diluent to form a solution that can be stored for a period of twenty-four hours or longer.

The anti-IL-12/23p40 (or anti-IL-23) antibody used in accordance with the present invention may be produced by recombinant methods, including from mammalian cells or transgenic preparations, or may be purified from other biological sources as described herein. document or as known to those skilled in the art.

The range of amounts of anti-IL-12/23p40 (or anti-IL-23) antibody includes amounts that, after dilution, in the case of a wet/dry system, reach concentrations from about 1.0 μg/mL to about 1000 mg/mL, although lower values are suitable. and high concentrations, and they depend on the intended carrier medium for administration, for example, solution compositions differ for transdermal patch, pulmonary, mucosal, or osmotic, or microdosing methods.

Preferably, the aqueous diluent optionally contains a pharmaceutically acceptable preservative. Preferred preservatives include those selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl paraben (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof. The concentration of preservative used in the composition must be sufficient to achieve antimicrobial activity. This concentration depends on the chosen preservative and is easily determined by a qualified specialist.

Preferably, other excipients are optionally added to the diluent, such as isotonic agents, buffers, antioxidants, and preservation enhancers. Isotonic agents such as glycerol are widely used at known concentrations. Preferably, a physiologically acceptable buffer is added to improve pH control. The compositions may cover a wide range of pH, such as from about pH 4 to about pH 10, with the preferred range from about pH 5 to about pH 9, and the most preferred range from about pH 6.0 to about pH 8.0. Preferably, the compositions of the present invention have a pH of about 6.8 to about 7.8. Preferred buffers include phosphate buffers, most preferably sodium phosphate, in particular phosphate buffered saline (PBS).

Other additives may optionally be added to formulations or compositions to reduce aggregation, such as pharmaceutically acceptable solubilizers, e.g. ) sorbitan monooleate), Pluronic F68 (block copolymers of polyoxyethylene and polyoxypropylene) and PEG (polyethylene glycol), or non-ionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or 188, Pluronic® polyols, other block copolymers, and chelating agents such as EDTA and EGTA. These additives are particularly useful if a pump or plastic container is used to administer the composition. The presence of a pharmaceutically acceptable surfactant reduces the propensity of the protein to aggregate.

The formulations can be prepared in a process that comprises mixing at least one anti-IL-12/23p40 (or anti-IL-23) antibody and a preservative selected from the group consisting of phenol, mcresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl paraben (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof in an aqueous diluent. Mixing of at least one antibody to IL-12/23p40 (or to IL23) and preservative in an aqueous diluent is carried out using conventional dissolution and mixing procedures. For example, to prepare a suitable formulation, a measured amount of at least one anti-IL-12/23p40 (or anti-IL-23) antibody in a buffer solution is combined with the desired preservative in the buffer solution in amounts sufficient to obtain the desired concentrations of protein and preservative. Variations of this process will be understood by one of ordinary skill in the art. For example, factors such as the order in which components are added, the addition of additional additives, and the temperature and pH at which the formulation is prepared can be varied to optimize for concentration and route of administration employed.

Formulations may be provided to patients as clear solutions or as two vials containing a vial of lyophilized anti-IL-12/23p40 (or anti-IL-23) antibody diluted with the second vial's water, preservative and/or excipients, preferably phosphate buffer and/or or saline and selected salt, in an aqueous diluent. Either one solution vial or two reconstituted vials can be used multiple times and are sufficient for one or more patient treatment cycles, which is more convenient than the current treatment regimen.

The present commercial products are suitable for administration either immediately or over a period of 24 hours or longer. Accordingly, the industrial products claimed herein provide significant benefits to patients. Compositions of the present invention optional

- 25 040272 can be safely stored at a temperature of from about 2 to about 40°C, retaining the biological activity of the protein for extended periods of time, and therefore a label is allowed on the package indicating that the solution can be stored and / or used for a period of 6 , 12, 18, 24, 36,

48, 72, or 96 hours or more. If a diluent with a preservative is used, then the label may indicate a shelf life of up to 1-12 months, six months, one and a half and / or two years.

Anti-IL-12/23p40 (or IL-23) antibody solutions can be prepared in a process that includes mixing at least one antibody in an aqueous diluent. Mixing is carried out using conventional dissolution and mixing procedures. For example, to prepare an acceptable diluent, a measured amount of at least one antibody in water or buffer is combined in amounts sufficient to obtain the desired concentrations of protein and, optionally, a preservative or buffer. Variations of this process will be understood by one of ordinary skill in the art. For example, factors such as the order in which components are added, the addition of additional additives, and the temperature and pH at which the formulation is prepared can be varied to optimize for concentration and route of administration employed.

The claimed products can be provided to patients in the form of clear solutions or two vials containing a vial of at least one lyophilized anti-IL-12/23p40 (or IL-23) antibody, which is diluted with the aqueous diluent contained in the second vial. Either one solution vial or two reconstituted vials can be used multiple times and are sufficient for one or more patient treatment cycles, which is more convenient than the current treatment regimen.

Claimed products can be provided to patients indirectly, but by supplying pharmacies, clinics or other such organizations and institutions with clear solutions or two vials containing a vial of at least one lyophilized anti-IL-12/23p40 (or anti-IL23) antibody, which is diluted contained in the second vial with an aqueous diluent. In this case, the volume of the clear solution can be up to one liter or even more, thereby providing a large vessel from which a solution of at least one antibody can be dosed in small portions in a pharmacy or clinic, single or multiple, for transfusion into smaller vials and providing to customers and/or patients.

Commonly recognized devices containing single vial systems include pen type injection devices such as BD Pens, BD Autojector®, Humaject®, NovoPen®, B-D®Pen, AutoPen® and OptiPen®, GenotropinPen®, Genotronorm Pen® , Humatro Pen®, Reco-Pen®, Roferon Pen®, Biojector®, Iject®, J-tip Needle-Free Injector®, Intraject®, Medi-Ject®, Smartject,® e.g. manufactured or developed by Becton Dickensen (Franklin Lake, NJ, USA, www.bectondickenson.com), Disetronic (Burgdorf, Switzerland, www.disetronic.com; Bioject, Portland, Oregon, USA (www.bioject.com); National Medical Products, Weston Medical (Peterborough, UK, www.weston-medical.com), Medi-Ject Corp. (Minneapolis, Minnesota, USA, www.mediject.com), and similar acceptable devices. of two vials, include such syringe-pen systems for reconstituting a lyophilized drug in a cartridge for administering a diluted solution, such as HumatroPen®. Examples of other acceptable devices include pre-filled syringes, auto-injectors, needleless injectors, and needleless IV sets.

Products include packaging material. In addition to information requested by regulatory authorities, the packaging material also indicates the conditions under which the product can be used. The packaging material of the present invention contains instructions for the patient, if applicable, to dilute at least one antibody to IL-12/23p40 (or to IL-23) in an aqueous diluent to obtain a solution, and to use the solution for a period of 2-24 h or longer in the case of two vials, wet/dry, with the product. In the case of a single vial of solution, pre-filled syringe or auto-injector product, the package indicates that such solution can be used for a period of 2-24 hours or longer. The products are intended for human use for pharmaceutical purposes.

The compositions used in the method of the present invention can be prepared in a process that comprises mixing an anti-IL-12/23p40 (or anti-IL-23) antibody and a selected buffer, preferably a phosphate buffer containing saline or a selected salt. Mixing of the anti-IL-12/23p40 (or anti-IL-23) antibody and buffer in aqueous diluent is carried out using conventional dissolution and mixing procedures. For example, to prepare an acceptable composition, a measured amount of at least one antibody in water or buffer is combined with the desired buffering agent in water in amounts sufficient to obtain the desired protein and buffer concentrations. Variations of this process will be understood by one of ordinary skill in the art. For example, factors such as the order in which components are added, the addition of additional additives, and the temperature and pH at which the formulation is prepared can be varied to optimize for the concentration and route of administration used.

- 26 040272

The method of the invention provides pharmaceutical compositions containing various formulations useful and acceptable for administration to a patient, human or animal. Such pharmaceutical compositions are prepared using standard water as diluent and by conventional methods well known to those of ordinary skill in the art. For example, buffer components such as histidine and histidine monohydrochloride hydrate may be provided first, followed by the addition of a suitable, non-final volume, aqueous diluent, sucrose and polysorbate-80 in standard form. The isolated antibody can then be added. Finally, the volume of the pharmaceutical composition is adjusted to the desired final volume under standard conditions by adding water as a diluent. Those skilled in the art will appreciate a number of other methods suitable for the preparation of pharmaceutical compositions.

Pharmaceutical compositions may be aqueous solutions or suspensions containing the specified weight of each component per unit volume of water or having the specified pH in the standard state. In the context of this document, the term reference state means a temperature of 25°C ± 2°C and a pressure of 0.1 megapascal (1 atmosphere). The term reference state is not used in the art to refer to a single recognized set of temperatures or pressures, but instead is a reference state that defines the temperature and pressure specified to describe a solution or slurry of a specific composition under reference reference conditions of the reference state. This is because the volume of the solution depends in part on temperature and pressure. Those skilled in the art will appreciate that pharmaceutical compositions equivalent to those described herein can be produced at other temperatures and pressures. Whether such pharmaceutical compositions are equivalent to those described herein should be determined under the standard conditions defined above (eg, temperature 25° C. ± 2° C. and pressure 0.1 megapascal (1 atm)).

It is important to note that such pharmaceutical compositions may contain component weights of about a certain value (eg, about 0.53 mg of L-histidine) per unit volume of the pharmaceutical composition, or have pH values of about a certain value. The weight of the component present in the pharmaceutical composition, or the pH value, is about a given numerical value if the isolated antibody present in the pharmaceutical composition is capable of binding a peptide chain containing residues 1-88 with the sequence of SEQ ID NO: 9, while in isolated antibody is present in the pharmaceutical composition, or after the isolated antibody has been removed from the pharmaceutical composition (eg, by dilution). In other words, the value, such as the weight value of the component or the pH value, is about a given numerical value when the binding activity of the isolated antibody is maintained and detected after the isolated antibody is placed in a pharmaceutical composition.

To determine if anti-IL-12/23p40 (or anti-IL-23) mAbs bind to similar or different epitopes and/or compete with each other, competitive binding assays are performed. The antibodies are applied individually to solid phase ELISA plates as a coating. Competing mAbs are added followed by biotinylated LrIL-12/23p40. For the positive control, the same mAb as for the coating is used as the competing mAb (self-competition). IL-12/23p40 binding is determined using streptavidin. These results indicate whether mAbs recognize similar or partially overlapping epitopes on IL-12/23p40.

One aspect of the method of the present invention comprises administering to a patient a pharmaceutical composition comprising an isolated antibody that binds to a peptide chain containing residues 1-88 of SEQ ID NO: 9; from about 0.27 to about 0.80 mg of L-histidine per milliliter of pharmaceutical composition; about 0.69 to about 2.1 mg of L-histidine monohydrochloride monohydrate per milliliter of pharmaceutical composition; about 0.02 to about 0.06 mg of polysorbate 80 per milliliter of pharmaceutical composition; and about 65 to about 87 mg of sucrose per milliliter of pharmaceutical composition; wherein the diluent is water in the standard state.

Another aspect of the present invention comprises administering a pharmaceutical composition comprising an isolated antibody having (i) the amino acid sequences of the heavy chain CDRs of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and (ii) the amino acid sequences of the light chain CDRs of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, wherein the isolated antibody binds to a peptide chain containing residues 1-88 of SEQ ID NO: 9; from about 0.27 to about 0.80 mg L histidine per milliliter of pharmaceutical composition; about 0.69 to about 2.1 mg of L-histidine monohydrochloride monohydrate per milliliter of pharmaceutical composition; from about 0.02 to about 0.06 mg of polysorbate-80 per milliliter of pharmaceutical composition and from about 65 to about 87 mg of sucrose per milliliter of pharmaceutical composition; wherein the diluent is water in the standard state.

Another aspect of the method of the present invention comprises administering to a patient a pharmaceutical composition comprising an isolated anti-IL-12/23p40 antibody having the heavy chain amino acid sequence of SEQ ID NO: 7 and the light chain amino acid sequence of SEQ ID NO: 8,

- 27 040272 wherein the isolated antibody binds to a peptide chain containing residues 1-88 with the sequence of SEQ ID NO: 9; from about 0.27 to about 0.80 mg of L-histidine per milliliter of pharmaceutical composition; about 0.69 to about 2.1 mg of L-histidine monohydrochloride monohydrate per milliliter of pharmaceutical composition; from about 0.02 to about 0.06 mg of polysorbate-80 per milliliter of pharmaceutical composition; and about 65 to about 87 mg of sucrose per milliliter of pharmaceutical composition; wherein the diluent is water in the standard state.

In one embodiment of pharmaceutical compositions, the concentration of isolated antibody is from about 77 to about 104 mg per milliliter of pharmaceutical composition. In another embodiment of the pharmaceutical compositions, the pH is from about 5.5 to about 6.5.

Another aspect of the method of the present invention comprises administering to a patient a pharmaceutical composition comprising an isolated anti-IL-12/23p40 antibody having (i) the amino acid sequences of the heavy chain CDRs of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and ( ii) the amino acid sequences of the light chain CDRs of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, wherein the isolated antibody binds to a peptide chain containing residues 1-88 of SEQ ID NO: 9; about 0.53 mg of L-histidine per milliliter of pharmaceutical composition; about 1.37 mg of L-histidine monohydrochloride monohydrate per milliliter of pharmaceutical composition; about 0.04 mg of polysorbate-80 per milliliter of pharmaceutical composition; and about 76 mg of sucrose per milliliter of pharmaceutical composition; wherein the diluent is water in the standard state.

An additional aspect of the method of the present invention comprises administering to a patient a pharmaceutical composition comprising an isolated anti-IL-12/23p40 antibody having the heavy chain amino acid sequence of SEQ ID NO: 7 and the light chain amino acid sequence of SEQ ID NO: 8, wherein the isolated antibody binds to the peptide chain containing residues 1-88 with the sequence of SEQ ID NO: 9; about 0.53 mg of L-histidine per milliliter of pharmaceutical composition; about 1.37 mg of L-histidine monohydrochloride monohydrate per milliliter of pharmaceutical composition; about 0.04 mg of polysorbate-80 per milliliter of pharmaceutical composition and about 76 mg of sucrose per milliliter of pharmaceutical composition; wherein the diluent is water in the standard state.

In one embodiment, the concentration of isolated antibody is about 90 mg per milliliter of the pharmaceutical composition. In another embodiment of these pharmaceutical compositions, the pH is about 6.0. Another aspect of the present invention is the administration of a pharmaceutical composition comprising an antibody that competes for binding with an anti-IL-12/23p40 antibody described herein, eg, binds to a peptide chain containing residues 1-88 of SEQ ID NO: 9; about 0.27 to about 0.80 mg of L-histidine per milliliter of pharmaceutical composition; about 0.69 to about 2.1 mg of L-histidine monohydrochloride monohydrate per milliliter of pharmaceutical composition; about 0.02 to about 0.06 mg of polysorbate 80 per milliliter of pharmaceutical composition; and about 65 to about 87 mg of sucrose per milliliter of pharmaceutical composition; wherein the diluent is water in the standard state.

Stable or canned formulations may be provided to patients as clear solutions or two vials containing a vial of at least one lyophilized anti-IL-12/23p40 antibody that is diluted with the preservative or buffer contained in the second vial and excipients in an aqueous diluent. Either one solution vial or two reconstituted vials can be used multiple times and are sufficient for one or more patient treatment cycles, which is more convenient than the current treatment regimen.

Other formulations or methods for stabilizing anti-IL-12/23p40 (or anti-IL-23) antibodies may result in an antibody-containing agent other than a clear lyophilized powder solution. Opaque solutions include, in particular, formulations containing suspended particles, which are compositions containing an antibody to IL-12/23p40 (or IL-23) in structures with varying sizes, and are known as microspheres, microparticles, nanoparticles, nanospheres or liposomes. Such relatively uniform, substantially spherical, particulate formulations containing the active agent can be formed by bonding an aqueous phase containing the active agent and a polymer to a non-aqueous phase, followed by evaporation of the non-aqueous phase and fusion of the particles from the aqueous phase, as described in the patent. US No. 4589330. Porous microparticles can be obtained by using the first phase containing the active substance and polymer dispersed in a continuous solvent, and removing said solvent from the suspension by freeze drying or dilution, extraction and precipitation, as described in US patent No. 4818542. Preferred polymers for such preparations are natural or synthetic copolymers or polymers selected from the group consisting of gelatin agar, starch, arabinogalactan, albumin, collagen, polyglycolic acid, polylactic acid, glycolide-L(-)-lactide, poly(epsilon-caprolactone) , poly(epsilon-caprolactone-CO-lactic acid), poly(epsilon -caprolactone-CO-glycolic acid), poly(B-hydroxybutyric acid), polyethylene oxide, polyethylene, poly(alkyl-2-cyanoacrylate), poly(hydroxyethyl methacrylate), polyamides, poly(amino acids), poly(2-hydroxyethyl-DL- aspart

- 28 040272 mid), poly(urea ether), poly(L-phenylalanine/ethylene glycol/1,6-diisocyanatehexane) and poly(methyl methacrylate). The most preferred polymers are polyesters such as polyglycolic acid, polylactic acid, β(-)-lactide glycolide, poly(epsilon-caprolactone), poly(epsilon-caprolactone-CO-lactic acid) and poly(epsilon-caprolactone-CO-glycolic acid). Suitable solvents for dissolving the polymer and/or active agent include: water, hexafluoroisopropanol, methylene chloride, tetrahydrofuran, hexane, benzene or hexafluoroacetone sesquihydrate. The process of dispersing the active substance-containing phase with the second phase may include forcibly passing the first phase through an orifice in the nozzle to form droplets.

Dry powder formulations can be prepared by methods other than lyophilization, such as spray drying, solvent evaporation, or precipitation of a crystalline composition followed by one or more aqueous or non-aqueous solvent removal steps. The preparation of an antibody preparation by spray drying is described in US Pat. Solvents may include polar compounds such as water and ethanol, which can be easily dried. The stability of the antibody can be enhanced by performing a spray drying procedure in the absence of oxygen, such as under nitrogen or using nitrogen as a drying gas. Another relatively dry formulation is a dispersion of a plurality of perforated microstructures dispersed in a suspension medium, typically containing hydrofluoroalkane propellant, as described in WO 9916419. The stabilized dispersions can be administered to the patient's lungs using a metered dose inhaler. The equipment used for industrial production of drugs by spray drying is manufactured by Buchi Ltd. or Niro Corp.

An anti-IL-12/23p40 (or anti-IL-23) antibody in the stable or preservative formulations or solutions described herein can be administered to a patient by a variety of delivery methods, including subcutaneous or intramuscular injection; transdermally, into the lungs, through the mucosa, via an implant, an osmotic dispenser, a cassette, a microdispenser, or other methods recognized by those skilled in the art, as is well known in the art.

Therapeutic use

The present invention also provides a method for modulating or treating at least one IL-12/23 associated disease in a cell, tissue, organ, animal, or patient known to those skilled in the art or described herein, using at least one anti-IL-12/23p40 (or anti-IL-23) antibody of the present invention, for example, by administering or exposing a therapeutically effective amount of an anti-IL-12/23p40 (or anti-IL-23) antibody to a cell, tissue, organ , animal or patient. The present invention also provides a method for modulating or treating at least one IL-12/23 related disease in a cell, tissue, organ, animal, or patient, including but not limited to at least one of obesity, immune disease , cardiovascular disease, infectious disease, malignant disease, or neurological disease.

The present invention also provides a method for modulating or treating at least one IL-12/23-associated immune disease in a cell, tissue, organ, animal, or patient, including, without limitation, at least one of the following: psoriasis , psoriatic arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, juvenile rheumatoid arthritis with systemic manifestations, ankylosing spondylitis, gastric ulcer, seronegative arthropathies, osteoarthritis, osteolysis, aseptic loosening of orthopedic implants, inflammatory bowel disease, ulcerative colitis, systemic lupus erythematosus, antiphospholipid syndrome, iridocyclitis/uveitis/optic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis/Wegener's granulomatosis, sarcoidosis, orchitis/reverse vasectomy procedures, allergic/atopic disease, asthma, allergic rhinitis, eczema, allergic contact dermatitis, allergic conjunctivitis, hypersensitivity pneumonitis, trans plantation, organ transplant rejection, graft-versus-host disease, systemic inflammatory response syndrome, sepsis syndrome, gram-positive sepsis, gram-negative sepsis, culture-negative sepsis, fungal sepsis, neutropenic fever, urosepsis, meningococcemia, trauma/bleeding, burns, exposure to ionizing radiation , acute pancreatitis, adult respiratory distress syndrome, rheumatoid arthritis, alcoholic hepatitis, chronic inflammatory conditions, sarcoidosis, Crohn's disease, sickle cell anemia, diabetes, nephrosis, atopic diseases, hypersensitivity reactions, allergic rhinitis, hay fever, prolonged rhinitis, conjunctivitis , endometriosis, asthma, urticaria, systemic anaphylaxis, dermatitis, pernicious anemia, hemolytic disease, thrombocytopenia, transplant rejection of any organ or tissue, kidney transplant rejection, heart transplant rejection, transplant rejection liver anthate, pancreas transplant rejection, lung transplant rejection, bone marrow transplant (BMT) rejection, ko allograft rejection

- 29 040272 zhi, cartilage graft rejection, bone graft rejection, small intestine graft rejection, fetal thymus implant rejection, parathyroid graft rejection, xenograft rejection of any organ or tissue, allograft rejection, antireceptor hypersensitivity reactions, Graves' disease, Raynaud's disease, insulin resistant sugar type B diabetes, asthma, myasthenia gravis, antibody-mediated cytotoxicity, type III hypersensitivity reactions, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin changes), polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin change syndrome, antiphospholipid syndrome , pemphigus, scleroderma, mixed connective tissue disease, idiopathic Addison's disease, diabetes mellitus, chronic active hepatitis, primary biliary cirrhosis, vitiligo, vasculitis, cardiotomy syndrome after myocardial infarction, hyperch Type IV sensitivity, contact dermatitis, hypersensitivity pneumonitis, allograft rejection, intracellular granulomas, metabolic/idiopathic drug sensitivity, Wilson's disease, hemachromatosis, alpha-1 antitrypsin deficiency, diabetic retinopathy, Hashimoto's thyroiditis, osteoporosis, hypothalamic assessment - pituitary-adrenal axis, primary biliary cirrhosis, thyroiditis, encephalomyelitis, cachexia, cystic fibrosis, chronic lung disease in newborns, chronic obstructive pulmonary disease (COPD), familial hemophagocytic lymphohistiocytosis, dermatological conditions, alopecia, nephrotic syndrome, nephritis, glomerulonephritis, acute renal insufficiency, hemodialysis, uremia, toxicity, preeclampsia, OKT3 therapy, anti-CD3 antibody therapy, cytokine therapy, chemotherapy, radiation therapy (for example, including, but not limited to, asthenia, anemia, cachexia, etc.), chronic salicylate intoxication, etc. n. See, n e.g., Merck Manual, 12th-17th Editions, Merck & Company, Rahway, NJ (1972, 1977, 1982, 1987, 1992, 1999), Pharmacotherapy Handbook, Wells et al., eds., Second Edition, Appleton and Lange, Stamford , Conn. (1998, 2000), each publication is incorporated herein by reference in its entirety. Such a method may optionally comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one anti-TNF antibody, or a specified portion or variant, to a cell, tissue, organ, animal, or patient in need of such modulation, treatment, or therapy. See, for example, Merck Manual, ^16th Edition, Merck & Company, Rahway, NJ (1992).

The present invention also provides a method for modulating or treating psoriasis, psoriatic arthritis, Crohn's disease, other inflammatory bowel diseases, lupus, sarcoidosis, AS or nrAxSpA, among other diseases listed above as being associated with IL-12/23, in a cell, tissue, organ, animal, or patient, including, without limitation, at least one of an immune disease, a cardiovascular disease, an infectious, malignant, and/or neurological disease. Such a method may optionally comprise administering an effective amount of at least one composition or pharmaceutical composition comprising an anti-IL-12/23p40 (or anti-IL-23) antibody to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.

Any method of the present invention may comprise administering an effective amount of an anti-IL-12/23p40 (or anti-IL-23) antibody composition or pharmaceutical composition to a cell, tissue, organ, animal, or patient in need of such modulation, treatment, or therapy. . Such a method may optionally further comprise the co-administration or use of a combination therapy for the treatment of such diseases or disorders, wherein the administration of said at least one antibody to IL-12/23p40 (or to IL-23), a specified portion or variant thereof, further comprises administering (before, simultaneously and/or after) at least one agent selected from at least one TNF antagonist (for example, without limitation, a chemical or protein TNF antagonist, an anti-TNF monoclonal or polyclonal antibody or fragment thereof, a soluble TNF receptor (for example, , p55, p70, or p85) or a fragment thereof, a fusion polypeptide thereof, or a small molecule TNF antagonist such as TNF-binding protein I or II (TBP-1 or TBP-II), nerlimonmab, infliximab, eternacept (Enbrel™), adalimulab (Humira ™), CDP-571, CDP-870, afelimomab, lenercept, etc.), antirheumatic drugs (for example, methotrexate, auranofin, aurothioglucose, azathioprine, zol ota-sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzin), muscle relaxant, narcotic drug, non-steroidal anti-inflammatory drug (NSAID), analgesic, anesthetic drug, sedative drug, local anesthetic drug, neuromuscular blocker, antimicrobial drug (eg, aminoglycoside, antifungal drug, antiparasitic drug, antiviral drug, carbapenem, cephalosporin, fluoroquinolone, macrolide, penicillin, sulfonamide, tetracycline, other antimicrobial drug), antipsoriatic drug, corticosteroid, anabolic steroid, drug for the treatment of diabetes mellitus, mineral, dietary drug, thyroid drug, vitamin , calcium regulating hormone, drug against diarrhea, drug against cough, antiemetic drug, drug against ulcer, laxative drug, anticoagulant, erythropoietin (for example, epoetin alfa), filgrastim (for example, G-CSF, Neupogen), sargramostim (GM-CSF, Leukine), immunization, immunoglobulin, immunosuppressant (eg, basiliximab, cyclosporine, daclizumab), growth hormone, hormone replacement therapy, estrogen receptor modulator, mid

- 30 040272 riatika, drug of cycloplegia, alkylating agent, antimetabolite, inhibitor of mitosis, radiopharmaceutical drug, antidepressant, drug against mania, antipsychotic drug, anxiolytic drug, hypnotic drug, sympathomimetic, excitatory drug, donepezil, tacrine, drug for asthma, beta-agonist, an inhaled steroid, a leukotriene inhibitor, methylxanthine, cromolyn, epinephrine, or an analogue thereof, dornase alfa (Pulmozyme), a cytokine, or a cytokine antagonist. Acceptable dosages are well known to those skilled in the art. See, for example, Wells et al., eds., Pharmacotherapy Handbook, ^2nd Edition, Appleton and Lange, Stamford, CT (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, CA (2000); Nursing 2001 Handbook of Drugs, ^21st edition, Springhouse Corp., Springhouse, PA, 2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc., Upper Saddle River, NJ, all of which are hereby incorporated by reference in their entirety.

Therapeutic treatments

Typically, the treatment of pathological conditions is carried out by administering an effective amount or dose of an anti-IL-12/23p40 (or anti-IL-23) antibody composition, which in total, on average, contains from at least about 0.01 to 500 mg of anti-IL antibody. -12 / 23p40 (or to IL-23) per kilogram of patient weight in a single dose, and preferably from at least about 0.1 to 100 mg of antibody per kilogram of patient weight in one or more injections, depending on the specific activity of the active agent, contained in the composition. Alternatively, an effective serum concentration may be 0.1-5000 μg/ml serum in one or more administrations. Suitable dosages are known to those skilled in the art and will, of course, depend on the particular disease state, the specific activity of the composition being administered, and the particular patient being treated. In some cases, to achieve the desired therapeutic amount, it may be necessary to perform repeated administration, i. repeated separate injections of a specific controlled or measured dose, and individual injections are repeated until the desired daily dose or effect is achieved.

Preferred dosages 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, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66,

67, 68, 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 100-500 mg/kg per administration, or any interval, value or part of this range, or amount to achieve a serum concentration of 0.1, 0.5, 0.9, 1, 0, 1.1, 1.2, 1.5, 1.9, 2.0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8, 9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 20, 12.5, 12.9, 13.0, 13, 5, 13.9, 14.0, 14.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 12, 12.5, 12.9, 13.0, 13.5, 13.9, 14, 14.5, 15, 15.5, 15.9, 16, 16.5, 16.9, 17, 17, 5, 17.9, 18, 18.5, 18.9, 19, 19.5, 19.9, 20, 20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 and/or 5000 µg/ml of serum for a single or multiple administration, or any interval, value or part of this range.

Alternatively, the doses administered may vary depending on known factors such as the pharmacodynamic properties of the particular agent, the mode and route of its administration; age, health status and weight of the recipient; nature and severity of symptoms, type of concomitant treatment, frequency of administration, and desired effect. Typically, the dose of the active ingredient is from about 0.1 to 100 mg per kilogram of body weight. Generally, 0.1 to 50 and preferably 0.1 to 10 mg per kilogram, in a single administration or in a sustained release dosage form, will be effective in achieving the desired results.

As a non-limiting example, the treatment of humans or animals can be carried out in the form of a single or periodic administration of at least one antibody of the present invention at a dose of from 0.1 to 100 mg/kg, for example, 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 mg/kg per day on at least one of days 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 weeks 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,

24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52, or alternatively or additionally, at least one of 1, 2, 3, 4, 5, b, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19 or 20, or any combination thereof, with the introduction of a single, infusion or repeated doses.

Dosage forms (composition) suitable for internal administration usually contain from about 0.001 to about 500 mg of the active ingredient per unit or container. In these pharmaceutical compositions, the active ingredient is usually present in an amount of about 0.5-99.999% by weight, based on the total weight of the composition.

For parenteral administration of an antibody, the dosage form may be a solution, suspension, emulsion, particle, powder, or lyophilized powder, together with or separately from a pharmaceutically acceptable carrier for parenteral administration. Examples of such carriers are water, saline, Ringer's solution, glucose solution, and human serum albumin 1-10%. Liposomes and anhydrous media, such as fixed oils, can also be used for this purpose. The carrier or lyophilized powder may contain additives to promote isotonicity (eg sodium chloride, mannitol) and chemical stability (eg buffers and preservatives). The composition is sterilized by known or acceptable methods.

Acceptable pharmaceutical carriers are described in the latest edition of Remington's Pharmaceutical Sciences, A. Osol, which is the standard reference in this field.

Alternative routes of administration

In accordance with the present invention for the introduction of pharmaceutically effective amounts of antibodies to IL-12/23p40 (or IL-23) can be applied to a variety of well-known and developed ways of conducting. The following describes the introduction through the lungs, however, in accordance with the present invention can also be applied to other methods of administration, giving acceptable results. Anti-IL12/23p40 (or IL-23) antibodies of the present invention may be delivered in a vehicle as a solution, emulsion, colloid, or suspension, or as a dry powder, using any of a variety of devices and methods suitable for administration by inhalation or other by the methods described herein or known to those skilled in the art.

Parenteral formulations and administration

Formulations for parenteral administration may contain sterile water, saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogenated naphthalenes, and the like as common excipients. Aqueous or oily injection suspensions can be prepared using a suitable emulsifier or humectant and suspending agent by known methods. For injection, a non-toxic diluting agent suitable for non-oral administration may be used, for example an aqueous solution, a sterile injectable solution, or a suspension in a solvent. As a suitable carrier medium or solvent, it is acceptable to use water, Ringer's solution, isotonic solution, etc.; a sterile fixed oil can be used as a conventional solvent or suspending solvent. For these purposes, you can use a fixed oil and fatty acid of any kind, including natural or synthetic or semi-synthetic fatty oils or fatty acids; natural or synthetic or semi-synthetic mono-, di- or triglycerides. Parenteral administration is known in the art and includes, without limitation, conventional injections, the pneumatic needleless injection device described in US Pat. No. 5,851,198, and the laser perforator described in US Pat.

Alternative shipping methods

The invention additionally relates to the administration of an antibody to IL-12/23p40 (or to IL-23) by parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraperitoneal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intraventricular, intracolon administration routes , intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, into the prostate, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, into the affected tissues, bolus, vaginal, rectal , buccal, sublingual, intranasal, or transdermal. An anti-IL-12/23p40 (or anti-IL-23) antibody composition can be formulated for parenteral (subcutaneous, intramuscular, or intravenous) or any other route of administration, particularly in the form of liquid solutions or suspensions; for use by the vaginal or rectal route, particularly in semi-solid forms such as, but not limited to, creams and suppositories; for buccal or sublingual administration, for example, without limitation, in the form of tablets or capsules; or for intranasal administration, for example, without limitation, in the form of powders, nasal drops or aerosols, or in the form of certain agents; or for transdermal administration, for example, without limitation, in the form of delivery systems in a gel, ointment, lotion, suspension or patch with chemical accelerators such as dimethyl sulfoxide, either to modify the structure of the skin, or to increase the concentration of the drug in the transdermal patch (Junginger, et al., In Drug Permeation Enhancement; Hsieh, D. S., Eds., pp. 5990 (Marcel Dekker, Inc. New York 1994, incorporated herein by reference in its entirety), or with oxidizing agents that facilitate application of formulations containing proteins and peptides, to the skin (WO 98/53847), or by using an electric field to create temporary delivery trajectories, for example, by electroporation, or to accelerate the movement of charged drugs through the skin, for example, by iontophoresis, or by the application of ultrasound, for example, sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) (The above publications and patents are hereby incorporated by reference in their entirety).

The above general description of the invention is further explained further by means of examples, which are presented by way of illustration and are not limiting. Additional details of the invention are illustrated by the following non-limiting

- 32 040272 examples. The disclosure of all quotations in the specification is expressly incorporated herein by reference.

Example 1 Cloning and Expression of an Anti-IL-12 Antibody in Mammalian Cells

A typical mammalian expression vector contains at least one promoter element mediating the initiation of mRNA transcription, an antibody coding sequence, and signals necessary for transcription termination and transcript polyadenylation. Additional elements include enhancers, Kozak sequences, and introns flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with early and late promoters from SV40, long terminal repeats (LTRs) from retroviruses such as RSV, HTLVI, HIVI, and the cytomegalovirus (CMV) early promoter. However, cell elements (eg, human actin promoter) can also be used. Suitable expression vectors for use in the practice of the present invention include, for example, pIRESIneo, pRetro-Off, pRetro-On, PLXSN or pLNCX (Clonetech Labs, Palo Alto, CA, USA), pcDNA3.1 ( +/-), pcDNA/Zeo (+/-) or pcDNA3.1/Hygro (+/-) (Invitrogen), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109). Mammalian cells that can be used include human Hela 2 93, H9 and Jurkat cells, mouse NIH3T3 and C127, Cos 1, Cos 7 and CV 1 cells, QC1-3 quail cells, mouse L cells and Chinese hamster ovary cells ( SNO). Alternatively, the gene can be expressed in stable cell lines containing the gene integrated into the chromosome. Co-transfection with a selectable marker such as dhfr, gpt, neomycin or hygromycin allows the identification and isolation of transfected cells.

The transfected gene can also be amplified to express large amounts of the encoded antibody. The marker DHFR (dihydrofolate reductase) is used to develop cell lines carrying several hundred or even several thousand copies of the gene of interest. Another useful selectable marker is the enzyme glutamine synthase (GS) (Murphy, et al., Biochem. J. 227: 277279 (1991); Bebbington, et al., Bio/Technology 10: 169-175 (1992)). Using such markers, mammalian cells are grown in a selective medium and cells with the highest resistance are selected. Such cell lines contain amplified(e) gene(s), integrated(e) into the chromosome. Chinese hamster ovary (CHO) cells and NSO cells are often used to produce antibodies.

Expression vectors pC1 and pC4 contain the Rous sarcoma virus strong promoter (LTR) (Cullen, et al., Molec. Cell. Biol. 5: 438-447 (1985)) with a CMV enhancer fragment (Boshart, et al., Cell 41: 521530 (1985)). Multiple cloning sites, such as BamHI, XbaI, and Asp718 restriction enzyme cleavage sites, facilitate cloning of the gene of interest. In addition to the 3'-intron, the vectors contain a polyadenylation signal and a termination signal for the rat preproinsulin gene.

Cloning and expression in CHO cells

The pC4 vector is used to express the anti-IL-12/23p40 antibody. Plasmid pC4 is a derivative of plasmid pSV2-dhfr (cat. no. ATCC 37146). The plasmid contains the mouse DHFR gene under the control of the SV40 early promoter. Ovary or other Chinese hamster cells lacking dihydrofolate activity transfected with these plasmids can be selected by growing the cells in a selective medium (e.g. alpha minus MEM, Life Technologies, Gaithersburg, MD, USA) supplemented with the chemotherapy drug methotrexate. Amplification of DHFR genes in methotrexate (MTX) resistant cells has been well described in the past (see, e.g., F. W. Alt, et al., J. Biol. Chem. 253: 1357-1370 (1978); J. L. Hamlin and C Ma, Biochem et Biophys Acta 1097: 107143 (1990); M. J. Page and M. A. Sydenham, Biotechnology 9: 64-68 (1991)). Cells grown at increasing concentrations of MTX develop resistance to this drug by overproduction of the target enzyme, DHFR, as a result of amplification of the DHFR gene. If a second gene is attached to the DHFR gene, as a rule, it is co-amplified and overexpressed. It is known to those skilled in the art that this approach can be used to develop cell lines carrying more than 1000 copies of the amplified gene(s). Then, when methotrexate is withdrawn, cell lines are obtained containing the amplified gene integrated into one or more chromosomes of the host cell.

Highly efficient promoters other than the strong long terminal repeat (LTR) promoter of Rous sarcoma virus, such as the human β-actin promoter, SV40 early or late promoters, or long terminal repeats from other retroviruses, such as HIV and HTLVI, can be used for expression. Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to regulate expression of IL-12 in mammalian cells (M. Gossen, and H. Bujard, Proc. Natl. Acad. Sci. USA 89: 5547- 5551 (1992)). Other signals can also be used for mRNA polyadenylation, for example from human growth hormone or globin genes. Stable cell lines carrying the gene of interest integrated into the chromosomes can also be selected after co-transfection with a selectable marker such as gpt, G418 or hygromycin. An advantage is the use of more than one selectable marker in the beginning, eg G418 plus methotrexate. Plasmid pC4 is digested with restriction enzymes and then dephosphorylated using calf intestinal phosphatase using procedures known to those skilled in the art. Then allocate

- 33 040272 1% agarose gel vector.

A DNA sequence encoding a complete IL-12/23p40 antibody corresponding to the HC and LC regions of an IL-12/23p40 antibody of the present invention is used, each according to known method steps. This construct also uses an isolated nucleic acid encoding an acceptable human constant region (ie, the HC and LC regions).

The DNA encoding the isolated variable and constant regions and the dephosphorylated vector are then ligated with T4 DNA ligase. The E. coli HB101 or XL-1 Blue cells are then transformed and bacteria containing the fragment inserted into the pC4 plasmid are identified by, for example, restriction enzyme analysis.

For transfection, Chinese hamster ovary (CHO) cells lacking DHFR gene activity are used. Expression plasmid pC4 (5 micrograms) was cotransfected with 0.5 micrograms of plasmid pSV2-neo with Lipofectin. The pSV2-neo plasmid contains a dominant selectable marker, the neo gene from Tn5, which encodes an enzyme conferring resistance to a group of antibiotics including G418. Cells are seeded in alpha minus MEM supplemented with 1 μg/ml G418. After 2 days, cells are trypsinized and plated in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml methotrexate and 1 μg/ml G418. Approximately 10-14 days later, individual clones are trypsinized and then plated in loaf Petri dishes or 10 ml vials supplemented with various concentrations of methotrexate (50, 100, 200, 400, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1, 2, 5, 10, 20 mM). The same procedure is repeated until clones growing at a concentration of 100-200 mM are obtained. Expression of the desired gene product is analyzed, for example, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, or by reverse phase HPLC analysis.

Example 2. Comparison of the therapeutic efficacy of antibodies to IL-12p35 and to IL-12/23p40 in a mouse model of experimental autoimmune encephalomyelitis (EAE)

Summary

This set of studies was conducted to investigate the therapeutic efficacy of specific neutralization of IL-12 or IL-12/IL-23 in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis.

Neutralizing rat monoclonal antibodies (mAbs) to mouse antigens specific for the p35 subunit of IL-12, or the p40 subunit shared by IL-12 and IL-23, were administered either before disease induction, before disease onset, or after disease progression. In all cases, only the anti-p40 antibody showed therapeutic potential. These data suggest that IL-23 is a predominant factor in the pathogenesis of the disease in this autoimmune model.

Abbreviations:

IL Interleukin mAb Monoclonal antibody

Experimental autoimmune EAE encephalomyelitis

Th Helper T cells

IFNu Interferon gamma cs Clinical evaluation score

MBR Myelin Basic Protein

FC Pharmacokinetics

Introduction

Biologically active IL-12 exists as a heterodimer consisting of 2 covalently linked subunits with a mass of 35 (p35) and 40 (p40) kilodaltons. Several lines of evidence have shown that IL-12 can induce robust Th1 immune responses, which are characterized by the production of IFNγ and IL-2 by CD4+ T cells. Inappropriate Th1 responses, and thus IL-12 expression, are thought to correlate with many immune-mediated diseases such as multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, insulin-dependent diabetes mellitus, and uveitis. In animal models, neutralization of IL-12 has been shown to alleviate immune-mediated disease. However, in these studies, IL-12 was neutralized via the p40 subunit. The recent description of IL-23(1), a heterodimeric cytokine that also has a p40 subunit, has shown the importance of determining whether the previous findings were due to IL-12 or IL-23 activity. To do this, we compared the specific neutralization of p35 and p40 in a mouse model of an autoimmune state - in experimental autoimmune encephalomyelitis (EAE).

- 34 040272

Neutralizing antibodies specific for IL-12p35 did not affect the progression of EAE.

In contrast, neutralization of IL-12 and IL-23 with an anti-p40 mAb suppressed the clinical signs of EAE, regardless of whether the antibody was administered before or after Th1 differentiation. These data suggest that the activity of anti-p40 antibody treatment in EAE is based solely on neutralization of IL-23.

Methods and Materials Mice

Pharmacokinetic analyzes used female C3H/HEB/FEJ mice (Jackson Laboratories, Bar Harbor, Maine, USA). For EAE studies, female B10.PL (H-2 ^u ) mice were purchased from Jackson Laboratories and used at 6-8 weeks of age. All animals were kept in accordance with the guidelines of the Organizing Committee for the Care and Use of Animals (IACUC), according to approved protocols.

Antibodies

Hybridomas C17.8 (rat anti-mouse IL-12/IL-23p40, IgG2a) and C18.2 (rat anti-mouse IL-12p35, IgG2a) were purchased from Dr. Giorgio Trinchieri and Wistar Institute (Philadelphia, Pennsylvania, USA). Ascitic fluid was obtained from Harlan Bioproducts (Indianapolis, IN, USA) and purified by protein G affinity purification.

FA in serum of rat antibodies to mouse antigens

Female C3H/HEB/FEJ mice weighing approximately 20-25 grams were individually weighed and injected intraperitoneally (ip) with a single dose of 5 mg/kg of antibody (C17.8, C18.2) labeled with ¹²⁵ I, while a constant volume dose per mouse of 10 ml/kg was administered. Anesthetized mice were bled from the retro-orbital region at 30 min, 6 and 24 h, 4, 7, 11 and 18 days. The blood samples were allowed to stand at room temperature for at least 30 minutes, but not more than 1 hour, after which they were centrifuged at a speed of approximately 2500-3500 rpm for 10-15 minutes. Aliquots of each serum sample of approximately 50 μl were counted ¹²⁵ I using a counter LKB Compugamma 1282 (Wallac, Gaithersburg, Maryland, USA). Aliquots of 10 ml injections were also counted. The average fraction of injected pulses at each time point was calculated and multiplied by the total mg of antibody administered to determine the total mg remaining in serum at each time point. Data are shown as mean mAb mg in serum ± st. off if there are 5-10 animals in each group.

EAE induction and scoring

To induce EAE, female B10.PL mice were injected subcutaneously at four sites on the back with a total of 100 μl of complete Freund's adjuvant (CFA) (containing 200 μg of Mycobacterium tuberculosis strain Jamaica) in combination with 200 μg of guinea pig myelin basic protein (MBP) (Sigma) . During immunization and 48 hours later, mice were also injected ip with 200 ng of pertussis toxin (List Biological, Campbell, CA) in 0.2 ml of phosphate buffered saline (PBS). On the designated days, mice received ip injections of monoclonal antibodies C17.8 (anti-IL-12p40) or C18.2 (anti-IL-12p35) diluted in PBS at a dose of 100 mg/kg (C18.2) or 20 mg/kg (C17.8). Control mice were injected with PBS or rat IgG (Biosource) at 20 mg/kg in PBS.

Animals showing clinical signs were clinically scored as follows: 1 - flaccid tail or limp gait with tail tone; 2 - limping gait with a sluggish tail (ataxia); 2.5 - ataxia with partial limb paralysis; 3 - complete paralysis of one limb; 3.5 - complete paralysis of one limb with partial paralysis of the second limb; 4 - complete paralysis of two limbs; 4.5 - dying; 5 - death. Animals with a score of 5 were not included in the average daily analysis of clinical signs for the remainder of the experiment. Daily CR was averaged for the group and described as mean frequency, day of onset, highest acute CR, cumulative CR, CR/day, number of relapses, and relapse severity ± SES (standard error of the mean). Cumulative CR values per group were calculated by averaging the sum of daily clinical scores for individual animals. CR/day was calculated by dividing the cumulative CR by the number of days the animal remained in the study. When determining the mean day of onset, animals that did not develop EAE were not included in the analysis. When determining the average maximum CR, mice that did not develop EAE were assigned a value of O and included in the analysis. Relapses were defined as a decrease in clinical score by a full score lasting for at least 2 days of observation, followed by an increase in clinical score by a full score for at least 2 days of observation.

Results and discussion

Antibodies to p35 and to p40 have the same pharmacokinetics.

In order to establish clearance values of anti-p40 antibody and anti-p35 antibody, normal mice were injected with a single dose of 5 mg/kg of antibodies labeled with ¹²⁵ I, and their circulating levels were measured for 11 days after administration of the antibodies. Anti-p35 antibody and anti-p40 antibody had overlapping pharmacokinetics, showing similar clearance rates in normal mice (2). Expected rate

- 35 040272 clearance for each mAb is approximately 7-10 days. Although this study is a single dose PK study, the findings support once weekly administration for in vivo studies.

Only administration of an anti-p40 antibody prior to EAE induction has a protective effect.

To determine the comparative roles of IL-12 and IL-23 in immune-mediated diseases, the authors used a mouse model of multiple sclerosis - relapsing experimental autoimmune encephalomyelitis (EAE). After induction of EAE by adjuvant administration of myelin basic protein (MBP) to B10.PL mice, they usually experience an initial episode of paralysis (acute illness), then a partial or complete recovery, and progression of multiple relapses and/or the development of chronic EAE. For a long time it was assumed that EAE depends on the expression of IL-12, since it was believed that IL-12 is the primary mediator of Th0 to Th1 differentiation. However, to reveal the potential role of IL-23 in inducing EAE, neutralizing concentrations of anti-p40 antibodies (IL-12 and IL-23) or anti-p35 antibodies (IL-12 only ). The onset of disease in animals may vary; therefore, the procedure was repeated later at 7 and 14 days to ensure that during Th1 differentiation, anti-p35 and anti-IL-p40 antibodies were present. Several in vitro neutralization studies have demonstrated that the p40 mAb is 5 times more effective at neutralizing IL-12 than the p35 mAb (data not shown). Therefore, the anti-p35 mAb dose in all EAE experiments was adjusted to be 5-fold higher than the anti-p40 antibody dose. In two separate experiments, mice treated with rat IgG isotype control antibody (20 mg/kg) or anti-p35 antibody (100 mg/kg) showed no protection against the disease. Importantly, peripheral administration of a non-specific control antibody (rat IgG) did not alter the clinical course of the disease compared to mice with EAE that did not receive such administration. In both studies, mice treated with anti-p40 mAb (20 mg/kg) showed almost complete inhibition of the clinical signs of EAE. Notably, disease suppression lasted longer than expected antibody clearance, up to 70 days after EAE induction. In each experiment, only one animal treated with p40 antibody exhibited clinical signs of EAE on two consecutive days, with each animal having a late onset and significantly lower clinical scores in the acute period, cumulative clinical scores, and no disease recurrence (Table 1). .1). These results indicate that neutralization of IL-12 and IL-23 through the common p40 subunit conferred almost complete protection against EAE. In contrast, specific neutralization of IL-12 alone by anti-p35 antibody was ineffective. These data clearly indicate that EAE is not mediated by IL-12.

Only administration of an antibody to p40 just before the onset of the disease has a protective effect.

Although prophylactic treatment completely protected mice from EAE, it remained to be determined whether specific neutralization of IL-12 would further protect once the in vivo Th1 population had been established. Therefore, in a separate set of experiments, ten days after EAE induction but before the onset of disease, mice were injected with either a control antibody (rat IgG) or anti-p35 or anti-p40 monoclonal antibodies. Since typical immune responses occur within 7 days, this time point should reflect the effect of the anti-IL-12 or anti-IL-23 mAb on differentiated Th1 cells. The onset of EAE in animals may vary, so the procedure was repeated later at 7 and 14 days to ensure that anti-p35 and anti-p40 antibodies were present at the time of onset. In two separate experiments, mice treated with rat isotype control antibody (20 mg/kg) or anti-p35 antibody (100 mg/kg) were not protected from disease compared to EAE mice that were not treated. However, mice treated with anti-p40 mAb (20 mg/kg) were largely protected from EAE. As shown in previously described studies, suppression of the disease was observed long before the time required for clearance of peripherally administered antibodies, and up to 70 days after the induction of EAE. Given that the antibody was not administered before Th1 differentiation occurred (day 10), it is not surprising that the incidence, day of onset, and highest clinical score during acute EAE did not differ between all groups (Table 2). However, in both experiments, mice treated with p40 antibody had significantly lower cumulative clinical scores, daily clinical scores, and relapse severity.

Only the introduction of antibodies to p40 during the established EAE has a protective effect.

The most difficult but clinically significant barrier to any therapy is the suppression of established disease.

Therefore, another set of experiments was carried out in which mice were immunized to develop EAE and then divided into treatment groups during the course of the disease. Approximately 30 days after EAE induction, the mice progressed to the acute phase of the disease. At this time, animals were divided into groups with comparable cumulative and daily clinical scores. The procedure was repeated later, after 7 and 14 days, to ensure the presence of antibodies at neutralizing concentrations during the transition of the disease from the acute phase to the chronic phase or to the remission and relapse phase. Only the introduction of antibodies to p40 (20 mg/kg) facilitated

- 36 040272 the course of the disease compared with the introduction of isotype control antibodies (20 mg/kg) or antibodies to p35 (100 mg/kg) into animals. Disease suppression was observed up to 80 days after EAE induction. In both experiments, analysis from day 1 of treatment to day 80 showed that mice treated with p40 antibody had lower cumulative clinical scores, daily clinical scores, and the lowest post-treatment maximum clinical score. These data suggest that IL-23 may not only mediate Th1 differentiation (Table 1) and EAE induction (Table 2), but that IL-23 also contributes to the effector phase of chronic immune-mediated (eg, autoimmune) responses. (Table 3). Therefore, administration of the p40 antibody can provide therapy at any time during the progression of immune-mediated diseases.

conclusions

Understanding the role of IL-12 in immune function has been based on studies of the p40 subunit of IL-12. Therefore, a side-by-side comparison of the neutralization of the p35 subunit specific for IL-12 and the p40 subunit shared by IL-12 and IL-23 was performed in an animal model of an autoimmune disease. Neutralization with p40 antibody significantly inhibited EAE when the mAb was administered at any time point. However, specific neutralization of IL-12 was completely ineffective. Therefore, the data obtained by the authors of the study indicate that IL-12 does not contribute to this autoimmune model, and IL-23 is expected to be a more prominent mediator of autoimmune T cell responses.

Example 3 Epitope for neutralizing p40

Summary

The epitope for a neutralizing antibody (anti-IL-12/23p40 mAb) against the p40 subunit of human IL-12 and IL-23 was determined based on the crystal structure of the Fab/IL-12 complex. The epitope is located on the D1 domain (residues 1-88) of the p40 subunit in human IL-12. This region is distant from the p40/p35 interface and is also expected to be available on the p40 subunit of IL-23. The residues involved in antigen-antibody binding are discontinuous (Table 4) and contain a unique conformational epitope. Antibodies to this epitope or parts thereof and adjacent regions will result in blocking of the functions of IL-12 and IL-23 mediated through this part of the p40 subunit.

Introduction

A fully human monoclonal antibody (anti-p40 mAb) directed against human IL-12/23p40 has been shown to be a potent neutralizer of IL-12 and IL-23 function. The p40 mAb has been shown to bind to the p40 subunit and block both cytokines from binding to their receptors. Because the p40 subunit is shared between IL-12 and IL-23, detailed interactions between IL-12 and anti-p40 mAbs define an important common neutralizing epitope, which in turn may shed light on cytokine-receptor interactions.

Crystal structure based epitope determination of p40 Fab/IL-12 anti-IL-12/IL-23p40 mAb was produced in culture from a mammalian cell line and purified on a protein A. anti-IL-12/IL-23p40 mAb column (70 mg) digested with papain (0.25 papain units per milligram IgG) in activation buffer (0.03 M sodium phosphate, 0.15 M NaCl, 0.01 M EDTA, 0.0072 M L-cysteine, pH 7.0) at 37 °C for 2 hours. Cleavage was monitored by surface laser desorption/ionization mass spectrometry (SELDI). Iodacetamide (0.5 M) was used to stop the cleavage. Fc was removed with immobilized protein G. Fab p40 was then purified by gel filtration on a Superdex 200 16/60 column. Received a total of 44 mg of purified Fab p40 and analyzed its purity using SDS-PAGE.

Recombinant human IL-12 was produced in culture of a stably transfected cell line overexpressing the p40 and p35 subunits and purified on a p40 mAb affinity column. Collected protein fractions and subjected to dialysis in 10 mm Tris, 100 mm NaCl, pH 7.4, and concentrated to 2.5 mg/ml. IL-12 was deglycosylated by incubation with several combinations of deglycosylation enzymes (PNG-ase F, sialadase, endo-O-glycosidase, α-, β-galactosidase, α-mannosidase, fucosidase [about 5 mU-10 U/100 µg of protein]) in for 3 days at 37°C in an argon atmosphere.

Deglycosylated IL-12 was mixed with excess Fab p40. The IL-12/Fab p40 complex was purified by size exclusion chromatography in 10 mM Tris, 50 mM NaCl, pH 7.4. The isolated complex was concentrated to approximately 4 mg/ml. The IL-12/Fab p40 complex was crystallized using the sessile drop vapor diffusion method by combining the above solution of the protein complex in a 1:1 v/v ratio with a reservoir solution of 50 mM Tris, pH 7.0, 16% PEG-3350. Within two weeks at 16° C., cubic, pyramidal or rod-shaped crystals appeared with a typical size of 50-150 µm.

The crystals were collected and soaked in a mother liquor supplemented with 30% ethylene glycol, and flash-frozen in liquid nitrogen to collect radiographic data. The best data set was collected (360°, 0.5°/frame, 10 s exposure per frame) up to a diffraction limit of 2.8 A in the Advanced Photon Radiation Source (APS), Argonne National Laboratory (Ax- 37 040272 as-ComCat ). Diffraction data were processed using Denzo and ScalePack software. (Otwinowski &

Minor, Methods Enzymol. 276: 307-326). The space group for this crystal form is P212121 with cell sizes a=116.8 A, b=55.77 A, c=182.96 A, and α=β=γ=90°. Available

141 independent reflections and the data was ~90% complete at 3.0 A (1/sigma=5.2, Rs _ym =9%.).

The crystal structure was solved by molecular substitution, as implemented in the CNX program (Accelrys, CA, USA). The search models were the published structure of IL12 (PDB code 1F45) for IL-12 and the homology model for Fab p40 based on the Fab crystal structure (PDB code 1VGE). Molecular models for IL-12 and Fab p40 were checked visually and corrected manually using the XtalView software. The structure was refined using the CNX program. Molecular models were verified using the Insightll program (Accelrys, CA, USA). In FIG. 1 shows the molecular structure of the bound IL-12/Fab p40 complex in ribbon representation.

In FIG. 2 shows the binding site of the p40 mAb (p40 epitope) on the surface of the molecules in surface and ribbon views. Binding epitope residues are defined as any exposed surface of p40 residues (relative solvent accessibility is 0.1 or greater) for any atoms within 4 A of any antibody atoms, according to common convention. Surface accessibility was calculated using ICM software (Molsoft, CA, USA) using default parameters. Residues containing the binding epitope mAb to p40 on IL-12 p40 are listed in table. 4, along with their open surface areas (sf) and relative accessibility scores (sf factor). Determining the open surface area of an amino acid residue in the context of a protein is generally accepted in the art. In particular, a water molecule with a radius of 1.4 A is rolled over the surface of the protein, and the size of the area obtained by this calculation for a particular residue is assigned to the open surface area of this residue. In addition, there is total surface area for an amino acid in a fully extended conformation. The surface accessibility (factor sf) is then the ratio of the exposed surface area to the standard surface area. These two values taken together give us an idea of whether a residue is open on the surface of the protein.

From Table. 4 and FIG. 2 (right panel) shows that the binding site on p40 is discontinuous and consists of a large number of residues exposed on the surface, which are distributed over an irregular surface. The antibody-antigen interaction covers a total of 1758 A ² available surfaces on IL-12 and anti-p40 mAb. Apparently, three salt bridges dominate in the interactions: K59(H)-E59(p40), K98(H)-E45(p40), and R99(H)-D62(p40). In addition, there is a contribution to the antibody-antigen interaction due to hydrophobic or van der Waals forces.

The residues on the antibody to IL-12p40 involved in binding to IL-12, which are identified in our study based on the co-crystal structure, are shown in table. 5 below. All surface-exposed residues in an anti-p40 antibody, with any atoms within 4 A of the p40 subunit, are considered part of these binding residues. Conservative changes to any one or more of these residues can produce mutant antibodies that have similar potency. Examples of such conservative substitutions include, without limitation, R59K, R98K, and R99K in VH (eg, in SEQ ID NO: 7) and D1E in VL (eg, in SEQ ID NO: 8).

In addition, at each position shown in Table 5, saturation mutagenesis (e.g., changing the amino acid sequence of the wild-type (WT) to any other amino acid, with the possible exception of cysteine) can be performed to identify mutations that cause increased, decreased, or substantially similar the activity of the resulting antibody (eg, binding). Binding of the resulting antibody to the p40 subunit can be tested according to any suitable binding assay. Saturation mutagenesis can be used, for example, to create a more or less effective antibody, or an equally effective antibody having different properties, that is, properties different from the effectiveness, resulting from a change in the sequence of the variable regions (for example, the size or other structural changes of the variable areas).

Saturation mutagenesis can also be carried out with more than one substitution, eg at two, three or more positions for each experiment. This can be done as single clones or libraries, followed by selection or screening in a suitable format, eg phage display format. In addition, single or combined mutations at the positions indicated in Table 5 that are desirable for regulating activity can be combined to generate additional combination mutants with similar or better potency.

Advantages

The mAb binding site on p40 is distant from the p40/p35 association site (FIG. 1). The p19 subunit of IL-23 is evolutionarily related to p35 of IL-12 with significant sequence homology. It is likely that p19 is associated with p40 in the same way as p35. Therefore, the mAb binding region on p40 is also distant from the site of p40/p19 interaction in IL-23. The epitope identified in the present invention is

- 38 040272 is equally available in both IL-12 and IL-23; thus, it is not surprising that an anti-p40 mAb can actively block the functions of both cytokines.

IL-12 (p40/p35) and IL-23 (p40/p19) interact with their respective receptors (IL-12Re1/e2 and IL-12Rβ1/IL-23R) in a similar manner. They induce similar signaling cascades. However, the details of the interaction between cytokines and receptors at the molecular level are not clearly defined. The newly identified p40 mAb epitope may represent a biologically important site of interaction between the IL-12 family of cytokines with their common IL-12Rβ1 receptor. Therefore, this epitope is an important target for therapeutic intervention using monoclonal antibodies, peptides, recombinant proteins, small molecules, and other natural or synthetic agents.

Example 4 Anti-p40 mAb Epitope Mapping for IL-12p40 Using Mutation Analysis

Summary

To confirm the binding epitope, the binding of IL-12p40 mutants to IL-12/IL-23p40 mAb was analyzed by solid phase ELISA. Based on the crystal structure of the p40 Fab/IL-12 complex, 7 single mutants and 2 double mutants were created. The mutated residues are located in the region of contact with Fab p40 in domain I (D1) of the p40 subunit. The relative binding affinity showed that three negatively charged residues - E45, E59 and D62 - contribute significantly to binding interactions with p40 mAb. The contribution of other residues -M23, L40 and S43 - is weaker, but is noticeable. This mutational analysis confirms that the p40 mAb recognizes domain I of the p40 subunit, and residues E45, E59, D62, M23, L40 and S43 are part of the binding epitope.

Materials and methods

The studies used seven single human p40 mutants: M23T, L40T, S43R, E45A, E45R, E59R, and D62R, and two double mutants: S43R/E45A and S43R/E45R. Wild type human p40 was used as a control. The p40 mutants were transiently expressed in HEK293E cells. Supernatants were used for binding assays.

Briefly, high binding MSD plates (Meso Scale Discovery, Maryland, USA) were coated with 5 µl capture monoclonal antibody (5 µg/ml) at room temperature for 1 hour. The capture monoclonal antibody recognizes the p40 subunit but does not compete with mAb to p40. One hundred fifty (150) µl of 5% MSD Blocker A blocking buffer was added to each well and cultured for 1 hour at room temperature. The plates were washed three times with 0.1 M HEPES buffer (pH 7.4). These protein-coated solid phase ELISA plates were incubated with 25 µl of supernatants (diluted 1:10 in 0.1 M HEPES buffer, pH 7.4) of various transiently expressed p40 mutants for 1 hour at room temperature. The plates were washed three times with 0.1 M HEPES buffer (pH 7.4). Twenty-five (25) μl of anti-p40 mAb labeled with MSD Sulfo-TAG were added to the microwells at various concentrations ranging from 0 to 20 μg/ml.

After 2 hours of incubation with shaking at room temperature, the plates were washed 3 times with 0.1 M HEPES buffer (pH 7.4). One hundred and fifty (150) µl of diluted MSD Read Buffer T was added to each well and the plates were analyzed using a SECTOR Reader (MSD).

results

As shown in FIG. 3, each of the E45R, E59R and D62R mutations significantly reduced the binding affinity of the p40 mAb mutant to p40 compared to wild-type p40. E45A had a significant but less strong effect on binding compared to E45R. Each of the M23T, L40T and S43R mutations had a moderate binding effect.

The relative binding affinity of the mAb for IL-12/IL-23p40 to various mutant proteins was further analyzed by a capture assay. As shown in FIG. 4, each of the E45R, E59R and D62R mutations almost completely abolished antigen binding. Each of the other mutations in the interface, M23T, L4 0T and S43R, reduced binding by approximately 40%. The E45A mutation reduced binding by about 70%.

It becomes clear that the application of the present invention in practice may differ from that given in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the foregoing teachings, and therefore remain within the scope of the appended claims.

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Table 1. EAE clinical scores with IL-12 and IL-23 neutralization initiated on day -1 (before Th1 differentiation)

Group R-2001- Incidence Mortality Day of onset highest KO in the acute period* Cumulative csl ko/ day Number of relapses Severity of relapse 060 Rat IgG 13/13 4/13 30.5± 3.2 3.6±0.3 71.4± 14.1 1.2 ± 0.2 1.3 ± 0.2 3.6± 0.2 Anti-r35 11/13 8/13 29.6± 3.4 3.5±0.5 45.5± 11.5 0.8± 0.2 1.2 ± 0.1 4.0 ± 0.3 Anti-r40 1/13 0/13 40.0 0.1 1.2 ± 0.5 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 R-2001- 079 Without procedure 6/7 0/7 24.7± 2.7 3.2±0.6 110.4± 20.4 1.7 ± 0.3 1.0 ± 0.4 3.8± 0.1 Rat IgG 9/9 2/9 29, 1 ± 2.9 3.8±0.2 90.6± 10.1 1.5± 0.1 0.3 ± 0.2 4.7± 0.3 Anti-r35 10/10 1/10 30.0 ± 2.6 3.9±0.2 94.9± 17.8 1.4+0 .2 0.7 ± 0.3 3.9± 0.2 Anti-r40 1/10 0/10 61.0 0.3 1.6 ± 1.1 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

^a clinical assessment (CR) ^b cumulative CR

Mice were administered as described above and clinical scores were analyzed from day 0 to day 70 post EAE induction. Data are shown as mean per group ± PCS.

Table 2 EAE Clinical Evaluations for IL-12 and IL-23 Neutralization Started on Day 10

Group Incidence (after differentiation Day Highest Mortal onset of KO in the acute period of the disease * Th1) Cumulative KO ¹² KO/ day Number of relapses Severity of relapse R-2001-037 Without procedure 7/8 0/8 30.6± 2.7 3.2±0.5 51.5± 14.4 0.8± 0.2 0.3 ± 0.2 3.3± 0.8 Rat IgG 9/10 0/10 25.9± 2.7 2.7±0.5 74.7± 15.8 1.2 ± 0.2 0.6± 0.2 3.7± 0.4 Anti-r35 9/10 0/10 25.8± 2.6 2.5±0.4 58.8± 15.6 1.0 ± 0.2 0.7 ± 0.3 3.2± 0.3 Anti-r40 6/7 0/7 34.7± 1.6±0.5 14.9± 0.2 ± 0.3 ± 1.5±

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6.3

7.5

0.1

0.2

0.5

R-2001-053 Without procedure 8/9 2/9 15, 8 2.2 ± 2.1 ± 0.6 56.4± 19.1 0.9 ± 0.3 0.6± 0.3 3.3± 0.5 Rat IgG 9/10 4/10 20.0 2.5 ± 3, 8 ± 0.5 70.1 ± 17.7 1.3 ± 0.2 0.3 ± 0.2 4.2± 0.4 Anti-r35 10/10 1/10 16.5 1.1 ± 3.2 + 0.3 93.8± 15.7 1.4 ± 0.2 0.8± 0.2 3.2± 0.3 Anti-r40 10/10 2/10 13, 6 1.1 ± 2.7 + 0.5 23.2± 7.9 0.4 ± 0.1 0.4 ± 0.3 2.0 ± 0.4

^a clinical assessment (CR) ^b cumulative CR

Mice were administered on days 10, 17 and 24 and clinical scores were analyzed from day 0 to day 70 post EAE induction. Data are shown as mean per group ± PCS.

Table 3. EAE Clinical Evaluations with IL-12 and IL-23 Neutralization Started on Day 30 (During Steady EAE)

From the first injection to 80 days after the induction of EAE

Cumul- Maxi- Mini- Quantity Gravity

Group Before . Tx ^{a Lethality} co/ day small small KO KO reci- divs reci- diva R-2002-01 Without procedure 2.7 ± 0.6 1/5 132.9± 29.3 3.3± 0.3 4.1±2.4± 0.2 0.5 0.6± 0.4 3.7± 0.0 Anti-r35 2.3 ± 0.7 1/5 135.9± 16.5 2.7 ± 0.3 3.8±1.8± 0.4 0.3 2.0 ± 0.4 3.7± 0.3 Anti-r40 2.0 ± 0.2 1/6 75.6± 16.1 1.9 ± 0.3 2.8±1.0± 0.5 0.4 0.7 ± 0.3 2.5± 1.0 R-2002-093 Anti-r35 Rat IgG 1.9 ± 0.7 1.7 ± 0.8 1/5 1/5 87.7± 16.4 98.2± 9.7 2.1 ± 0.2 2.2± 0.1 3.7±1.2± 0.4 0.5 3.7 ± 1, 0.4 0 1.5± 0.5 4 ± ,4 3.8± 1.0 1.5± 0.3 3.3± 0.2 Anti-r40 2.4 ± 0.8 0/5 71.7± 21, 1.5± 0.4 2.9 ± 0, 0.6 0 8± _f5 1.3 ± 0.3 2.7 ± 0.6

a b

average clinical score in the group on the first day of administration (Tx) clinical score (CR)

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Table 4. Residues contributing to the binding of the anti-p40 mAb epitope to IL-12/p40

Number Remainder sf coefficient sf 15 Tgr 94.2 0.36 17 Pro 69.3 0.46 18 asp 132.9 0.86 19 Ala 27.7 0.22 20 Pro 131.5 0.88 21 gly 18.7 0.21 23 Met 142.4 0.66 40 Leu 32.7 0.16 41 asp 69 0.45 42 gin 145.8 0.77 43 Ser 58.2 0.46 45 Glu 139.2 0.74 46 Vai 95.8 0.57 47 Leu 95 0.48 54 Thr 77.2 0.51 55 He 32.7 0.17 56 gin 111 0.58 58 Lys 69.2 0.32 59 Glu 83.2 0.44 60 Phe 113, 3 0.51 61 gly 68.2 0.77 62 asp 41.3 0.27

Table 5. Anti-p40 mAb residues involved in binding to the IL-12/p40 epitope

Example 5 Clinical Study Results with Increasing Dose Intervals

Ustekinumab was studied in a randomized, double-blind, multicentre, active-controlled phase 3b study with a 4-week screening period, an open-label lead-in period from week 0 to week 28, a double-blind treatment period from week 28 to week 104, a after treatment until week 116 and follow-up for reliability by telephone conversation or visit to the study center at week 124 (see Fig. 5: Design of the study). Study Title: Randomized, double-blind, multicentre, phase 3b, active-drug-controlled study to evaluate individualized maintenance dose management in subjects with moderate to severe plaque psoriasis (PSTELLAR), protocol number: CNTO1275PSO3009. In FIG. 5: NR=patient with no effect of treatment at a given point in time; P = randomization of patients with treatment effect [defined as having a PGA score < 2] at week 28 (subjects not responding to treatment [defined as having a PGA score < 2] at week 28 will be discontinued from further injections of study agent; they will be observed for reliability, for at least 20 weeks after the last injection of investigational agent, after which they will be withdrawn from the study); UST=ustekinumab; Nd=weeks.

All subjects included in the study were exposed to ustekinumab. At each injection visit up to a week, 28 subjects with a baseline body weight of 100 kg received one injection (ustekinumab 45 mg) and subjects with a baseline body weight of >100 kg received 2 injections (ustekinumab 45 mg + ustekinumab 45 mg). At each injection visit from week 28 to week 104, subjects with baseline body weight < 100 kg received one injection (ustekinumab 45 mg or placebo) and subjects with baseline body weight > 100 kg received 2 injections (ustekinumab 45 mg + ustekinumab 45 mg, or placebo+placebo) depending on allocation to treatment option and injection visit (see Table 7: Study Agent Administration Schedule). During the course of the study, a dose of study agent was administered, which was determined based on baseline body weight. At week 28 patients with treatment effect, defined as subjects with PGA with clearance (0) or minimal manifestations (1), were randomized 4 to 1 into 2 groups, and all patients with no effect (PGA score > 2) were withdrawn from research.

Group 1 (n=76): Beginning at week 28, subjects received a maintenance dose of ustekinumab

- 42 040272 at fixed intervals of 1 p/12 d until the end of the study (i.e. at weeks 28, 40, 52, 64, 76, 88 and

100).

Group 2 (n=302): Beginning at week 28, subjects were given a maintenance dose at individually adjusted intervals. The period between injections of the study drug was extended with placebo injections to determine an individually adjusted interval between maintenance doses. The interval between doses of study drug for each subject was determined by assessing the retention of effect of treatment without receiving an injection of study drug in 4-week increments until loss of effect was observed as assessed by PGA. At this point in time for subjects, the interval between drug administrations was taken, defined as the longest interval during which the effect was maintained according to the PGA assessment (i.e., 4 weeks shorter than the time point when the loss of effect was noted), or the maximum interval between doses of 24 weeks if the effect persisted for 24 weeks. Thus, four different dosing intervals were studied in subjects from Group 2, with the respective treatment groups designated as Groups 2a, 2b, 2c, and 2d.

a) Subjects with a first loss of effect (PGA score < 2) 16 weeks post-injection at week 16 (week 32 visit) will receive ustekinumab 1 r/12 d at dosing visits from week 32 to week 104 ( i.e. at weeks 32, 44, 56, 68, 80, 92 and 104).

b) Subjects with a first loss of effect (PGA score < 2) 20 weeks post-injection at week 16 (week 36 visit) will receive ustekinumab 1 p/16 wd at dosing visits from week 36 to week 104 (t .e. at weeks 36, 52, 68, 84 and 100).

c) Subjects with a first loss of effect (PGA score < 2) 24 weeks post-injection at week 16 (week 40 visit) will receive ustekinumab 1 r/20 nwd at dosing visits from week 40 to week 104 (t i.e. at weeks 40, 60, 80 and 100).

d) Subjects with no loss of effect (PGA score < 2) for 24 weeks post-injection at week 16 (week 40 visit) will receive ustekinumab 1 p/24 wd at dosing visits from week 40 to week 104 (i.e. at weeks 40, 64 and 88).

During the double-blind treatment period, subjects in Groups 1 and 2 received placebo injections for as long as necessary to maintain dosing interval masking. Subjects in Arm 2 were required to receive at least 3 cycles of maintenance doses at individually adjusted intervals during the randomized study period (see Table 7: Study Agent Administration Schedule).

Randomization: At week 28, subjects with a static PGA score with clearance (0) or minimal manifestations (1) were randomized, by random block permutation, in a 1:4 ratio, or to group 1 (approved maintenance schedule of 1 p/12 n.d.). ), or in group 2 (maintenance dose regimen with individually selected intervals). Randomization was stratified by subject's baseline weight [<100 kg or >100 kg] and PGA score [0 or 1] at week 28.

Duration of treatment/test duration:

Subjects were administered study agent from week 0 to week 104.

Thereafter, subjects were followed for at least an additional 20 weeks, with the last study visit at week 124.

Primary Efficacy Assay Set: The primary efficacy analysis was performed on all subjects randomized at week 28 to one of two treatment groups (Group 1 or Group 2).

Primary Performance Parameters/Primary Time

The primary endpoint is the number of visits at which subjects achieved a static PGA score with clearance (0) or minimal symptoms (1) between week 88 and week 112 (score interval), for subjects randomized at week 28. Evaluation visits were performed every 4 weeks during the evaluation interval.

Main secondary performance parameters

The proportion of subjects with a static PGA score clearing (0) or minimal manifestations (1) and its 95% CI at visits from week 28 to week 112, for subjects randomized at week 28.

Number of visits to which subjects randomized at week 28 achieved a PASI 75 treatment response between week 88 and week 112.

The proportion of subjects with a PASI score of 75 and its 95% confidence interval at visits from week 28 to week 112, for subjects randomized at week 28.

Expected effect size and planned sample size

Based on data from the PHOENIX 1 and PHOENIX 2 trials, it was expected that approximately 35% of the 500 subjects enrolled in the study would not meet the criteria for randomization at week 28, due to withdrawal from the study before week 28 or lack of efficacy. that at week 28 as a PGA score clearing (0) or minimal manifestations (1). A predicted 325 subjects eligible for randomization at week 28 in a 1:4 ratio would provide approximately 65 subjects in group 1 (maintenance dose 1 r/12 n.d.) and approximately 260 subjects in group 2 (maintenance dose administered at individually adjusted intervals) .

The study was designed to evaluate the frequency of effects in the 2 maintenance arms of ustekinumab in more than 300 subjects. A total of approximately 325 randomized subjects would provide a 95% confidence interval of 64.9 to 85.9% around the 75% effect in group 1 (n=65; administration of a maintenance dose of 1 r/12 wd) and a 95% confidence interval of 54.0 % to 66.0% around the 60% effect in group 2 (n=260; administration of a maintenance dose at individually adjusted intervals) during the evaluation period between weeks 8-8 and 112.

The main objective:

The primary aim of this study was to evaluate the effect of extending the maintenance dose interval to longer than every 12 weeks (1 p/12 wk) on the clinical efficacy of ustekinumab.

Brief overview of the main results

CNTO1275PSO3009 is a randomized, double-blind, multicentre, phase 3v, active-drug-controlled trial in adults >18 to <80 years of age with moderate to severe plaque psoriasis, defined by a PGA score >3, and involving body surface area (BSA). ) at least 10% who were candidates for phototherapy or systemic treatment of psoriasis.

The study began with the identification of the first subject on March 8, 2012. A total of 611 subjects were identified, of which 478 subjects were recruited into the study, and at week 28, 378 subjects were randomized into two treatment option arms: one received a maintenance dose on a fixed interval regimen. 1 r / 12 weeks (group 1), and in the second, a maintenance dose was administered according to the scheme with individually selected intervals (group 2). The study was conducted at 42 centers in the USA. The final database includes data collected up to week 124 for all subjects recruited into the study. The first subject was dosed on March 22, 2012. The first subject was randomized on October 4, 2012, and the last subject was randomized on September 16, 2013.

A total of 611 subjects were identified and 478 subjects were finally recruited into the study. A total of 378 patients with a treatment effect (PGA 0/1) were randomized at week 28 (randomization rate is 80%). This randomization rate was higher than expected, which may be due in part to the subjective nature of the PGA score at week 28. The final database included data up to week 124 for all enrolled subjects.

Allocation of subjects to treatment groups and the ability to evaluate subjects

A total of 478 subjects were recruited into the study and received ustekinumab treatment during the open-label lead-in period of weeks 0-28 in accordance with US approved body weight-based dosing guidelines (ustekinumab 45 mg, n=308; ustekinumab 90 mg , n=170). A total of 378 subjects were randomized at week 28 into 2 groups: a maintenance dose group at a fixed interval every 12 weeks (group 1), and a maintenance dose group at individually adjusted intervals (group 2). With a randomization ratio of 1:4, a total of 76 subjects were randomized to group 1 and 302 subjects were randomized to group 2. The distribution of patients in group 2 receiving a maintenance dose at each of the 4 possible dosing intervals was as follows: 1 p/ 12 nd, n=84; 1 р/16 nd, n=61; 1 p/20 nd, n=51 and 1 p/24 nd, n=84.

Subject Demographics

Of all subjects recruited into the study, the majority were Caucasian (84.9%), 63.0% of participants were male, and the median age was 46 years. Of all randomized subjects, the majority were Caucasian (76.7% in group 1 and 89.4% in group 2) and male (57.9% in group 1 and 63.6% in group 2). The median age was 42 years in group 1 and 46 years in group 2.

Disease characteristics in subjects

For all recruited subjects, at baseline, the median duration of psoriasis was 13.3 years, the median percentage of body surface area (BSA) involved was 19.0%, and the median PASI score was 16.0. A total of 35.8% of subjects had a PGA score >4 points, consistent with severe disease; thus, the majority of subjects had moderate disease, as defined by a baseline PGA score of 3. Baseline disease characteristics in randomized subjects in groups 1 and 2 were substantially comparable and similar to disease characteristics in the entire study population. However, subjects in Group 1 had, on average, a longer disease duration (17.5 years) and a greater baseline BSA of affected skin (21.0%) compared to subjects in Group 2 (11.8 years and 17.0% BSA, respectively) .

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History of medical treatment of psoriasis in subjects

Of all subjects included in the study, 32.4% had previously received phototherapy, 35.4% had previously received conventional systemic therapy (including PUVA, methotrexate, acitretin, cyclosporine, mycophenolate mofetil), and 31.0% had previously received biologic therapy. As such, among subjects randomized to groups 1 and 2, similar proportions were observed. Of all subjects included in the study, 69.0% had not previously received biologic therapy. Previous use of conventional systemic agents was slightly higher in subjects in group 1 (39.5%) compared to group 2 (31.1%). A higher proportion of subjects in group 2 (74.5%) compared to group 1 (64.5%) had not previously received biologic therapy.

Discontinuation of Investigational Agent

During the open period (week 0 to week 28), 20.9% (100/478) of enrolled subjects discontinued study agent. Substantially similar proportions of subjects discontinued study agent in the 45 mg dose group (19.8%) and in the 90 mg dose group (22.9%). The most common reason for drug discontinuation was failure to achieve a static PGA clearance score (0) or minimal symptoms (1) at week 28 [12.1% (58/478)] to match the transition to the randomized portion of the study. During the randomized dosing period (week 28 to week 104), 22.2% (84/378) of randomized subjects discontinued study agent. The most common reason for discontinuation of study agent was failure to follow up subjects (7.9%) in group 1 and adverse event (5.0%) or withdrawal of consent (5.0%) in subjects in group 2.

During the randomized treatment period (week 28 to week 104), 22.2% (84/378) of randomized subjects discontinued study agent. The discontinuation rates of the drug in groups 1 and 2 were similar (table 8). The most common reason for discontinuation of study agent was failure to follow up subjects (7.9%) in group 1 and adverse event (5.0%) or withdrawal of consent (5.0%) in subjects in group 2.

Efficacy Conclusions Primary Endpoint

Group 2 had fewer visits (mean difference -0.46) at which subjects received a PGA clearance (0) or minimal (1) score (PGA 0/1) in the score interval from week 88 to week 112, compared with group 1 (on average, group 1 had 4.5 visits with a PGA 0/1 score, and group 2 had 4.1 visits with a PGA 0/1 score).

Of note, during this interval, group 1 (55.3%) had a higher proportion of subjects with a PGA score of 0/1 at all 7 visits in the assessment interval compared to group 2 (38.1%).

Secondary Endpoints

Group 2 had fewer visits (mean difference was -0.32) where subjects received a PASI 75 score between week 88 and week 112 compared to group 1 (mean, group 1 had 5.8 visits with PASI score of 75 and Group 2 had 5.4 visits with a PASI score of 75). The number of visits on which subjects benefited from PASI 75 treatment during the evaluation interval was similar in group 1 (69.7%) and group 2 (66.9%).

Treatment effects over time after randomization

During the dosing interval period (week 28 to week 40), the frequency of PGA 0/1 effects decreased in both groups 1 and 2. As expected, a greater decrease was observed in group 2 compared to group 1 because the determination of individually adjusted dosing intervals was based on the deterioration of subjects in group 2. After week 40, the PGA-assessed effect rates in both groups were substantially maintained until week 112. The effect rates at most visits were slightly higher in group 1 compared to group 2.

In general, from week 28 to week 112, the PASI 75 effect models reflected the PGA effects.

The proportions of subjects achieving effects on PGA clearance (0), PASI 90, or PASI 100 scores by week 112 were substantially higher in group 1 compared to group 2. The magnitude of the difference between groups 1 and 2 was higher for these endpoints compared to with PGA 0/1 and PASI 75 endpoints.

Safety Conclusions

For randomized study participants of 378 subjects, from week 28 to week 124:

The proportion of subjects experiencing 1 or more adverse events (AEs) was comparable between the two treatment groups (72.4% in group 1 and 72.8% in group 2).

The most frequently reported AEs were in the system organ class (SOC) of infectious and parasitic diseases, both for group 1 (46.1%) and for group 2 (48.7%; 48.1% combined for groups 1 and 2); the most common AEs in this SOC were upper respiratory infections (URTIs) (27.6% in group 1 and 19.5% in group 2) and nasopharyngitis (9.2% in group 1 and 13.2% in group 2) .

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The proportion of subjects with one or more serious adverse events (SAEs) was

9.2% in group 1 and 7.0% in group 2.

The proportions of subjects who discontinued the study agent due to one or more AEs (POAEs) were 6.6% in group 1 and 5.6% in group 2.

The proportions of subjects with one or more infections were comparable between the two groups (48.7% in group 1 and 45.7% in group 2). The most common infections in the pooled group were URTI (27.6% in group 1 and 19.5% in group 2) and nasopharyngitis (9.2% in group 1 and 13.2% in group 2).

Group 1 subjects did not have serious infections; serious infections were noted in 3 subjects in group 2.

Investigators reported two severe cardiovascular AEs (SAEs): 1 myocardial infarction (in a subject treated with a dose of 90 mg in group 2 with a dose interval of 1 p/24 weeks) and 1 stroke (in a subject treated with a dose of 45 mg per day). group 2 with an interval between doses of 1 r/24 weeks).

Of the 378 randomized subjects, 10 had at least one malignancy: 6 subjects had non-melanoma skin cancer (NMSC) and an additional 4 subjects had other malignancies.

Subjects in group 1 did not experience injection site reactions (ISRs). In the group, 22.0% of injections of placebo and 0.4% of injections of the drug at a dose of 45 mg were accompanied by ISR; they were all of low intensity.

Significant deviations from the norm of general and biochemical blood tests were rare.

For the total population of 478 subjects recruited into the study, from week 0 to week 124:

Two deaths were recorded during the study. 1 death was due to natural causes and the other due to acute myeloid leukemia (AML). No deaths were recorded during the open period (week 0 to week 28).

Up to week 124, 39 subjects (8.2% of the total study population) experienced one or more SAEs.

Serious infections were reported in 7 subjects (1.5%).

No opportunistic infections or cases of active tuberculosis (TB) were reported.

Up to week 124, 7.3% of all subjects recruited into the study discontinued study agent due to 1 or more AEs.

During the study period, no possible anaphylactic or possible painful serum reactions associated with the use of ustekinumab were reported.

A total of 12 subjects (2.5%) recruited into the study had one or more malignancies (including NMSC and other malignancies): 5 subjects had basal cell carcinoma (BCC), 4 subjects had squamous cell carcinoma of the skin (SCC), 6 subjects had other malignancies.

Three MACEs (0.6%) were reported in 3 subjects: myocardial infarction in 2, and stroke in 1.

conclusions

Efficacy was generally better maintained in patients responding to treatment at week 28 randomized to a fixed maintenance dose interval (1 r/12 wd) compared to those randomized to an individually adjusted maintenance dose group (1 r /12nd, 1p/16nd, 1p/20nd, or 1p/24nd), especially for high performance endpoints (PGA 0, PASI 90, or PASI 100).

No new safety signals were observed during the study period, and safety findings were similar between the two randomized groups.

Other assays to better profile the subjects in group 2 who maintained a sustained treatment response at longer dosing intervals through RNA and DNA analyzes are ongoing.

Degree of impact

A summary of the cumulative dose of ustekinumab received by randomized subjects from week 28 to week 124 is provided. The mean number of administrations of study drug was as expected for each treatment regimen and time period (6.1 for group 1 and 4.1 for group 2) .

Primary Endpoint Analysis

Primary Endpoint

The primary endpoint of this study is the number of visits at which subjects had a static PGA clearance score (0) or minimal symptoms (1) between week 88 and week 112 (score interval), for subjects randomized at week 2 to 8 into groups 1 and 2 .

The evaluation interval included a total of 7 visits every 4 weeks. The median number of visits (95% CI) in which subjects had a static PGA score clearing (0) or minimal manifestations (1) during the assessment interval was calculated for Group 1 and Group 2. Assuming normal distributions, a 95% CI difference was provided.

- 46 040272 primary endpoint averages.

The number of visits at which subjects achieved a PGA score (0) or minimal manifestations (1) between week 88 and week 112 is summarized in Table 1. 9 below.

Group 2 had fewer visits (mean difference was -0.46) at which subjects had a PGA clearance score (0) or minimal manifestations (1) during the assessment interval, compared to group 1. In addition, group 1 ( 55.3%) had a higher proportion of subjects with a PGA score clearing (0) or minimal manifestations (1) during all 7 visits during the assessment interval compared to group 2 (38.1%). Interestingly, during this interval, group 1 (55.3%) had a higher proportion of subjects with a PGA score of 0/1 on all 7 visits during the assessment interval compared to group 2 (38.1%). In addition, the proportions of subjects who had no PGA-scored treatment effect clearance (0) or minimal symptoms (1) during the evaluation interval were similar for group 1 (22.4%) and group 2 (24.2 %).

Subgroup Analysis

In general, there was no significant difference in the number of visits at which subjects achieved a static PGA score clearing (0) or minimal manifestations (1) (PGA 0/1) during the score interval in the different analyzed subgroups of groups 1 and 2. Subgroups were defined on based on baseline demographic characteristics, baseline disease characteristics, and medical history. Little variability was observed in the number of visits at which subjects in some subgroups achieved a PGA score of 0/1 between week 88 and week 112; the observed variability may be due to the limited sample size within each subgroup.

Analysis of primary secondary endpoint(s)

Primary secondary analyzes of the proportion of subjects (95% CI) with a treatment effect as measured by PGA clearance (0) or minimal symptoms (1) or as measured by PASI 75 over time were performed based on efficacy in eligible subjects randomized at week 28, at according to the allocation to the treatment option group, regardless of the actual treatment received. For the primary secondary endpoints, the number of visits at which subjects achieved a treatment response as measured by PASI 75 during the evaluation interval between weeks 88 and week 112, the same missing data handling rules were applied as in the primary analysis so that all randomized subjects.

Treatment effects as assessed by PGA clearing (0) or minimal manifestations (1) from week 28 to week 112 in a population of randomized subjects

Treatment effects as assessed by PGA clearance (0) or minimal manifestations (1) over time from week 28 to week 112 for groups 1 and 2 are summarized in FIG. 6. During the dosing interval period (week 28 to week 40), the frequency of PGA 0/1 effects decreased in both groups (FIG. 6; data not published). As expected, a greater reduction was observed in group 2 compared to group 1, as the determination of individually adjusted dosing intervals was based on the deterioration of subjects in group 2. At week 40, the proportion of subjects achieving a PGA score of 0/1 was 67 .1% for group 1 and 56.4% for group 2. After week 40, there was some variability in effects based on variation in time points between 1 p/12 week injections, as seen in previous ustekinumab clinical trials, with a more pronounced in arm 1. However, effect rates were generally maintained at visits between 1 p/12 weeks (eg, weeks 40, 52, 64, etc.) at which the next ustekinumab injection was administered. The effect rates for group 2 were also substantially maintained over time until week 112. The effect rates at most visits were slightly higher in group 1 compared to group 2 (FIG. 6).

Number of visits to which subjects randomized at week 28 achieved a PASI 75 treatment response between week 88 and week 112.

The number of visits during the assessment interval on which subjects had a treatment effect as measured by PASI 75 is summarized in Table 1. 10 below. Similar to the respective analyzes based on treatment effects on the PGA score, group 2 had slightly fewer visits where subjects had an effect on the PASI score of 75 compared to group 1 (mean difference was -0.32 visits); on average, group 1 had 5.8 visits with a PASI score of 75, and group 2 had 5.4 visits with a PASI score of 75. Moreover, there were similar proportions of patients with an effect on a PASI score of 75 in group 1 (69.7%) and group 2 (66.9%) for each possible number of visits with an effect (ranging from 0 to 7).

Similar results for the number of visits during the evaluation interval on which subjects were affected by the PASI 75 score were observed in subgroup analyses.

Proportion of subjects responding to treatment as assessed by PASI 75 across visits from week 28 to week 112 for subjects randomized at week 28

The frequency of treatment effects as assessed by PASI 75 over time from week 28 to week 112 is summarized in FIG. 7. In general, the pattern of treatment effects as measured by PASI 75 over time from week 28 to week 112 is comparable to the effects as measured by PGA 0/1 (FIG. 6; data not published). However

- 47 040272 The reduction in PASI 75 effect rates during the dosing interval period from week 28 to week 40 is less comparable to the observations for PGA effects during this period. After week 40, the effect rates for group 1 and group 2 were essentially the same until week 112. The effect rates in group 1 at most visits from week 44 to week 112 were slightly higher in group 1 compared to group 2. It was noted that the administration pattern with a frequency of 1 p/12 weeks for the effects according to the PASI 75 assessment, which was more pronounced for group 1, similar to what was observed for the effects according to the PGA 0/1 assessment. Because the PASI score includes both body surface area and qualitative elements (erythema, scaling, induration), it can provide a better representation of the overall burden of disease over time than using the PGA score alone, which takes into account only the qualitative features of psoriasis. .

Other secondary PGA treatment effect endpoints

PGA score, clearance (0) and PGA score mild or improvement (< 2) from week 28 to week 112 for subjects randomized at week 28

The frequency of treatment effects as assessed by PGA clearance (0) over time from week 28 to week 112 is summarized in FIG. 8. The separation between the treatment response curves for group 1 (ustekinumab 1 r/12 wd maintenance schedule) and group 2 (ustekinumab maintenance dose schedule at individually adjusted intervals) was already noticeable at the first visit after randomization (week 32), with the frequency of effects over time for group 1 was consistently higher than that for group 2. Differences in PGA 0 effects between the two groups were greater than for PGA 0/1 or PASI 75 effects, especially at later time points. . PGA scores of mild or improved (PGA < 2) over time from week 28 to week 112 were comparable for both groups and were generally stable over time.

Treatment Effects by PGA Score from Week 28 to Week 112 for Subjects Administered at a Maintenance Dose Schedule at Individually Adjusted Intervals (Arm 2) PGA Clearance Scores (0), PGA Clearance Scores (0) or Minimal Events (1), and PGA Scores mild or improvement (< 2) from week 28 to week 112 was determined in group 2 (data not published). In terms of PGA effects, clear (0) and clear or minimal (0 or 1), as expected, subjects in group 2, the 1 r/20 weeks and 1 r/24 weeks subgroups showed better efficacy, than in subjects of group 2, in the subgroups of drug use 1 p / 12 weeks and 1 p / 16 weeks. In addition, the data show that in more than 25% of the initially responding patients randomized to group 2, the interval between doses was eventually increased to 1 p/24 weeks, and in the majority of these subjects, the effect was maintained over time.

Other secondary endpoints of treatment effect as assessed by PASI

Treatment effects as assessed by PASI from week 28 to week 112 for subjects who received a maintenance dose schedule at individually adjusted intervals (group 2):

Here, PASI 50, PASI 75, PASI 90 and PASI 100 scores are determined over time from week 28 to week 112 for group 2 (data not published). In terms of effects as assessed by PASI 7 5 , PASI 9 0 and PASI 100 , as expected, subjects in group 2 in the 1 p/24 week treatment subgroup had better efficacy than subjects in the other subgroups of group 2.

Number of visits for which subjects on an individually adjusted interval maintenance dose schedule (Arm 2) had a treatment effect as assessed by PASI 75 between week 88 and week 112

The results for the number of visits where subjects from group 2 had an effect on the PASI 75 score between week 28 and week 112 are summarized in Table 1. 11 below. The average number of visits on which subjects had an effect, as assessed by PASI 75 between week 88 and week 112, increased in parallel with the lengthening of the interval between doses.

Treatment effects as assessed by PASI 90 from week 28 to week 112 for subjects randomized at week 28

Effect rates as assessed by PASI 90 from week 28 to week 112 are summarized in FIG. 9 (data not published). Separation between the treatment response curves for group 1 (ustekinumab 1 r/12 wd maintenance schedule) and group 2 (ustekinumab maintenance dose schedule at individually adjusted intervals) was also observed from the first visit after randomization (week 32), remaining until weeks 108 (Fig. 9). The proportion of subjects who achieved and sustained a PASI 90 effect was higher in group 1 than in group 2. The separation between the PASI 90 effect curves for group 1 and group 2 is more pronounced compared with the results for group 2. PGA 0/1 and PASI 75 effect curves.

Safety

Safety evaluations are focused on the double-blind part of the study lasting from week 28 to week 124 because comparisons can be made between randomized groups by treatment option. Ancillary data up to week 124 are also presented. 12 below provides an overview of key safety findings from week 28 to week 124.

All adverse events

In treated subjects randomized at week 28, from week 28 to week 124:

The proportions of subjects experiencing 1 or more AEs were similar in group 1 (72.4%) and group 2 (72.8%); see table. 12 (data not published). The most common AEs were in the system organ class (SOC) of infectious and parasitic diseases, both for group 1 (46.1%) and for group 2 (48.7%; 48.1% combined for groups 1 and 2).

The most common AEs in this SOC were URTI (27.6% in group 1 and 19.5% in group 2) and nasopharyngitis (9.2% in group 1 and 13.2% in group 2).

For all enrolled subjects, from week 0 to week 124:

A total of 72.6% of subjects experienced 1 or more AEs. Similarly, the most frequently reported AEs were in the SOC of infectious and parasitic diseases (49.8%), with the most frequently reported AEs being URTI (19.9%) and nasopharyngitis (13.8%).

Deaths, other serious adverse events and other significant adverse events

Deaths

There were two deaths before week 124. One subject (001009010) died of natural causes. This subject received ustekinumab 90 mg and was randomized to group 1 with a fixed dosing interval of 1 p/12 weeks.

Another subject (001029005) died due to acute myeloid leukemia (AML). This subject had an elevated platelet count prior to study entry and was diagnosed with AML 2 years after the first dose of study drug. This subject was administered ustekinumab at a dose of 90 mg and was included in group 2, a subgroup with an individually adjusted interval of 1 p/12 weeks.

Other serious adverse events

The proportion of treated subjects randomized at week 28 who experienced one or more serious adverse events (SAEs) up to week 124 was low in both group 1 (9.2%) and group 2 (7.0%) ( Table 5). No specific pattern of SAEs was observed and most SAEs were reported as single and isolated events. Of all subjects recruited into the study, 8.2% (39/478) experienced 1 or more SAEs between week 0 and week 124 (data not published).

Adverse events leading to discontinuation of study agent

Of treated randomized subjects, the proportions of subjects who discontinued study agent between week 28 and week 124 due to one or more AEs (POAEs) were low (6.6% in group 1, 5.6% in group 2) [cm. tab. 12]. No specific pattern of AE was observed that led to discontinuation of the drug, and most events were reported as single events.

Of all subjects recruited into the study, 7.3% discontinued study agent due to 1 or more AEs between week 0 and week 124 (data not published).

Infections, Serious Infections, and Infections Requiring Treatment Of treated randomized subjects, comparable proportions of subjects in groups 1 and 2 (48.7% and 45.7%, respectively; Table 5) experienced one or more infections from week 28 to week 124 The most common infections were URTI (27.6% of subjects in group 1 and 19.5% in group 2) and nasopharyngitis (9.2% of subjects in group 1 and 13.2% in group 2). No serious infections were reported in group 1, and 3 serious infections were reported in subjects in group 2 (Table 12). Reported serious infections included 1 case of bacterial infection, 1 case of cystitis, and 1 case of urinary tract infection. Infections requiring treatment with oral or parenteral antimicrobials were reported in 18 of 7 6 (23.7%) subjects in group 1 and 70 of 302 (23.2%) subjects in group 2 (see Table 12). Overall, the most common type of infection requiring treatment was URTI (5.3% in group 1; 5.0% in group 2).

Of all subjects recruited into the study, 47.3% experienced 1 or more infections between week 0 and week 124 (data not published). The most frequently reported infections were URTI (19.9%) and nasopharyngitis (11.7%). The proportion of subjects with one or more serious infections was 1.5% (data not published). No cases of active TB or opportunistic infections were reported before week 124.

Reactions at the injection site

From week 28 to week 124, no injection site reactions (ISRs) were reported among subjects in group 1, either with placebo or with the 45 mg dose. Among group 2 subjects, there were 6 ISRs associated with placebo administration (2.0%) and 1 ISR with ustekinumab 45 mg (0.4%). All registered ISRs were mild (data not published).

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Possible anaphylactic or possible painful serum reactions associated with the use of ustekinumab

No subject prior to week 124 experienced a possible anaphylactic reaction or a possible painful serum reaction associated with the use of the study agent.

Malignant neoplasms

Of the treated randomized subjects, 10 had at least one malignancy between week 28 and week 124 (Table 12). Six of 378 (1.6%) subjects had non-melanoma skin cancer (NMSC), including 2 of 76 (2.6%) subjects in Group 1 and 4 of 302 subjects in Group 2 (1.3%).

Four of 378 (1.1%) treated randomized subjects had other types of malignancies. One of 76 (1.3%) subjects in Group 1 had TCC (45 mg dose). Three of 302 (1.0%) subjects in group 2 had malignancy, including 1 each of pancreatic cancer (45 mg dose), acute myeloid leukemia (90 mg dose), and chronic myeloid leukemia (90 mg dose) .

Of all subjects with malignancies (including NMSC and other malignancies) recruited into the study, 2.5% (12/478) had malignancies between week 0 and week 124. In addition to the above, there were three cases of NMSC and 2 cases of other malignancies (1 case of colon cancer (90 mg dose) and 1 case of prostate cancer (90 mg dose)).

Cardiovascular events

Investigators reported two MACEs in treated randomized patients from week 28 to week 124. One subject in group 2 (subject 001050002) who received ustekinumab 90 mg in the subgroup at 1 p/24 weeks interval experienced a myocardial infarction, and 1 subject from group 2 (subject 001027002) who received ustekinumab at a dose of 45 mg, also in the subgroup with an interval of 1 p/24 weeks, had a stroke. Investigators reported another MACE, myocardial infarction, in a subject receiving ustekinumab 45 mg (Subject 001029007) that occurred before week 28.

Laboratory indicators

Some subjects had significant CBC laboratory abnormalities, but the incidence of significant laboratory abnormalities was essentially low and comparable between groups 1 and 2 from week 28 to week 124 (data not published). The most frequently reported significant laboratory abnormality in a complete blood count was a decrease in the number of lymphocytes (5.3% [20/378]). Significant CBC laboratory abnormalities occurring in more than 1 case were observed only in group 2 and included an increase in the number of leukocytes (0.3%), a decrease in the number of lymphocytes (1.3%) and an increase in the number of eosinophils (0, 3%).

Some subjects also showed significant abnormalities in blood chemistry (data not published). The frequency of significant laboratory abnormalities was essentially low in both groups 1 and 2. The only significant abnormality that occurred in more than 1 case was an increase in alkaline phosphatase, ALT, AST, and total bilirubin, all of which were observed at low frequency in subjects in group 2. The most common significant abnormality observed in both groups 1 and 2 was increased ALT activity (3.2% [12/378]).

Immunogenicity

In the ustekinumab-treated population, of the samples to be tested up to week 124, 63 of 455 (13.8%) patients tested positive for ustekinumab antibodies. This percentage is similar among patients who received the 45 mg dose (n=41; 13.9%) and those who received the 90 mg dose (n=22; 13.7%) as in group 1 (n=7; 9, 2%) and in group 2 (n=32; 10.6%). The majority of patients with antibodies (33 of 63) had titers < 1:800. Most of the antibodies produced (in antibody positive patients with sufficient serum samples) were able to neutralize ustekinumab's in vitro biological activity (47 of 62, 75.8%). When testing dosing intervals that allowed ustekinumab concentrations to be reduced between injections below quantifiable levels (which approached multiple cycles of withdrawal and resumption), no increased ability to form anti-drug antibodies was found. These results indicate that there is no increased risk of immunogenicity as the interval between doses increases to 24 weeks.

Predicted PGA=0 Week 28

Patients in group 2, who consistently maintained clinical effects of treatment over time at a 24-week interval between injections, as a rule, showed high levels of effect according to the most stringent criteria during the initial treatment lead-in period. Because a high proportion of patients in the 24-week dosing interval subgroup had

- 50 040272 PGA 0 score (Table 6), the usefulness of this treatment effect parameter as a predictive marker of the ability to maintain clinical effect over this interval was recognized. Receiving a PGA score of 0 at week 28 correlated with a PPV of 60% for maintaining a PGA score of 0 or 1 with any dosing interval greater than 12 weeks (e.g., 16 weeks, 20 weeks, 24 weeks) for < five of seven visits at assessment period from week 88 to week 112. In addition, a PPV of 44% was consistent with maintaining a PGA score of 0 or 1 at any dosing interval greater than 12 weeks at all seven visits. Receiving a PGA=0 score at week 28 correlated with a PPV of 44% and 32%, respectively, for PGA 0 for < five of the seven visits in the assessment period from week 88 to week 112, and all seven visits. Sensitivity and specificity for these definitions varied in the range of 61-75%, as shown in table.6.

Table 6. Possibility of PGA=0 at week 28 to predict the long-term effect of treatment at a dose of 1 p/12-24 wd, depending on the effect among all patients randomized at week 28

Visits with a PGA score < 2 from week 88 to week 112

Sensitivity

Specificity

interval between maintenance doses (TPR) ^a (FPR) ^b PPV ^{C NPVd} introduction of a maintenance dose 1 r / 24 nd > 5 visits 75% 72% 44% ^a 91% ^b 7 visits 75% 69% 32% ^a 93% ^b introduction of a maintenance dose 1 r/20 nd or 1 r/24 nd > 5 visits 66% 74% 53% 83% 7 visits 69% 70% 41% 88% administration of a maintenance dose > 1 r/12 n ^e > 5 visits 61% 75% 60% 76% 7 visits 63% 70% 44% 88%

^a Sensitivity, or true positive rate (TPR), is the proportion of patients, excluding the 1 r/12 wd or 1 r/24 wd maintenance subgroups, that can be correctly identified by a predictive marker or criterion.

^b Specificity, or true negative rate (TNR), is the proportion of patients to be excluded from the predicted group, which is assumed if the predictive marker is negative.

^c PPV represents the probability of a given outcome, if the predictor is positive, that is, the probability of a positive result after analysis. As an additional explanation, these specific PPVs are based on the observation that 117/302 patients from group 2 had a PGA score of 0 at week 28, and 51/117 (44%) patients were on maintenance doses of 1 r/24 wd and had a PGA score. < 2 at five or more visits from week 88 to week 112. Similarly, 38/117 (32%) of these patients from group 2 with 1 q/24 wd dosing had a PG score >

^d NPV represents the probability of a negative outcome if the predictive marker is negative, i.e. the probability of a negative outcome after analysis. As an additional explanation, these specific NPVs are derived from the observation that 17/185 patients from group 2, with a PGA score of 1 at week 28, were treated with maintenance doses of 1 r/24 wd, and had a PGA score of < 2 at week 28. five or more visits (NPV=[185-17)]/185=91%), and 13/185 patients had a PGA < 2 at all seven visits (NPV=[185-13]/185=93%), from a week 88 to week 112.

^e > 1 r/12 nd includes the interval between administrations of the maintenance dose 1 r/16 nd, 1 r/20 nd, 1 r/24 nd.

FPR - False Positive Rate, NPV Negative Predictive Value, PGA - Physician Global Score, PPV - Positive Predictive Value, 1 p/12/16/20/24 wd - every 12/16/20/24 weeks, TPR - True Positive Rate results

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Table 7. Study Agent Administration Schedule

Open introductory period Subjects with a static PGA score clearing (0) or minimal manifestations (1) will be randomized at week 28 to 1 of 2 treatment option troupes. Group 1 will receive the drug in accordance with the approved maintenance dose regimen. In group 2, the maintenance dose regimen will be determined by the time to loss of treatment effect (defined as the PGA 2 score). ^A Double-blind Treatment Period (Weeks) weeks 0 4 16 28 ^b 32 ^b 36 ^b 40 ^b 44 48 52 56 60 64 68 72 76 80 84 88 92 96 100 104 A A A Group 1: administration of a maintenance dose in accordance with the approved regimen (ustekinumab 1 r/12 d, starting from weeks 28) A R R A R A R R A R A R R A R R A A A Group 2a: If loss of treatment effect occurs by week 16 after the injection at week 16 (i.e. by the visit to week 32), then subjects will receive ustekinumab 1 p/12 d starting at week 32 R A R R A R A R R A R A R R A R A A A A Group 2b: If loss of treatment effect occurs by week 20 after the injection at week 16 (i.e. by the visit for week 36), then subjects will receive ustekinumab 1 p/16 wd starting at week 36 Group 2a: If loss of treatment effect occurs by week 24 after the injection at week 16 (i.e. by the follow-up visit week 40), then subjects will receive ustekinumab 1 r/20 wd starting at week 40 Group 2d: If loss of treatment effect does not occur by week 24 after injection at week 16 (i.e. by week 40 visit), then subjects will receive ustekinumab 1 p/24 wd starting at week 40 R R R R R R R R R A R R R R R R R R R A R R R R R R R R R A R R R R R R R R A R R R R

= Treatment with active drug ustekinumab 45 mg (for subjects weighing < 100 kg) or 90 mg (for subjects weighing > 100 kg) = Placebo = No administration ^of study agent (0) or minimal (1) at week 28, or who discontinued investigational agent before week 28, who did not receive all 3 injections of investigational agent before week 2-8 (i.e., injections at weeks 0, 4, 16) will be further injections of investigational agent have been discontinued, they will be monitored for reliability for at least 20 weeks after their last injection of investigational agent, after which they will be withdrawn from the study.

^b At these visits, the PGA score must be entered into the IVRS/IWRS program. At these visits, particular attention should be paid to the timing of the visits to allow administration of the investigational agent based on disease activity.

Note. At each applicable visit, subjects will receive 1 to 2 injections. Subjects < 100 kg will receive 1 injection (ustekinumab 45 mg or placebo) and subjects > 100 kg will receive 2 injections (ustekinumab 45 mg + ustekinumab 45 mg or placebo + placebo), depending on the treatment option group to which they are distributed, and from visiting. The study will use a dose of study agent based on baseline body weight.

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Table 8

Table 8 Number of subjects who discontinued study agent from week 28 to week 104; all subjects randomized at week 28 (study CNTO1275PSO3009)

Group 2: ustekinumab maintenance dose regimen with individually adjusted

intervals Group 1: scheme All introductions maintenance dose randomiziro bathrooms 1 1/ R/ 'nd 1 r/ 16 nd 1 r/ 2 0 nd 1 r/ 2 4 nd Total ustekinumab 1 p/12 n.d. subjects Sample for analysis: all subjects randomized at week 28 76 302 84 61 51 84 378 Subjects who discontinued study agent application 17 ( 22.4%) 67 (22.2%) 17 (20.2%) 7 (11.5%) 7 (13.7%) 14 (16.7%) 84 (22.2%) Reason for termination adverse event 4 (5.3%) 15 (5.0%) 4 (4.8%) 3 (4.9%) 2 (3.9%) 3 (3.6%) 19 (5.0%) Death 1 (1.3%) 0 0 0 0 0 1 (0.3%) Lack of effectiveness 2 (2.6%) 8 (2.6%) 4 (4.8%) 2 (3.3%) 1 (2.0%) 1 (1.2%) 10 (2.6%) impossibility follow-up 6 (7.9%) 12 (4.0%) 3 (3.6%) 0 3 (5.9%) 3 (3.6%) 18 (4.8%) Pregnancy 0 3 (1.0%) 1 (1.2%) 0 0 0 3 (0.8%) protocol violation 0 4 (1.3%) 1 (1.2%) 0 0 0 4 (1.1%) Termination of the study by the sponsor 0 0 0 0 0 0 0 Withdrawal of consent 2 (2.6%) 15 (5.0%) 3 (3.6%) 1 (1.6%) 0 6 (7.1%) 17 (4.5%) Other 2 (2.6%) 10 (3.3%) 1 (1.2%) 1 (1.6%) 1 (2.0%) 1 (1.2%) 12 (3.2%)

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Table 9

Number of visits at which subjects achieved a PGA score clearing (0) or minimal manifestations (1) between week 88 and week 112; all subjects randomized at week 28 (study

CNTO1275PSO3009) Group 1: scheme Group 2: administration scheme introductions maintenance dose maintenance dose ustekinumab with ustekinumab individually 1 r / 12 nd hand-picked Sample for analysis: all subjects, 76 intervals 302 randomized at week 28 N ^a 76 302 Average (Std. off.) 4.5 (3.07) 4.1 (2.93) 95% confidence (3.81; 5.21) (3.72; 4.39) mean interval ^Median 7.0 50 Interval (0; 7) (0; 7) IQ interval (1.0; 7.0) (1.0; 7.0) Difference between groups by treatment option 95% confidence ^interval -0.46 (-1.20; 0.29)

Number of visits at which subjects achieved PGA score clearance (0) or minimal manifestations (1) 0 17 (22.4%) 73 (24.2%)

6 (7.9%) 19 (6.3%)

2 (2.6%) 17 (5.6%)

3 (3.9%) 17 (5.6%)

2 (2.6%) 16 (5.3%)

2 (2.6%) 20 (6.6%)

2 (2.6%) 23 (7.6%)

42 (55.3%) 117 (38.7%) a After treatment failure rules were applied, subjects with missing data between weeks 88 and 112 were treated as follows:

(1) If the missed visit was intermediate, then the missing value was replaced by a weighted average, depending on the distance of the available values before and after the missed visit (linear relationships were assumed for visits). The resulting score was rounded to the nearest whole number.

(2) If the missed visit was not an intermediate visit, i.e. Since there was no data available after the missed visit, the method of carrying forward the last observation was used to replace the missing data.

^b 95% confidence interval was based on a normal fit.

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Table 10

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Table 10 Number of visits for which subjects achieved treatment response as assessed by PASI 75 between week 88 and week 112; all subjects randomized at week 28 (study CNTO1275PSO3009)

Group 1: regimen Group 2: maintenance dose regimen maintenance dose of ustekinumab with ustekinumab 1 p/12 at individually selected intervals

Sample for analysis: all subjects randomized at week 28 N ^a 76

Medium (Std.

5.8 (2.31) off)

95% confidence (5.23; 6.29) interval of the mean ^b

Median 7.0

Interval (0; 7)

IQ interval (6.0,-7.0)

Difference between groups by treatment option 95% confidence ^interval

302

302

5.4 (2.61) (5.15; 5.74)

7.0 (0; 7) (5.0; 7.0)

-0.32 (-0.96; 0.33)

Number of visits for which subjects had the effect of PASI treatment 75

0 7 (9.2%) 43 (14.2%: 1 2 (2.6%) 7 (2.3%) 2 0 8 (2.6%) 3 5 (6.6%) 5 (1.7%) 4 1 (1.3%) 9 (3.0%) 5 2 (2.6%) 11 (3.6%) 6 6 (7.9%) 17 (5.6%) 7 53 (69.7%) 202 (66.9%

^a After applying the treatment failure rules, subjects with missing data between weeks 88 and 112 were processed as follows:

(1) If the missed visit was intermediate, then the missing value was replaced by a weighted average, depending on the distance of the available values before and after the missed visit (linear relationships were assumed for visits).

(2) If the missed visit was not an intermediate visit, i.e. Since there was no data available after the missed visit, the method of carrying forward the last observation was used to replace the missing data.

^b 95% confidence interval was based on a normal fit.

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Table 11

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Table 11 Number of visits for which subjects achieved treatment response as assessed by PASI 75 from week 88 to week 112; Subjects Randomized at Week 28 to a Customized Interval Maintenance Dose of Ustekinumab (Study CNTO1275PSO3009)

Group 2: maintenance dose schedule of ustekinumab at individually adjusted intervals

All randomize-

1 r / 12 nd 1 r / 16 nd 1 r/20 1 r/24 shaped subjects nd nd Sample for analysis: subjects divided into week 28 to the regimen group 84 61 51 84 302 maintenance dose of ustekinumab at individually adjusted intervals N ^a 84 61 51 84 302 Medium (Std. 4.7 5.2 5, 6 6, 3 off) (3.03) (2.73) (2.37) (1.67) 5.4 (2.61) 95% confidence (4.07; (4.53; (4.92; (5.97; mean ^b interval 5, 38) 5, 93) 6.25) 6, 70) (5.15; 5.74) Median 7.0 7.0 7.0 7.0 7.0 Interval (0; 7) (0; 7) (0; 7) (0; 7) (0; 7) IQ interval (1.0; 7.0) (3.0; 7.0) (5.0; 7.0) (7.0; 7.0) (5.0; 7.0)

Number of visits, to which subjects had an effect treatment PASI 75 0 20 (23.8%) 9 (14.8%) 5 (9.8%) 3 (3.6%) 43 (14.2%: 1 3 (3.6%) 1 (16%) 2 (3.9%) 1 (1.2%) 7 (2.3%) 2 3 (3.6%) 4 (6.6%) 1 (2.0%) 0 8 (2.6%) 3 0 2 (3.3%) 0 3 (3.6%) 5 (1.7%) 4 2 (2.4%) 3 (4.9%) 2 (3.9%) 2 (2.4%) 9 (3.0%) 5 2 (2.4%) 0 4 (7.8%) 5 (6.0%) eleven (3.6%) 6 8 (9.5%) 2 (3.3%) 6 (11.8%) 1 (1.2%) 17 (5.6%) 7 46 (54.8%) 40 (65.6%) 31 (60.8% 69 (82.1% 202 (66.9%

) ) ^a After the treatment failure rules were applied, subjects with missing data between weeks 88 and 112 were processed as follows:

- 56 040272 (1) If the missed visit was intermediate, then the missing value was replaced by a weighted average, depending on the distance of the available values before and after the missed visit (linear relationships are assumed for visits).

(2) If the missed visit was not an intermediate one, i.e. Since there was no data available after the missed visit, the method of carrying forward the last observation was used to replace the missing data.

^b 95% confidence interval was based on a normal fit.

Table 12

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Table 12 Summary of key safety findings from week 28 to week 124; Treated Subjects Randomized at Week 28 (Study CNTO1275PSO3009)

Group 2: maintenance dose schedule of ustekinumab at individually adjusted intervals

Group 1: administration scheme All

supporting randomization 1 р/16 1 r/20 1 r/24 1 r / 12 nd dosed _________ nd nd Total nd ________

ustekinumab 1 subjects p/12 n.d.

Sample for analysis: subjects who received 302 84 61 51 84 378 treatment, randomized at week 28 76 Average duration follow-up (n.d.) Average impact (number 85.9 84.2 87.2 89.9 90.1 90.9 84.6 injections) injections before and after week 28 Subjects who discontinued study agent due to 1 or more adverse events 14.6 5 (6.6%) 14.6 17 (5.6%) 15, 3 5 (6.0% 15, 7 ) 3 (4.9% 15.7 15.9 14.6 ) 2 (3.9%) 3 (3.6%) 22 (5.8%

Subjects with 1 or more: Adverse events 55 (72.4%) 220 (72.8%) 62 (73.8%) 54 (88.5%) 35 (68.6%) 62 (73.8%) 275 (72.8%) Serious adverse phenomena 7 (9.2%) 21 (7.0%) 6 (7.1%) 2 (3.3%) 2 (3.9%) 9 (10.7%) 28 (7.4%) Common infections 37 (48.7%) 138 (45.7%) 45 (53.6%) 38 (62.3%) 19 (37.3%) 35 (41.7%) 175 (46.3%) - Serious infections 0 3 (1.0%) 2 (2.4%) 0 1 (2.0%) 0 3 (0.8%) - Requiring treatment 18 (23.7%) 70 (23.2%) 22 (26.2%) 20 (32.8%) 12 (23.5%) 15 (17.9%) 88 (23.3%) malignant neoplasm 3 (3.9%) 7 (2.3%) 3 (3.6%) 1 (1.6%) 1 (2.0%) 1 (1.2%) 10 (2.6%) MACE * 0 2 (0.7%) 0 0 0 2 (2.4%) 2 (0.5%) * MACE: myocardial infarction (MI), stroke or death due to cardiovascular disease

- 57 040272

SEQUENCES

SEQ ID NO: 1 < 211>5 < 212> PRT < 213> Homo sapiens < 400>1

Thr Tyr Trp Leu Gly

SEQ ID NO: 2 <211>17 < 212>PRT < 213> Homo sapiens < 400>2 lie Met Ser Pro Vai Asp Ser Asp lie Arg Tyr Ser Pro Ser Phe Gin

1015

gly

SEQ ID NO: 3 <211>10 <212> PRT <213> Homo sapiens

- 58 040272 <400>

Arg Arg Pro Gly Gin Gly Tyr Phe Asp Phe 1510

SEQ ID NO: 4 <211>11 <212>PRT <213> Homo sapiens <400>4

Arg Ala Ser Gin Gly He Ser Ser Trp Leu Ala 1510

SEQ ID NO: 5 < 211>7 < 212> PRT < 213> Homo sapiens < 400>5

Ala Ala Ser Ser Leu Gin Ser 15

SEQ ID NO: 6 <211>9 <212> PRT <213> Homo sapiens <400>6

Gin Gin Tyr Asn He Tyr Pro Tyr Thr 15

SEQ ID NO: 7 <2H>119 < 212>PRT < 213> Homo sapiens < 400>7

Glu Vai Gin Leu Vai Gin Ser Gly Ala Glu Vai Lys Lys Pro Gly Glu 15 1015

Ser Leu Lys He Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr 20 2530

Trp Leu Gly Trp Vai Arg Gin Met Pro Gly Lys Gly Leu Asp Trp lie 35 4045

Gly He Met Ser Pro Vai Asp Ser Asp He Arg Tyr Ser Pro Ser Phe 50 5560

Gin Gly Gin Vai Thr Met Ser Vai Asp Lys Ser He Thr Thr AlaTyr

70 7580

Leu Gin Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr TyrCys

9095

- 59 040272

Ala Arg Arg Arg Pro Gly Gin Gly Tyr

100105

Thr Leu Vai Thr Vai Ser Ser

115

SEQ ID NO: 8 <2H>108 < 212>PRT < 213> Homo sapiens < 400>8

Asp He Gin Met Thr Gin Ser Pro Ser 15

Asp Arg Vai Thr He Thr Cys Arg Ala 2025

Leu Ala Trp Tyr Gin Gin Lys Pro Glu 3540

Tyr Ala Ala Ser Ser Leu Gin Ser Gly 5055

Ser Gly Ser Gly Thr Asp Phe Thr Leu 6570

Glu Asp Phe Ala Thr Tyr Tyr Cys Gin 8590

Thr Phe Gly Gin Gly Thr Lys Leu Glu

100105

SEQ ID NO: 9

Phe Asp Phe Trp Gly Gin Gly

110

Ser Leu Ser Ala Ser Vai Gly

15

Ser Gin Gly He Ser Ser Trp

Lys Ala Pro Lys Ser Leu He

Vai Pro Ser Arg Phe Ser Gly

Thr lie Ser Ser Leu Gin Pro

80

Gin Tyr Asn lie Tyr Pro Tyr

He Lys Arg

- 60 040272 <2H>503 <212>PRT <213> Homo sapiens <400>9

Arg Asn Leu Pro Vai Ala Thr Pro

His His Ser Gin Asn Leu Leu Arg

Ala Arg Gin Thr Leu Glu Phe Tyr

3540

His Glu Asp lie Thr Lys Asp Lys

5055

Pro Leu Glu Leu Thr Lys Asn Glu

6570

Ser Phe lie Thr Asn Gly Ser Cys

Met Met Ala Leu Cys Leu Ser Ser

100

Gin Vai Glu Phe Lys Thr Met Asn

115120

Arg Gin lie Phe Leu Asp Gin Asn

130135

Met Gin Ala Leu Asn Phe Asn Ser

145150

Leu Glu Glu Pro Asp Phe Tyr Lys

165

Asp Pro Gly Met Phe Pro Cys Leu

1015

Ala Vai Ser Asn Met Leu Gin Lys

2530

Pro Cys Thr Ser Glu Glu He Asp

Thr Ser Thr Vai Glu Ala Cys Leu

Ser Cys Leu Asn Ser Arg Glu Thr 7580

Leu Ala Ser Arg Lys Thr Ser Phe

9095

He Tyr Glu Asp Leu Lys Met Tyr

105110

Ala Lys Leu Leu Met Asp Pro Lys

125

Met Leu Ala Vai He Asp Glu Leu

140

Glu Thr Vai Pro Gin Lys Ser Ser

155 160

Thr Lys He Lys Leu Cys He Leu

170 175

-

Claims (2)

Vai Thr He Asp Arg Vai Met Ser 185 190 Leu Lys Lys Asp Vai Tyr Vai Vai 205 Pro Gly Glu Met Vai Vai Leu Thr 220 He Thr Trp Thr Leu Asp Gin Ser 235240 Thr Leu Thr He Gin Vai Lys Glu 250255 Cys His Lys Gly Gly Glu Vai Leu 265270 Lys Lys Glu Asp Gly He Trp Ser 285 Glu Pro Lys Asn Lys Thr Phe Leu 300 Gly Arg Phe Thr Cys Trp Trp Leu 315320 Phe Ser Vai Lys Ser Ser Arg Gly 330335 Cys Gly Ala Ala Thr Leu Ser Ala 345350 Glu Tyr Glu Tyr Ser Vai Glu Cys 365 Ala Glu Glu Ser Leu Pro He Glu 380 Leu Lys Tyr Glu Asn Tyr Thr Ser 395400 Lys Pro Asp Pro Pro Lys Asn Leu 410415 Arg Gin Vai Glu Vai Ser Trp Glu 425430 His Ser Tyr Phe Ser Leu Thr Phe 445 Lys Arg Glu Lys Lys Asp Arg Vai 460 Vai He Cys Arg Lys Asn Ala Ser 475 480 Tyr Tyr Ser Ser Ser Trp Ser Glu 490 495 Leu His Ala Phe Arg lie Arg Ala 180 Tyr Leu Asn Ala Ser lie Trp Glu 195200 Glu Leu Asp Trp Tyr Pro Asp Ala 210215 Cys Asp Thr Pro Glu Glu Asp Gly 225230 Ser Glu Vai Leu Gly Ser Gly Lys 245 Phe Gly Asp Ala Gly Gin Tyr Thr 260 Ser His Ser Leu Leu Leu Leu His 275280 Thr Asp lie Leu Lys Asp Gin Lys 290295 Arg Cys Glu Ala Lys Asn Tyr Ser 305310 Thr Thr lie Ser Thr Asp Leu Thr 325 Ser Ser Asp Pro Gin Gly Vai Thr 340 Glu Arg Vai Arg Gly Asp Asn Lys 355360 Gin Glu Asp Ser Ala Cys Pro Ala 370375 Vai Met Vai Asp Ala Vai His Lys 385390 Ser Phe Phe lie Arg Asp lie lie 405 Gin Leu Lys Pro Leu Lys Asn Ser 420 Tyr Pro Asp Thr Trp Ser Thr Pro 435440 Cys Vai Gin Vai Gin Gly Lys Ser 450455 Phe Thr Asp Lys Thr Ser Ala Thr 465470 lie Ser Vai Arg Ala Gin Asp Arg 485 Trp Ala Ser Vai Pro Cys Ser 500 CLAIM 1. A method of treating a patient with psoriasis using an increasing interval between doses or a maintenance dose, comprising administering to the patient an antibody to IL-12 and IL-23, where the antibody contains the amino acid sequence of the heavy chain variable region of SEQ ID NO: 7 and the amino acid sequence of the variable region light chain SEQ ID NO: 8, at the initial dose, the dose 4 weeks after the initial dose, and the dose once every 12 weeks for 24 weeks after the initial dose and increasing the interval between doses 28 weeks after the initial dose to interval between dosing every 24 weeks after identifying the patient as responding to antibody treatment 28 weeks after the initial dose, where the dose is 45 or 90 mg. 2. The method of claim 1, wherein the step of identifying a patient as responsive to antibody treatment comprises determining and identifying a patient as having a prevalence and severity index. -