Methods for producing anti-IL12 / IL23 antibody compositions

Abstract

Manufacturing methods for producing anti-IL-12 / IL-23p40 antibodies in CHO, such as the anti-IL-12 / IL-23p40 antibody ustekinumab, and certain pharmaceutical compositions of the antibodies are useful for treating a variety of diseases.

Description

[Technical field]

[0001] (Reference to electronically submitted sequence listing) This application contains a sequence listing that has been submitted electronically via EFS-Web as an ASCII format sequence listing with the file name "JBI6056WOPCT1SEQLIST.txt" created on March 5, 2020, and has a size of 14,000 bytes. The sequence listing submitted via EFS-Web is a part of the present specification and is incorporated herein by reference in its entirety.

[0002] FIELD OF THEINVENTION The present invention relates to manufacturing methods for producing anti-IL12 / IL23p40 antibodies, such as the anti-IL12 / IL23p40 antibody ustekinumab, and to certain pharmaceutical compositions of the antibodies. [Background technology]

[0003] Interleukin (IL)-12 is a secreted heterodimeric cytokine composed of two disulfide-linked glycosylated protein subunits (designated p35 and p40 for their approximate molecular weight). IL-12 is produced primarily by antigen-presenting cells and promotes cellular immunity by binding to a two-chain receptor complex expressed on the surface of T cells or natural killer (NK) cells. The IL-12 receptor beta-1 (IL-12Rβ1) chain binds to the p40 subunit of IL-12, providing the primary interaction between IL-12 and its receptor. However, it is IL-12p35 ligation of the second receptor chain, IL-12Rβ2, that confers intracellular signaling (e.g., STAT4 phosphorylation) and activation of receptor-bearing cells (Presky et al, 1996). IL-12 signaling in parallel with antigen presentation is thought to trigger T cell differentiation towards a T helper 1 (Th1) phenotype characterized by interferon gamma (IFN-γ) production (Trinchieri, 2003). Th1 cells are thought to promote immunity against several intracellular pathogens, generate complementary antibody isotypes, and contribute to tumor immunosurveillance. This makes IL-12 a critical component of the host defense immune mechanism.

[0004] It was discovered that the p40 protein subunit of IL-12 can also associate with a separate protein subunit designated p19 to form a new cytokine, IL-23 (Oppman, et al. 2000). IL-23 also signals through a two-chain receptor complex. Since the p40 subunit is shared between IL-12 and IL-23, it follows that the IL-12Rβ1 chain is also shared between IL-12 and IL-23. However, it is IL-23p19 ligation of the second component of the IL-23 receptor complex, IL-23R, that confers IL-23-specific intracellular signaling (e.g., STAT3 phosphorylation) and subsequent IL-17 production by T cells (Parham et al. 2002; Aggarwal et al. 2003). Recent studies have shown that the biological functions of IL-23 and IL-12 are distinct, despite the structural similarities between these two cytokines (Langrish et al. 2005).

[0005] Abnormal regulation of IL-12 and Th1 cell populations has been linked to many immune-mediated diseases, as neutralization of IL-12 by antibodies has been effective in treating animal models of psoriasis, multiple sclerosis (MS), rheumatoid arthritis, inflammatory bowel disease, insulin-dependent (type 1) diabetes mellitus, and uveitis (Leonard et al., 1995; Hong et al., 1999; Malfait et al., 1998; Davidson et al., 1998). IL-12 has also been shown to play an important role in the pathogenesis of systemic lupus erythematosus in two independent mouse models (Kikawada et al., 2003; Dai et al., 2007).

[0006] Systemic lupus erythematosus (SLE) is a complex, chronic, heterogeneous autoimmune disease of unknown etiology that can affect almost any organ system and follows a waxing and waning disease course. Systemic lupus erythematosus occurs much more frequently in women than men, up to nine times more frequently in some studies, and often presents during the childbearing years of 15-45 years. The disease is more prevalent in Afro-Caribbean, Asian, and Hispanic populations. In SLE, the immune system attacks the body's cells and tissues, resulting in inflammation and tissue damage that can harm the heart, joints, skin, lungs, blood vessels, liver, kidneys, and nervous system. Approximately half of subjects are diagnosed with SLE presenting with organ-threatening disease, but it may take years to diagnose subjects without organ involvement. Some of the main symptoms in newly diagnosed lupus patients are arrhythmias (62%) and skin symptoms (new photosensitivity; 20%), followed by persistent fever and fatigue. The estimated annual incidence of lupus varies from 1.8 to 7.6 cases per 100,000 people, with worldwide prevalence ranging from 14 to 172 cases per 100,000 people. Patients with mild disease have primarily skin rash and joint pain and require non-aggressive therapy. Regimens include nonsteroidal anti-inflammatory drugs (NSAIDs), antimalarials (e.g., hydroxychloroquine, chloroquine, or quinacrine), and / or low-dose corticosteroids. Patients with more severe disease may experience a range of severe conditions depending on the organ system involved, including potential renal failure, endocarditis or myocarditis, pneumonia, pregnancy complications, stroke, neurological complications, vasculitis, and cytopenias with associated risks of bleeding or infection. Common treatments for more severe disease include methotrexate (MTX), azathioprine, cyclophosphamide, cyclosporine, high-dose corticosteroids, biological B-cell cytotoxic agents or B-cell modulators, and other immunomodulators. Patients with severe SLE have a shortened life expectancy of 10 to 30 years, primarily due to complications of the disease, of standard treatment therapies, and / or of accelerated atherosclerosis. In addition, SLE has a substantial impact on quality of life, work productivity, and medical costs.Existing therapies for SLE are generally either cytotoxic or immunomodulatory, and may have significant safety risks.Newer therapies for SLE provide only minor benefits over standard treatment therapies.Therefore, there is a great unmet need for new alternative therapies that can provide significant benefits in this disease without incurring high safety risks. Summary of the Invention [Means for solving the problem]

[0007]

[0013] Embodiments of the invention are defined by the independent and dependent claims appended hereto, each of which is incorporated herein by reference for brevity. Other embodiments, features, and advantages of various aspects of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings.

[0008] In a particular embodiment, the present invention provides an anti-IL-12 / IL-23p40 antibody expressed in Chinese Hamster Ovary cells (CHO cells). An "anti-IL-12 / IL-23p40 antibody" as defined by the present invention includes an antibody having (i) a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 10 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 11, (ii) a heavy chain variable domain amino acid sequence of SEQ ID NO: 7 and a light chain variable domain amino acid sequence of SEQ ID NO: 8, and (iii) an amino acid sequence selected from the group consisting of heavy chain CDR amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and light chain CDR amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, expressed in Chinese Hamster Ovary cells (CHO cells).

[0009] In certain embodiments, the oligosaccharide profile of the anti-IL-12 / IL-23p40 antibody comprises >99.0% total neutral oligosaccharide species and <1.0% total charged oligosaccharide species. In other embodiments, (i) the oligosaccharide profile of the anti-IL-12 / IL-23p40 antibody comprises >99.0% total neutral oligosaccharide species, <1.0% total charged oligosaccharide species, and individual neutral oligosaccharide species G0F>70.0%, G1F<20.0%, and G2F<5.0%. (ii) The oligosaccharide profile comprises >99.0% total neutral oligosaccharide species and <1.0% total charged oligosaccharide species, and the peak 3 area % of the capillary isoelectric focusing (cIEF) electropherogram of the anti-IL-12 / IL-23p40 antibody is >70.0%. (iii) the anti-IL-12 / IL-23p40 antibody is free of disialylated glycan species as measured by high performance liquid chromatography (HPLC) or reduced mass spectrometry (RMA), (iv) the anti-IL-12 / IL-23p40 antibody has a longer half-life compared to the anti-IL-12 / IL-23p40 antibody expressed in Sp2 / 0 cells, and / or (v) the anti-IL-12 / IL-23p40 antibody is a follow-on biologic (antibody dependent on regulatory approval and / or data generated with ustekinumab) (sold as Stelara® by Janssen Biotech, Inc.).

[0010] In a particular embodiment, the present invention provides a manufacturing method for producing an anti-IL-12 / IL-23p40 antibody, the method comprising: a. culturing Chinese hamster ovary cells (CHO cells); b. expressing an anti-IL-12 / IL-23p40 antibody in the CHO cells; and c. purifying the anti-IL-12 / IL-23p40 antibody, wherein (i) the oligosaccharide profile of the anti-IL-12 / IL-23p40 antibody comprises >99.0% total neutral oligosaccharide species and <1.0% total charged oligosaccharide species, and (ii) the oligosaccharide profile of the anti-IL-12 / IL-23p40 antibody comprises >99.0% total neutral oligosaccharide species, <1.0% total charged oligosaccharide species, and individual neutral oligosaccharide species G0F>70.0%, G1F<20.0%, and G2F<5.0%. (iii) the oligosaccharide profile contains >99.0% total neutral oligosaccharide species and <1.0% total charged oligosaccharide species and the peak 3 area % of the capillary isoelectric focusing (cIEF) electropherogram of the anti-IL-12 / IL-23p40 antibody is >70.0%, (iv) the anti-IL-12 / IL-23p40 antibody does not contain disialylated glycan species as measured by high performance liquid chromatography (HPLC) or reduced mass spectrometry (RMA), (v) the anti-IL-12 / IL-23p40 antibody has a longer half-life compared to the anti-IL-12 / IL-23p40 antibody expressed in Sp2 / 0 cells, and / or (vi) the anti-IL-12 / IL-23p40 antibody is a follow-on biologic to ustekinumab.

[0011] In certain embodiments, the invention provides a composition comprising an anti-IL-12 / IL-23p40 antibody, wherein (i) the oligosaccharide profile of the anti-IL-12 / IL-23p40 antibody comprises >99.0% total neutral oligosaccharide species and <1.0% total charged oligosaccharide species, or (ii) the oligosaccharide profile of the anti-IL-12 / IL-23p40 antibody comprises >99.0% total neutral oligosaccharide species, <1.0% total charged oligosaccharide species, and individual neutral oligosaccharide species G0F>70.0%, G1F<20.0%, and G2F<5.0%. (iii) the oligosaccharide profile contains >99.0% total neutral oligosaccharide species and <1.0% total charged oligosaccharide species and the peak 3 area % of the capillary isoelectric focusing (cIEF) electropherogram of the anti-IL-12 / IL-23p40 antibody is >70.0%, (iv) the anti-IL-12 / IL-23p40 antibody does not contain disialylated glycan species as measured by high performance liquid chromatography (HPLC) or reduced mass spectrometry (RMA), (v) the anti-IL-12 / IL-23p40 antibody has a longer half-life compared to the anti-IL-12 / IL-23p40 antibody expressed in Sp2 / 0 cells, and / or (vi) the anti-IL-12 / IL-23p40 antibody is a follow-on biologic to ustekinumab. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows an overview of the 10 steps of the ustekinumab manufacturing process. [Diagram 2] FIG. 1 shows a flow diagram of the manufacturing process for Stage 1 of the pre-culture and growth steps, including in-process control and process monitoring tests. [Diagram 3] FIG. 1 illustrates a flow diagram of the manufacturing process steps for Stage 2, including in-process control and process monitoring testing. [Figure 4] FIG. 1 shows a representative HPLC chromatogram of oligosaccharide analysis of ustekinumab produced in Sp2 / 0 cells. [Diagram 5] FIG. 1 shows a representative deconvoluted mass spectrum of IRMA analysis of ustekinumab produced in Sp2 / 0 cells. [Figure 6] FIG. 1 shows a representative cIEF electropherogram profile of ustekinumab expressed in Sp2 / 0 cells. A diagram depicting the general relationship between cIEF peaks and decreasing negative charge / degree of sialylation is also shown, with peaks A, B, 1, 2, 3, and C labeled. [Figure 7] Figure 1 shows an overview of some of the major N-linked oligosaccharide species of ustekinumab IgG. The role of some enzymes in the glycosylation maturation process and the role of some divalent cations (e.g., Mn2+ as a cofactor and Cu2+ as an inhibitor of GalTI) are also shown (see, e.g., BiotechnolBioeng. 2007 Feb 15;96(3):538-49; Curr Drug Targets. 2008 Apr;9(4):292-309; J Biochem Mol Biol. 2002 May 31;35(3):330-6). Note that the species containing terminal sialic acid (S1 and S2) are charged species, whereas the species lacking terminal sialic acid (G0F, G1F, and G2F) are neutral species, but the generation of charged species depends on the presence of galactose in G1F and G2F added by the GalT1 enzyme. [Figure 8] FIG. 1 shows a representative HPLC chromatogram of oligosaccharide analysis of ustekinumab produced in CHO cells. The hash marks indicate all peaks above the baseline identified by the analysis software, and the labeled brackets indicate groups of peaks representing all neutral, all charged, and monosialylated oligosaccharide species. [Figure 9] FIG. 1 shows a representative cIEF electropherogram profile of ustekinumab expressed in CHO cells. A diagram depicting the general relationship between cIEF peaks and decreasing negative charge / degree of sialylation is also shown, with peaks 1, 2, 3, and C labeled. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] As used herein, "anti-IL-12 antibodies," "anti-IL-23 antibodies," "anti-IL-12 / 23p40 antibodies," "anti-IL-12 / IL-23p40 antibodies," "IL-12 / 23p40 antibodies," "IL-12 / IL-23p40 antibodies," ""antibody portion," or "antibody fragment," and / or "antibody variants," and the like, include any protein- or peptide-containing molecule that can be incorporated into an antibody of the invention, including a molecule comprising at least a portion of an immunoglobulin molecule, such as, but not limited to, at least one complementarity determining region (CDR) of a heavy or light chain or ligand binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework region, or any portion thereof, or at least a portion of an IL-12 and / or IL-23 receptor or binding protein. Such antibodies optionally further affect a particular ligand, including, but not limited to, such antibodies modulate, decrease, increase, antagonize, agonize, reduce, mitigate, block, inhibit, abrogate, and / or interfere with at least one IL-12 / 23 activity or binding, or IL-12 / 23 receptor activity or binding in vitro, in situ, and / or in vivo. By way of non-limiting example, a suitable anti-IL-12 / 23p40 antibody, specified portion, or variant of the invention can bind to at least one IL-12 / 23 molecule, or a specified portion, variant, or domain thereof. A suitable anti-IL-12 / 23p40 antibody, specified portion, or variant can also optionally affect at least one of IL-12 / 23 activities or functions, such as, but not limited to, RNA, DNA, or protein synthesis, IL-12 / 23 release, IL-12 / 23 receptor signaling, membrane IL-12 / 23 cleavage, IL-12 / 23 activity, IL-12 / 23 production, and / or synthesis.

[0014] As used herein, the term "antibody" or "antibodies" includes biosimilar antibody molecules approved under the Biologics Price Competition and Innovation Act of 2009 (BPCI Act) and similar global laws and regulations. Under the BPCI Act, an antibody may be demonstrated to be biosimilar if data show that it is "highly similar" to the reference product, despite minor differences in clinically inactive components, and is "expected to produce equivalent clinical results" as the reference product in terms of safety, purity, and potency (Endocrine Practice: February 2018, Vol. 24, No. 2, pp. 195-204). These biosimilar antibody molecules offer an abbreviated approval pathway, whereby applicants rely on the clinical data of the innovator's reference product to secure regulatory approval. Compared to the original innovator reference antibody approved by the FDA based on successful clinical trials, biosimilar antibody molecules are referred to herein as "follow-on biologics." As presented herein, STELARA® (ustekinumab) is the original innovator reference anti-IL-12 / IL-23p40 antibody approved by the FDA based on successful clinical trials. Ustekinumab has been marketed in the United States since 2009.

[0015] The term "antibody" is further intended to encompass antibodies, digested fragments thereof, specified portions, and variants, including antibody mimetics or portions of antibodies that mimic the structure and / or function of antibodies, such as single chain antibodies and fragments thereof, or specified fragments or portions thereof. Functional fragments include antigen-binding fragments that bind to mammalian IL-12 / 23. Antibody fragments capable of binding to IL-12 / 23 or portions thereof, including, but not limited to, Fab (e.g., by papain digestion), Fab' (e.g., by pepsin digestion and partial reduction), and F(ab')2 (e.g., by pepsin digestion), facb (e.g., by plasmin digestion), pFc' (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsin digestion, partial reduction, and reassembly), Fv, or scFv (e.g., by molecular biology techniques) fragments, are encompassed by the present invention (see, e.g., Colligan, Immunology, supra).

[0016] Such fragments can be produced by enzymatic cleavage, synthetic or recombinant techniques as known in the art and / or as described herein. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a combination of genes encoding a F(ab')2 heavy chain portion can be produced by inserting the C(A) codon into the heavy chain. H The antibody can be designed to contain DNA sequences encoding a single domain and / or hinge region. The various portions of the antibody can be joined chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.

[0017] As used herein, the term "human antibody" refers to an antibody that is humanized in a manner that comprises substantially all parts of the protein (e.g., CDRs, frameworks, C L , C H Domain (e.g., C H 1. C H 2. C H 3), Hinge (V L, V H )) is substantially non-immunogenic in humans with only minor sequence changes or mutations. A "human antibody" may also be an antibody derived from or closely corresponding to a human germline immunoglobulin sequence. A human antibody may contain amino acid residues not encoded by germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). In many cases, this means that the human antibody is substantially non-immunogenic in humans. Human antibodies have been classified into groups based on the similarity of their amino acid sequences. Thus, using sequence similarity searches, antibodies with similar linear sequences can be selected as templates for creating human antibodies. Similarly, antibodies with names including primates (monkeys, baboons, chimpanzees, etc.), rodents (mouse, rats, rabbits, guinea pigs, hamsters, etc.) and other mammals designate specific antibodies of such species, subgenus, genus, subfamily, and family. Furthermore, chimeric antibodies may contain any combination of the above. Such changes or mutations optionally and preferably retain or reduce immunogenicity in humans or other species compared to the unaltered antibody. Thus, a human antibody is distinct from a chimeric or humanized antibody.

[0018] It is noted that human antibodies can be produced by non-human animals, or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy and / or light chain) genes. Moreover, when a human antibody is a single-chain antibody, it may contain a linker peptide not found in naturally occurring human antibodies. For example, an Fv may contain a linker peptide, such as 2 to about 8 glycine or other amino acid residues, connecting the variable region of the heavy chain and the variable region of the light chain. Such a linker peptide is considered to be of human origin.

[0019] Anti-IL-12 / 23p40 antibodies (also referred to as IL-12 / 23p40 antibodies) (or antibodies against IL-23) useful in the methods and compositions of the invention may optionally be characterized by high affinity binding to IL-12 / 23p40 (or IL-23), and optionally and preferably low toxicity. In particular, antibodies of the invention, specified fragments or variants thereof, in which the individual components such as the variable region, constant region, and framework, individually and / or collectively, optionally and preferably, have low immunogenicity, are useful in the present invention. Antibodies that can be used in the present invention may optionally be characterized by their ability to treat patients for extended periods with measurable alleviation of symptoms and low and / or acceptable toxicity. Low or acceptable immunogenicity and / or high affinity, as well as other favorable properties, may contribute to the therapeutic results obtained. "Low immunogenicity" is defined herein as a significant increase in HAHA, HACA or HAMA responses in less than about 75%, or preferably less than about 50%, of treated patients, and / or a low titer increase (less than about 300, preferably less than about 100, as measured by double antigen enzyme immunoassay) in treated patients (Elliott et al, Lancet 344:1125-1127 (1994), incorporated herein by reference in its entirety). "Low immunogenicity" may also be defined as the incidence of titer levels of antibodies to anti-IL-12 antibodies in patients treated with anti-IL-12 antibodies as occurring in less than 25% of treated patients, preferably less than 10% of treated patients, at the recommended dose during the recommended course of therapy during the treatment period.

[0020] As used herein, the term "human antibody" refers to an antibody that is humanized in a manner that comprises substantially all parts of the protein (e.g., CDRs, frameworks, C L , C H Domain (e.g., C H 1. C H 2, and C H 3), hinge (VL, V H)) refers to antibodies that are substantially non-immunogenic in humans with only minor sequence changes or mutations. Similarly, antibodies designated primates (monkeys, baboons, chimpanzees, etc.), rodents (mouse, rats, rabbits, guinea pigs, hamsters, etc.), and other mammalian animals refer to antibodies specific for such species, subgenus, genus, subfamily, and family. Furthermore, chimeric antibodies include any combination of the above. Such changes or mutations optionally and preferably retain or reduce immunogenicity in humans or other species compared to the unmodified antibody. Thus, a human antibody is distinct from a chimeric antibody or a humanized antibody. It is noted that a human antibody can be produced by a non-human animal or a prokaryotic or eukaryotic cell capable of expressing functionally rearranged human immunoglobulin (e.g., heavy and / or light chain) genes. Moreover, when a human antibody is a single chain antibody, it can include a linker peptide not found in a naturally occurring human antibody. For example, an Fv can include a linker peptide, such as 2 to about 8 glycine or other amino acid residues, connecting the heavy chain variable region and the light chain variable region, such a linker peptide being considered to be of human origin.

[0021] Bispecific (e.g., DuoBody®), heterospecific, heterobinding, or similar antibodies may also be used, which are monoclonal, preferably human or humanized, antibodies with binding specificities for at least two different antigens. Methods for producing bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537 (1983)). Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a possible mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, usually performed by affinity chromatography steps, can be laborious with low product yields, so different approaches have been developed to facilitate the production of bispecific antibodies.

[0022] Full-length bispecific antibodies can be generated using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies, for example, in a cell-free environment in vitro or using co-expression, by introducing substitutions in the heavy chain CH3 interface in each half molecule to favor heterodimer formation of two antibody half molecules with different specificities. The Fab arm exchange reaction is the result of a disulfide bond isomerization reaction and dissociation-association of the CH3 domains. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibody are reduced. The resulting free cysteine ​​of one of the parent monospecific antibodies forms an intra-heavy chain disulfide bond with a cysteine ​​residue of the second parent monospecific antibody molecule, while the CH3 domain of the parent antibody is released and reformed by dissociation-association. The CH3 domain of the Fab arm may be modified to favor heterodimer formation over homodimer formation. The resulting product is a bispecific antibody with two Fab arms or half molecules, each capable of binding a different epitope.

[0023] As used herein, "homodimerization" refers to the interaction of two heavy chains with identical CH3 amino acid sequences. As used herein, "homodimer" refers to an antibody having two heavy chains with identical CH3 amino acid sequences.

[0024] As used herein, "heterodimerization" refers to the interaction of two heavy chains with non-identical CH3 amino acid sequences. As used herein, "heterodimer" refers to an antibody having two heavy chains with non-identical CH3 amino acid sequences.

[0025] A "knob-in-hole" strategy (see, for example, WO 2006 / 028936) can be used to generate full-length bispecific antibodies. Briefly, selected amino acids that form the interface of the CH3 domain in human IgG can be mutated at positions that affect CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into the heavy chain of an antibody that specifically binds to a first antigen, and an amino acid with a large side chain (knob) is introduced into the heavy chain of an antibody that specifically binds to a second antigen. After co-expression of the two antibodies, a heterodimer is formed as a result of the preferential interaction of the heavy chain with the "hole" and the heavy chain with the "knob". Exemplary pairs of CH3 substitutions that form knobs and holes are T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V (represented as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain).

[0026] Other strategies, such as promoting heavy chain heterodimer formation using electrostatic interactions by substituting positively charged residues on one CH3 surface and negatively charged residues on the second CH3 surface, may be used as described in U.S. Patent Application Publication Nos. 2010 / 0015133, 2009 / 0182127, 2010 / 028637, or 2011 / 0123532. In another strategy, heterodimerization can be achieved by the following substitutions: L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T36 ... The modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain can be promoted by K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W (represented as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain).

[0027] In addition to the methods described above, bispecific antibodies can be generated in an in vitro cell-free environment by introducing asymmetric mutations in the CH3 regions of two monospecific homodimeric antibodies and forming a bispecific heterodimeric antibody from two parent monospecific homodimeric antibodies under reducing conditions that cause disulfide bonds to isomerize, according to the method described in WO 2011 / 131746. In this method, a first monospecific bivalent antibody and a second monospecific bivalent antibody are modified to have certain substitutions in the CH3 domain that promote the stability of the heterodimer, and these antibodies are incubated together under reducing conditions sufficient to cause the cysteines in the hinge region to isomerize the disulfide bonds, thereby generating the bispecific antibody by Fab arm exchange. The incubation conditions can be optimally returned to non-reducing conditions. Exemplary reducing agents that may be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and beta-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. For example, incubation at a temperature of at least 20° C., in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol, at a pH of 5-8, e.g., pH 7.0 or pH 7.4, for at least 90 minutes may be used.

[0028] The terms "efficacy" and "effective" as used herein in the context of a dose, dosing regimen, treatment or method refer to the effectiveness of a particular dose, dosing, treatment regimen. Efficacy can be measured based on changes in the course of a disease in response to an agent of the invention. For example, an anti-IL12 / 23p40 or anti-IL23 antibody of the invention (e.g., anti-IL12 / 23p40 antibody ustekinumab) is administered to a subject in an amount and for a time sufficient to cause an improvement, preferably a sustained improvement, in at least one indicator reflecting the severity of the disorder being treated. To determine whether the amount and time of treatment is sufficient, various indicators reflecting the extent of the disease, disorder or condition of the subject can be evaluated. Such indicators include, for example, clinically recognized indicators of disease severity, symptoms, or manifestations of the disorder being treated. The degree of improvement is determined overall by the physician, who can base this determination on signs, symptoms, biopsies, or other test results, and can also employ questionnaires administered to the subject, such as quality of life questionnaires developed for a given disease.

[0029] The term "safety" refers to a favorable risk profile with respect to a dose, dosing regimen, treatment or method with an anti-IL12 / 23p40 or anti-IL23 antibody of the invention (e.g., the anti-IL12 / 23p40 antibody ustekinumab). It refers to a favorable risk:benefit ratio with an acceptable frequency and / or severity of treatment-emergent adverse events (also called adverse events, AEs or treatment-emergent adverse events, TEAEs) compared to a standard of care or another comparator. An adverse event is an untoward medical occurrence in a patient administered a medicinal product. In particular, safety associated with a dose, dosing regimen or treatment with an anti-IL12 / 23p40 or anti-IL23 antibody of the invention refers to an acceptable frequency and / or acceptable severity of adverse events associated with administration of the antibody, where the adverse event is considered possible, likely or highly likely to be due to the use of the anti-IL12 / 23p40 or anti-IL23 antibody.

[0030] usefulness The isolated nucleic acids of the present invention may be used to produce at least one anti-IL-12 / 23p40 (or anti-IL-23) antibody or specified variant thereof, which may be measured or acted on a cell, tissue, organ, or animal (including mammals and humans) to diagnose, monitor, regulate, treat, alleviate, help prevent the occurrence of, or reduce the symptoms of at least one IL-12 / 23 condition selected from, but not limited to, at least one of an immune disorder or disease, a cardiovascular disorder or disease, an infectious, malignant and / or neurological disorder or disease, or other known or specified IL-12 / 23 related condition.

[0031] Such methods may include administering to a cell, tissue, organ, animal, or patient in need of such modulation, treatment, amelioration, prevention, or reduction of a symptom, effect, or mechanism an effective amount of a composition or pharmaceutical composition comprising at least one anti-IL-12 / 23p40 (or anti-IL-23) antibody. An effective amount may include an amount of about 0.001-500 mg / kg per single (e.g., bolus), multiple, or continuous administration, or an amount that achieves a serum concentration of 0.01-5000 μg / mL per single, multiple, or continuous administration, or any effective range or value therein, as determined using known methods, as described herein or known in the relevant art.

[0032] References All publications or patents cited herein, whether specifically stated or not, are incorporated herein by reference in their entirety to indicate the prior art at the time of the invention and / or to describe and enable the invention. A publication refers to any scientific publication or patent publication or any other information available in any media format, including all recorded in electronic or printed format. The following publications are incorporated by reference in their entirety: Ausubel, et al., eds., 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).

[0033] Antibodies of the Invention - Production and Purification At least one anti-IL-12 / 23p40 (or anti-IL-23) used in the methods of the invention can optionally be produced by a cell line, a mixed cell line, an immortalized cell, or a clonal population of immortalized cells, as known in the art. See, e.g., Ausubel, et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, NY (1989); Harlow and Lane, antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), each of which is incorporated by reference in its entirety.

[0034] A preferred anti-IL-12 / 23p40 antibody is ustekinumab (STELARA®), which has a heavy chain variable region amino acid sequence of SEQ ID NO:7, a light chain variable region amino acid sequence of SEQ ID NO:8, and has heavy chain CDR amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and light chain CDR amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. A preferred anti-IL-23 antibody is guselkumab (also referred to as CNTO1959). Other anti-IL-23 antibodies have the sequences listed herein and are described in U.S. Patent No. 7,935,344, the entire contents of which are incorporated herein by reference.

[0035] Human antibodies specific for human IL-12 / 23p40 or IL-23 proteins or fragments thereof can be raised against suitable immunogenic antigens, such as isolated IL-12 / 23p40 proteins, IL-23 proteins, and / or portions thereof (including synthetic molecules such as synthetic peptides). Other specific or general mammalian antibodies can be raised as well. Preparation of immunogenic antigens and generation of monoclonal antibodies can be carried out using any suitable technique.

[0036] In one approach, a suitable immortal 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, MLA144, ACT IV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA144, NAMALWA, NEURO Myeloma cell lines such as 2A, or heteromyelomas, fusion products thereof, or any cells or fusion cells derived therefrom, or any other suitable cell line known in the art) (see, e.g., www.atcc.org, www.lifetech.com, etc.), antibody producing cells such as, but not limited to, isolated or cloned spleen, peripheral blood, lymph, tonsil, or other immune or B cell containing cells, or recombinant or endogenous, viral, bacterial, algal, progenitor, or progenitor cells, as endogenous or heterologous nucleic acids. The hybridomas are produced by fusing with any other cell expressing the heavy or light chain constant or variable, or framework or CDR sequences, either as genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triple stranded, hybridized, etc., or as any combination thereof, from any mammalian, amphibian, insect, reptile, fish, mammal, rodent, horse, ovine, caprine, ovine, primate, eukaryote, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triple stranded, hybridized, etc., to produce hybridomas. See, e.g., Ausubel, supra, and Colligan, supra, Immunology, Chapter 2, both of which are incorporated herein by reference in their entireties.

[0037] Antibody producing cells can also be obtained from the peripheral blood, or preferably the spleen or lymph nodes, of humans or other suitable animals immunized with the antigen of interest. Any other suitable host cells can also be used to express heterologous or endogenous nucleic acid encoding the antibodies, specified fragments or variants thereof of the invention. Fused cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other suitable known methods and cloned by limiting dilution or cell sorting or other known methods. Cells producing antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).

[0038] Select recombinant antibodies from peptide or protein libraries (e.g., but not limited to, bacteriophage, ribosomal, oligonucleotide, RNA, cDNA, and other display libraries, e.g., Cambridge Antibody Technologies, Cambridgeshire, UK, MorphoSys, Martinsreid / Planegg, DE, Biovation, Aberdeen, Scotland, UK, BioInvent, Lund, Sweden, Dyax Corp., Enzon, Affymax / Biosite, Xoma, Berkeley, CA, Ixsys. See, for example, European Patent No. 368,684, International Application No. PCT / GB91 / 01134, International Application No. PCT / GB92 / 01755, International Application No. PCT / GB92 / 002240, International Application No. PCT / GB92 / 00883, International Application No. PCT / GB93 / 00605, U.S. Patent Application Publication No. 08 / 350260 (5 / 12 / 94), International Application No. PCT / GB94 / 01422, ... Application No. PCT / GB94 / 02662, International Application No. PCT / GB97 / 01835, (CAT / MRC), International Publication No. WO 90 / 14443, International Publication No. WO 90 / 14424, International Publication No. WO 90 / 14430, International Application No. PCT / US94 / 1234, International Publication No. WO 92 / 18619, International Publication No. WO 96 / 07754 (Scripps), International Publication No. WO 96 / 13583, International Publication No. 97 / 08320 (MorphoSys), WO 95 / 16027 (BioInvent), WO 88 / 06630, WO 90 / 3809 (Dyax), U.S. Patent No. 4,704,692 (Enzon), International Application PCT / US91 / 02989 (Affymax), WO 89 / 06283, EP 371998, EP 550 400, (Xoma), EP 229046, International Application PCT / US91 / 07149 (Ixsys), or stochastically generated peptides or proteins - U.S. Pat. Nos. 5,723,323, 5,763,192, 5,814,476, 5,817,483, 5,824,514, 5,976,862, WO 86 / 05803, EP 590 689 (Ixsys, a predecessor to Applied Molecular Evolution (AME), each of which is incorporated herein by reference in its entirety), or by immunization of transgenic animals capable of producing a repertoire of human antibodies as known in the art and / or described herein (e.g., SCID mice, Nguyen et al., Microbiol. Immunol. 41:901-907 (1997); Sandhu et al., Crit. Rev. Biotechnol.Other suitable methods for producing or isolating antibodies of the required specificity can be used, including, but not limited to, the methods described in Immunol. 16:95-118 (1996); Eren et al., Immunol. 93:154-161 (1998) (each of which is incorporated by reference in its entirety), and related patents and applications. Such techniques include ribosome display (Hanes et al., Proc. Natl. Acad. Sci. USA, 94:4937-4942 (May 1997); Hanes et al., Proc. Natl. Acad. Sci. USA, 95:14130-14135 (Nov. 1998)), single cell antibody generation techniques (e.g., the selected lymphocyte antibody method ("SLAM") (U.S. Pat. No. 5,627,052; Wen et al., J. Immunol. 17:887-892 (1987); Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-7848 (1996)), gel microdroplets and flow cytometry (Powell et al., Biotechnol. 8:333-337 (1990); One Cell Systems, Cambridge, MA; Gray et al., J. Imm. Meth. 182:155-163 (1995); Kenny et al., Bio / Technol. 13:787-790 (1995); B cell selectors (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 BV, Amsterdam, Netherlands (1988)).

[0039] Methods for engineering or humanizing non-human or human antibodies can also be used and are well known in the art. Typically, a humanized or modified antibody has one or more amino acid residues from a non-human source, such as, but not limited to, mouse, rat, rabbit, non-human primate, or another mammalian source. These non-human amino acid residues are often replaced by residues referred to as "import" residues. Such "import" residues are typically taken from an "import" variable, constant, or other domain of a known human sequence.

[0040] Known human Ig sequences are disclosed, e.g. www.ncbi.nlm.nih.gov / entrez / query.fcgi; www.ncbi.nih.gov / igblast; www.atcc.org / phage / hdb.html; www.mrc-cpe.cam.ac.uk / ALIGNMENTS.php; www.kabatdatabase.com / top.html; ftp.ncbi.nih.gov / repository / kabat; www.sciquest.com; www.abcam.com; www.antibodyresource.com / onlinecomp.html; www.public.iastate.edu / ~pedro / research_tools.html; www.whfreeman.com / immunology / CH05 / kuby05.htm; 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.appliedbiosystems.com; www.nal.usda.gov / awic / pubs / antibody; www.m.ehime-u.ac.jp / ~yasuhito / Elisa.html; www.biodesign.com; www.cancerresearchuk.org; www.biotech.ufl.edu; www.isac-net.org;baserv.uci.kun.nl / ~jraats / links1.html、 www.recab.uni-hd.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 / humanisation / TAHHP.html; www.ibt.unam.mx / vir / structure / stat_aim.html; www.biosci.missouri.edu / smithgp / index.html; www.jerini.de; Kabat et al., Sequences of Proteins of Immunological Interest, USDept. Health (1983), each of which is incorporated herein by reference in its entirety.

[0041] Such imported sequences can be used to reduce immunogenicity or to reduce, enhance or modify binding, affinity, association rate constant, dissociation rate constant, avidity, specificity, half-life, or any other suitable property as known in the art. Generally, CDR residues directly and most substantially affect antigen binding. Thus, non-human sequences of variable and constant regions can be replaced with human amino acids or other amino acids while maintaining some or all of the non-human or human CDR sequences.

[0042] Antibodies may optionally be humanized, or human antibodies may be engineered while retaining high affinity for the antigen and other favorable biological properties. To this end, humanized (or human) antibodies may optionally be prepared by a process of analysis of the parental sequences and various theoretical humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformations of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that affect the antigen-binding ability of the candidate immunoglobulin. In this way, framework (FR) residues can be selected and combined from the consensus and import sequences such that desired antibody properties, such as enhanced affinity for the target antigen(s), are achieved.

[0043] In addition, human anti-IL-12 / 23p40 (or anti-IL-23) specific antibodies used in the methods of the invention may comprise a human germline light chain framework. In certain embodiments, the light chain germline 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, the light chain human germline framework is selected from V1-11, V1-13, V1-16, V1-17, V1-18, 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.

[0044] In other embodiments, the human anti-IL-12 / 23p40 (or anti-IL-23) specific antibody used in the methods of the invention may comprise a human germline heavy chain framework. In certain embodiments, the heavy chain human germline framework is selected from the group consisting of 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.

[0045] In certain embodiments, the light chain variable region and / or the heavy chain variable region comprises a framework region, or at least a portion of a framework region (e.g., comprising two or three subregions, such as FR2 and FR3). In certain 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 sequence (e.g., human germline) or comprises a human consensus sequence for a particular framework (which are readily available at the sources of known human Ig sequences mentioned above). In other embodiments, at least FRH1, FRH2, FRH3, or FRH4 is a germline sequence (e.g., human germline) or comprises a human consensus sequence for a particular framework. In preferred embodiments, the framework regions are fully human framework regions.

[0046] Humanization or engineering of the antibodies of the invention has been described by 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. USA 89:4285 (1992), Presta et al. al., J.Immunol.151:2623 (1993), U.S. Patent No. 5723323, U.S. Patent No. 5976862, U.S. Patent No. 5824514, U.S. Patent No. 5817483, U.S. Patent No. 5814476, U.S. Patent No. 5763192, U.S. Patent No. 5723323, U.S. Patent No. 5 , 766886, 5714352, 6204023, 6180370, 5693762, 5530101, 5585089, 5225539, 4816567, International Application PCT / :US98 / 1 This can be done using any known method, such as, but not limited to, those described in US96 / 18978, US91 / 09630, US91 / 05939, US94 / 01234, GB89 / 01334, GB91 / 01134, GB92 / 01755, WO 90 / 14443, WO 90 / 14424, WO 90 / 14430, EP 229246 (each of which is incorporated by reference in its entirety, including the references cited therein).

[0047] In certain embodiments, the antibody comprises an altered (e.g., mutated) Fc region. For example, in some embodiments, the Fc region is altered to reduce or enhance the effector function of the antibody. In some embodiments, the Fc region is an isotype selected from IgM, IgA, IgG, IgE, or other isotypes. Alternatively, or in addition, it may be useful to combine the amino acid modification with one or more further amino acid modifications that alter the C1q binding and / or complement dependent cytotoxicity function of the Fc region of the IL-23 binding molecule. Starting polypeptides of particular interest may be those that bind C1q and exhibit complement dependent cytotoxicity (CDC). Polypeptides that have existing C1q binding activity, and optionally further have the ability to mediate CDC, may be modified to enhance one or both of these activities. Amino acid modifications that alter C1q and / or modify its complement dependent cytotoxicity function are described, for example, in WO 0042072, incorporated herein by reference.

[0048] As disclosed above, the Fc region of the human anti-IL-12 / 23p40 (or anti-IL-23) specific antibody of the present invention can be engineered with altered effector functions, for example, by modifying C1q binding and / or FcγR binding, thereby altering complement-dependent cytotoxicity (CDC) and / or antibody-dependent cell-mediated cytotoxicity (ADCC) activity. An "effector function" serves to activate or reduce a biological activity (e.g., in a subject). Examples of effector functions include, but are not limited to, C1q binding, CDC, Fc receptor binding, ADCC, phagocytosis, downregulation of cell surface receptors (e.g., B cell receptor, BCR), and the like. Such effector functions may require the Fc region to bind to a binding domain (e.g., an antibody variable domain) and can be assessed using a variety of test methods (e.g., Fc binding assays, ADCC assays, CDC assays, and the like).

[0049] For example, variant Fc regions of human anti-IL-12 / 23p40 (or anti-IL-23) antibodies can be generated that have improved C1q binding and improved FcγRIII binding (e.g., that have both improved ADCC activity and improved CDC activity). Alternatively, where it is desired to reduce or eliminate effector function, variant Fc regions can be engineered to have reduced CDC activity and / or reduced ADCC activity. In other embodiments, only one of these activities may be enhanced, optionally with the other activity reduced at the same time (e.g., to generate Fc region variants with improved ADCC activity and reduced CDC activity, and vice versa).

[0050] Fc mutations can also be engineered and introduced to alter interactions with the fetal Fc receptor (FcRn) and improve their pharmacokinetic properties. A collection of human Fc mutants with improved binding to FcRn has been described (Shields et al., 2001). High-resolution mapping of the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn, and design of IgG1 mutants with improved binding to FcγR, (J. Biol. Chem. 276:6591-6604).

[0051] Another type of amino acid substitution serves to alter the glycosylation pattern of the Fc region of a human anti-IL-12 / 23p40 (or anti-IL-23) specific antibody. Glycosylation of the Fc region is typically either N-linked or O-linked. N-linked refers to the addition of a carbohydrate moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of one of the sugars, N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. The recognition sequences for enzymatic addition of a carbohydrate moiety to an asparagine side chain peptide sequence 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 provides a potential glycosylation site.

[0052] The glycosylation pattern can be altered, for example, by deleting one or more glycosylation site(s) found in the polypeptide and / or adding one or more glycosylation sites not present in the polypeptide. Addition of glycosylation sites to the Fc region of a human IL-23-specific antibody is conveniently accomplished by altering the amino acid sequence to include one or more of the above tripeptide sequences (for N-linked glycosylation sites). An exemplary glycosylation variant has an amino acid substitution of residue Asn297 of the heavy chain. This alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the original polypeptide sequence (for O-linked glycosylation sites). In addition, changing Asn 297 to Ala can remove one of the glycosylation sites.

[0053] In certain embodiments, the human anti-IL-12 / 23p40 (or anti-IL-23) specific antibodies of the invention are expressed in cells expressing beta(1,4)-N-acetylglucosaminyltransferase III (GnT III), such that GnT III adds GlcNAc to the human anti-IL-12 / 23p40 (or anti-IL-23) antibody. Methods for producing antibodies in such a manner are provided in WO 9954342, WO 03011878, Patent Publication No. 20030003097(A1), and Umana et al., Nature Biotechnology, 17:176-180, Feb. 1999, all of which are specifically incorporated herein by reference in their entirety.

[0054] Human anti-IL-12 / 23p40 (or anti-IL-23) antibodies can also be optionally produced by immunization of transgenic animals (e.g., mice, rats, hamsters, non-human primates, etc.) capable of producing a repertoire of human antibodies, as described herein and / or known in the art. Cells producing human anti-IL-12 / 23p40 (or anti-IL-23) antibodies can be isolated from such animals and immortalized using suitable methods, such as those described herein.

[0055] Transgenic mice capable of producing a repertoire of human antibodies that bind to human antigens can be produced by known methods (see, for example, but not limited to, U.S. Pat. Nos. 5,770,428, 5,569,825, 5,545,806, 5,625,126, 5,625,825, 5,633,425, 5,661,016, and 5,789,650 issued to Lonberg et al.; WO 98 / 50433 to Jakobovits et al.; WO 98 / 24893 to Jakobovits et al.; WO 98 / 24884 to Lonberg et al.; See, for example, WO 97 / 13852 to Lonberg et al., WO 94 / 25585 to Lonberg et al., WO 96 / 34096 to Kucherlapate et al., EP 0463151(B1) to Kucherlapate et al., EP 0710719(A1) to Kucherlapate et al., U.S. Pat. No. 5,545,807 to Surani et al., WO 90 / 04036 to Bruggemann et al., EP 0438474(B1) to Bruggemann et al., EP 0814259(A2) to Lonberg et al., UK Patent 2272440(A) to Lonberg et al., and U.S. Pat. No. 5,545,807 to Surani et al. 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. 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.Generally, these mice contain at least one transgene that comprises DNA derived from at least one human immunoglobulin locus that has been functionally rearranged, or is capable of undergoing functional rearrangement. The endogenous immunoglobulin loci of such mice can be disrupted or deleted to eliminate the ability of the mice to produce antibodies encoded by endogenous genes.

[0056] Screening of antibodies for specific binding to similar proteins or fragments can be successfully accomplished using peptide display libraries. This method involves screening a large collection of peptides for individual members with the desired function or structure. Antibody screening of peptide display libraries is well known in the art. The displayed peptide sequences can be 3-5000 or more amino acids in length, frequently 5-100 amino acids long, and often about 8-25 amino acids long. In addition to direct chemical synthesis methods for creating peptide libraries, several recombinant DNA methods have also been described. One type involves the display of peptide sequences on the surface of bacteriophages or cells. Each bacteriophage or cell contains a nucleotide sequence that codes for a particular displayed peptide sequence. Such methods are described in WO 91 / 17271, WO 91 / 18980, WO 91 / 19818, and WO 93 / 08278.

[0057] Other systems for generating peptide libraries include aspects of both in vitro chemical synthesis and recombinant methods. See International Patent Publication Nos. WO 92 / 05258, WO 92 / 14843, and WO 96 / 19256. See also U.S. Patent Nos. 5,658,754 and 5,643,768. Peptide display libraries, vectors, and screening kits are commercially available from sources such as Invitrogen (Carlsbad, CA) and Cambridge antibody Technologies (Cambridgeshire, UK). See, e.g., U.S. Patent Nos. 4,704,692, 4,939,666, 4,946,778, 5,260,203, 5,455,030, 5,518,889, 5,534,621, 5,656,730, 5,763,733, 5,767,260, and 5,856,456 all assigned to Enzon; ​​U.S. Patent Nos. 5,223,409, 5,403,484, 5,571,698, and 5,837,500 all assigned to Dyax; U.S. Patent Nos. 5,427,908 and 5,580,717 all assigned to Affymax; Cambridge antibody See U.S. Patent No. 5,885,793 assigned to Sigma Technologies, U.S. Patent No. 5,750,373 assigned to Genentech, U.S. Patent Nos. 5,618,920, 5,595,898, 5,576,195, 5,698,435, 5,693,493, 5,698,417 assigned to Xoma, Colligan, supra, Ausubel, supra, or Sambrook, supra, each of which is incorporated herein by reference in its entirety.

[0058] The antibodies used in the methods of the present invention can also be prepared using at least one anti-IL-12 / 23p40 (or anti-IL-23) antibody encoding nucleic acid to provide a transgenic animal or mammal, such as a goat, cow, horse, sheep, rabbit, etc., that produces such antibodies in its milk. Such animals can be prepared using known methods. See, for example, but not limited to, U.S. Patent Nos. 5,827,690, 5,849,992, 4,873,316, 5,849,992, 5,994,616, 5,565,362, 5,304,489, etc. (each of which is incorporated herein by reference in its entirety).

[0059] The antibodies used in the methods of the invention can further be prepared using at least one anti-IL-12 / 23p40 (or anti-IL-23) antibody encoding nucleic acid to provide transgenic plants and cultured plant cells (e.g., but not limited to, tobacco and corn) that produce such antibodies, specified portions, or variants in plant parts or cells cultured therefrom. As a non-limiting example, transgenic tobacco leaves expressing recombinant proteins using, for example, an inducible promoter have been successfully used to provide large amounts of recombinant proteins. See, e.g., Cramer et al., Curr. Top. Microbol. Immunol. 240:95-118 (1999) and references cited therein. Transgenic corn has also been used to express mammalian proteins at commercial production levels with biological activity equivalent to proteins produced in other recombinant systems or purified from natural sources. See, e.g., Hood et al., Adv. Exp. Med. Biol. 464:127-147 (1999) and references cited therein. Antibodies have also been produced in large quantities from transgenic plant seeds, such as tobacco seeds and potato tubers, including antibody fragments, such as single chain antibodies (scFv). See, e.g., Conrad et al., Plant Mol. Biol. 38:101-109 (1998) and references cited therein. Thus, the antibodies of the present invention can also be produced using transgenic plants according to known methods. See, e.g., Fischer et al., Biotechnol. Appl. Biochem. 30:99-108 (Oct., 1999), Ma et al., Trends Biotechnol. 13:522-7 (1995), Ma et al., Plant Physiol. 109:341-6 (1995), Whitelam et al., Biochem. Soc. Trans. 22:940-944 (1994) and references cited therein. Each of the above documents is incorporated herein by reference in its entirety.

[0060] The antibodies used in the methods of the present invention have a wide range of affinities (K D ) can bind human IL-12 / IL-23p40 or IL-23. In a preferred embodiment, the human mAb can optionally bind human IL-12 / IL-23p40 or IL-23 with high affinity. For example, the human mAb can bind human IL-12 / IL-23p40 or IL-23 with approximately 10 -7 M or less, for example, but not limited to, 0.1 to 9.9 (or any range or value therein) x 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 K, or any range or value therein D can be combined with

[0061] The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method. (See, e.g., 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), and methods described therein.) The affinity measured for a particular antibody-antigen interaction may differ when measured under different conditions (e.g., salt concentration, pH). Thus, affinity and other antigen binding parameters (e.g., K D、 K a , K d Measurements of ) are preferably made using standard solutions of antibody and antigen, and standard buffers, such as those described herein.

[0062] nucleic acid molecule Using the information provided herein, e.g., a nucleotide sequence encoding at least 70-100% of the contiguous amino acids of at least one of the light or heavy chain variable or CDR regions described herein, an identified fragment, variant or consensus sequence thereof, or a deposited vector containing at least one of these sequences, among other sequences disclosed herein, a nucleic acid molecule of the invention encoding at least one IL-12 / IL-23p40 or IL-23 antibody can be obtained using methods described herein or known in the art.

[0063] 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 but not limited to cDNA and genomic DNA obtained by cloning or produced synthetically, or any combination thereof. The DNA may be triple-stranded, double-stranded or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA may be the coding strand, also known as the sense strand, or the non-coding strand, called the antisense strand.

[0064] The isolated nucleic acid molecules used in the methods of the present invention may include open reading frames (ORFs), optionally having one or more introns, for example, nucleic acid molecules comprising at least one specified portion of at least one CDR, such as, but not limited to, CDR1, CDR2, and / or CDR3 of at least one heavy or light chain, nucleic acid molecules comprising coding sequences of anti-IL-12 / IL-23p40 or IL-23 antibodies or variable regions, as well as nucleic acid molecules that are substantially different from the above-mentioned nucleic acid molecules, but that, due to the degeneracy of the genetic code, still encode at least one anti-IL-12 / IL-23p40 or IL-23 antibody described herein and / or known in the art. Of course, the genetic code is well known in the art. Thus, it would be routine for one of skill in the art to generate such degenerate nucleic acid variants that encode the specific anti-IL-12 / IL-23p40 or IL-23 antibodies used in the methods of the present invention. See, for example, Ausubel et al., supra, and 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.

[0065] As described herein, nucleic acid molecules comprising nucleic acids encoding anti-IL-12 / IL-23p40 or IL-23 antibodies can include, but are not limited to, those that themselves encode the amino acid sequence of an antibody fragment, sequences encoding full length antibodies or portions of antibodies, coding sequences for antibodies, fragments or portions, and additional sequences, such as at least one intron, with or without the aforementioned additional coding sequences, including, but not limited to, non-coding 5' and 3' sequences, e.g., transcribed non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals (e.g., ribosome binding and stability of mRNA), as well as coding sequences for at least one signal leader or fusion peptide, additional coding sequences encoding additional amino acids, e.g., amino acids that provide additional functions. Thus, the antibody coding sequence can be fused to a marker sequence, e.g., a sequence encoding a peptide that facilitates purification of the antibody comprising the antibody fragment or portion to which it is fused.

[0066] Polynucleotides that selectively hybridize to the polynucleotides described herein The method of the present invention uses isolated nucleic acids that hybridize under selective hybridization conditions to the polynucleotides disclosed herein. Thus, the polynucleotides of the present embodiment can be used to isolate, detect, and / or quantify nucleic acids that contain 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 or cDNA sequences that are isolated or otherwise complementary to cDNAs from a human or mammalian nucleic acid library.

[0067] Preferably, the cDNA library contains at least 80% of full-length sequences, preferably at least 85% or 90% of full-length sequences, more preferably at least 95% of full-length sequences. The cDNA library can be normalized to increase the representation of rare sequences. Low or medium stringency hybridization conditions are typical, but not limited to, using sequences with low sequence identity to the complementary sequence. Optionally, medium and high stringency conditions can be used for sequences with higher identity. Low stringency conditions allow selective hybridization of sequences with about 70% sequence identity and can be used to identify orthologous or paralogous sequences.

[0068] Optionally, the polynucleotide encodes at least a portion of an antibody. The polynucleotide comprises a nucleic acid sequence that is available for selective hybridization to a polynucleotide encoding an antibody of the invention. See, e.g., Ausubel, supra; Colligan, supra. Each is incorporated herein by reference in its entirety.

[0069] Nucleic acid construction An isolated nucleic acid can be produced using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and / or (d) combinations thereof, as are well known in the art.

[0070] The nucleic acid can conveniently contain sequences in addition to the polynucleotide of the invention. For example, a multiple cloning site containing one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in the isolation of the polynucleotide. Also, a translatable sequence can be inserted to aid in the isolation of the translated polynucleotide of the invention. For example, a hexahistidine marker sequence provides a convenient means for purifying the protein of the invention. The nucleic acid of the invention (excluding the coding sequence) is optionally a vector, adapter, or linker for cloning and / or expression of the polynucleotide of the invention.

[0071] Additional sequences can be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, to aid in the isolation of polynucleotides, or to improve the introduction of polynucleotides into cells. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra, or Sambrook, supra.)

[0072] Recombinant methods for constructing nucleic acids Isolated nucleic acid compositions, 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 of skill in the art. In some embodiments, oligonucleotide probes that selectively hybridize under stringent conditions to the polynucleotides of the present invention are used to identify desired sequences in a cDNA or genomic DNA library. Isolation of RNA and construction of cDNA and genomic libraries are well known to those of skill in the art. (See, e.g., Ausubel, supra, or Sambrook, supra.)

[0073] Nucleic Acid Screening and Isolation Methods Probes based on the sequences of the polynucleotides used in the methods of the invention, such as those disclosed herein, can be used to screen cDNA or genomic libraries. 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 various degrees of hybridization stringency can be used in the assay, and that either the hybridization or the wash medium can be made more stringent. The more stringent the hybridization conditions, the greater the degree of complementarity between the probe and the target at which duplex formation occurs. The degree of stringency can be controlled by one or more of temperature, ionic strength, pH, and the presence of a partially denaturing solvent such as formamide. For example, the stringency of hybridization is successfully altered by changing the polarity of the reaction solution, for example, by manipulating the formamide concentration in the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding varies depending on the stringency of the hybridization medium and / or the wash medium. The degree of complementarity is optimally 100%, or 70-100%, or any range or value therein, however, it should be understood that minor differences in sequence in the probe and primers can be compensated for by reducing the stringency of the hybridization and / or wash medium.

[0074] Methods for amplifying RNA or DNA are well known in the art and can be used in accordance with the present invention without undue experimentation, based on the teachings and guidance provided herein.

[0075] Known methods of DNA or RNA amplification include the polymerase chain reaction (PCR) and related amplification processes (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, and 4,965,188 to Mullis et al., U.S. Pat. Nos. 4,795,699 and 4,921,794 to Tabor et al., U.S. Pat. No. 5,142,033 to Innis, U.S. Pat. No. 5,122,464 to Wilson et al., U.S. Pat. No. 5,091,310 to Innis, U.S. Pat. No. 5,062,222 to Gyllensten et al., and U.S. Pat. No. 5,122,464 to Innis, and U.S. Pat. No. 5,062,222 to Gyllensten et al.). No. 6,584, U.S. Patent No. 4,889,818 to Gelfand et al., U.S. Patent No. 4,994,370 to Silver et al., U.S. Patent No. 4,766,067 to Biswas, and U.S. Patent No. 4,656,134 to Ringold), and RNA-mediated amplification (U.S. Patent No. 5,130,238 to Malek et al., under the trade name NASBA) using antisense RNA to a target sequence as a template for double-stranded DNA synthesis (the entire contents of which are incorporated herein by reference). (See, e.g., Ausubel, supra, or Sambrook, supra).

[0076] For example, polymerase chain reaction (PCR) technology can be used to amplify the sequences of polynucleotides and related genes used in the methods of the present invention directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods can also be useful, for example, for cloning nucleic acid sequences encoding proteins to be expressed, for generating nucleic acids to be used as probes for detecting the presence of desired mRNA in a sample, for sequencing nucleic acids, or for other purposes. Examples of techniques sufficient to guide the skilled artisan through in vitro amplification methods can be found in Berger, Sambrook, and Ausubel, supra, as well as U.S. Patent No. 4,683,202 to Mullis et al. (1987), and Innis, et al., PCR Protocols A Guide to Methods and Applications, Eds., Academic Press Inc, San Diego, CA (1990). Commercial kits for genomic PCR amplification are known in the art. See, for example, Advantage-GC Genomic PCR Kit (Clontech). In addition, for example, the T4 gene 32 protein (Boehringer Mannheim) can be used to improve yields of long PCR products.

[0077] Synthetic methods for constructing nucleic acids The isolated nucleic acid used in the method of the present invention can also be prepared by direct chemical synthesis by known methods (see, for example, Ausubel et al., supra). Chemical synthesis generally produces a single-stranded oligonucleotide that can be converted into double-stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template. Those skilled in the art will recognize that although chemical synthesis of DNA can be limited to sequences of about 100 or more bases, longer sequences can be obtained by ligation of shorter sequences.

[0078] Recombinant Expression Cassettes The present invention uses recombinant expression cassettes that contain nucleic acids. A nucleic acid sequence, e.g., a cDNA or genomic sequence encoding an antibody used in the methods of the present invention, can be used to construct a recombinant expression cassette that can be introduced into at least one desired host cell. A recombinant expression cassette typically contains a polynucleotide that is operably linked to a transcription initiation regulatory sequence that directs transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be utilized to direct the expression of the nucleic acid.

[0079] In some embodiments, isolated nucleic acids that function as promoters, enhancers, or other elements can be introduced into a suitable location (upstream, downstream, or within an intron) of a non-heterologous form of a polynucleotide of the invention to up- or down-regulate expression of the polynucleotide. For example, endogenous promoters can be altered by mutation, deletion, and / or substitution, either in vivo or in vitro.

[0080] Vectors and host cells The present invention also relates to vectors containing the isolated nucleic acid molecules, host cells engineered with the recombinant vectors, and the production of at least one anti-IL-23 antibody by recombinant techniques well known in the art (see, e.g., Sambrook et al., supra; Ausubel et al., supra, each of which is incorporated herein by reference in its entirety).

[0081] The polynucleotide can be optionally linked to a vector containing a selectable marker for propagation in a host. Generally, the plasmid vector is introduced into a precipitate, such as a calcium phosphate precipitate, or into a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into a host cell.

[0082] The DNA insert should be operably linked to a suitable promoter. The expression construct further comprises a transcription initiation site, a transcription termination site, and, within the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct preferably comprises translation beginning with a start and stop codon (e.g., UAA, UGA, or UAG) appropriately positioned at the end of the mRNA to be translated, with UAA and UAG being preferred for expression in mammalian or eukaryotic cells.

[0083] The expression vector preferably includes at least one selectable marker, although this is optional. Such markers include, for example, methotrexate (MTX) for eukaryotic cell culture, dihydrofolate reductase (DHF) for eukaryotic cell culture, and the like. reductase, DHFR, U.S. Pat. Nos. 4,399,216, 4,634,665, 4,656,134, 4,956,288, 5,149,636, 5,179,017, ampicillin, neomycin (G418), mycophenolic acid or glutamine synthetase (GS) (U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739) resistance genes, and tetracycline or ampicillin resistance genes for cultivation in E. coli and other bacteria or prokaryotes (the above patents are incorporated herein by reference in their entireties). (Appropriate culture media and conditions for the above host cells are known in the art. Suitable vectors will be readily apparent to one of skill in the art. Introduction of the vector construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other known methods. Such methods are described in the art, such as Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, 16.

[0084] At least one antibody used in the method of the invention may be expressed in modified form, such as a fusion protein, and may contain not only secretion signals, but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the antibody to improve stability and persistence in the host cell during purification or during subsequent processing and storage. Peptide moieties may also be added to the antibody of the invention to facilitate purification. Such regions may be removed prior to final preparation of the antibody or at least one fragment thereof. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74, Ausubel, supra, Chapters 16, 17 and 18.

[0085] Those skilled in the art are familiar with the many expression systems available for expressing the nucleic acid encoding the protein used in the method of the present invention. Alternatively, the nucleic acid can be expressed in a host cell by switching on (by engineering) the endogenous DNA encoding the antibody in the host cell. Such methods are well known in the art, for example, as described in U.S. Patent Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, which are incorporated herein by reference in their entirety.

[0086] Useful cells for the production of antibodies, specified portions or variants thereof are mammalian cells. Mammalian cell lines are often cultured in the form of a monolayer of cells, but the cells can also be adapted to grow in suspension, for example in shake flasks or bioreactors. Many suitable host cell lines capable of expressing intact glycosylated proteins have been developed in the art, including COS-1 (e.g., ATCC® CRL1650), COS-7 (e.g., ATCC® CRL-1651), HEK293, BHK21 (e.g., ATCC® CCL-10), BSC-1 (e.g., ATCC® CCL-26), Chinese Hamster Ovary (CHO), HeP G2, P3X63Ag8.653, Sp2 / 0-Ag14, HeLa cells, and the like, which are readily available, for example, from the American Type Culture Collection, Manassas, Va. (www.atcc.org). In certain embodiments, host cells include CHO cells and cells of lymphoid origin, such as myeloma and lymphoma cells, e.g., CHO-K1 cells, P3X63Ag8.653 cells (ATCC® CRL-1580), and Sp2 / 0-Ag14 cells (ATCC® CRL-1581).

[0087] Expression vectors for these cells can include one or more of the following expression control sequences, such as, but not limited to, an origin of replication, a promoter (e.g., late or early SV40 promoters, CMV promoter (U.S. Pat. Nos. 5,168,062, 5,385,839), HSV tk promoter, pgk (phosphoglycerate kinase) promoter, EF-1α promoter (U.S. Pat. No. 5,266,491), at least one human immunoglobulin promoter, an enhancer, and / or processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., SV40 large T Ag polyaddition site), and transcription termination sequences. See, e.g., Ausubel et al., supra; Sambrook et al., supra. Other cells useful for producing the nucleic acids or proteins of the invention are known and / or available, for example, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (www.atcc.org) or other known or commercial sources.

[0088] When eukaryotic host cells are used, typically polyadenylation or transcription termination sequences are incorporated into the vector. An example of a termination sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript can also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)). In addition, gene sequences for controlling replication in the host cell can be incorporated into the vector, as is known in the art.

[0089] CHO cell line Despite the availability of several other mammalian cell lines, the majority of recombinant therapeutic proteins currently produced are made in Chinese Hamster Ovary (CHO) cells (see Jayapal KP et al. Recombinant protein therapeutics from CHO cells-20 years and counting. Chem. Eng. Prog. 2007;103:40-47; Kunert R, Reinhart D. Advances in recombinant antibody manufacturing. Appl Microbiol Biotechnol. 2016;100(8):3451-61). Their strengths include, for example, robust growth in adherent cells or in suspension, adaptability to serum-free and chemically defined media, high productivity, and an established history of regulatory approval for therapeutic recombinant protein production. They are also highly amenable to genetic modification, and methods for cell transfection, recombinant protein expression, and clonal selection are well characterized. CHO cells can also offer post-translational modifications that are compatible with humans. As used herein, "CHO ​​cells" include, but are not limited to, for example, CHO-DG44, CHO-Kl, CHO-M, CHO-S, CHO GS knockout, and modifications and derivatives thereof.

[0090] Cloning and expression in CHO cells. One vector commonly used for expression in CHO cells is pC4. Plasmid pC4 is a derivative of plasmid pSV2-dhfr (ATCC® 37146). This plasmid contains the mouse DHFR gene under the control of the SV40 early promoter. Chinese hamster ovary cells or other cells transfected with these plasmids that lack dihydrofolate activity can be selected by growing the cells in selective medium (e.g., α-MEM, Life Technologies, Gaithersburg, MD) supplemented with the chemotherapeutic drug methotrexate. Amplification of the DHFR gene in cells resistant to methotrexate (MTX) has been well documented (see, for example, FWAlt, et al., J. Biol. Chem. 253:1357-1370 (1978); JL Hamlin and C. Ma, Biochem. et Biophys. Acta 1097:107-143 (1990); and MJ Page and MASydenham, Biotechnology 9:64-68 (1991)). Cells grown in increased MTX concentrations develop resistance to the drug due to overproduction of the target enzyme, DHFR, as a result of amplification of the DHFR gene. When a second gene is linked to the DHFR gene, it is usually co-amplified and overexpressed. It is known in the art that this approach can be used to develop cell lines with more than 1,000 copies of the amplified gene(s). Subsequently, upon recovery of methotrexate, cell lines are obtained which contain the amplified gene integrated into one or more chromosome(s) of the host cell.

[0091] Plasmid pC4 contains the strong promoter of the long terminal repeat (LTR) of Rous sarcoma virus (Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985)) as well as the fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart, et al., Cell 41:521-530 (1985)) to express the gene of interest. Downstream of the promoter are BamHI, XbaI and Asp718 restriction enzyme cleavage sites that allow integration of the gene. After these cloning sites, the plasmid contains the 3' intron and polyadenylation site of the rat preproinsulin gene. Other highly efficient promoters, such as the human β-actin promoter, the SV40 early or late promoters, or the long terminal repeats from other retroviruses, such as HIV and HTLVI, can also be used for expression. Also, Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express proteins in a regulated manner in mammalian cells (M. Gossen, and H. Bujard, Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992)). For polyadenylation of mRNA, other signals, for example from human growth hormone or globin genes, can also be used. Stable cell lines carrying the gene of interest integrated into the chromosome can also be selected upon cotransfection with a selection marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selection marker, for example G418, plus methotrexate at the outset.

[0092] Antibody purification Anti-IL-12 / IL-23p40 or IL-23 antibodies can be recovered and purified from recombinant cell cultures by well-known methods, including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. High performance liquid chromatography ("high performance liquid chromatography, HPLC") can also be used for purification. See, e.g., Colligan, Current Protocols in Immunology or Current Protocols in Protein Science, John Wiley & Sons, NY, NY (1997-2001), e.g., chapters 1, 4, 6, 8, 9, 10, each of which is incorporated herein by reference in its entirety.

[0093] Antibodies for use in the methods of the invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant methods from eukaryotic hosts, including, for example, yeast, higher plants, insect, and mammalian cells. Depending on the host utilized in the recombinant production procedure, the antibodies may be glycosylated or non-glycosylated, but are preferably glycosylated. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18, and 20; Colligan, Protein Science, supra, Chapters 12-14, all of which are incorporated herein by reference in their entirety.

[0094] Anti-IL-12 / IL-23p40 or IL-23 antibody The anti-IL-12 / 23p40 or IL-23 antibodies of the present invention comprise at least a portion of an immunoglobulin molecule, such as, but not limited to, at least one ligand binding portion (LBP), such as, but not limited to, a heavy or light chain complementarity determining region (CDR) or a ligand binding portion thereof, a heavy or light chain variable region, a framework region (e.g., FR1, FR2, FR3, FR4, or a fragment thereof, and optionally, including at least one substitution, insertion, or deletion), a heavy or light chain constant region (e.g., at least one CDR or CDR fragment thereof), a heavy or light chain variable region, ... H 1, Hinge 1, Hinge 2, Hinge 3, Hinge 4, C H 2 or C H 3, or a fragment thereof, and optionally including at least one substitution, insertion, or deletion), or any portion thereof. The antibody may include or be derived from any mammal, such as, but not limited to, human, mouse, rabbit, rat, rodent, primate, or any combination thereof.

[0095] The isolated antibodies used in the methods of the invention include the amino acid sequences of the antibodies disclosed herein encoded by any suitable polynucleotide, or any isolated or prepared antibody. Preferably, the human antibodies or antigen-binding fragments bind to human IL-12 / IL-23p40 or IL-23, thereby partially or substantially neutralizing at least one biological activity of the protein. An antibody or specified portion or variant thereof that partially or preferably substantially neutralizes at least one biological activity of an IL-12 / IL-23p40 or IL-23 protein or fragment can bind to the protein or fragment, thereby inhibiting an activity of IL-12 / IL-23p40 or IL-23 mediated through binding to the IL-12 and / or IL-23 receptor, or through other IL-12 / IL-23p40 or IL-23-dependent or mediated mechanisms. As used herein, the term "neutralizing antibody" refers to an antibody capable of inhibiting IL-12 / IL-23p40 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 assay. The ability of an anti-IL-12 / IL-23p40 or IL-23 antibody to inhibit IL-12 / IL-23p40 or IL-23 dependent activity is preferably assessed by at least one suitable IL-12 / IL-23p40 or IL-23 protein or receptor assay described herein and / or known in the art. Human antibodies may be of 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 comprises an IgG heavy chain or a defined fragment, such as at least one of the following isotypes: IgG1, IgG2, IgG3, or IgG4 (e.g., gamma 1, gamma gamma 2, gamma 3, 4).Antibodies of this type can be prepared by utilizing transgenic mice or other non-human transgenic mammals that contain at least one human light chain (e.g., IgG, IgA, and IgM) transgene as described herein and / or known in the art. In another embodiment, the anti-IL-23 human antibody comprises an IgG1 heavy chain and an IgG1 light chain.

[0096] The antibody binds to at least one defined epitope specific for at least one IL-12 / IL-23p40 or IL-23 protein, subunit, fragment, portion, or any combination thereof, which may comprise at least one antibody binding region that comprises at least a portion of the protein, and which preferably comprises at least one extracellular, soluble, hydrophilic, exoportion, or cytoplasmic portion of the protein.

[0097] Generally, a human antibody or antigen-binding fragment comprises an antigen-binding region that comprises at least one human complementarity determining region (CDR1, CDR2, and CDR3) or variant of at least one heavy chain variable region, and at least one human complementarity determining region (CDR1, CDR2, and CDR3) or variant of at least one light chain variable region. The CDR sequences can be derived from human germline sequences or can match exactly to germline sequences. For example, CDRs from a synthetic library derived from the original non-human CDRs can be used. These CDRs can be formed by incorporation of conservative substitutions from the original non-human sequence. In another specific embodiment, the antibody or antigen-binding portion or variant can have an antigen-binding region that comprises at least a portion of at least one light chain CDR (i.e., CDR1, CDR2, and / or CDR3) with the corresponding CDR1, 2, and / or 3 amino acid sequence.

[0098] Such antibodies can be prepared by preparing and expressing a nucleic acid molecule (i.e., one or more) encoding the antibody using conventional techniques involving recombinant DNA technology, or by chemically linking together the various portions of the antibody (e.g., CDRs, framework) using conventional techniques, or by using any other suitable methods.

[0099] An anti-IL-12 / IL-23p40 or IL-23 specific antibody may comprise at least one of a heavy or light chain variable region having a defined amino acid sequence. For example, in a preferred embodiment, an anti-IL-12 / IL-23p40 or IL-23 antibody comprises an anti-IL-12 / IL-23p40 antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. An anti-IL-12 / IL-23p40 or IL-23 specific antibody may also comprise at least one of a heavy or light chain having a defined amino acid sequence. In another preferred embodiment, an anti-IL-12 / IL-23p40 or IL-23 antibody comprises an anti-IL-12 / IL-23p40 antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 10 and a light chain comprising the amino acid sequence of SEQ ID NO: 11. Antibodies that bind human IL-12 / IL-23p40 or IL-23 and comprise defined heavy or light chain variable regions can be prepared using suitable methods, such as those employing phage display (Katsube, Y., et al., Int J Mol. Med, 1(5):863-868 (1998)) or transgenic animals, as known in the art and / or described herein. For example, a transgenic mouse comprising a functionally rearranged human immunoglobulin heavy chain transgene and a transgene comprising DNA from a human immunoglobulin light chain locus capable of undergoing functional rearrangement can be immunized with human IL-12 / IL-23p40 or IL-23 or a fragment thereof to elicit the production of antibodies. If desired, antibody-producing cells can be isolated and hybridomas or other immortalized antibody-producing cells can be prepared as described herein and / or known in the art. Alternatively, the antibody, specified portion or variant can be expressed using the encoding nucleic acid, or a portion thereof, in a suitable host cell.

[0100] The present invention also relates to antibodies, antigen-binding fragments, immunoglobulin chains and CDRs that comprise amino acids in sequences that are substantially the same as the amino acid sequences described herein. Preferably, such antibodies or antigen-binding fragments and antibodies that comprise such chains or CDRs have high affinity (e.g., K D About 10 -9 M or less). Amino acid sequences that are substantially the same as those described herein include sequences containing conservative amino acid substitutions as well as amino acid deletions and / or insertions. A conservative amino acid substitution refers to the replacement of a first amino acid with a second amino acid that has chemical and / or physical properties (e.g., charge, structure, polarity, hydrophobicity / hydrophilicity) similar to those of the first amino acid. Conservative substitutions include, but are not limited to, replacing one amino acid with another amino acid within the following groups: lysine (K), arginine (R), and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D, and E; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine ​​(C), and glycine (G); F, W, and Y; C, S, and T.

[0101] Amino acid code The amino acids that make up the anti-IL-12 / IL-23p40 or IL-23 antibodies of the invention are often abbreviated. Amino acid designations can be provided by designating the amino acid by its one-letter code, its three-letter code, name, or three nucleotide codon(s) and are well understood in the art (see Alberts, B., et al., Molecular Biology of The Cell, Third Ed., Garland Publishing, Inc., New York, 1994).

[0102] [Table 1]

[0103] array Exemplary Anti-IL-12 / IL-23p40 Antibody Sequence—STELARA® (ustekinumab) Amino acid sequence of anti-IL-12 / IL-23p40 antibody complementarity determining region heavy chain 1 (CDRH1): (SEQ ID NO:1) TYWLG

[0104] Amino acid sequence of anti-IL-12 / IL-23p40 antibody complementarity determining region heavy chain 2 (CDRH2): (SEQ ID NO:2) IMSPVDSDIRYSPSFQG

[0105] Amino acid sequence of anti-IL-12 / IL-23p40 antibody complementarity determining region heavy chain 3 (CDRH3): (SEQ ID NO:3) RRPGQGYFDF

[0106] Amino acid sequence of anti-IL-12 / IL-23p40 antibody complementarity determining region light chain 1 (CDRL1): (SEQ ID NO:4) RASQGISSWLA

[0107] Amino acid sequence of anti-IL-12 / IL-23p40 antibody complementarity determining region light chain 2 (CDRL2): (SEQ ID NO:5) AASSLQS

[0108] Amino acid sequence of anti-IL-12 / IL-23p40 antibody complementarity determining region light chain 3 (CDRL3): (SEQ ID NO:6) QQYNIYPYT

[0109] Amino acid sequence of the variable heavy chain region of anti-IL-12 / IL-23p40 antibody (underlined CDRs): (SEQ ID NO: 7)

[0110] [1] TIFF0007660067000002.tif9142

[0111] Amino acid sequence of the variable light chain region of anti-IL-12 / IL-23p40 antibody (underlined CDRs): (SEQ ID NO: 8)

[0112] [2] TIFF0007660067000003.tif9142

[0113] Amino acid sequence of anti-IL-12 / IL-23p40 antibody heavy chain (CDRs underlined): (SEQ ID NO: 10)

[0114] [3] TIFF0007660067000004.tif32144

[0115] Amino acid sequence of anti-IL-12 / IL-23p40 antibody light chain (CDRs underlined): (SEQ ID NO: 11)

[0116] [4] TIFF0007660067000005.tif17144

[0117] Amino acid sequence of IL-12 Amino acid sequence of human interleukin (IL)-12 having α and β subunits: (SEQ ID NO:9)

[0118] [5] TIFF0007660067000006.tif36144

[0119] The anti-IL-12 / IL-23p40 or IL-23 antibodies used in the methods of the invention may contain one or more amino acid substitutions, deletions, or additions, either by natural mutation or by human manipulation, as specified herein.

[0120] The number of amino acid substitutions that one of skill in the art may make will depend on many factors, including those described above. Generally speaking, the number of amino acid substitutions, insertions or deletions in a given anti-IL-12 / IL-23p40 or IL-23 antibody, fragment or variant will not exceed 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, e.g., 1-30, or any range or value therein, as specified herein.

[0121] Amino acids within an anti-IL-12 / IL-23p40 or IL-23 specific antibody that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (e.g., Ausubel, supra, Chapter 8, 15; Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as, for example and without limitation, at least one IL-12 / IL-23p40 or IL-23 neutralizing activity. Sites critical for antibody binding can also be identified by structural analyses such as crystallization, nuclear magnetic resonance, or photoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992) and de Vos, et al., Science 255:306-312 (1992)).

[0122] An anti-IL-12 / IL-23p40 or IL-23 antibody can include, but is not limited to, at least one portion, sequence, or combination selected from from 5 to all of at least one of the contiguous amino acids of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 10, or 11.

[0123] The IL-12 / IL-23p40 or IL-23 antibody or specified portion or variant may include, but is not limited to, at least one portion, sequence, or combination selected from at least 3 to 5 contiguous amino acids of the above SEQ ID NO:, 5 to 17 contiguous amino acids of the above SEQ ID NO:, 5 to 10 contiguous amino acids of the above SEQ ID NO:, 5 to 11 contiguous amino acids of the above SEQ ID NO:, 5 to 7 contiguous amino acids of the above SEQ ID NO:, or 5 to 9 contiguous amino acids of the above SEQ ID NO:.

[0124] An anti-IL-12 / IL-23p40 or IL-23 antibody can further optionally comprise at least one polypeptide that is 70-100% of the contiguous amino acids of SEQ ID NO:5, 17, 10, 11, 7, 9, 119, 108, 449, or 214 above. In one embodiment, the amino acid sequence of an immunoglobulin chain, or a portion thereof (e.g., variable region, CDR) has about 70-100% identity (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or any range or value therein) to the amino acid sequence of the corresponding chain of at least one of the above SEQ ID NOs. For example, the amino acid sequence of the light chain variable region can be compared to the above SEQ ID NOs, or the amino acid sequence of the heavy chain CDR3 can be compared to the above SEQ ID NOs. Preferably, 70-100% amino acid identity (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or any range or value therein) is determined using a suitable computer algorithm, as known in the art.

[0125] As known in the art, "identity" is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between such linear sequences. "Identity" and "similarity" can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, 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). Additionally, percent identity values ​​can be obtained from amino acid and nucleotide sequence alignments generated using default settings in AlignX, a component of Vector NTI Suite 8.0 (Informax, Frederick, MD).

[0126] Preferred methods for determining identity are designed to obtain the highest degree of match between the sequences tested. Methods for determining identity and similarity are codified in publicly available computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387(1984)), BLASTP, BLASTN, and FASTA (Atschul, SF et al., J.Molec.Biol.215:403-410(1990)). BLAST X programs are 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 may also be used to determine identity.

[0127] Preferred parameters for polypeptide sequence comparisons 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. Sci, USA. 89: 10915-10919 (1992) Gap penalty: 12 Gap length penalty: 4 A program useful with these parameters is publicly available as the "Gap" program from the Genetics Computer Group, Madison Wis. The aforementioned parameters are the default parameters for peptide sequence comparisons (as well as no penalty for end gaps).

[0128] Preferred parameters for polynucleotide comparisons include the following: (1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48:443-453 (1970) Comparison matrix: match = +10, mismatch = 0 Gap penalty: 50 Gap length penalty: 3 Available as the "Gap" program from the Genetics Computer Group, Madison Wis. These are the default parameters for nucleic acid sequence comparisons.

[0129] As an example, a polynucleotide sequence may be identical to another sequence, i.e., 100% identical, or may contain up to a certain integer number of nucleotide changes compared to a reference sequence. Such changes may be selected from the group consisting of at least one nucleotide deletion, substitution (including transitions and transversions), or insertion, and the changes may occur at the 5' or 3' terminal position of the reference nucleotide sequence, or anywhere between these terminal positions, and may be interspersed between nucleotides of the reference sequence, either individually, or in one or more adjacent groups within the reference sequence. The number of nucleotide changes may be determined by multiplying the total number of nucleotides in the sequence by the numerical percentage of the corresponding percent identity (divided by 100) and subtracting the product from the total number of nucleotides in the sequence, or is determined by n.sub.n.ltorsim.x.sub.n-(x.sub.ny), 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 product of x.sub.n and y is rounded down to the nearest integer before subtraction from x.sub.n.

[0130] Alterations in the polynucleotide sequence encoding the SEQ ID NO can create nonsense, missense, or frameshift mutations in the coding sequence, thereby altering the polypeptide encoded by the polynucleotide after such alterations. Similarly, the polypeptide sequence can be identical to the reference sequence of the SEQ ID NO, i.e., 100% identical, or can contain up to a certain integer number of amino acid alterations compared to the reference sequence such that the percent identity is less than 100%. Such alterations are selected from the group consisting of deletion, substitution (including conservative and non-conservative substitutions), or insertion of at least one amino acid, and the alterations can occur at the amino or carboxy terminal position of the reference polypeptide sequence or anywhere between these terminal positions, and can be interspersed between the amino acids of the reference sequence either individually or in one or more adjacent groups within the reference sequence. The number of amino acid alterations for a given percent identity can be calculated by multiplying the total number of amino acids in the SEQ ID NO by the numerical percent of the respective percent identity (divided by 100), and then subtracting the product from the total number of amino acids in the SEQ ID NO, or n.sub.a.ltorsim.x.sub.a-(x.sub.ay) where n.sub.a is the number of amino acid changes, x.sub.a is the total number of amino acids in the sequence identified above, and y is, for example, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, etc., and any non-integer product of x.sub.a and y is rounded to the nearest integer before subtracting it from x.sub.a.

[0131] Exemplary heavy and light chain variable region sequences, and portions thereof, are set forth in the SEQ ID NOs. An antibody of the invention, or specified variant thereof, can include any number of contiguous amino acid residues from an antibody of the invention, the number being selected from the group of integers consisting of 10-100% of the number of contiguous residues in an anti-IL-12 / IL-23p40 or IL-23 antibody. Optionally, this subsequence of contiguous amino acids is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, or more amino acids in length, or any range or value therein. Additionally, the number of such subsequences can be any integer selected from the group consisting of 1-20, such as at least 2, 3, 4, or 5.

[0132] As will be apparent to one of skill in the art, the present invention includes at least one biologically active antibody of the present invention. A biologically active antibody has a specific activity that is at least 20%, 30%, or 40%, preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95% to 100% or more (including but not limited to up to 10-fold the specific activity) of that of a natural (non-synthetic), endogenous, or related and known antibody. Methods for assaying and quantitatively measuring enzymatic activity and substrate specificity are well known to those of skill in the art.

[0133] In another aspect, the invention relates to human antibodies and antigen-binding fragments described herein that are modified by the covalent attachment of an organic moiety. Such modifications can generate antibodies or antigen-binding fragments with improved pharmacokinetic properties (e.g., increased serum half-life in vivo). The organic moiety can be a linear or branched hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. In certain embodiments, the hydrophilic polymer group can be a polyalkane glycol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), a carbohydrate polymer, an amino acid polymer, or polyvinylpyrrolidone, with a molecular weight of about 800 to about 120,000 daltons, and the fatty acid group or fatty acid ester group can contain about 8 to about 40 carbon atoms.

[0134] As defined herein, the term "half-life" indicates that the plasma concentration of a drug (e.g., the therapeutic anti-IL-12 / IL-23p40 antibody ustekinumab) is halved after one elimination half-life. Thus, with each subsequent half-life, less drug is eliminated. After one half-life, the amount of drug remaining in the body is 50%, after two half-lives, 25%, and so on. The half-life of a drug depends on its clearance and volume of distribution. The elimination half-life is believed to be independent of the amount of drug in the body.

[0135] The modified antibodies and antigen-binding fragments may contain one or more organic moieties that are directly or indirectly covalently attached to the antibody. Each organic moiety attached to the antibody or antigen-binding fragment of the present invention may be independently a hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. As used herein, the term "fatty acid" includes monocarboxylic and dicarboxylic acids. As used herein, the term "hydrophilic polymer group" refers to an organic polymer that is more soluble in water than octane. For example, polylysine is more soluble in water than octane. Thus, antibodies modified by the covalent attachment of polylysine are encompassed by the present invention. Suitable hydrophilic polymers for modifying the antibodies of the present invention may be linear or branched, and include, for example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG, etc.), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides, etc.), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartic acid, etc.), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide, etc.), and polyvinylpyrrolidone. Preferably, the hydrophilic polymers for modifying the antibodies of the present invention have a molecular weight of about 800 to about 150,000 daltons as individual molecular entities. For example, PEG 5000 and PEG 20,000 can be used, where the subscript is the average molecular weight of the polymer in Daltons. The hydrophilic polymer group can be substituted with 1 to about 6 alkyl groups, fatty acid groups, or fatty acid ester groups. Hydrophilic polymers substituted with fatty acid or fatty acid ester groups can be prepared by utilizing a suitable method. For example, a polymer containing an amine group can be linked to a carboxylate of a fatty acid or fatty acid ester, and an activated carboxylate on the fatty acid or fatty acid ester (e.g., activated with N,N-carbonyldiimidazole) can be linked to a hydroxyl group on the polymer.

[0136] Fatty acids and fatty acid esters suitable for modifying antibodies of the invention may be saturated or may contain one or more units of unsaturation. Fatty acids suitable for modifying antibodies of the invention include, for example, n-dodecanoate (C 12 , laurate), n-tetradecanoate (C 14 , myristate), n-octadecanoate (C 18 , stearate), n-eicosanoate (C 20 , arachidate), n-docosanoate (C 22 , behenic acid), n-triacontanoate (C 30 ), n-tetracontanoate (C 40 ), cis-Δ9-octadecanoate (C 18 oleate), all cis-Δ5,8,11,14-eicosatetraenoate (C 20 , arachidonate), octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like. Suitable fatty acid esters include monoesters of dicarboxylic acids containing a straight or branched chain lower alkyl group. The lower alkyl group may contain from 1 to about 12, preferably from 1 to about 6, carbon atoms.

[0137] Modified human antibodies and antigen-binding fragments can be prepared using suitable methods, such as by reaction with one or more modifying agents. As used herein, the term "modifying agent" refers to a suitable organic group (e.g., hydrophilic polymer, fatty acid, fatty acid ester) that includes an activating group. An "activating group" is a chemical moiety or functional group that can react with a second chemical group under appropriate conditions, thereby forming 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, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. Aldehyde functional groups can be linked to amine- or hydrazide-containing molecules, and azide groups can react with trivalent phosphorus groups to form phosphoramidate or phosphorimide bonds. Suitable methods for introducing activating groups into molecules are known in the art (see, for example, Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, CA (1996)). Activating groups can be attached directly to organic groups (e.g., hydrophilic polymers, fatty acids, fatty acid esters) or to linker moieties, such as divalent C1-C 12The linker moiety can be linked via a group (wherein one or more carbon atoms can be replaced with a heteroatom such as oxygen, nitrogen, or sulfur). Suitable linker moieties include, for example, tetraethylene glycol, -(CH2)3-, -NH-(CH2)6-NH-, -(CH2)2-NH-, and -CH2-O-CH2-CH2-O-CH2-CH2-O-CH-NH-. Modifiers containing a linker moiety can be generated by forming an amide bond between a free amine and a fatty acid carboxylate, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine that can be coupled to another carboxylate as described, or it can be reacted with maleic anhydride and the resulting product cyclized to generate an activated maleimide derivative of a fatty acid (see, e.g., Thompson et al., International Publication No. WO 92 / 16221, the teachings of which are incorporated herein by reference in their entirety).

[0138] Modified antibodies can be produced by reacting a human antibody or antigen-binding fragment with a modifying agent. For example, organic moieties can be attached to the antibody in a non-site-specific manner using an amine-reactive modifying agent, e.g., an NHS ester of PEG. Modified human antibodies or antigen-binding fragments can also be prepared by reducing disulfide bonds (e.g., intrachain disulfide bonds) of an antibody or antigen-binding fragment. The reduced antibody or antigen-binding fragment can then be reacted with a thiol-reactive modifying agent to produce the modified antibody of the invention. Modified human antibodies and antigen-binding fragments containing organic moieties attached to specific sites of the antibodies of the invention can be prepared using suitable methods such as reverse proteolysis (based on Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci. 6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1):59-68 (1996); Capellas et al., Biotechnol. Bioeng., 56(4):456-463 (1997)) and those described in Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, CA (1996).

[0139] The methods of the invention also employ anti-IL-12 / IL-23p40 or IL-23 antibody compositions comprising at least one, at least two, at least three, at least four, at least five, at least six or more anti-IL-12 / IL-23p40 or IL-23 antibodies provided in a non-naturally occurring composition, mixture or form as described herein and / or known in the art. Such compositions include non-naturally occurring compositions comprising at least one or two full-length, C- and / or N-terminal deletion variants, domains, fragments, or specified variants of an anti-IL-12 / IL-23p40 or IL-23 antibody amino acid sequence selected from the group consisting of 70-100% of the contiguous amino acids of said sequences, or specified fragments, domains, or variants thereof. A preferred anti-IL-12 / IL-23p40 or IL-23 antibody composition comprises at least one or two full-length, fragments, domains, or variants of at least one CDR- or LBP-containing portion of the anti-IL-12 / IL-23p40 or IL-23 antibody sequences described herein, e.g., 70-100% of the above SEQ ID NOs., or specified fragments, domains, or variants thereof. Further preferred compositions comprise, e.g., 40-99% of at least one of the above SEQ ID NOs., etc., or specified fragments, domains, or variants thereof. Percentages of such compositions are by weight, volume, concentration, molarity, or molarity as liquid or dry solutions, mixtures, suspensions, emulsions, particles, powders, or colloids, as known in the art or as described herein.

[0140] Antibody compositions containing further therapeutically active ingredients The antibody compositions used in the methods of the present invention can optionally further comprise an effective amount of at least one compound or protein selected from at least one of the following: anti-infective agents, cardiovascular (CV) agents, central nervous system (CNS) agents, autonomic nervous system (ANS) agents, airway agents, gastrointestinal (GI) tract agents, hormonal agents, fluid or electrolyte balancing agents, hemodynamic agents, antineoplastic agents, immunomodulatory agents, ophthalmic, otic or nasal agents, topical agents, nutritional agents, etc. Such agents are well known in the art (see, e.g., Nursing 2001 Handbook of Drugs, 21), including their respective formulations, indications, dosages, and administration as set forth herein. st edition, Springhouse Corp., Springhouse, PA, 2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc., Upper Saddle River, NJ; Pharmacology Handbook, Wells et al., Appleton & Lange, Stamford, CT, each of which is incorporated herein by reference).

[0141] As examples of drugs that can be combined with the antibodies of the method of the present invention, the anti-infective drug can be at least one selected from amebicide or at least one antiprotozoal drug, anthelmintic drug, antifungal drug, antimalarial drug, antituberculous drug or at least one antilepromycotic drug, aminoglycoside, penicillin, cephalosporin, tetracycline, sulfonamide, fluoroquinolone, antiviral drug, macrolide anti-infective drug, and miscellaneous anti-infective drugs. The hormonal drug can be at least one selected from corticosteroid, androgen, or at least one anabolic steroid, estrogen, or at least one progestin, gonadotropin, antidiabetic drug, or at least one glucagon, thyroid hormone, thyroid hormone antagonist, pituitary hormone, and parathyroid mimetic drug. The at least one cephalosporin can be at least one selected from cefaclor, cefadroxil, cefazolin sodium, cefdinir, cefepime hydrochloride, cefixime, cefmetazole sodium, cefonicid sodium, cefoperazone sodium, cefotaxime sodium, cefotetan disodium, cefoxitin sodium, cefpodoxime proxetil, cefprozil, ceftazidime, ceftibuten, ceftizoxime sodium, ceftriaxone sodium, cefuroxime axetil, cefuroxime sodium, cephalexin hydrochloride, cephalexin monohydrate, cephradine, and loracarbef.

[0142] The at least one corticosteroid can be at least one selected from betamethasone, betamethasone acetate or betamethasone sodium phosphate, betamethasone sodium phosphate, cortisone acetate, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, fludrocortisone acetate, hydrocortisone, hydrocortisone acetate, hydrocortisone cypionate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebutate, prednisone, triamcinolone, triamcinolone acetonide, and triamcinolone diacetate. The at least one androgenic or anabolic steroid can be at least one selected from danazol, fluoxymesterone, methyltestosterone, nandrolone decanoate, nandrolone phenpropionate, testosterone, testosterone cypionate, testosterone enanthate, testosterone propionate, and a testosterone transdermal system.

[0143] The at least one immunosuppressant can be at least one selected from azathioprine, basiliximab, cyclosporine, daclizumab, lymphocyte immunoglobulin, muromonab-CD3, mycophenolate mofetil, mycophenolate mofetil hydrochloride, sirolimus, 6-mercaptopurine, methotrexate, mizoribine, and tacrolimus.

[0144] The at least one topical anti-infective agent can 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, tioconazole, and tolnaftate. The at least one scabicide or pediculicide can be at least one selected from crotamiton, lindane, permethrin, and pyrethrins. The at least one topical corticosteroid can be at least one selected from betamethasone dipropionate, betamethasone valerate, clobetasol propionate, desonide, desoximetasone, dexamethasone, dexamethasone sodium phosphate, diflorasone diacetate, fluocinolone acetonide, fluocinonide, flurandrenolide, fluticasone propionate, halcionide, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone valerate, mometasone furoate, and triamcinolone acetonide. (See, e.g., pages 1098-1136 of Nursing 2001 Drug Handbook.)

[0145] Anti-IL-12 / IL-23p40 or IL-23 antibody compositions comprise at least one anti-IL-12 / IL-23 antibody that is contacted or administered to a cell, tissue, organ, animal, or patient in need of such modulation, treatment, or therapy, and optionally at least one TNF antagonist (such as, but not limited to, a TNF chemical or protein antagonist, a TNF monoclonal or polyclonal antibody or fragment, a soluble TNF receptor (e.g., p55, p70, or p85) or fragment, a fusion polypeptide thereof, or a small molecule TNF antagonist, such as TNF binding protein I or II (TBP-1 or TBP-II), nerelimonumab (n The composition may further comprise at least one of any suitable and effective amount of compositions or pharmaceutical compositions further comprising at least one selected from the group consisting of antirheumatic drugs (e.g., methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, sodium aurothiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalazine), immunization, immunoglobulins, immunosuppressants (e.g., basiliximab, cyclosporine, daclizumab), cytokines, or cytokine antagonists. Non-limiting examples of such cytokines include, but are not limited to, any of IL-1 to IL-23, etc. (e.g., IL-1, IL-2, etc.). Suitable dosages are well known in the art. For example, Wells et al., eds., Pharmacotherapy Handbook, 2 nd Edition, Appleton and Lange, Stamford, CT (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, CA (2000), each of which is incorporated herein by reference in its entirety.

[0146] The anti-IL-12 / IL-23p40 or IL-23 antibody compounds, compositions, or mixtures used in the methods of the invention may further comprise at least one of any suitable auxiliary agent, such as, but not limited to, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, etc. Pharmaceutically acceptable auxiliary agents are preferred. Non-limiting examples of and methods for preparing such sterile solutions are well known in the art and are described, for example, in Gennaro, Ed., Remington's Pharmaceutical Sciences, 1896, ed., "Preparation of Sterile Solutions of IL-12 / IL-23p40 or IL-23 Antibody Compounds," ed., "Preparation of Sterile Solutions of IL-12 / IL-23p40 ... th Edition, Mack Publishing Co. (Easton, PA), 1990. As is well known in the art or described herein, pharma- ceutically acceptable carriers suitable for the mode of administration, solubility, and / or stability of the anti-IL-23 antibody, fragment, or variant composition can be routinely selected.

[0147] Pharmaceutical excipients and additives useful in the present compositions include, but are not limited to, proteins, peptides, amino acids, lipids, and carbohydrates (e.g., saccharides including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides, derivatized sugars such as alditols, aldonic acids, esterified sugars, and polysaccharides or sugar polymers), which may be present alone or in combination and comprise 1-99.99% by weight or volume, alone or in combination. Exemplary protein excipients include serum albumins such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acids / antibody components that may also function in a buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. One preferred amino acid is glycine.

[0148] Suitable carbohydrate excipients for use in the present invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc., disaccharides such as lactose, sucrose, trehalose, cellobiose, etc., polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starches, etc., alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), myo-inositol, etc. Preferred carbohydrate excipients for use in the present invention are mannitol, trehalose, and raffinose.

[0149] The anti-IL-12 / IL-23p40 or IL-23 antibody compositions may also include a buffer or pH adjusting agent, typically a salt prepared from an organic acid or base. Exemplary buffers include organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid, Tris, tromethamine hydrochloride, or phosphate buffers. A preferred buffer for use in the present compositions is an organic acid salt such as citric acid.

[0150] In addition, anti-IL-12 / IL-23p40 or IL-23 antibody compositions may include polymeric excipients / additives such as polyvinylpyrrolidone, Ficoll (a polymeric sugar), dextrates (e.g., cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin), polyethylene glycol, flavorings, antimicrobials, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "TWEEN 20" and "TWEEN 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

[0151] These and additional known pharmaceutical excipients and / or additives suitable for use in the anti-IL-12 / IL-23p40 or IL-23 antibody, portion or variant compositions according to the invention are known in the art and are described, for example, in Remington: The Science & Practice of Pharmacy, 19th ed., Williams & Williams, (1995), and Physician's Desk Reference, 52 nd ed., Medical Economics, Montvale, NJ (1998), the disclosures of which are incorporated herein by reference in their entireties. Preferred carrier or excipient materials are carbohydrates (e.g., monosaccharides and alditols) and buffers (e.g., citric acid) or polymeric agents. Exemplary carrier molecules are mucopolysaccharides, hyaluronic acid, which may be useful for intra-articular delivery.

[0152] formulation As mentioned above, the present invention preferably provides stable formulations comprising saline or a phosphate buffer with a selected salt, as well as preservative-containing preservative solutions and formulations, and versatile preserved formulations suitable for pharmaceutical or veterinary use comprising at least one anti-IL-12 / IL-23p40 or IL-23 antibody in a pharma- ceutically acceptable formulation. The preserved formulations contain at least one known preservative, optionally selected from the group consisting of at least one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrate, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof, in an aqueous diluent. As known in the art, the range of concentrations may be from 0.001 to 5%, or 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 range or value therein may be used.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. 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like.

[0153] As discussed above, the methods of the invention employ articles of manufacture comprising packaging and at least one vial containing a solution of at least one anti-IL-12 / IL-23p40 or IL-23 antibody, optionally with a buffer and / or preservative formulated in an aqueous diluent, the packaging comprising a label indicating that such solution can be maintained 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 more. The invention further employs articles of manufacture comprising packaging and a first vial containing a lyophilized anti-IL-12 / IL-23p40 or IL-23 antibody and a second vial containing an aqueous diluent of the formulated buffer or preservative, the packaging comprising a label instructing the patient to reconstitute the anti-IL-12 / IL-23p40 or IL-23 antibody with the aqueous diluent to form a solution that can be maintained for a period of 24 hours or more.

[0154] The anti-IL-12 / IL-23p40 or IL-23 antibodies used in accordance with the present invention may be produced by recombinant means, such as from mammalian cells or transgenic preparations, or may be purified from other biological sources, as described herein or known in the art.

[0155] Anti-IL-12 / IL-23p40 or IL-23 antibody ranges are included in amounts to provide a concentration of about 1.0 μg / mL to about 1000 mg / mL upon reconstitution for wet / dry systems, although lower and higher concentrations are workable and depend on the intended delivery vehicle, e.g., for solution formulations, as opposed to transdermal patch, pulmonary, transmucosal, or osmotic or micropump methods.

[0156] Preferably, the aqueous diluent optionally further comprises a pharma- ceutically acceptable preservative. Preferred preservatives include those selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof. The concentration of the preservative used in the formulation is sufficient to produce an antimicrobial effect. Such concentration will vary depending on the preservative selected and can be easily determined by one skilled in the art.

[0157] Other excipients, such as isotonicity agents, buffers, antioxidants, and preservative enhancers, can be optionally and preferably added to the diluent. An isotonicity agent, such as glycerin, is commonly used at a known concentration. Preferably, a physiologically tolerable buffer is added to provide improved pH control. The formulation can cover a wide range of pH, such as from about pH 4 to about pH 10, and preferably from about pH 5 to about pH 9, and most preferably from about 6.0 to about 8.0. Preferably, the formulation of the present invention has a pH of about 6.8 to about 7.8. Suitable buffers include phosphate buffers, most preferably sodium phosphate, especially phosphate buffered saline (PBS).

[0158] Other additives, such as pharma- ceutically acceptable solubilizers, such as Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block copolymer), and PEG (polyethylene glycol), or non-ionic surfactants, such as polysorbate 20 or 80, or poloxamer 184 or 188, Pluronic® polyls, other block copolymers, and chelating agents, such as EDTA and EGTA, can be optionally added to the formulation or composition to reduce aggregation. These additives are particularly useful when pumps or plastic containers are used to administer the formulation. The presence of pharma-ceutically acceptable surfactants reduces the tendency of proteins to aggregate.

[0159] The formulations can be prepared by a process that includes mixing at least one anti-IL-12 / IL-23p40 or IL-23 antibody with a preservative selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal or mixtures thereof in an aqueous diluent. Mixing at least one anti-IL-12 / IL-23p40 or IL-23 specific antibody with a preservative in an aqueous diluent is performed using conventional dissolution and mixing procedures. To prepare a suitable formulation, for example, a quantity of at least one anti-IL-12 / IL-23p40 or IL-23 antibody in a buffer solution is combined with the desired preservative in a sufficient amount of buffer solution to provide the desired concentration of protein and preservative. Variations of this process will be recognized by those skilled in the art. For example, the order of addition of the components, whether or not additional additives are used, the temperature and pH at which the formulation is prepared are all factors that can be optimized for the dosage concentration and means of administration used.

[0160] The formulations can be provided to patients as clear solutions or as dual vials containing a vial of lyophilized anti-IL-12 / IL-23p40 or IL-23 specific antibody to be reconstituted with a second vial containing water, preservatives and / or excipients, preferably phosphate buffer and / or saline, and the selected salt in an aqueous diluent. Either the single solution vial or the dual vial requiring reconstitution can be reused multiple times to satisfy single or multiple patient treatment cycles, thus providing a more convenient treatment regimen than is currently available.

[0161] The product is useful for administration over a period ranging from immediate to 24 hours or more. Thus, the products claimed by the present invention provide significant benefits to patients. The formulations of the present invention can optionally be safely stored at temperatures between about 2° C. and about 40° C. and retain biological activity of the protein for extended periods of time, and thus the package label can indicate that the solution can be stored and / or used for periods of 6, 12, 18, 24, 36, 48, 72, or 96 hours or more. When a preserved diluent is used, such label can include use up to 1-12 months, half a year, one and a half years, and / or up to two years.

[0162] Solutions of anti-IL-12 / IL-23p40 or IL-23 specific antibodies can be prepared by a process that includes mixing at least one antibody in an aqueous diluent. Mixing is performed using conventional dissolution and mixing procedures. To prepare a suitable diluent, for example, a quantity of at least one antibody in water or buffer is combined in an amount sufficient to provide the desired concentration of protein, and optionally a preservative or buffer. Variations of this process will be recognized by those skilled in the art. For example, the order of addition of the components, whether or not additional additives are used, the temperature and pH at which the formulation is prepared are all factors that can be optimized for the administration concentration and means of administration used.

[0163] The claimed products can be provided to patients as clear solutions or as combination vials containing a vial of at least one lyophilized anti-IL-12 / IL-23p40 or IL-23 specific antibody that is reconstituted with a second vial containing an aqueous diluent. Either the single solution vial or the dual vial requiring reconstitution can be reused multiple times to satisfy single or multiple patient treatment cycles, thus providing a more convenient treatment regimen than is currently available.

[0164] The claimed products can be provided indirectly to patients by providing a pharmacy, clinic, or other such institution and facility with a combination vial containing a clear solution or a vial of at least one lyophilized anti-IL-12 / IL-23p40 or IL-23 specific antibody reconstituted with a second vial containing an aqueous diluent, where the clear solution can be up to a liter or even more in volume, from which smaller amounts of the at least one antibody solution can be removed one or more times from the larger container and transferred to smaller vials and provided to customers and / or patients by the pharmacy or clinic.

[0165] Approved devices that contain single vial systems include BD Pens, BD Autojector®, Humaject®, NovoPen®, BD® Pen, AutoPen®, and pen-type injector devices for solution delivery such as OptiPen®, GenotropinPen®, Genotronorm Pen®, Humatro Pen®, Reco-Pen®, Roferon Pen®, Biojector®, Iject®, J-tip Needle-Free Injector®, Intraject®, Medi-Ject®, and Smartject® (e.g., Becton Dickensen (Franklin Lakes, NJ, www.bectondickenson.com), Disetronic (Burgdorf, Switzerland, www.disetronic.com), Bioject, Portland, Oregon (www.bioject.com); National Medical Suitable devices include those manufactured or developed by Eppendorf Products, Weston Medical (Peterborough, UK, www.weston-medical.com), Medi-Ject Corp (Minneapolis, MN, www.mediject.com), and similar suitable devices. Approved devices that include dual vial systems include pen-type injector systems for reconstituting lyophilized medications in cartridges for delivery of the reconstituted solution, such as the HumatroPen®. Examples of suitable other devices include pre-filled syringes, auto-injectors, needleless syringes, and needleless IV infusion sets.

[0166] The product may include packaging material. The packaging material provides the conditions under which the product may be used, in addition to any information required by regulatory agencies. The packaging material of the present invention provides instructions to the patient, if applicable, to reconstitute at least one anti-IL-12 / IL-23p40 or IL-23 antibody with an aqueous diluent to form a solution and use this solution for a period of 2-24 hours or more in a wet / dry two vial product. In the case of a single vial solution product, pre-filled syringe, or auto-injector, the label indicates that such solution may be used for a period of 2-24 hours or more. The product is useful for human pharmaceutical product applications.

[0167] The formulations used in the methods of the invention can be prepared by a process that includes mixing an anti-IL-12 / IL-23p40 or IL-23 antibody with a selected buffer, preferably saline or phosphate buffer containing a selected salt. Mixing the anti-23 antibody with the buffer in an aqueous diluent is performed using conventional dissolution and mixing procedures. To prepare a suitable formulation, for example, a quantity of at least one antibody in water or buffer is combined with the desired buffer in a sufficient amount of water to provide the desired concentration of protein and buffer. Variations of this process will be recognized by those skilled in the art. For example, the order of addition of the components, whether or not additional additives are used, the temperature and pH at which the formulation is prepared are all factors that can be optimized for the administration concentration and administration means used.

[0168] The method of the present invention provides pharmaceutical compositions including various formulations useful and acceptable for administration to human or animal patients. Such pharmaceutical compositions are prepared using "standard conditions" water as a diluent and routine methods well known to those skilled in the art. For example, buffer components such as histidine and histidine monohydrochloride hydrate may be provided first, followed by the addition of an appropriate non-final volume of "standard conditions" water diluent, sucrose, and polysorbate 80. The isolated antibody may then be added. Finally, the volume of the pharmaceutical composition is adjusted to the desired final volume under "standard conditions" using water as a diluent. Those skilled in the art will recognize several other methods suitable for the preparation of pharmaceutical compositions.

[0169] A pharmaceutical composition may be an aqueous solution or suspension containing the indicated mass of each component per volume unit of water or having the indicated pH at "standard conditions". As used herein, the term "standard conditions" refers to a temperature of 25°C ± 2°C and a pressure of 1 atmosphere. The term "standard conditions" is not used in the art to refer to a single art-recognized set of temperatures or pressures, but is instead a reference condition that specifies the temperature and pressure used to describe a solution or suspension containing a particular composition under reference "standard conditions" conditions. This is because the volume of a solution is in part a function of temperature and pressure. Those skilled in the art will recognize that pharmaceutical compositions equivalent to those disclosed herein can be produced at other temperatures and pressures. The equivalence of such pharmaceutical compositions to those disclosed herein should be determined under "standard conditions" conditions defined above (e.g., 25°C ± 2°C and a pressure of 1 atmosphere).

[0170] Importantly, such pharmaceutical compositions may contain "approximately" a certain value of component mass (e.g., "about 0.53 mg of L-histidine") or have a certain value of pH value per unit volume of the pharmaceutical composition. The component mass or pH value present in the pharmaceutical composition is "about" a given numerical value when the isolated antibody is present in the pharmaceutical composition or when the isolated antibody is present in the pharmaceutical composition after it is removed from the pharmaceutical composition (e.g., by dilution) and can bind to a peptide chain. That is, the value, such as the component mass value or pH value, is "about" a given numerical value when the binding activity of the isolated antibody is maintained and detectable after the isolated antibody is placed in the pharmaceutical composition.

[0171] Competitive binding analysis is performed to determine whether IL-12 / IL-23p40 or IL-23 specific mAbs bind similar or different epitopes and / or compete with each other. Abs are individually coated onto ELISA plates. Competing mAbs are added, followed by biotinylated hrIL-12 or IL-23. For positive controls, the same mAb for coating can be used as a competing mAb ("self-competition"). IL-12 / IL-23p40 or IL-23 binding is detected using streptavidin. These results indicate whether the mAbs recognize similar or partially overlapping epitopes on IL-12 / IL-23p40 or IL-23.

[0172] One aspect of the methods of the invention involves administering a pharmaceutical composition to a patient.

[0173] In one embodiment of the pharmaceutical composition, the isolated antibody concentration is about 77 to about 104 mg per mL of pharmaceutical composition. In another embodiment of the pharmaceutical composition, the pH is about 5.5 to about 6.5.

[0174] The stable or preserved formulations can be provided to patients as clear solutions or as combination vials containing a vial of at least one lyophilized anti-IL-23 antibody that is reconstituted with a second vial containing a preservative or buffer and excipients in an aqueous diluent. Either the single solution vial or the dual vial requiring reconstitution can be reused multiple times to satisfy single or multiple patient treatment cycles, thus providing a more convenient treatment regimen than is currently available.

[0175] Other formulations or methods of stabilizing anti-IL-23 antibodies may be other than clear solutions of lyophilized powders containing the antibodies. Non-clear solutions include formulations containing particulate suspensions, which are compositions containing anti-IL-23 antibodies in structures of various sizes known variously as microspheres, microparticles, nanoparticles, nanospheres, or liposomes. Such relatively homogeneous essentially spherical particulate formulations containing active agents can be formed by contacting an aqueous phase containing active agents and polymers with a non-aqueous phase, as taught in U.S. Pat. No. 4,589,330, and then evaporating the non-aqueous phase to cause coalescence of particles from the aqueous phase. Porous microparticles can be prepared by using a first phase containing active agents and polymers dispersed in a continuous solvent, and removing the solvent from the suspension by lyophilization or dilution-extraction-precipitation, as taught in U.S. Pat. No. 4,818,542. Preferred polymers for such preparations are 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(β-hydroxybutyric acid), polyethylene oxide, polyethylene, poly(alkyl-2-cyanoacrylate), poly(hydroxyethyl methacrylate), polyamide, poly(amino acids), poly(2-hydroxyethyl DL-aspartamide), poly(ester urea), poly(L-phenylalanine / ethylene glycol), poly( ... The preferred polymers are natural or synthetic copolymers or polymers selected from the group consisting of poly(methyl methacrylate), poly(phenylene / 1,6-diisocyanatohexane), and poly(methyl methacrylate). Particularly preferred polymers are polyesters such as polyglycolic acid, polylactic acid, glycolide-L(-)lactide poly(epsilon-caprolactone), poly(epsilon-caprolactone-co-lactic acid), and poly(epsilon-caprolactone-co-glycolic acid). Solvents useful 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-containing phase into the second phase can include forcing the first phase through orifices in a nozzle under pressure to affect droplet formation.

[0176] Dry powder formulations may also result from processes other than freeze-drying, such as, for example, spray drying, or solvent extraction by evaporation or precipitation of a crystalline composition followed by one or more steps to remove the aqueous or non-aqueous solvent. The preparation of spray-dried antibody formulations is taught in U.S. Pat. No. 6,019,968. Antibody-based dry powder compositions can be produced by spray drying a solution or slurry of antibody and, optionally, excipients in a solvent under conditions to provide a respirable dry powder. Solvents include polar compounds that are easily dried, such as water and ethanol. Antibody stability can be enhanced by performing the spray-drying procedure in the absence of oxygen, for example under a nitrogen blanket, or by using nitrogen as the drying gas. Another relatively dry formulation is a dispersion of multiple porous microstructures dispersed in a suspension medium, typically containing a hydrofluoroalkane propellant, as taught in WO 9916419. The stabilized dispersion can be administered to the patient's lungs using a metered dose inhaler. Equipment useful in the commercial production of spray dried drugs is manufactured by Buchi Ltd. or Niro Corp.

[0177] Anti-IL-23 antibodies in any of the stable or preserved formulations or solutions described herein can be administered to a subject according to the invention via a variety of delivery methods, as known in the art, such as SC or IM injection, transdermal, transpulmonary, transmucosal, implants, osmotic pumps, cartridges, micropumps, or other means well known in the art and understood by the skilled artisan.

[0178] therapeutic application The present invention also provides methods for modulating or treating lupus in a cell, tissue, organ, animal, or patient using at least one IL-23 antibody of the invention, e.g., by administering to or contacting the cell, tissue, organ, animal, or patient with a therapeutically effective amount of an IL-12 / IL-23p40 or IL-23 specific antibody, as known in the art or described herein.

[0179] Any of the methods of the invention may comprise administering to a cell, tissue, organ, animal, or patient in need of such modulation, treatment, or therapy an effective amount of a composition or pharmaceutical composition comprising an anti-IL-23 antibody. Such methods may optionally further comprise co-administration or combination therapy for the treatment of such a disease or disorder, wherein administering the at least one anti-IL-23 antibody, specified portion, or variant thereof, is administered in combination with at least one TNF antagonist (such as, but not limited to, a chemical or proteinaceous TNF antagonist, a TNF monoclonal or polyclonal antibody or fragment, a soluble TNF receptor (e.g., p55, p70, or p85) or fragment, a fusion polypeptide thereof, or a small molecule TNF antagonist, such as TNF antagonist, ... Binding protein I or II (TBP-1 or TBP-II), nerelimonumab, infliximab, etanercept (Enbrel™), adalimumab (Humira™), CDP-571, CDP-870, afelimomab, lenercept, etc.), antirheumatic drugs (e.g., methotrexate, auranofin, aurothioglucose, azathioprine, sodium aurothiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalazine), muscle relaxants, narcotics, non-steroidal anti-inflammatory drugsdrugs, NSAIDs), analgesics, anesthetics, sedatives, local anesthetics, neuromuscular blockers, antibiotics (e.g., aminoglycosides, antifungals, antiparasitics, antivirals, carbapenems, cephalosporins, fluoroquinolones, macrolides, penicillins, sulfonamides, tetracyclines, other antibiotics), psoriasis medications, corticosteroids, anabolic steroids, diabetes medications, minerals, nutritional medications, thyroid medications, vitamins, calcium-related hormones, antidiarrheals, antitussives, antiemetics, antineoplastics, laxatives, anticoagulants, erythropoietin (e.g., epoetin alfa), filgrastim (e.g., G-CSF, Neupogen), sargramostim (GM-CSF, Leukine), immunosuppressants, The method further comprises administering, before, simultaneously and / or after administration of at least one of the following: immunosuppressants, immunoglobulins, immunosuppressants (e.g., basiliximab, cyclosporine, daclizumab), growth hormones, hormone replacement drugs, estrogen receptor modulators, mydriatics, cycloplegics, alkylating agents, antimetabolites, mitotic inhibitors, radiopharmaceuticals, antidepressants, antimanics, antipsychotics, anxiolytics, hypnotics, sympathomimetics, stimulants, donepezil, tacrine, asthma medications, beta agonists, inhaled steroids, leukotriene inhibitors, methylxanthines, cromolyn, epinephrine or analogs, dornase alfa (Pulmozyme), cytokines or cytokine antagonists. Suitable dosages are well known in the art. See, for example, Wells et al., eds., Pharmacotherapy Handbook, 2006, 144:131-135. nd 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, 21 stedition, Springhouse Corp., Springhouse, PA, 2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc., Upper Saddle River, NJ, each of which is incorporated herein by reference in its entirety.

[0180] Treatment Typically, the treatment of lupus is effected by administering an effective amount or dose of anti-IL-12 / 23p40 or anti-IL-23 antibody composition, on average, in the range of at least about 0.01 to 500 milligrams of anti-IL-12 / 23p40 or anti-IL-23 antibody per kg of patient body weight per dose, and preferably at least about 0.1 to 100 milligrams of antibody per kg of patient body weight per single or multiple doses, depending on the specific activity of the active agent contained in the composition. Alternatively, an effective serum concentration may include a serum concentration of 0.1 to 5000 μg / mL per single or multiple doses. Suitable dosages are known to medical practitioners and will, of course, depend on the specific disease state, the specific activity of the composition administered, and the specific patient undergoing treatment. In some cases, it may be necessary to provide repeated administrations, i.e., repeated individual administrations of a specific monitored amount or fixed amount, to obtain the desired therapeutic dose, where the individual administrations are repeated until the desired daily dose or effect is obtained.

[0181] Preferred doses are optionally 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, 7 0, 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 / dose, or any range, value or fraction thereof, or 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, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 9.1, 9.2, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 9.9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 9.9, 9.1, 9.2, 9.4, 9.5, 9.6, .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, or any range, value or fraction thereof.

[0182] Alternatively, the dose administered may vary depending on known factors such as the pharmacodynamic characteristics of the particular agent and its method and route of administration, the age, health condition and weight of the recipient, the nature and extent of the condition, the type of concurrent treatment, the frequency of treatment, and the desired effect. The dosage of the active ingredient may typically be about 0.1 to 100 milligrams per kilogram of body weight. Typically, 0.1 to 50, preferably 0.1 to 10 milligrams per kilogram per administration, or in sustained release form, is effective to obtain the desired results.

[0183] As non-limiting examples, treatment of humans or animals may be performed using a single dose, intravenous administration, or multiple doses for 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 days. or additionally, on at least one day of eyes 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, 61, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 11 0.1 to 100 mg / kg per day, for example, 0.5, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 1.9, 1.10, 1.20, 1.40, 1.65, 1.80, 1.95, 1.99, 1.99, 1.06, 1.07, 1.08, 1.09, 1.10, 1.11, 1.12, 1.13, 14, 15, 16, 17, 18, 19, or 20 years, or any combination thereof. The antibody may be provided as a single or periodic dose of 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 of at least one antibody of the invention.

[0184] Dosage forms (compositions) suitable for internal administration generally contain about 0.001 milligrams to about 500 milligrams of active ingredient per unit or container. In these pharmaceutical compositions, the active ingredient is usually present in an amount of about 0.5 to 99.999% by weight based on the total weight of the composition.

[0185] For parenteral administration, the antibody may be formulated as a solution, suspension, emulsion, particle, powder, or lyophilized powder, provided together with or separately from a pharma- ceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 1-10% human serum albumin. Non-aqueous vehicles such as liposomes and fixed oils can also be used. The vehicle or lyophilized powder may contain additives that maintain isotonicity and chemical stability (e.g., sodium chloride, mannitol for isotonicity; buffers and preservatives for chemical stability). The formulation is sterilized by known or suitable techniques.

[0186] Suitable pharmaceutical carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field.

[0187] Alternative Administration Many known and developed modes can be used in accordance with the present invention to administer a pharma- ceutically effective amount of an anti-IL-23 antibody. Although pulmonary administration is used in the following description, other modes of administration may be used in accordance with the present invention to obtain suitable results. The IL-12 / IL-23p40 or IL-23 antibodies of the present invention can be delivered in a carrier, as a solution, emulsion, colloid or suspension, or as a dry powder, by inhalation, or using any of a variety of devices and methods suitable for administration by other modes described herein or known in the art.

[0188] Parenteral Formulation and Administration Preparations for parenteral administration may contain sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogenated naphthalenes, etc. as common excipients. Aqueous or oily suspensions for injection can be prepared by using appropriate emulsifiers or wetting agents and suspending agents according to known methods. Injections may be non-toxic parenterally administrable diluents such as aqueous solutions, sterile injections, or suspensions in solvents. Usable vehicles or solvents include water, Ringer's solution, isotonic saline, etc., and sterile fixed oils can be used as common solvents or suspension solvents. For these purposes, any kind of fixed oils and fatty acids, including natural, synthetic, or semi-synthetic fatty oils or fatty acids, natural, synthetic, or semi-synthetic monoglycerides, diglycerides, or triglycerides, can be used. Parenteral administration is known in the art and includes, but is not limited to, conventional injection means, gas pressurized needleless injection devices such as those described in U.S. Pat. No. 5,851,198, and laser perforator devices such as those described in U.S. Pat. No. 5,839,446, which are incorporated herein by reference in their entireties.

[0189] alternative delivery The invention further relates to administration of anti-IL-12 / IL-23p40 or IL-23 antibodies by parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intraabdominal, intracapsular, intrachondral, intrasinus, intracavity, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, intralesional, bolus, intravaginal, rectal, buccal, sublingual, intranasal or transdermal means. The anti-IL-12 / IL-23p40 or IL-23 antibody compositions may be administered parenterally (subcutaneously, intramuscularly, or intravenously) or any other way, particularly for use in the form of liquid solutions or suspensions, particularly in semi-solid forms such as, but not limited to, creams and suppositories, for use in vaginal or rectal administration, in forms such as, but not limited to, tablets or capsules, for buccal or sublingual administration, or intranasally, in forms such as, but not limited to, powders, nasal drops or aerosols, or certain drugs, or with chemical enhancers such as dimethylsulfoxide to either modify the skin structure or increase the drug concentration in transdermal patches (Junginger, et al. In "Drug Permeation Enhancement"; Hsieh, DS, Eds., pp. 59-90 (Marcel Dekker, Inc. New York 1994, incorporated herein by reference in its entirety), or application of protein and peptide containing formulations to the skin (WO 98 / 53847), or application of an electric field to create a transient transport pathway, such as electroporation, or to increase the mobility of a charged drug through the skin, such as iontophoresis, or application of ultrasound, such as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402), transdermally in the form of, but not limited to, a gel, ointment, lotion, suspension, or patch delivery system, using oxidizing agents that allow for this (the above publications and patents are incorporated herein by reference in their entirety).

[0190] Having generally described the present invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting. Further details of the present invention are illustrated by the following non-limiting examples. The disclosures of all citations herein are expressly incorporated herein by reference.

[0191] Example: Manufacturing Process for Producing STELARA® (Ustekinumab) background STELARA® (ustekinumab) is a fully human G1 kappa monoclonal antibody that binds with high affinity and specificity to the shared p40 subunit of the human interleukin (IL)-12 and IL-23 cytokines. Ustekinumab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 10, and a light chain comprising the amino acid sequence of SEQ ID NO: 11, a heavy chain variable domain amino acid sequence of SEQ ID NO: 7, and a light chain variable domain amino acid sequence of SEQ ID NO: 8, heavy chain CDR amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and light chain CDR amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. Binding of ustekinumab to the IL-12 / IL-23 p40 subunit inhibits natural killer and CD4 + It blocks the binding of IL-12 or IL-23 to the IL-12Rβ1 receptor on the surface of T cells, inhibiting IL-12- and IL-23-specific intracellular signaling and subsequent activation and cytokine production. Aberrant regulation of IL-12 and IL-23 is associated with multiple immune-mediated diseases.

[0192] To date, ustekinumab has received marketing approval worldwide, including in countries in North America, Europe, South America, and the Asia-Pacific region, for the treatment of adult patients, including those with chronic moderate to severe plaque psoriasis and / or active psoriatic arthritis, Crohn's disease (CD), and ulcerative colitis (UC). Ustekinumab is also being evaluated in Phase 3 trials for the treatment of active systemic lupus erythematosus (SLE).

[0193] Manufacturing process overview STELARA® (ustekinumab) is manufactured in a 10-step process involving continuous perfusion cell culture followed by purification. An overview of the manufacturing process is shown in Figure 1.

[0194] As used herein, the terms "culture", "cultivate", "cultured", and "cell culture" refer to a population of cells suspended in a medium under conditions suitable for the survival and / or growth of the population of cells. As will be clear to those of skill in the art from the context, these terms as used herein also refer to a combination comprising a population of cells and the medium in which the population is suspended. A cell culture includes cells grown, for example, by batch, fed-batch, or perfusion cell culture methods. In certain embodiments, the cell culture is a mammalian cell culture.

[0195] Cell lines for use in the present invention include, but are not limited to, Chinese hamster ovary cells (CHO cells), human embryonic kidney cells (HEK cells), baby hamster kidney cells (BHK cells), mouse myeloma cells (e.g., NS0 cells and Sp2 / 0 cells), and human retinal cells (e.g., PER.C6 cells).

[0196] As used herein, the terms "chemically defined media", "chemically defined media", "chemically defined hybridoma media", or "chemically defined hybridoma media" refer to synthetic growth media in which the identity and concentration of all components are known. Synthetic media, i.e., chemically defined media, do not contain bacteria, yeast, animal, or plant extracts, animal serum or plasma, but may or may not contain individual plant or animal derived components (e.g., proteins, polypeptides, etc.). Chemically defined media may contain inorganic salts such as phosphates, sulfates, etc., necessary to support growth. The carbon source is defined, usually a sugar such as glucose, lactose, galactose, or other compounds such as glycerol, lactate, acetate, etc. Certain chemically defined media also use phosphate as a buffer, but other buffers such as citrate, triethanolamine, etc. may be used. Examples of commercially available chemically defined media include Thermo Fisher's CD Hybridoma Medium and CD Hybridoma AGT™ Medium, various Dulbecco's Modified Eagle (DME) media (Sigma-Aldrich Co; SAFC Bioscienses, Inc), Ham's Nutrient Mix (Sigma-Aldrich Co; SAFC Bioscienses, Inc), combinations thereof, etc. Methods for preparing chemically defined media are known in the art, for example, U.S. Pat. Nos. 6,171,825 and 6,936,441, WO 2007 / 077217, and U.S. Patent Application Publication Nos. 2008 / 0009040 and 2007 / 0212770.

[0197] As used herein, the term "bioreactor" refers to any vessel useful for the growth of cell cultures. Bioreactors may be of any size as long as they are useful for culturing cells. In certain embodiments, such cells are mammalian cells. Typically, bioreactors are at least 1 liter and may be 10, 100, 250, 500, 1,000, 2,500, 5,000, 8,000, 10,000, 12,000 liters or more, or any volume in between. During the culture period, the internal conditions of the bioreactor, including but not limited to pH and temperature, can be optionally controlled. Bioreactors can be constructed of any material suitable for holding a mammalian cell culture suspended in a medium under the culture conditions of the present invention, including glass, plastic, or metal. As used herein, the term "production bioreactor" refers to a final bioreactor used for the production of a polypeptide or glycoprotein of interest. The volume of the production bioreactor is typically at least 500 liters, and may be 1,000, 2,500, 5,000, 8,000, 10,000, 12,000 liters or more, or any volume in between. One of skill in the art would be able to select a suitable bioreactor for use in practicing the present invention.

[0198] Preculture, growth, and production of ustekinumab are carried out in stages 1 and 2. In stage 1, preculture is started from a single working cell bank vial of transfected Sp2 / 0 cells expressing the HC and LC sequences of ustekinumab, and the cells are grown in culture flasks, disposable culture bags, and 100L seed bioreactors. The cells are cultured until the cell density and volume required for inoculation of the 500L production bioreactor are obtained. In stage 2, the cell culture is perfused in the 500L production bioreactor using an alternating tangential flow (ATF) hollow fiber filter cell retention system. The cell culture permeate (harvest) is collected from the ATF system, the cells are retained in the bioreactor, and the culture is replenished with fresh medium. The harvest from one or more 500L production bioreactors can be combined in stage 3. The harvest is purified using MabSelect Protein A resin affinity chromatography. The resulting direct product capture (DPC) eluate is frozen until further processing.

[0199] Purification of ustekinumab from DCP is accomplished in stages 4-8 by a combination of ion exchange chromatography steps and steps to inactivate or remove potential viral contamination (solvent / detergent treatment and virus removal filtration). In stage 4, the DPC eluate is thawed, pooled, and filtered, and in stage 5, incubated with tri-n-butyl phosphate (TNBP) and polysorbate 80 / D treatment to inactivate any lipid enveloped viruses potentially present. In stage 6, SPXL® Sepharose cation exchange resin chromatography is used to remove TNBP and polysorbate 80 reagents, aggregates, and impurities from ustekinumab. In stage 7, ustekinumab is further purified to remove DNA, viruses, and impurities using QXL® Sepharose anion exchange resin chromatography. In stage 8, the purified ustekinumab product is diluted and filtered through a virus-retentive filter (NFP®).

[0200] Preparation of ustekinumab preformulated bulk (PFB) and formulated bulk (FB) is performed in steps 9 and 10, respectively. In step 9, an ultrafiltration step concentrates the ustekinumab product, and a diafiltration step adds formulation excipients and removes buffer salts in the process. In step 10, polysorbate 80 is added to the ustekinumab PFB to obtain the FB. The FB is filtered into polycarbonate containers for frozen storage. The frozen FB is packaged in insulated containers with dry ice for transportation to the drug product manufacturing site.

[0201] A detailed description of cell culture in large-scale manufacturing processes Phase 1 Pre-culture and proliferation The first step in the production of ustekinumab is the initiation of a pre-culture from a working cell bank (WCB) vial of transfected Sp2 / 0 cells expressing the HC and LC sequences of ustekinumab and expansion in culture flasks, disposable culture bags, and 100 L seed bioreactors. The cells are cultured until the cell density and volume required for inoculation of a 500 L production bioreactor is obtained. A flow diagram showing the pre-culture and expansion process is shown in Figure 2.

[0202] Manufacturing procedure Thaw one or more cryovials of WCB and dilute with CD (chemically defined) hybridoma medium (CDH-A) supplemented with 6 mM L-glutamine, 0.5 mg / L mycophenolic acid, 2.5 mg / L hypoxanthine, and 50 mg / L xanthine. Culture viability should be ≥ 45%. Dilute the cells further in CDH-A in culture flasks to a concentration of 0.2–0.5 × 10 6 Further dilute to a seeding density of 0.6 x 10 viable cells (VC) / mL. Precultures are maintained in a humidified CO2 incubator with controlled temperature, CO2 concentration, and agitation within ranges defined in the batch record. Precultures are maintained at 0.6 x 10 6The culture is incubated for ≦3 days until a minimum cell density of VC / mL and a culture viability of ≧50% is obtained. As a scale-up mechanism for inoculation of a 100 L seed bioreactor, the preculture is serially expanded in a series of culture flasks and then cultured in culture bags. During the culture expansion phase, each incubation step is performed for ≦3 days to achieve passaging conditions, with ≧0.6×10 6 A cell density of 0.2–0.5 × 10 VC / mL and a culture viability of ≥80% are required. The seeding density for each passage is 0.2–0.5 × 10 VC / mL in culture flasks. 6 VC / mL, and 0.2–0.6 × 10 in culture bags 6 Each passage is sampled for viable cell density (VSD), culture viability, and microscopy. Prior to inoculation of the 100 L seed bioreactor, the preculture is sampled for bioburden.

[0203] Precultures may be maintained for up to 30 days after thawing. Precultures not used within 30 days are discarded. A back-up preculture, grown as described above and subject to the same in-process monitoring, control tests, and process parameters as the primary preculture, can be maintained and used to inoculate another 100 L seed bioreactor, if necessary.

[0204] Once the preculture met the inoculation criteria, the contents of the culture bag were transferred to a 100 L seed bioreactor containing CDH-A and ≥ 0.3 × 10 6 A seeding density of ≥ 1.5 x 10 VC / mL is targeted. Seed bioreactor culture pH, temperature, and dissolved oxygen concentration are controlled within ranges defined in the batch record. 6 Cultures are grown until a cell density of VC / mL and ≧80% culture viability is obtained. Cultures are sampled for VCD, culture viability, and microscopy throughout the seed bioreactor process. Precultures are sampled for bioburden prior to inoculation of the 500 L production bioreactor.

[0205] VCD of seeded bioreactor cultures ≥ 1.5 x 106 Once VC / mL is reached, the culture may be used to inoculate a 500 L production bioreactor. Alternatively, a portion of the culture can be removed from the 100 L seed bioreactor and the remaining culture diluted with fresh medium. Following this "remove and fill" process, the culture is grown to sufficient cell density to inoculate a 500 L production bioreactor. The maximum duration of the 100 L seed bioreactor culture is 9 days after inoculation.

[0206] Phase 2 Bioreactor Production In stage 2, the cell culture is continuously perfused in a 500 L production bioreactor using an alternating tangential flow (ATF) hollow fiber filter cell retention system. The cell culture permeate (harvest) is collected from the ATF system, the cells are returned to the bioreactor, and the culture is replenished with fresh medium. A flow diagram showing the bioreactor production process is shown in Figure 3.

[0207] Manufacturing procedure Inoculation of the 500 L production bioreactor is performed by transferring the contents of the 100 L seed bioreactor into the 500 L production bioreactor containing CD (chemically defined) hybridoma medium (CDH-A) supplemented with 6 mM L-glutamine, 0.5 mg / L mycophenolic acid, 2.5 mg / L hypoxanthine, and 50 mg / L xanthine. The transferred volume is ≥ 0.3 x 10 6 The required seeding density should be sufficient to target 10 ...

[0208] Continuous perfusion is initiated to remove culture from the 500 L bioreactor to the ATF system and separate cells from the permeate. The permeate is filtered through a 0.2 μm ATF filter and collected as harvest in the bioprocess container (BPC). Cells are returned to the bioreactor and fed with fresh CDH-A to maintain a constant culture volume. Viable cell count (VCD), culture viability, pH, DO, temperature and immunoglobulin G (IgG) content are monitored during the production run. The perfusion rate is gradually increased proportional to the VCD until a target rate of approximately 1 bioreactor volume per day is reached. The perfusion rate is controlled to not exceed 1.20 bioreactor volumes per day. Retention in the ATF system is monitored to facilitate shutdown of the ATF filter before IgG retention across the filter exceeds 50%.

[0209] The VCD in the 500 L bioreactor was 8.0 × 10 6 The pH target is reduced to 7.2 to 7.1 at day 20 or when 12.0 x 10 VC / mL is reached, whichever occurs first. 6 When the VCD of VC / mL is reached, whichever occurs first, is initiated. Biomass is removed from the 500 L production bioreactor to the BPC at a rate of up to 20% of the bioreactor volume per day. Each harvest is sampled for bioburden.

[0210] The perfusion cell culture operation in the 500 L production bioreactor continues for up to 46 days after inoculation. At the end of production, the culture is sampled for mycoplasma and enveloped virus testing. The harvest can be stored at 2-8°C for ≦30 days after disconnection from the bioreactor.

[0211] Description of the small-scale production of ustekinumab expressed in CHO cells Generation of CHO cells expressing ustekinumab CHO cell lines were originally generated from adult Chinese hamster ovaries by TTPuck. CHO-K1 (ATCC® CCL-61) is a subclone of the parent CHO cell line lacking the proline synthesis gene. CHO-K1 has also been deposited at the European Collection of Cell Cultures, CHO-K1 (ECACC 85051005). A master cell bank (MCB) of CHO-K1, 024 M, was established at Celltech Biologics (now Lonza Biologics) and used to adapt CHO-K1 to suspension culture and serum-free medium. The adapted cell line was named CHOK1SV. The CHOK1SV cell line was further adapted to protein-free medium to generate an MCB of cells called 269-M. Cells derived from the 269-M MCB were transfected as follows to generate a CHO cell line expressing ustekinumab.

[0212] Cell lines were generated, grown, and maintained in a humidified incubator at 37°C and 5% CO2 using cell culture plates and shake flasks. The usual seeding density in shake flasks was 3 × 10 per mL. 5 Viable cells (vc / mL). All shake flask cultures were maintained at 130 revolutions per minute (rpm) with a 25 mm orbit, and 96 deep-well (DW, Thermo Scientific, Waltham, MA, Cat. #278743) cultures were maintained at 800 rpm with a 3 mm orbit.

[0213] CHO clones expressing ustekinumab are generated using a medium identified as MACH-1, an in-house developed, chemically defined medium for CHO cell culture. The basal medium for routine passage of CHO host cell lines is MACH-1 supplemented with 6 mM L-glutamine (Invitrogen, Carlsbad, CA, Cat. #25030-081). CHO cells transfected with the glutamine synthetase (GS) gene were grown in MACH-1+MSX unless otherwise noted, which is MACH-1 supplemented with 25 μM L-methionine sulfoximine (MSX, Sigma, St. Louis, MO, Cat. #M5379-1G) to inhibit glutamine synthetase function. For bolus fed-batch shake flask and bioreactor experiments, cells were cultured in MACH-1+F8, which is MACH-1 supplemented with 8 g / kg F8 (a proprietary growth enhancer supplement) to further support cell growth and antibody production. A proprietary feed medium was used for shake flask and bioreactor experiments.

[0214] DNA encoding the gene of interest was cloned into a glutamine synthetase (GS) dual gene expression plasmid (Lonza Biologics). Expression of the heavy chain (HC) and light chain (LC) genes was driven by separate human cytomegalovirus (hCMV-MIE) promoters. The GS gene selection marker, driven by the Simian virus SV40 promoter, allows the selection of transfected cells in glutamine-free medium in the presence of MSX.

[0215] Prior to each transfection, one aliquot of plasmid DNA containing both the HC and LC coding regions of ustekinumab was linearized by restriction enzyme digestion. A 15 μg aliquot of linearized DNA was then diluted to 1x10 using a BTX ECM 830 electrocell manipulator (Harvard Apparatus, Holliston, MA). 7An aliquot of cells was transfected. Cells were electroporated three times at 250V with a pulse length of 15 ms and an interpulse interval of 5 seconds in a cuvette with an electrode gap of 4 mm. Transfected cells were transferred to MACH-1 + L-glutamine in a shake flask and incubated for 1 day. Transfectants were centrifuged and resuspended in MACH-1 + 25uM MSX for selection, transferred to shake flasks and incubated for 6 days.

[0216] After chemical selection, cells were seeded in single-cell suspension in custom glutamine-free Methocult medium containing 2.5% (w / v) methylcellulose in Dulbecco's Modified Eagle's Medium (DMEM) basal medium (Methocult, StemCell Technologies, Inc., Vancouver, BC, Cat. #03899). The working solution also contained 30% (v / v) gamma-irradiated dialyzed fetal bovine serum (dFBS.IR, Hyclone, Logan, UT, Cat. #SH30079.03), 1xGS supplement (SAFC, St. Louis, MO, Cat. #58672-100M), 1.5 mg of animal component-free Protein G Alexa Fluor 488 conjugate (Protein G, Invitrogen, Carlsbad, CA, Cat. #C47010), 25 M MSX, Dulbecco's modified Eagle's medium with μM F12 (DMEM / F12, Gibco / Invitrogen, Carlsbad, CA, Cat. #21331-020), and the cell suspension.

[0217] Protein G recognizes human monoclonal antibodies and binds to IgG secreted by cells. Protein G is conjugated to the fluorescent label AlexaFluor 488, so cell colonies secreting the most antibody show the highest levels of fluorescence. After 12–18 days of incubation, colonies with the highest levels of fluorescence were picked using a ClonePix FL colony picking device (Molecular Devices, Sunnyvale, CA) into 100 μL MACH-1+MSX containing phenol red in 96-well plates and incubated without shaking for 5–7 days. After 5–7 days, cells from the 96-well plates (Thermo Scientific, Waltham, MA, Cat. #278743) were grown in 50–100 μL MACH-1+MSX containing phenol red in 96DW plates and shaken at 800 rpm with a 3 mm orbit. 96DW plates were fed and titrated via Octet (ForteBio, Menlo Park, CA) 7 days after 96DW seeding. The top 10 cultures corresponding to the highest batch 96DW growth titers were expanded and frozen cell banks were generated by shaking flasks in MACH-1+MSX and suspending the cells in MACH-1+MSX medium containing 10% DMSO.

[0218] Cell culture for small scale production For the small-scale production of ustekinumab expressed in Chinese hamster ovary cells (CHO cells) as well as for the large-scale production of ustekinumab expressed in Sp2 / 0 cells, preculture, cell growth, and cell production are carried out in stages 1 and 2. In stage 1, the preculture is initiated from a single cell bank vial of transfected CHO cells expressing the HC and LC sequences of ustekinumab, and the cells are grown in culture flasks. The cells are cultured until the cell density and volume required for inoculation of the 10-L production bioreactor are obtained. In stage 2, the cell culture is run in the 10-L production bioreactor in fed-batch culture mode. During the 15-day bioreactor run, the culture is fed with concentrated glucose-based and amino acid-based feeds as needed. Upon completion of the production bioreactor run, the cell culture harvest is clarified to remove biomass and filtered for further processing.

[0219] Purification for small scale production The purification steps for small-scale production of ustekinumab are the same as the large-scale manufacturing process, except that the step 8 viral filtration step has been omitted for small-scale production. Briefly, for small-scale production, purification of ustekinumab from the cell culture harvest is carried out in steps 3-7 by a combination of affinity and ion exchange chromatography steps and steps to inactivate or remove possible viral contamination (solvent / detergent treatment and virus removal). In step 3, the harvested and / or pooled harvest is clarified and purified using Protein A affinity chromatography. The resulting direct product capture (DPC) eluate is frozen until further processing. In step 4, after thawing, the DPC eluate is filtered and pooled, and then in step 5, treated with tri-n-butyl phosphate (TNBP) and polysorbate 80 (PS80) to inactivate any lipid-enveloped viruses that may be present.

[0220] In step 6, cation exchange chromatography is used to remove TNBP and PS80 reagents and impurities from the ustekinumab product. The ustekinumab product is further purified in step 7 using anion exchange chromatography to remove DNA, possible viruses, and impurities. As noted above, filtration in step 8 through a virus-retaining filter is omitted from the purification process for the CHO-derived ustekinumab product.

[0221] Final preparation of ustekinumab preformulated bulk (PFB) and formulated bulk (FB) is carried out in stages 9 and 10, respectively (see large scale stages). In stage 9, an ultrafiltration process concentrates the ustekinumab product, and a diafiltration step adds formulation excipients and removes buffer salts in the process. Polysorbate 80 is added to the ustekinumab PFB in stage 10 to obtain the FB, which is filtered into polycarbonate containers and stored frozen.

[0222] method How to determine viable cell density (VCD) and viability Total cells / ml, viable cells / ml (VCD), and % viability are typically determined on a Beckman Coulter Vi-CELL-XR cell viability analyzer using the manufacturer's protocols, software, and reagents. Alternatively, a CEDEX automated cell counting system has also been used. However, it should be noted that those skilled in the art are familiar with other methods for determining VCD and % viability, such as using a hemocytometer and trypan blue exclusion.

[0223] Bioactivity assays The biological activity of ustekinumab is determined by neutralization of IL-12-induced interferon gamma (IFN-γ) production by the IL-12-responsive human natural killer cell line NK-92MI (ATCC® CRL-2408). Ustekinumab binds to the p40 subunit of IL-12 and inhibits its interaction with IL-12Rβ1 on the cell surface of NK cells. This inhibits IL-12-mediated production of IFN-γ (see Aggeletopoulou I, et al. Interleukin 12 / interleukin 23 pathway: biological basis and therapeutic efficacy in patients with Crohn's disease. World J Gastroenterol. 2018;24(36):4093-4103). Briefly, the assay involves incubating NK-92MI cells with recombinant human IL-12 (rhIL-12) and comparing the levels of IFN-γ secreted by the cells in the presence and absence of ustekinumab. Levels of IFN-γ are quantified by enzyme-linked immunosorbent assay (ELISA) using anti-IFN-γ antibodies (see, for example, Jayanthi S, et al. Modulation of interleukin-12 activity in the presence of heparin. Sci Rep. 2017;7(1):5360).

[0224] Methods for determining oligosaccharide composition Oligosaccharide composition by HPLC The N-linked oligosaccharide composition of ustekinumab is measured by normal phase anion exchange HPLC method with fluorescence detection using an Agilent 1100 / 1200 series HPLC system with Chemstation / Chemstore software. To quantify the relative amount of glycans, N-linked oligosaccharides are first cleaved from the reduced and denatured test material using N-glycanase (PNGase F). The released glycans are labeled with anthranilic acid, purified by filtration using a 0.45 μm nylon filter, and analyzed by HPLC with fluorescence detection. The HPLC chromatogram serves as a map that can be used to identify and quantify the relative amount of N-linked oligosaccharides present in the sample. Glycans are identified by co-elution with oligosaccharide standards and retention times according to previous results from extensive characterization. A representative HPLC chromatogram of ustekinumab is shown in FIG. 4.

[0225] The amount of each glycan was quantified by peak area integration and expressed as a percentage of the total glycan peak area (Peak Area%). Results can be reported for G0F, G1F, G2F, total neutral glycans, and total charged glycans. Other neutral glycans is the sum of all integrated peaks between 17-35 min, excluding peaks corresponding to G0F, G1F, and G2F. Total neutral glycans is the sum of G0F, G1F, G2F, and other neutral glycans. Total charged glycans is the sum of all monosialylated glycan peaks eluting between 42-55 min and all disialylated glycan peaks eluting between 78-90 min.

[0226] A mixture of oligosaccharide standards (G0F, G2F, G2F + N-acetylneuraminic acid (NANA), and G2F + 2NANA) is analyzed in parallel as a positive control for the labeling reaction, as a standard for peak identification, and as a measure of system suitability. Oligosaccharides reconstituted from prozymes, G0F (Cat. No. GKC-004301), G2F (Cat. No. GKC-024301), SA1F (Cat. No. GKC-124301), and SA2F (Cat. No. GKC-224301), or equivalent, are used as reference standards. A method blank negative control and a pre-labeled G0F standard are also run for system suitability purposes. To obtain valid results, the following system suitability and assay (test article) acceptance criteria are applied during the oligosaccharide mapping procedure:

[0227] System suitability criteria: The resolution (USP) between the G0F and G2F peaks of the oligosaccharide standard should be ≥ 3.0. The theoretical plate number (tangent method) of the G0F peak of the oligosaccharide standard must be ≥ 5000. The total glycan peak area of ​​the ustekinumab reference standard should be ≥ 1.5 times the major glycan peak area of ​​the pre-labeled G0F. If the reference standard glycan peak is out of scale, the reference standard is re-injected with a smaller injection volume. The retention time of the G0F peak of the ustekinumab reference standard should be within 0.4 minutes of the G0F retention time of the oligosaccharide standard.

[0228] Assay pass criteria: The method blank should have no detectable peaks co-eluting with the oligosaccharide peaks assigned to ustekinumab. · The total glycan peak area of ​​each test article should be ≥ 1.5 times the major glycan peak area of ​​the pre-labeled G0F standard. If a sample glycan peak is off-scale, the sample is reinjected with a smaller injection volume along with pre-labeled G0F, oligosaccharide standards, method blanks, and normal amounts of reference standards. The retention time of the G0F peak of each test sample must be within 0.4 minutes of the retention time of the G0F peak of the oligosaccharide standard. If the assay fails to meet the acceptance criteria, the assay is invalid.

[0229] Oligosaccharide composition by IRMA The IdeS-RMA (IRMA) method allows differentiation of the major glycoforms by reduced mass spectrometry (RMA) after enzymatic treatment of immunoglobulin G (IgG) with FabRICATOR®, an IgG-degrading enzyme from Streptococcus pyogenes (IdeS), available from Genovis AB (SKU: A0-FR1-050). See also, for example, U.S. Pat. No. 7,666,582. Reduced mass spectrometry (RMA) involves reduction of the disulfide bonds of the antibody followed by intact mass analysis of the antibody heavy chain and its attached glycan moieties. Some antibodies exhibit considerable heterogeneity due to the presence of N-terminal modifications such as pyroglutamic acid formation and carboxylation. As a result, disulfide reduction and mass measurement of the heavy chain results in a complex pattern of deconvoluted peaks. Therefore, in some applications, proteolytic generation of antibody fragments is preferred over generation of light and heavy chains using reducing agents such as dithiothreitol (DTT). Traditionally, papain and pepsin are used to generate antibody fragments, all of which are laborious processes. Pepsin cleavage of IgG requires extensive optimization and is performed at low acidic pH. Papain requires an activator and can obtain both F(ab')2 and Fab depending on the reaction conditions, resulting in a heterogeneous pool of fragments. These drawbacks can be circumvented by using a new enzyme, FabRICATOR®. The cleavage procedure is very fast, simple, and importantly does not require optimization. It is performed at neutral pH and produces precise F(ab')2 and Fc fragments. No further degradation or overdigestion is observed, as commonly associated with other proteolytic enzymes such as pepsin and papain. Importantly, FabRICATOR® cleaves only the C-terminus of the disulfide bridges of the heavy chain, so no reduction step is required, resulting in intact F(ab')2 and two residual Fc fragments.

[0230] definition H: Hexose (mannose, glucose, galactose) Man5: Mannose 5 N: N-acetylhexosamine (N-acetylglucosamine and N-acetylgalactosamine) ·F: Fucose S: Sialic acid (N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA)) G0: Asialo-agalacto-defucosylated biantennary oligosaccharides G0F: Asialo-agalacto-fucosylated biantennary oligosaccharides G1: Asialo-monogalactosylated-defucosylated biantennary oligosaccharides G1F: asialo-monogalactosylated-fucosylated biantennary oligosaccharides G2: Asialo-digalactosylated, defucosylated biantennary oligosaccharides G2F: asialo-digalactosylated-fucosylated biantennary oligosaccharides GlcNAc: N-acetyl-D-glucosamine Lys: Lysine -Lys: truncated heavy chain (no C-terminal lysine residue present) +Lys: heavy chain containing C-terminal lysine ·ppm: parts per million

[0231] Device ·Thermo Scientific Q Exactive(Plus) mass spectrometer Agilent 1200 HPLC system ·Applied Biosystems POROSR2 / 10 2.1mmD×100mmL column Thermo Scientific Q ExactiveTune software Thermo Scientific Protein Deconvolution Software Analytical balance capable of measuring 0.01 mg A vortex mixer, any suitable model A water bath or heating block, any suitable model Calibrated thermometer -10~110°C, any suitable model Calibrated pipettes A microcentrifuge, any suitable model

[0232] treatment IdeS digestion of samples Sample (equivalent to 50 μg IgG). Add 1μl (50 units) of IdeS enzyme to 50μg of IgG, vortex briefly, spin down, and incubate at 37℃ for 30 minutes (stock enzyme @ 5000 units / 100μl). 1 unit of enzyme will completely digest 1μg of IgG at 37℃ for 30 minutes) ·Spin down the sample and transfer it to an LC-MS vial, then place the sample vial into the Agilent 1200 autosampler.

[0233] LC / MS method Preparation of solutions Mobile phase A (0.1% formic acid (FA) in ultrapure water) - Add 999 mL of ultrapure water to a 1 L HPLC mobile phase bottle, then add 1 mL of FA and stir. This solution can be stored at RT for 2 months. Mobile phase B (0.1% FA, 99.9% acetonitrile) - Add 999 mL of acetonitrile to a 1 L HPLC mobile phase bottle, then add 1 mL of FA and stir. This solution can be stored at RT for 2 months.

[0234] LC Method Column: Applied Biosystems POROS R2 / 10 2.1mmD×100mmL Column temperature: 60℃ Autosampler temperature: 4℃ ·Flow rate: 300μL / min ·Injection volume: 5μL Mobile phase A: 0.1% FA in ultrapure water Mobile phase B: 0.1% FA in acetonitrile

[0235] [Table 2]

[0236] MS method Scan parameters: Scan Type: Full MS Scan range: 700~3500m / z Fragmentation: In-source CID 35.0 eV Resolution: 17500 Polarity: Positive Lock mass: On, m / z 445.12002 AGC target: 3e6 ·Maximum injection time: 250

[0237] HESI Source: Sheath gas flow rate: 32 Auxiliary gas flow rate: 7 Sweep gas flow rate: 0 Spray voltage (|kV|): 4.20 Capillary temperature (℃): 280 S Lens RF Level: 55.0 Heater temperature (℃): 80

[0238] 4. Data Analysis The relative content of each detected glycan species is recorded based on the analysis of the deconvoluted mass spectrum. Figure 5 shows a representative deconvoluted mass spectrum of the IRMA analysis of ustekinumab produced in Sp2 / 0 cells. The main structures determined by IRMA analysis include, for example, G0 (H3N4), G0F (H3N4F1), G1F-GlcNAc (H4N3F1), H5N3, G1 (H4N4), H5N3F1, G1F (H4N4F1), G2 (H5N4), G2F (H5N4F1), G1FS (H4N4F1S1), H6N4F1, G2FS (H5N4F1S1), H7N4F1, H6N4F1S1, G2FS2 (H5N4F1S2). The percentage of each of these structures is monitored. The measured peak intensities represent the percentage of each structure (% of the total assigned) after normalization. Glycans whose observed masses are outside the mass deviation threshold of 100 ppm are not included in the calculation. For example, ( * G1F-GlcNAc-Lys,* H5N3-Lys, * G1-Lys, * H5N3F1-Lys, and * G2-Lys. As mentioned above, these are denoted by asterisks ( * "). Also, Man5-Lys is not always detected in the spectra due to its very low intensity, but when present it is considered and included in the calculation. The percentage of glycans is calculated as being detected in both isoforms of the Fc fragment with and without the terminal lysine. For example, the percentage G0F is (%G0F-Lys+%G0F+Lys). Structures detected in only one of the heavy chain isoforms are indicated by a double asterisk (" ** "). For example, ** G1F-GlcNAc-Lys, ** H5N3-Lys, ** H5N4-Lys, and ** H5N3F1+Lys. Most of these structures are low in abundance and either cannot be resolved from neighboring peaks of higher intensity or are below the detection power of the method.

[0239] * NOTE: Differences between the HPLC and IRMA methods (e.g., see Table 2 below) may be due to coelution of species in HPLC and possible underestimation of some sialylated species by IRMA because their intensities are very close to the limit of detection capability of the IRMA method.

[0240] [Table 3]

[0241] Capillary isoelectric focusing Capillary isoelectric focusing (cIEF) separates proteins based on their overall charge or isoelectric point (pI). This method is used to monitor the charge-based isoform distribution of ustekinumab. Unlike gel-based IEF procedures, cIEF provides a quantitative measurement of the charged species present. Furthermore, cIEF has improved resolution, sensitivity, and reproducibility compared to gel-based methods. The assay is performed on a commercially available imaging cIEF analyzer equipped with an autosampler capable of maintaining a sample temperature of ≤10.5 °C, such as the Alcott autosampler (GP Instruments, Inc.), in an ambient environment of ≤30 °C. The analysis uses an inner-wall coated silica capillary without an outer-wall polyimide coating to allow detection throughout the column. Additionally, an anolyte of dilute phosphoric acid and methylcellulose, a catholyte of sodium hydroxide and methylcellulose, and a defined mixture of wide-range (pH 3-10) and narrow-range (pH 8-10.5) ampholytes are used. In this assay, both the test article and the reference standard (RS) are pretreated with carboxypeptidase B (CPB) to remove the C-terminal lysine of the heavy chain to eliminate ambiguity caused by the presence of multiple C-terminal variants. A representative cIEF electropherogram of ustekinumab expressed in Sp2 / 0 cells is shown in Figure 6, and a representative cIEF electropherogram of ustekinumab expressed in CHO cells is shown in Figure 9.

[0242] Prior to each analysis, the autosampler temperature setpoint is set to 4°C, the autosampler is pre-cooled for at least 30 minutes, and the ambient laboratory room temperature is maintained at ≤30°C. The pretreated test articles and RS, sample vials, vial inserts, and reagents used in the assay, including purified water and parent solution containing N,N,N',N'-tetramethylethylenediamine (TEMED) (to optimize focusing in the capillary), ampholytes, pI 7.6 and 9.5 markers for internal standards, and methylcellulose (MC), are kept on ice for at least 30 minutes before starting sample preparation. Samples are prepared on ice, and the time of addition of the parent solution is recorded and exposure to TEMED is controlled. The assay must be completed within 180 minutes of this addition. The system suitability control is injected once, and the test articles and RS are injected twice according to the sequence table below (Table 3).

[0243] [Table 4]

[0244] After the sample is injected into the capillary by a syringe pump, an electric field (3 kV) is applied across the capillary for 8 min to create a pH gradient and separate the charge-based isoforms of ustekinumab according to their isoelectric points (pI). Protein isoforms in the capillary are detected by imaging the entire capillary at 280 nm and data are displayed in the form of electropherograms as a function of pI values ​​and A280. pI values ​​are assigned by comparison to internal pI standards (pI 7.6 and 9.5) using the instrument software and peak areas are determined from the electropherograms using standard data acquisition software. The average pI and percentage of average peak area from duplicate injections of all peaks above the LOD, ΔpI values ​​compared to the reference standard, and percent area peaks are reported.

[0245] Manufacturing control strategy overview In large-scale commercial production, manufacturing control strategies are deployed to maintain consistent drug substance (DS) and drug product (DP) properties of therapeutic proteins (e.g., therapeutic antibodies such as ustekinumab) with respect to oligosaccharide profile, biological activity (potency), and / or other properties of the DS and DP (see, e.g., properties identified in Tables 4 and 5). For example, glycosylation of ustekinumab is monitored as an in-process control of the formulated bulk (FB) at stage 10 of the manufacturing process by setting upper and lower specifications for the average % total neutral oligosaccharides, % total charged oligosaccharides, and % individual neutral oligosaccharide species, G0F, G1F, and G2F. As used herein, the terms "drug substance" (abbreviated as "DS") and "drug product" (abbreviated as "DP") refer to compositions for use, e.g., in clinical trials or as a commercial drug, such as a marketed drug. A DS is an active ingredient intended to provide pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure or any function of the human body. The formulated bulk (FB) produced in the manufacturing process is the drug substance (DS). A DP (also called a medicinal product, drug product, medicine, or pharmaceutical agent) is a drug used in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure or any function of the human body. A DP is a DS formulated as a medicinal product for sale and / or administration to patients.

[0246] As shown in Table 4, there are only minor differences in % monomer, % purity, and % bioactivity of ustekinumab produced in Sp2 / 0 and CHO cells. However, there are substantial differences in the cIEF profiles, mainly caused by the difference in the oligosaccharide profiles of ustekinumab produced in Sp2 / 0 and CHO cells. See also, e.g., Figures 6 and 9 for a comparison of the cIEF profiles of ustekinumab produced in Sp2 / 0 and CHO cells.

[0247] [Table 5]

[0248] Oligosaccharide profile of ustekinumab Ustekinumab is N-glycosylated at a single site on each heavy chain at asparagine 299. These N-linked oligosaccharide structures can be any of a family of biantennary oligosaccharide structures attached to proteins via the primary amines of asparagine residues, but in ustekinumab they are primarily composed of biantennary core-fucosylated species with heterogeneity of galactose and sialic acid. The major individual oligosaccharide species include, for example, "G0F", asialo, agalacto core-fucosylated biantennary glycan, "G1F", asialo, monogalacto core-fucosylated biantennary glycan, and "G2F", asialo, digalacto core-fucosylated biantennary glycan. A schematic diagram of some of the major N-linked oligosaccharide species of ustekinumab IgG is shown in Figure 7. The role of some divalent cations (e.g., Mn) in these enzymatic processes is important. 2+ and Cu 2+ The role of several enzymes in the glycosylation maturation process has also been demonstrated, including the role of

[0249] HPLC is the analytical procedure deployed to analyze the glycosylation of ustekinumab during the manufacturing process. In the HPLC analysis, the glycans are first enzymatically cleaved from the heavy chain and then labeled and detected with a fluorescent label. In this method, the uncharged peaks of G0F, G1F, and G2F and a subset of smaller neutral peaks can be distinguished. Furthermore, peaks of differentially sialylated material can also be observed (Figure 4). Another method of oligosaccharide analysis is IRMA, a reduced mass spectrometry (RMA) method using immunoglobulin G (IgG)-degrading enzyme from Streptococcus pyogenes (IdeS), which allows the differentiation of the major glycoforms after enzymatic digestion of IgG. Figure 5 shows a representative deconvoluted mass spectrum of the IRMA analysis of ustekinumab produced in Sp2 / 0 cells. In the case of ustekinumab, there is also a direct relationship between the degree of sialylation of the oligosaccharide structures and the charge heterogeneity as determined by cIEF, IRMA, or HPLC (see, e.g., Figures 4, 5, 6, 8, and 9).

[0250] Control of oligosaccharide profile Control of the oligosaccharide profile of recombinant monoclonal antibodies is important because changes in the oligosaccharide profile can have a significant impact on the biological functions of the antibody. For example, biological studies have shown that the distribution of various glycoforms in the Fc region can have a significant impact on the efficacy, stability, and effector functions of antibodies (J.Biosci.Bioeng.2014 117(5):639-644; Bio-Process Int.2011,9(6):48-53; Nat.Rev.Immunol.2010,10(5):345-352). In particular, defucosylation (J.Mol.Biol.368:767-779) and galactosylation (Biotechnol.Prog.21:1644-1652) can play a major role in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). These are two important mechanisms by which antibodies mediate target cell killing via immune function. Furthermore, high mannose levels have been shown to negatively impact efficacy by increasing antibody clearance (Glycobiology. 2011, 21(7):949-959), and sialic acid content can affect anti-inflammatory activity (Antibodies. 2013 2(3):392-414). As a result of these biological effects of altered oligosaccharide profiles, regulatory agencies need to control the glycosylation pattern of antibodies to ensure compliance with lot release specifications for a consistent, safe, and effective product.

[0251] Oligosaccharide profile – effect of expression in different cells Two commonly used rodent host cell lines for recombinant expression of antibodies are Chinese hamster ovary cells (CHO) and mouse myeloma cells (e.g., Sp2 / 0 cells). CHO cells express recombinant antibodies that can be substantially devoid of sialic acid glycans, and the glycans can be up to 99% fucosylated. In contrast, mouse myeloma cells express recombinant antibodies that can contain up to 50% sialic acid and generally have reduced fucose. These differences can have a significant impact on antibody activity in vivo, for example, it has been shown that such differences can affect the structure of the Fc portion of the molecule, thereby altering antibody effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (see, e.g., U.S. Pat. No. 8,975,040). For example, decreased ADCC activity has been observed with increased sialylated (charged) Fc glycans (Scallon et al., Mol Immunol 2007;44:1524-34), and increased ADCC activity has been reported in antibodies that are deficient in fucose (Shields et al., J Biol Chem. 2002;277:26733-26740; Shinkawa et al., J Biol Chem. 2003;278:3466-3473).

[0252] Furthermore, antibodies produced in CHO and Sp2 / 0 cells may have significant differences in the levels of two glycan epitopes, galactose-α-1,3-galactose (α-gal) and the sialylated N-glycan Neu5Gc-α-2-6-galactose (Neu5Gc). For example, it has been shown that CHO cells can express antibodies with undetectable or trace levels of α-Gal and Neu5Gc, whereas Sp2 / 0 cells can express much higher levels of the two glycan structures (Yu et al., Sci Rep. 2016 Jan 29;7:20029). In contrast, humans are genetically deficient in the genes for biosynthesis of α-gal, and the genes involved in the production of Neu5Gc are irreversibly mutated in all humans. As a result, α-Gal and Neu5Gc are not produced in humans. Furthermore, the presence of these non-human glycan epitopes on therapeutic antibodies may induce undesirable immune responses in certain human populations due to high levels of pre-existing antibodies against α-Gal and Neu5Gc. For example, anti-α-gal IgE-mediated anaphylactic reactions have been reported for cetuximab (Chung, CH et al., N Engl J Med. 2008 Mar 13;358(11):1109-17), and the presence of circulating anti-Neu5Gc antibodies has been reported to promote the clearance of cetuximab (Ghaderi et al., Nat Biotechnol. 2010 Aug;28(8):863-7).

[0253] It is also reported that ustekinumab expressed in Sp2 / 0 cells contains high levels of Neu5Gc compared to many other antibodies. Western blot analysis showed highly monospecific anti-Neu5Gc antibody preparations against Neu5Gc bound to ustekinumab, but not to ustekinumab treated with PNGase F, which removes nearly 100% of the N-glycans (Yu et al., Sci Rep. 2016 Jan 29;7:20029). Further analysis also shows that anti-Neu5Gc antibody preparations cannot bind ustekinumab with only one Neu5Gc (monosialylated in one Fc region), but can bind to antibodies with two to four Neu5Gc. It remains to be determined whether anti-Neu5Gc antibodies can bind to two Neu5Gc (monosialylated in both Fc regions) in two different Fc regions of the same antibody or only to disialylated N-glycans in one Fc region of the antibody, but it is determined that at least two FcNeu5Gc residues are required for binding to anti-Neu5Gc antibodies, regardless of their distribution.

[0254] Oligosaccharide profile of ustekinumab expressed in Sp2 / 0 and CHO cells Compiled HPLC data from multiple commercial manufacturing runs of ustekinumab showed that DS or DP produced in Sp2 / 0 cells contained 64.8% to 85.4% total neutral oligosaccharide species, 14.4% to 35.6% total charged oligosaccharide species, and 11.5% to 40.2% individual neutral oligosaccharide species G0F, 29.9% to 40.6% G1F, and 4.1% to 11.3% G2F. Additionally, the peak 3 area % of the capillary isoelectric focusing (cIEF) electropherogram of ustekinumab produced in Sp2 / 0 cells is 39.8% to 64.4%. As shown in Tables 5 and 6, based on IRMA or HPLC analysis, ustekinumab produced in CHO cells has a very different oligosaccharide profile with total neutral, total charged, and individual neutral oligosaccharide species G0F, G1F, and G2F compared to ustekinumab produced in Sp2 / 0 cells. These differences are evident in the representative HPLC chromatograms of ustekinumab produced in Sp2 / 0 and CHO cells, as shown in Figures 4 and 8, respectively. Compared to ustekinumab produced in Sp2 / 0 cells, the oligosaccharide profile of ustekinumab produced in CHO cells is shifted to very low levels of charged glycans and high levels of neutral glycans, mainly G0F. The oligosaccharide profile of ustekinumab produced in CHO cells includes >99.0% total neutral oligosaccharide species, <1.0% total charged oligosaccharide species, and individual neutral oligosaccharide species G0F>70.0%, G1F<20.0%, and G2F<5.0%. The peak 3 area% of the capillary isoelectric focusing (cIEF) electropherogram of ustekinumab produced in CHO cells is >70.0%. Furthermore, disialylated glycan species of ustekinumab produced in CHO cells were not detected by IRMA or HPLC, and monosialylated glycan species were at very low levels based on HPLC analysis and were not detectable by IRMA analysis (see, e.g., Table 5 and Figure 8).

[0255] [Table 6]

[0256] [Table 7]

[0257] conclusion Therefore, as mentioned above, manufacturing control strategies are deployed to maintain consistent drug substance (DS) and drug product (DP) properties of therapeutic proteins in terms of oligosaccharide profile and / or other properties of the DS or DP (e.g., DS and / or DP containing the therapeutic antibody ustekinumab). In particular, it is important to control the oligosaccharide profile of therapeutic antibodies, since changes in the oligosaccharide profile can have a significant impact on the biological function of the antibody. The key to controlling the oligosaccharide profile of therapeutic antibodies is the selection of the cell host for expression of the therapeutic antibody. As provided herein, ustekinumab expressed in Sp2 / 0 cells comprises an anti-IL-12 / IL-23p40 antibody having a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 10, and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 11, comprising a heavy chain variable domain amino acid sequence of SEQ ID NO: 7 and a light chain variable domain amino acid sequence of SEQ ID NO: 8, heavy chain CDR amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and light chain CDR amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and the oligosaccharide profile of the anti-IL-12 / IL-23p40 antibody comprises: total neutral oligosaccharide species of between 64.8% and 85.4%, total charged oligosaccharide species of between 14.4% and 35.6%, and individual neutral oligosaccharide species G0F between 11.5% and 40.2%, G1F between 29.9% and 40.6%, and G2F between 4.1% and 11.3%. Furthermore, the peak 3 area % of the capillary isoelectric focusing (cIEF) electropherogram of the anti-IL-12 / IL-23p40 antibody produced in Sp2 / 0 cells is 39.8% or more and 64.4% or less.

[0258] In contrast, for ustekinumab produced in CHO cells, the oligosaccharide profile is shifted towards very low levels of charged glycans and high levels of neutral glycans, mainly G0F. The oligosaccharide profile of ustekinumab produced in CHO cells comprises an anti-IL-12 / IL-23p40 antibody having a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 10 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 11, comprising a heavy chain variable domain amino acid sequence of SEQ ID NO: 7 and a light chain variable domain amino acid sequence of SEQ ID NO: 8, heavy chain CDR amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and light chain CDR amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and the oligosaccharide profile of the anti-IL-12 / IL-23p40 antibody comprises >99.0% total neutral oligosaccharide species, <1.0% total charged oligosaccharide species, and the individual neutral oligosaccharide species G0F>70.0%, G1F<20.0%, and G2F<5.0%. The peak 3 area% of the capillary isoelectric focusing (cIEF) electropherogram of ustekinumab produced in CHO cells is >70.0%. Furthermore, the disialylated glycan species of ustekinumab produced in CHO cells is not detected by IRMA or HPLC, and the monosialylated glycan species is at very low levels based on HPLC analysis and is not detectable by IRMA analysis. The reduction of sialylated species in general, and the reduction of Neu5Gc specific to ustekinumab produced in CHO cells, can provide benefits by reducing undesirable immunogenic responses when administered to humans. For example, particularly for patient populations with high levels of anti-Neu5Gc antibodies, the reduced levels of Neu5Gc can reduce clearance, resulting in a longer half-life of anti-IL-12 / 23p40 antibodies produced in CHO cells compared to anti-IL-12 / 23p40 antibodies expressed in Sp2 / 0 cells. The present invention may include the following aspects. [1] An isolated anti-IL-12 / IL-23p40 antibody comprising: (I) a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 10 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 11; (ii) a heavy chain variable domain amino acid sequence of SEQ ID NO: 7 and a light chain variable domain amino acid sequence of SEQ ID NO: 8; and (iii) an amino acid sequence selected from the group consisting of heavy chain CDR amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and light chain CDR amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, The anti-IL-12 / IL-23p40 antibody is expressed in Chinese Hamster Ovary cells (CHO cells); The anti-IL-12 / IL-23p40 antibody. [2] The anti-IL-12 / IL-23p40 antibody according to [1] above, wherein the oligosaccharide profile of the anti-IL-12 / IL-23p40 antibody comprises >99.0% total neutral oligosaccharide species and <1.0% total charged oligosaccharide species. [3] The anti-IL-12 / IL-23p40 antibody according to [2] above, wherein the oligosaccharide profile of the anti-IL-12 / IL-23p40 antibody further comprises the individual neutral oligosaccharide species G0F>70.0%, GIF<20.0%, and G2F<5.0%. [4] The anti-IL-12 / IL-23p40 antibody according to the above-mentioned [2], wherein the peak 3 area % of the anti-IL-12 / IL-23p40 antibody in a capillary isoelectric focusing (cIEF) electropherogram is >70.0%. [5] The anti-IL-12 / IL-23p40 antibody according to [2] above, wherein the anti-IL-12 / IL-23p40 antibody does not contain disialylated glycan species as measured by high performance liquid chromatography (HPLC) or reduced mass spectrometry (RMA). [6] The anti-IL-12 / IL-23p40 antibody according to any one of [1] to [5] above, which has a longer half-life than an anti-IL-12 / IL-23p40 antibody having the same amino acid heavy chain and light chain sequences expressed in Sp2 / 0 cells. [7] The anti-IL-12 / IL-23p40 antibody according to any one of [1] to [5] above, wherein the anti-IL-12 / IL-23p40 antibody comprises follow-on biologics. [8] A method for producing an anti-IL-12 / IL-23p40 antibody comprising: (i) a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 10 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 11; (ii) a heavy chain variable domain amino acid sequence of SEQ ID NO: 7 and a light chain variable domain amino acid sequence of SEQ ID NO: 8; and (iii) an amino acid sequence selected from the group consisting of heavy chain CDR amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and light chain CDR amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, comprising: a. culturing Chinese hamster ovary cells (CHO cells) with nucleotides encoding an anti-IL-12 / IL-23p40 antibody; b. expressing the anti-IL-12 / IL-23p40 antibody in the CHO cells; c. purifying the anti-IL-12 / IL-23p40 antibody; The manufacturing method comprising the steps of: [9] The method of producing the anti-IL-12 / IL-23p40 antibody according to [8] above, wherein the oligosaccharide profile of the antibody comprises >99.0% total neutral oligosaccharide species and <1.0% total charged oligosaccharide species.

[10] The method of producing the anti-IL-12 / IL-23p40 antibody according to the above-mentioned [9], wherein the oligosaccharide profile of the anti-IL-12 / IL-23p40 antibody further comprises the individual neutral oligosaccharide species G0F>70.0%, GIF<20.0%, and G2F<5.0%.

[11] The method for production described in [9] above, wherein the peak 3 area % of the anti-IL-12 / IL-23p40 antibody in a capillary isoelectric focusing (cIEF) electropherogram is >70.0%.

[12] The method of producing the anti-IL-12 / IL-23p40 antibody according to [9] above, wherein the anti-IL-12 / IL-23p40 antibody is free of disialylated glycan species as measured by high performance liquid chromatography (HPLC) or reduced mass spectrometry (RMA).

[13] The method for producing the anti-IL-12 / IL-23p40 antibody according to any one of [8] to

[12] above, wherein the anti-IL-12 / IL-23p40 antibody has a longer half-life than an anti-IL-12 / IL-23p40 antibody having the same amino acid heavy chain and light chain sequences expressed in Sp2 / 0 cells.

[14] The method according to any one of the above [8] to

[12] , wherein the anti-IL-12 / IL-23p40 antibody is a follow-on biologic.

[15] A composition comprising: (i) a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 10 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 11; (ii) a heavy chain variable domain amino acid sequence of SEQ ID NO: 7 and a light chain variable domain amino acid sequence of SEQ ID NO: 8; and (iii) an amino acid sequence selected from the group consisting of heavy chain CDR amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and light chain CDR amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, wherein the anti-IL-12 / IL-23p40 antibody is expressed in Chinese hamster ovary cells (CHO cells).

[16] The composition according to

[15] above, wherein the oligosaccharide profile of the anti-IL-12 / IL-23p40 antibody comprises >99.0% total neutral oligosaccharide species and <1.0% total charged oligosaccharide species.

[17] The composition described in

[16] above, wherein the oligosaccharide profile of the anti-IL-12 / IL-23p40 antibody further comprises the individual neutral oligosaccharide species G0F>70.0%, GIF<20.0%, and G2F<5.0%.

[18] The composition described in

[16] above, wherein the peak 3 area % of the anti-IL-12 / IL-23p40 antibody in a capillary isoelectric focusing (cIEF) electropherogram is >70.0%.

[19] The composition described in

[16] above, wherein the anti-IL-12 / IL-23p40 antibody is free of disialylated glycan species as measured by high performance liquid chromatography (HPLC).

[20] The composition according to any of the above

[15] to

[19] , wherein the anti-IL-12 / IL-23p40 antibody has a longer half-life than an anti-IL-12 / IL-23p40 antibody having the same amino acid heavy chain and light chain sequences expressed in Sp2 / 0 cells.

[21] The composition according to any one of the above

[15] to

[19] , wherein the anti-IL-12 / IL-23p40 antibody is a follow-on biologic.

Claims

1. (I) an isolated anti-IL-12 / IL-23p40 antibody comprising a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 10 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 11; (ii) a heavy chain variable domain amino acid sequence of SEQ ID NO: 7 and a light chain variable domain amino acid sequence of SEQ ID NO: 8; and (iii) an amino acid sequence selected from the group consisting of heavy chain CDR amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and light chain CDR amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, the oligosaccharide profile of said anti-IL-12 / IL-23p40 antibody comprises >99.0% total neutral oligosaccharide species and <1.0% total charged oligosaccharide species; The oligosaccharide profile of said anti-IL-12 / IL-23p40 antibody comprises individual neutral oligosaccharide species G0F>70.0%, G1F<20.0%, and G2F<5.0%.

2. The anti-IL-12 / IL-23p40 antibody of claim 1, wherein the peak 3 area % of the capillary isoelectric focusing (cIEF) electropherogram of the anti-IL-12 / IL-23p40 antibody is >70.0%.

3. The anti-IL-12 / IL-23p40 antibody of claim 1, wherein the anti-IL-12 / IL-23p40 antibody is free of disialylated glycan species as measured by high performance liquid chromatography (HPLC) or reduced mass spectrometry (RMA).

4. The anti-IL-12 / IL-23p40 antibody of any one of claims 1 to 3, wherein the anti-IL-12 / IL-23p40 antibody has a longer half-life compared to an anti-IL-12 / IL-23p40 antibody having the same amino acid heavy and light chain sequences expressed in Sp2 / 0 cells.

5. The anti-IL-12 / IL-23p40 antibody of any one of claims 1 to 3, wherein the anti-IL-12 / IL-23p40 antibody is a follow-on biologic.

6. 1. A method for producing an anti-IL-12 / IL-23p40 antibody comprising: (i) a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 10 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 11; (ii) a heavy chain variable domain amino acid sequence of SEQ ID NO: 7 and a light chain variable domain amino acid sequence of SEQ ID NO: 8; and (iii) heavy chain CDR amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and light chain CDR amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, comprising: a) culturing Chinese hamster ovary cells (CHO cells) with nucleotides encoding an anti-IL-12 / IL-23p40 antibody; b. expressing the anti-IL-12 / IL-23p40 antibody in the CHO cells; c. purifying the anti-IL-12 / IL-23p40 antibody; Including, the oligosaccharide profile of said anti-IL-12 / IL-23p40 antibody comprises >99.0% total neutral oligosaccharide species and <1.0% total charged oligosaccharide species; The method of producing said anti-IL-12 / IL-23p40 antibody, wherein the oligosaccharide profile comprises individual neutral oligosaccharide species G0F>70.0%, G1F<20.0%, and G2F<5.0%.

7. The method according to claim 6, wherein the anti-IL-12 / IL-23p40 antibody has a peak 3 area % of >70.0% in a capillary isoelectric focusing (cIEF) electropherogram.

8. The method of claim 6, wherein the anti-IL-12 / IL-23p40 antibody is free of disialylated glycan species as measured by high performance liquid chromatography (HPLC) or reduced mass spectrometry (RMA).

9. The method according to any one of claims 6 to 8, wherein the anti-IL-12 / IL-23p40 antibody has a longer half-life than an anti-IL-12 / IL-23p40 antibody having the same amino acid heavy and light chain sequences expressed in Sp2 / 0 cells.

10. The method according to any one of claims 6 to 8, wherein the anti-IL-12 / IL-23p40 antibody is a follow-on biologic.

11. 1. A pharmaceutical composition comprising an anti-IL-12 / IL-23p40 antibody comprising: (i) a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 10 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 11; (ii) a heavy chain variable domain amino acid sequence of SEQ ID NO: 7 and a light chain variable domain amino acid sequence of SEQ ID NO: 8; and (iii) an amino acid sequence selected from the group consisting of heavy chain CDR amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and light chain CDR amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, the oligosaccharide profile of said anti-IL-12 / IL-23p40 antibody comprises >99.0% total neutral oligosaccharide species and <1.0% total charged oligosaccharide species; The pharmaceutical composition, wherein the oligosaccharide profile of the anti-IL-12 / IL-23p40 antibody comprises individual neutral oligosaccharide species G0F>70.0%, G1F<20.0%, and G2F<5.0%.

12. The pharmaceutical composition according to claim 11, wherein the anti-IL-12 / IL-23p40 antibody has a peak 3 area % of >70.0% in a capillary isoelectric focusing (cIEF) electropherogram.

13. The pharmaceutical composition of claim 11, wherein the anti-IL-12 / IL-23p40 antibody is free of disialylated glycan species as measured by high performance liquid chromatography (HPLC).

14. The pharmaceutical composition of any one of claims 11 to 13, wherein the anti-IL-12 / IL-23p40 antibody has a longer half-life compared to an anti-IL-12 / IL-23p40 antibody having the same amino acid heavy and light chain sequences expressed in Sp2 / 0 cells.

15. The pharmaceutical composition according to any one of claims 11 to 13, wherein the anti-IL-12 / IL-23p40 antibody is a follow-on biologic.

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