RECOMBINANT ISOLATED ANTIGEN BINDING PROTEINS THAT BIND IL-23, PHARMACEUTICAL COMPOSITION, RECOMBINA

专利摘要:
human il-23 antigen binding proteins. antigen binding proteins that bind to human il-23 protein are provided. nucleic acids encoding the antigen binding protein, vectors, and cells encoding the same as well as the use of il-23 antigen binding proteins for diagnostic and therapeutic purposes are also provided.
公开号:BR112012009854B1
申请号:R112012009854-3
申请日:2010-10-26
公开日:2020-12-15
发明作者:Jennifer E. Towne；Janet D. Cheng；Jason C. O'neill；Yu Zhang；Yu Sun；Heather Cerne；Derek E. Piper；Randal R. Ketchem
申请人:Amgen Inc；
IPC主号:

专利说明:

Cross-referencing related orders
This application claims the benefit under 35 USC 119 (e) of United States patent application number 61 / 254,982, filed on October 26, 2009, and United States patent application number 61 / 381,287, filed on September 9, 2009 2010, which are incorporated by reference. Foundation
Interleukin 23 (IL-23), a heterodimeric cytokine, is a potent inducer of pro-inflammatory cytokines. IL-23 is related to the heterodimeric cytokine Interleukin 12 (IL-12), both sharing a common p40 subunit. In IL-23, a single p19 subunit is covalently linked to the p40 subunit. In IL-12, the single subunit is p35 (Oppmann et al, Immunity, 2000, 13: 713-715). The heterodimeric protein of IL-23 is secreted. Like IL-12, IL-23 is expressed by antigen presenting cells (such as dendritic cells and macrophages) in response to activation stimuli such as CD40 binding, Toll-like receptor agonists and pathogens. IL-23 binds to a heterodimeric receptor that comprises an IL-12Rβ1 subunit (which is shared with the IL-12 receptor) and a single receptor subunit, IL-23R. The IL-12 receptor consists of IL-12Rβ1 and IL-12Rβ2. IL-23 binds to its heterodimeric receptor and signals through JAK2 and Tyk2 to activate STAT1, 3, 4 and 5 (Parham et al., J. Immunol. 2002, 168: 5.699-708). The receptor subunits are predominantly co-expressed in activated or memory T cells and natural killer cells and also at lower levels in dendritic cells, monocytes, macrophages, microglia, keratinocytes and synovial fibroblasts. IL-23 and IL-12 act on different subsets of T cell and have substantially different roles in vivo.
IL-23 acts on activated T cells and memory and promotes the survival and expansion of the T cell subset, Th17. Th17 cells produce proinflammatory cytokines that include IL-6, IL-17, TNFα, IL-22 and GM-CSF. IL-23 also acts on natural killer cells, dendritic cells and macrophages to induce pro-inflammatory cytokine expression. Unlike IL-23, IL-12 induces differentiation of CD4 + T cells in effector cells that produce mature Th1 IFNY, and induces the function of NK cells and cytotoxic T cells by stimulating IFNY production. It was previously believed that IL-12-directed Th1 cells were the subset of pathogenic T cell in several autoimmune diseases, however, more recent animal studies in models of inflammatory bowel disease, psoriasis, inflammatory arthritis and multiple sclerosis, in which the individual contributions of IL-12 versus IL-23 were assessed, established that IL-23, and not IL-12, is the key controller in autoimmune / inflammatory disease (Ahern et al., Immun. Rev. 2008 226: 147-159 ; Cua et al., Nature 2003 421: 744-748; Yago et al, Arthritis Res and Ther. 2007 9 (5): R96). IL-12 is believed to play a critical role in the development of innate and adaptive protective immune responses to various intracellular pathogens and viruses and in tumor immune surveillance. See Kastelein, et al., Annual Review of Immunology, 2007, 25: 221-42; Liu, et al., Rheumatology, 2007, 46 (8): 1266-73; Bowman et al., Current Opinion in Infectious Diseases, 2006 19: 245-52; Fieschi and Casanova, Eur. J. Immunol. 2003 33: 1.461-4; Meeran et al, Mol. Cancer Ther. 2006 5: 825-32; Langowski et al., Nature 2006 442: 461-5. As such, specific inhibition of IL-23 (sparing IL-12 or the shared p40 subunit) should have a potentially superior safety profile compared to double inhibition of IL-12 and IL-23.
Therefore, the use of specific IL-23 antagonists that inhibit human IL-23 (such as antibodies that bind at least the single p19 subunit or bind both the p19 subunit and the p40 subunit of IL-23) that spares IL-12 should provide efficacy equal to or greater than that of IL-12 antagonists or p40 antagonists without the potential risks associated with IL-12 inhibition. Murine, humanized and phage-presenting antibodies selected for inhibition of recombinant IL-23 have been described; see, for example, US Patent 7,491,391, WIPO Publications WO1999 / 05280, WO2007 / 0244846, WO2007 / 027714, WO 2007/076524, WO2007 / 147019, WO2008 / 103473, WO 2008/103432, WO2009 / 043933 and WO2009 / 082624 . However, there is a need for fully human therapeutic agents that are capable of inhibiting native human IL-23. Such therapies are highly target specific, particularly in vivo. Complete inhibition of the target in vivo may result in formulations with lower, less frequent doses and / or more effective dosage which, in turn, results in reduced cost and increased efficiency. The present invention provides such IL-23 antagonists. summary
Antigen binding proteins that bind IL-23, particularly native human IL-23, are provided. Human IL-23 antigen binding proteins can reduce, inhibit, interfere with, and / or modulate at least one of the biological responses related to IL-23, and, as such, are useful in improving the effects of diseases or disorders related to IL-23. IL-23 antigen-binding proteins can be used, for example, to reduce, inhibit, interfere with and / or modulate IL-23 signaling, IL-23 activation of Th17 cells, IL-23 activation of NK cells, or inducing pro-inflammatory cytokine production.
Expression systems, including cell lines, are also provided for the production of IL-23 antigen binding proteins and methods of diagnosing and treating diseases related to human IL-23.
Some of the IL-23 binding antigen binding proteins that are provided comprise at least one heavy chain variable region comprising a CDRH1, a CDRH2 and a CDRH3 selected from the group consisting of a CDRH1 that differs by no more than a substitution amino acid, insertion or deletion of a CDRH1 as shown in Table 3; a CDRH2 that differs by no more than three, two or one amino acid substitutions, insertions and / or deletions from a CDRH2 as shown in Table 3; a CDRH3 that differs by no more than three, two or one amino acid substitutions, insertions and / or deletions from a CDRH3 as shown in Table 3; and comprising at least one light chain variable region comprising a CDRL1, a CDRL2 and a CDRL3 selected from the group consisting of: a CDRL1 that differs by no more than three, two or one amino acid substitutions, insertions and / or deletions a CDRL1 as shown in Table 3; a CDRL2 that differs in no more than an amino acid substitution, insertion or deletion from a CDRL2 as shown in Table 3; a CDRL3 that differs in no more than an amino acid substitution, insertion or deletion of a CDRL3 as shown in Table 3. In one embodiment, isolated antigen binding protein is provided comprising: a CDRH1 selected from the group consisting of Id. of Seq. No.: 91, 94, 97, 100 and 103; a CDRH2 selected from the group consisting of Seq Id. No.: 92, 95, 98, 101, 104, 107 and 110; a CDRH3 selected from the group consisting of Seq Id. No.: 93, 96, 99, 102 and 105; a CDRL1 selected from the group consisting of Seq Id. No. 62, 65, 68, 71 and 74; a CDRL2 selected from the group consisting of Seq Id. No. 63, 66, 69, 72, 75 and 78; and a CDRL3 selected from the group consisting of Seq Id. No. 64, 67, 70 and 73. In another embodiment, an isolated antigen binding protein is provided which comprises: a CDRH1 selected from the group consisting of Seq. No.: 91, 106, 109, 112 and 115; a CDRH2 selected from the group consisting of Seq Id. No. 113, 116, 118, 120, 121 and 122; a CDRH3 selected from the group consisting of Seq Id. No. 108, 111, 114, 117 and 119; a CDRL1 selected from the group consisting of Seq Id. No.: 77, 80, 83, 85, 86, 87, 88, 89 and 90; a CDRL2 is Seq. No.: 81; and a CDRL3 selected from the group consisting of Seq Id. No. 76, 79, 82 and 84. In another embodiment, an isolated antigen binding protein is provided that comprises at least one heavy chain variable region and at least one light chain variable region. In yet another embodiment, an isolated antigen-binding protein as described above is provided that comprises at least two heavy chain variable regions and at least two light chain variable regions. In yet another embodiment, an isolated antigen-binding protein is provided in which the antigen-binding protein is attached to a labeling group.
Also provided are isolated antigen binding proteins that bind IL-23 selected from the group consisting of: a) an antigen binding protein that has CDRH1 of Seq. No. 129, Seq. No. 132, Seq. No. 136, and CDRL1 of Seq Id. No.: 123, CDRL2 of Seq Id. No. 81, and CDRL3 of Seq Id. N °: 76; b) an antigen binding protein that has CDRH1 of Seq. No. 131, Seq. No. 134, Seq. No.: 137 and CDRL1 of Seq Id. No. 124, Seq Id. CDRL2. No.: 126 and CDRL3 of Seq Id. No.: 128; c) an antigen binding protein that has CDRH1 of Seq. No. 130, Seq. No. 133, Seq Id. CDRH3. No.: 99 and CDRL1 of Seq Id. No. 68, Seq Id. CDRL2. No. 69, and CDRL3 of Seq Id. No. 67; and d) an antigen binding protein that has CDRH1 Id. of Seq. No. 91, CDRH2 Seq. No. 135, CDRH3 Seq. No.: 138 and CDRL1 Seq. No. 125, CDRL2 Seq. No. 127 and CDRL3 Seq. N °: 64.
Also provided are isolated antigen binding proteins that bind IL-23 that comprise at least one heavy chain variable region and at least one light chain variable region, selected from the group consisting of: a heavy chain variable region comprising residues amino acid 31-35, 50-65 and 99-113 of Seq. No.: 31; and a light chain variable region comprising amino acid residues 23-36, 52-58 and 91-101 of Seq. No.: 1; a heavy chain variable region comprising amino acid residues 31-35, 50-65 and 99-110 of Seq. No. 34 and heavy chain variable region comprising amino acid residues 31-35, 50-66 and 99-110 of Seq. No.: 36; and a light chain variable region comprising amino acid residues 23-36, 52-62 and 97-105 of Seq. No.: 4; a heavy chain variable region comprising amino acid residues 31-35, 50-66 and 99-114 of Seq. No. 38; and a light chain variable region comprising amino acid residues 23-34, 50-61 and 94-106 of Seq. No.: 7; a heavy chain variable region comprising amino acid residues 31-35, 50-66 and 99-114 of Seq. No.: 40; and a light chain variable region comprising amino acid residues 24-34, 50-56 and 94-106 of Seq. No.: 9; a heavy chain variable region comprising amino acid residues 31-35, 50-66 and 99-114 of Seq. No.: 42; and a light chain variable region comprising amino acid residues 23-34, 50-61 and 94-106 of Seq. No.: 11; a heavy chain variable region comprising amino acid residues 31-35, 50-65 and 98-107 of Seq. No. 44; and a light chain variable region comprising amino acid residues 24-34, 50-56 and 89-97 of Seq. No.: 13; a heavy chain variable region comprising amino acid residues 31-37, 52-67 and 100-109 of Seq. No. 46 or Seq. No.: 153; and a light chain variable region comprising amino acid residues 24-34, 50-56 and 89-97 of Seq. N °: 15; a heavy chain variable region comprising amino acid residues 31-37, 52-67 and 100-109 of Seq. No. 48; and a light chain variable region comprising amino acid residues 24-34, 50-56 and 89-97 of Seq. N °: 17; a heavy chain variable region comprising amino acid residues 31-37, 52-67 and 101-109 of Seq. No. 50; and a light chain variable region comprising amino acid residues 24-34, 50-56 and 89-97 of Seq. N °: 19; a heavy chain variable region comprising amino acid residues 31-35, 50-65 and 98-107 of Seq. No. 52; and a light chain variable region comprising amino acid residues 24-34, 50-56 and 98-107 of Seq. No.: 21; a heavy chain variable region comprising amino acid residues 31-37, 52-67 and 100-109 of Seq. No. 54; and a light chain variable region comprising amino acid residues 24-34, 50-56 and 89-97 of Seq. No.: 23; a heavy chain variable region comprising amino acid residues 31-37, 52-67 and 100-109 of Seq. No.: 56; and a light chain variable region comprising amino acid residues 24-34, 50-56 and 89-97 of Seq. No.: 25; and a heavy chain variable region comprising amino acid residues 3137, 52-57 and 100-109 of Seq. No. 58; and a light chain variable region comprising amino acid residues 24-34, 500-56 and 89-97 of Seq. N °: 27.
An isolated antigen-binding protein that binds IL-23 comprising a heavy chain variable region and a light chain variable region is provided herein, where the heavy chain variable region sequence differs by no more than 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions, additions and / or deletions of a heavy chain variable region sequence as shown in Table 2; and in which a light chain variable region sequence differs by no more than 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions, additions and / or deletions of a light chain variable region sequence as shown in Table 1.
Also provided is an isolated antigen-binding protein that binds IL-23 selected from the group consisting of: a) a heavy chain variable region of Seq. No. 140 and a variable region of light chain of Seq. No.: 30; b) a heavy chain variable region of Seq Id. N °: 141 and a variable region of light chain of Seq Id. No.: 61; c) a heavy chain variable region of Seq Id. No. 142 and a variable region of light chain of Seq. No.: 4; and d) a heavy chain variable region of Seq Id. No. 143 and a variable region of Seq Id. N °: 139.
An isolated antigen binding protein is also provided which comprises a heavy chain variable region comprising an amino acid sequence that has at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to Seq. N °: 31, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56 and 58; and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to the Id. of Seq. N °: 1, 4, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27. Another modality is an isolated antigen binding protein that comprises a heavy chain variable region selected from the group which consists of Seq. N °: 44, 46, 48, 50, 52, 54, 56, 58 and 153, and a light chain variable region selected from the group consisting of Seq. No. 13, 15, 17, 19, 21, 23, 25 and 27. In yet another embodiment it is an isolated antigen binding protein comprising a heavy chain variable region selected from the group consisting of Seq Id. N °: 31, 34, 36, 38, 40 and 42, and a variable region of light chain selected from the group consisting of Seq. N °: 1, 4, 7, 9 and 11.
An isolated antigen-binding protein that binds IL-23 is also provided which comprises a heavy chain variable region and a light chain variable region selected from the group consisting of: a) a heavy chain variable region of Seq. . N °: 31 and a light chain variable region of Seq Id. No.: 1; b) a heavy chain variable region of Seq Id. No. 34 or 36 and a light chain variable region of Seq. No.: 4; c) a heavy chain variable region of Seq Id. No. 38 and a variable region of light chain of Seq. No.: 7; d) a heavy chain variable region of Seq Id. No. 40 and a variable region of light chain of Seq. No.: 9; e) a heavy chain variable region of Seq Id. No. 42 and a variable region of light chain of Seq. No.: 11; f) a heavy chain variable region of Seq Id. No. 44 and a variable region of light chain of Seq Id. No.: 13; g) a heavy chain variable region of Seq Id. No. 46 or Seq. No. 153 and a variable region of light chain of Seq Id. N °: 15; h) a heavy chain variable region of Seq Id. No. 48 and a variable region of light chain of Seq Id. N °: 17; i) a heavy chain variable region of Seq Id. No. 50 and a variable region of light chain of Seq. N °: 19; j) a heavy chain variable region of Seq Id. N °: 52 and a light chain variable region of Seq Id. No.: 21; k) a heavy chain variable region of Seq Id. N °: 54 and a light chain variable region of Seq Id. No.: 23; I) a heavy chain variable region of Seq Id. N °: 56 and a light chain variable region of Seq Id. No.: 25; and m) a heavy chain variable region of Seq Id. No. 58 and a variable region of light chain of Seq. N °: 27.
An isolated antigen binding protein that binds human IL-23 is also provided, wherein the covered patch formed when the antigen binding protein is bound to human IL-23 comprises residue contacts 30, 31, 32, 49, 50 , 52, 53, 56, 92 and 94 of Seq. N °: 15, where the waste contacts have a difference value greater than or equal to 10 A2 as determined by surface area exposed to the solvent. In one embodiment, the waste contacts comprise residues 31-35, 54, 58-60, 66, and 101105 of Seq. No. 46.
An isolated antigen binding protein that binds human IL-23 is also provided, wherein the covered patch formed when the antigen binding protein is bound to human IL-23 comprises residue contacts 31-34, 51, 52, 55 , 68, 93 and 98 of Seq. N °: 1, where the waste contacts have a difference value greater than or equal to 10 A2 as determined by surface area exposed to the solvent. In one embodiment, the waste contacts comprise residues 1, 26, 28, 31, 32, 52, 53, 59, 76, 101, 102 and 104-108 of Seq. N °: 31.
An isolated antigen-binding protein that binds human IL-23 is also provided, where, when the antigen-binding protein is bound to human IL-23, the antigen-binding protein is 5 A or less of residues 32- 35, 54, 58-60, 66 and 101-105 of Seq. No. 46 as determined by X-ray crystallography. In one embodiment, the antigen binding protein is 5 A or less of residues 31-35, 54, 56, 58-60, 66 and 101-105 of Id. of Seq. No. 46.
An isolated antigen binding protein that binds human IL-23 is also provided, where, when the antigen binding protein is bound to human IL-23, the antigen binding protein is 5 A or less of the 30- 32, 49, 52, 53, 91-94 and 96 of Seq. N °: 15, as determined by X-ray crystallography. In one embodiment, the antigen binding protein is 5 A or less of residues 30-32, 49, 50, 52, 53, 56, 91-94 and 96 of Seq. N °: 15.
An isolated antigen binding protein that binds human IL-23 is also provided, where, when the antigen binding protein is bound to human IL-23, the antigen binding protein is 5 A or less of residues 26- 28, 31, 53, 59, 102 and 104-108 of Seq. No. 31, as determined by X-ray crystallography. In one embodiment, the antigen binding protein is 5 A or less from residues 1, 26-28, 30-32, 52, 53, 59, 100, and 102 -108 of Seq Id. N °: 31.
An isolated antigen binding protein that binds human IL-23 is also provided, wherein, when said antigen binding protein is bound to human IL-23, said antigen binding protein is 5 A or less of the residues 31-34, 51, 52, 55, 68 and 93 of Seq. No.: 1 as determined by X-ray crystallography. In one embodiment, the antigen binding protein is 5 A or less of residues 29, 31-34, 51, 52, 55, 68, 93 and 100 of Id. Seq. N °: 1.
An isolated antigen-binding protein as described above is also provided, wherein the antigen-binding protein is an antibody. In one embodiment, an isolated antigen binding protein is provided in which the antibody is a monoclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment thereof. In another embodiment, an isolated antigen binding protein is provided in which the antibody fragment is a Fab fragment, a Fab 'fragment, an F (ab') 2 'fragment, an Fv fragment, a diabody, or a molecule of single chain antibody. In yet another embodiment, an isolated antigen-binding protein is provided in which the antigen-binding protein is a human antibody. In yet another embodiment, an isolated antigen binding protein is provided in which the antigen binding protein is a monoclonal antibody. In another embodiment, an isolated antigen-binding protein is provided in which the antigen-binding protein is of the type IgG1, IgG2, IgG3 or IgG4. In yet another embodiment, an isolated antigen-binding protein is provided in which the antigen-binding protein is of the IgG1 or IgG2 type.
An isolated nucleic acid molecule encoding an antigen-binding protein, as described above, is also provided. In one embodiment, an isolated nucleic acid molecule is provided in which at least one variable region of the heavy chain is encoded by an isolated nucleic acid molecule selected from the group consisting of Seq Id. Nos: 32, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59 and 152, and at least one light chain variable region is encoded by an isolated nucleic acid molecule selected from the group consisting of Seq Id. Nos: 2, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28. In another embodiment, a nucleic acid molecule is provided in which the nucleic acid molecule is operationally linked to a control sequence. In another embodiment, a vector is provided that comprises a nucleic acid molecule as described above. In yet another embodiment, a host cell is provided which comprises the nucleic acid molecule as described above. In another embodiment, a host cell is provided which comprises the vector described above. In yet another embodiment, an isolated polynucleotide sufficient for use as a hybridization marker, PCR primer or sequencing primer which is a fragment of the nucleic acid molecule as described above or its complement is provided.
Also provided is a method of making the antigen-binding protein as described above, which comprises the step of preparing said antigen-binding protein from a host cell that secretes said antigen-binding protein.
An isolated antigen binding protein that binds human IL-23 is also provided, in which the covered patch formed when the antigen binding protein is bound to human IL-23 comprises a residue contact on residues 46-58, a contact residue at residues 112-120, and a residue contact at residues 155-163 of the p19 subunit of human IL-23 as described in Id. of Seq. N °: 145, where the residue contact has a difference value greater than or equal to 10A2 as determined by surface area exposed to the solvent. A modality is provided in which the covered patch formed when the antigen binding protein is bound to human IL-23 comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or thirteen residue contacts in residues 46-58, one, two, three, four, five, six, seven, eight, nine or ten residue contacts in residues 112-120, and one, two, three, four, five, six, seven, eight or nine residue contacts at residues 155-163 of the human IL-23 p19 subunit as described in Seq Id. No. 145. A modality is provided in which the covered patch formed when the antigen binding protein binds to human IL-23 comprises a residue contact at residues 121-125 of the human IL-23 p40 subunit as described in Seq. No. 147. A modality is provided in which the covered patch formed when the antigen binding protein is bound to human IL-23 comprises one, two, three, four or five residue contacts in residues 121-125 of the p40 subunit of human IL-23 as described in Seq. No. 147. A modality is provided in which the covered patch formed when the antigen-binding protein is bound to human IL-23 comprises residue contacts 46, 47, 49, 50, 53, 112-116, 118, 120 , 155, 156, 159, 160 and 163 of Seq. No. 145. Another embodiment is provided in which the covered patch formed when the antigen-binding protein is bound to human IL-23 comprises residue contacts 46, 47, 49, 50, 53, 112-118, 120, 155 , 156, 159, 160 and 163 of Seq. No. 145. Another embodiment is provided in which the covered patch formed when the antigen-binding protein is bound to human IL-23 comprises residues 46, 47, 49, 50, 53-55, 57, 58, 112-116 , 118-120, 155, 156, 159, 160, 162 and 163 of Seq. No. 145. A related embodiment is provided in which the covered patch formed when the antigen-binding protein is bound to human IL-23 comprises contact of residue 122 of the human IL-23 p40 subunit as described in Seq Id. . No. 147. Another related embodiment is provided in which the covered patch formed when the antigen-binding protein is bound to human IL-23 comprises residue contacts 122 and 124 of the human IL-23 p40 subunit as described in Id. of Seq. No. 147. Another related modality is provided in which the covered patch formed when the antigen-binding protein is bound to human IL-23 comprises contact residue 121-123 and 125 of the human IL-23 p40 subunit as described in the Seq. No. 147. An additional embodiment provided in which the covered patch formed when the antigen binding protein is bound to human IL-23 comprises contact residue 121-123, 125 and 283 of the human IL-23 p40 subunit as described in the Seq. N °: 147.
An isolated antigen-binding protein is also provided that binds human IL-23, wherein, when said antigen-binding protein is bound to human IL-23, said antigen-binding protein is 5A or less than one residue at residues 46-58, a residue at residues 112123, and a residue at residues 155-163 of the p19 subunit of human IL-23 as described in Seq Id. No. 145, as determined by X-ray crystallography. In one embodiment, when the antigen binding protein is bound to human IL-23, the antigen binding protein is 5A or less than one, two, three, four , five, six, seven, eight, nine, ten, eleven, twelve or thirteen residues in residues 46-58, one, two, three, four, five, six, seven, eight, nine or ten residues in residues 112 -123, and one, two, three, four, five, six, seven, eight, or nine residues in residues 155-163 of the p19 subunit of human IL-23 as described in Seq Id. No. 145. In another embodiment, when the antigen-binding protein is bound to human IL-23, the antigen-binding protein is 5A or less of residues 46-50, 113-116, 120, 156, 159, 160 and 163 of Seq. No. 145. In another embodiment, when the antigen binding protein is bound to human IL-23, the antigen binding protein is 5A or less of residues 46-50, 112-120, 156, 159, 160 and 163 of Seq. No. 145. In a related embodiment, when the antigen binding protein is bound to human IL-23, the antigen binding protein is 5A or less of residues 46-50, 53, 112-120, 156, 159 , 160 and 163 of Seq. No. 145. In another embodiment, when the antigen-binding protein is bound to human IL-23, the antigen-binding protein is 5A or less of residues 46-50, 53-55, 58, 113116, 120, 121, 156, 159, 160, 162 and 163 of Seq. No. 145. In a related embodiment, when the antigen binding protein is bound to human IL-23, the antigen binding protein is 5A or less of residues 46-51, 53-55, 57, 58, 112 -116, 118-121, 123, 155, 156, 159, 160, 162 and 163 of Seq. No. 145. In an additional embodiment, when the antigen binding protein is bound to human IL-23, the antigen binding protein is 5A or less than one residue in residues 121-125, of the IL-p40 subunit 23 human as described in Seq Id. No. 147, as determined by X-ray crystallography. In a related embodiment, when the antigen binding protein is bound to human IL-23, said antigen binding protein is 5 A or less from residues 122 and 124 of Seq. No. 147. In another embodiment, when the antigen-binding protein is bound to human IL-23, the antigen-binding protein is 5 A or less from residues 121-123 and 125 of Seq. N °: 147.
An isolated antigen-binding protein as described above is also provided, wherein the antigen-binding protein has at least one property selected from the group consisting of: a) reduction of human IL-23 activity; b) reduction in the production of a pro-inflammatory cytokine; c) binding to human IL-23 with a KD <5x10-8 M; d) that has a Koff rate <5x10-6 1 / s; and e) which has an IC 50 <400 M.
A pharmaceutical composition is provided which comprises at least one antigen binding protein as described above and a pharmaceutically acceptable excipient. In one embodiment, a pharmaceutical composition is provided that also comprises a labeling group or an effector group. In yet another embodiment, a pharmaceutical composition is provided in which the labeling group is selected from the group consisting of isotopic labels, magnetic labels, active redox moieties, optical dyes, biotinylated groups and predetermined polypeptide epitopes recognized by a secondary reporter. In yet another embodiment, a pharmaceutical composition is provided in which the effector group is selected from the group consisting of a radioisotope, radionuclide, a toxin, a therapeutic group and a chemotherapeutic group.
Also provided is a method for the treatment or prevention of a condition associated with IL-23 in a patient, which comprises administering to a patient in need of an effective amount of at least one isolated antigen binding protein as described above. In one embodiment, a method is provided in which the condition is selected from the group consisting of an inflammatory disorder, a rheumatic disorder, an autoimmune disorder, an oncological disorder and a gastrointestinal disorder. In yet another modality, a method is provided in which the condition is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, cancer, psoriasis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, disseminated lupus erythematosus, psoriatic arthritis, autoimmune myocarditis ; type 1 diabetes and ankylosing spondylitis. In yet another embodiment, a method is provided in which the isolated antigen binding protein is administered alone or as a combination therapy.
Also provided is a method of reducing IL-23 activity in a patient which comprises administering an effective amount of at least one antigen-binding protein as described above. In one embodiment, a method of reducing IL-23 activity is provided, in which said IL-23 activity is the induction of the production of a pro-inflammatory cytokine. Brief description of the drawings
Figure 1A: Results of reporter assay for STATluciferase using recombinant human IL-23. All antibodies completely inhibited recombinant human IL-23
Figure 1B: Results of reporter assay for STATluciferase using native human IL-23. Only half of those antibodies that completely inhibited recombinant human IL-23 were able to completely inhibit native human IL-23 Detailed Description
The present invention provides compositions, kits and methods related to IL-23 antigen binding proteins, which include molecules that antagonize IL-23, such as anti-IL-23 antibodies, antibody fragments, and antibody derivatives, for example, anti-IL-23 antagonist antibodies, antibody fragments, or antibody derivatives. Polynucleotides, and derivatives and fragments thereof, are also provided, which comprise a nucleic acid sequence that encodes all or a portion of a polypeptide that binds IL-23, for example, a polynucleotide that encodes all or part of an anti- IL-23, antibody fragment, or antibody derivative, plasmids and vectors comprising such nucleic acids, and cells or cell lines comprising such polynucleotides and / or vectors and plasmids. The methods provided include, for example, methods of making, identifying, or isolating IL-23 antigen binding proteins, such as anti-IL-23 antibodies, methods of determining whether the molecule binds to IL-23, methods for determining whether the molecule antagonizes IL-23, methods of making compositions, such as pharmaceutical compositions, that comprise an IL-23 antigen binding protein, and methods for administering an IL-23 antigen binding protein to an individual, for example, methods for treating an IL-23 mediated condition, and for antagonizing biological activity of IL-23, in vivo or in vitro.
Unless otherwise defined in this specification, the scientific and technical terms used in connection with the present invention must have the meanings that are commonly understood by those of ordinary skill in the art. In addition, unless otherwise required by context, singular terms must include pluralities and plural terms must include the singular. Generally, nomenclatures and technical terms used with cell and tissue culture, molecular biology, immunology, microbiology, genetics and chemistry of protein and nucleic acid and hybridization described here are those well known and commonly used in the art. The methods and techniques of the present invention are generally carried out in accordance with conventional methods well known in the art and as described in various general and more specific references which are cited and discussed throughout the present specification, unless otherwise indicated. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990). The enzymatic reactions and purification techniques are performed according to the manufacturer's specifications, as commonly performed in the technique or as described herein. The terminology used and the laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and patient release and treatment.
All patents and other identified publications are expressly incorporated herein by reference in their entirety for the purpose of description and disclosure, for example, of the methodologies described in such publications that should be used in conjunction with information described herein.
The polynucleotide and protein sequences of the human IL-23 p19 subunit (Seq. Nos: 144 and 145), the shared p40 subunit (Seq. Nos: 146 and 147), the hererodimeric subunits of Human IL-23 IL-12Rβ1 (Seq. Nos: 150 and 151) and IL-23R (Seq. Nos: 148 and 149) are known in the art; see, for example, Nos. GenBank Access Code AB030000; M65272, NM_005535, NM_144701, as are those of other species of mammal. Recombinant IL-23 and IL-23 receptor proteins that include single chain and Fc proteins, as well as cells that express the IL-23 receptor, have been described or are available from commercial sources (see, for example, Oppmann et al ., Immunity, 2000, 13: 713-715; R&D Systems, Minneapolis, Minnesota; United States Biological, Swampscott, Massachusetts; WIPO Publication No. WO 2007/076524). Native human IL-23 can be obtained from human cells as dendritic cells using methods known in the art that include those described herein.
IL-23 is a heterodimeric cytokine comprising a single p19 subunit that is covalently linked to a shared p40 subunit. The p19 subunit comprises four a-propellers, "A", "B", "C" and "D" in an "up-down-down" motif connected by three intra-propeller loops between the propellers A and B, between propellers B and C and between propellers C and D; see Oppmann et al., Immunity, 2000, 13: 713-715 and Beyer, et al., J Mol Biol, 2008. 382 (4): 942-55. Helices A and D of 4 helical beam cytokines are believed to be involved with receptor binding. The p40 subunit comprises three beta sheet sandwich domains, D1, D2 and D3 (Lupardus and Garcia, J. Mol. Biol., 2008, 382: 931-941).
The term "polynucleotide" includes single-stranded and double-stranded nucleic acids and includes genomic DNA, RNA, mRNA, cDNA, or synthetic origin, or any combination thereof that is not associated with sequences normally found in nature. Isolated polynucleotides comprising specified sequences may include, in addition to the specified sequences, coding sequences for ten or even twenty other proteins or portions thereof, or may include operably linked regulatory sequences that control the expression of the coding region of the aforementioned nucleic acid sequences, and / or may include vector strings. The nucleotides that comprise the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of each type of nucleotide. The modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2 ', 3'-dideoxyribose, and internucleotide binding modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodisellenoate, phosphoranilioate, phosphoraniladate and phosphoramidate.
The term "oligonucleotide" means a polynucleotide that comprises 100 or less nucleotides. In some embodiments, the oligonucleotides are 10 to 60 bases in length. In other embodiments, the oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 nucleotides in length. Oligonucleotides can be single-stranded or double-stranded, for example, for use in the construction of a mutant gene. Oligonucleotides can be sense or antisense oligonucleotides. An oligonucleotide can include a detectable label, such as a radiolabel, a fluorescent label, a hapten or an antigenic label, for detection assays. Oligonucleotides can be used, for example, as PCR primers, cloning primers or hybridization markers.
The term "polypeptide" or "protein" means a macromolecule that has a sequence of amino acids from a native protein, that is, a protein produced by a naturally occurring and non-recombinant cell; or it is produced by a genetically constructed or recombinant cell, and comprises molecules that have a sequence of amino acids from the native protein, or molecules that have one or more deletions, insertions, and / or substitutions of the amino acid residues of the native sequence. The term also includes amino acid polymers in which one or more amino acids are chemical analogues of a corresponding naturally occurring amino acid and polymers. The terms "polypeptide" and "protein" encompass IL-23 antigen binding proteins (such as antibodies) and sequences that have one or more deletions, additions, and / or substitutions for the amino acid residues of the antigen binding protein sequence . The term "polypeptide fragment" refers to a polypeptide that has an amino terminal deletion, a carboxyl terminal deletion, and / or an internal deletion when compared to native full-length protein. Such fragments can also contain modified amino acids when compared to the native protein. In certain embodiments, fragments are about five to 500 amino acids long. For example, fragments can be at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 70, 100, 110, 150 , 200, 250, 300, 350, 400 or 450 amino acids in length. Useful polypeptide fragments include immunologically functional fragments of antibodies, which include binding domains. In the case of an IL-23 antigen binding protein, such as an antibody, useful fragments include, without limitation, one or more regions of CDR, a variable domain of a heavy chain or light chain, a portion of a chain of antibody, a portion of a variable region that includes less than three CDRs, and others.
"Amino acid" includes its normal meaning in the art. The twenty naturally occurring amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis, 2nd Edition, (E. S. Golub and D. R. Gren, eds.), Sinauer Associates: Sunderland, Mass. (1991). Stereoisomers (eg, D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as [alpha] -, [alpha] - disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids can also be suitable components for polypeptides. Examples of unconventional amino acids include: 4-hydroxyproline, [gamma] - carboxyglutamate, [epsilon] -N, N, N-trimethyl lysine, [epsilon] -N-acetyl lysine, O-phosphoserine, N-acetylserine, N-formylmethionine , 3-methylhistidine, 5-hydroxylysine, [sigma] -N-methylaryginine, and other similar amino acids and imino acids (for example, 4-hydroxyproline). In the polypeptide denomination used here, the left direction is the direction of the amino terminal and the right direction is the direction of the carboxyl terminal, according to standard usage and convention.
The term "isolated protein" refers to a protein, such as an antigen-binding protein (an example of which may be an antibody), which is purified from proteins or polypeptides or other contaminants that may interfere with its use or therapeutic research , diagnosis, prophylactic. As used herein, "substantially pure" means that the described species of the molecule is the predominant species present, that is, on a molar basis it is more abundant than any other individual species in the same mixture. In certain embodiments, a substantially pure molecule is a composition in which the species comprises at least 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition will comprise at least 80%, 85%, 90%, 95% or 99% of all macromolecular species present in the composition. In certain embodiments, an essentially homogeneous substance has been purified to such a degree that contaminating species cannot be detected in the composition by conventional methods of detection and thus the composition consists of a single detectable macromolecular species.
A "variant" of a polypeptide (for example, an antigen-binding protein such as an antibody) comprises an amino acid sequence in which one or more amino acid residues are inserted, deleted and / or substituted in the amino acid sequence relative to another sequence polypeptide. Variants include fusion proteins. A "derivative" of a polypeptide is a polypeptide that has been chemically modified in some way other than insertion, deletion, or substitution variants, for example, by conjugation to another chemical fraction.
The terms "naturally occurring" or "native" as used throughout the specification with biological materials such as polypeptides, nucleic acids, host cells, and others, refer to materials that are found in nature, such as native human IL-23 . In certain respects, recombinant antigen binding proteins that bind to native IL-23 are provided. In this context, a "recombinant protein" is a protein made using recombinant techniques, that is, through the expression of a recombinant nucleic acid as described herein. Methods and techniques for producing recombinant proteins are well known in the art.
The term "antibody" refers to an intact immunoglobulin of any isotype, or a fragment thereof that can compete with the intact antibody for specific binding to the target antigen, and includes, for example, chimeric, humanized, fully human, and bispecific antibodies . An antibody as such is a kind of antigen-binding protein. Unless otherwise indicated, the term "antibody" includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments and muteins thereof, examples of which are described below. An intact antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but in some cases it may include fewer chains such as naturally occurring antibodies in camelids that may comprise only heavy chains. The antibodies can be derived only from a single source, or they can be "chimeric", that is, different portions of the antibody can be derived from two different antibodies as described later below. Antigen-binding proteins, antibodies, or binding fragments can be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies.
The term "functional fragment" (or simply "fragment") of an antibody or immunoglobulin chain (heavy or light chain), as used herein, is an antigen-binding protein that comprises a portion (regardless of how that portion is obtained or synthesized) of an antibody that lacks at least some of the amino acids present in a full-length chain, but that is capable of specifically binding to an antigen. Such fragments are biologically active, as they specifically bind to the target antigen and can compete with other antigen binding proteins, which include intact antibodies, for specific binding to a given epitope. In one aspect, such a fragment will retain at least one CDR present in the full length light chain or heavy chain, and in some embodiments it will comprise a single heavy chain and / or light chain or portion thereof. These biologically active fragments can be produced by recombinant DNA techniques, or they can be produced by enzymatic or chemical cleavage of antigen binding proteins, which include intact antibodies. Fragments include, without limitation, immunologically functional fragments such as Fab, Fab ', F (ab') 2, Fv, domain antibodies and single chain antibodies, and can be derived from any mammalian source, including, without limitation, human , mouse, rat, camelid or rabbit. It is further contemplated that a functional portion of the antigen-binding proteins disclosed herein, for example, one or more CDRs, can be covalently linked to a second protein or small molecule to create a therapeutic agent directed at a particular target in the body, having bifunctional therapeutic properties, or having a prolonged serum half-life.
The term “competes” when used in the context of antigen binding proteins (for example, neutralizing antigen binding proteins or neutralizing antibodies) means competition between antigen binding proteins as determined by an assay in which the binding protein antigen (for example, antibody or immunologically functional fragment thereof) under test prevents or inhibits specific binding of a reference antigen binding protein (for example, a linker, or a reference antibody) to a common antigen (for example , an IL-23 protein or a fragment thereof). Numerous types of competitive binding assay can be used, for example: direct or indirect solid phase radioimmunoassay (RIA), direct or indirect solid phase enzyme immunoassay (EIA), sandwich competition assay (see, for example, Stahli and cols., 1983, Methods in Enzymology 92: 242-253); Direct biotin-avidin solid phase EIA (see, for example, Kirkland et al., 1986, J. Immunol. 137: 3.614-3.619) direct solid phase labeled assay, direct solid phase labeled sandwich assay (see, for example, example, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); Direct solid phase labeled RIA using the I125 label (see, for example, Morel et al., 1988, Molec. Immunol. 25: 7-15); Direct solid phase biotin-avidin EIA (see, for example, Cheung, et al., 1990, Virology 176: 546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells carrying any of these, an unlabeled test antigen binding protein and a labeled reference antigen binding protein.
Competitive inhibition is measured by determining the amount of marker attached to the solid surface or cells in the presence of the test antigen binding protein. The test antigen-binding protein is commonly present in excess. The antigen binding proteins identified by competition assay (competition antigen binding proteins) include antigen binding proteins that bind to the same epitope as the reference antigen binding proteins and antigen binding proteins that they bind to an adjacent epitope close enough to the epitope bound by the reference antigen binding protein for steric hindrance to occur. Commonly, when a competing antigen binding protein is present in excess, it will inhibit the specific binding of a reference antigen binding protein to a common antigen by at least 40%, 45%, 50%, 55%, 60 %, 65%, 70% or 75%. In some cases, binding is inhibited by at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more.
The term "epitope" or "antigenic determinant" refers to a site in an antigen to which an antigen-binding protein binds. Epitopes can be formed from contiguous amino acids or non-contiguous amino acids juxtaposed by the tertiary fold of a protein. Epitopes formed from contiguous amino acids are typically retained on display to denature solvents, while epitopes formed by tertiary fold are typically lost in treatment with denaturing solvents. Epitope determinants may include chemically active surface clusters of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics, and / or specific charge characteristics. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 amino acids in one unique spatial conformation. Epitopes can be determined using methods known in the art. IL-23 antigen binding proteins
An "antigen binding protein" as used herein means a protein that specifically binds to a specific target antigen; the antigen as provided herein is IL-23, particularly human IL-23, including native human IL-23. The antigen binding proteins as provided herein interact with at least a portion of the unique IL-23 p19 subunit, which detectably binds IL-23; but does not bind significantly to IL-12 (for example, IL-12 p40 and / or p35 subunits), thus "saving IL-12". As a consequence, the antigen binding proteins provided herein are able to alter the activity of IL-23 without the potential risks that inhibition of IL-12 or the shared p40 subunit can cause. Antigen binding proteins can alter the ability of IL-23 to interact with its receptor, for example, by altering binding to the receptor, such as by interfering with receptor association. In particular, such antigen-binding proteins totally or partially reduce, inhibit, interfere with, or modulate one or more biological activities of IL-23. Such inhibition or neutralization disrupts a biological response in the presence of the antigen binding protein compared to the response in the absence of the antigen binding protein and can be determined using assays known in the art and described herein. The antigen-binding proteins provided herein inhibit IL-23-induced proinflammatory cytokine production, for example, IL-23-induced IL-22 production in whole blood cells and IL-23-induced IFNY expression in NK and whole blood cells. The reduction in biological activity can be about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97% 98%, 99% or more.
An antigen-binding protein can comprise a portion that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that promotes the binding of the antigen-binding protein to the antigen . Examples of antigen binding proteins include antibodies, antibody fragments (e.g., an antigen binding portion of an antibody), antibody derivatives, and antibody analogs. The antigen binding protein may comprise an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives. Such scaffolds include, without limitation, antibody-derived scaffolds that comprise mutations introduced, for example, to stabilize the three-dimensional structure of the antigen binding protein, as well as completely synthetic scaffolds that comprise, for example, a biocompatible polymer. See, for example, Korndorfer et al, Proteins: Structure, Function, and Bioinformatics, (2003) Volume 53, Issue 1: 121-129; Roque et al, Biotechnol. Prog., 2004, 20: 639-654. In addition, peptide antibody mimetics ("PAMs") can be used, as well as scaffolds based on antibody mimics that use fibronectin components as a scaffold.
Certain antigen binding proteins described herein are antibodies or are derived from antibodies. Such antigen binding proteins include, without limitation, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies, antibody mimetics, chimeric antibodies, humanized antibodies, human antibodies, antibody fusions, antibody conjugates, chain antibodies single, and fragments thereof, respectively. In some cases, the antigen binding protein is an immunological fragment of an antibody (for example, a Fab, Fab ', F (ab') 2, or scFv). The various structures are also described and defined here.
Certain antigen binding proteins that are provided can comprise one or more CDRs as described herein (for example, 1, 2, 3, 4, 5, 6 or more CDRs). In some cases, the antigen binding protein comprises (a) a polypeptide structure and (b) one or more CDRs that are inserted and / or joined to the polypeptide structure. The structure of the polypeptide can take a variety of different forms. For example, it can be, or comprise, the framework of a naturally occurring antibody, or fragment or variant thereof, or it can be completely synthetic in nature. Examples of various polypeptide structures are also written below.
An antigen binding protein of the invention is said to "specifically bind" to its target antigen when the dissociation equilibrium constant (KD) is <10-8 M. The antigen binding protein specifically binds to the antigen with “High affinity” when KD is <5 x 10-9 M, and “very high affinity” when KD is <5 x 10-10 M. In one embodiment, the antigen binding protein will bind to IL- 23 human with a KD of 5 x 10-12 M, and in yet another embodiment it will bind with a KD <5 x 10-13 M. In another embodiment of the invention, the antigen binding protein has a KD <5 x 10-12 M and a Koff of about <5x10-6 1 / s. In another modality, the Koff is <5x10-71 / s.
Another aspect provides an antigen-binding protein that has a half-life of at least one day in vitro or in vivo (for example, when administered to a human). In one embodiment, the antigen-binding protein has a half-life of at least three days. In another embodiment, the antibody or portion thereof has a half-life of four days or longer. In another embodiment, the antibody or portion thereof has a half-life of eight days or longer. In another embodiment, the antibody or antigen binding portion of it is derivatized or modified so that it has a longer half-life when compared to the non-derivatized or unmodified antibody. In another embodiment, the antigen binding protein contains point mutations to increase serum half-life, as described in WIPO Publication No. WO 00/09560.
In modalities where the antigen-binding protein is used for therapeutic applications, an antigen-binding protein can reduce, inhibit, interfere with or modulate one or more biological activities of IL-23, such as inducing the production of pro-inflammatory cytokines. IL-23 has several distinct biological effects, which can be measured in several different assays on different types of cells; examples of such assays are known and are provided herein.
Some of the antigen binding proteins that are provided have the structure typically associated with naturally occurring antibodies. The structural units of these antibodies typically comprise one or more tetramers, each composed of two identical pairs of polypeptide chains, although some species of mammals also produce antibodies that have only a single heavy chain. In a typical antibody, each pair or pair includes a full-length "light" chain (in some embodiments, about 25 kDa) and a full-length "heavy" chain (in some embodiments, about 50-70 kDa). Each individual immunoglobulin chain is made up of several "immunoglobulin domains", each consisting of about 90 to 110 amino acids, and express a characteristic fold pattern. These domains are the basic units of which the antibody polypeptides are composed. The amino-terminal portion of each chain typically includes a variable region that is responsible for antigen recognition. The carboxy-terminal portion is more evolutionarily conserved than the other end of the chain, and is referred to as the "constant region" or "C region". Human light chains are generally classified as kappa and lambda light chains, each of which contains a variable region and a constant domain (CL1) .z. Heavy chains are typically classified as mu, delta, gamma, alpha or epsilon chains, and these define the antibody isotype as IgM, IgD, IgG, IgA and IgE, respectively. IgG has several subtypes, including, without limitation, IgG1, IgG2, IgG3 and IgG4. IgM subtypes include IgM and IgM2. IgA subtypes include IgA1 and IgA2. In humans, the IgA and IgD isotypes contain four heavy chains and four light chains; the IgG and IgE isotypes contain two heavy chains and two light chains; and the IgM isotype contains five heavy chains and five light chains. The heavy chain constant (CH) region typically comprises one or more domains that may be responsible for effector function. The number of heavy chain constant region domains will depend on the isotype. The IgG heavy chains, for example, each contain three CH region domains known as CH1, CH2 and CH3. The antibodies that are provided can have any of these isotypes and subtypes, for example, the IL-23 antigen binding protein is of the IgG1, IgG2 or IgG4 subtype. If an IgG4 is desired, it may also be desirable to introduce a point mutation (CPSCP-> CPPCP) in the hinge region as described in Bloom et al, 1997, Protein Science 6: 407) to alleviate a tendency to form disulfide bonds intra-chain H which can lead to heterogeneity in IgG4 antibodies. The antibodies provided here that are of one type can be changed to a different type using subclass exchange methods. See, for example, Lantto et al., 2002, Methods Mol. Biol. 178: 303-316.
In light and heavy chains of full length, the variable and constant regions are joined by a "J" region of about twelve or more amino acids, with the heavy chain also including a "D" region of about ten or more amino acids. See, for example, Fundamental Immunology, 2nd ed., C. 7 (Paul, W., ed.) 1989, New York: Raven Press. The variable regions of each light / heavy chain pair typically form the antigen binding site. Variable regions
Several of the heavy and light chain variable regions (or domains) provided here are shown in Tables 1 and 2. Each of these variable regions can be attached, for example, to the heavy and light chain constant regions described above. In addition, each of the heavy and light chain sequences thus generated can be combined to form a complete antigen-binding protein structure.
Antigen binding proteins are provided that contain at least one heavy chain variable region (VH) selected from the group consisting of VH1, VH2, VH3, VH4, VH5, VH6, VH7, VH8, VH9, VH10, VH11, VH12, VH13, VH14, VH15 and VH16, and / or at least one light chain variable region (VL) selected from the group consisting of VL1, VL2, VL3, VL4, VL5, VL6, VL7, VL8, VL9, VL10, VL11, VL12, VL13, VL14, VL15 and VL16, as shown in Tables 1 and 2 below.
Each of the heavy chain variable regions listed in Table 2 can be combined with any of the light chain variable regions shown in Table 1 to form an antigen binding protein. In some cases, the antigen binding protein includes at least one heavy chain variable region and / or a light chain variable region from those listed in Tables 1 and 2. In some cases, the antigen binding protein includes at least two different heavy chain variable regions and / or light chain variable regions from those listed in Tables 1 and 2. The various combinations of heavy chain variable regions can be combined with any of the various combinations of light chain variable regions.
In other cases, the antigen binding protein contains two identical light chain variable regions and / or two identical heavy chain variable regions. As an example, the antigen binding protein can be an immunologically functional antibody or fragment that comprises two light chain variable regions and two heavy chain variable regions in combinations of light chain variable region pairs and variable chain region pairs. heavy as listed in Tables 1 and 2. Examples of such antigen binding proteins that comprise two identical heavy chain and light chain variable regions include: Antibody A VH14 / VL14; VH9 / VL9 Antibody; VH10 / VL10 C antibody; Antibody D VH15 / VL15; Antibody E VH1 / VL1, Antibody F VH11 / VL11; VH12 / VL12 G antibody; VH13 / VL13 H antibody; Antibody I VH8 / VL8; Antibody J VH3 / VL3; VH7 / VL7 Antibody; L VH4 / VL4 antibody; Antibody M VH5 / VL5 and Ant. N VH6 / VL6.
Some antigen binding proteins that are provided comprise a heavy chain variable region and / or a light chain variable region that comprises an amino acid sequence that differs from the sequence of a heavy chain variable region and / or a variable chain region selected from Tables 1 and 2 in just 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues, where each sequence difference is independently a deletion, insertion or substitution of an amino acid. The heavy and light chain variable regions in some antigen-binding proteins comprise amino acid sequences that have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequences provided in Table 1 and 2. Still other antigen binding proteins, for example, immunologically functional antibodies or fragments, also include variant forms of the heavy chain region and / or light chain region as described herein.
The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more polynucleotides, as determined by sequence alignment and comparison. “Percentage of identity” means the percentage of identical residues between amino acids or nucleotides in the compared molecules and is calculated based on the size of the largest of the molecules being compared. TABLE 1
TABLE 2

For these calculations, gaps in alignments (if any) must be controlled by a particular mathematical model or computer program (ie, an "algorithm"). Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A. M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48: 1,073.
In calculating the percent identity, the strings being compared are aligned in a way that provides the greatest combination of strings. The computer program used to determine the identity percentage is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl Acid Res. 12: 387; Genetics Computer Group, University of Wisconsin, Madison, WI). The GAP computer algorithm is used to align the two polypeptides or polynucleotides for which the percentage of sequence identity is to be determined. The sequences are aligned to optimally match their respective amino acid or nucleotide (the "matched span", as determined by the algorithm). An open gap penalty (which is calculated as 3x the average diagonal, where the “average diagonal” is the mean of the diagonal of the comparison matrix being used; the “diagonal” is the score or number determined for each perfect amino acid combination by the particular comparison matrix) and a gap extension penalty (which is commonly 1/10 times the open gap penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 is used in conjunction with the algorithm. In certain embodiments, a standard comparison matrix (see Dayhoff et al, 1978, Atlas of Protein Sequence and Structure 5: 345-352 for the PAM 250 comparison matrix; Henikoff et al, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm.
The recommended parameters for determining the identity percentage for polypeptide or nucleotide sequences using the GAP program are as follows: Algorithm: Needleman et al, 1970, J. Mol. Biol. 48: 443453; Comparison matrix: BLOSUM 62 by Henikoff et al, 1992, supra; Gap penalty: 12 (but no penalty for end gaps), Gap length penalty: 4, Similarity threshold: 0. Certain alignment schemes to align two amino acid sequences can result in a combination of just a short region of the two sequences and that small aligned region can have very high sequence identity, although there is no significant relationship between the two full-length sequences. Therefore, the selected alignment method (GAP program) can be adjusted if so desired to result in an alignment that transposes at least 50 contiguous amino acids from the target polypeptide.
The variable heavy and light chain regions disclosed herein include consensus sequences derived from groups of related antigen binding proteins. The amino acid sequences of the variable regions of heavy and light chain were analyzed for similarities. Four groups emerged, one group that has variable regions of kappa light chain, (VH9 / VL9, VH10 / VL10, VH11 / VL11, VH13 / VL13, VH14 / VL14 and VH15 / VL15) and three groups that have variable regions of light chain lambda: lambda group 1 (VH5 / VAS, VH6 / VL6 and VH7 / VII), lambda group 2 (VH3 / VL3 and VH4 / VL4), and lambda group 3 (VH1 / VL1 and VH2 / VL2). The light chain germ lines represented include VK1 / A30 and VK1 / L19 The light chain lambda germ lines represented include VL1 / 1e, VL3 / 3p, VL5 / 5c and VL9 / 9a. The heavy chain germ lines represented include VH3 / 3-30, VH3 / 3-30.3, VH3 / 3-33, VH3 / 3-48, VH4 / 4-31 and VH4 / 4-59. As used herein, a "consensus sequence" refers to amino acid sequences that have conserved amino acids common among numerous variable sequences and amino acids that vary with given amino acid sequences. The consensus sequences can be determined using standard phylogenetic analysis of the variable regions of heavy and light chain that correspond to the IL-23 antigen binding proteins disclosed herein.
The light chain variable region consensus sequence for the kappa group is DX1QX2TQSPSSVSASVGDRVTITCRASQGX3X4SX5WX6AWYQQKPGX7APX8LLIYAAS SLQSGVPSR GSX9SGTX10FTLTISSLQPX11DFATYX12CQQANSFPFTFGPGTKVDX13K FS (SEQ ID NO:.. 30), wherein Xi is selected from R or S; X2 is selected from M or L; X3 is selected from G or V and X4 is selected from S, F or I; X5 is selected from S or G; X6 is selected from F or L; X7 is selected from K or Q; X8 is selected from K, N or S; X9 is selected from G or V; X10 is selected from D or E, X11 is selected from E or A; X12 is selected from Y or F; and X13 is selected from I, V or F.
The variable region consensus sequence for light chain lambda 1 group is QPX1 LTQPPSASASLGASVTLTCTLX2SGYS DYKVDWYQX3RPG KGPRFVMRVGTGGX4VGSKGX5G PDRFSVLGSGLNRX6LTIKNIQEEDESDYHCGADHGSGX7NFVYVFGTGTKVTVL I (SEQ ID NO:.. 61), wherein Xi is selected from V or E; X2 is selected from N or S; X3 is selected from Q or L and X4 is selected from I or T; X5 is selected from D or E; X6 is selected from Y or S; and X7 is selected from S or N.
The variable region consensus sequence for light chain lambda 3 group is QSVLTQPPSVSGAPGQRVTISCTGSSSNXiGAGYDVHWYQQX2PGTAPKLLIYGSX3NR PSGVPDRF SKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTX4RLTVL SG (SEQ ID NO:.. 139), wherein Xi is selected from T or I; X2 is selected from V or L; X3 is selected from G or N; and X4 is selected from R or K.
The consensus sequence for the heavy chain variable region for the kappa group is QVQLQESGPGLVKPSQTLSLTCTVSGGSIXiSGGYYWX2WIRQHPGKGLEWIGX31X4YS GX5X6YYNP SLK SRX7TX8SVDTSX9NQFSTAXYLTTVTGTQVTGTQVTGTQTGTQVTGTGTXTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGG logo logo dist. X2 is selected from S or T; X3 is selected from Y or H and X4 is selected from Y or H; X5 is selected from Y or N; X6 is selected from S or T; X7 is selected from V or I; X8 is selected from I or M; X9 is selected from K or Q; X10 is selected from K or S, X11 is selected from R or K; X12 is selected from D or N; and X13 is selected from H, F or Y.
The consensus sequence of the heavy chain variable region for lambda group 1 is EVQLVESGGGLVQPGGSLRLSCX1X2SGFTFSX3X4SMNWVRQAPGKGLEWVSYISSX5S STX6YX7AD SV KGRFTISRDNAKNSLYLQMNSLRDEDTAVYYXAR; X2 is selected from A or V; X3 is selected from T or S and X4 is selected from Y or F; X5 is selected from S or R; X6 is selected from R or I; X7 is selected from H, Y or I; X8 is selected from P or G; X9 is selected from W or F; X10 is selected from G or H; and X11 is selected from M or L.
The consensus sequence of the heavy chain variable region to lambda 2 is QVQLVESGGGVVQPG group RSLRLSCAASG FTFSSYX1M HWVRQAPG KGLEWX2X3VISX4DGSX5KYYAD KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARERTTLSGSYFDYWGQGTLVTVSS SV (SEQ ID NO:.. 142), wherein Xi is selected from G or A; X2 is selected from V or L; X3 is selected from A or S and X4 is selected from F or H; and X5 is selected from L or I.
The consensus sequence of the heavy chain variable region for lambda group 3 is QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNX1 YYADSV KG RFTISRDNSKNTLYLQMNSLRAEDTAVYYWARDTYYYYY is selected. Complementarity determination regions
The complementarity determination regions or "CDRs" are embedded in a framework in the variable regions of heavy and light chain in which they constitute the regions responsible for binding and recognition of the antigen. Variable domains of immunoglobulin chains of the same species, for example, generally exhibit a similar general structure; comprising relatively conserved structure regions (FR) joined by hypervariable CDR regions. An antigen-binding protein can have 1, 2, 3, 4, 5, 6 or more CDRs. The variable regions discussed above, for example, typically comprise three CDRs. The CDRs of variable regions of variable regions of heavy and light chain are typically aligned by the structure regions to form a structure that specifically binds to a target antigen (for example, IL-23). From terminal N to terminal C, variable regions of naturally occurring heavy and light chains typically fit the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The CDR and FR regions of example light chain variable domains are shown in Tables 1 and 2. It is recognized that the limits of the CDR and FR regions can vary from those indicated. Numbering systems have been developed to assign numbers to the amino acids that occupy positions in each of these domains. The complementarity determining regions and structure regions of a given antigen binding protein can be identified using these systems. Numbering systems are defined in Kabat et al, Sequences of Proteins of Immunological Interest, 5th ed., “US Dept. of Health and Human Services ”, PHS, NIH, NIH Publication No. 91-3242, 1991, or Chothia & Lesk, 1987, J. Mol. Biol. 196: 901-917; Chothia et al., 1989, Nature 342: 878-883.
Other numbering systems for amino acids in immunoglobulin chains include IMGT® (“the international ImMunoGeneTics information system”; Lefranc et al, Dev. Comp. Immunol. 2005, 29: 185-203); and AHo (Honegger and Pluckthun, J. Mol. Biol. 2001, 309 (3): 657-670). The CDRs provided herein can be used not only to define the antigen binding domain of a traditional antibody structure, but can be embedded in a variety of other polypeptide structures, as described herein.
The antigen binding proteins disclosed herein are polypeptides into which one or more CDRs can be grafted, inserted, fitted and / or joined. An antigen binding protein can have, for example, a heavy chain CDR1 (“CDRH1”), and / or a heavy chain CDR2 (“CDRH2”), and / or a heavy chain CDR3 (“CDRH3”) , and / or a light chain CDR1 (“CDRL1”), and / or a light chain CDR2 (“CDRL2”), and / or a light chain CDR3 (“CDRL3”). Some antigen-binding proteins include both a CDRH3 and a CDRL3. Specific modalities generally use combinations of CDRs that are non-repetitive, for example, antigen binding proteins are generally not made with two regions of CDRH2 in a variable region of heavy chain etc. Antigen binding proteins can comprise one or more amino acid sequences that are identical to or differ from the amino acid sequences of one or more of the CDRs shown in Table 3 in just 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues, each of which such sequence differences is independently a deletion, insertion or substitution of an amino acid. CDRs in some antigen binding proteins comprise amino acid sequences that have at least 80%, 85%, 90%, 91%, 92, 93%, 94%, 95%, 96%, 97%, 98% or 99 % sequence identity to the CDR sequences listed in Table 3. In some antigen binding proteins, the CDRs are embedded in a framework region, which orientates the CDR (s) so that the proper binding properties of the antigen of the CDR (s) are reached. Table 3 Examples of CDRH and CDRL Sequences




Here, regions of CDR1 are provided that comprise amino acid residues 23-34 of Seq. Nos: 7 and 11; amino acid residues 24-34 of Seq. Nos: 9, 13, 15, 17, 19 21, 23, 25, 27 and 29; amino acid residues 23-36 of Seq. Nos: 1, 3 and 4; amino acid residues 31-35 of Seq. Nos: 31, 33, 34, 38, 40, 44, 52 and 60; and amino acid residues 31-37 or Seq. Nos: 46, 48, 50, 54, 56 and 58.
Here, regions of CDR2 are provided that comprise amino acid residues 50-56 of Seq Id. Nos: 9, 13, 15, 17, 19, 21, 23, 25, 27 and 29; amino acid residues 50-61 of Seq. Nos: 7 and 11; amino acid residues 52-62 of Seq. No.: 4; amino acid residues 50-65 of Seq. Nos: 31, 33, 44 and 52; amino acid residues 50-66 of Seq Id. Nos: 36, 38, 40, 42 and 60; amino acid residues 52-58 of Seq. Nos: 1 and 3; and amino acid residues 52-67 of Seq. Nos: 46, 48, 50, 54, 56 and 58.
The regions of CDR3 that comprise amino acid residues 89-97 of Seq. Nos: 13, 15, 17, 19, 21, 23, 25, 27 and 29; amino acid residues 91-101 of Seq. Nos: 1 and 3; amino acid residues 94-106 of Seq. Nos: 7, 9 and 11; amino acid residues 98-107 of Seq. Nos: 44 and 52; amino acid residues 97-105 of Seq. No.: 4; amino acid residues 99-110 of Seq Id. Nos: 34 and 36; amino acid residues 99-112 of Seq. No. 112; amino acid residues 99-113 of Seq. Nos: 31 and 33; amino acid residues 99-114 of Seq. Nos: 38, 40 and 42; amino acid residues 100-109 of Seq. Nos: 46, 48, 54, 56 and 58; and amino acid residues 101-019 of Seq. No. 50; are also provided.
The CDRs disclosed herein include consensus sequences derived from groups of related sequences. As previously described, four groups of variable region sequences were identified, one kappa group and three lambda groups. The consensus sequence of CDRL1 of the kapa group consists of RASQX1X2SX3WX4A (Seq. No.: 123) where X1 is selected from G or V; X2 is selected from I, F or S; X3 is selected from S or G and X4 is selected from F or L. The consensus sequence of CDRL1 of the lambda group 1 consists of TLX1SGYSDYKVD (Seq. No.: 124) where X1 is selected from N or S. The consensus sequences for CDRL1 of the lambda 3 group consist of TGSSSNX1GAGYDVH (Seq. ID: 125) where X1 is selected from I or T.
The consensus sequence for CDRL2 of the lambda group 1 consists of VGTGGX1VGSKGX2 (Seq. ID: 126) where X1 is selected from I or T and X2 is selected from D or E. The consensus sequence of CDRL2 from the group lambda 3 consists of GSX1NRPS (Seq. No.: 127) where X1 is selected from N or G.
CDRL3 consensus strings include GADHGSGX1NFVYV (Seq. No.: 128) where X1 is S or N.
The consensus sequence of CDRH1 of the kapa group consists of SGGYYWXi (Seq. ID: No. 129) where Xi is selected from S or T. The consensus sequence of CDRH1 of the lambda group 1 consists of X1X2SMN (Id. Of Seq. No. 131) where Xi is selected from S or T and X2 is selected from Y or F. The consensus sequence of CDRH1 of the lambda group 2 consists of SYX1MH (Seq. No.: 130). where X1 is selected from G or A.
The consensus sequence of CDRH2 of the kappa group consists of X1IX2YSGX3X4YYNPSLKS (Seq. ID: 132) where X1 is selected from Y or H; X2 is selected from Y or H; X3 is selected from S or N and X4 is selected from T or S. The consensus sequence of the lambda group 1 consists of YISSX1SSTX2YX3ADSVKG (Seq. No.: 134) where X1 is selected from R or S, X2 is selected from I or R, X3 is selected from I, H or Y. The consensus sequence of the lambda group 2 consists of VISX1DGSX2KYYADSVKG (Seq. No.: 133) where X1 is F or H and X2 is L or T. The lambda 3 CDRH2 consensus sequence consists of VIWYDGSNX1YYADSVKG (Seq. No.: 135) where X1 is selected from K or E.
The kapa group CDRH3 consensus sequence consists of X1 RGX2YYGMDV (Seq. ID: 136) where X1 is selected from N or D and X2 is selected from H, Y or F. The CDRH3 consensus sequence of the lambda group 1 consists of RIAAAGX1X2X3YYYAX4DV (Seq. ID: No. 137) where X1 is selected from G or P; X2 is selected from F or W; X3 is selected from H or G and X4 is selected from L and M. The consensus sequence of CDRH3 of the lambda group 3 consists of DRGYX1SSWYPDAFDI (Seq. No.: 138) where X1 is selected from S or T. Monoclonal antibodies
The antigen binding proteins that are provided include monoclonal antibodies that bind to IL-23. Monoclonal antibodies can be produced using any known technique, for example, by immortalizing splenic cells collected from the transgenic animal after the end of the immunization schedule. Splenic cells can be immortalized using any known technique, for example, by fusing them with myeloma cells to produce hybridomas. Myeloma cells for use in hybridoma-producing fusion procedures are preferably non-antibody producing, have high fusion efficiency, and enzyme deficiencies that make them unable to grow in certain selective media that support the growth of only the desired fused cells (hybridomas). Examples of cell lines suitable for use in mouse fusions include Sp-20, P3-X63 / Ag8, P3-X63-Ag8.653, NS1 / 1.Ag 4 1, Sp210-Ag14, FO, NSO / U, MPC -11, MPC11-X45-GTG 1.7 and S194 / 5XXO Bul; examples of cell lines used in mouse fusions include R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210. Other cell lines useful for cell fusions are U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6.
In some cases, a hybridoma cell line is produced by immunizing an animal (for example, a transgenic animal that has human immunoglobulin sequences) with an IL-23 immunogen; with collection of splenic cells from the immunized animal; fusion of the splenic cells collected to a myeloma cell line, thus generating hybridoma cells; establishing hybridoma cell lines from hybridoma cells, and identifying a hybridoma cell that produces an antibody that binds to an IL-23 polypeptide while sparing IL-12. Such hybridoma cell lines, and anti-IL-23 monoclonal antibodies produced by them, are aspects of the present application.
Monoclonal antibodies secreted by a hybridoma cell line can be purified using any known technique. Hybridomas or mAbs can also be screened to identify mAbs with particular properties, such as the ability to inhibit IL-23-induced activity. Chimeric and humanized antibodies
Chimeric and humanized antibodies based on the following sequences are also provided. Monoclonal antibodies for use as therapeutic agents can be modified in a number of ways before use. An example is a chimeric antibody, which is an antibody composed of protein segments of different antibodies that are covalently joined to produce immunoglobulin functional heavy or light chains or immunologically functional portions thereof. Generally, a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in antibodies derived from a particular species or that belong to a particular class or subclass of antibody, while the rest of the chain is identical or homologous to a corresponding sequence in antibodies derived from another species or belonging to another class or subclass of antibody. For methods regarding chimeric antibodies, see, for example, US Patent No. 4,816,567; and Morrison et al, 1985, Proc. Natl. Acad. Sci. USA 81: 6.851-6.855. The CDR graft is described, for example, in US Patent Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101.
A useful type of chimeric antibody is a "humanized" antibody. Generally, a humanized antibody is produced from a monoclonal antibody that first appears in a non-human animal. Certain amino acid residues in that monoclonal antibody, typically from antigen unrecognized portions of the antibody, are modified to be homologous to corresponding residues in a human antibody of the corresponding isotype. Humanization can be accomplished, for example, with the use of various methods by replacing at least a portion of a variable rodent region with the corresponding regions of a human antibody (see, for example, US Patent Nos. 5,585,089, and No. 5,693,762; Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988, Nature 332: 323-27; Verhoeyen et al., 1988, Science 239: 1,534-1,536). In certain embodiments, the constant regions of different species of humans can be used together with the human variable region to produce hybrid antibodies. Fully human antibodies
Fully human antibodies are also provided. Methods are available for making specific fully human antibodies for a given antigen without exposing humans to the antigen (“fully human antibodies”). A specific means provided for the implementation of the production of fully human antibodies is the "humanization" of the humoral immune system of the mouse. The introduction of a human immunoglobulin (Ig) locus in mice in which endogenous Ig genes have been inactivated is a means of producing fully human monoclonal antibodies (mAbs) in mice, an animal that can be immunized with any desirable antigen. The use of fully human antibodies can minimize the immunogenic and allergic responses that can sometimes be caused by administering mouse or mouse-derived mAbs to humans as therapeutic agents.
Fully human antibodies can be produced by immunizing transgenic animals (commonly mice) that are capable of producing a variety of human antibodies in the absence of endogenous immunoglobulin production. Antigens for this purpose typically have six or more contiguous amino acids, and are optionally conjugated to a carrier, such as a hapten. See, for example, Jakobovits et al, 1993, Proc. Natl. Acad. Sci. USA 90: 2.551-2.555; Jakobovits et al, 1993, Nature 362: 255-258; and Bruggermann et al., 1993, Year in Immunol. 7:33. In an example of such a method, transgenic animals are produced by disabling the locus of mouse endogenous immunoglobulin that encodes the mouse light and heavy immunoglobulin chains, and insertion into the mouse genome of large fragments of human genome DNA containing locus that encode human heavy and light chain proteins. Partially modified animals, which have less than the total complement of human immunoglobulin locus, are then interlocked to obtain an animal that has all the desired immune system modifications. When administered an immunogen, these transgenic animals produce antibodies that are immunospecific for the immunogen, but have human amino acid sequences instead of murine, including variable regions. For further details of such methods, see, for example, Patent Publications WIPO WO96 / 33735 and WO94 / 02602. Additional methods in relation to transgenic mice for making human antibodies are described in US Patent Nos. 5,545,807; 6,713,610; 6,673,986; 6,162,963; 5,545,807; 6,300,129; 6,255,458; 5,877,397; 5,874,299 and 5,545,806; in WIPO patent publications WO91 / 10741, WO90 / 04036, and in EP 546073B1 and EP 546073A1.
The transgenic mice described above contain a human immunoglobulin gene miniloccus that encodes heavy chain immunoglobulin ([mu] and [gamma]) and [kappa] and non-rearranged human light chain sequences, along with target mutations that inactivate the locus endogenous [mu] and [kappa] chains (Lonberg et al., 1994, Nature 368: 856-859). Therefore, mice exhibit reduced expression of mouse IgM or [kappa] and in response to immunization, and introduced human heavy and light chain transgenes undergo class change and somatic mutation to generate high-level human IgG [kappa] antibodies affinity (Lonberg et al., supra; Lonberg and Huszar, 1995, Intern. Rev. Immunol. 13: 65-93; Harding and Lonberg, 1995, Ann. NY Acad. Sci. 764: 536-546). The preparation of such mice is described in detail in Taylor et al, 1992, Nucleic Acids Research 20: 6.287-6.295; Chen et al, 1993, International Immunology 5: 647-656; Tuaillon et al, 1994, J. Immunol. 152: 2,912-2,920; Lonberg et al., 1994, Nature 368: 856-859; Lonberg, 1994, Handbook of Exp. Pharmacology 113: 49-101; Taylor et al, 1994, International Immunology 6: 579-591; Lonberg and Huszar, 1995, Intern. Rev. Immunol. 13: 65-93; Harding and Lonberg, 1995, Ann. N.Y Acad. Sci. 764: 536-546; Fishwild et al, 1996, Nature Biotechnology 14: 845-85. See, additional United States Patents No. 5,545,806; No. 5,569,825; No. 5,625,126; No. 5,633,425; No. 5,789,650; No. 5,877,397; No. 5,661,016; No. 5,814,318; No. 5,874,299; and No. 5,770,429; as well as United States Patent No. 5,545,807; WIPO Publications Nos. WO 93/1227; WO 92/22646; and WO 92/03918. The technologies used for the production of human antibodies in these transgenic mice are also disclosed in WIPO Publication No. WO 98/24893, and Mendez et al, 1997, Nature Genetics 15: 146-156. For example, the HCo7 and HCo12 transgenic mouse strains can be used to generate anti-IL-23 antibodies.
With the use of hybridoma technology, human mAbs specific for antigen with the desired specificity can be produced and selected from transgenic mice such as those described above. Such antibodies can be cloned and expressed using a suitable vector and host cell, or the antibodies can be collected from cultured hybridoma cells.
Fully human antibodies can also be derived from phage display libraries (as disclosed in Hoogenboom et al., 1991, J. Mol. Biol. 227: 381; Marks et al., 1991, J. Mol. Biol. 222: 581; WIPO Publication No. WO 99/10494). Phage display techniques mimic immune selection by presenting antibody repertoires on the filamentous bacteriophage surface, and subsequent phage selection by binding to an antigen of choice. Bi-specific or bifunctional antigen binding proteins
A bispecific, doubly specific or bifunctional antigen binding protein or antibody is an antigen binding antibody or hybrid antibody, respectively, which has two different antigen binding sites, such as one or more CDRs or one or more variable regions as described above. In some cases, they are an artificial hybrid antibody that has two different heavy / light chain pairs and two different binding sites. The multi-specific antigen-binding protein or "multi-specific antibody" is one that targets more than one antigen or epitope. Bi-specific antigen-binding proteins and antibodies are a kind of multi-specific antigen-binding protein antibody and can be produced by various methods that include, without limitation, fusion of hybridomas or binding of Fab 'fragments. See, for example, Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79: 315-321; Kostelny et al, 1992, J. Immunol. 148: 1.547-1.553. Immunological fragments
Antigen binding proteins also include immunological fragments of an antibody (for example, a Fab, Fab ', F (ab') 2 or scFv). A "Fab fragment" comprises a light chain (the light chain variable region (VL) and its corresponding constant domain (CO) and a heavy chain (the heavy chain variable region (VH) and first constant domain (CH1)). The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A “Fab fragment” contains a light chain and a portion of a heavy chain that also contains the region between the CH1 and CH2 domains , so that a disulfide bond between chains can be formed between the two heavy chains of the two Fab 'fragments to form an F (ab') 2 molecule. An “F (ab ') 2 fragment” thus consists of two Fab 'fragments that are held together by a disulfide bond between the two heavy chains. An "Fv fragment" consists of the light chain variable region and the heavy chain variable region of a single arm of an antibody. The single chain antibodies " scFv ”are molecules in which the variable regions of heavy and light chains were connected by a flexible linker to form a single polypeptide chain, which forms an antigen binding region. Single chain antibodies are discussed in detail in WIPO Publication No. WO 88/01649, U.S. Patent Nos. 4,946,778 and No. 5,260,203; Bird, 1988, Science 242: 423; Huston et al, 1988, Proc. Natl. Acad. Sci. U.S.A. 85: 5,879; Ward et al., 1989, Nature 334: 544, de Graaf et al., 2002, Methods Mol Biol. 178: 379-387; Kortt et al, 1997, Prot. Eng. 10: 423; Kortt et al., 2001, Biomol. Eng. 18: 95-108 and Kriangkum et al., 2001, Biomol. Eng. 18: 31-40. An "Fc" region contains two heavy chain fragments that comprise the CH1 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.
Also included are domain antibodies, immunologically functional immunoglobulin fragments that contain only the variable region of a heavy chain or the variable region of a light chain. In some cases, two or more VH regions are covalently joined with a peptide linker to create a divalent domain antibody. The two VH regions of a divalent domain antibody can target the same or different antigens. Diabodiession are bivalent antibodies that comprise two polypeptide chains, where each polypeptide chain comprises VH and VL domains joined by a linker that is too short to allow pairing between two domains on the same chain, thus allowing each domain to pair with a complementary domain in another polypeptide chain (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6.444-48, 1993 and Poljak et al., Structure 2: 1121-23, 1994). Similarly, tribodies and tetrabodi are antibodies that comprise three and four polypeptide chains, respectively, and that form three and four antigen binding sites, respectively, which may be the same or different. Maxibodies comprise bivalent scFvs covalently attached to the IgG1 Fc region, (see, for example, Fredericks et al., 2004, Protein Engineering, Design & Selection, 17: 95-106; Powers et al., 2001, Journal of Immunological Methods, 251: 123-135; Shu et al., 1993, Proc. Natl. Acad. Sci. USA 90: 7.995-7,999; Hayden et al., 1994, Therapeutic Immunology 1: 3-15). Various Other Shapes
Variant forms of the antigen binding proteins disclosed above are also provided; some of the antigen binding proteins have, for example, one or more conservative amino acid substitutions in one or more of the heavy or light chains, variable regions or CDRs listed in Tables 1 and 2.
Naturally occurring amino acids can be divided into classes based on common side-chain properties: hydrophobic (norleucine, Met, Ala, Val, Leu, Ile); neutral hydrophilics (Cys, Ser, Thr, Asn, Gln); acids (Asp, Glu); basic (His, Lys, Arg); residues that influence the orientation of the chain (Gly, Pro); and aromatics (Trp, Tyr, Phe).
Conservative amino acid substitutions may involve exchanging a member of one of these classes for another member of the same class. Conservative amino acid substitutions may include non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reverse or inverted forms of amino acid moieties. Such substantial modifications in the functional and / or biochemical characteristics of the antigen binding proteins described herein can be achieved by creating substitutions in the amino acid sequence of the heavy and light chains that differ significantly in their effect on the maintenance (a) of the structure of the skeleton molecular in the substitution area, for example, as a leaf or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain.
Non-conservative substitutions may involve exchanging a member of one of the above classes for a member of another class. Such substituted residues can be introduced into regions of the antibody that are homologous to human antibodies, or into non-homologous regions of the molecule.
In making such changes, according to certain modalities, the hydropathic index of amino acids can be considered. The hydropathic profile of a protein is calculated by assigning a numerical value to each amino acid ("hydropathy index") and then repeatedly averaging those values along the peptide chain. Each amino acid was assigned a hydropathic index based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
The importance of the hydropathic profile in conferring interactive biological function on a protein is understood in the art (see, for example, Kyte et al., 1982, J. Mol. Biol. 157: 105-131). It is known that certain amino acids can be replaced by other amino acids that have a similar hydropathic index and still retain a similar biological activity. In making changes based on a hydropathic index, in certain modalities, the substitution of amino acids whose hydropathic indexes are within ± 2 is included. In some respects, those that are within ± 1 are included, and in other respects, those that are within ± 0.5 are included.
It is also understood in the art that the replacement of similar amino acids can be done effectively based on hydrophilicity, particularly when the biologically functional protein or peptide thus created is intended for use in immunological modalities, as in the present case. In certain modalities, the highest average local hydrophilicity of a protein, as directed by the hydrophilicity of its adjacent amino acids, is correlated with its immunogenicity and antigen binding or immunogenicity, that is, with the biological property of the protein.
The following hydrophilic values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+ 3.0 ± 1); glutamate (+ 3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). In making changes based on similar hydrophilic values, in certain modalities, the substitution of amino acids whose hydrophilic values are within ± 2 is included; in other modalities, those that are in ± 1 are included, and in still other modalities, those that are in ± 0.5 are included. In some cases, a person can also identify epitopes from primary amino acid sequences based on hydrophilia. These regions are also referred to as “epitopic nucleus regions”.
Examples of conservative amino acid substitutions are shown in Table 4. Table 4 Conservative amino acid substitutions

Residue = Original Residue / Sub = Replacement example
A skilled practitioner will be able to determine suitable variants of polypeptides as presented herein using well known techniques. A person skilled in the art can identify suitable areas of the molecule that can be altered without destroying the activity by targeting regions that are not important for the activity. The skilled professional will also be able to identify residues and portions of the molecules that are conserved among similar polypeptides. In additional modalities, even areas that may be important for biological activity or structure can be subjected to conservative amino acid substitutions without destroying biological activity or without adversely affecting the structure of the polypeptide.
Additionally, a person skilled in the art can review a structure-function study that identifies residues in similar polypeptides that are important for the activity or structure. In view of such a comparison, a person can predict the importance of amino acid residues in a protein that correspond to amino acid residues important for the activity or structure in similar proteins. A person skilled in the art can opt for chemically similar amino acid substitutions for such important amino acid residues.
A person skilled in the art can also analyze the three-dimensional structure and sequence of amino acids in relation to that structure in similar polypeptides. In view of such information, a person skilled in the art can predict the alignment of an antibody's amino acid residues with respect to its three-dimensional structure. A person skilled in the art can choose not to make radical changes to the amino acid residues expected to be on the protein's surface, since such residues may be involved in important interactions with other molecules. In addition, a person skilled in the art can generate test variants that contain a single amino acid substitution at each desired amino acid residue. These variants can then be screened using assays for IL-23 activity, (see examples below), thereby producing information regarding which amino acids can be changed and which should not be. In other words, based on information collected from such routine experiments, a person skilled in the art can easily determine the amino acid positions at which additional substitutions should be avoided alone or in combination with other mutations.
Countless scientific publications have been dedicated to the forecast of secondary structure. See Moult, 1996, Curr. Op. In Biotech. 7: 422-427; Chou et al., 1974, Biochem. 13: 222-245; Chou et al., 1974, Biochemistry 113: 211-222; Chou et al., 1978, Adv. Enzymol. Report Areas Mol. Biol. 47: 45-148; Chou et al, 1979, Ann. Rev. Biochem. 47: 251276; and Chou et al., 1979, Biophys. J. 26: 367-384. In addition, computer programs are currently available to assist with forecasting secondary structures. A distinct secondary structure forecasting method is based on homology modeling. For example, two polypeptides or proteins that have a sequence identity greater than 30%, or similarity greater than 40%, often have similar structural topologies. The recent growth of the structural protein database (PDB) has provided increased secondary structure predictability, including the potential number of folds in a polypeptide or protein structure. See Holm et al., 1999, Nucl. Acid. Res. 27: 244-247. It has been suggested (Brenner et al, 1997, Curr. Op. Struct. Biol. 7: 369-376) that there is a limited number of folds in a given polypeptide or protein and that, once a critical number of structures has been resolved , the structural forecast will be dramatically more accurate.
Additional secondary structure forecasting methods include “threading” (Jones, 1997, Curr. Opin. Struct. Biol. 7: 377-387; Sippl et al, 1996, Structure 4: 15-19), “profile analysis” (Bowie et al., 1991, Science 253: 164-170; Gribskov et al., 1990, Meth. Enzym. 183: 146-159; Gribskov et al., 1987, Proc. Nat. Acad. Sci. 84: 4.355 -4,358) and “evolutionary link” (See, Holm, 1999, supra; and Brenner, 1997, supra).
In some embodiments, amino acid substitutions are made that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) change the binding affinity to form protein complexes, (4) change the affinity of binding of the ligand or antigen, and / or (4) confer or modify other physicochemical or functional properties in such polypeptides, such as maintaining the structure of the molecular skeleton in the substitution area, for example, as a leaf or helical conformation; maintaining or altering the charge or hydrophobicity of the molecule at the target site, or maintaining or altering the volume of a side chain.
For example, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) can be made in the naturally occurring sequence. Substitutions can be made in that portion of the antibody that lies outside the domain (s) forming intermolecular contacts. In such embodiments, conservative amino acid substitutions that do not substantially alter the structural characteristics of the parent sequence can be used (for example, one or more replacement amino acids that do not disrupt the secondary structure that characterizes the native or parent antigen binding protein). Examples of secondary and tertiary polypeptide structures recognized in the art are described in Proteins, Structures and Molecular Principles (Creighton, Ed.), 1984, W. H. New York: Freeman and Company; Introduction to Protein Structure (Branden and Tooze, eds.), 1991, New York: Garland Publishing; and Thornton et al., 1991, Nature 354: 105.
Additional variants include cysteine variants in which one or more cysteine residues in the parent or native amino acid sequence are deleted or replaced with another amino acid (for example, serine). Cysteine variants are useful, among others, when antibodies (for example) must be refolded into a biologically active conformation. Cysteine variants may have less cysteine residues than native protein, and typically have an equal number to minimize interactions that result from unpaired cysteines.
The heavy and light chain variable region and CDRs that are disclosed can be used to prepare antigen binding proteins that contain an antigen binding region that can specifically bind to an IL-23 polypeptide. “Antigen-binding region” means a protein, or a portion of a protein, that specifically binds to a specified antigen, such as the region that contains the amino acid residues that interact with an antigen and confer on the antigen-binding protein its specificity and affinity for the target antigen. An antigen binding region can include one or more CDRs and certain antigen binding regions also include one or more framework regions. For example, one or more of the CDRs listed in Table 3 can be incorporated into a molecule (for example, a polypeptide) covalently or non-covalently to perform an immunoadhesion. An immunoadhesion can incorporate the CDR (s) as part of a larger polypeptide chain, can covalently attach the CDR (s) to another polypeptide chain, or can incorporate the CDR (s) non-covalently. CDR (s) allows immunoadhesion to specifically bind to an antigen of particular interest (for example, an IL-23 polypeptide).
Other antigen binding proteins include mimetics (for example, "peptide mimetics" or "peptidomimetics") based on the variable regions and CDRs that are described herein. Such analogs can be peptides, non-peptides or combinations of peptide and non-peptide regions. Fauchere, 1986, Adv. Drug Res. 15:29; Veber and Freidinger, 1985, TINS p. 392; and Evans et al., 1987, J. Med. Chem. 30: 1229. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce a similar therapeutic or prophylactic effect. Such compounds are often developed with the help of computerized molecular modeling. Generally, peptidomimetics are proteins that are structurally similar to an antigen-binding protein that has a desired biological activity, such as the ability to bind to IL-23, but peptidomimetics have one or more peptide bonds optionally substituted by a selected link from , for example: - CH2NH-, -CH2S-, -CH2-CH2-, -CH-CH- (cis and trans), -COCH2-, - CH (OH) CH2- and -CH2SO-, by methods well known in the art. technical. Systematic substitution of one or more amino acids in a consensus sequence with a D-amino acid of the same type (for example, D-lysine in place of L-lysine) can be used in certain modalities to generate more stable proteins. In addition, limited peptides comprising a consensus sequence or a variation of substantially identical consensus sequence can be generated by methods known in the art (Rizo and Gierasch, 1992, Ann. Rev. Biochem. 61: 387), for example, by addition of internal cysteine residues capable of forming intramolecular disulfide bridges that cyclize the peptide.
Derivatives of the antigen binding proteins that are described herein are also provided. Derivatized antigen-binding proteins can comprise any molecule or substance that imparts a desired property to the antigen or fragment-binding protein, such as increased half-life in a particular use. The derivatized antigen-binding protein can comprise, for example, a detectable portion (or label) (for example, a radioactive, colorimetric, antigenic or enzymatic molecule, a detectable globule (such as a magnetic or electrode dense globule (for example, gold) ), or a molecule that binds to another molecule (for example, biotin or streptavidin)), a therapeutic or diagnostic portion (for example, a radioactive, cytotoxic, or pharmaceutically active portion), or a molecule that increases protein compatibility of antigen binding for a particular use (for example, administration to an individual, such as a human being, or other uses in vivo or in vitro). Examples of molecules that can be used to derivatize an antigen-binding protein include albumin (for example, human serum albumin) and polyethylene glycol (PEG). Derivatives linked to albumin and PEGylates of antigen binding proteins can be prepared using techniques well known in practice. In one embodiment, an antigen-binding protein is conjugated or linked to transthyretin (TTR) or a variant of TTR. The TTR or TTR variant can be chemically modified, for example, with a chemical agent selected from the group consisting of dextran, poly (n-vinyl pyrrolidone), polyethylene glycols, propropylene glycol homopolymers, polypropylene oxide / oxide copolymers of ethylene, polyoxyethylated polyols and polyvinyl alcohols.
Other derivatives include covalent or aggregating conjugates of IL-23 antigen binding proteins with other proteins or polypeptides, such as by expression of recombinant fusion proteins comprising heterologous polypeptides fused to the N or C terminal of an antigen binding protein. IL-23. For example, the conjugated peptide can be a heterologous signal polypeptide (or leader), for example, the yeast alpha factor leader, or a peptide as an epitope tag. Fusion proteins that contain IL-23 antigen binding protein may comprise added peptides to facilitate the purification or identification of the IL-23 antigen binding protein (e.g., poly-His). An IL-23 antigen binding protein can also be linked to the FLAG peptide as described in Hopp et al, 1988, Bio / Technology 6: 1204; and US Patent No. 5,011,912. The FLAG peptide is highly antigenic and provides an epitope reversibly linked by a specific monoclonal antibody (mAb), allowing for rapid analysis and easy purification of expressed recombinant protein. Reagents useful for the preparation of fusion proteins in which the FLAG peptide is fused to a given polypeptide are commercially available (Sigma, St. Louis, MO).
Oligomers that contain one or more IL-23 antigen binding proteins can be used as IL-23 antagonists. Oligomers can be in the form of covalently bonded or non-covalently bonded dimers, trimers or higher oligomers. Oligomers comprising two or more IL-23 antigen binding proteins are contemplated for use, with one example being a homodimer. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, etc. Oligomers comprising multiple IL-23 binding proteins joined together through covalent or non-covalent interactions between the peptide moieties fused to IL-23 antigen binding proteins are also included. Such peptides can be peptide ligands (spacers), or peptides that have the property of promoting oligomerization. Suitable peptide linkers include those described in US Patent Nos. 4,751,180 and 4,935,233. Leucine zippers and certain antibody-derived polypeptides are among the peptides that can promote the oligomerization of IL-23 antigen binding proteins attached to them. Examples of leucine zipper domains suitable for the production of soluble oligomeric proteins are described in WIPO Publication No. WO 94/10308; Hoppe et al, 1994, FEBS Letters 344: 191; and Fanslow et al., 1994, Semin. Immunol. 6: 267-278. In one approach, recombinant fusion proteins comprising an IL-23 antigen binding protein fragment or derivative fused to a leucine zipper peptide are expressed in suitable host cells, and the protein binding fragments or derivatives of soluble oligomeric IL-23 antigens that form are recovered from the culture supernatant.
Such oligomers can comprise two to four IL-23 antigen binding proteins. The IL-23 antigen-binding protein portions of the oligomer can be in any of the forms described above, for example, variants or fragments. Preferably, the oligomers comprise IL-23 antigen binding proteins that have IL-23 binding activity. Oligomers can be prepared using polypeptides derived from immunoglobulins. The preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, for example, by Ashkenazi et al, 1991, Proc. Natl. Acad. Sci. USA 88: 10,535; Byrn et al., 1990, Nature 344: 677; and Hollenbaugh et al, 1992 “Construction of Immunoglobulin Fusion Proteins”, in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11.
Also included are dimers that comprise two fusion proteins created by fusing an IL-23 antigen binding protein to the Fc region of an antibody. The dimer can be made, for example, by inserting a gene fusion that encodes the fusion protein into a suitable expression vector, which expresses the gene fusion in host cells transformed with the recombinant expression vector, and allows the Expressed fusion protein gathers like the antibody molecules, and therefore interchain disulfide bonds are formed between the Fc portions to produce the dimer. Such Fc polypeptides include native and mutein forms of polypeptides derived from the Fc region of an antibody. Truncated forms of such polypeptides that contain the hinge region that promotes dimerization are also included. Fusion proteins comprising Fc portions (and oligomers formed therefrom) offer the advantage of easy purification by affinity chromatography on Protein A or Protein G columns. A suitable Fc polypeptide, described in WIPO Publication No. WO 93/10151 and US Patent Nos. 5,426,048 and 5,262,522, is a single chain polypeptide that extends from the N-terminal fold region to the native C-terminal of the Fc region of a human IgG1 antibody. Another suitable Fc polypeptide is the Fc mutein described in US Patent No. 5,457,035, and in Baum et al, 1994, EMBO J. 13: 3,992-4,001. The amino acid sequence of this mutein is identical to that of the native Fc sequence presented in WIPO Publication No. WO 93/10151, except that amino acid 19 has been switched from Leu to Ala, amino acid 20 has been exchanged from Leu to Glu, and the amino acid 22 have been switched from Gly to Ala. Mutein exhibits reduced affinity for Fc receptors. Glycosylation
The antigen-binding protein may have a glycosylation pattern that is different or altered from that found in native species. As is known in the art, glycosylation patterns may depend on the sequence of the protein (for example, the presence or absence of particular glycosylation amino acid residues, discussed below), or the host cell or organism in which the protein is produced. Particular expression systems are discussed below.
Glycosylation of polypeptides is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate portion to the side chain of an asparagine residue. The asparagine-X-serine and asparagine-X-threonine tri-peptide sequences, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of these tri-peptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the N-acetylgalactosamine, galactose, or xylose sugars to a hydroxyamino acid, more commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylisine can also be used.
The addition of glycosylation sites to the antigen binding protein is conveniently accomplished by altering the amino acid sequence so that it contains one or more of the above described tri-peptide sequences (for N-linked glycosylation sites). The change can also be made by adding, or replacing, one or more serine or threonine residues to the starting sequence (for O-linked glycosylation sites). To make it easier, the amino acid sequence of the antigen-binding protein can be altered through changes in the DNA level, particularly by mutating the DNA encoding the target polypeptide on pre-selected bases so that codons are generated that will translate into the desired amino acids .
Another way to increase the number of carbohydrate portions in the antigen-binding protein is by chemically or enzymatically linking glycosides to the protein. These procedures are advantageous because they do not require the production of the protein in a host cell that has glycosylation capabilities for N and O-linked glycosylation. Depending on the binding mode used, sugar can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in PCT Publication No. WO 87/05330, and in Aplin and Wriston, 1981, CRC Crit. Rev, Biochem., Pp. 259-306.
The removal of carbohydrate portions present in the initial antigen binding protein can be carried out chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all of the sugars except the binding sugar (N-acetylglycosamine or N-acetylgalactosamine), while leaving the polypeptide intact. Chemical deglycosylation is described by Hakimuddin et al, 1987, Arch. Biochem. Biophys. 259: 52 and by Edge et al, 1981, Anal. Biochem. 118: 131. Enzymatic cleavage of carbohydrate moieties into polypeptides can be accomplished by using various endo and exoglycosidases as described by Thotakura et al., 1987, Meth. Enzymol. 138: 350. Glycosylation at potential glycosylation sites can be prevented by using the compound tunicamycin as described by Duskin et al, 1982, J. Biol. Chem. 257: 3,105. Tunicamycin blocks the formation of protein-N-glycoside bonds.
Therefore, aspects include the glycosylation variants of the antigen binding proteins in which the number and / or type of glycosylation sites has changed compared to the amino acid sequences of the parent polypeptide. In certain embodiments, variants of the antigen-binding protein comprise a greater or lesser number of N-linked glycosylation sites than the parent polypeptide. Substitutions that eliminate or alter this sequence will prevent the addition of an N-linked carbohydrate chain present in the parent polypeptide. For example, glycosylation can be reduced by deleting an Asn or replacing Asn with a different amino acid. Antibodies typically have an N-linked glycosylation site in the Fc region. Labels and effector groups
Antigen binding proteins can comprise one or more labels. The term "label" or "labeling group" refers to any detectable label. In general, labels can be of various classes, depending on the assay in which they will be detected: a) isotopic labels, which can be radioactive or heavy isotopes; b) magnetic labels (for example, magnetic particles); c) active redox portions; d) optical dyes; enzyme groups (for example, horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); e) biotinylated groups; and f) predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.). In some embodiments, the labeling group is linked to the antigen binding protein by means of spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art. Examples of suitable labeling groups include, without limitation, the following: radioisotopes or radionuclides (for example, 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent groups (for example, FITC, rhodamine, phosphorus lanthanide), enzyme groups (for example, strong root peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups, or predetermined polypeptide epitopes recognized by a secondary reporter (for example, zipper pair sequences leucine, binding sites for secondary antibodies, metal binding domains, epitope tags).
In some embodiments, the labeling group is linked to the antigen binding protein by means of spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used as appropriate.
The term "effector group" means any group attached to an antigen-binding protein that acts as a cytotoxic agent. Examples for suitable effector groups are radioisotopes or radionuclides (for example, 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I). Other suitable groups include toxins, therapeutic groups, or chemotherapeutic groups. Examples of suitable groups include calicheamicin, auristatins, geldanamycin and maytansine. In some embodiments, the effector group is linked to the antigen binding protein by means of spacer arms of various lengths to reduce potential steric hindrance. Polynucleotides encoding IL-23 antigen binding proteins
Polynucleotides encoding the antigen binding proteins described herein, or portions thereof, are also provided, including polynucleotides that encode one or both chains of an antibody, or a fragment, derivative, mutein, or variants thereof, polynucleotides that encode variable regions heavy chain or just CDRs, enough polynucleotides for use as hybridization markers, PCR primers or sequencing primers for the identification, analysis, mutation or amplification of a polynucleotide encoding a polypeptide, antisense nucleic acids for inhibiting expression polynucleotide, and complementary sequences from the previous ones. Polynucleotides can be of any length. They can have, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 85, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400 , 450, 500, 750, 1,000, 1,500, 3,000, 5,000 or more nucleic acids in length, including all values in between, and / or may comprise one or more additional sequences, for example, regulatory sequences, and / or be part of of a larger polynucleotide, for example, a vector. Polynucleotides may be single-stranded or double-stranded and may comprise RNA and / or DNA nucleic acids and artificial variants thereof (e.g., peptide nucleic acids).
Polynucleotides encoding certain antigen-binding proteins, or portions thereof (for example, full-length antibody, heavy or light chain, variable domain, or a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3) can be isolated from mouse B cells that have been immunized with IL-23 or an immunogenic fragment thereof. The polynucleotide can be isolated by conventional procedures such as polymerase chain reaction (PCR). Phage display is another example of a known technique in which derivatives of antibodies and other antigen binding proteins can be prepared. In one approach, polypeptides that are components of an antigen binding protein of interest are expressed in any suitable recombinant expression system, and the expressed polypeptides assemble to form molecules of the antigen binding protein. Phage display is also used to derive antigen binding proteins that have different properties (ie, variable affinities for the antigen to which they bind) through chain shuffling, see Marks et al, 1992, BioTechnology 10: 779 .
Due to the degeneracy of the genetic code, each of the polypeptide sequences shown here are also encoded by a large number of other polynucleotide sequences in addition to those provided. For example, heavy chain variable domains provided here can be encoded by sequences of polynucleotides of Seq. Nos: 32, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or 59. The light chain variable domains can be encoded by Seq Id polynucleotide sequences. Nos: 2, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28. A person of ordinary skill in the art will realize that the present application therefore provides an appropriate written description and enabled for each degenerate nucleotide sequence that encodes each antigen-binding protein.
One aspect also provides polynucleotides that hybridize to other polynucleotide molecules under particular conditions of hybridization. Methods for nucleic acid hybridization, basic parameters that affect the choice of hybridization conditions and a guide for proper condition planning are well known in the art. See, for example, Sambrook, Fritsch, and Maniatis (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY and Current Protocols in Molecular Biology, 1995, Ausubel et al., Eds., John Wiley & Sons, Inc .. As defined herein, a hybridization condition of moderate stringency uses a prewash solution containing 5x sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50% formamide, 6x SSC, and a hybridization temperature of 55 ° C (or other similar hybridization solutions, such as one containing about 50% formamide, with a hybridization temperature of 42 ° C), and washing conditions of 60 ° C, in 0.5x SSC, 0.1% SDS A strict hybridization condition hybridizes in 6x SSC at 45 ° C, followed by one or more washes 0.1x SSC, 0.2% SDS at 68 ° C. In addition, a person skilled in the art can manipulate hybridization and / or wash conditions to increase or decrease the stringency of hybridization so that polynucleotides that comprise nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to each other, including all values in between, typically remain hybridized to each other.
Mutations can be introduced by mutation in a polynucleotide, thereby leading to changes in the amino acid sequence of a polypeptide (for example, an antigen binding protein or derived antigen binding protein) that it encodes. Mutations can be introduced using any known technique, such as site-directed mutagenesis and random mutagenesis. Mutant polypeptides can be expressed and selected for a desired property. Mutations can be introduced into a polynucleotide without significantly altering the biological activity of a polypeptide that it encodes. For example, substitutions in non-essential amino acid residues. Alternatively, one or more mutations can be introduced into a polynucleotide that selectively alters the biological activity of a polypeptide that it encodes. For example, the mutation can alter quantitatively or qualitatively biological activity, such as increasing, reducing or eliminating activity and altering the antigen specificity of an antigen-binding protein.
Another aspect provides polynucleotides that are suitable for use as primers or hybridization markers for the detection of nucleic acid sequences. A polynucleotide can comprise only a portion of a nucleic acid sequence that encodes a full-length polypeptide, for example, a fragment that can be used as a marker or primer or a fragment that encodes an active portion (for example, a portion of IL-23 binding) of a polypeptide. Markers based on the sequence of a nucleic acid can be used to detect nucleic acid or similar nucleic acids, for example, transcripts that encode a polypeptide. The marker may comprise a labeling group, for example, a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such markers can be used to identify a cell that expresses the polypeptide. Methods of expressing antigen binding proteins
The antigen binding proteins provided herein can be prepared by any of a number of conventional techniques. For example, IL-23 antigen binding proteins can be produced by recombinant expression systems, which use any technique known in practice. See, for example, Monoclonal antibodies, Hybridomas: A New Dimension in Biological Analyzes, Kennet et al (eds.) Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988).
Expression systems and constructs in the form of plasmids, expression vectors, transcription or expression cassettes that comprise at least one polynucleotide as described above are also provided herein, as well as host cells that comprise such expression systems or constructs. As used herein, "vector" means any molecule or entity (for example, nucleic acid, plasmid, bacteriophage or virus) suitable for use to transfer protein-coding information in a host cell. Examples of vectors include, without limitation, plasmids, viral vectors, non-episomal mammalian vectors and expression vectors, for example, recombinant expression vectors. Expression vectors, like recombinant expression vectors, are useful for the transformation of a host cell and contain nucleic acid sequences that direct and / or control (together with the host cell) the expression of one or more operatively linked heterologous coding regions and they. An expression construct can include, without limitation, sequences that affect or control transcription, translation and, if introns are present, affect RNA splicing of a coding region operatively linked to it. “Operationally linked” means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions. For example, a control sequence, for example, a promoter, in a vector that is "operationally linked" to a protein coding sequence are arranged so that the normal activity of the control sequence leads to the transcription of the protein resulting in recombinant expression of the encoded protein.
Another aspect provides host cells in which an expression vector, such as a recombinant expression vector, has been introduced. A host cell can be any prokaryotic cell (for example, E. coli) or eukaryotic cell (for example, yeast, insect, or mammalian cells (for example, CHO cells)). Vector DNA can be introduced into prokaryotic or eukaryotic cells using conventional transformation or transfection techniques. For stable transfection of mammalian cells, it is known that, depending on the expression vector and the transfection technique used, only a small fraction of cells can integrate the entrained DNA in their genome. To identify and select these members, a gene that encodes a selectable marker (for example, for antibiotic resistance) is usually introduced into host cells along with the gene of interest. Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced polynucleotide can be identified by drug selection (for example, cells that have the selectable marker gene will survive, while the other cells will die), among other methods.
Antigen binding proteins can be expressed in hybridoma cell lines (for example, in particular antibodies can be expressed in hybridomas) or in cell lines other than hybridomas. The expression constructs that encode antigen-binding proteins can be used to transform a mammalian, insect or microbial host cell. Transformation can be performed using any known method of introducing polynucleotides into a host cell, including, for example, packaging the polynucleotide into a virus or bacteriophage and transducing a host cell with the construction by transfection procedures known in the art , as exemplified by US Patent Nos. 4,399,216; 4,912,040; 4,740,461; 4,959,455. The optimal transformation procedure used will depend on what type of host cell is being transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include, without limitation, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, polynucleotide (s) encapsulation in liposomes, mixture of nucleic acid with positively charged lipids, and direct microinjection of DNA into the nuclei.
Recombinant expression constructs typically comprise a polynucleotide that encodes a polypeptide. The polypeptide can comprise one or more of the following: one or more CDRs as provided herein; a light chain variable region; a heavy chain variable region; a light chain constant region; a heavy chain constant region (for example, CH1, CH2 and / or CH3); and / or another scaffold portion of an IL-23 antigen binding protein. These nucleic acid sequences are inserted into a suitable expression vector using standard binding techniques. In one embodiment, the heavy or light chain constant region is attached to the C-terminus of a heavy or light chain variable region provided here and is linked into an expression vector. The vector is typically selected to be functional in the particular host cell employed (that is, the vector is compatible with the machinery of the host cell, allowing amplification and / or expression of the gene to occur). In some embodiments, vectors are used that employ protein-fragment complementation assays that use protein reporters, such as dihydrofolate reductase (see, for example, U.S. Pat. No. 6,270,964). Suitable expression vectors can be purchased, for example, from Invitrogen Life Technologies (Carlsbad, CA) or BD Biosciences (San Jose, CA). Other useful vectors for the cloning and expression of antibodies and fragments include those described in Bianchi and McGrew, 2003, Biotech. Biotechnol. Bioeng. 84: 439-44. Additional suitable expression vectors are discussed, for example, in Methods Enzymol., Vol. 185 (D. V. Goeddel, ed.), 1990, New York: Academic Press.
Typically, the expression vectors used in any of the host cells will have sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as "flanking sequences" in certain embodiments will typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, an intron sequence complete containing a donated junction site and an acceptor, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of the polynucleotide encoding the polypeptide to be express, and a selectable marker element. The expression vectors that are provided can be constructed from an initiation vector as a commercially available vector. Such vectors may or may not contain all of the desired flanking sequences. When one or more of the flanking sequences described here are not already present in the vector, they can be individually obtained and linked in the vector. Methods used to obtain each of the flanking sequences are well known to a person skilled in the art.
Optionally, the vector can contain a sequence that encodes a "tag", that is, an oligonucleotide molecule located at the 5 'or 3' end of the sequence that encodes IL-23 antigen binding protein; the oligonucleotide sequence encodes polyHis (like hexaHis), or another copmo FLAG® tag, HA (hemagglutinin influenza virus), or myc, for which antibodies are commercially available. This tag is typically fused to the polypeptide under expression of the polypeptide, and can serve as a means for affinity purification or detection of the IL-23 antigen binding protein from the host cell. Affinity purification can be performed, for example, by column chromatography using antibodies against the tag as an affinity matrix. Optionally, the tag can subsequently be removed from the purified IL-23 antigen binding protein by various means such as by using certain peptidases for cleavage.
Flanking sequences can be homologous (that is, of the same species and / or strain as the host cell), heterologous (that is, of a species other than the host cell species or strain), hybrids (that is, a combination of flanking sequences from more than one source), synthetic or native. As such, the source of a flanking sequence can be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, as long as the flanking sequence is functional, and can be activated by the machinery of the host cell.
The flanking sequences useful in the vectors can be obtained by any of several methods well known in the art. Typically, useful flanking sequences have been previously identified by mapping and / or by restriction endonuclease digestion and can thus be isolated from the appropriate tissue source using the appropriate restriction endonucleases. In some cases, the total nucleotide sequence of a flanking sequence may be known. Here, the flanking sequence can be synthesized using the methods described herein for nucleic acid synthesis or cloning.
If all or only a portion of the flanking sequence is known, it can be obtained by using the polymerase chain reaction (PCR) and / or by scanning a genomic library with a suitable marker such as an oligonucleotide and / or fragment of flanking sequence of the same or another species. When the flanking sequence is not known, a fragment of DNA that contains a flanking sequence can be isolated from a large piece of DNA that can contain, for example, a coding sequence or even another gene or genes. Isolation can be performed by restriction endonuclease digestion to produce the appropriate DNA fragment followed by isolation using agarose gel purification, Qiagen® column chromatography (Qiagen, Chatsworth, CA), or other methods known to the practitioner Experient. The selection of enzymes suitable for this purpose will be readily apparent to a person of ordinary skill in the art.
An origin of replication is typically a part of those commercially acquired prokaryotic expression vectors, and the origin assists in amplifying the vector in a host cell. If the vector of choice does not contain a site of origin of replication, one can be chemically synthesized based on a known sequence, and ligated into the vector. For example, the origin of replication of plasmid pBR322 (New England Biolabs, Beverly, MA) is suitable for most gram-negative bacteria, and various viral sources (for example, SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV ), or papillomaviruses such as HPV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (for example, the SV40 origin is often used only because it also contains the virus's early promoter).
A transcription termination sequence is typically located 3 'at the end of a polypeptide coding region and serves to terminate transcription. Commonly, the transcription termination sequence in prokaryotic cells is a fragment rich in G-C followed by a poly-T sequence. although the sequence is easily cloned from a library or even commercially acquired as part of a vector, it can also be easily synthesized using methods for nucleic acid synthesis such as those described herein.
A selectable marker gene encodes a protein necessary for the survival and growth of a host cell that grows in a selective culture medium. Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, for example, ampicillin, tetracycline, or kanamycin for prokaryotic host cells; (b) complement the cell's auxotrophic deficiencies; or (c) supply critical nutrients not available from a complex or defined medium. Specific selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. Advantageously, a neomycin resistance gene can also be used for selection in prokaryotic and eukaryotic host cells.
Other selectable genes can be used to amplify the gene that will be expressed. Amplification is the process in which the genes that are necessary for the production of a protein critical to cell growth or survival are reiterated in tandem on the chromosomes of successive generations of recombinant cells. Examples of selectable markers suitable for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase genes without a promoter. Mammalian cell transformants are placed under selection pressure in which only the transformants are uniquely adapted to survive due to the selectable gene present in the vector. The selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of the selection agent in the medium is successively increased, thus leading to the amplification of the selectable gene and the DNA encoding another gene, such as an antigen binding protein that binds to IL-23. As a result, increased amounts of a polypeptide as an antigen-binding protein are synthesized from the amplified DNA.
A ribosome binding site is commonly necessary for the initiation of mRNA translation and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). The element is typically located 3 'to the promoter and 5' to the coding sequence for the polypeptide to be expressed.
In some cases, such as when glycosylation is desired in a eukaryotic host cell expression system, a person can manipulate the various pre- or pro-sequences to improve glycosylation or yield. For example, a person can alter the peptidase cleavage site of a particular signal peptide, or add prosequences, which can also affect glycosylation. The final product of the protein may have, in position -1 (in relation to the first amino acid of the mature protein), one or more additional amino acids for expression, which may not have been completely removed. For example, the final protein product may have one or two amino acid residues found at the peptidase cleavage site, attached to the amino terminus. Alternatively, the use of some enzyme cleavage sites can result in a slightly truncated form of the desired polypeptide, if the enzyme cuts in such an area in the mature polypeptide.
Expression and cloning will typically contain a promoter that is recognized by the host organism and operably linked to the molecule encoding an IL-23 antigen binding protein. Promoters are non-transcribed sequences located upstream (i.e., 5 ') to the start codon of a structural gene (usually at about 100 to 1,000 bp) that control the transcription of the structural gene. Promoters are conventionally grouped into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of DNA transcription under their control in response to changes in culture conditions, such as the presence or absence of a nutrient or a change in temperature. Constitutive promoters, on the other hand, uniformly transcribe a gene to which they are operationally linked, that is, with little or no control over gene expression. A large number of promoters, recognized by a variety of potential host cells, are well known. A suitable promoter is operably linked to DNA encoding a heavy chain variable region or a light chain variable region of an IL-23 antigen binding protein by removing the source DNA promoter by restriction enzyme digestion and insertion of the desired promoter sequence in the vector.
Promoters suitable for use with yeast hosts are also well known in the art. Yeast enhancers are advantageously used with yeast promoters. Promoters suitable for use with mammalian host cells are well known and include, without limitation, those obtained from virus genomes such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus , hepatitis-B virus and simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, for example, heat-shock promoters and the actin promoter.
Additional promoters that may be of interest include, without limitation: SV40 early promoter (Benoist and Chambon, 1981, Nature 290: 304-310); CMV promoter (Thornsen et al, 1984, Proc. Natl. Acad. U.S.A. 81: 659-663); the promoter contained in the 3 'long terminal repeat of Rous sarcoma virus (Yamamoto et al, 1980, Cell 22: 787797); herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78: 1,444-1,445); promoter and regulatory sequences of the metallothionine gene (Prinster et al, 1982, Nature 296: 39-42); and prokaryotic promoters such as the beta-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75: 3.727-3.731); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80: 21-25). Also of interest are the following animal transcription control regions, which exhibit tissue specificity and have been used in transgenic animals: the elastase I gene control region that is active in pancreatic acinar cells (Swift et al, 1984, Cell 38: 639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50: 399409; MacDonald, 1987, Hepatology 7: 425-515); the insulin gene control region that is active in pancreatic beta cells (Hanahan, 1985, Nature 315: 115-122); the immunoglobulin gene control region that is active in lymphoid cells (Grosschedl et al., 1984, Cell 38: 647-658; Adames et al., 1985, Nature 318: 533-538; Alexander et al, 1987, Mol. Cell, Biol. 7: 1.436-1.444); the control region of mamariod and mouse tumor viruses that is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45: 485-495); the albumin gene control region that is active in the liver (Pinkert et al, 1987, Genes and Devel. 1: 268-276); the alpha-fetoprotein gene control region that is active in the liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5: 1.639-1.648; Hammer et al., 1987, Science 253: 53-58 ); the alpha 1-antitrypsin gene control region that is active in the liver (Kelsey et al., 1987, Genes and Devel. 1: 161-171); the beta-globin gene control region that is active in myeloid cells (Mogram et al., 1985, Nature 315: 338-340; Kollias et al., 1986, Cell 46: 89-94); the myelin basic protein gene control region that is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48: 703-712); the control region of the myosin light chain-2 gene that is active in skeletal muscle (Sani, 1985, Nature 314: 283-286); and the control region of the gonadotropic release hormone gene that is active in the hypothalamus (Mason et al., 1986, Science 234: 1372-1378).
An enhancer sequence can be inserted into the vector to increase transcription by higher eukaryotes. Intenifiers are cis-acting elements of DNA, commonly about 10-300 bp in length, that act on the promoter to increase transcription. Intenifiers are relatively independent of orientation and position, which were found in positions 5 'and 3' to the transcription unit. Several enhancer sequences available from mammalian genes are known (for example, globin, elastase, albumin, alpha-fetus-protein and insulin). Typically, however, a virus intensifier is used. The SV40 enhancer, the cytomegalovirus precocious enhancer, the polyoma enhancer, and the adenovirus enhancers known in the art are examples of enhancement elements for the activation of eukaryotic promoters. Although an enhancer can be positioned in the vector 5 'or 3' to a coding sequence, it is typically located at a 5 'site of the promoter. A sequence encoding a suitable native or heterologous signal sequence (leader sequence or signal peptide) can be incorporated into an expression vector, to promote extracellular secretion of the antibody. The choice of the signal or leader peptide depends on the type of host cells in which the antibody is to be produced, and a heterologous signal sequence can replace the native signal sequence. Examples of signal peptides that are functional in mammalian host cells include the following: the signal sequence for interleukin-7 described in US Patent No. 4,965,195; the signal sequence for interleukin-2 receptor described in Cosman et al, 1984, Nature 312: 768; the interleukin-4 receptor signal peptide described in EP Patent No. 0367 566; the type I interleukin-1 receptor signal peptide described in U.S. Patent No. 4,968,607; the type II interleukin-1 receptor signal peptide described in EP Patent No. 0 460 846.
After the vector has been constructed, the complete vector can be inserted into a host cell suitable for amplification and / or expression of the polypeptide. Transformation of an expression vector into an antigen-binding protein and a selected host cell can be accomplished by well-known methods that include transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-mediated transfection -dextran, or other known techniques. The selected method will be partly a function of the type of host cell to be used. Such methods and other suitable methods are well known to the skilled practitioner and are presented, for example, in Sambrook et al, Molecular Cloning: A Laboratory Manual, 3aed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001).
A host cell, when grown under suitable conditions, synthesizes protein that can subsequently be collected from the culture medium (if the host cell secretes it in the medium) or directly from the host cell that produces it (if it is not secreted). The selection of a suitable host cell will depend on several factors, such as the desired levels of expression, modifications of polypeptide that are desirable or necessary for activity (such as glycosylation or phosphorylation) and ease of folding into a biologically active molecule.
Mammalian cell lines available as hosts for expression are well known in the art and include, without limitation, immortalized cell lines available from the American Type Culture Collection (ATCC), including, without limitation, Chinese hamster ovary (CHO) cells ), HeLa cells, hamster cub kidney (BHK) cells, mamque kidney cells (COS), humanp hepatocellular carcinoma cells (eg, Hep G2), and numerous other cell lines. In certain embodiments, cell lines can be selected by determining which cell lines have high levels of expression and constitutively produce antigen binding proteins with IL-23 binding properties. In another embodiment, a cell line of the B cell line does not produce its own antibody, but it has an ability to produce and secrete a heterologous antibody can also be selected. Use of human IL-23 antigen binding proteins for diagnostic and therapeutic purposes
Antigen-binding proteins are useful for detecting IL-23 in biological samples and identifying cells or tissues that produce IL-23. Antigen-binding proteins that specifically bind IL-23 can be used in the diagnosis and / or treatment of IL-23-related diseases in a patient in need of that. IL-23 antigen binding proteins can be used in diagnostic assays, for example, binding assays to detect and / or quantify IL-23 expressed in blood, serum, cells or tissue. In addition, IL-23 antigen binding proteins can be used to reduce, inhibit, interfere with or modulate one or more biological activities of IL-23 in a cell or tissue. Thus, antigen-binding proteins that bind to IL-23 may have therapeutic use in ameliorating IL-23-related diseases. Indications
The present invention also relates to the use of IL-23 antigen binding proteins for use in the prevention or therapeutic treatment of medical disorders, such as those disclosed herein. IL-23 antigen binding proteins are useful for treating a variety of conditions in which IL-23 is associated with or has a role in contributing to the underlying disease or disorder or contributes to a negative symptom.
Conditions effectively treated by IL-23 antigen binding proteins play a role in the inflammatory response. Such inflammatory disorders include periodontal disease; lung disorders like asthma; skin disorders such as psoriasis, atopic dermatitis, contact dermatitis; rheumatic disorders such as rheumatoid arthritis, progressive systemic sclerosis (scleroderma); disseminated lupus erythematosus; spondyloarthritis including ankylosing spondylitis, psoriatic arthritis, enteropathic arthritis and reactive arthritis. Uveitis is also contemplated, which includes Vogt-Koyanagi-Harada disease, idiopathic anterior and posterior uveitis, and uveitis associated with spondyloarthritis. The use of IL-23 antigen binding proteins is also contemplated for the treatment of autoimmune disorders that include multiple sclerosis; autoimmune myocarditis; type 1 diabetes and autoimmune thyroiditis.
Degenerative conditions of the gastrointestinal system are treatable or preventable with IL-23 antigen binding proteins. Such gastrointestinal disorders include inflammatory bowel disease: Crohn's disease, ulcerative colitis and Celiac disease.
Also included are the use of IL-23 antigen binding proteins in treatments for graft-versus-host disease, and complications such as graft rejection, which result from implantation of a solid organ transplant, such as heart, liver, skin, kidney , lung or other transplants, including bone marrow transplants.
Also provided here are methods for using IL-23 antigen binding proteins to treat various oncological disorders that include various forms of cancer including colon, stomach, prostate, kidney, cervical and ovarian cancer, and lung cancer (SCLC and NSCLC). Also included are solid tumors, which include sarcoma, osteosarcoma, and carcinoma, such as adenocarcinoma and squamous cell carcinoma, esophageal cancer, gastric cancer, gallbladder carcinoma, leukemia, including acute myelogenous leukemia, chronic myelogenous leukemia, myeloid leukemia, myeloid leukemia, myeloid leukemia chronic or acute and hair cell leukemia, and multiple myeloma. Diagnostic methods
The described antigen-binding proteins can be used for diagnostic purposes to detect, diagnose or monitor diseases and / or conditions associated with IL-23. Examples of methods useful in detecting the presence of IL-23 include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
For diagnostic applications, the antigen-binding protein will typically be labeled with a detectable labeling group. Suitable labeling groups include, without limitation, the following: radioisotopes or radionuclides (for example, 3H, 14C 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent groups (for example, FITC, rhodamine, lanthanide matches ), enzyme groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups or predetermined polypeptide epitopes recognized by a secondary reporter (for example, leucine zipper pair sequences , binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the labeling group is coupled to the antigen-binding protein by means of spacer arms of various lengths to reduce potential steric impediment. Various methods for labeling proteins are known in the art and can be used.
Other diagnostic methods are provided for the identification of a cell or cells that express IL-23. In a specific embodiment, the antigen-binding protein is labeled with a labeling group and the binding of the labeled antigen-binding protein to IL-23 is detected. In an additional specific embodiment, the binding of the antigen-binding protein to IL-23 is detected in vivo. In an additional specific embodiment, the IL-23 antigen binding protein is isolated and measured using methodologies known in the art. See, for example, Harlow and Lane, 1988, “Antibodies: A Laboratory Manual”, New York: Cold Spring Harbor (ed. 1991 and periodic supplements); John E. Coligan, ed., 1993, Current Protocols In Immunology, New York: John Wiley & Sons.
Other methods allow the detection of the presence of a test molecule that competes for binding to IL-23 with the supplied antigen binding proteins. An example of such an assay would involve detecting the amount of free antigen binding protein in a solution containing an amount of IL-23 in the presence or absence of the test molecule. An increase in the amount of free antigen-binding protein (i.e., the antigen-binding protein not bound to IL-23) would indicate that the test molecule is capable of competing for binding of IL-23 with the binding protein of IL-23 antigen. In one embodiment, the antigen-binding protein is labeled with a labeling group. Alternatively, the test molecule is labeled and the amount of free test molecule is monitored in the presence and absence of an antigen-binding protein. Treatment methods: pharmaceutical formulations, routes of administration
Pharmaceutical compositions are provided that comprise a pharmaceutically effective amount of one or more antigen binding proteins and a pharmaceutically acceptable excipient, diluent, vehicle, solubilizer, emulsifier, preservative and / or adjuvant. In addition, methods of treating a patient by administering this pharmaceutical composition are included. The term "patient" includes human patients. The terms "treat" and "treatment" encompass the relief or prevention of at least one symptom or other aspect of a disorder, or reduction in the severity of the disease, and the like. The term "pharmaceutically effective amount" or "effective amount" refers to the amount of an IL-23 antigen binding protein determined to produce any therapeutic response in a mammal. Such pharmaceutically effective amounts are easily verified by those skilled in the art.
An antigen-binding protein does not need to complete a cure, or eradicate all symptoms or manifestations of a disease, to constitute a viable therapeutic agent. As it is recognized in the pertinent field, drugs used as therapeutic agents can reduce the severity of a certain disease state, it is not necessary to abolish all manifestations of the disease to be considered as useful therapeutic agents.
Similarly, a prophylactically administered treatment need not be completely effective in preventing the onset of a condition in order to constitute a viable prophylactic agent. Simply reducing the impact of a disease (for example, by reducing the number or severity of six symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or by reducing the likelihood that the disease occur or worsen in an individual is sufficient. Certain methods provided herein comprise administering to an patient an IL-23 antagonist (for example, the antigen binding proteins disclosed herein) in an amount and for a time sufficient to induce sustained improvement over the baseline level of a indicator that reflects the severity of the particular disorder.
As is understood in the relevant field, pharmaceutical compositions comprising the molecules of the invention are administered to a patient in a manner suitable for the indication. The pharmaceutical compositions can be administered by any suitable technique, including, without limitation, parenterally, topically or by inhalation. If injected, the pharmaceutical composition can be administered, for example, by intra-articular, intravenous, intramuscular, intralesional, intraperitoneal or subcutaneous route, by bolus injection or continuous infusion. Localized administration, for example, in a place of disease or injury, is contemplated, as are transdermal and sustained release by implants. Inhalation delivery includes, for example, nasal or oral inhalation, the use of a nebulizer, inhalation of the aerosolized antagonist, and the like. Other alternatives include eye drops; oral preparations, including pills, syrups, lozenges or chewing gum; and topical preparations such as lotions, gels, sprays and ointments.
The use of antigen binding proteins in ex vivo procedures is also contemplated. For example, a patient's blood or other body fluid can be brought into contact with an antigen-binding protein that binds IL-23 ex vivo. The antigen binding protein can be attached to any suitable insoluble matrix or solid support material.
Advantageously, antigen binding proteins are administered in the form of a composition that comprises one or more additional components such as, for example, a physiologically acceptable vehicle, excipient or diluent. Optionally, the composition additionally comprises one or more physiologically active agents for combination therapy. A pharmaceutical composition can comprise an IL-23 antigen-binding protein together with one or more substances selected from the group consisting of a buffer, an antioxidant, such as ascorbic acid, a low molecular weight polypeptide (such as those that have less than 10 amino acids), a protein, an amino acid, a carbohydrate such as glucose, sucrose or dextrins, a chelating agent such as EDTA, glutathione, a stabilizer and an excipient. Neutral buffered saline or saline solution mixed with specific serum albumin are examples of suitable diluents. According to the appropriate industry standards, preservatives, such as benzyl alcohol, can also be added. The composition can be formulated as a lyophilizate using appropriate excipient solutions (for example, sucrose) as diluents. Suitable components are non-toxic to the recipients at the dosages and concentrations used. Additional examples of components that can be used in pharmaceutical formulations are presented in any edition of "Remington's Pharmaceutical Sciences", including 2nd Edition (2005), Mack Publishing Company, Easton, PA.
Kits for use by medical professionals include an IL-23 antigen binding protein and a label or other instructions for use in the treatment of any of the conditions discussed here. In one embodiment, the kit includes a sterile preparation of one or more IL-23 binding antigen binding proteins, which can be in the form of a composition, as disclosed above, and can be in one or more vials.
Dosages and frequency of administration may vary according to factors such as the route of administration, the particular antigen binding proteins employed, the nature and severity of the disease to be treated, whether the condition is acute or chronic and the size and general condition of the individual. Appropriate dosages can be determined by procedures known in the relevant art, for example, in clinical experiments that may involve dose escalation studies.
A typical dosage can range from about 0.1 μg / kg to about 30 mg / kg or more, depending on the factors mentioned above. In specific embodiments, the dosage can vary from 0.1 μg / kg to about 30 mg / kg, optionally from 1 μg / kg to about 30 mg / kg, optionally from 10 μg / kg to about 10 mg / kg , optionally from about 0.1 mg / kg to 5 mg / kg or, optionally, from about 0.3 mg / kg to 3 mg / kg.
The dosing frequency will depend on the pharmacokinetic parameters of the particular human IL-23 antigen binding protein used in the formulation. Typically, a physician will administer the composition until a dosage is reached that achieves the desired effect. The composition can, therefore, be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion through an implantation device or catheter. Appropriate dosages can be verified using appropriate dose-response data. An IL-23 antigen binding protein of the invention can be administered, for example, once or more than once, for example, at regular intervals over a period of time. In particular embodiments, an IL-23 antigen-binding protein is administered over a period of at least a month or more, for example, for one, two, or three months or even indefinitely. For the treatment of chronic conditions, long-term treatment is usually the most effective. However, for the treatment of acute conditions, administration for shorter periods, for example, one to six weeks, may be sufficient. In general, the antigen-binding protein is administered until the patient shows a medically relevant degree of improvement over the baseline level for the chosen indicator or indicators.
It is contemplated that an IL-23 antigen-binding protein can be administered to the patient in an amount and for a time sufficient to induce an improvement, preferably a sustained improvement, in at least one indicator that reflects the severity of the disorder being treated. Various indicators that reflect the extent of the patient's illness, disease or condition can be assessed to determine whether the amount and timing of treatment is sufficient. These indicators include, for example, clinically recognized indicators of disease severity, symptoms or manifestations of the disorder in question. In one embodiment, an improvement is considered to be sustained if the individual exhibits improvement on at least two occasions separated by two to four weeks. The degree of improvement is usually determined by a doctor, who makes this determination based on signs, symptoms, biopsies or other test results, and who can also use questionnaires that are applied to the individual, for example, developed quality of life questionnaires for a certain disease.
Particular embodiments of methods and compositions of the invention involve the use of an IL-23 antigen binding protein and one or more additional IL-23 antagonists, for example, two or more antigen binding proteins of the invention, or a protein antigen binding agents of the invention and one or more other IL-23 antagonists. Also provided are IL-23 antigen binding proteins administered alone or in combination with other agents useful for treating the condition that the patient suffers from. Examples of these agents include both proteinaceous and non-proteinaceous drugs. These agents include therapeutic moieties that have anti-inflammatory properties (for example, non-steroidal anti-inflammatory agents, steroids, immunomodulators and / or other cytokine inhibitors such as those that antagonize, for example, IFN-Y, GM-CSF, IL- 6, IL-8, IL-17, IL-22 and TNFs), or an IL-23 antigen binding protein and one or more other treatments (for example, surgery, ultrasound or effective treatment to reduce inflammation ). When multiple therapeutic substances are co-administered, dosages can be adjusted accordingly, as is recognized or known in the relevant art. Useful agents that can be combined with IL-23 antigen binding proteins include those used to treat, for example, Crohn's disease or ulcerative colitis, for example, aminosalicylate (for example, mesalamine), corticosteroids (including prednisone), antibiotics , for example, metronidazole or ciprofloxacin (or other antibiotics useful for the treatment, for example, of patients suffering from fistulas), and immunosuppressants such as, for example, azathioprine, 6-mercaptopurine, methotrexate, tacrolimus and cyclosporine. These agents can be administered orally or by another route, for example, through suppository or enema. Agents that can be combined with IL-23 binding proteins in the treatment of psoriasis include corticosteroids, calcipotriene and other derivatives of vitamin D, acetretine and other derivatives of retinoic acid, methotrexate, tacrolimus and cyclosporine used topically or systemically. These agents can be administered simultaneously, consecutively, alternatively or according to any other regimen that allows the total evolution of the therapy to be effective.
In addition to human patients, IL-23 antigen binding proteins are useful in the treatment of non-human animals, for example, pets (dogs, cats, birds, primates, etc.), farm animals (horses, cattle, sheep, pigs, birds, etc.). In such cases, an appropriate dose can be determined according to the animal's body weight. For example, a dose of 0.2-1 mg / kg can be used. Alternatively, the dose is determined according to the animal's surface area, with an exemplary dose ranging from 0.1-20 mg / m2, or more preferably, 5-12 mg / m2. For small animals, for example, dogs or cats, an appropriate dose is 0.4 mg / kg. The IL-23 antigen binding protein (preferably constructed from genes derived from the recipient species) is administered by injection or by another suitable route one or more times a week until the condition of the animal is improved, or it can be improved. administered indefinitely.
The following examples, which include the experiments performed and the results obtained, are provided for illustrative purposes only, and should not be considered as limiting the scope of the appended claims. EXAMPLES Example 1 Generation of antibodies to human IL-23
XenoMouseTM technology (Amgen, Thousand Oaks, CA) has been used to develop human monoclonal antibodies that recognize and inhibit activity of native human IL-23, while sparing human IL-12. The antibodies also recognize and inhibit recombinant Cynomolgus IL-23, but do not recognize murine or rat IL-23.
Antibodies were selected for recognition and complete inhibition of native human IL-23 obtained from human monocyte-derived dendritic cells (MoDCs) using the STAT-luciferase reporter assay described below. Human monocytes were isolated from peripheral blood mononuclear cells from healthy donors using negative selection (“Monocyte Isolation Kit II”, Miltenyi Biotec, Auburn, CA). MoDCs were generated by monocyte culture with human GM-CSF (50 ng / ml) and human IL-4 (100 ng / ml) for 7 days in RPMI 1640 with 10% fetal bovine serum. MoDCs were then washed twice with PBS followed by stimulation with human CD40L (1 μg / ml) for an additional 48 hours. The supernatant of MoDCs stimulated with CD40L contains IL-23, IL-12 and IL-12 / 23p40. ELISAs are used to determine the amount of IL-12p70 (R&D System, Minneapolis, MN), IL-23 (eBiosciences, San Diego, CA) and IL-12 / 23p40 (R&D Systems). The STAT-luciferase assay responds to IL-23 and not to IL-12 or free IL-12 / 23p40 and therefore the assay could be used with crude supernatants to assess IL-23 activity. For use in the NK cell assay, described below, the crude supernatant of native human IL-23 was purified using an IL-23 affinity column followed by size exclusion chromatography. The concentration was determined using an ELISA specific for IL-23 (eBiosciences).
Purified antibody supernatants were also tested against recombinant human IL-23 (rhu) and recombinant Cynomolgus IL-23 (cyno) in the STAT-luciferase assay. Of the antibodies tested that completely inhibited recombinant human IL-23, only half of those antibodies recognized and completely inhibited native human IL-23. Recognition and complete inhibition of recombinant human IL-23 were not predictive, nor were they correlated with recognition and complete inhibition of native human IL-23. As shown in FIGURES 1A and 1B, of the antibody supernatants that completely inhibited recombinant human IL-23, only half of those antibodies completely inhibited native human IL-23. Those antibodies that recognized and completely inhibited native human IL-23 were selected for further characterization. EXAMPLE 2 Functional tests a) STAT-luciferase assay
IL-23 is known to bind to its heterodimeric receptor and signal through JAK2 and Tyk2 to activate STAT 1, 3, 4 and 5. In this assay, cells transfected with a STAT / luciferase reporter gene are used to assess the ability of IL-23 antibodies to inhibit IL-23-induced bioactivity.
Chinese hamster ovary cells expressing human IL-23 receptor are transiently transfected with STAT-luciferase reporter overnight. IL-23 antibodies are serially diluted (12 points of 1: 4 serial dilutions starting at 37.5 μg / ml) in 96-well plates. Native human IL-23 (the preparation method is described in Example 1) is added to each well at a concentration of 2 ng / ml and incubated at room temperature for 15-20 minutes. The transiently transfected cells are added (8 x 103 cells) to a final volume of 100 μl / well and incubated for 5 hours at 37 ° C, 10% CO2. After incubation, cells are lysed using 100 μl / well of Glo lysis buffer (1x) (Promega, Madison, Wisconsin) at room temperature for 5 minutes. Fifty microliters of cell lysate are added to a 96-well plate along with 50 μl of Bright-Glo luciferase substrate (Promega) and read on a luminometer.
Statistical analysis can be performed using the GraphPad PRISM software (GraphPad Software, La Jolla, CA). The results can be expressed as the mean ± standard deviation (SD).
As noted in TABLE 5, all IL-23 antibodies potently and completely inhibited STAT / luciferase induced by human native IL-23 in a dose-dependent manner. The antibodies also potently and completely inhibited recombinant human IL-23 (rhu) and recombinant Cynomolgus IL-23 (cyno). All antibodies had IC 50 values in the picomolar range. TABLE 5. Table of mean IC50 values (pM) for IL-23 antibodies in the STAT-luciferase assay.




b) NK cell assay
IL-23 is known to act on natural killer cells to induce expression of pro-inflammatory cytokines, for example, interferon y (IFNy). In that assay, primary human natural 5 killer (NK) cells are used to assess the ability of IL-23 antibodies to inhibit IL-23-induced IFNy activity in cells that express the native receptor for human IL-23.
NK cells are isolated from multiple human donors 10 by means of negative selection (“NK Cell Isolation Kit”, Miltenyi Biotec, Auburn, CA). Purified NK cells (1 x 106 cells / ml) are added to 6-well plates in RPMI 1640 plus complete medium of 10% fetal bovine serum supplemented with recombinant human IL-2 (10 ng / ml, R&D Systems, 15 Minneapolis, MN), up to a final volume of 10 ml / well. The cells are cultured for 7 days at 37 ° C, 5% CO2. IL-2 activated NK cells are then stimulated with rhuIL-23 or cyno IL-23 (10 ng / ml) and recombinant human IL-18 (20 ng / ml, R&D Systems, Minneapolis, MN) in the presence of serial dilutions (11 points of 1: 3 serial dilutions starting at 3 μg / ml) of IL-23 antibodies for 24 hours. IFNY levels are measured in the supernatant by ELISA for IFNY (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.
Statistical analysis can be performed using the GraphPad PRISM software. The results can be expressed as the mean ± standard deviation (SD).
As seen in TABLE 6, all antibodies potently inhibited the expression of IFNY induced by rhuIL-23 and cyno IL-23 in NK cells in a dose-dependent manner. All antibodies had IC 50 values in the picomolar range. The assay was performed on a subset of antibodies using native human IL-23 (30 μg / ml; the preparation method is described in Example 1) and rhuIL-18 (40 ng / ml, R&D Systems) and generated the results shown in TABLE 6. Consistent with the selection for IL-23-specific antibodies, these anti-IL-23 antibodies had no effect on IL-12-stimulated IFNY production in NK cells using the assay described above, while an IL neutralizing antibody -12p35-specific, mAb219 (R&D Systems, Minneapolis, MN), potently inhibited recombinant human IL-12. TABLE 6. Table of mean IC50 values (pM) for IL-23 antibodies in the NK cell assay.

c) Human whole blood assay
Human whole blood is collected from multiple healthy donors using Refludan® (Bayer Pittsburgh, PA) as an anticoagulant. The final concentration of Refludan® in whole blood is 10 μg / ml. A stimulation mixture of rhuIL-23 or cyno IL-23 (final concentration of 1 ng / ml) + rhuIL-18 (final concentration of 20 ng / ml) + rhuIL-2 (final concentration of 5 ng / ml) in RPMI 1640 + 10% FBS is added to a 96-well plate, with a final volume of 20 μl / well. Serially diluted IL-23 antibodies (11 points of 1: 3 serial dilutions starting from 3 μg / ml) are added at 20 μl / well and incubated with the stimulation mixture for 30 minutes at room temperature. Whole blood is then added (120 μl / well) and the final volume adjusted to 200 μl / well with RPMI 1640 + 10% FBS. The final concentration of whole blood is 60%. The plates are incubated for 24 hours at 37 ° C, 5% CO2. Cell-free supernatants are collected and IFNY levels are measured from the supernatants by ELISA for IFNY (R&D Systems) according to the manufacturer's instructions.
Statistical analysis can be performed using the GraphPad PRISM software. The results can be expressed as the mean ± standard deviation (SD).
As seen in TABLE 7, all antibodies potently inhibited the expression of IFNY induced by rhuIL-23 and induced by cyno-IL-23 in whole blood cells in a dose-dependent manner. All antibodies had IC 50 values in the picomolar range. TABLE 7. Table of mean IC50 values (pM) for IL-23 antibodies in the IFNy human whole blood assay
d) IL-22 assay
IL-23 is known to be a potent inducer of pro-inflammatory cytokines. IL-23 acts on activated and memory T cells and promotes the survival and expansion of Th17 cells that produce pro-inflammatory cytokines, including IL-22. In this assay, human whole blood is used to assess the ability of IL-23 antibodies to inhibit IL-23-induced IL-22 production.
The whole blood assay is performed in the same manner as described above, with the modification of the use of rhuIL-23 or cyno IL-23 at 1 ng / ml and rhuIL-18 at 10 ng / ml to induce the production of IL-22. The concentration of IL-22 is determined by ELISA for IL-22 (R&D Systems, Minneapolis, MN).
As seen in TABLE 8, the antibodies potently inhibited the production of IL-22 induced by rhuIL-23- and cyano-induced IL-23 in whole blood cells in a dose-dependent manner. All antibodies had IC 50 values in the picomolar range. TABLE 8. Table of mean IC50 values (pM) for IL-23 antibodies in the human IL-22 whole blood assay.
Example 3 Determination of equilibrium dissociation constant (KD) for anti-IL-23 antibodies using KinExA technology
The binding affinity of rhuIL-23 to IL-23 antibodies is assessed using a kinetic exclusion assay (KinExA assay, Sapidyne Instruments, Inc., Boise, ID). Sepharose 4 fast flowing blood cells activated by normal human serum (NHS) (Amersham Biosciences, part of GE Healthcare, Uppsala, Sweden), are pre-coated with rhuIL-23 and blocked with 1 M 10 mg / ml Tris buffer of BSA. Fifty pM of IL-23 antibody is incubated with rhuIL-23 (12 points of 1: 2 dilutions starting from 800 pM) at room temperature for 72 hours, before being processed through the rhuIL-coated Sepharose globules. 23. The amount of antibody bound to the globule was quantified by goat anti-human Fc antibody with fluorescent labeling (Cy5) (Jackson Immuno Research, West Grove, PA). The binding signal is proportional to the amount of free antibody in equilibrium.
The equilibrium dissociation constant (KD) and the association rate (Kon) are obtained by adjusting the curve using the KinExA Pro software. The dissociation rate (Koff) is derived from: KD = Koff / Kon.
As noted in TABLE 9, antibodies have high affinity for binding to human IL-23. All had KD values in the low to sub-pM range. TABLE 9. KD (pM), Kon (1 / MS) and Koff (1 / s) rates

Example 4 Structure determination using X-ray crystallography
One way to determine the structure of an antigen-antibody complex is by using X-ray crystallography; see, for example, Harlow and Lane: “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990), page 23. The crystal structure of IL-23 has been determined (see Lupardus and Garcia, J. Mol. Biol., 2008, 382: 931-941) and the crystal structure of an IL-23 / Fab complex was revealed (see Beyer et al J. Mol. Biol., 2008. 382 (4): 942 -55). Structural determination of IL-23 with Fab antibody fragments claimed here was obtained using X-ray crystallography. Protein for crystallization
A recombinantly derived human IL-23 heterodimer was used for crystallization studies (see Beyer et al, supra). The sequence of the human p19 subunit was composed of residues 20-189 of the Seq Id. N °: 145, by the signal sequence of the Seq. No. 154 and a 6-His tag from the C Id. Terminal of Seq. No. 155. The human p40 subunit sequence was mutated from asparagine to glutamine at position 222 of the Seq. N °: 147 in order to avoid glycosylation at that site (Beyer, et al, supra).
Fabs derived from Antibody B and Antibody E were expressed in an IgG1 framework that incorporated a caspase cleavage site. Fabs were processed by means of protease cleavage. Complex formation and crystallization
The Antibody B IL-23-Fab complex was made by mixing a 2X molar excess of antibody B Fab with the human heterodimeric IL-23 described above. The complex was purified by size exclusion chromatography to remove excess Antibody B Fab and concentrated to approximately 12 mg / ml for crystallization. The IL-23-Fab complex of Antibody B crystallized from 0.1 M Hepes pH 7, 8% PEG 8000.
The IL-23-Fab Antibody E complex was made by mixing a 2X molar excess of antibody E Fab with the human heterodimeric IL-23 described above. The complex was methylated using a JBS Methylation Kit according to the manufacturer's instructions (Jena Bioscience, Jena, Germany). The complex was then treated with PNGase to deglycosylate the protein. After these treatments, the complex was purified by size exclusion chromatography to remove excess Antibody E Fab and concentrated to 13.5 mg / ml for crystallization. The IL-23-Fab complex of Antibody E crystallized from 0.1 M Tris pH 8.5, 0.2 M magnesium chloride, PEG 400015%. Data collection and structure determination
IL-23-Fab antibodies from Antibody B grew in the P21 space group with unit cell dimensions a = 70.93, b = 71.27, c = 107.37 Â, β = 104.98 ° and refract until resolved of 2.0 Â. The structure of IL-23-Fab from Antibody B was solved by molecular substitution with the MOLREP program (“CCP4, The CCP4 suite: programs for protein crystallography”. Acta Crystallogr. D. Biol. Crystallogr., 1994. 50 (Part 5 ): pages 760-3) using the IL-23 framework (Beyer et al supra) as the starting research model. Keeping the IL-23 solution fixed, an antibody variable domain was used as a research model. Keeping the IL-23-antibody variable domain solution fixed, an antibody constant domain was used as a research model. The complete structure was improved with several rounds of construction of the model with Quanta and refinement with cnx (Brunger, et al., Acta Crystallogr. D. Biol. Crystallogr., 1998, 54 (Part 5): pages 905-21).
The distances between atoms of the protein were calculated using the PyMOL program (DeLano, W.L. “The PyMOL Graphics System”. Palo Alto, 2002) (Schrodinger, LLC; New York, NY)). Amino acids were chosen if at least one atom was located within the distance threshold required for the partner protein.
Helix A, B, C and D boundaries of the p19 subunit of IL-23 when bound to antibody B Fab include residues 28-47 of helix A, residues 86-105 of helix B, residues 119-134 of helix C and residues 154-187 of helix D of Seq. No. 145.
The regions of interaction in the IL-23 p19 subunit when bound to antibody B Fab include residues within Ser46-Glu58, Glu112-Glu123 and Pro155-Phe163 of Seq Id. No. 145.
Amino acid residues of the p19 subunit of IL-23 with atoms of 4 Å or less of antibody B Fab include Ser46, Ala47, His48, Pro49, Leu50, His53, Met54, Asp55, Glu58, Pro113, Ser114, Leu115, Leu116, Pro120 , Val121, Trp156, Leu159, Leu160, Arg162 and Phe163 of the Seq. No. 145. Amino acid residues of IL-23 p19 with atoms between 4 Å and 5 Å of antibody B Fab include Val51, Arg57, Glu112, Asp118, Ser119, Gln123, Pro155 of Seq Id. No. 145.
Amino acid residues of IL-23 p40 subunit with atoms 4 µm or less of antibody B Fab include Glu122 and Lys 124 of Seq Id. N °: 147.
Amino acid residues of antibody heavy Fab B chain with atoms of 4 Å or less of the IL-23 heterodimer include Gly32, Gly33, Tyr34, Tyr35, His54, Asn58, Thr59, Tyr60, Lys66, Arg101, Gly102, Phe103, Tyr104 and Tyr105 from Seq. No. 46. Amino acid residues of antibody heavy Fab B with atoms <5 Å from the IL-23 heterodimer include Ser31, Gly32, Gly33, Tyr34, Tyr35, His54, Ser56, Asn58, Thr59, Tyr60, Lys66 , Arg101, Gly102, Phe103, Tyr104 and Tyr105 from Seq. No. 46.
Amino acid residues of antibody B Fab light chain with atoms of 4 Å or less of the IL-23 heterodimer include Ser30, Ser31, Trp32, Tyr49, Ser52, Ser53, Ala91, Asn92, Ser93, Phe94 and Phe96 of Id. of Seq. N °: 15. Amino acid residues of antibody B Fab light chain with <5 Â atoms of the IL-23 heterodimer include Ser30, Ser31, Trp32, Tyr49, Ala50, Ser52, Ser53, Ser56, Ala91, Asn92, Ser93 , Phe94 and Phe96 from the Seq. N °: 15
The crystals of the IL-23-Fab complex of Antibody E grew in the space group P2221 with unit cell dimensions a = 61.60, b = 97.59, c = 223.95 Â and refract to a resolution of 3.5 THE. The structure of the IL-23-Fab complex of Antibody E was solved by molecular substitution with the Phaser program (CCP4, supra) using the structure of IL-23, an antibody variable domain and an antibody constant domain as the three models starting search, as described above. The complete structure was improved with several rounds of construction of the model with Quanta and refinement with cnx (Brunger, et al., Supra). The constant domain of antibody E Fab was left out of the final refined structure due to the very low electron density for that portion of the protein.
The regions of interaction on the IL-23 p19 subunit identified when bound to antibody E Fab include residues within Ser46-His53, Glu112-Val120 and Trp156-Phe163 of Seq Id. No. 145.
Amino acid residues of IL-23p19 with atoms of 4 µm or less of antibody E Fab include Ser46, Ala47, His48, Pro49, Leu50, Glu112, Pro113, Ser114, Leu115, Leu116, Pro117, Asp118, Ser119, Pro120, Trp156 , Leu159, Leu160 and Phe163 of the Seq. No. 145. The amino acid residues of IL-23p19 with atoms between 4 Šand 5 Šof antibody E Fab include His53 of Seq Id. No. 145.
Amino acid residues of IL-23p40 with atoms of 4 µm or less of antibody E Fab include Lys121, Glu122, Pro123 and Asn 125 of Seq Id. N °: 147.
Amino acid residues of the antibody E Fab heavy chain with atoms of 4 Å or less of the IL-23 heterodimer include Gly26, Phe27, Thr28, Ser31, Tyr53, Tyr59, Tyr102, Ser104, Ser105, Trp106, Tyr107 and Pro108 do Seq. No. 31. Amino acid residues from antibody E heavy Fab with atoms <5 Å from the IL-23 heterodimer include Gln1, Gly26, Phe27, Thr28, Ser30, Ser31, Tyr32, Trp52, Tyr53, Tyr59, Arg100 , Tyr102, Thr103, Ser104, Ser105, Trp106, Tyr107 and Pro108 of the Seq. N °: 31.
Amino acid residues of antibody E Fab light chain with atoms of 4 Å or less of the IL-23 heterodimer include Ala31, Gly32, Tyr33, Asp34, Tyr51, Gly52,
Asn55, Lys68 and Tyr93 from Seq. No.: 1. Antibody Fab light chain amino acid residues with <5 Â atoms of the IL-23 heterodimer include Thr29, Ala31, Gly32, Tyr33, Asp34, Tyr51, Gly52, Asn55, Lys68, Tyr93 and Trp100 of the Seq. N °: 1. Example 5 Determination of contact residues of the IL-23-Antibody complex by means of differences in surface area accessible to the solvent
The contact residues in the paratope (the portion of the antibody that recognizes the antigen) and the portion of the antigen that binds when bound by the paratope in a human IL-23-Antibody Fab complex and an IL- 23 human-Fab Antibody E were determined using differences in the surface area accessible by the solvent. Calculations of the surface area accessible to the solvent were performed using “Molecular Operating Environment” (Chemical Computing Group, Montreal, Quebec).
The differences in the solvent accessible surface area of the paratope residues in the IL-23-Fab complex of Antibody B were calculated by adjusting the residues of the antibody B Fab as the desired set. The structural information obtained in Example 4 for the Antibody B IL-23-Fab complex was used and the surface areas accessible to the solvent from the residue of the antibody B Fab amino acid residues in the presence of the IL-23 heterodimer were calculated and represent the “boundary areas” for the group.
The surface areas accessible to the solvent from the residue of each of the Fab B antibody residues in the absence of IL-23 antigen were calculated and represent the “free areas” of the set.
The “boundary areas” were then subtracted from the “free areas” resulting in the “difference in surface area exposed to the solvent” for each residue in the set. Residues of antibody B Fab that had no change in surface area, or a zero difference, had no contact with residues of IL-23 antigen when complexed. Residues of antibody B Fab that had a difference value> 10 A2 were considered to be in significant contact with residues in the IL-23 antigen such that these Antibody B Fab residues were at least partially to completely occluded when antibody B Fab was linked to human IL-23. This set of Antibody Fab residues constitutes the “covered stretch”, the residues involved in the interface structure when Antibody Fab B is linked to human IL-23; see Tables 10 and 11. Antibody Fab residues in that covered stretch may not be involved in binding interactions with IL-23 antigen residues, but mutating any single residue within the covered stretch could introduce energy differences that would impact Antibody B Fab binding to human IL-23. With the exception of Tyr49, all residues are located in the CDR regions of antibody B Fab light and heavy chains. These residues were also within 5 Å or less of the IL-23 antigen when bound to antibody B Fab, such as described in Example 4. TABLE 10. Differences in surface area of solvent accessibility for Antibody Fab light chain B. AHO number of Residue position Area difference
TABLE 11. Differences in surface area of solvent accessibility for Fab heavy chain Antibody B.


The differences in the solvent accessible surface area of the residues in the IL-23-Fab complex of Antibody E were calculated as described above. Residues of antibody E Fab that had a difference value> 10 Â2 were considered to be in significant contact with residues in the IL-23 antigen and those Antibody E Fab residues were at least partially to completely occluded when Fab antibody E was linked to human IL-23. This set of Antibody E Fab residues constitutes the covered stretch, the residues involved in the interface structure when antibody E Fab is linked to human IL-23; see Tables 12 and 13. The residues of antibody E Fab in that covered stretch may not be involved in binding interactions with IL-23 antigen residues, but mutating any single residue within the covered stretch could introduce energy differences that would impact the binding of Antibody E Fab to human IL-23. For the most part, these residues from the covered stretch were located within the CDR regions of the heavy and light chains of antibody E Fab. These residues were also within 5Å or less of the IL-23 antigen when bound to antibody E Fab , as described in Example 4. TABLE 12. Differences in surface area of solvent accessibility for Antibody E Fab light chain.
TABLE 13. Differences in the surface area of solvent accessibility for Antibody E Fab heavy chain.

The differences in the solvent-accessible surface area of the IL-23 heterodimer-bound portion of the antibody B Fab paratope were calculated by adjusting the residues of the IL-23 heterodimer as the desired set. The structural information obtained in Example 4 for the Fab complex of antibody B-IL-23 was used and the surface area accessible to the solvent from the residue of the amino acid residues of the IL-23 heterodimer in the presence of the antibody B Fab was calculated and represent the boundary areas for the whole.
The surface areas accessible to the residue solvent of each of the residues of the IL-23 heterodimer in the absence of antibody B Fab were calculated and represent the free areas of the pool.
As described above, the boundary areas were subtracted from the free areas, resulting in the difference in surface area exposed to the solvent for each IL-23 residue. Residues of the IL-23 heterodimer that had no change in surface area, or a zero difference, had no contact with residues of antibody B Fab when complexed. Residues of the IL-23 heterodimer that had a difference value Â2 were considered to be in significant contact with residues of antibody B Fab and those residues of the IL-23 heterodimer were at least partially to completely occluded when the IL heterodimer -23 human was linked to antibody B Fab. This set of residues from the IL-23 heterodimer constitutes the covered stretch, the residues involved in the interface structure when the human IL-23 heterodimer is linked to antibody E Fab; see Table 14. Not all residues of the IL-23 heterodimer in that covered stretch may be involved in binding interactions with residues on antibody B Fab, but mutating any single residue within the covered stretch could introduce energy differences that would impact Antibody B Fab binding to human IL-23. These residues are also within 4 Å or less of antibody B Fab, as described in Example 4. TABLE 14. Solvent accessibility surface area differences for IL-23 heterodimer residues.

The differences in the solvent accessible surface area of the IL-23 heterodimer portion bound by the antibody E Fab paratope were calculated as described above. Residues of the IL-23 heterodimer that had a difference value> 10 Â2 were considered to be in significant contact with residues of antibody E Fab, and those residues of the IL-23 heterodimer were at least partially to completely occluded when the human IL-23 heterodimer was linked to antibody E Fab. This set of residues from the IL-23 heterodimer constitutes the covered stretch, the residues involved in the interface structure when the human IL-23 heterodimer is linked to the Fab of antibody E; see Table 15. Not all residues of the IL-23 heterodimer on this covered stretch can be involved in binding interactions with residues on antibody E Fab, but mutating any single residue within the covered stretch could introduce energy differences that would impact the binding of Antibody E Fab to human IL-23. These residues are also within 5 Å or less of antibody E Fab, as described in Example 4. TABLE 15. Solvent accessibility surface area differences for IL-23 heterodimer residues.

权利要求:
Claims (12)
[0001]
1. Recombinant isolated antigen-binding protein that binds IL-23 characterized by comprising at least one heavy chain variable region comprising: a CDRH1 of Seq Id. No.: 91; a CDRH2 of Seq Id. No. 92; and a CDRH3 of Seq Id. No.: 93; and at least one light chain variable region comprising: a CDRL1 of Seq Id. No. 62; a CDRL2 of Seq Id. No.: 63; and a CDRL3 of Seq Id. N °: 64.
[0002]
2. Recombinant isolated antigen-binding protein that binds IL-23 characterized by comprising a heavy chain variable region comprising amino acid residues 31-35, 50-65 and 99-113 of Seq. No.: 31; and a light chain variable region comprising amino acid residues 23-36, 52-58 and 91-101 of Seq. N °: 1.
[0003]
3. Isolated recombinant antigen-binding protein binding IL-23 characterized by comprising a heavy chain variable region of Seq. No.: 31; and a light chain variable region of Seq Id. N °: 1.
[0004]
4. Recombinant isolated antigen binding protein according to any one of claims 1 to 3, characterized by the fact that said antigen binding protein has at least one property selected from the group consisting of: a) reduction of antigen activity Human IL-23; b) reduction in the production of a pro-inflammatory cytokine; c) binding to human IL-23 with a KD <5x10-8 M; d) have a Koff rate <5 x 10—6 1 / s; and e) have an IC 50 <400 pM.
[0005]
Pharmaceutical composition characterized by comprising at least one recombinant antigen binding protein, as defined in any one of claims 1 to 3, and a pharmaceutically acceptable excipient.
[0006]
6. Pharmaceutical composition characterized by comprising at least one recombinant antigen binding protein, as defined in claim 4, and a pharmaceutically acceptable excipient.
[0007]
7. Recombinant isolated nucleic acid molecule characterized by the fact that the nucleic acid encoding the variable region of the heavy chain is Seq. No. 32 and the nucleic acid encoding the variable region of the light chain is Seq. N °: 2.
[0008]
8. Recombinant isolated nucleic acid molecule according to claim 7, characterized by the fact that said nucleic acid molecule is operably linked to a control sequence.
[0009]
9. Recombinant vector characterized by comprising a nucleic acid molecule as defined in claim 7.
[0010]
10. Method of making the recombinant antigen binding protein that binds IL-23, as defined in any one of claims 1 to 4, characterized by comprising the step of collecting said recombinant antigen binding protein from a host cell, wherein the host cell comprises 5 nucleic acid that encodes both variable regions of the heavy chain and the light chain of the antigen binding protein.
[0011]
11. Use of at least one isolated recombinant antigen binding protein, as defined in any one of claims 1 to 4, characterized by 10 being for the manufacture of a medicament for the treatment or prevention of a condition associated with IL-23 in a patient.
[0012]
12. Use of at least one recombinant antigen-binding protein, as defined in any one of claims 1 to 4, characterized by being for the manufacture of a medicament for reducing IL-23 activity in a patient.

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法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-04-24| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-06-25| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2019-11-05| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-05-26| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2020-09-15| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-12-15| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 10 (DEZ) ANOS CONTADOS A PARTIR DE 15/12/2020, OBSERVADAS AS CONDICOES LEGAIS. |

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2021-05-25| B16C| Correction of notification of the grant [chapter 16.3 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 26/10/2010 OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF |

优先权:

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申请号 | 申请日 | 专利标题

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US25498209P| true| 2009-10-26|2009-10-26|

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US61/254,982|2009-10-26|

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US38128710P| true| 2010-09-09|2010-09-09|

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US61/381,287|2010-09-09|

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PCT/US2010/054148|WO2011056600A1|2009-10-26|2010-10-26|Human il-23 antigen binding proteins|

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