Anti-interleukin-23 p19 antibodies and methods of use thereof

Abstract

summary The present disclosure provides antibodies and antibody fragments thereof that bind to IL-23p19. The antibodies and antibody fragments thereof of the present disclosure can modulate the biological activity of the IL-23 receptor signaling axis and are therefore useful for treating immune-mediated inflammatory disorders.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This international patent application claims priority to U.S. Provisional Application No. 62 / 951,231, filed December 20, 2019, all of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on December 16, 2019, is named "122863-5002-WO_NVRB-004-001_ST25.TXT" and is 13 kilobytes in size.

[0003] Field The present disclosure relates generally to antibodies and antibody fragments thereof that bind to the p19 subunit of interleukin-23. The antibodies are useful for treating immune-mediated inflammatory disorders, autoimmune diseases, or cancer. [Background technology]

[0004] The interleukin-12 (IL-12) family of regulatory cytokines includes a unique group of cytokines (IL-12, IL-23, IL-27, IL-35, and IL-39) that contain covalently linked heterodimeric subunits. Heterodimeric IL-12 family cytokine members consist of an α chain (p19, p28, or p35) and a β chain (p40 or Ebi3).

[0005] IL-23 is a heterodimeric cytokine containing a unique p19 subunit linked to a p40 subunit shared with IL-12. The primary sources of IL-23 are tissue-resident or recruited dendritic cells and macrophages. The biological effects of IL-23 are hypothesized to occur through a receptor complex composed of two parts: i.) IL-12Rβ1, a portion shared with IL-12, and ii.) IL-23R, a portion unique to IL-23.

[0006] Members of the IL-12 family of cytokines act as immunological command centers by directing innate and adaptive immune responses. These regulatory cytokines act by inducing the development of T cell subpopulations and by altering the function and fate of many immune cell populations, directing adaptive immune responses to infection, inflammation, and the outcome of autoimmune diseases. IL-12 and IL-23 are primarily proinflammatory / prostimulatory cytokines, which play roles in the development of Th1 and Th17 cells, respectively.

[0007] The functional IL-23 receptor is a heterodimer of the IL-12Rβ1 subunit, which is shared with the IL-12 receptor and partners with the signaling chain IL-23R (p19 subunit binding). The IL-23 receptor constitutively associates with Janus kinase 2 (Jak2) and primarily activates STAT3. IL-23 receptor expression is detected primarily on memory T cells and NK cells. Monocytes, macrophages, and dendritic cells also express IL-23 receptor at low levels.

[0008] There is considerable evidence that IL-23-responsive cells are associated with autoimmune inflammatory diseases and cancer, and that modulating IL-23 activity could provide potential therapeutic options. In particular, dysregulation of IL-23 has been linked to immune-mediated inflammatory diseases (IMIDs), such as psoriasis, psoriatic arthritis, Crohn's disease, and ulcerative colitis. In addition, the balance of proinflammatory cytokines, including IL-23 and IL-12, plays a key role in shaping the development of antitumor or protumor immunity.

[0009] The IL-23 / IL-12 pathway is linked to cellular mechanisms involved in the pathophysiology of multiple inflammatory diseases. While several therapeutic strategies are designed to inhibit IL-23 activity, there remains a need for therapeutic agents that target the proinflammatory IL-23 / IL-23 receptor signaling axis for the treatment of immune-mediated inflammatory disorders. More specifically, there remains a need for selective IL-23p19 antagonist antibodies that bind with high affinity to the p19 subunit of IL-23, particularly human IL-23, but do not bind to the p40 subunit of the related cytokine family member, IL-12. Summary of the Invention

[0010] The present disclosure addresses the above needs by providing antibodies and antibody fragments that bind to the cytokine p19 subunit of IL-23. The antibodies and antibody fragments are useful for the treatment of immune-mediated inflammatory diseases (IMIDs) (e.g., autoimmune diseases and inflammatory disorders), either alone (e.g., as monotherapy) or in combination with other immunotherapeutic agents.

[0011] In some embodiments, the anti-IL-23p19 antibody or antibody fragment thereof binds to the cytokine p19 subunit of human IL-23. In further embodiments, the antibody is fully human.

[0012] In some embodiments, the anti-IL-23p19 antibody or antibody fragment thereof comprises a heavy chain variable region comprising CDR1: SEQ ID NO: 9, CDR2: SEQ ID NO: 10, and CDR3: SEQ ID NO: 11; and / or a light chain variable region comprising CDR1: SEQ ID NO: 12, CDR2: SEQ ID NO: 13, and CDR3: SEQ ID NO: 14.

[0013] In some embodiments, the anti-IL-23p19 antibody or antibody fragment thereof comprises a heavy chain variable region comprising CDR1: SEQ ID NO: 15, CDR2: SEQ ID NO: 16, and CDR3: SEQ ID NO: 17; and / or a light chain variable region comprising CDR1: SEQ ID NO: 18, CDR2: SEQ ID NO: 19, and CDR3: SEQ ID NO: 20.

[0014] In some embodiments, the anti-IL-23p19 antibody or antibody fragment thereof comprises a heavy chain variable region comprising CDR1: SEQ ID NO: 21, CDR2: SEQ ID NO: 22, and CDR3: SEQ ID NO: 23; and / or a light chain variable region comprising CDR1: SEQ ID NO: 24, CDR2: SEQ ID NO: 25, and CDR3: SEQ ID NO: 26.

[0015] In some embodiments, the anti-IL-23p19 antibody or antibody fragment thereof comprises a heavy chain variable region comprising CDR1: SEQ ID NO: 27, CDR2: SEQ ID NO: 28, and CDR3: SEQ ID NO: 29; and / or a light chain variable region comprising CDR1: SEQ ID NO: 30, CDR2: SEQ ID NO: 31, and CDR3: SEQ ID NO: 32.

[0016] In some embodiments, the anti-IL-23p19 antibody or antibody fragment thereof comprises a variable heavy chain sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7.

[0017] In other embodiments, the anti-IL-23p19 antibody or antibody fragment thereof comprises a variable light chain sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8.

[0018] In other embodiments, the anti-IL-23p19 antibody or antibody fragment thereof comprises a variable heavy chain sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7, and a variable light chain sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8.

[0019] In some embodiments, the anti-IL-23p19 antibody or antibody fragment is a combination of: (a) a variable heavy chain sequence comprising SEQ ID NO: 1 and a variable light chain sequence comprising SEQ ID NO: 2; (b) a variable heavy chain sequence comprising SEQ ID NO:3 and a variable light chain sequence comprising SEQ ID NO:4; (c) a variable heavy chain sequence comprising SEQ ID NO:5 and a variable light chain sequence comprising SEQ ID NO:6; and (d) a variable heavy chain sequence comprising SEQ ID NO:7 and a variable light chain sequence comprising SEQ ID NO:8 The variable heavy and light chain sequences are selected from:

[0020] In some embodiments, an anti-IL-23p19 antibody (eg, an antagonist antibody) binds with high affinity to the p19 subunit of IL-23 and does not bind to the p40 subunit of the related cytokine family member, IL-12.

[0021] In some embodiments, the anti-IL-23p19 antibody or antibody fragment thereof exhibits one or more of the following characteristics: (a) is specific for human IL-23p19 and has the ability to block IL-23 binding to its receptor (IL-23R); (b) inhibits, interferes with, or modulates IL-23p19 interaction with IL-23 receptor signaling; (c) inhibits STAT3 activation induced by IL-23 in DB cells; (d) suppresses IL-17 production induced by human IL-23 in mouse splenocytes. (e) inhibits human IL-23-induced IL-17 production in activated human PBMCs; (f) does not inhibit IL-23 interaction with IL-12Rβ1 signaling; (g) does not inhibit human IL-12-induced interferon gamma production in human activated T cells (PBMCs); (h) does not inhibit cynomolgus monkey IL-12-induced interferon gamma production in human activated T cells (PBMCs); and (i) inhibits skin inflammation induced by human IL-23 in a mouse psoriasis-like model.

[0022] In one embodiment, the antibodies and isolated antigen binding agents of the present disclosure can be used to inhibit IL-23p19-induced IL-23 receptor signaling networks (e.g., of the inflammatory microenvironment that promotes autoimmune disease).

[0023] An anti-IL-23p19 antibody or antibody fragment thereof may exhibit one or more of the following properties: (a) specific for human IL-23p19 and capable of blocking IL-23 binding to the IL-23 receptor, which is the receptor for IL-23 (e.g., a blocker); (b) inhibiting, interfering with, or modulating IL-23 / IL-23 receptor-mediated signaling; (c) blocking IL-23-induced STAT3 activation induced by IL-23 in DB cells; (d) inhibiting IL-23-induced IL-17 production in mouse splenocytes; (e) inhibits IL-23-induced IL-17 production in human PBMCs; (f) does not inhibit IL-23 interaction with IL-12Rβ1 signaling; (g) does not block human IL-12-induced interferon-γ production in human PBMCs; (h) does not inhibit cynomolgus monkey IL-12-induced interferon gamma production in human PBMCs; and (i) Inhibits IL-23-induced skin inflammation in a mouse psoriasis-like model.

[0024] In some embodiments, the anti-IL-23p19 antibody or antibody fragment thereof comprises a combination of CDR sequences from a variable heavy chain sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7, and a variable light chain sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8.

[0025] In some embodiments, the anti-IL-23p19 antibodies and antibody fragments thereof comprise one or more heavy chain variable region CDRs disclosed in Table 1 and / or one or more light chain variable region CDRs disclosed in Table 2.

[0026] In some embodiments, the anti-IL-23p19 antibody or antibody fragment is a recombinant antibody (e.g., a chimeric or humanized antibody) and comprises six (6) CDRs, all derived from the VH or VL domain of a single anti-IL-23p19 antibody disclosed herein. For example, the binding agent can comprise all six CDR regions of the anti-IL-23p19 antibody designated Hu-2.18006B (for a human antibody). In a representative example, the antibody or antibody fragment thereof can comprise the amino acid sequences of SEQ ID NOs: 9-11 and 12-14, which represent CDR1, CDR2, and CDR3 of the variable heavy chain region and CDR1, CDR2, and CDR3 of the variable light chain region of the Hu-2.18006B antibody.

[0027] In some embodiments, the anti-IL-23p19 antibody is a full-length antibody.

[0028] In some embodiments, the anti-IL-23p19 antibody is an antibody fragment. In further embodiments, the antibody fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fv, scFv and scFv-Fc fragments, single chain antibodies, minibodies, and diabodies.

[0029] In some embodiments, the anti-IL-23p19 antibody is a monoclonal antibody.

[0030] In some embodiments, the anti-IL-23p19 antibody is a human antibody. In some embodiments, the anti-IL-23p19 antibody is a murine antibody.

[0031] In some embodiments, the anti-IL-23p19 antibody is a chimeric antibody. In some embodiments, the anti-IL-23p19 antibody is a bispecific antibody. In some embodiments, the anti-IL-23p19 antibody is a humanized antibody.

[0032] Anti-IL-23p19 antibodies and antibody fragments thereof can be used to treat or prevent immune-mediated inflammatory diseases (IMIDs), such as autoimmune diseases or inflammatory disorders, or cancer. Such methods for treating or preventing IMIDs or cancer include administering a composition or formulation containing an anti-IL-23p19 antibody or antibody fragment thereof to a subject in need thereof. In further embodiments, the anti-IL-23p19 antibody or antibody fragment thereof can be administered alone (e.g., as monotherapy) or in combination with other immunotherapeutic agents and / or chemotherapy. The IMID can be selected from the group consisting of psoriasis, psoriatic arthritis, inflammatory bowel disease (e.g., ulcerative colitis or Crohn's disease), ankylosing spondylitis, systemic lupus erythematosus, hidradenitis suppurativa, atopic dermatitis, asthma, and familial adenomatous polyposis (FAP).

[0033] The foregoing summary, as well as the following detailed description, of the present disclosure will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present disclosure, there are shown in the drawings embodiments which are presently preferred. It is to be understood, however, that the disclosure is not limited to the precise arrangements, examples, and instrumentalities shown. [Brief explanation of the drawings]

[0034] [Figure 1-1] (FIGS. 1A and 1B) provide the amino acid sequences of the VH and VL domains of anti-IL-23p19 antibodies and their respective CDR sequences. Sequence identifiers are provided, and the CDRs are underlined in the context of the variable domain sequences. [Figure 1-2] (FIGS. 1C and 1D) Amino acid sequences of the VH and VL domains of anti-IL-23p19 antibodies and their respective CDR sequences. Sequence identifiers are provided, and the CDRs are underlined in the context of the variable domain sequences. [Figure 2A] FIG. 1 shows the binding profiles of anti-IL-23p19 antibodies to human IL-23, recombinant cytokines comprising the human p19 subunit and mouse p40 subunit, human IL-12 and human p40 subunit as determined by BIAcore. [Figure 2B] FIG. 1 shows the binding profiles of anti-IL-23p19 antibodies to human IL-23, recombinant cytokines comprising the human p19 subunit and mouse p40 subunit, human IL-12 and human p40 subunit as determined by BIAcore. [Figure 2C] FIG. 1 shows the binding profiles of anti-IL-23p19 antibodies to human IL-23, recombinant cytokines comprising the human p19 subunit and mouse p40 subunit, human IL-12 and human p40 subunit as determined by BIAcore. [Figure 2D]FIG. 1 shows the binding profiles of anti-IL-23p19 antibodies to human IL-23, recombinant cytokines comprising the human p19 subunit and mouse p40 subunit, human IL-12 and human p40 subunit as determined by BIAcore. [Figure 2E] FIG. 1 shows the binding profiles of anti-IL-23p19 antibodies to human IL-23, recombinant cytokines comprising the human p19 subunit and mouse p40 subunit, human IL-12 and human p40 subunit as determined by BIAcore. [Figure 3-1] 3A and 3B: Dose-dependent binding of selected representative IL-23p19 antibodies to recombinant cytokines, including human IL-23, as determined by ELISA. [Figure 3-2] Figure 3C: Dose-dependent binding of selected representative IL-23p19 antibodies to recombinant cytokines containing the human p19 subunit and mouse p40 subunit as determined by ELISA. Figures 3D and 3E: Absence of binding of selected representative anti-IL-23p19 antibodies to human IL-12 and human p40 subunit as determined by ELISA. [Figure 4] FIG. 1 shows blockade of IL-23 / IL-23 receptor interaction by four IL-23p19 antibodies as determined by ELISA. [Figure 5] FIG. 1 shows two representative IL-23p19 antibodies that do not block the IL-23 / IL-12 receptor β1 interaction. [Figure 6] (FIGS. 6A and 6B) Inhibition of IL-23-induced IL-17 production by three representative anti-IL-23p19 antibodies in a mouse splenocyte assay (MSA). [Figure 7] FIG. 1 shows inhibition of IL-23-induced STAT3 activation in a reporter cell assay by two representative anti-IL-23p19 antibodies. [Figure 8]FIG. 1 shows that two representative anti-IL-23p19-specific antibodies do not inhibit human 1L-12-induced IFN-γ production in human PBMCs. [Figure 9] FIG. 1 shows that two representative anti-IL-23p19 antibodies do not inhibit cynomolgus monkey 1L-12-induced IFN-γ production in human PBMCs. [Figure 10] FIG. 1 shows in vivo inhibition of IL-23-mediated inflammatory responses (ear thickness) by two representative anti-IL-23p19 antibodies in a murine skin inflammation model, as described in Example 7. [Figure 11] (Figures 11A, 11B, 11C, and 11D) Figures 11A, 11B, 11C, and 11D provide a graphical representation of the pathology score (H&E staining of frozen ear tissue) effect from two anti-IL-23p19 antibodies at day 8 post-treatment from treated mice in the mouse skin inflammation model presented in Example 7. [Figure 12] (Figures 12A, 12B, 12C, and 12D) Representative photographs of hematoxylin and eosin (H&E) staining of frozen ear tissues taken on the final day (day 8) of the in vivo study from treated mice in the mouse skin inflammation model presented in Example 7. DETAILED DESCRIPTION OF THE INVENTION

[0035] IL-23 is a proinflammatory heterodimeric cytokine that contains a p19 subunit and binds to the IL-23 receptor. Targeting the proinflammatory IL-23 / IL-23 receptor signaling axis is an area of ​​intense therapeutic exploration. The present disclosure provides antibodies and antibody fragments thereof that inhibit the human IL-23 / IL-23 receptor signaling axis and can be used to treat or prevent IMID. Advantageously, the anti-IL-23p19 antibodies disclosed herein allow for complete inhibition of IL-23p19, resulting in lower-dose formulations, allowing for less frequent and / or more effective dosing, leading to reduced costs and improved efficiency.

[0036] The anti-IL-23p19 antibodies and antibody fragments thereof disclosed herein specifically bind to human IL-23p19 and antagonize the IL-23 / IL-23 receptor signaling axis. In one embodiment, the antibodies and antibody fragments thereof of the present disclosure bind to human IL-23 with high affinity and prevent its interaction with IL-23R, thereby blocking the downstream signaling cascade. In a specific embodiment, the antibodies or antibody fragments thereof inhibit IL-23-stimulated production of IL-17 from mouse splenocytes and from human PBMCs. In another embodiment, the antibodies or antibody fragments thereof neither bind to nor antagonize IL-12.

[0037] In order that the present disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0038] The following abbreviations are used throughout this disclosure: mAb or Mab or MAb - Monoclonal antibody. CDR - Complementarity determining region of an immunoglobulin variable region. VH or VH - immunoglobulin heavy chain variable region. VL or VL - immunoglobulin light chain variable region. FR - antibody framework region, immunoglobulin variable region excluding the CDR regions.

[0039] As used herein, the term "interleukin-23" (used interchangeably with IL-23) refers to a human IL-23 heterodimer, including, for example, a human IL-23 heterodimer comprising or consisting of a protein subunit having the amino acid sequence as provided in UniProt entry UniProtKB-Q9NPF7, identified as IL-23 subunit (p40), disulfide-linked to a protein subunit having the amino acid sequence as provided in UniProt entry UniProtKB-P29460, identified as interleukin-23 subunit alpha (p19).

[0040] As used herein, the terms "IL-12R complex" and "IL-12R" refer to the high-affinity IL-12 cytokine receptor complex comprising the IL-12Rβ1 and IL-12Rβ2 subunits.

[0041] As used herein, the terms "IL-23R complex" and "IL-23R" refer to the high-affinity IL-23 cytokine receptor, which comprises the IL-12Rβ1 (in common with the IL-12R complex) subunit and the IL-23R subunit.

[0042] As used herein, the term "interleukin-12" (used interchangeably with IL-12 throughout this disclosure) refers to a human IL-12 heterodimer, including, for example, a human IL-12 heterodimer comprising or consisting of a protein subunit having the amino acid sequence provided in UniProt entry UniProtKB-P29459 (identified as interleukin-12 subunit alpha) disulfide-linked to a protein subunit comprising the amino acid sequence provided in UniProt entry UniProtKB-P29460 (identified as interleukin-12 subunit beta (p40)). The term includes a heterodimeric protein comprising a 35 kD subunit (p35) and a 40 kD subunit (p40), both of which are linked together by a disulfide bridge. This heterodimeric protein is referred to as the "p70 subunit." The structure of human IL-12 is further described in, for example, Kobayashi, et al. (1989) J. Exp Med. 170:827-845 and Ling, et al. (1995) J. Exp Med. 154:116-127. The term human IL-12 is intended to include recombinant human IL-12 (rh IL-12), which can be prepared by standard recombinant expression methods.

[0043] As used herein, the term "interleukin-17," also referred to as "IL-17" or "IL-17A," refers to a 20-30 kD glycosylated homodimeric protein, including homodimeric proteins comprising or consisting of protein subunits, for example, having the amino acid sequence provided in UniProt entry UniProtKB-Q16552. The human IL-17 gene encodes a 155-amino acid protein with a 19-amino acid signal sequence and a 136-amino acid mature segment. IL-17 is secreted by activated T cells at sites of inflammation but is not normally present in the systemic circulation. IL-17 binds to a type I transmembrane receptor, designated IL-17R, which is a large, ubiquitously expressed protein that does not display significant sequence identity to other known cytokine receptors. Human IL-17 exhibits 62.5% and 58% amino acid sequence identity with the mouse and rat IL-17 sequences, respectively. Human IL-17 shows 97.4% amino acid sequence identity with cynomolgus monkey IL-17.

[0044] The term "antibody" as used herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies).

[0045] An exemplary antibody, such as an IgG, comprises two heavy chains and two light chains. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0046] The hypervariable region generally encompasses amino acid residues from about amino acid residues 24-34 (LCDR1; "L" indicates light chain), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable region, and amino acid residues around about amino acid residues 31-35B (HCDR1; "H" indicates heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), and / or those residues forming the hypervariable loops (e.g., residues 26-32 (LCDR1), residues 50-52 (LCDR2), and residues 91-96 (LCDR3) in the light chain variable region, and residues 26-32 (HCDR1), residues 53-55 (HCDR2), and residues 96-101 (HCDR3) in the heavy chain variable region); Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.

[0047] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variant antibodies. For example, variant antibodies may contain naturally occurring mutations or arise during the production of monoclonal antibody preparations, with such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the nature of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any method. For example, monoclonal antibodies to be used in accordance with the present invention can be made by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0048] The term "chimeric" antibody refers to a recombinant antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0049] A "human antibody" is an antibody that has an amino acid sequence corresponding to that of an antibody produced by a human and / or that has been produced using any of the techniques for producing human antibodies known to those skilled in the art. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including those described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol, 147(I):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol, 5: 368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal that has been engineered to produce such antibodies in response to antigen challenge, and whose endogenous gene locus has been disabled, e.g., immunized HuMab mice (see, e.g., Nils Lonberg et al., 1994, Nature 106:141-144, for a description of HuMab mice). 368:856-859, WO 98 / 24884, WO 94 / 25585, WO 93 / 1227, WO 92 / 22645, WO 92 / 03918 and WO 01 / 09187), Xenomouse (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology), or Trianni mice (see, e.g., WO 2013 / 063391, WO 2017 / 035252 and WO 2017 / 136734).

[0050] The term "humanized antibody" refers to an antibody that has been modified to contain one or more human framework regions in the variable regions together with non-human (e.g., mouse, rat, or hamster) complementarity-determining regions (CDRs) of the heavy and / or light chains. In certain embodiments, a humanized antibody contains sequences that are completely human except for the CDR regions. Humanized antibodies are typically less immunogenic in humans than non-humanized antibodies, and therefore provide therapeutic benefit in certain situations. Those skilled in the art are familiar with humanized antibodies and with techniques suitable for producing them. See, for example, Hwang, WYK, et al., Methods 36:35, 2005; Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033, 1989; Jones et al., Nature, 321:522-25, 1986; Riechmann et al., Nature, 332:323-27, 1988; Verhoeyen et al., Science, 239:1534-36, 1988; Orlandi et al., Proc. Natl. Acad. Sci. USA, 86:3833-37, 1989, each of which is incorporated herein by reference in its entirety. 1989; U.S. Patent Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370; and Selick et al., WO 90 / 07861.

[0051] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the various classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0052] The term "antigen-binding domain" of an antibody (or simply "binding domain") or similar terms refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen complex. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH domains; (iv) a Fv fragment consisting of the VL and VH domains of a single antibody arm; (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341: 544-546); (vi) an isolated complementarity-determining region (CDR); and (vii) a combination of two or more isolated CDRs, optionally linked by a synthetic linker.

[0053] As used herein, the term "complementarity determining region" or "CDR" refers to short polypeptide sequences within the variable regions of both heavy and light chain polypeptides that are primarily responsible for mediating specific antigen recognition. L and each V H There are three CDRs within the CDR (termed CDR1, CDR2, and CDR3).

[0054] As will be understood by those skilled in the art, the exact numbering and arrangement of CDRs may vary among various numbering systems. However, it should be understood that the disclosure of a variable heavy sequence and / or variable light sequence includes the disclosure of the associated CDRs. Thus, the disclosure of each variable heavy region is a disclosure of the vhCDRs (e.g., vhCDR1, vhCDR2, and vhCDR3), and the disclosure of each variable light region is a disclosure of the vlCDRs (e.g., vlCDR1, vlCDR2, and vlCDR3).

[0055] In certain embodiments, the CDRs of an antibody can be determined according to the IMGT numbering system described in Lefranc MP, (1999) The Immunologist 7: 132-136 and Lefranc MP et al, (1999) Nucleic Acids Res 27: 209-212, each of which is incorporated by reference in its entirety. Unless otherwise specified herein, references to residue numbers in the variable domain of an antibody refer to residue numbering according to the IMGT numbering system.

[0056] In other embodiments, antibody CDRs can be determined according to MacCallum RM et al. (1996) J Mol Biol 262: 732-745, which is incorporated herein by reference in its entirety. See also, for example, Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001), which is incorporated herein by reference in its entirety. In other embodiments, antibody CDRs can be determined according to the AbM numbering scheme, which refers to AbM hypervariable regions, which represent a compromise between Kabat CDRs and Chothia structural loops, and is used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.), which is incorporated herein by reference in its entirety.

[0057] "Framework" or "Framework region" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4.

[0058] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda, Md. (1991), Vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup II as in Kabat et al., supra.

[0059] The "hinge region" is generally defined as the stretch from 216 to 238 (EU numbering) or 226 to 251 (Kabat numbering) of human IgG1. The hinge can be further divided into three distinct regions: the upper, middle (e.g., core), and lower hinge.

[0060] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0061] A "blocking" or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Certain blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0062] The term "effector function" refers to effector biological activities attributable to the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent T-cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B-cell receptors), and B-cell activation.

[0063] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that contacts an overlapping set of amino acid residues of an antigen compared to the reference antibody or blocks the binding of the reference antibody to the antigen by 50% or more in a competition assay. The amino acid residues of an antibody that contact the antigen can be determined, for example, by determining the crystal structure of the antibody complexed with the antigen or by performing hydrogen / deuterium exchange. In some embodiments, residues of an antibody that are within 5 Å of the antigen are considered to contact the antigen. In some embodiments, an antibody that binds to the same epitope as a reference antibody blocks the binding of the reference antibody to the antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks the binding of the antibody to the antigen by 50% or more in a competition assay.

[0064] The term "antibody fragment" refers to a molecule distinct from an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; and single-chain antibody molecules (e.g., scFv). Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment, a designation reflecting the ability to readily crystallize. The Fab fragment consists of an entire light (L) chain along with the variable region domain (VH) of the heavy (H) chain and the first constant domain (CH1) of one heavy chain. Pepsin treatment of an antibody yields a single large F(ab)2 fragment, roughly corresponding to two disulfide-linked Fab fragments with divalent antigen-binding activity and still capable of cross-linking antigen. Fab fragments differ from Fab' fragments in that they have a few additional residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0065] An "Fv" consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. The folding of these two domains results in six hypervariable loops (three from each of the H and L chains) that provide the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody.

[0066] A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising a VH antibody domain and a VL antibody domain connected in a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0067] The term "antigen-binding domain" of an antibody (or simply "binding domain") or similar terms refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen complex. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH domains; (iv) a Fv fragment consisting of the VL and VH domains of a single antibody arm; (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341: 544-546); (vi) an isolated complementarity-determining region (CDR); and (vii) a combination of two or more isolated CDRs, optionally linked by a synthetic linker.

[0068] The term "multispecific antibody" is used in the broadest sense and particularly covers antibodies comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), where the VH-VL unit has multiple epitope specificity (e.g., can bind to two different epitopes on one biological molecule or to each epitope on a different biological molecule). Such multispecific antibodies include, but are not limited to, full-length antibodies, antibodies with two or more VL and VH domains, bispecific diabodies, and triabodies. "Multiple epitope specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different target(s).

[0069] "Dual specificity" or "bispecificity" refers to the ability to specifically bind to two different epitopes on the same or different target(s). However, in contrast to bispecific antibodies, dual-specific antibodies have two antigen-binding arms with identical amino acid sequences, with each Fab arm capable of recognizing two antigens. Dual specificity allows an antibody to interact with two different antigens with high affinity as a single Fab or IgG molecule. According to one embodiment, a multispecific antibody in the IgG1 format binds to each epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0.1 μM to 0.001 pM. "Monospecificity" refers to the ability to bind to only one epitope. Multispecific antibodies can have a structure similar to that of a complete immunoglobulin molecule and include an Fc region, e.g., an IgG Fc region. Such structures may include, but are not limited to, IgG-Fv, IgG-(scFv)2, DVD-Ig, (scFv)2-(scFv)2-Fc, and (scFv)2-Fc-(scFv)2. In the case of IgG-(scFv)2, the scFv can be attached to either the N- or C-terminus of either the heavy or light chain.

[0070] As used herein, the term "bispecific antibody" refers to a monoclonal antibody, often a human or humanized antibody, that has binding specificities for at least two different antigens. In the present invention, one of the binding specificities can be directed to IL-12 or IL-23, and the other can be directed to any other antigen, such as a cell surface protein, a receptor, a receptor subunit, a tissue-specific antigen, a virus-derived protein, a virus-encoded envelope protein, a protein derived from bacteria, or a bacterial surface protein.

[0071] As used herein, the term "diabody" refers to a bivalent antibody comprising two polypeptide chains, each of which contains a VH domain and a VL domain connected by a linker (e.g., a linker of five amino acids) that is too short to allow intramolecular association between the VH and VL domains on the same peptide chain. This arrangement forces each domain to pair with a complementary domain on another polypeptide chain to form a homodimeric structure. Accordingly, the term "triabody" refers to a trivalent antibody comprising three peptide chains, each of which contains one VH domain and one VL domain connected by a linker (e.g., a linker of one to two amino acids) that is too short to allow intramolecular association between the VH and VL domains on the same peptide chain.

[0072] The term "isolated antibody," as used to describe various antibodies disclosed herein, refers to an antibody that has been identified and separated and / or recovered from the cell or cell culture in which it is expressed. Contaminant components of its natural environment are substances that would normally interfere with diagnostic or therapeutic uses of the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as determined, for example, by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse-phase HPLC) approaches. For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007). In a preferred embodiment, the antibody is purified (1) sufficiently to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequencer, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain.

[0073] With respect to the binding of an antibody to a target molecule, the term "specific binding," or "specifically binds to" or is "specific for" a particular polypeptide or epitope on a particular polypeptide target, refers to binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule. For example, specific binding can be determined by competition with a control molecule that is similar to the target, e.g., an excess of unlabeled target. In this case, specific binding is indicated when the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. As used herein, "specific binding," or the term "specifically binds to" or is "specific for" a particular polypeptide or epitope on a particular polypeptide target, may be indicated, for example, by a molecule having a Kd for the target of 10 M or less, alternatively 10 M or less, alternatively 10 M or less, alternatively 10 M or less, alternatively 10 M or less, alternatively 10 M or less, alternatively 10 M or less, alternatively 10 M or less, alternatively 10 M or less, or alternatively 10 M or less, or a molecule having a Kd in the range of 10 M to 10 M, or 10 M to 10 M, or 10 M to 10 M. As one of skill in the art will appreciate, affinity and KD values ​​are inversely proportional. High affinity for an antigen is measured by a low KD value. In one embodiment, the term "specific binding" refers to binding in which a molecule binds to a particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes. As used herein, the terms "specific binding," "specifically binds," and "selectively binds" refer to the binding of an antibody to an epitope of human interleukin-23p19.

[0074] The term "affinity" as used herein refers to the binding strength of an antibody to an epitope. The affinity of an antibody is given by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant Ka is defined as 1 / Kd. Methods for determining the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which are incorporated herein by reference in their entireties. One standard method well known in the art for determining the affinity of mAbs is the use of surface plasmon resonance (SPR) screening (eg, by analysis on a BIAcore™ SPR analyzer).

[0075] "Epitope" is a technical term that designates the site(s) of interaction between an antibody and its antigen(s). (Janeway, C, Jr., P. Travers, et al. (2001). Immunobiology: The Immune System in Health and Disease. Part II, Sections 3-8. New York, Garland Publishing, Inc.): As stated by: "Antibodies generally recognize only small regions on the surface of large molecules such as proteins..." It is likely that [a particular epitope] is composed of amino acids from different parts of the [antigen] polypeptide chain, held together by protein folding. This type of antigenic determinant is known as a conformational or discontinuous epitope, because the recognized structure is composed of protein segments that are discontinuous in the antigen's amino acid sequence but held together in their three-dimensional structure. In contrast, epitopes composed of a single segment of a polypeptide chain are called continuous or linear epitopes (Janeway, C. Jr., P. Travers, et al. (2001). Immunobiology: the immune system in health and disease. Part II, Sections 3-8. New York, Garland Publishing, Inc.).

[0076] The term "KD," as used herein, is intended to refer to the dissociation constant of a particular antibody-antigen interaction, which is calculated according to the formula: Koff / Kon=KD.

[0077] The term "IC50", as used herein, is intended to refer to the effective concentration of an antibody of the invention required to neutralize 50% of the biological activity of IL-23 on human lymphoma DB cells in the bioassay described in Example 5: Inhibition of STAT3 Activation in Human DB Cell Assay.

[0078] "EC50" with respect to a drug and a particular activity (e.g., binding to cells, inhibiting enzyme activity, activating or inhibiting immune cells) refers to the effective concentration of that drug that produces 50% of the drug's maximum response or effect with respect to that activity. "EC100" with respect to a drug and a particular activity refers to the effective concentration of that drug that produces substantially the drug's maximum response with respect to that activity.

[0079] As used herein, the terms "antibody-based immunotherapy" and "immunotherapy" are used broadly to refer to any form of therapy that relies on the targeting specificity of an anti-IL-23p19 antibody, bispecific molecule, multispecific molecule, binding agent, or fusion protein containing an IL-23p19-specific binding agent to mediate a direct or indirect effect on cells characterized by aberrant expression of IL-23p19. The terms are intended to encompass methods of treatment using naked antibodies, bispecific antibodies (including T cell-, NK cell-, and other immune cell / effector cell-binding modes), antibody-drug conjugates, T cells (CAR-T) or NK cells (CAR-NK) engineered to contain an IL-23p19-specific chimeric antigen receptor, and cell therapy using oncolytic viruses containing an IL-23p19-specific binding agent, and gene therapy by delivering the antigen-binding sequence of an anti-IL-23p19 antibody and expressing the corresponding antibody fragment in vivo.

[0080] As used herein, the term "immune-mediated inflammatory disease" or "IMID" includes a group of seemingly unrelated diseases that share common inflammatory pathways and are caused or result from dysregulated functioning of the innate and adaptive immune systems. These conditions include, but are not limited to, psoriasis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, ankylosing spondylitis, hidradenitis suppurativa, atopic dermatitis, and asthma. Any organ system can be affected by IMID, and individuals can experience significantly reduced quality of life, significant morbidity, and shortened lifespan (Bunte, K and Beikler, T, Int. J. Mol. Sci., 20: 3394 (2019)). Please note that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0081] IL-12 / IL-23 receptor signaling axis The p19 subunit of IL-23 (also referred to herein as "IL-23p19" and "p19 subunit") is a 189 amino acid polypeptide containing a 21 amino acid leader sequence (Oppmann et al. Immunity 13:715 (2000)). The biological activity of the p19 subunit is detected only when the p19 subunit partners with the IL-12 p40 subunit to form IL-23. Both IL-12 and IL-23 exist only as secreted heterodimeric cytokines, and neither the IL-12 p35 subunit nor the IL-23 p19 subunit is secreted without intracellular covalent association with p40. Although the p40 subunit shared by the IL-12 and IL-23 cytokines binds to the common IL-12Rβ1 component of their receptors, signaling specificity is determined by the unique p35 (IL-12) and p19 (IL-23) subunits that bind to the IL-12Rβ2 and IL-23R components of their respective high-affinity receptors. The interaction of IL-12 and IL-23 with their cognate receptors forms part of a complex regulatory network that coordinates innate and adaptive immune responses.

[0082] It has been hypothesized that IL-12 may play a crucial role in the development of protective immune responses against many intracellular pathogens and viruses and in tumor immunosurveillance. 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: 1461-4; Meeran et al., Mol. Cancer. Ther. 2006, 5: 825-32; Langowski et al., Nature, 2006, 442: 461-5. Therefore, IL-23-specific inhibition (sparing IL-12 or the shared p40 subunit) may have a potentially superior safety profile compared with dual inhibition of IL-12 and IL-23.

[0083] The IL-23 receptor contains the IL-12Rβ1 subunit, which partners with a unique subunit called IL-23R, which is shared with the IL-12 receptor (Parham et al. J. Immunol. 168:5699 (2002)). IL-23R has been reported to bind IL-23 with high affinity (KD = 44 ± 3 nM). In contrast, IL-23 binds to the IL-12β1 subunit with lower affinity (KD = 2 ± 1 uM). Binding of IL-23R to IL-23 facilitates the binding of IL-12Rβ1 to IL-23 with very high affinity (KD = 25 ± 5 nM) (Bloch et al, Immunity, 48, 45-58 (2018)). IL-23R is expressed by a wide range of cells (natural killer cells, macrophages, dendritic cells, memory T cells, and keratinocytes). IL-23 production induces IL-23R expression, creating a positive feedback loop that enhances IL-23 expression.

[0084] IL-23 is produced by activated antigen-presenting cells and binds to the IL-23 receptor complex expressed on NK cells and T cells. IL-23, alone or in combination with other cytokines (e.g., IL-1β), has been shown to promote the production of IL-17A, IL-17F, IL-6, and tumor necrosis factor alpha (TNFα), which are proinflammatory cytokines known to contribute to the inflammatory response in IMID disease.

[0085] Binding of IL-23p19 to the IL-23R leads to a remodeling process of the IL-23p19 helical domain, which allows IL-12 p40 to bind to IL-12Rβ1 (Bloch, Y et al. Immunity. 2018; 48(1):45-58). This process activates JAK2 and TYK2, leading to the formation of STAT3 and STAT4, which ultimately function as transcription factors (Parham, C. et al., Immunol. 168(11):5699-5708 (2002)). IL-23 is a key player in the late differentiation of naive CD4+ T cells into Th17 cells (Gaffen, SL et al., Nat Rev Immunol. 14(9):585-600 (2014)). Naive T cells lack IL-23R and therefore require other cytokines, such as transforming growth factor (TGF)-β and IL-6, to modulate the early stages of differentiation. These cytokines induce the expression of the retinoic acid receptor-related orphan receptor-γt, a transcription factor that promotes IL-23R expression. Immature Th17 cells induced by TGF-β and IL-6 require exposure to IL-23 to acquire pathogenic potential. Upon maturation, Th17 cells can produce IL-17 and TNF-α (Kashani, A et al., Gastroenterology & Hepatology 15(5):255-265 (2019)).

[0086] Despite the structural similarities between the two cytokines, the biological activity / function of IL-23 is distinct from that of IL-12. IL-23 supports the differentiation and maintenance of naive CD4+ T cells into a new subset of cells called Th17 cells, which are distinct from classical Th1 and Th2 cells. Th17 cells produce interleukin-17A (IL-17A) and interleukin-17F (IL-17F). Th17 cells produce a variety of other factors known to drive inflammatory responses, including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), CXCL1, and CCL20. Innate lymphoid cells, such as NK cells and lymphoid tissue inducer (LTi)-like cells, express the IL-23 receptor and retinoic acid-related orphan receptor (ROR) gamma and produce IL-17 in response to IL-23. IL-1β and IL-23 also costimulate gamma-delta T cells to induce IL-17 production without T cell receptor engagement.

[0087] Importantly, IL-23 sustains the differentiation and proliferation of naive T cells into the distinct Th17 cell lineage. In the absence of IL-23, the Th17 phenotype is lost. IL-23 has been described as a "master regulator" of the immune inflammatory response in IMID due to its pivotal role in maintaining cytotoxic Th17 cells that generate a proinflammatory cytokine profile. IL-23 pathogenesis is partially dependent on the dysregulated production of IL-17A, IL-17F, and IL-22, thus providing a rationale for targeting the IL-23 / IL-23R axis for immunotherapy.

[0088] Targeting the proinflammatory IL-23 / IL-23 receptor signaling axis Anti-IL-12 / IL-23 antibodies reported to confer therapeutic effects in vivo include the antibodies ustekinumab (CNT01275) and briakinumab (ABT-874), both of which target the common IL-12 p40 subunit in a region of the p40 subunit that is critical for IL-12Rβ1 binding (Clarke, A. et al. mAbs 2(5):539-549 (2010)).

[0089] Anti-IL-23 selective antibodies that have been reported to confer therapeutic efficacy in vivo include guselkumab (TREMFYA®), tildrakizumab (ILUMYA®), risankizumab (SKYRIZI®), brazikumab (MEDI2070), and mirakizumab (Ly3074828); all of these are specific for the p19 subunit of IL-23. Data from randomized, placebo- and active-controlled phase 3 trials indicate that tildrakizumab, guselkumab, and risankizumab have favorable risk-benefit profiles in patients with moderate-to-severe psoriasis. No significant safety concerns have been observed with any of these IL-23p19 inhibitors.

[0090] IL-12-driven Th1 cells were previously thought to be the pathogenic T cell subset in many 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, have established that IL-23, but not IL-12, is the key driver of autoimmune / inflammatory disease (Ahern et al., Immun. Rev. 226:147-159 (2008); Cua et al., Nature421:744-748 (2003); Yago et al., Arthritis Res and Ther. 9(5): R96 (2007)).

[0091] The role of IL-23 in immune-mediated inflammatory responses is also supported by genetic studies. Genome-wide association studies (GWAS) have linked IL-23R polymorphisms to susceptibility to autoimmune conditions such as psoriasis and psoriatic arthritis (Liu et al., PLoS Genet. 4(3)e1000041 (2008), Reveille, et al., Nat. Genet. 42(2): 123-127 (2010), and Duerr et al., Science 314(5804):1461-1463 (2006)). An association between a single nucleotide polymorphism (SNP) in the IL-23R gene, rsl1209026, and CD has been established (Reveille, JD et al.). This variant has been shown to be protective against CD and UC. The protective properties of rsl1209026 were confirmed in a meta-analysis that showed that carriage of this SNP variant reduced disease risk in a cohort of over 75,000 cases and controls (Jostins, L. Nature 491(7422):119-124 (2012)). This SNP variant, along with several other coding variants in IL-23R, led to decreased expression of IL-23R, thereby reducing immune responses mediated by the IL-23 axis (J Biol Chem. 291(16):8673-8685 (2016)).

[0092] Cytokines such as IL-6 and TGF-β1 can promote the differentiation of RORγt+ Th17 cells from naive CD4+ T cells, whereas IL-23 is required for the full inflammatory function of these cells. In addition, binding of IL-23 to the IL-23 receptor on activated RORγt+ Th17 cells induces further expression of the IL-23 receptor (IL-23R), thus providing a feed-forward loop for the maintenance and proliferation of these cells (Singh, S, et al., MAbs 7(4):1493-1503 (2015)).

[0093] There is strong evidence that the IL-23 / IL-17 axis plays a key role in the development of chronic inflammation, and genetic studies have revealed a potential association between the IL-23 receptor (IL-23R) or its ligands and several inflammatory diseases, including psoriasis, inflammatory bowel disease, and graft-versus-host disease. Targeting the IL-23 / IL-17 axis is an area of ​​intense therapeutic exploration in IMIDs, including psoriasis, psoriatic arthritis, inflammatory bowel disease (ulcerative colitis and Crohn's disease), ankylosing spondylitis, and systemic lupus erythematosus (SLE).

[0094] Generally speaking, IL-23-specific antibodies, such as guselkumab, tildrakizumab, risankizumab, brazikumab, or mirakizumab, selectively bind to IL-23p19 and inhibit IL-23 binding to its receptor; thereby antagonizing the action of IL-23, thereby inducing and maintaining T helper (Th)17 cells, innate lymphoid cells, γδ T cells, and natural killer (NK) cells, which are involved in the tissue inflammation, destruction, and / or abnormal tissue repair associated with IMID.

[0095] Plaque psoriasis, or psoriasis vulgaris (PsO), is a chronic inflammatory, T-cell-mediated skin disorder characterized by complex pathophysiology. Its incidence in developed countries is 1-4%. Psoriasis is the most prevalent autoimmune disease in the United States, affecting approximately 7.5 million people. Plaque psoriasis is the most common form of psoriasis, affecting 80-90% of patients. While the pathogenesis of psoriasis is not fully understood, multiple environmental factors, T cells, dendritic cells, numerous cytokines, and 45 identified genetic loci all interact to create a systemic psoriatic disease state and ultimately psoriatic plaques (Nestle FO, et al., N Engl J Med. 361(5):496-509 (2009); Mahil SK, et al., Dermatol Clin. 33(1):1-11 (2015)). The synergistic effects of genetic and environmental factors, along with the interplay between innate and adaptive immunity, ultimately lead to abnormal keratinocyte proliferation and the formation of psoriatic lesions (Chan, J. R., et al., J. Exp. Med, 203(12)2577-2587 (2006)).

[0096] PsO plaques are typically well-demarcated, erythematous, scaly skin lesions characterized by epidermal thickening. Involved keratinocytes activate dendritic cells, which migrate to local lymph nodes and release several cytokines, including interleukins IL-12 and IL-23, which activate type 1 T helper cells (Th1) and type 17 T helper cells (Th17), respectively. T lymphocytes and other cell types release additional cytokines, including tumor necrosis factor (TNF)-α, IL-22, and IL-17, resulting in increased keratinocyte activation and the initiation of a self-propelling cycle of inflammation (Lowes MA et al., Trends Immunol. 34(4):174-81 (2013)). Histologically, there is marked epidermal hyperplasia with parakeratosis and a mixed dermal infiltrate containing CD4+ T cells, dendritic cells, macrophages, and mast cells.

[0097] Early publications reported the presence of elevated levels of tumor necrosis factor-α and the p40 subunit of IL-12, accompanied by overexpression of IL-12 p40 and IL-23 p40 messenger RNA in psoriatic skin lesions. These findings suggested that inhibiting IL-12 and IL-23 using neutralizing antibodies against the IL-12 / 23 p40 subunit proteins might provide an effective therapeutic approach for the treatment of psoriasis (Piskin G, et al., J Immunol 2006, 176: 1908-15). Psoriasis was initially considered a Th-1-mediated disease (based on a cytokine secretion profile characteristic of T helper type 1 cells: interleukin-2, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ).

[0098] The fundamental role of IL-23 in the pathogenesis of psoriasis has been elucidated and is related to the biology of the Th17 lineage. Initial differentiation of naive T lymphocytes into Th17 requires the presence of TGF-β1, IL-6, and IL-1, whereas IL-23 is required for the activation and maintenance of Th17 to secrete the proinflammatory cytokines IL-17, IL-22, IL-21, and tumor necrosis factor-α, which ultimately contribute to the formation of psoriatic skin lesions (Fotaidou, C. et al., Psoriasis: Targets and Therapy 8: 1-5 (2018)).

[0099] Thus, although both IL-12 and IL-23 are known to contribute to the development of Th1 immune responses in psoriasis, IL-23 is now recognized as a key driver of the differentiation and survival of Th17 cells. The primary cytokines produced by Th17 cells are members of the proinflammatory IL-17 family, including IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. IL-17A and IL-17F are similar and bind to the same IL-17 receptor, a heterodimer composed of IL-17RA and IL-17RC subunits.

[0100] While early therapeutic strategies targeted Th1 cells as the central cell type in the pathogenesis of psoriasis, newer models are focusing on the IL-23 / Th17 axis (Lowes MA, et al. Trends Immunol. 34(4):174-81(2013)). The rationale for this new focus is premised on the belief that IL-17 is a key player in the pathogenesis of psoriasis and the recognition that IL-23 drives the activation of Th17 cells. Furthermore, IL-23 stimulates the production of other Th17 cytokines (e.g., IL-22) by other cell types, including innate lymphoid type 3 cells and γδ T cells (Ward, NL, J Investig Dermatol. 134: 2305-2307). (2014)). It was suggested that inhibiting IL-23 would block the downstream production of IL-17A and IL-22 by Th17 cells, and that this effect would manifest itself in antagonism of the immunopathogenesis of psoriasis.

[0101] The IL-23 / IL-17 axis is now believed to be crucial in the pathogenesis of psoriasis, and selective IL-23p19 inhibition may offer some advantages over IL-12 / 23 p40 inhibition or distal blockade of IL-17A or its receptor (Torres, T Drugs 77:1493-1503 (2017)). To date, three IL-23p19-subunit-specific monoclonal antibodies (i.e., guselkumab, tildrakizumab, and risankizumab) have been approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of moderate to severe plaque psoriasis in adults who are candidates for systemic therapy or phototherapy. In July 2020, guselkumab was also approved by the FDA for the treatment of adults with active psoriatic arthritis.

[0102] The high efficacy of IL-23 blockade in psoriasis has been demonstrated in early proof-of-concept studies and phase I clinical trials. A phase I study showed that a single dose of guselkumab resulted in significant clinical responses in patients with moderate to severe plaque psoriasis (Sofen H, et al., J Allergy Clin Immunol; 133:1032-1040 (2014)). A phase I study also reported that selective antagonism of interleukin-23 with guselkumab resulted in clinical improvement of psoriasis characterized by decreased epidermal thickness, T cell and dendritic cell infiltration, psoriasis-related gene expression, and decreased serum IL-17A levels. The reported findings of measurable clinical responses in patients with moderate to severe psoriasis after a single dose of guselkumab support the emerging theory that selective neutralization of IL-23 is a potential therapeutic option.

[0103] A rapid onset of guselkumab activity was also observed in a Phase II dosing trial (NCT01483599) evaluating the use of guselkumab over a wide range of doses and at two different dosing intervals for up to 40 weeks of continuous treatment. Efficacy was evident at the earliest assessment (week 4). Some guselkumab regimens were associated with significantly better response rates than those associated with adalimumab, a biologic agent commonly used to treat psoriasis (Gordon, KB et al., N Engl J Med 373:136-144 (2015)). Guselkumab efficacy continued to improve beyond week 16 (the primary endpoint assessment) and was maintained through week 40. Furthermore, the majority of patients in the 100-mg guselkumab group experienced complete elimination of psoriasis, as indicated by a PGA score of 0 (in 62% of patients) and a 100% improvement from baseline in the PASI score (in 54% of patients) after 40 weeks of continuous treatment. Regulatory approval by the FDA and EMA was based in part on findings from three pivotal Phase III trials: VOYAGE 1 (Blauvelt, A et al. J. Am. Acad. Dermatol. 76: 405-417 (2017)), VOYAGE (Reich, K et al. J Am. Acad. Dermatol.,76: 418-431 (2017)), and NAVIGATE (Langley, RG et al., Brit. J. Dermatol. 178:114-123 (2017)).

[0104] VOYAGE 1 (NCT02207231) was a phase III, randomized, double-blind, placebo- and active-controlled trial conducted at 101 global sites (December 2014–April 2016). The trial included an active-treatment period (weeks 0–48) comparing guselkumab with adalimumab and a placebo-controlled period (weeks 0–16). Patients receiving placebo subsequently underwent a crossover period in which guselkumab was administered through week 48. Guselkumab was superior to placebo and / or adalimumab for the coprimary endpoint and all key secondary endpoints (all P<.001). Compared with placebo, significantly higher proportions of patients receiving guselkumab achieved IGA 0 / 1 (6.9% vs. 85.1%) and PASI 90 (2.9% vs. 73.3%) at week 16. Similarly, the PASI 100 responses in the guselkumab group were significantly better than those in the adalimumab group at weeks 24 and 48 (P<0.001). After initiating guselkumab at week 16, patients in the placebo crossover group achieved responses similar to those observed in the guselkumab group. VOYAGE 1 confirms the role of IL-23 in the pathogenesis of psoriasis. Compared with TNF-α blockade, selective targeting of the IL-23 pathway allows for psoriasis-specific cytokine inhibition with a higher degree of efficacy while maintaining a favorable safety profile (Blauvelt, A, et al., J Investig Dermatol., 135: 1946-1953 (2015)).

[0105] VOYAGE 1 was an extension-label trial that followed patients for four years after the initial trial. Patients were initially randomized to receive either Tremfya or placebo, but all received Tremfya at week 16. The VOYAGE 1 study found that in the combined groups of individuals who initially received Tremfya or placebo and then crossed over to Tremfya at week 16, 82% of patients receiving Tremfya demonstrated at least a 90% improvement in the Psoriasis Area Severity Index (PASI 90) and cleared (0) or minimal disease (1) on the Investigator's Global Assessment (IGA) score at week 204, four years after the initial trial.

[0106] Psoriatic arthritis (PsA) is a chronic inflammatory musculoskeletal disease that occurs in up to 40% of patients with psoriasis. As a result, PsA can be considered a disease within a disease, sharing many common pathogenic pathways with psoriasis. Psoriasis typically precedes PsA in 70% of patients, inflammatory skin and joint disease co-occur in 15% of patients, and inflammatory arthritis precedes skin disease in the remaining patients. Although nearly all patients with PsA eventually develop psoriasis, the clinical presentation and course of PsA are quite heterogeneous, and five distinct patterns of PsA based on the distribution of affected joints have been described (Dobbin-Sears, I et al. Ther Adv Chronic Dis,. 9(10) 191-198 (2018)).

[0107] PsA is a heterogeneous condition with articular and extra-articular manifestations, including peripheral arthritis, axial disease, enthesitis, dactylitis, and a combination of skin and nail disease (Quireo, R and Coto-Sequra, P, Expert Opinion On Biological Therapy, 18:9, 931-935 (2018)). Genetic, immunological, and environmental factors that activate both innate and adaptive immune responses appear to play an important role in the pathogenesis of PsA. As the disease progresses, patients may present with multiple patterns and are not limited to one subset of arthritis. Approximately two-thirds of PsA patients will experience progressive joint damage, often accompanied by loss and disability of function.

[0108] The proinflammatory IL-23 / IL-23 receptor signaling axis is involved in both PsO and PsA. In particular, the Th-17 axis (inhibited by IL-23) is thought to play a key role in the immunopathogenesis of both psoriasis and PsA. IL-23 / IL-23-R interaction induces IL-23-dependent differentiation and activation of Th-17 cells, leading to the production and secretion of IL-17 and IL-22, ultimately resulting in synovial and cutaneous inflammation and bone remodeling. Of particular relevance to the pathology of PsA, IL-17 promotes bone erosion through upregulation of RANKL. Results from a pooled data analysis indicated that patients treated with ustekinumab (regardless of dose) significantly inhibited radiographic progression of joint damage in patients with active PsA ((Kavanaugh, A et al. Ann. Rheum. Dis. 73(6):1000-1006 (2014)). This supports a role for IL-23 and the downstream Th17 pathway in the radiographic damage that occurs in most PsA patients.

[0109] Both Crohn's disease (CD) and ulcerative colitis (UC), the primary inflammatory bowel diseases (IBD) in humans, are chronic, relapsing disorders characterized by chronic tissue inflammation that alters intestinal integrity and function. Elevated levels of interleukin (IL)-23 and T helper (Th)17 cell cytokines are found in the intestinal mucosa, plasma, and serum of patients with inflammatory bowel diseases (IBD), such as Crohn's disease (CD) and ulcerative colitis (UC).

[0110] Variants in several genes encoding elements of the IL-23 and IL-17 cellular pathways are associated with IBD risk. In particular, loss-of-function variants in the IL-23 receptor gene encoding an amino acid change from arginine to glutamine at position 381 have been observed to reduce the risk of IBD due to reduced STAT3 signaling and reduced Th17 cell responses upon exposure to IL-23 (Barrett, JC et al. Nat. Genet. 40:955-962 (2008); Duerr, RH et al. Science 314:1461-1463 (2006); Allocca, M et al. Best Practice & Res. Clin. Gastro. 32-33:95-102 (2018)).

[0111] Crohn's disease (CD) is a chronic immune-mediated condition characterized by a relapsing nature and involvement of the gastrointestinal system. CD is characterized by dysregulation of both innate and adaptive immune responses. Although the pathophysiological mechanisms are not fully understood, the disease appears to be the result of an interaction between the intestinal flora and the host's microbial defenses in genetically predisposed individuals, resulting in a transmural inflammatory response in Crohn's disease (Deepak, P and Loftus, E, Drug Design, Development and Therapy (10) 3685-3698) (2016)). Over the long term, persistent transmural inflammatory responses often lead to the development of strictures and / or fistulas, requiring hospitalization and / or surgery. Following the discovery of the IL-23 / IL-17 pathway, the treatment paradigm for CD shifted from nonspecific immunosuppressive therapy (i.e., methotrexate) to immunotherapy targeting the IL-2 and / or IL-17 pathways.

[0112] UC is a chronic, relapsing-remitting inflammatory bowel disease (UC) that causes ongoing mucosal inflammation of the large intestine, resulting in the development of small open wounds or ulcers that produce pus and mucosa. It is estimated that nearly 1 million people with UC live in the United States and 2.6 million people in Europe. While the disease is more common among Caucasians, it can affect people of any racial or ethnic group, and men are more likely to be diagnosed than women. The pathogenesis of UC is poorly understood, but it is thought to be due in part to an abnormal immune response to the microbiota (microflora and pathogens) in genetically predisposed subjects, leading to chronic inflammation of the colon. Ulcerative colitis is known to exhibit a Th2-type cytokine profile.

[0113] IL-23-specific p19 antagonists under clinical investigation for IBD include brazikumab (MEDI2070), risankizumab (BI 655066), mirikizumab (LY3074828), and guselkumab (Tremfya, Janssen). To date, the anti-p19 (anti-IL-23) antibodies, brazikumab and risankizumab, have been reported to be effective in moderate to severe CD in phase II trials.

[0114] Mirikizumab was recently shown to be effective for moderate-to-severe UC in a phase II clinical trial. Across all doses studied, between 11.5% and 22.6% of patients treated with mirikizumab achieved clinical remission compared with 4.8% of patients treated with placebo. In addition, a greater proportion of patients treated with mirikizumab achieved endoscopic and symptomatic remission at week 12 compared with placebo. Currently, no p19-selective antibodies are approved for the treatment of IBD. Phase II and III trials of anti-p19 agents (risankizumab, brazikumab, and guselkumab) are ongoing, and these trials will provide further information not only on their efficacy and safety per se, but also on their efficacy in direct comparison with existing biologics and on the evolving therapeutic concept of combination treatment with multiple biologics.

[0115] Ankylosing spondylitis (AS) is another spondyloarthropathy that, like psoriatic arthritis, is genetically linked to the IL-23 pathway; it is a painful condition involving inflammation of the spine that can lead to irreversible spinal fusion. AS generally does not respond to traditional disease-modifying antirheumatic drugs (DMARDs), and systemic treatment for AS consists of nonsteroidal anti-inflammatory drugs (NSAIDs) and tumor necrosis factor inhibitors.

[0116] Several lines of evidence have identified IL-23 as a potential therapeutic target for AS (Paine A, et al. Curr. Opin. Rheumatol. 28:359-67 (2016)). At the genetic level, a case-control genome-wide association study demonstrated that IL-23 receptor (IL-23R) polymorphisms are associated with an increased risk of developing AS (Reveille JD, et al. Genet 42:123-7 (2010)). In addition, a protective effect of the IL-23R R381Q polymorphism is observed in AS (Sarin R, et al. Proc Natl Acad Sci USA; 108:9560-58 (2011)). Increased numbers of IL-23-producing cells have been found in the facet joints of patients with AS (Appel H, et al. Arthritis Rheum; 65:1522-9 (2013)), while the numbers of IL-23-responsive T helper (Th)22 (Th22), Th17, and gamma / delta T cells are elevated in the blood from patients with AS (Zhang L, et al. PLoS One (7):e31000 (2012)).

[0117] The recent approval of the IL-17A inhibitor secukinumab for the treatment of AS (Baeten D. et al., N Engl. J. Med. 373:2534-48 (2015)) supports the clinical hypothesis that direct and specific inhibition of IL-23 may be therapeutic for patients with AS. However, a recent publication (NCT02047110) reporting the results of a randomized, double-blind, placebo-controlled, proof-of-concept, dose-ranging phase 2 trial evaluating the efficacy of risankizumab in patients with active AS concluded that treatment with risankizumab did not meet the study's primary endpoint and did not demonstrate any evidence of clinically meaningful improvement compared with placebo in patients with active AS (Baeten D, et al., Annals of the Rheumatic Diseases 77: 1295-1302 (2018)).

[0118] IL-23p19 antagonist The IL-23 receptor complex consists of IL-12Rβ1 partnered with the signaling chain IL-23R (p19 subunit binding). IL-23 mediates cellular activities through sequential binding to the two receptor chains expressed on the surface of T cells and natural killer (NK) cells as the IL-12Rβ1 / IL-23R receptor complex.

[0119] Murine, humanized and phage-displayed antibodies selected for the inhibition of recombinant IL-23 have been described; see, e.g., U.S. Pat. No. 7,491,391, WIPO publications WO 1999 / 05280, WO 2007 / 0244846, WO 2007 / 027714, WO 2007 / 076524, WO 2007 / 147019, WO 2008 / 103473, WO 2008 / 103432, WO 2009 / 043933 and WO 2009 / 082624.

[0120] Monoclonal antibodies or their antigen-binding domains that bind with high affinity to the p19 subunit of the IL-23 cytokine can neutralize the activity of the IL-23 cytokine and thereby block its downstream effects. To date, three anti-p19 antibodies, guselkumab (TREMFYA®), tildrakizumab (ILUMYA®), and risankizumab (SKYRIZI®), have been approved by the FDA for the treatment of IMIDs. Two other IL-23p19 subunit-specific antibodies are currently in late-stage clinical development: MEDI2070 (brazikumab, Astrazeneca / Medimmue) and Ly3074828 (mirikizumab, Eli Lilly). Mirikizumab is a humanized IgG4 monoclonal antibody. By blocking IL-23, anti-p19 specific antibodies inhibit the release of pro-inflammatory cytokines and chemokines, thereby dampening the inflammatory response.

[0121] This group of IL-23 antagonists targets the p19 subunit of IL-23, but not the p40 subunit, and therefore does not affect IL-12 activity. This property distinguishes them from ustekinumab (STELARA), which targets the common p40 subunit shared by IL-12 and IL-23. Despite the efficacy and favorable safety profile of ustekinumab, drug development for IMIDs has shifted its focus toward the development of agents that selectively antagonize the IL-23 / IL-17 pathway.

[0122] Guselkumab (CNTO1959) is a fully human monoclonal IgG1,λ antibody that binds with high affinity to the p19 subunit of human IL-23. Guselkumab is the first FDA-approved anti-p19-specific antibody / IL-23 antagonist. It was approved on July 13, 2017, after accelerated regulatory review, for the treatment of adults with moderate to severe plaque psoriasis. It is also approved in Canada, the European Union, Japan, and several other countries worldwide. Guselkumab is marketed by Janssen as TREMFYA (U.S. Patent Nos. 7,935,344 and 7,993,645).

[0123] Guselkumab inhibits the biological activity of human IL-23 by preventing IL-23 from binding to the IL-23 receptor protein expressed on the surface of immune cells. More specifically, guselkumab binds to the human IL-23 cytokine via the p19 subunit, preventing the formation of the IL-23-IL-23R complex and subsequent intracellular signaling of the partner receptor chain.

[0124] The TREMFYA® development program currently includes a Phase III trial evaluating the efficacy of TREMFYA® to treat active psoriatic arthritis, a Phase Ib / III trial in Crohn's disease, a Phase IIb / III trial in ulcerative colitis, and a separate clinical trial evaluating guselkumab for hidradenitis suppurativa.

[0125] Janssen recently announced plans to further expand the clinical development of guselkumab into familial adenomatous polyposis (FAP), a gastrointestinal disease. Janssen has initiated a Phase 1b proof-of-concept trial (NCT03649971) evaluating the efficacy and safety of guselkumab versus placebo in approximately 72 patients. FAP syndrome is the most common adenomatous polyposis syndrome. It is an autosomal dominant genetic disorder characterized by the early development of hundreds to thousands of adenomatous polyps throughout the colon. FAP has an incidence of approximately 1 in 8,300 births worldwide and manifests equally in both men and women. If left untreated, patients with this syndrome will almost certainly develop colorectal cancer. In addition, there is an increased risk of developing other malignancies. Removal of the colon is currently the only way to prevent colorectal cancer in these patients.

[0126] Tildrakizumab (MK322) is a humanized monoclonal IgG1,κ antibody marketed by Merck & Co. / Sun Pharmaceutical as ILUMYA (U.S. Patent No. 8,404,813). Tildrakizumab selectively binds to the p19 subunit, thereby inhibiting IL-23 interaction with its receptor and thus inhibiting IL-23-mediated proinflammatory cytokine release. Tildrakizumab was first approved worldwide by the FDA in March 2018 for use in adult patients with moderate to severe plaque psoriasis.

[0127] Risankizumab (BI 655066) is a humanized monoclonal IgG1,κ marketed by AbbVie / Boehringer Ingelheim as SKYRIZI (U.S. Patent No. 8,778,346). Risankizumab was developed as a high-affinity antibody antagonist of IL-23.

[0128] Risankizumab selectively binds to the p19 subunit of interleukin-23 (IL-23p19) with high affinity (dissociation constant <10 pmol / L) (Singh, S, et al., MAbs 7(4)77-791 (2015)). It selectively targets the p19 subunit of IL-23 and potently inhibits IL-23-induced (human IL-23 produced by THP-1 cells) IL-17 production in a mouse splenocyte assay with an IC50 value of approximately 2 pM (Singh, S, et al., MAbs 7(4)77-791 (2015)). The framework region of risankizumab has been modified with two mutations in the Fc region to reduce FcγR receptor and complement binding. More specifically, the Fc portion of risankizumab has two substitution mutations (Leu234Ala and Leu235Ala) to reduce Fcγ receptor and complement binding. Risankizumab was approved by the FDA in April 2019 for the treatment of moderate to severe plaque psoriasis in adults.

[0129] Anti-IL-23p19 antibody The anti-IL-23p19 antibodies of the present disclosure bind to the p19 subunit of IL-23. Preferably, such antibodies are fully human and do not bind to the p40 subunit of IL-12.

[0130] In one embodiment, an anti-IL-23p19 antibody or antibody fragment thereof comprises a VH having a set of CDRs (HCDR1, HCDR2, and HCDR3) disclosed in Table 1. For example, an anti-IL-23p19 antibody or antibody fragment thereof can comprise a set of CDRs corresponding to those CDRs in one or more anti-IL-23p19 antibodies disclosed in Table 1 (e.g., the CDRs of the Hu-2.18006B antibody).

[0131] In another embodiment, the anti-IL-23p19 antibody comprises a VL having a set of CDRs (LDCR1, LDCR2, and LDCR3) disclosed in Table 2. For example, an anti-IL-23p19 antibody or antibody fragment thereof can comprise a set of CDRs corresponding to those CDRs in one or more anti-IL-23p19 antibodies disclosed in Table 2 (e.g., the CDRs of the Hu-2.18006B antibody).

[0132] In an alternative embodiment, the anti-IL-23p19 antibody or antibody fragment thereof comprises a VH having the set of CDRs (HCDR1, HCDR2, and HCDR3) disclosed in Table 1, and a VL having the set of CDRs (LCDR1, LCDR2, and LCDR3) disclosed in Table 2.

[0133] [Table 1]

[0134] [Table 2]

[0135] In one embodiment, the antibody can be a monoclonal, chimeric, humanized or human antibody (or an antigen-binding portion thereof) that specifically binds to human IL-23p19.

[0136] In one embodiment, the anti-IL-23p19 antibody or antibody fragment thereof is (i) CDR1: SEQ ID NO: 9, CDR2: SEQ ID NO: 10, CDR3: SEQ ID NO: 11; (ii) CDR1: SEQ ID NO: 15, CDR2: SEQ ID NO: 16, CDR3: SEQ ID NO: 17; (iii) CDR1: SEQ ID NO: 21, CDR2: SEQ ID NO: 22, CDR3: SEQ ID NO: 23; and (iv) CDR1: SEQ ID NO: 27, CDR2: SEQ ID NO: 28, CDR3: SEQ ID NO: 29 The VH comprises a series of complementarity determining regions (CDR1, CDR2, and CDR3) selected from the group consisting of:

[0137] In another embodiment, the anti-IL-23p19 antibody or antibody fragment thereof is (i) CDR1: SEQ ID NO: 12, CDR2: SEQ ID NO: 13, CDR3: SEQ ID NO: 14; (ii) CDR1: SEQ ID NO: 18, CDR2: SEQ ID NO: 19, CDR3: SEQ ID NO: 20; (iii) CDR1: SEQ ID NO: 24, CDR2: SEQ ID NO: 25, CDR3: SEQ ID NO: 26; and (iv) CDR1: SEQ ID NO: 30, CDR2: SEQ ID NO: 31, CDR3: SEQ ID NO: 32 The VL has a series of complementarity determining regions (CDR1, CDR2, and CDR3) selected from the group consisting of:

[0138] In another embodiment, the anti-IL-23p19 antibody or antibody fragment thereof is (a) (i) CDR1: SEQ ID NO: 9, CDR2: SEQ ID NO: 10, CDR3: SEQ ID NO: 11; (ii) CDR1: SEQ ID NO: 15, CDR2: SEQ ID NO: 16, CDR3: SEQ ID NO: 17; (iii) CDR1: SEQ ID NO: 21, CDR2: SEQ ID NO: 22, CDR3: SEQ ID NO: 23; and (iv) CDR1: SEQ ID NO: 27, CDR2: SEQ ID NO: 28, CDR3: SEQ ID NO: 29, VH having a series of complementarity determining regions (CDR1, CDR2, and CDR3) selected from the group consisting of: (b) (i) CDR1: SEQ ID NO: 12, CDR2: SEQ ID NO: 13, CDR3: SEQ ID NO: 14; (ii) CDR1: SEQ ID NO: 18, CDR2: SEQ ID NO: 19, CDR3: SEQ ID NO: 20; (iii) CDR1: SEQ ID NO: 24, CDR2: SEQ ID NO: 25, CDR3: SEQ ID NO: 26; and (iv) CDR1: SEQ ID NO: 30, CDR2: SEQ ID NO: 31, CDR3: SEQ ID NO: 32, A VL having a series of complementarity determining regions (CDR1, CDR2, and CDR3) selected from the group consisting of: Includes:

[0139] In one embodiment, the antibody (i) VH: CDR1: SEQ ID NO: 9, CDR2: SEQ ID NO: 10, CDR3: SEQ ID NO: 11, VL: CDR1: SEQ ID NO: 12, CDR2: SEQ ID NO: 13, CDR3: SEQ ID NO: 14; (ii) VH: CDR1: SEQ ID NO: 15, CDR2: SEQ ID NO: 16, CDR3: SEQ ID NO: 17, VL: CDR1: SEQ ID NO: 18, CDR2: SEQ ID NO: 19, CDR3: SEQ ID NO: 20; (iii) VH: CDR1: SEQ ID NO: 21, CDR2: SEQ ID NO: 22, CDR3: SEQ ID NO: 23, VL: CDR1: SEQ ID NO: 24, CDR2: SEQ ID NO: 25, CDR3: SEQ ID NO: 26; and (iv) VH: CDR1: SEQ ID NO: 27, CDR2: SEQ ID NO: 28, CDR3: SEQ ID NO: 29, VL: CDR1: SEQ ID NO: 30, CDR2: SEQ ID NO: 31, CDR3: SEQ ID NO: 32 The combination of VH and VL has a set of complementarity determining regions (CDR1, CDR2 and CDR3) selected from the group consisting of:

[0140] In one embodiment, the anti-IL-23p19 antibody or antibody fragment thereof comprises a variable heavy chain sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7, and / or a variable light chain sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8.

[0141] In one embodiment, the anti-IL-23p19 antibody or antibody fragment thereof comprises a pair of variable heavy and variable light chain sequences selected from the following combinations: a variable heavy chain sequence comprising SEQ ID NO: 1 and a variable light chain sequence comprising SEQ ID NO: 2; a variable heavy chain sequence comprising SEQ ID NO: 3 and a variable light chain sequence comprising SEQ ID NO: 4; a variable heavy chain sequence comprising SEQ ID NO: 5 and a variable light chain sequence comprising SEQ ID NO: 6; and a variable heavy chain sequence comprising SEQ ID NO: 7 and a variable light chain sequence comprising SEQ ID NO: 8. Those of skill in the art will further appreciate that the variable light and variable heavy chains can be independently selected or mixed and matched to prepare anti-IL-23p19 antibodies comprising variable heavy and variable light chain combinations distinct from the pairings identified above.

[0142] In one embodiment, the anti-IL-23p19 antibody or antibody fragment thereof comprises a pair of variable heavy and variable light chain sequences selected from the following combinations: a variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO:1 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO:2; a variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO:3 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO:4; a variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO:5 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO:6; and a variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO:7 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO:8. Those of ordinary skill in the art will further understand that variable light and variable heavy chains can be independently selected or mixed and matched to prepare anti-IL-23p19 antibodies comprising variable heavy and light chain combinations distinct from the pairings identified above.

[0143] In some embodiments, an anti-IL-23p19 antibody (eg, an antagonist antibody) binds with high affinity to the p19 subunit of IL-23 and does not bind to the p40 subunit of the related cytokine family member, IL-12.

[0144] In some embodiments, the antibody is a full-length antibody. In other embodiments, the antibody is an antibody fragment, including, for example, an antibody fragment selected from the group consisting of Fab, Fab', F(ab)2, Fv, domain antibody (dAb), and complementarity-determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, minibodies, and polypeptides containing at least a portion of an immunoglobulin sufficient to confer IL-23 specific binding thereto.

[0145] In some embodiments, the variable region domain of an anti-IL-23p19 antibody disclosed herein can be covalently attached at its C-terminal amino acid to at least one other antibody domain or fragment thereof. Thus, for example, a VH domain present in the variable region domain can be linked to an immunoglobulin CH1 domain or fragment thereof. Similarly, a VL domain can be linked to a CK domain or fragment thereof. Thus, for example, an antibody can be a Fab fragment, in which the antigen-binding domain contains associated VH and VL domains, which are covalently linked at their C-termini to the CH1 and CK domains, respectively. The CH1 domain can be extended with additional amino acids to provide, for example, a hinge region or part of a hinge region domain, as found in a Fab fragment, or to provide additional domains, such as the CH2 and CH3 domains of an antibody.

[0146] In some embodiments, the variable region domain of an anti-IL-23p19 antibody can be covalently attached at its C-terminal amino acid to at least one other antibody domain or fragment thereof. Thus, for example, a VH domain present in the variable region domain can be linked to an immunoglobulin CH1 domain or fragment thereof. Similarly, a VL domain can be linked to a CK domain or fragment thereof. Thus, for example, an antibody can be a Fab fragment, in which the antigen-binding domain contains associated VH and VL domains, which are covalently linked at their C-termini to the CH1 and CK domains, respectively. The CH1 domain can be extended with additional amino acids to provide, for example, a hinge region or part of a hinge region domain, as found in a Fab' fragment, or to provide additional domains, such as the CH2 and CH3 domains of an antibody.

[0147] Thus, in one embodiment, an antibody fragment comprises at least one CDR described herein. An antibody fragment may comprise at least two, three, four, five, or six CDRs described herein. An antibody fragment may further comprise at least one variable region domain of an antibody described herein. The variable region domain may be of any size or amino acid composition, but will collectively comprise at least one CDR sequence responsible for binding to human IL-23p19, e.g., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3 described herein, which is adjacent to or in-frame with one or more framework sequences.

[0148] In some embodiments, the anti-IL-23p19 antibody is a monoclonal antibody. In some embodiments, the anti-IL-23p19 antibody is a human antibody. In alternative embodiments, the anti-IL-23p19 antibody is a murine antibody. In some embodiments, the anti-IL-23p19 antibody is a chimeric antibody, a bispecific antibody, or a humanized antibody.

[0149] In further embodiments, the anti-IL-23p19 antibody or antibody fragment thereof exhibits one or more of the following properties: (a) specific for human IL-23p19 and capable of blocking IL-23 binding to its receptor (IL-23R); (b) inhibiting, disrupting, or modulating IL-23p19 interaction with IL-23 receptor signaling; (c) inhibiting IL-23-induced STAT3 activation; (d) inhibiting IL-17 production induced by human IL-23 in mouse splenocytes; (e) inhibiting IL-17 production induced by human IL-23 in activated human T cells in PBMCs; (f) does not inhibit IL-23 interaction with IL-12Rβ1 signaling; (g) does not inhibit human IL-12-induced interferon gamma production in human activated T cells (PBMC); (h) does not inhibit cynomolgus monkey IL-12-induced interferon gamma production in human activated T cells (PBMC); and (i) inhibits skin inflammation induced by human IL-23 in a mouse psoriasis-like model.

[0150] In one embodiment, anti-IL-23p19 antibodies or antibody fragments thereof can reduce, inhibit, block, and / or modulate at least one biological response associated with IL-23 and are therefore useful for ameliorating the effects of an IL-23-associated disease or disorder. Such antibodies and antibody fragments thereof can be used, for example, to reduce, inhibit, block, and / or modulate IL-23 signaling, IL-23 activation of Th17 cells, IL-23 activation of NK cells, or inducing the production of proinflammatory cytokines.

[0151] In some embodiments, an anti-IL-23p19 antibody or antibody fragment thereof contains one or more conservative amino acid substitutions. Those skilled in the art will understand that a conservative amino acid substitution is a substitution of one amino acid with another amino acid having similar structural or chemical properties, such as a similar side chain. Exemplary conservative substitutions are described in the art, for example, in Watson et al., Molecular Biology of the Gene, The Benjamin / Cummings Publication Company, 4th Ed. (1987).

[0152] "Conservative modifications" refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Conservative modifications include amino acid substitutions, additions, and deletions. A conservative substitution is one in which an amino acid is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are well defined, including amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine), and sulfur-containing side chains (cysteine, methionine). Furthermore, any naturally occurring residue in the polypeptide can be substituted with alanine, as previously described for alanine scanning mutagenesis (MacLennan et al. (1998) Acta Physiol Scand Suppl 643: 55-67; ​​Sasaki et al. (1998) Adv Biophys 35: 1-24). Amino acid substitutions in the antibodies of the invention can be made by known methods, for example, by PCR mutagenesis (U.S. Pat. No. 4,683,195).

[0153] In one embodiment, the anti-IL-23p19 antibody or antibody fragment thereof comprises all six of the CDR regions of Hu-2.18006B, Hu-4.18006B, Hu-5.18006B, or Hu-6.18006B antibodies formatted as a chimeric or humanized antibody. In other embodiments, the anti-IL-23p19 antibody or antibody fragment thereof comprises all six of the CDR regions of one of the disclosed fully human antibodies.

[0154] How to generate antibodies Anti-IL-23p19 antibodies or antibody fragments thereof can be produced by any method known in the art. For example, a recipient can be immunized with soluble recombinant human IL-23 protein, or a fragment thereof, or a peptide thereof conjugated to a carrier protein. Any suitable immunization method can be used. Such methods can include the use of adjuvants, other immunostimulants, repeated booster immunizations, and one or more immunization routes.

[0155] Any suitable source of human IL-23 can be used as the immunogen for the generation of non-human or human anti-IL-23p19 antibodies in the compositions and methods disclosed herein.

[0156] Various forms of IL-23 antigen can be used to generate antibodies sufficient to produce biological activity. Thus, the inducing IL-23 antigen can be a single epitope, multiple epitopes, or the entire protein, alone or in combination with one or more immunogenicity enhancing agents. In some embodiments, the inducing antigen is an isolated, soluble, full-length protein, or a soluble protein containing less than the full-length sequence (e.g., immunizing with a peptide containing a specific portion or epitope of IL-23). ​​As used herein, the term "portion" refers to the minimum number of amino acids or nucleic acids that constitute an immunogenic epitope of the antigen of interest, as appropriate. Any genetic vector suitable for transforming the cells of interest can be used, including, but not limited to, adenoviral vectors, plasmids, and non-viral vectors such as cationic lipids.

[0157] It is desirable to prepare monoclonal antibodies (mAbs) from various mammalian hosts, including mice, rodents, primates, humans, etc. Descriptions of techniques for preparing such monoclonal antibodies can be found, for example, in Sties et al. (eds.) BASIC AND CLINICAL IMMUNOLOGY (4th ed.) Lance Medical Publication, Los Altos, CA, and references cited therein; Harlow and Lane (1988) ANTIBODIES: A LABORATORY MANUAL CSH Press; Goding (1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2nd ed.) Academic Press, New York, NY. Typically, spleen cells from an animal immunized with a desired antigen are immortalized, usually by fusion with a myeloma cell. See Kohler and Milstein (1966) Eur. J. Immunol. 6:511-519. Alternative methods for immortalization include transformation with Epstein-Barr virus, oncogenes, or retroviruses, or other methods known in the art. See, for example, Doyle et al. (eds. 1994 and periodic supplements) CELL AND TISSUE CULTURE: LABORATORY PROCEDURES, John Wiley and Sons, New York, NY. Colonies arising from single immortalized cells are screened for the production of antibodies of the desired specificity and affinity to the antigen. The yield of monoclonal antibodies produced by such cells can be increased by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, DNA sequences encoding monoclonal antibodies or antigen-binding fragments thereof can be isolated by screening a DNA library derived from human B cells, following the general protocol outlined in, for example, Huse et al. (1989) Science 246: 1275-1281.Thus, antibodies can be obtained by a variety of techniques well known to the skilled worker in the art.

[0158] Other suitable techniques involve the selection of antibody libraries in phage, yeast, viral, or similar vectors. See, e.g., Huse et al., supra; and Ward et al., (1989) Nature 341:544-546. The polypeptides and antibodies disclosed herein, including chimeric or humanized antibodies, can be used with or without modification. Often, polypeptides and antibodies are labeled by joining, either covalently or noncovalently, a substance that provides a detectable signal. A wide variety of labels and conjugation techniques are known and widely reported in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents that teach the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Recombinant immunoglobulins can also be produced, see Cabilly U.S. Pat. No. 4,816,567; and Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86: 10029-10023; or can be made in transgenic mice, see Nils Lonberg et al., (1994), Nature 368:856-859; and Mendez et al. (1997) Nature Genetics 15: 146-156; TRANSGENIC ANIMALS AND METHODS OF USE (WO 2012 / 62118), Medarex, Trianni, Abgenix, Ablexis, OminiAb, Harbour, and other techniques.

[0159] In some embodiments, the ability of the produced antibodies to bind to IL-23p19 can be assessed using standard binding assays, such as surface plasmon resonance (SPR), octet (BLI), ELISA, Western blot, immunofluorescence, flow cytometry analysis, chemotaxis assays, and cell migration assays. In some aspects, the produced antibodies can also be assessed for their ability to prevent IL-23 from blocking IL-23 receptor β1 signaling and to inhibit the sequential effects of IL-23p19 and / or the IL-23p19-mediated inflammatory microenvironment, including inhibiting IL-23-induced Stat3 phosphorylation, IL-17 production, and / or IFN-γ production.

[0160] Antibody compositions prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the common purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (see, e.g., Lindmark et al., 1983 J. Immunol. Meth. 62:1-13). Protein G is recommended for all mouse isotypes and for human γ3 (see, e.g., Guss et al., 1986 EMBO J. 5:1567-1575). The matrix to which the affinity ligand is attached is most often agarose, although other matrices are also available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on ion-exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion- or cation-exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available, depending on the antibody to be recovered.

[0161] Following any preliminary purification steps, the mixture containing the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography, typically performed at low salt concentrations (e.g., from about 0-0.25 M salt), but using an elution buffer at a pH between about 2.5 and 4.5.

[0162] Also included are nucleic acids that hybridize to all or a portion (e.g., a portion encoding a variable region) of the nucleotide sequence represented by the isolated polynucleotide sequence encoding the antibody or antibody fragment of the present disclosure under low, medium, and high stringency conditions, as defined herein. The hybridizing portion of the hybridizing nucleic acid is typically at least 15 (e.g., 20, 25, 30, or 50) nucleotides in length. The hybridizing portion of the hybridizing nucleic acid is at least 80%, e.g., at least 90%, at least 95%, or at least 98% identical to the sequence of part or all of a nucleic acid encoding an anti-IL-23p19 polypeptide (e.g., a heavy chain variable region or a light chain variable region) or its complementary strand. Hybridizing nucleic acids of the type described herein can be used, for example, as cloning probes, primers, e.g., PCR primers, or diagnostic probes.

[0163] Polynucleotides, vectors and cells Other embodiments include isolated polynucleotides comprising sequences encoding anti-IL-23p19 antibodies or antibody fragments thereof, vectors and cells comprising such polynucleotides, and recombinant techniques for producing such antibodies. The isolated polynucleotides can encode any desired form of anti-IL-23p19 antibody, including, for example, full-length monoclonal antibodies, Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, miniantibodies, and multispecific antibodies formed from antibody fragments.

[0164] Some embodiments include an isolated polynucleotide comprising a sequence encoding a light chain variable region of an antibody or antibody fragment having the amino acid sequence of any of SEQ ID NOs: 2, 4, 6, and 8. Some embodiments include an isolated polynucleotide comprising a sequence encoding a heavy chain variable region of an antibody or antibody fragment having the amino acid sequence of SEQ ID NOs: 1, 3, 5, and 7.

[0165] In one embodiment, the isolated polynucleotide sequence is (a) a variable heavy chain sequence of SEQ ID NO: 1 and a variable light chain sequence of SEQ ID NO: 2; (b) a variable heavy chain sequence of SEQ ID NO: 3 and a variable light chain sequence of SEQ ID NO: 4; (c) a variable heavy chain sequence of SEQ ID NO: 5 and a variable light chain sequence of SEQ ID NO: 6; or (d) the variable heavy chain sequence of SEQ ID NO: 7 and the variable light chain sequence of SEQ ID NO: 8 The antibody or antibody fragment encodes an antibody or antibody fragment having a light chain variable region and a heavy chain variable region comprising the amino acid sequence of

[0166] In another embodiment, the isolated polynucleotide sequence is (a) a variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 1 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 2; (b) a variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO:3 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO:4; (c) a variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO:5 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO:6; or (d) a variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO:7 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO:8 The antibody or antibody fragment encodes an antibody or antibody fragment having a light chain variable region and a heavy chain variable region comprising the amino acid sequence of

[0167] A polynucleotide containing a sequence encoding an anti-IL-23p19 antibody or antibody fragment thereof can be fused to one or more regulatory or control sequences known in the art and contained in an appropriate expression vector or cell known in the art. Each polynucleotide molecule encoding a heavy chain variable domain or a light chain variable domain can be independently fused to a polynucleotide sequence encoding a constant domain, such as a human constant domain, thereby enabling the production of an intact antibody. Alternatively, polynucleotides or portions thereof can be fused to each other, resulting in a template for the production of a single-chain antibody.

[0168] For recombinant production, a polynucleotide encoding the antibody is inserted into a replicable vector for cloning (amplification of the DNA) or expression. Many vectors suitable for expressing recombinant antibodies are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0169] Anti-IL-23p19 antibodies or antibody fragments thereof may also be produced as fusion polypeptides, in which the antibody or fragment is fused to a heterologous polypeptide, such as a signal sequence, or other polypeptide having a specific cleavage site at the amino terminus of the mature protein or polypeptide. The selected heterologous signal sequence is typically one that is recognized and processed by the cell (i.e., cleaved by a signal peptidase). In the case of prokaryotic cells that do not recognize and process the anti-IL-23p19 antibody signal sequence, the signal sequence can be replaced with a prokaryotic signal sequence. The signal sequence can be, for example, an alkaline phosphatase, penicillinase, lipoprotein, or heat-stable enterotoxin II leader, etc. For secretion in yeast, the native signal sequence can be substituted with leader sequences derived from, for example, yeast invertase alpha-factor (including the alpha-factor leaders of Saccharomyces and Kluyveromyces), acid phosphatase, C. albicans glucoamylase, or the signals described in WO 90 / 13646. In mammalian cells, mammalian signal sequences can be used, as well as viral secretory leaders, such as the herpes simplex gD signal. DNA for such a precursor region is ligated in reading frame to DNA encoding the anti-IL-23p19 antibody.

[0170] Expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected cells. Generally, the sequence in cloning vectors is one that enables the vector to replicate independently of the host chromosomal DNA and includes an origin of replication or autonomously replicating sequence. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2-u. plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV, and BPV) are useful for cloning vectors in mammalian cells. Generally, origin of replication components are not needed for mammalian expression vectors (the SV40 origin may commonly be used only because it contains the early promoter).

[0171] Expression and cloning vectors can contain a gene encoding a selectable marker to facilitate identification of expression. Typical selectable marker genes encode proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, or alternatively complement auxotrophic deficiencies. Alternatively, the selectable marker gene may supply a particular nutrient not present in complex media, for example, a gene encoding D-alanine racemase for Bacilli.

[0172] Non-therapeutic uses The anti-IL-23p19 antibodies or antibody fragments described herein are useful as affinity purification agents. In this method, the antibody is immobilized on a solid phase, such as a protein A resin, using methods well known in the art. The immobilized antibody is contacted with a sample containing the IL-23p19 protein (or a fragment thereof) to be purified, and the support is then washed with a suitable solvent that will substantially remove all substances in the sample other than the IL-23p19 protein bound to the immobilized antibody. Finally, the support is washed with another suitable solvent, which will release the IL-23p19 protein from the antibody.

[0173] Anti-IL-23p19 antibodies or antibody fragments are also useful in diagnostic assays, e.g., to detect IL-23p19 expression in specific cells, tissues, or serum, and to detect and / or quantify IL-23p19 protein. Anti-IL-23p19 antibodies can be used diagnostically, e.g., as part of a clinical trial procedure to monitor the development or progression of a disease, thereby determining, for example, the effectiveness of a given treatment and / or prophylactic regimen. Detection can be facilitated by coupling anti-IL-23p19 antibodies to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, various positron-emitting metals using positron emission tomography, and non-radioactive paramagnetic metal ions. See, e.g., U.S. Pat. No. 4,741,900 for metal ions that can be conjugated to antibodies for diagnostic use according to the present disclosure.

[0174] Anti-IL-23p19 antibodies or antibody fragments can be used in methods for diagnosing IL-23p19-associated disorders (e.g., disorders characterized by aberrant expression of IL-23p19) or for determining whether a subject has an elevated risk of developing an IL-23p19-associated disorder. Such methods include contacting a biological sample from a subject with an anti-IL-23p19 antibody or antibody fragment thereof and detecting binding of the antibody to IL-23p19. By "biological sample" is intended any biological sample obtained from an individual cell line, tissue culture, or other source of cells potentially expressing IL-23p19. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.

[0175] In some embodiments, the method may further include comparing the level of IL-23p19 in the patient sample with a control sample (e.g., a subject without an IL-23p19-associated disorder) to determine whether the patient has or is at risk of developing an IL-23p19-associated disorder.

[0176] In some embodiments, for example, for diagnostic purposes, it may be advantageous to label the antibody with a detectable moiety. Numerous detectable labels are available, including radioisotopes, fluorescent labels, enzyme substrate labels, and the like. The label can be indirectly conjugated to the antibody using various known techniques. For example, the antibody can be conjugated with biotin, and any of the three broad categories of labels described above can be conjugated with avidin, or vice versa. Biotin selectively binds to avidin, and thus the label can be conjugated to the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label to the antibody, the antibody can be conjugated with a small hapten (e.g., digoxin), and one of the various types of labels described above is conjugated with an anti-hapten antibody (e.g., anti-digoxin antibody). This allows for indirect conjugation of the label to the antibody.

[0177] Exemplary radioisotope labels include: 35 S, 14 C. 125 I, 3 H, and 131 I. Antibodies can be labeled with radioisotopes using, for example, the techniques described in Current Protocols in Immunology, Volumes 1 and 2, 1991, Coligen et al., Ed. Wiley-Interscience, New York, NY, Pubs. Radioactivity can be measured, for example, by scintillation counting.

[0178] Exemplary fluorescent labels include those derived from rare earth chelators (europium chelators), or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin, and Texas Red are available. Fluorescent labels can be conjugated to antibodies via known techniques, such as those disclosed in Current Protocols in Immunology. Fluorescence can be quantified using a fluorometer.

[0179] There are a variety of well-characterized enzyme-substrate labels known in the art (see, e.g., U.S. Pat. No. 4,275,149). The enzyme generally catalyzes a chemical change in a chromogenic substrate that can be measured using a variety of techniques. For example, the change can be a change in the hue of the substrate, which can be measured spectrophotometrically. Alternatively, the enzyme can alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying changes in fluorescence are described above. The chemiluminescent substrate can be electrically excited by a chemical reaction and then emit light, which can be measured using, for example, a chemiluminometer, or donate energy to a fluorescent acceptor.

[0180] Examples of enzyme labels include luciferases, such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidases, such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are described, for example, in O'Sullivan et al., 1981, Methods for the Preparation of Enzyme-Antibody Conjugates for Use in Enzyme Immunoassay, in Methods in Enzym. (J. Langone & H. Van Vunakis, eds.), Academic Press, NY, 73: 147-166.

[0181] Examples of enzyme-substrate combinations include, for example, horseradish peroxidase (HRPO) with hydrogen peroxidase as the substrate, where the hydrogen peroxidase oxidizes a dye precursor such as orthophenylenediamine (OPD) or 3,3,5,5-tetramethylbenzidine hydrochloride (TMB); alkaline phosphatase (AP) with para-nitrophenyl phosphate as the chromogenic substrate; and β-D-galactosidase (β-D-Gal) with a chromogenic substrate such as p-nitrophenyl-β-D-galactosidase or the fluorogenic substrate 4-methylumbelliferyl-β-D-galactosidase.

[0182] In another embodiment, the anti-IL-23p19 antibody or antibody fragment thereof is used unlabeled and detected with a labeled antibody that binds to the anti-IL-23p19 antibody or antibody fragment thereof.

[0183] The antibodies and antibody fragments thereof described herein can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. See, e.g., Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

[0184] Anti-IL-23p19 antibodies or antibody fragments thereof can be used to inhibit the binding of a ligand to the IL-23 receptor. Such methods include administering an anti-IL-23p19 antibody to a cell (e.g., a mammalian cell) or cellular environment, thereby inhibiting IL-23 receptor-mediated signal transduction. These methods can be carried out in vitro or in vivo. "Cellular environment" refers to the tissue, culture medium, or extracellular matrix surrounding the cell.

[0185] Compositions and methods of treatment The present disclosure also provides compositions, including, for example, pharmaceutical compositions, comprising anti-IL-23p19 antibodies or antibody fragments thereof, which have numerous therapeutic uses for the treatment, prevention, or amelioration of diseases or disorders, such as immune-mediated inflammatory disorders or autoimmune diseases (e.g., diseases or disorders involving biological activity mediated by the IL-23 / IL-23 receptor signaling axis).

[0186] The anti-IL-23p19 antibodies or antibody fragments thereof disclosed herein are useful for treating various diseases or disorders, such as immune-mediated inflammatory disorders (IMIDs) or autoimmune diseases. A method for treating an IL-23-associated disorder comprises administering a therapeutically effective amount of an anti-IL-23p19 antibody or antibody fragment thereof to a subject in need thereof. The IMID can be selected from the group consisting of psoriasis, psoriatic arthritis, inflammatory bowel disease (e.g., ulcerative colitis or Crohn's disease), ankylosing spondylitis, systemic lupus erythematosus, hidradenitis suppurativa, atopic dermatitis, and asthma.

[0187] The present disclosure also provides methods for treating or preventing IMID, comprising administering to a subject in need thereof a composition or formulation comprising an anti-IL-23p19 antibody or antibody fragment thereof, and optionally another immune-based therapy.

[0188] The antibodies of the present disclosure are also useful in methods of treating cancer, either alone (eg, as monotherapy) or in combination with other immunotherapeutic agents and / or chemotherapy.

[0189] The antibodies can be administered either alone or in combination with other compositions useful for treating immune-mediated inflammatory disorders or autoimmune diseases. In some embodiments, compositions, including, for example, pharmaceutical compositions comprising anti-IL-23p19 antibodies, can further comprise a therapeutic agent, which may or may not be conjugated to a binding agent.

[0190] In some embodiments, compositions, e.g., pharmaceutical compositions, comprising one or more antibodies disclosed herein are provided. Pharmaceutical compositions can be formulated with pharmaceutically acceptable carriers or diluents, as well as any other known adjuvants and excipients, according to conventional techniques, such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995.

[0191] Typically, the composition for administration by injection is a solution in a sterile isotonic aqueous buffer.If necessary, the medicine may also contain a solubilizer and a local anesthetic such as lignocaine to alleviate pain at the injection site.Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example, as a lyophilized powder or a water-free concentrate in a sealed container such as an ampoule or sachet indicating the amount of active ingredient.When administered by infusion, the medicine can be dispensed using a dropper bottle containing sterile pharmaceutical-grade water or saline.When administered by injection, the medicine can be provided with an ampoule of sterile water for injection or saline so that the ingredients can be mixed before administration.

[0192] As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compounds, i.e., antibodies, bispecific and multispecific molecules, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0193] The composition can be administered by various methods known in the art.As those skilled in the art will understand, the route and / or mode of administration varies depending on the desired results.The active compound can be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches and microencapsulated delivery systems.Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used.Methods for preparing such formulations are generally known to those skilled in the art.For example, see Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0194] Dosage levels of the active ingredient in a pharmaceutical composition can be varied to provide an amount of the active ingredient that is non-toxic to the subject and effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration. The selected dosage level will depend on various pharmacokinetic factors, including factors well known in the medical arts, such as the activity of the particular composition employed, the route of administration, the time of administration, the excretion rate of the particular compound employed, the duration of treatment, other drugs, compounds, and / or substances used in combination with the particular composition employed, and the age, sex, weight, condition, overall health, and previous medical history of the patient being treated.

[0195] The pharmaceutical compositions described herein can be administered in an effective amount. "Effective amount" refers to an amount that achieves a desired response or a desired effect, either alone or together with further doses. In the case of treating a specific disease or a specific condition, the desired response is preferably related to inhibiting the progression of the disease. This includes delaying the progression of the disease, and in particular, halting or reversing the progression of the disease.

[0196] In some aspects, the compositions described herein are administered to a patient, e.g., in vivo, to treat or prevent various disorders, such as those described herein. Preferred patients include human patients having a disorder that can be corrected or ameliorated by administering an agent that modulates the biological activity of the IL-23 / IL-23 receptor signaling axis.

[0197] In some aspects, conventional viral and non-viral gene transfer methods can be used to introduce nucleic acids encoding the antibodies or derivatives thereof described herein into mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding antibodies to cells in vitro. In some embodiments, nucleic acids encoding antibodies or derivatives thereof are administered for in vivo or ex vivo gene therapy applications. In other embodiments, gene delivery techniques are used to study antibody activity in cell-based or animal models. Non-viral vector delivery systems include DNA plasmids, naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes. Viral vector delivery systems include DNA and RNA viruses that have either episomal or integrated genomes after delivery to cells. Such methods are well known in the art.

[0198] Non-viral delivery methods for nucleic acids encoding modified polypeptides of the present disclosure include lipofection, microinjection, gene guns, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and drug-enhanced uptake of DNA. Lipofection methods and lipofection reagents are well known in the art (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those described in Felgner, WO 91 / 17424, and WO 91 / 16024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration). The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those skilled in the art.

[0199] The use of RNA or DNA virus-based systems for delivery of nucleic acids encoding the antibodies described herein takes advantage of well-developed methods for targeting viruses to specific cells in the body and transporting the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo), or they can be used to treat cells in vitro and the modified cells are then administered to patients (ex vivo). Conventional viral-based systems for delivery of the polypeptides of the present disclosure include retroviral, lentiviral, adenoviral, adeno-associated herpes virus, and herpes simplex virus vectors for gene transfer. Viral vectors are currently the most efficient and versatile method for gene transfer in target cells and tissues. Integration into the host genome is possible using retroviral, lentiviral, and adeno-associated virus gene transfer methods, often resulting in long-term expression of the inserted transgene. In addition, high transduction efficiencies have been observed in many different cell types and target tissues. All patents and publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, methodologies described in such publications that might be used in connection with the present disclosure. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0200] Unless previously indicated, it will be understood by those skilled in the art that any of the various embodiments described and illustrated herein may be further modified to incorporate features shown in any of the other embodiments disclosed herein.

[0201] The broad scope of the present disclosure is best understood with reference to the following examples, which are not intended to limit the disclosure to the specific embodiments. The specific embodiments described herein are offered by way of example only, and the present disclosure is limited by the terms of the appended claims, including the full scope of equivalents to which such claims are entitled. [Example]

[0202] General method Protein purification methods, including immunoprecipitation, chromatography, and electrophoresis, are described in Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York. Chemical analysis, chemical modifications, post-translational modifications, production of fusion proteins, and protein glycosylation are also described. See, for example, Coligan et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, NJ, pp. 384-391. Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described. Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra.

[0203] Hybridoma supernatants were purified using a HiTrap Protein G column (GE, catalog no. 17040401) according to the manufacturer's protocol. Briefly, the Protein G column was equilibrated with 5 CV of DPBS (Gibco, catalog no. 14190-136), and the hybridoma supernatant was loaded via a syringe / infusion pump (Legato 200, KDS) at ambient temperature with a 3-minute residence time. The column was washed with 5 CV of DPBS, and elution was performed with 4 CV of pH 2.8 elution buffer (Fisher Scientific, catalog no. PI21004). The elution was fractionated, and fractions were neutralized with 1 M Tris-HCl, pH 8.5 (Fisher Scientific, catalog no. 50-843-270) and assayed by A280 (DropSense96, Trinean). Peak fractions were pooled and buffer exchanged into DPBS. Centrifugal filters (EMD Millipore, catalog number UFC803024) were equilibrated in DPBS at 4,000 x g for 2 minutes. The purified sample was loaded, DPBS was added, and the sample was spun at 4,000 x g for 5-10 minutes until the total DPBS volume was ≥ 6 DV. The final pool was analyzed by A280.

[0204] Standard methods of molecular biology are described in Maniatis et al. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif. Standard methods can also be found in Ausbel et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describe cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugate and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0205] The sequences of the heavy and light chain variable regions of the hybridoma clones were determined as follows: Total RNA was purified using the RNeasy Plus Mini Kit from Qiagen (Germantown, MD, USA) at 1–2 × 10 6The cDNA was extracted from 100 hybridoma cells. cDNA was generated by performing a 5' RACE reaction using the SMARTer RACE 5' / 3' Kit from Takara (Mountainview, CA, USA). PCR was performed using Q5 High-Fidelity DNA Polymerase from NEB (Ipswitch, MA, USA) to amplify the variable regions from the heavy and light chains using the Takara Universal Primer mix in combination with gene-specific primers for the 3' mouse constant region of the appropriate immunoglobulin. The amplified variable regions for the heavy and light chains were run on a 2% agarose gel, and the appropriate bands were excised and gel-purified using the Mini Elute Gel Extraction Kit from Qiagen. The purified PCR products were cloned using the Zero Blunt PCR Cloning Kit from Invitrogen (Carlsbad, CA, USA), transformed into Stellar Competent E. coli cells from Takara, and plated on LB agar + 50 μg / mL kanamycin plates. Direct colony Sanger sequencing was performed using GeneWiz (South Plainfield, NJ, USA). The resulting nucleotide sequences were analyzed using IMGT V-QUEST to identify productive rearrangements and to analyze the translated protein sequences. CDR determination was based on IMGT numbering.

[0206] Flow cytometry methods are available, including fluorescence-activated cell sorting detection systems (FACS®). See, for example, Owens et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2nd ed.; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ. Suitable fluorescent reagents for modifying nucleic acids, including nucleic acid primers and probes, polypeptides, and antibodies, are available, for example, for use as diagnostic reagents. Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.

[0207] Positive control (PC1 and PC2), IL-23p19 and IL-12 / IL-23 p40-specific antibodies, were from Biointron. Control antibodies can be prepared by any suitable expression method, for example, by cloning the heavy and light chain variable regions of the antibodies into the 293F or ExpiCHO™ Expression System (ThermoFisher Scientific, Waltham, MA). These antibodies were used as controls to establish the binding and functional assays described in the Examples and tested in parallel with the newly created anti-IL-23p19-specific antibodies disclosed herein. "PC1" refers to a reference antibody, which was synthesized based on the VH and VL sequences reported in U.S. Patent No. 7,935,344 (VH SEQ ID NO: 106 and VL SEQ ID NO: 116 of the '344 patent) and is known to be specific for the human IL-23p19 subunit (Biointron, Lot No.: 20180926A04). The term "PC2" refers to a reference, which was synthesized based on the VH and VL sequences reported in U.S. Pat. No. 6,902,734 (VH SEQ ID NO: 7 and VL SEQ ID NO: 8 of the '734 patent) and is known to be specific for the human IL-12 / IL-23 p40 subunit (BIOINTRON, Lot No: 20180925A07).

[0208] Control antibodies can be produced by standard methods. For example, a plasmid containing the sequence of a control antibody can be transfected using a mammalian system (293F or ExpiCHO™) (catalog number: A29133, ThermoFisher Scientific, USA) according to the manufacturer's protocol. The cells were cultured at 37°C and 8% CO2 on day 1, and then at 32°C and 5% CO2 after transfection in the medium provided in the kit. The antibody was purified by clarifying the ExpiCHO™ culture medium by centrifugation at 1,000g for 10 minutes, followed by centrifugation at 5,000g for 30 minutes. The supernatant was then filtered using a 0.45µm filter, followed by a 0.22µm filter. The supernatant was then subjected to affinity purification using Protein A / G resin (Life Technologies, Carlsbad, CA; catalog number 20424) according to the manufacturer's protocol. Prior to ELISA purification, the antibody titer in the culture medium is roughly determined to ensure that the amount of medium loaded occupies less than 80% of the resin's binding capacity. After incubation, the resin is washed with PBS and eluted with Elution Buffer (Life Technologies, catalog number 21004). The elution fraction is immediately adjusted to physiological pH by adding Tris buffer, pH 8.0. The purified antibody is then subjected to buffer exchange and protein concentration using an Amicon Ultra-15 Centrifugal Filter Unit (Life Technologies, catalog number UFC900324) in PBS buffer. The antibody concentration is determined by BCA Protein Assay. SDS-PAGE and Coomassie staining are performed to test the antibody purity. The purified protein is aliquoted and stored at -80°C for long-term storage or kept at 4°C for immediate use.

[0209] The integrity of the antibody can be verified by SDS-PAGE followed by Coomassie staining under non-reducing versus reducing conditions; under non-reducing conditions, one dominant band of approximately 150 kDa is observed, whereas under reducing conditions, two bands of 50 kDa and 25 kDa are observed. Standard techniques for characterizing ligand / receptor interactions are available. See, for example, Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York. Standard methods for antibody functional characterization suitable for characterizing antibodies with specific mechanisms of action are also well known to those skilled in the art.

[0210] For example, software packages and databases are available for determining antigenic fragments, leader sequences, protein folds, functional domains, CDR annotations, glycosylation sites, and sequence alignments.

[0211] [Example 1] Generation of anti-IL-23p19 antibodies Human anti-IL-23p19 specific antibodies were generated by immunizing human Ig transgenic mice (see, eg, WO 2013 / 063391, TRIANNI® mice).

[0212] Immunization: TRIANNI® mice were immunized with human IL-23 recombinant protein or a combination of human IL-23 protein and a heterodimer of human p19 and mouse p40 protein by intraperitoneal (IP), subcutaneous (SC), or via the footpad or base of the tail. Immune responses were monitored by retroorbital bleeding. Plasma was screened for activity in binding to the human IL-23 heterodimer by ELISA (as described below). Mice with sufficient titers were used for fusions. Mice were boosted with immunogen, then sacrificed, and the spleen and draining lymph nodes were removed.

[0213] Selection of mice producing anti-IL-23p19 antibodies: To select Trianni mice producing p19-specific antibodies, sera from immunized mice were screened by ELISA for binding to recombinant human IL-23. Briefly, ELISA plates coated with recombinant human IL-23 were incubated with dilutions of sera from immunized mice for 1 hour at room temperature, the assay plates were washed, and specific antibody binding was detected using an HRP-labeled anti-mouse IgG antibody. The plates were read using an ELISA reader (Biotek).

[0214] Hybridoma Generation: To generate hybridomas producing the human antibodies of the present disclosure, splenocytes and draining lymph node cells collected from immunized mice were fused to an appropriate immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas were screened for the production of p19-specific antibodies. For example, single-cell suspensions of splenocytes and lymph node cells from immunized mice were fused to an equal number of Sp2 / 0 mouse IgG non-secreting myeloma cells (ATCC, CRL1581) by electrofusion. The cells were seeded into flat-bottom 96-well tissue culture plates, followed by incubation in selective medium (HAT medium) for approximately two weeks and then switched to hybridoma culture medium. Approximately 10–14 days after cell seeding, supernatants from individual wells were screened by ELISA as described above. Antibody-secreting hybridomas were transferred to 24-well plates and screened again, and if still positive for anti-p19 activity, the hybridomas were subcloned by limiting dilution or sorting using a single-cell sorter. Stable subclones were then cultured in vitro to generate small amounts of antibody for purification and characterization.

[0215] Hybridoma screening: Hybridoma supernatants were tested for IL-23-specific binding using human IL-23, human IL-12, and human p19 / mouse p40 by ELISA using the same assay used to monitor the immune response of immunized mice, as described above.

[0216] [Example 2] Anti-IL-23p19 specific antibody binding The binding of anti-IL-23p19 specific antibodies to IL-23 protein was analyzed by surface plasmon resonance (SPR) as determined by BIAcore. Briefly, serial dilutions of anti-IL-23p19 antibodies or control antibodies were captured onto anti-mouse or anti-human Fc chips immobilized on a CM5 chip using an amine coupling kit (GE Healthcare, Cat. No.: BR-1000-50, Lot No.: 2087295).

[0217] Control antibodies used in the BIAcore binding assays included: PC1 (known to be a p19-specific antibody, Biointron, lot number: 20180926A04); PC2 (reference antibody with known specificity for the p40 subunit of human IL-12 and IL-23, Biointron, lot number: 20180925A07); as negative controls, a human IgG isotype control (Invitrogen, catalog number: 02-7102, lot number: TJ276309); a mouse IgG2a isotype control (Novarock Biotherapeutics) and a human IgG4 isotype control (Dendritics, catalog number: DDXCH04P-100; batch: DDXCH04-028).

[0218] Next, serial dilutions of IL-23 recombinant protein, human p19 / mouse p40 heterodimer protein, human IL-12 protein, and human p40 subunit protein in a running buffer containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.005% Tween 20, pH 7.4, were injected over the immobilized antibody at 50 μl / min for 1 min, followed by a 2-min dissociation period. Each injection was followed by a regeneration step with a 60-s pulse of 10 mM glycine-HCl, pH 1.7 buffer. The experimental data were fitted using BIAevaluation software (GE Healthcare) to a Langmuir 1:1 model to determine apparent binding.

[0219] The binding profiles of the purified antibodies are illustrated in Figure 2. Figure 2A shows the binding profile of Hu-2 18006B (purified from hybridoma). Figure 2B shows the binding profile of Hu-5 18006B (purified from hybridoma). Figure 2C shows the binding profile of Hu-6 18006B. * Figure 2D shows the binding profile of Hu-4 18006B (recombinant mIgG2a). * ( * Figure 2E shows the binding profile of Hu-4 18006B (recombinant, mIgG2a). ** ( ** Figure 3 shows the binding profiles of anti-IL-23p19 antibodies and their binding specificities to human IL-23 and human p19 / mouse p40 heterodimeric proteins by BIAcore. Recombinant antibodies are designated with an asterisk in the table.

[0220] [Table 3]

[0221] The results show that the anti-IL-23p19 antibody binds to human IL-23 and the human p19 / mouse p40 heterodimer, but not to human IL-12 or the human p40 subunit (Figure 2 and Table 3).

[0222] PC1 was positive for human IL-23, human p19 / mouse p40, and negative for human IL-12 and the human p40 subunit (data not shown). PC2 was positive for human IL-23, human IL-12, and the human p40 subunit, and negative for the human p19 / mouse p40 heterodimer. The isotype controls mIgG2a and hIgG did not bind to hIL-23, hp19 / mp40, hIL-12, or the hp40 subunit.

[0223] Results: Anti-IL-23p19-specific antibodies were characterized by their binding to a recombinant protein containing human IL-23 and a heterodimer consisting of the human p19 and mouse p40 subunits. These antibodies did not bind to human IL-12 and the human p40 subunit by BIAcore.

[0224] The binding specificity of the anti-IL-23p19 antibody of the present disclosure was also evaluated by ELISA. Briefly, biotinylated IL-23 was captured via streptavidin-coated ELISA plates. Human p19 / mouse p40, human IL-12, and human p40 subunits were directly coated onto the ELISA plates. Purified antibodies were then added to the plates, followed by detection with goat anti-mouse IgG-HRP (Jackson ImmunoResearch, Catalog No.: 115-036-071, Lot No.: 147271). After adding ABTS substrate (Moss Inc., Catalog No.: ABTS-1000, Lot No.: 03086202), the ELISA plate was read using an ELISA plate reader (Biotek). Controls illustrated in Figure 3: PC1 refers to a reference antibody (known to be a p19-specific antibody, Biointron, lot number: 20180926A04); PC2 refers to a reference antibody (known to be a p40-specific antibody, Biointron, lot number: 20180925A07); PC3 refers to a reference antibody (MT155, known to be a p19-specific antibody from Mabtech, catalog number: 3457-6-100, code: 3457-6-1000), negative controls are human IgG4 (Dendritics, catalog number: DDXCHO4P-100, lot number: DDXCH04-028) and mouse IgG2a (produced by Novarock Biotherapeutics).

[0225] Figure 3 shows the binding activity of p19-specific antibodies of the present disclosure. Figures 3A and 3B show Hu-4 18006B ** (hIgG4), Hu-5 18006B (mIgG2b), Hu-6 18006B * (mIgG2a), Hu-4 18006B *(mIgG2a) and Hu-2 18006B (mIgG1) bind to human IL-23 in a dose-dependent manner; the positive control PC1 binds to IL-23 in a dose-dependent manner (Figure 3A). Figure 3C shows that these selected representative anti-IL-23p19 antibodies, H-2 18006B (mIgG1), Hu-4 18006B (mIgG2c), and Hu-4 18006B * (mIgG2a), and H-6 18006B * Figure 3D shows that these anti-IL-23p19 antibodies do not bind to hIL-12, whereas the positive control antibody PC2 binds to hIL-12 in a dose-dependent manner. Figure 3E shows that these anti-IL-23p19 antibodies do not bind to the human p40 subunit, whereas the positive control PC2 binds to the human p40 subunit in a dose-responsive manner.

[0226] The results from Figures 3A-3E indicate that anti-IL-23p19-specific antibodies were characterized by ELISA for binding to human IL-23 and to a recombinant protein containing a heterodimer consisting of human p19 combined with the mouse p40 subunit. These antibodies do not bind to human IL-12 or the human p40 subunit.

[0227] To ensure accurate measurement of KD and IC50 endpoints in binding and functional assays, antibodies were purified from hybridoma culture supernatants prior to testing. The binding kinetics of the disclosed anti-IL-23p19-specific antibody to recombinant human IL-23 was determined by surface plasmon resonance (SPR) using a BIAcore 3000 system docked with a CM5 chip pre-immobilized via amine coupling chemistry with an anti-mouse IgG antibody (GE catalog no. BR-1008-38). Flow cell 1 was left unmodified and served as a reference cell for subtraction of systematic instrument noise and drift. Fc2-1 detection was performed using double blanking (Fc1 and blank analyte buffer). Antibody samples were diluted to 50 μg / mL in HBS-EP (GE catalog no. BR1001-88) and injected for 1 minute at a flow rate of 10 μL / min. Next, hIL-23 (R&D Systems, Catalog No. 1290-IL / CF) diluted to 0.156–40 nM was injected at 50 μL / min for 2 min, followed by a 10-min dissociation period. Data were analyzed using BIAEvalution software (GE Healthcare) with a global fit and a 1:1 binding model to determine apparent binding kinetics.

[0228] Binding kinetics data for the anti-IL-23p19 antibodies of the present disclosure are provided in Table 4. The results indicate that anti-IL-23p19 specific antibodies bind to human recombinant IL-23 with KDs ranging from 3.84E-11 to 6.62E-11 M. PC1 (known to be a p19 specific antibody, Biointron, Lot No: 20180926A04) had KD values ​​ranging from 3.77E-10 to 1.10E-11 across multiple runs.

[0229] [Table 4]

[0230] [Example 3] Blocking IL-23 interaction with the IL-23 receptor The ability of the disclosed p19-specific antibody to block IL-23 binding to its cognate high-affinity IL-23 receptor was determined by ELISA. Briefly, human IL-23 receptor was coated onto a 96-well plate (2 μg / ml), and serial dilutions of purified anti-IL-23p19 antibody premixed with recombinant human IL-23 (50 ng / mL) were then added to the plate. After a 30-minute incubation, the plate was washed. Biotinylated anti-p40 antibody (Invitrogen ref: 13-7129-85, lot: 2028761, 1 / 3000 dilution) was then added to the plate. After a 30-minute incubation, the plate was washed, followed by detection with streptavidin-HRP. After the addition of ABTS substrate, the plate was read using a plate reader (OD 405 nM). The positive control antibody PC1 used in this blocking assay was a reference antibody (known to be p19-specific, Biointron, lot number: 20180926A04). The negative controls were mouse IgG1 (Novus, catalog number: NBP1-97005, lot number: 35613), mIgG2a (NovaRock Biotherapeutics), and hIgG4 (Dendritics, catalog number: DDXCHO4P-100, lot number: DDXCH04-028).

[0231] Results: The data in Figure 4 show that the anti-p19 antibodies of the present disclosure (Hu-2 18006B, Hu-6 18006B) * , Hu-5 18006B and Hu-4 18006B ** ) blocked the interaction of human IL-23 with the human IL-23 receptor in a dose-dependent manner. The positive control PC1 also blocked the IL-23 / IL-23 receptor interaction in a dose-responsive manner. The negative controls hIgG4 and mIgG2a were negative in the assay.

[0232] To demonstrate the specificity of the p19-specific antibodies of the present disclosure, a blocking assay was designed to assess the ability of the antibodies to block IL-23 binding to IL-12 receptor β1.

[0233] Briefly, human IL-12 receptor β1 was coated onto a 96-well plate (2 μg / ml), and then serial dilutions of purified anti-IL-23p19 antibody premixed with recombinant human IL-23 (50 ng / ml) were added to the plate. The positive control antibody used in the blocking assay was PC2 (a reference antibody with known specificity for the p40 subunit of IL-12 / IL-23 p40, Biointron, lot number: 20180925A07). The negative control was mouse IgG1 (Novus, catalog number: NBP1-97005, lot 35613).

[0234] After 30 minutes of incubation, the plate was washed. Then, biotinylated anti-p40 antibody (Invitrogen ref: 13-7129-85, lot: 2028761, 1 / 3000 dilution) was added to the wells containing anti-IL-23p19 antibody; biotinylated anti-p19 antibody (Mabtech, catalog number: MT155, code: 3457-6-1000) was added to the wells containing PC2 antibody. After 30 minutes of incubation, the plate was washed, followed by detection with streptavidin-HRP. After adding ABTS substrate, the plate was read using a plate reader (OD 405 nM).

[0235] Results: The data in Figure 5 show that the three selected p19-specific antibodies (Hu-6 18006 B * , Hu-4 18006B * and Hu-4 18006 B ** ) but did not block the IL-23 / IL-12 receptor β1 binding interaction. Similarly, the PC1 antibody did not block the IL-23 / IL-12 receptor β1 interaction (data not shown). However, the p40 control antibody (PC2) blocked the IL-23 / IL-12 β1 interaction, as expected.

[0236] [Example 4] Inhibition of IL-17 production in a mouse splenocyte assay It is widely known that human IL-23 binds to mouse IL-23R and induces mouse IL-17 production in mouse splenocytes. Human IL-23 in the presence of IL-2 stimulates IL-17 production in mouse splenocytes at extremely low (picomolar) concentrations, but this production can be inhibited by co-incubation with inhibitors against either p40 or p19 (Aggarwal, S., et al., 2003, J Biol Chem; 278: 1910-4; Singh et al., 2015, MAbs, July-Aug; 7(4): 778-791).

[0237] The ability of anti-IL-23p19 specific antibodies of the present disclosure to inhibit human IL-23-induced IL-17 production was evaluated in a mouse splenocyte assay (MSA). The potency of inhibition of human-IL-23-induced IL-17 production was determined.

[0238] Briefly, mouse splenocytes were isolated from C57 / BL-6 mice using a glass homogenizer and a Ficoll-Pague cell isolation kit (Ge Healthcare, Cat. No. 17-5442-02) according to the manufacturer's protocol. Splenocytes were incubated with IL-2 (5 × 10 6 The splenocytes were activated with IL-23 (20 ng / ml per cell / ml) for 5 minutes, and then human IL-23 (1.5 ng / ml) was added to the splenocytes. The activated splenocytes were seeded into a 96-well plate, 100 ul per well. Four purified p19 antibodies, hu-6 18006B, * (mIgG2a), Hu-4 18006B * (mIgG2a), Hu-4 18006 ** (hIgG4) and Hu-2 18006B (mIgG1), 100 ul / well, were added to the plate.

[0239] After 72 hours of incubation, supernatants were transferred from the plates for IL-17 quantification assays using a Quantikine ELISA Kit (R&D, M1700 or SM1700). Controls included in the IL-17 MSA included: PC1 (a reference antibody known to be p19-specific, Biointron, lot number 20180926A04) as a positive control; mouse IgG1 (Novus, catalog number NBP 1-97005) and human IgG4 (Dendritics, catalog number DDXCHO4P-100, lot number DDXCH04-028) as negative controls.

[0240] Results: The data presented in Figures 6A and 6B support the conclusion that the antibodies of the present disclosure selectively neutralize the binding of IL-23 to IL-23R and therefore inhibit IL-17 production in a dose-dependent manner.

[0241] [Example 5] Inhibition of STAT3 activation by reporter cell assay The receptor for IL-23 contains the IL-12Rβ1 subunit, which is shared with the IL-12 receptor and known to partner with IL-23R. IL-23p19 selectively binds to IL-23R, and signaling through IL-23R induces Janus kinase 2 (JAK2), which activates STAT3, resulting in upregulation of RORγgt and subsequent increased production of the proinflammatory cytokine IL-17 (Parham et al., J. Immunol. 168:5699-5708, 2002). To determine whether the anti-pI9 antibody of the present disclosure can inhibit STAT3 activation, the antibody was evaluated for IL-23-induced STAT3 activation by a reporter cell assay. Human IL-23 induces STAT3 phosphorylation upon binding to IL-23R on the surface of human lymphoma DB cells (Example 13 of U.S. Patent No. 2013 / 0172272 and Desmet, J. et al., Nat. Commun. 5:5237 (2014)).

[0242] DB cells were derived from a human B-cell lymphoma cell line that expresses endogenous IL-23 receptor and STAT3, resulting in a fully functional IL-23 signaling pathway. DB assay cells were generated by stable transfection of DB cells with pGL4.47[luc2p / SIE / Hybro], which allows for quantitative detection of bioactive human IL-23 using a luciferase reporter system.

[0243] This DB assay measures the inhibitory activity of anti-IL-23 antibodies of the present disclosure against human IL-23-induced STAT3 activation. Briefly, DB cells (ATCC, CRL-2289) were cultured in growth medium (RPMI + 10% FBS) for 2 days. On the day of the experiment, the cells were harvested and resuspended in growth medium. Serial dilutions of the test antibody were prepared in growth medium in a low-binding 384-well plate (ThermoScientific 264574), followed by the addition of human IL-23 and incubation at room temperature for 30 minutes. The mixture of test antibody and human IL-23 was then added to the plate. The signaling assay plate was incubated in a humidified incubator at 37°C / 5% CO2 for 16 hours. OneGlo reagent was added, and the mixture was incubated at room temperature for 2 minutes. Luminescence was read on a BioTek Neo2 (BioTek.Winooski.VT) and IC50 values ​​were determined using GraphPad® software (GraphPad Software Inc., San Diego, California, USA) by plotting ratios against log-transformed antibody concentrations and determining IC50 values ​​using a sigmoidal dose-response nonlinear regression (curve fit). Control antibodies used in the STAT3 activation assay included PC1 (PC1 is a reference antibody known to be specific for p19, Biointron, lot number: 20180926A04) as a positive control and mIgG2a (generated in-house) as a negative control.

[0244] Results: As shown in Table 5, the anti-IL-23p19-specific antibodies evaluated in the assay inhibited STAT3 activation with IC50 values ​​ranging from 35.2 pM to 264.6 pM, with up to 99-100% inhibition. The positive control (PC1) had IC50 values ​​ranging from 24.30 pM to 117.20 pM, with 98-99% inhibition from multiple experiments.

[0245] [Table 5]

[0246] The results from Figure 7 indicated that the four selected anti-IL-23p19 antibodies of the present disclosure (hu-4 18006B, hu-4-18006B * , hu-6 18006B and hu-6 18006B * ) inhibited STAT3 activation in a dose-dependent manner. The positive control (PC1) also showed inhibition of STAT3 activation in a dose-responsive manner, as expected.

[0247] [Example 6] Inhibition of IL-12-dependent IFN-γ production by human PBMCs IL-12 stimulation of PBMCs is known to stimulate IFN-γ production by NK cells and T cells. To determine whether the anti-p19 antibodies of the present disclosure can inhibit IFN-γ production, representative human anti-p19 antibodies of the present disclosure were analyzed in a PBMC IL-12 stimulation assay.

[0248] Briefly, human PBMCs were thawed from frozen stocks and resuspended in RPMI + 10% FBS containing 50 ng / ml of IL-18 (R&D, 9124-IL / CF) in 384-well plates. Serial dilutions of test antibodies were prepared in growth medium in low-binding 384-well plates. Human IL-12 (25 ng / ml) or cynomolgus IL-12 (25 ng / ml) was transferred to each well, followed by a 30-minute incubation at room temperature. The mixture (test antibody + IL-12) was seeded onto PBMC cell plates. Cells were incubated for 48 hours in a humidified incubator at 37°C / 5% CO2. IFN-γ production was measured by AlphaLISA (PerkinElmer, AL217C) according to the manufacturer's protocol.

[0249] Control antibodies used in the human PBMC assay: PC1 (reference antibody known to be a p19-specific antibody, Biointron, lot number: 20180926A04), PC2 (reference antibody with known specificity for the p40 subunit of IL-12 and IL-23, Biointron, lot number: 20180925A07), anti-IL-23 p40 subunit Mab (Hu-19 18006 * , generated in-house); as negative control antibodies, mIgG2a (produced by NovaRock Biotherapeutics) and human IgG4 (Dendritics, catalog: DDXCHO4P-100, lot: DDXCH04-028).

[0250] Results: As shown in Figures 8 and 9, the anti-IL-23p19 antibody (Hu-6 18006B * and Hu-4 18006B * and PC1) did not inhibit human IL-12-mediated (Fig. 8) or cynomolgus monkey IL-12-mediated (Fig. 9) IFN-γ production by human PBMCs, whereas the positive controls PC2 and Hu-19 18006 *inhibited p19 in human (Figure 8) and cynomolgus monkey (Figure 9) PBMCs in a dose-dependent manner, as expected. This data supports the conclusion that the p19 antibodies of the present disclosure are specific for p19.

[0251] [Example 7] In vivo efficacy of anti-p19 specific antibody in an IL-23-induced mouse skin inflammation model The role of the IL-23 / IL-17 pathway as a key driver of human psoriasis (PsO) has been well characterized and clinically validated. Animal models of psoriasis (PsO) are important for our understanding of the pathophysiology of the human disease. Intradermal injection of IL-23 has been used to study the IL-23 pathway in rodents and can be used to evaluate the pharmacology of novel small molecules / biologics in the treatment of PsO (Stephen B. Gauld et al., J. Dermatological Science, 92 (2018) 45-53).

[0252] Human IL-23 is known to bind to the mouse IL-23 receptor and induce mIL-17 production and inflammation in mice. Intradermal injection of human IL-23 into the ears of mice to induce ear inflammation has been used as a psoriasis model to characterize biological drugs for human psoriasis (PsO) (Aggarwal et al., J Biol Chem 2003; 278: 1910-4; Singh et al., MAbs 2015 July-Aug; 7(4): 77-791).

[0253] Hu-4 18006 B (mIgG2c), Hu-4 18006 B ** (hIgG4), hu-5 18006B (mIgG2b) and Hu-6 18006 B *To assess the ability of each p19-specific antibody (mIgG2a) to block IL-23 function in vivo, we tested these antibodies in a human IL-23-induced mouse skin inflammation model. These representative antibodies were evaluated for their ability to reduce the inflammatory response.

[0254] In this model, recombinant human IL-23 (3 μg / 10 μl / mouse / day) was injected intradermally into the skin of the right ear of mice for 8 consecutive days (D0-D7) to induce a psoriasis-like inflammatory skin reaction characterized by erythema and induration, along with histological evidence of epidermal hyperplasia, parakeratosis, and focal inflammatory infiltrates.

[0255] IL-23p19 antibodies (hu-4 18006B, hu-4 18006B) were used according to two protocols. ** , hu-5 18006B, or hu-6 18006B * Mice were treated twice by intraperitoneal (ip) injection of either PC1, a reference antibody known to be a p19-specific antibody (Biointron, lot number: 20180926A04). In the first protocol, mice were administered a PBS control (vehicle) or antibody. The first injection was the day before IL-23 injection, and the second injection was the third day after IL-23 injection. In the second protocol, mice were administered a PBS control or antibody. The first injection was 1 hour before IL-23 injection, and the second injection was the third day after IL-23 injection (Figure 10).

[0256] Mice were measured daily for weight, ear thickness, and ear inflammation score. Ear inflammation scores were calculated on days 0, 2, 4, 6, and 8 based on the following criteria: ear shape (relatively normal - 0; minimal change - 1; moderate to significant change - 2; severe swelling and deformation - 3), skin color (relatively normal - 0; minimal hyperplasia - 1; mild hyperplasia - 2; severe hyperplasia - 3), and white scale (relatively normal - 0; minimal - 1; mild - 2; obvious - 3). The right ear of each mouse was measured and photographed on days 0, 2, 4, 6, and 8.

[0257] On the last day of the experiment (day 8), the animals were euthanized with carbon dioxide, blood samples were collected, and serum was separated (stored in a -80°C freezer). Modeling ears were harvested and cut into two pieces: one piece was fixed in 10% neutral buffered formalin, and the other piece was frozen in liquid nitrogen and stored in a -80°C freezer.

[0258] Data are presented as mean±SEM. A P value of less than 0.05 was considered statistically significant.

[0259] The data provided in Tables 6 and 7 summarize the total score of the injected ears (by adding the scores for pinna shape, skin color, microvascular changes, and white scale). The results indicated that mice treated with a representative p19-specific antibody experienced a reduced inflammatory response compared to the IL-23-injected model group. The effect began on day 4 and continued through day 8. The effect was statistically significant. This conclusion is evident from both the summary score values ​​and the ear thickness values.

[0260] [Table 6]

[0261] [Table 7]

[0262] As shown in Tables 8 and 9, mouse ear thickness was reduced by treatment with selected anti-IL-23p19 antibodies compared to the model (IL-23 treatment). The therapeutic effect (in vivo inhibition of cutaneous inflammatory immune responses) began on day 4 and continued through day 8, with the effect being statistically significant (p<0.001 vs. model, IL-23 treatment).

[0263] [Table 8]

[0264] [Table 9]

[0265] The data presented in Figure 10 demonstrate that the anti-p19 specific antibody hu-4 18006B of the present disclosure ** and hu-6 18006B * caused a statistically significant reduction in ear thickness compared to untreated controls (models receiving only human IL-23 treatment).

[0266] Figures 11A, 11B, 11C and 11D provide data demonstrating the effect of anti-p19 specific antibodies on inflammatory skin reactions as determined by H&E pathology staining scores, such as epidermal thickness (Figure 11A), dermal thickness (Figure 11B), inflammatory cell infiltration (Figure 11C) and hyperkeratosis or dysfunction (Figure 11D), obtained over the course of the experiment and represented by the following scoring system:

[0267] Briefly, on day 8, mouse ears were harvested and examined microscopically. Tissues were fixed in 10% neutral buffered formalin. After fixation, tissues were trimmed, dehydrated, embedded, sectioned onto slides, and stained with hematoxylin / eosin (H&E) according to the relevant SOP. Histopathological evaluation was performed by a pathologist using a light microscope. A five-point scoring system (relatively normal, minimal, mild, moderate to severe, severe) was used to grade microscopic findings.

[0268] Results: Treatment with the anti-p19 antibody hu4 18006B resulted in a significant reduction in both the swelling response and inflammation score induced by IL-23 injection compared to the model. Hu-4 18006B showed superior inhibitory effects compared to PC1 on three of the scores: epidermal thickness (Figure 11A), dermal thickness (Figure 11B), and inflammatory cell infiltration (Figure 11C). Hu-4 also inhibited hyperkeratosis (Figure 11D).

[0269] Figure 12 provides representative photographs of H&E stained ear sections obtained 8 days after treatment. The H&E staining procedure was as described above.

[0270] Results: Selected anti-IL-23p19 antibody treatment of the present disclosure (Hu-4 18006B) significantly inhibited inflammation in the skin of mice compared to the model.

[0271] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and the like used in the specification and claims should be understood to be modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0272] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical values, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0273] As used in the context of describing this disclosure (particularly in the context of the claims below), the terms "a," "an," "the," and similar referents are intended to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually listed herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any examples or exemplary language (e.g., "such as") provided herein is intended merely to better illustrate the disclosure and does not impose limitations on the scope of the disclosure unless specifically stated. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0274] Groupings of alternative elements or embodiments of the disclosure disclosed herein should not be construed as limiting. The members of each group can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification includes the modified group and thus satisfies the written specification of the entire Markush group as used in the appended claims.

[0275] Certain embodiments of the present disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Of course, variations on those described embodiments will become apparent to those skilled in the art upon reading and understanding the foregoing description. The inventors contemplate that those skilled in the art will employ such variations as appropriate, and it is intended that the present disclosure be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the present disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

[0276] Specific embodiments disclosed herein may be further limited in the claims using the phrases "consisting of" or "consisting essentially of." When used in a claim, whether as filed or added by amendment, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of a claim to the specified materials or steps, and those that do not materially affect the basic and novel characteristics. Embodiments of the present disclosure so claimed are essentially or explicitly described herein and are effective herein.

[0277] It will be understood that the embodiments of the present disclosure disclosed herein are illustrative of the principles of the present disclosure. Other modifications that may be employed are within the scope of the present disclosure. Thus, by way of example, and not of limitation, alternative configurations of the present disclosure may be utilized in accordance with the teachings herein. Accordingly, the present disclosure is not limited to that precisely as shown and described.

[0278] Although the present disclosure has been described and illustrated herein by reference to various specific materials, procedures, and examples, it is understood that the disclosure is not limited to the particular combination of materials and procedures selected for that purpose. Numerous variations of such details may be implicit, as will be understood by those skilled in the art. It is intended that the specification and examples be considered exemplary only, with the true scope and spirit of the present disclosure being indicated by the following claims. All references, patents, and patent applications referenced in this application are incorporated herein by reference in their entirety. The present invention includes the following embodiments. [1] (a) a heavy chain variable region comprising CDR1: SEQ ID NO: 9, CDR2: SEQ ID NO: 10, and CDR3: SEQ ID NO: 11; and a light chain variable region comprising CDR1: SEQ ID NO: 12, CDR2: SEQ ID NO: 13, and CDR3: SEQ ID NO: 14; (b) a heavy chain variable region comprising CDR1: SEQ ID NO: 15, CDR2: SEQ ID NO: 16, and CDR3: SEQ ID NO: 17; and a light chain variable region comprising CDR1: SEQ ID NO: 18, CDR2: SEQ ID NO: 19, and CDR3: SEQ ID NO: 20; (c) a heavy chain variable region comprising CDR1: SEQ ID NO: 21, CDR2: SEQ ID NO: 22, and CDR3: SEQ ID NO: 23; and a light chain variable region comprising CDR1: SEQ ID NO: 24, CDR2: SEQ ID NO: 25, and CDR3: SEQ ID NO: 26; or (d) a heavy chain variable region comprising CDR1: SEQ ID NO: 27, CDR2: SEQ ID NO: 28, and CDR3: SEQ ID NO: 29; and a light chain variable region comprising CDR1: SEQ ID NO: 30, CDR2: SEQ ID NO: 31, and CDR3: SEQ ID NO: 32. an anti-IL-23p19 antibody comprising [2] (a) a heavy chain variable region sequence of SEQ ID NO: 1 and a light chain variable region sequence of SEQ ID NO: 2; (b) a heavy chain variable region sequence of SEQ ID NO: 3 and a light chain variable region sequence of SEQ ID NO: 4; (c) a heavy chain variable region sequence of SEQ ID NO: 5 and a light chain variable region sequence of SEQ ID NO: 6; or (d) the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8 The anti-IL-23p19 antibody according to [1] above, comprising: [3] The anti-IL-23p19 antibody according to [1] above, which is an anti-human IL-23p19 antibody. [4] The anti-IL-23p19 antibody according to [1] above, which is a full-length antibody. [5] The anti-IL-23p19 antibody according to [1] above, which is an antibody fragment. [6] The anti-IL-23p19 antibody according to [4] above, wherein the antibody fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fd, Fv, scFv and scFv-Fc fragments, single-chain antibodies, minibodies, and diabodies. [7] The anti-IL-23p19 antibody according to [1] above, which is a monoclonal antibody. [8] The anti-IL-23p19 antibody according to [1] above, which is a human antibody. [9] The anti-IL-23p19 antibody according to [1] above, which is a mouse antibody.

[10] The anti-IL-23p19 antibody according to [1] above, which is a chimeric antibody.

[11] The anti-IL-23p19 antibody according to [1] above, which is a bispecific antibody.

[12] The anti-IL-23p19 antibody according to [1] above, which is a humanized antibody.

[13] The anti-IL-23p19 antibody according to [1] above, which does not bind to the p40 subunit of IL-12.

[14] A pharmaceutical composition comprising the antibody according to [1] above and a pharmaceutically acceptable carrier.

[15] A method for treating or preventing an immune-mediated inflammatory disease, comprising the step of administering the antibody described in [1] above to a patient in need thereof.

[16] An isolated polynucleotide comprising a sequence encoding the anti-IL-23p19 antibody described in [1] above.

[17] The isolated polynucleotide according to

[16] above, which encodes the sequence set forth in any one of SEQ ID NOs: 1, 3, 5, or 7.

[18] A vector comprising the polynucleotide described in

[16] above.

[19] A cell comprising the polynucleotide according to

[16] above and / or the vector according to

[17] above.

[20] A method for producing the anti-IL-23p19 antibody according to [1] above, comprising the step of culturing the cell according to

[19] above.

Claims

1. (a) a heavy chain variable region comprising CDR1: SEQ ID NO: 9, CDR2: SEQ ID NO: 10, and CDR3: SEQ ID NO: 11; and a light chain variable region comprising CDR1: SEQ ID NO: 12, CDR2: SEQ ID NO: 13, and CDR3: SEQ ID NO: 14; (b) a heavy chain variable region comprising CDR1: SEQ ID NO: 15, CDR2: SEQ ID NO: 16, and CDR3: SEQ ID NO: 17; and a light chain variable region comprising CDR1: SEQ ID NO: 18, CDR2: SEQ ID NO: 19, and CDR3: SEQ ID NO: 20; (c) a heavy chain variable region comprising CDR1: SEQ ID NO: 21, CDR2: SEQ ID NO: 22, and CDR3: SEQ ID NO: 23; and a light chain variable region comprising CDR1: SEQ ID NO: 24, CDR2: SEQ ID NO: 25, and CDR3: SEQ ID NO: 26; or (d) a heavy chain variable region comprising CDR1: SEQ ID NO: 27, CDR2: SEQ ID NO: 28, and CDR3: SEQ ID NO: 29; and a light chain variable region comprising CDR1: SEQ ID NO: 30, CDR2: SEQ ID NO: 31, and CDR3: SEQ ID NO:

32. An anti-IL-23p19 antibody comprising:

2. (a) a heavy chain variable region sequence of SEQ ID NO: 1 and a light chain variable region sequence of SEQ ID NO: 2; (b) a heavy chain variable region sequence of SEQ ID NO: 3 and a light chain variable region sequence of SEQ ID NO: 4; (c) a heavy chain variable region sequence of SEQ ID NO: 5 and a light chain variable region sequence of SEQ ID NO: 6; or (d) a heavy chain variable region sequence of SEQ ID NO: 7 and a light chain variable region sequence of SEQ ID NO: 8 The anti-IL-23p19 antibody of claim 1, comprising:

3. The anti-IL-23p19 antibody according to claim 1, which is an anti-human IL-23p19 antibody.

4. The anti-IL-23p19 antibody of claim 1, which is a full-length antibody.

5. An anti-IL-23p19 antibody described in claim 1, which is an antigen-binding fragment.

6. The antigen-binding fragments include Fab, Fab', F(ab') ２ 6. The anti-IL-23p19 antibody of claim 5, which is selected from the group consisting of Fv, scFv and scFv-Fc, single chain antibodies, minibodies, and diabodies.

7. The anti-IL-23p19 antibody of claim 1, which is a monoclonal antibody.

8. The anti-IL-23p19 antibody of claim 1, which is a human antibody.

9. The anti-IL-23p19 antibody of claim 1, which is a bispecific antibody.

10. The anti-IL-23p19 antibody of claim 1, which does not bind to the p40 subunit of IL-12.

11. A pharmaceutical composition comprising the antibody of claim 1 and a pharmaceutically acceptable carrier.

12. A pharmaceutical composition for treating an immune-mediated inflammatory disease, comprising the anti-IL-23p19 antibody of claim 1.

13. An isolated polynucleotide comprising a sequence encoding the anti-IL-23p19 antibody of claim 1.

14. The anti-IL-23p19 antibody encoding sequence comprising: (a) comprising a sequence encoding the heavy chain variable region sequence of SEQ ID NO: 1 and a sequence encoding the light chain variable region sequence of SEQ ID NO: 2; (b) comprising a sequence encoding the heavy chain variable region sequence of SEQ ID NO: 3 and a sequence encoding the light chain variable region sequence of SEQ ID NO: 4; (c) comprising a sequence encoding the heavy chain variable region sequence of SEQ ID NO:5 and a sequence encoding the light chain variable region sequence of SEQ ID NO:6; or (d) the isolated polynucleotide of claim 13, comprising a sequence encoding the heavy chain variable region sequence of SEQ ID NO: 7 and a sequence encoding the light chain variable region sequence of SEQ ID NO:

8.

15. A vector comprising the polynucleotide of claim 13.

16. A cell comprising the polynucleotide of claim 13 and / or the vector of claim 15.

17. A method for producing the anti-IL-23p19 antibody according to claim 1, comprising the step of culturing the cell according to claim 16.

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