Antibodies against interleukin-23 p19 and methods of use thereof

Abstract

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

Description

[0001] Cross Reference to Related Applications

[0002] This international patent application claims priority to U.S. Provisional Application Serial No. 62 / 951,231, filed December 20, 2019, which is hereby incorporated by reference in its entirety.

[0003] SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format via EFS-Web and is hereby incorporated 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 13,000 bytes in size. TECHNICAL FIELD

[0005] The present disclosure relates generally to antibodies and antibody fragments thereof that bind the p19 subunit of interleukin-23. These antibodies are useful for treating immune-mediated inflammatory diseases, autoimmune diseases, or cancer. BACKGROUND

[0006] The interleukin-12 (IL-12) family of regulatory cytokines includes a unique set of cytokines (IL-12, IL-23, IL-27, IL-35, and IL-39) that comprise covalently linked heterodimeric subunits. The heterodimeric IL-12 family cytokine members are composed of an alpha chain (p19, p28, or p35) and a beta chain (p40 or Ebi3).

[0007] IL-23 is a heterodimeric cytokine that comprises a unique p19 subunit linked to a p40 subunit (IL-12 shares this subunit). The primary sources of IL-23 are tissue-resident or recruited dendritic cells and macrophages. The biological action of IL-23 is hypothesized to occur through a receptor complex composed of two parts: i.) IL-12Rβ1, a part shared by IL-12, and ii.) IL-23R, a part specific to IL-23.

[0008] Members of the IL-12 family of cytokines act as immunological playmakers by directing innate and adaptive immune responses. These regulatory cytokines act by inducing the development of T cell subsets and altering the function and fate of many immune cell populations that direct adaptive immune responses to infection, inflammation, and autoimmune disease outcomes. IL-12 and IL-23 are the primary pro-inflammatory / pro-stimulatory cytokines that act in the development of Th1 and Th17 cells, respectively.

[0009] The functional IL-23 receptor is a heterodimer of the IL-12Rβ1 subunit (shared with the IL-12 receptor) partnered with the signaling chain IL-23R (which binds the pl9 subunit). The receptor for IL-23 constitutively associates with Janus kinase 2 (Jak2), primarily activating STAT3. Expression of the IL-23 receptor is detected primarily on memory T cells and NK cells. Monocytes, macrophages, and dendritic cells also express IL-23 receptor at low levels.

[0010] There is substantial evidence that IL-23-responsive cells are implicated in autoimmune inflammatory diseases and cancer, and modulation of IL-23 activity can provide promising therapies. In particular, aberrant regulation of IL-23 is implicated in 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 critical role in shaping the development of anti-tumor or pro-tumor immunity.

[0011] The IL-23 / IL-12 pathway is involved in cellular mechanisms implicated in the pathophysiology of a variety of inflammatory diseases. Several therapeutic strategies have been designed to inhibit IL-23 activity, and there is a continuing need for therapeutic agents that target the proinflammatory IL-23 / IL-23 receptor signaling axis to treat immune-mediated inflammatory disorders. More particularly, there remains a need for selective IL-23 pl9 antagonist antibodies that bind with high affinity to the pl9 subunit of IL-23, particularly human IL-23, and do not bind the p40 subunit of the related cytokine family member IL-12. SUMMARY

[0012] The present disclosure addresses the aforementioned needs by providing antibodies and antibody fragments that bind to the cytokine pl9 subunit of IL-23. The antibodies and antibody fragments can be used alone (e.g., as monotherapy) or in combination with other immunotherapeutic agents for the treatment of immune-mediated inflammatory diseases (IMIDs) (e.g., autoimmune and inflammatory diseases).

[0013] In some embodiments, the anti-IL-23 pl9 antibody or antibody fragment thereof binds to the cytokine pl9 subunit of human IL-23. In further embodiments, the antibody is a fully human antibody.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] In some embodiments, the anti-IL-23p19 antibody or antibody fragment comprises a variable heavy chain and a variable light chain sequence selected from the following combinations:

[0022] (a) a variable heavy chain sequence comprising SEQ ID NO: 1 and a variable light chain sequence comprising SEQ ID NO: 2;

[0023] (b) a variable heavy chain sequence comprising SEQ ID NO: 3 and a variable light chain sequence comprising SEQ ID NO: 4;

[0024] (c) a variable heavy chain sequence comprising SEQ ID NO: 5 and a variable light chain sequence comprising SEQ ID NO: 6; and

[0025] (d) a variable heavy chain sequence comprising SEQ ID NO: 7 and a variable light chain sequence comprising SEQ ID NO: 8.

[0026] In some embodiments, the anti-IL-23p19 antibody (e.g., an antagonist antibody) binds with high affinity to the pl9 subunit of IL-23, but not to the p40 subunit of the related cytokine family member IL-12.

[0027] 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-23pl9 and has the ability to block the binding of IL-23 to its receptor (IL-23R); (b) inhibits, interferes with, or modulates the interaction of IL-23pl9 with IL-23 receptor signaling; (c) inhibits the activation of STAT3 induced by IL-23 in DB cells; (d) inhibits IL-17 production induced by human IL-23 in mouse splenocytes; (e) inhibits IL-17 production induced by human IL-23 in activated human PBMCs; (f) does not inhibit the interaction of IL-23 with IL-12Rpl signaling; (g) does not inhibit the production of interferon gamma induced by human IL-12 in human activated T cells (PBMCs); (h) does not inhibit the production of interferon gamma induced by cynomolgus IL-12 in human activated T cells (PBMCs); and (i) inhibits skin inflammation induced by human IL-23 in a murine psoriasis model.

[0028] In one aspect, the disclosed antibodies and isolated antigen binding agents are useful for inhibiting the IL-23pl9-induced IL-23 receptor signaling network (e.g., the IL-23 receptor signaling network that promotes the inflammatory microenvironment of autoimmune diseases).

[0029] The anti-IL-23p19 antibody or antibody fragment thereof can exhibit one or more of the following properties:

[0030] (a) is specific for human IL-23p19 and has the ability to block the binding of IL-23 to its receptor, the IL-23 receptor (e.g., a blocker);

[0031] (b) inhibits, interferes with, or modulates IL-23 / IL-23 receptor-mediated signal transduction;

[0032] (c) blocks IL-23-induced STAT3 activation (induced by IL-23 in DB cells);

[0033] (d) inhibits IL-23-induced IL-17 production in mouse splenocytes;

[0034] (e) inhibits IL-23-induced IL-17 production in human PBMCs;

[0035] (f) does not inhibit the interaction of IL-23 with IL-12Rβ1 signal transduction;

[0036] (g) does not block human IL-12-induced interferon-γ production in human PBMCs;

[0037] (h) does not inhibit cynomolgus IL-12-induced interferon-γ production in human PBMCs; and

[0038] (i) inhibits IL-23-induced skin inflammation in murine psoriasis models.

[0039] In some embodiments, the anti-IL-23p19 antibody or antibody fragment thereof comprises a combination of CDR sequences derived 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.

[0040] In some embodiments, the anti-IL-23p19 antibody 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.

[0041] In some embodiments, the anti-IL-23p19 antibody or antibody fragment is a recombinant antibody (e.g., a chimeric antibody or a humanized antibody) and comprises six (6) CDRs, all of which are 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 an anti-IL-23p19 antibody designated Hu-2.18006B (for human antibodies). In representative examples, the antibody or antibody fragment thereof can comprise the amino acid sequences of SEQ ID NOs: 9-11 and SEQ ID NOs: 12-14, representing 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.

[0042] In some embodiments, the anti-IL-23p19 antibody is a full-length antibody.

[0043] 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: a Fab, a Fab', a F(ab')2, a Fd, a Fv, a scFv and a scFv-Fc fragment, a single-chain antibody, a minibody, and a diabody.

[0044] In some embodiments, the anti-IL-23p19 antibody is a monoclonal antibody.

[0045] In some embodiments, the anti-IL-23p19 antibody is a human antibody. In some embodiments, the anti-IL-23p19 antibody is a murine antibody.

[0046] 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.

[0047] The anti-IL-23p19 antibodies and antibody fragments thereof can be used to treat or prevent an immune-mediated inflammatory disease (IMID) (such as an autoimmune disease or an inflammatory disease) or a cancer. Such a method for treating or preventing an IMID or a cancer comprises administering to a subject in need thereof a composition or formulation comprising an anti-IL-23p19 antibody or antibody fragment thereof. In further embodiments, the anti-IL-23p19 antibody or antibody fragment thereof can be administered alone (e.g., as a monotherapy) or in combination with other immunotherapeutics and / or chemotherapeutics. The IMID can be selected from the group consisting of psoriasis, psoriatic arthritis, inflammatory bowel disease (i.e., ulcerative colitis or Crohn’s disease), ankylosing spondylitis, systemic lupus erythematosus, hidradenitis suppurativa, atopic dermatitis, asthma, and familial adenomatous polyposis (FAP). BRIEF DESCRIPTION OF DRAWINGS

[0048] The foregoing summary, as well as the following detailed description of the disclosure, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosure, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the disclosure is not limited to the precise arrangements, examples, and instrumentalities shown.

[0049] Figures 1A-1D Amino acid sequences of the VH and VL domains of anti-IL-23p19 antibodies and their respective CDR sequences are provided. Sequence identifiers are provided and the CDRs are underlined in the variable domain sequences.

[0050] Figure 2A Figure 2B Figure 2C Figure 2D and Figure 2E shows the binding profile of anti-IL-23p19 antibodies to human IL-23, recombinant cytokine comprising human pl9 and murine p40 subunits, human IL-12, and human p40 subunit as determined by BIAcore.

[0051] Figure 3A Figure 3B and Figure 3C shows the dose-dependent binding of selected representative IL-23p19 antibodies to human IL-23 and recombinant cytokine comprising human pl9 and murine p40 subunits as determined by ELISA.

[0052] Figure 3D and Figure 3E shows the lack of binding of selected representative anti-IL-23p19 antibodies to human IL-12 and human p40 subunit as determined by ELISA.

[0053] Figure 4 shows the blocking of IL-23 / IL-23 receptor interaction by four IL-23p19 antibodies as determined by ELISA.

[0054] Figure 5 shows two representative IL-23p19 antibodies that do not block IL-23 / IL-12 receptor βl interaction.

[0055] Figure 6A and Figure 6B shows the inhibition of IL-23-induced IL-17 production by three representative anti-IL-23p19 antibodies in a mouse splenocyte assay (MSA).

[0056] Figure 7 shows the inhibition of IL-23-induced STAT3 activation by two representative anti-IL-23p19 antibodies in a reporter cell assay. ​​​​

[0057] Figure 8 Two representative anti-IL-23p19 specific antibodies were shown not to inhibit human IL-12 induced IFN-g production in human PBMCs.

[0058] Figure 9 Two representative anti-IL-23p19 antibodies were shown not to inhibit cynomolgus IL-12 induced IFN-g production in human PBMCs.

[0059] Figure 10 Two representative anti-IL-23p19 antibodies were shown to inhibit IL-23 mediated inflammatory responses in vivo (ear thickness) in the murine skin inflammation model explained in Example 7.

[0060] Figure 11A Figure 11B Figure 11C and Figure 11D The graphical representation provided shows the effect on the histopathology score (H&E staining of frozen ear tissue) of treated mice on day 8 after treatment with two anti-IL-23p19 antibodies in the murine skin inflammation model shown in Example 7.

[0061] Figure 12A Figure 12B Figure 12C and Figure 12D The representative photographs of hematoxylin and eosin (H&E) staining of frozen ear tissue collected from treated mice on the last day of the in vivo study (day 8) in the murine skin inflammation model shown in Example 7 are shown. DETAILED DESCRIPTION

[0062] IL-23 is a proinflammatory heterodimeric cytokine comprising a pl9 subunit and binds to the IL-23 receptor. Targeting the proinflammatory IL-23 / IL-23 receptor signaling axis is an intense area of therapeutic exploration. The present disclosure provides antibodies and antibody fragments thereof that inhibit the human IL-23 / IL-23 receptor signaling axis, which are useful in the treatment or prevention of IMIDs. Advantageously, the anti-IL-23pl9 antibodies disclosed herein allow for complete inhibition of IL-23pl9, leading to lower dosages of the agent, leading to lower dosing frequency and / or more effective dosing, and leading to reduced costs and increased efficiency.

[0063] ​​​​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 aspect, the disclosed antibodies and antibody fragments thereof bind to human IL-23 with high affinity and prevent its interaction with IL-23R, thereby blocking the downstream signaling cascade. In a particular aspect, the antibodies or antibody fragments thereof inhibit IL-23-stimulated IL-17 production from mouse splenocytes and human PBMCs. In another aspect, the antibodies or antibody fragments thereof neither bind nor antagonize IL-12.

[0064] For the purposes of the present disclosure, 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 in the field of the present disclosure.

[0065] Throughout this disclosure, the following abbreviations will be used:

[0066] mAb or Mab or MAb—monoclonal antibody.

[0067] CDR—complementarity determining region in an immunoglobulin variable region.

[0068] VH or VH—immunoglobulin heavy chain variable region.

[0069] VL or VL—immunoglobulin light chain variable region.

[0070] FR—antibody framework region, an immunoglobulin variable region that does not comprise a CDR region.

[0071] As used herein, the term "interleukin-23" (which can be 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 provided by UniProt entry UniProtKB-P29460 (identified as IL-23 subunit (p40)), linked by a disulfide bond to a protein subunit having the amino acid sequence provided by UniProt entry UniProtKB-Q9NPF7 (identified as interleukin-23 subunit alpha (pl9)).

[0072] As used herein, the terms "IL-12R complex" and "IL-12R" refer to the high affinity IL-12 cytokine receptor complex comprising IL-12Rβl and IL-12Rβ2 subunits.

[0073] As used herein, the terms "IL-23R complex" and "IL-23R" refer to the high affinity IL-23 cytokine receptor comprising IL-12Rβl (which the IL-12R complex also has) and the IL-23R subunit.

[0074] As used herein, the term "interleukin-12" (which can be used interchangeably with IL-12 in the present disclosure) refers to the human IL-12 heterodimer, including, for example, the human IL-12 heterodimer comprising or consisting of: a protein subunit having the amino acid sequence provided by UniProt entry UniProtKB-P29459 (identified as interleukin-12 subunit alpha), linked by a disulfide bond to a protein subunit having the amino acid sequence provided by UniProt entry UniProtKB-P29460 (identified as interleukin-12 subunit beta (p40)). The term includes the heterodimeric protein comprising a 35 kD subunit (p35) and a 40 kD subunit (p40) linked together by disulfide bonds. The heterodimeric protein is referred to as the "p70 subunit." The structure of human IL-12 is further described, for example, in 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 L-12), which can be produced by standard recombinant expression methods.

[0075] As used herein, the term "interleukin-17" also referred to as "IL-17" or "IL-17A" is a 20-30 kD glycosylated homodimeric protein, including, for example, the homodimeric protein comprising or consisting of: a protein subunit having the amino acid sequence provided by 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 generally not found in the systemic circulation. IL-17 binds to a type I transmembrane receptor called IL-17R, which is a widely expressed protein with no significant sequence similarity to other known cytokine receptors. Human IL-17 shows 62.5% and 58% amino acid sequence identity to mouse and rat amino acid IL-17 sequences, respectively. Human IL-17 shows 97.4% amino acid sequence identity to cynomolgus monkey IL-17.

[0076] The term "antibody" as used herein is used in the broadest sense, and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies).

[0077] Exemplary antibodies, such as IgGs, comprise two heavy chains and two light chains. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain comprises 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, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed 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.

[0078] The hypervariable region generally comprises amino acid residues 24-34 (LCDR1; "L" denotes light chain), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable region and amino acid residues 31-35B (HCDR1; "H" denotes 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 that form a hypervariable loop (e.g., residues 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3) in the light chain variable region and residues 26-32 (HCDR1), 53-55 (HCDR2), and 96-101 (HCDR3) in the heavy chain variable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.

[0079] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope except for possible variants that can arise during production of the monoclonal antibody, such variants typically 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 character of the antibody as not being a polyclonal mixture in which antibodies of different specificities can be present. For example, a monoclonal antibody to use according to the present application can be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, as described herein.

[0080] 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.

[0081] A "human antibody" is one that possesses an amino acid sequence that corresponds to that of an antibody produced by a human and / or has been made using any of the techniques for making human antibodies known to those skilled in the art. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known to those skilled in the art, including the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al. J. Immunol, 147(l):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 the antigen to a transgenic animal that has been modified to repose to an antigen challenge with the production of human antibodies, but whose endogenous loci have been disabled to prevent endogenous antibody production, e.g., an immunized HuMab mouse (see, e.g., Nils Lonber et al., Nature 368:856-859, WO 98 / 24884, WO 94 / 25585, WO 93 / 1227, WO 92 / 22645, WO 92 / 03918, and WO 01 / 09187 regarding HuMab mice), a xenomouse (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 regarding xenomice of Genpharm) or a Trianni mouse (see, e.g., WO 2013 / 063391, WO 2017 / 035252, and WO 2017 / 136734 regarding Trianni mice). TM U.S. Patent Nos. 6,075,181 and 6,150,584) or Trianni mouse (see, e.g., WO 2013 / 063391, WO 2017 / 035252, and WO 2017 / 136734).

[0082] The term "humanized antibody" refers to an antibody that has been engineered to contain one or more human framework regions in the variable region, as well as 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 entirely human sequences except for the CDR regions. Humanized antibodies are generally less immunogenic in humans than non-humanized antibodies, and can therefore provide therapeutic benefits in certain situations. Those skilled in the art will be aware of humanized antibodies, and will also be aware of suitable techniques for their production. See, e.g., Hwang, W. Y. K. 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; 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, each of which is incorporated herein by reference in its entirety.

[0083] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively.

[0084] The term "antigen binding domain" of an antibody, or antibody "binding domain" for short, or similar terms, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigenic 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 VHand CH domains; (iv) a Fv fragment consisting of the VLand VHdomains of a single arm of an antibody, (v) a dAb fragment (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, which can optionally be joined by a synthetic linker.

[0085] The term "complementarity determining region" or "CDR" as used herein refers to short polypeptide sequences within the variable region of the heavy and light chain polypeptides that are primarily responsible for mediating specific antigen recognition. There are three CDRs in each of the V L and each of the V H Each of the V

[0086] As will be appreciated by those skilled in the art, the exact numbering and placement of CDRs can differ in different numbering systems. However, it will be understood that the disclosure of a variable heavy chain sequence and / or a variable light chain sequence includes the disclosure of the relevant CDRs. Thus, the disclosure of each variable heavy chain region is the disclosure of vhCDRs (e.g., vhCDRl, vhCDR2, and vhCDR3), and the disclosure of each variable light chain region is the disclosure of vlCDRs (e.g., vlCDRl, vlCDR2, and vlCDR3).

[0087] In certain embodiments, the CDRs of an antibody can be determined according to the IMGT numbering system described in Lefranc M-P, (1999) The Immunologist 7: 132-136 and Lefranc M-P et al. (1999) Nucleic Acids Res 27: 209-212, each of which is incorporated herein by reference in its entirety. Unless otherwise noted herein, reference to residue numbering in the variable domains of an antibody refers to residue numbering by the IMGT numbering system.

[0088] In other embodiments, the CDRs of an antibody can be determined according to MacCallum RM et al. (1996) J Mol Biol 262:732-745, which is incorporated by reference herein in its entirety. See, e.g., Martin A "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, eds. Kontermann and Diibel, Chapter 31, pp. 422-439, Springer- Verlag, Berlin (2001), which is incorporated by reference herein in its entirety. In other embodiments, the CDRs of an antibody 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 are used by the AbM antibody modeling software of Oxford Molecular (Oxford Molecular Group, Inc.), which is incorporated by reference herein in its entirety.

[0089] "Framework" or "framework region" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4.

[0090] A "human consensus framework" is a framework which represents the most commonly occurring amino acid residues in the framework positions of human immunoglobulin VL or VH sequences. Generally, the selection of amino acid residues in a human consensus framework is based on the analysis of a large number of human immunoglobulin VL or VH sequences. Typically, the sequences analyzed are from a subgroup of variable domain sequences. Generally, the subgroup 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 the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al., supra.

[0091] A "hinge region" is generally defined as extending from 216-238 (EU numbering) or 226-251 (Kabat numbering) in human IgGl. The hinge can be further divided into three distinct regions, upper hinge, middle hinge (e.g., core), and lower hinge.

[0092] The term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of the constant region. The 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 carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region can or can not be present. Unless otherwise indicated, amino acid residue positions of an Fc region or constant region are numbered according to the EU numbering system, also called the EU index, described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0093] A "blocking" antibody or an "antagonist" antibody is one which inhibits or decreases the biological activity of its bound antigen. Certain blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0094] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq 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 receptor); and B cell activation.

[0095] An antibody that "binds to the same epitope" as a reference antibody refers to an antibody that contacts a overlapping set of amino acid residues of an antigen as compared to the reference antibody, or an antibody that blocks 50% or more of the binding of the reference antibody to its antigen in a competition assay. For example, the amino acid residues of an antibody that contact an antigen can be determined by determining the crystal structure of the antibody in complex with the antigen or by performing hydrogen / deuterium exchange. In some embodiments, antibody residues that are within 3.5 A of any atom 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 50% or more of the binding of the reference antibody to its antigen in a competition assay, and conversely, the reference antibody blocks 50% or more of the binding of the antibody to its antigen in a competition assay. An antibody that "binds to the same epitope" as a reference antibody refers to an antibody that contacts a overlapping set of amino acid residues of an antigen as compared to the reference antibody, or an antibody that blocks 50% or more of the binding of the reference antibody to its antigen in a competition assay. For example, the amino acid residues of an antibody that contact an antigen can be determined by determining the crystal structure of the antibody in complex with the antigen or by performing hydrogen / deuterium exchange. In some embodiments, antibody residues that are within 3.5 A of any atom 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 50% or more of the binding of the reference antibody to its antigen in a competition assay, and conversely, the reference antibody blocks 50% or more of the binding of the antibody to its antigen in a competition assay.

[0096] The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to an antigen that 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; 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 its ability to crystallize readily. The Fab fragment consists of an entire light (L) chain and the variable region domain (VH) of the heavy (H) chain, and one heavy chain constant domain (CH1). Pepsin treatment of an antibody yields a single large F(ab)2fragment which roughly corresponds to two disulfide linked Fab fragments having divalent antigen binding activity and is still capable of cross-linking antigen. Fab fragments differ from Fab' fragments by the presence of an additional few residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is herein designated as Fab' where the cysteine residue(s) of the constant domains carry(s) free thiol groups. F(ab')2antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0097] "Fv" is a dimer of one heavy and one light chain variable region domain in tight, non-covalent association. From the folding of these two domains arises the three antigen binding loops (one loop for each of the H and L chains) that contribute the amino acid residues involved in antigen binding and give rise to the antigen binding specificity of antibodies.

[0098] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody fragments that comprise VHand VLantibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VHand VLdomains which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0099] The term "antigen binding domain" of an antibody, or 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 antigenic 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 arm of an antibody, (v) a dAb fragment, which consists of a 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, which can optionally be joined by a synthetic linker.

[0100] The term "multispecific antibody" is used in the broadest sense, and specifically covers an antibody comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH-VL unit has polyepitopic specificity (i.e., is capable of binding two different epitopes on one biological molecule or each epitope on different biological molecules). 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. "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different targets.

[0101] “Dual specificity” or “bispecificity” refers to the ability to specifically bind to two different epitopes on the same or different targets. However, in contrast to bispecific antibodies, dual specific antibodies have two antigen binding arms that are identical in amino acid sequence, and each Fab arm is capable of recognizing two antigens. Dual specificity allows the antibody to interact with two different antigens with high affinity as a single Fab or IgG molecule. According to one embodiment, the multispecific antibody in the IgGl format binds to each epitope with an affinity of 5 pM to 0.001 pM, 3 pM to 0.001 pM, 1 pM to 0.001 pM, 0.5 pM to 0.001 pM, or 0.1 pM to 0.001 pM. “Monospecific” refers to the ability to bind to only one epitope. Multispecific antibodies can have a similar structure to intact immunoglobulin molecules and include an Fc region, such as that of an IgG. Such structures 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 the N- or C-terminus of the heavy or light chain.

[0102] The term “bispecific antibody” as used herein refers to a monoclonal antibody, typically a human or humanized antibody, that has binding specificities for at least two different antigens. In the present application, one of the binding specificities can be for IL-12 or IL-23 and the other can be for any other antigen, such as a cell surface protein, receptor, receptor subunit, tissue-specific antigen, virus-derived protein, virus-encoded envelope protein, bacteria-derived protein, or bacterial surface protein, etc.

[0103] The term “diabody” as used herein refers to a bivalent antibody comprising two polypeptide chains, wherein each polypeptide chain comprises a VH and a VL domain connected by a linker that is too short (e.g., a linker consisting of five amino acids) to allow intramolecular binding of the VH and VL domains on the same polypeptide chain. This configuration forces each domain to pair with a complementary domain on the other polypeptide chain, thereby forming a homodimeric structure. Accordingly, the term “triabody” refers to a trivalent antibody comprising three peptide chains, each comprising one VH domain and one VL domain connected by a linker that is too short (e.g., a linker consisting of 1-2 amino acids) to allow intramolecular binding of the VH and VL domains within the same peptide chain.

[0104] The term "isolated antibody," when used to describe the various antibodies disclosed herein, refers to an antibody that has been identified and separated and / or recovered from the cell or cells from which it was expressed. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses of the polypeptide, and can include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. See, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007) for a review of methods for assessing antibody purity. In preferred embodiments, the antibody is purified to: (1) a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) homogeneity by SDS-PAGE under non-reducing or reducing conditions, using Coomassie blue or, preferably, silver staining.

[0105] With respect to the binding of an antibody to a target molecule, the term "specific binding" or "specifically binds to" or "specific for" a particular polypeptide or an epitope on a particular polypeptide of interest means binding that is measurably different from non-specific interaction. For example, specific binding can be measured 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 similar to the target (e.g., an excess of unlabeled target). In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by the excess of unlabeled target. As used herein, the term "specific binding" or "specifically binds to" or "specific for" a particular polypeptide or an epitope on a particular polypeptide of interest can be manifested, for example, by a Kd of the molecule for the target of 10"4M or less, or 10"5M or less, or 10"6M or less, or 10"7M or less, or 10"8M or less, or 10"9M or less, or 10"10M or less, or 10"11M or less or 10"12M or less, or a Kd in the range of 10"4M to 10"6M, or 10"6M to 10"10M or 10"7M to 10"9M. Those skilled in the art will appreciate that affinity and KD values are inversely related. High affinity is measured by low KD values. 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 polypeptide or polypeptide epitope. 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.

[0106] The term "affinity" as used herein refers to the strength of binding 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 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 mAb affinity can be found in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993) and Muller, Meth. Enzymol. 92:589-601 (1983), the references of which are incorporated herein by reference in their entirety. One standard method for determining mAb affinity that is well known in the art is to use surface plasmon resonance (SPR) screening (e.g., by using a BIAcore TM SPR analysis equipment) to analyze.

[0107] An "epitope" is a term of art that refers to one or more sites on an antigen that interact with an antibody. See (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.): "Antibodies usually recognize only a small region of a macromolecule (such as a protein)... [some epitopes] can be composed of amino acids from different parts of a polypeptide chain brought together by protein folding. Such antigenic determinants are called conformational or discontinuous epitopes, because the structure recognized is composed of segments of a protein that are not contiguous in the amino acid sequence but come together in a 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.).

[0108] As used herein, the term "KD" is intended to refer to the dissociation constant of a particular antibody-antigen interaction. It is calculated from the following equation: Koff / Kon= KD.

[0109] As used herein, the term "IC50" is intended to refer to the effective concentration of an antibody of the application required to neutralize 50% of the biological activity of IL-23 in human lymphoma DB cells in the biological assay described in Example 5 (inhibition of STAT3 activation in human DB cells assay).

[0110] "EC50" in reference to an agent and a particular activity (e.g., binding to a cell, inhibiting enzyme activity, activating or inhibiting an immune cell) refers to the effective concentration of an agent that produces 50% of its maximum response or effect with respect to such activity. "EC100" in reference to an agent and a particular activity refers to the effective concentration of an agent that produces essentially the maximum response with respect to such activity.

[0111] As used herein, the terms "antibody-based immunotherapy" and "immunotherapy" are used to refer broadly to any form of therapy that relies on the targeted specificity of an anti-IL-23pl9 antibody, bispecific molecule, multispecific molecule, binding agent, or fusion protein comprising an IL-23pl9 specific binding agent to mediate a direct or indirect effect on cells characterized by aberrant expression of IL-23pl9. The term is meant to encompass therapeutic methods using naked antibodies, bispecific antibodies (including T cell engaging, NK cell engaging and other immune cell / effect cell engaging formats), antibody drug conjugates, cell therapies using T cells (CAR-T) or NK cells (CAR-NK) engineered to comprise an IL-23pl9 specific chimeric antigen receptor, oncolytic viruses comprising an IL-23pl9 specific binding agent, and gene therapies that deliver an antigen binding sequence of an anti-IL-23pl9 antibody and express the corresponding antibody fragment in vivo.

[0112] 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 triggered or caused by dysregulation of innate and adaptive immune system functions. 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 IMIDs, and the quality of life of individuals can be significantly decreased, with high morbidity and shortened life span (Bunte, K, and Beikler, T, Int. J. Mol. Sci., 20: 3394 (2019)). It is noted herein that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0113] IL-12 / IL-23 receptor signaling axis

[0114] The pl9 subunit of IL-23 (also referred to herein as "IL-23pl9" and "pl9 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 pl9 subunit is only detectable when it is associated 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 nor the IL-23 pl9 subunit is secreted unless covalently bound to p40 intracellularly. Both IL-12 and IL-23 cytokines have the p40 subunit bind to the common IL-12Rβl component of their receptors, with the unique p35 (IL-12) and pl9 (IL-23) subunits determining the specificity of signaling through 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 constitutes part of a complex regulatory network that orchestrates the innate and adaptive immune response.

[0115] IL-12 is thought to play a key role in the development of protective immune responses against many intracellular pathogens and viruses, as well as 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. Thus, IL-23 specific inhibition (spares IL-12 or the p40 subunit common to both) can have a potentially superior safety profile compared to dual inhibition of IL-12 and IL-23.

[0116] The receptor for IL-23 comprises an IL-12Rβ1 subunit (the IL-12 receptor also has this subunit) in association with a unique subunit called IL-23R (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 the IL-12Rβ1 subunit with low affinity (KD = 2 ± 1 uM). Binding of IL-23R to IL-23 promotes 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 variety of cells (natural killer cells, macrophages, dendritic cells, memory T cells, keratinocytes). Production of IL-23 induces expression of IL-23R, creating a positive feedback loop that enhances IL-23 expression.

[0117] IL-23 is produced by activated antigen-presenting cells and binds to an 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 (TNFa), which are proinflammatory cytokines known to contribute to the inflammatory response in IMID disorders.

[0118] Binding of IL-23pl9 to IL-23R results in a reorganization process of the IL-23pl9 helical domain that enables IL-12p40 to bind to IL-12Rpl (Bloch, Y, et al. Immunity. 2018; 48(1): 45-58). This process activates JAK2 and TYK2, resulting in STAT3 and STAT4 formation, which ultimately act as transcription factors (Parham, C, et al. Immunol. 168(11): 5699-5708 (2002). IL-23 is a key player in the late stage of naive CD4+ T cell differentiation into Thl7 cells (Gaffen, SL, et al. Nat Rev Immunol. 14(9): 585-600 (2014). In the absence of IL-23R, naive T cells require other cytokines, such as transforming growth factor (TGF)-beta and IL-6, to regulate early stages of differentiation. These cytokines induce retinoic acid receptor-related orphan receptor-gamma t expression as a transcription factor, which promotes IL-23R expression. Immature Thl7 cells induced by TGF-beta and IL-6 require exposure to IL-23 to gain pathogenicity. When mature, Thl7 cells are capable of producing IL-17 and TNF-alpha (Kashani, A, et al. Gastroenterology & Hepatology 15(5): 255-265 (2019)).

[0119] Despite structural similarities between the two cytokines, the biological activities / functions of IL-23 are different from those of IL-12. IL-23 supports the differentiation of naive CD4+ T cells into a new subset of cells, known as Thl7 cells, and maintains them, which is different from the classic Thl and Th2 cells. Thl7 cells produce interleukin 17A (IL-17A) and interleukin 17F (IL-17F). Thl7 cells produce a range of other factors known to drive inflammatory responses, including tumor necrosis factors known to drive inflammatory responses, including tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), CXCL1, and CCL20. NK cells and innate lymphoid cells, such as lymphoid tissue inducer (LTi)-like cells, express IL-23 receptor and retinoic acid-related orphan receptor (ROR) gamma and respond to IL-23, producing IL-17. IL-1 beta and IL-23 also co-stimulate gamma-delta cells to induce IL-17 production, and do not require T cell receptor involvement.

[0120] Importantly, IL-23 sustains the differentiation of naive T cells into a distinct Thl7 cell lineage and expansion. In the absence of IL-23, the Thl7 phenotype is lost. IL-23 has been described as the "master regulator" of the immunoinflammatory response in IMIDs, as it plays a critical role in sustaining cytotoxic Thl7 cells that produce a proinflammatory cytokine profile. The pathogenic component of IL-23 depends on the dysregulation of IL-17A, IL-17F, and IL-22 production, which provides a rationale for immunotherapy targeting the IL-23 / IL-23R axis.

[0121] Targeting the proinflammatory IL-23 / IL-23 receptor signaling axis

[0122] Anti-IL-12 / IL-23 antibodies reported to impart therapeutic benefit in vivo include antibodies ustekinumab (CNTO 1275) and briakinumab (ABT-874). Both antibodies target the common IL-12p40 subunit, in a region of the p40 subunit critical for IL-12Rβ1 binding (Clarke, A. et al. mAbs 2(5): 539-549 (2010)).

[0123] Anti-IL-23 selective antibodies reported to have therapeutic benefit in vivo include guselkumab (MAYVZTMI) and risankizumab (SKYRATMI). Tildrakizumab (MK-3222) Risankizumab (SKYRATMI) Brazikumab (MEDI2070) and mirakizumab (Ly3074828); all of which are specific to the pl9 subunit of IL-23. Data from phase 3 clinical trials in moderate-to-severe psoriasis patients with randomization, placebo, and active-controll show that tildrakizumab, guselkumab, and risankizumab have a favorable risk-benefit profile. No major safety concerns have been observed for these IL-23 pl9 inhibitors.

[0124] Thl cells driven by IL-12 were previously thought to be the pathogenic T cell subset in many autoimmune diseases, however, recent animal studies in models of inflammatory bowel disease, psoriasis, inflammatory arthritis and multiple sclerosis have assessed the respective contributions of IL-12 and IL-23 and determined that it is IL-23, not IL-12, that is the key driver of autoimmune / inflammatory disease (Ahern et al., Immun. Rev. 226: 147-159 (2008); Cua et al., Nature 421 :744-748 (2003); Yago et al., Arthritis Res and Ther. 9(5):R96 (2007).

[0125] 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) el 000041 (2008), Reveille et al., Nat. Genet. 42(2): 123-127 (2010) and Duerr et al., Science 314(5804): 1461-1463 (2006). An association between rs11209026 (a single nucleotide polymorphism (SNP) in the IL-23R gene) and CD has been established (Reveille, JD et al.). This variant shows a protective effect for CD and UC. The protective nature of rs11209026 was confirmed in a meta-analysis which showed that carrying this SNP variant reduced the risk of disease in a group of over 75,000 cases and controls (Jostins, L. Nature 491(7422): 119-124 (2012). This SNP variant, along with some of the other coding IL-23R variants, together result in reduced IL-23R expression, leading to reduced immune responses mediated through the IL-23 axis (J Biol Chem. 291(16): 8673-8685 (2016).

[0126] While cytokines such as IL-6 and TGF-β1 can promote the differentiation of RORyt+Thl7 cells from naive CD4+T cells, IL-23 is necessary for these cells to exert their full inflammatory function. In addition, IL-23 binds to the IL-23 receptor on activated RORyt+Thl7 cells, further inducing expression of IL-23 receptor (IL-23R), providing a feed-forward loop for the maintenance and proliferation of these cells (Singh, S, et al. MAbs 7(4): 1493-1503 (2015).

[0127] There is substantial evidence that the IL-23 / IL-17 axis plays an important role in the development of chronic inflammation, and genetic studies have revealed a potential link between the IL-23 receptor (IL-23R) or its ligand 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 intensive therapeutic exploration in IMIDs, including psoriasis, psoriatic arthritis, inflammatory bowel disease (ulcerative colitis and Crohn’s disease), ankylosing spondylitis, and systemic lupus erythematosus (SLE).

[0128] In general, IL-23 specific antibodies, such as guselkumab, tildrakine, risankizumab, brecuizumab, or mirakizumab, selectively bind IL-23pl9 and inhibit IL-23 binding to its receptor; thereby antagonizing the role of IL-23 in inducing and maintaining T helper (Th) 17 cells, innate lymphocytes, gd T cells, and natural killer (NK) cells responsible for tissue inflammation, destruction, and / or abnormal tissue repair associated with IMIDs.

[0129] Psoriasis or psoriasis (PsO) is a chronic inflammatory, T-cell mediated skin disorder characterized by a complex pathophysiology. Its prevalence is 1-4% in developed countries. Psoriasis is the most common autoimmune disease in the United States, affecting approximately 7.5 million people. Plaque psoriasis is the most common form of psoriasis, affecting 80% to 90% of patients. Although the pathogenesis of psoriasis is not fully understood, multiple environmental factors, T cells, dendritic cells, numerous cytokines, and 45 established genetic loci interact to cause systemic psoriasis severity, ultimately forming 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 influence of genetic and environmental factors, and the interaction of innate and adaptive immunity ultimately lead to abnormal keratinocyte proliferation and formation of psoriatic lesions (Chan, J.R. et al., J. Exp. Med, 203(12) 2577-2587 (2006).

[0130] PsO plaques are typically well-demarcated erythematous, scaly lesions characterized by epidermal thickening. Affected 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 (Thl) and type 17 T helper (Thl7) cells, respectively. T lymphocytes and other cell types release additional cytokines, including tumor necrosis factor (TNF)-a, IL-22, and IL-17, leading to increased keratinocyte activation and the initiation of a self-perpetuating 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, including CD4+ T cells, dendritic cells, macrophages, and mast cells.

[0131] Early publications reported elevated levels of tumor necrosis factor a and the p40 subunit of IL-12 in psoriatic lesions, accompanied by overexpression of IL-12p40 and IL-23p40 messenger RNA. These findings suggested that inhibition of IL-12 and IL-23 with neutralizing antibodies against the IL-12 / 23 p40 subunit protein could provide an effective treatment for psoriasis (Piskin G et al., J Immunol 2006, 176:1908-15). Psoriasis was initially considered a Th-1 mediated disease (based on the cytokine profile secreted by T helper type 1 cells: interleukin 2, tumor necrosis factor (TNF) a, and interferon (IFN) g).

[0132] The fundamental role of IL-23 in psoriasis pathogenesis has been elucidated in relation to the biology of the Th17 lineage. The presence of TGF-βΙ, IL-6, and IL-1 is required for initial T lymphocyte commitment to Th17 differentiation, while IL-23 is essential for the activation and maintenance of Th17 to secrete the proinflammatory cytokines IL-17, IL-22, IL-21, and tumor necrosis factor-a, ultimately contributing to the formation of psoriatic lesions (Fotaidou, C. et al. Psoriasis: Targets and Therapy 8: 1-5 (2018)).

[0133] Thus, while 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 the key driver of Th17 cell differentiation and survival. The major cytokines produced by Th17 cells are the proinflammatory IL-17 family of cytokines, 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, which is a heterodimer comprising IL-17RA and IL-17RC subunits.

[0134] While early therapeutic strategies targeted Th1 cells as the central cell type in psoriasis pathogenesis, more recent models have focused on the IL-23 / Th17 axis (Lowes MA, et al. Trends Immunol. 34(4): 174-81 (2013). The rationale for the new focus is based on the view that IL-17 is a key player in psoriasis pathogenesis, and the knowledge that IL-23 drives Th17 cell activation. In addition, IL-23 stimulates other cell types, including innate lymphoid type 3 cells and gd T cells, to produce other Th17 cytokines (e.g., IL-22) (Ward, N.L., J Investig Dermatol. 134:2305-2307 (2014). It has been proposed that inhibition of IL-23 will block the downstream production of IL-17A and IL-22 by Th17 cells, translating this effect into an antagonistic effect on psoriasis immune pathogenesis.

[0135] The IL-23 / IL-17 axis is currently believed to be central to the pathogenesis of psoriasis, and selective IL-23p19 inhibition can offer several advantages over IL-12 / 23p40 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, tildrakine, 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 as a systemic therapy or a phototherapy candidate. In July 2020, guselkumab was also approved by the FDA for the treatment of adult patients with active psoriatic arthritis.

[0136] The high efficacy of IL-23 blockade in psoriasis was demonstrated in early proof-of-concept testing and Phase I clinical trials. Phase 1 studies showed that a single dose of guselkumab produced a significant clinical response in patients with moderate-to-severe plaque psoriasis (Sofen H, et al., J Allergy Clin Immunol; 133: 1032-1040 (2014). Phase 1 studies also reported that selective antagonism of interleukin-23 with guselkumab led to clinical improvement in psoriasis characterized by reduced epidermal thickness, infiltration of T cells and dendritic cells, expression of psoriasis-associated genes, and levels of serum IL-17A. It was reported that a measurable clinical response was observed in patients with moderate-to-severe psoriasis following a single dose of guselkumab, which supports the emerging theory that selective neutralization of IL-23 is a promising treatment option.

[0137] A rapid onset of activity of secukinumab was also observed in a Phase II dosing study (NCT01483599) that evaluated the use of secukinumab for continuous treatment for up to 40 weeks at a broad range of doses and two different dosing intervals. Efficacy was evident in the earliest assessments (week 4). The response rates associated with secukinumab regimens were much better than those associated with adalimumab, a biologic commonly used to treat psoriasis (Gordon, KB, et al. N Engl J Med 373: 136-144 (2015). Efficacy of secukinumab continued to increase after week 16 (primary endpoint assessment) and persisted through week 40. Moreover, after 40 weeks of continuous treatment, most patients in the 100 mg secukinumab group had complete clearance of psoriasis, as indicated by a PGA score of 0 (62% of patients) and a 100% improvement from baseline in PASI score (54% of patients). Regulatory approval by the FDA and EMA was contingent, in part, on the results of three pivotal Phase III clinical 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).

[0138] VOYAGE 1 (NCT02207231) was a phase III, randomized, double-blind, placebo- and active-controlled trial conducted at 101 sites globally (December 2014 to April 2016). The study included an active-comparator phase (Weeks 0-48) comparing guselkumab to adalimumab and a placebo-controlled phase (Weeks 0-16), after which patients receiving placebo crossed over to receive guselkumab through Week 48. Gusekumab was superior to placebo and / or adalimumab for both coprimary endpoints and all major secondary endpoints (all P <.001). Patients receiving guselkumab had significantly greater proportions of patients achieving IGA 0 / 1 (6.9% vs 85.1%) and PASI 90 (2.9% vs 73.3%) at Week 16 compared to placebo. Also, at Week 24 and Week 48, PASI 100 responses were significantly greater in the guselkumab group than in the adalimumab group (P <.001). Patients in the placebo crossover group achieved responses similar to those observed in the guselkumab group after starting guselkumab at Week 16. VOYAGE 1 confirmed the role of IL-23 in the pathogenesis of psoriasis. Selective targeting of the IL-23 pathway provides inhibition of more psoriasis-specific cytokines and has higher efficacy compared to TNF-a blockade, while maintaining a favorable safety profile (Blauvelt, A, et al., J Investig Dermatol., 135: 1946-1953 (2015).

[0139] VOYAGE 1 was an extended-label trial that followed patients for four years after the initial trial. Patients were initially randomized to receive Tremfya or placebo, but everyone received Tremfya at Week 16. The VOYAGE 1 study found that in the combined group of individuals who initially received Tremfya or placebo and then crossed over to receive Tremfya at Week 16, 82% of patients receiving Tremfya showed at least 90% improvement in Psoriasis Area Severity Index (PASI 90) and scored clear (0) or minimal disease (1) on Investigator’s Global Assessment (IGA) at Week 204 (i.e., four years).

[0140] Psoriatic arthritis (PsA) is a chronic inflammatory musculoskeletal disease that occurs in up to 40% of psoriasis patients. PsA can therefore be considered a disease within a disease, with many common pathogenic pathways with psoriasis. In 70% of patients, psoriasis usually precedes PsA, with simultaneous inflammatory skin and joint disease in 15% of patients, and inflammatory arthritis occurring before the skin disease in the remaining patients. Finally, while almost all PsA patients develop psoriasis, the clinical presentation and course of PsA are quite heterogeneous, with five different PsA patterns based on the distribution of affected joints having been described (Dobbin-Sears, I, et al. Ther Adv Chronic Dis,. 9(10) 191-198 (2018)).

[0141] PsA is a heterogeneous condition with both joint and extra-articular manifestations, including a combination of peripheral arthritis, axial disease, enthesitis, dactylitis, and 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 innate and adaptive immune responses appear to play an important role in the pathogenesis of PsA. As the disease progresses, patients can exhibit multiple patterns and are not limited to one subset of arthritis. Approximately two-thirds of PsA patients experience progressive joint damage, which is often associated with loss of function and disability.

[0142] 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 an important role in the immunopathogenesis of psoriasis and PsA. IL-23 / IL-23-R interaction induces differentiation and activation of IL-23-dependent Th-17 cells and production and secretion of IL-17 and IL-22, ultimately leading to synovial and skin inflammation and bone remodeling. Of particular relevance to PsA pathology, IL-17 promotes bone erosion by upregulating RANKL. The results of a pooled data analysis indicated that ustekinumab-treated patients (independent 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 downstream Th17 pathways in radiographic damage occurring in most PsA patients.

[0143] Crohn's disease (CD) and ulcerative colitis (UC) (the human major inflammatory bowel diseases (IBD)) are chronic relapsing diseases characterized by chronic tissue inflammation that alters the 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 disease (IBD) (e.g., Crohn's disease (CD) and ulcerative colitis (UC)).

[0144] Several variants of genes encoding elements of the IL-23 and IL-17 cell pathways are associated with IBD risk. In particular, loss-of-function variants of 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, attributed to reduced STAT3 signaling and abrogation of the response of Th17 cells 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).

[0145] Crohn's disease (CD) is a chronic immune-mediated condition characterized by relapsing and gastrointestinal system involvement. CD is characterized by dysregulation of innate and adaptive immune responses. Although the pathophysiological mechanisms are not fully understood, the disease can be the result of an interaction between the intestinal microbiota and host microbial defense in genetically susceptible individuals, leading to a transmural inflammatory response in Crohn's disease (Deepak, P and Loftus, E, Drug Design, Development and Therapy (10) 3685-3698) (2016). In the long term, the persistent transmural inflammatory response usually leads to the development of strictures and / or fistulas, which require hospitalization and / or surgery. Following the discovery of the IL-23 / IL-17 pathway, the treatment model for CD shifted from nonspecific immunosuppressive therapy (i.e., methotrexate) to immunotherapy targeting the IL-2 and / or / IL-17 pathway.

[0146] UC is a chronic, relapsing-remitting inflammatory bowel disease that results in persistent mucosal inflammation of the colon that develops into tiny open sores or ulcers that produce pus and mucus. It is estimated that nearly 1 million patients with ulcerative colitis live in the United States, and UC affects 2.6 million people in Europe. Although the disease can affect people of any race or ethnicity, it is more common in Caucasians and men are more likely to be diagnosed than women. The etiology of UC is poorly understood, but it is believed to be due in part to an abnormal immune response to the microbiota (microbial flora and pathogens) in subjects with a genetic predisposition to chronic inflammation of the colon. Ulcerative colitis is known to exhibit a Th2-type cytokine profile.

[0147] In clinical studies for IBD, IL-23 specific p19 antagonists include Brazikumab (MEDI2070), Risankizumab (BI 655066), Mirikizumab (LY3074828), and Guselkumab (Tremfya, Janssen). To date, it has been reported that the antibodies against p19 (anti-IL-23), Brazikumab and Risankizumab, were effective for moderate to severe CD in phase II trials.

[0148] Recently, in a phase II trial, Mirikizumab was shown to be effective for moderate to severe UC. In all doses studied, 11.5% to 22.6% of patients receiving Mirikizumab achieved clinical remission, compared to 4.8% of patients receiving placebo. In addition, a higher proportion of patients receiving Mirikizumab achieved endoscopic and symptomatic remission within 12 weeks compared to placebo. No p-19 selective antibody has been approved for the treatment of IBD to date. Phase 2 and 3 clinical trials of anti-p19 agents (Risankizumab, Brazikumab, Guselkumab) are ongoing, which will not only provide further information on their efficacy and safety per se, but also provide head-to-head efficacy compared to existing biologics, as well as provide evolving treatment concepts in combination with multiple biologics.

[0149] Ankylosing spondylitis (AS) is another spondyloarthropathy genetically associated with the IL-23 pathway, similar to psoriatic arthritis; it is a painful condition involving inflammation of the spine that can lead to irreversible fusion of the spine. AS is generally unresponsive to traditional disease-modifying antirheumatic drugs (DMARDs), and systemic therapies for AS include nonsteroidal anti-inflammatory drugs (NSAIDs) and tumor necrosis factor inhibitors.

[0150] There are several lines of evidence that IL-23 is a promising therapeutic target in AS (Paine A et al. Curr. Opin. Rheumatol. 28: 359-67 (2016). At the genetic level, case-control genome-wide association studies have implicated IL-23 receptor (IL-23R) polymorphisms in 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 has been observed in AS (Sarin R et al. Proc Natl Acad Sci USA; 108: 9560-58 (2011). An increased number of IL-23 producing cells were found in the facet joints of AS patients (Appel H et al., Arthritis Rheum; 65: 1522-9 (2013), and the number of IL-23 responsive T helper (Th)22, Th17 and γ / δ T cells were elevated in the blood of AS patients (Zhang L, et al. PLoS One (7): e31000 (2012)).

[0151] An IL-17A inhibitor, secukinumab, was recently approved for the treatment of AS (Baeten D. et al., N Engl. J. Med. 373: 2534-48 (2015)), which supports the clinical hypothesis that direct and specific inhibition of IL-23 would benefit AS patients. However, a recent publication reported the results of a randomized, double-blind, placebo-controlled, proof-of-concept, dose-finding phase 2 study that evaluated the efficacy of risankizumab in patients with active AS (NCT02047110), concluding that treatment with risankizumab did not meet the study’s primary endpoint and did not show a clinically meaningful improvement in patients with active AS compared with placebo (Baeten D, et al. Annals of the Rheumatic Diseases 77: 1295-1302 (2018)).

[0152] IL-23p19 antagonists

[0153] The IL-23 receptor complex consists of IL-12Rβ1 in association with the signaling chain IL-23R (p19 subunit binding). IL-23 mediates cellular activity by sequential binding to 2 receptor chains expressed on the surface of T cells and natural killer (NK) cells as an IL-12Rβ1 / IL-23R receptor complex.

[0154] Murine, humanized, and phage-displayed antibodies have been described that are selected for inhibition of recombinant IL-23; see, e.g., U.S. Patent 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.

[0155] Monoclonal antibodies or antigen-binding domains thereof that bind with high affinity to the pl9 subunit of the IL-23 cytokine can neutralize the activity of the cytokine, thereby blocking its downstream effects. To date, three (3) anti-pl9 antibodies, guselkumab tildrakizumab and risankizumab have been approved by the FDA for the treatment of IMIDs. Two other antibodies specific for the IL-23 pl9 subunit are currently in late-stage clinical development, MEDI2070 (brazikumab, Astrazeneca / Medimmune) and Ly3074828 (mirikizumab, Eli Lilly). Mirikizumab is a humanized IgG4 monoclonal antibody. By blocking IL-23, anti-pl9 specific antibodies inhibit the release of proinflammatory cytokines and chemokines, thereby suppressing the inflammatory response.

[0156] Because this group of IL-23 antagonists targets the pl9 subunit of IL-23 and not the p40 subunit, the IL-23 antagonists do not affect IL-12 activity. This feature distinguishes IL-23 receptor antagonists from ustekinumab (STELARA), which targets the common p40 subunit shared by both IL-12 and IL-23. While ustekinumab has efficacy and a good safety profile, drug development for IMIDs has shifted focus to the development of agents that selectively antagonize the IL-23 / IL-17 pathway.

[0157] Guselkumab (CNTO 1959) is a fully human monoclonal IgGl, lambda antibody that binds with high affinity to the pl9 subunit of human IL-23. Guselkumab is the first anti-pl9 specific antibody / IL-23 antagonist approved by the FDA. After expedited regulatory review, guselkumab was approved on July 13, 2017 for the treatment of adult patients with moderate-to-severe plaque psoriasis. Guselkumab is also approved in Canada, the European Union, Japan, and several other countries globally. Guselkumab is marketed by Janssen as TREMFYA (U.S. Patent Nos: 7,935,344 and 7,993,645).

[0158] Guselkumab inhibits the biological activity of human IL-23 by preventing the binding of IL-23 to the IL-23 receptor protein expressed on the surface of immune cells. More particularly, guselkumab binds to the p19 subunit of the human IL-23 cytokine, thereby preventing the formation of the IL-23-IL-23R complex and the subsequent intracellular signaling of the partner receptor chain.

[0159] The development program currently includes evaluation of a Phase III trial for efficacy in the treatment of active psoriatic arthritis, a Phase IIb / III study in Crohn’s disease, a Phase IIb / III trial in ulcerative colitis, and another clinical study evaluating guselkumab for hidradenitis suppurativa.

[0160] Janssen recently announced plans to further expand the clinical development of guselkumab to familial adenomatous polyposis (FAP), a gastrointestinal disease. Janssen has initiated a Phase lb proof-of-concept clinical trial (NCT03649971) that evaluates the efficacy and safety of guselkumab compared to placebo in approximately 72 patients. FAP syndrome is the most common adenomatous polyposis syndrome. FAP syndrome is an autosomal dominant genetic disease characterized by the early onset of hundreds to thousands of adenomatous polyps throughout the colon. FAP has a global birth incidence of approximately 1 in 8,300 people, equally in men and women, and if left untreated, patients with the syndrome will most likely develop colorectal cancer. In addition, the risk of developing other malignancies is also increased. Currently, colectomy is the only method to prevent these patients from developing colorectal cancer.

[0161] Tildrakizumab (MK322) is a humanized monoclonal IgGl, kappa antibody, marketed by Merck & Co. / Sun Pharmaceutical as ILUMYA (U.S. Patent No. 8,404,813). Tildrakizumab selectively binds the p19 subunit, thereby inhibiting the interaction of IL-23 with its receptor, and in turn inhibiting IL-23-mediated release of proinflammatory cytokines. Tildrakizumab was first globally approved by the FDA in March 2018 for adult patients with moderate-to-severe plaque psoriasis.

[0162] Risankizumab (BI 655066) is a humanized monoclonal IgGl, kappa, marketed by AbbVie / Boehringer Ingelheim as SKYRIZI (U.S. Patent No. 8,778,346). Risankizumab has been developed as a high-affinity antibody that antagonizes IL-23.

[0163] Risankizumab selectively binds to the pl9 subunit of interleukin-23 (IL-23pl9) with high affinity (dissociation constant < 10 pmol / L) (Singh, S, et al., MAbs 7(4) 77-791 (2015). Risankizumab selectively targets the pl9 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 regions of risankizumab are engineered with two mutations in the Fc region to reduce FcyR receptor and complement binding. More particularly, the Fc portion of risankizumab has two substitution mutations (Leu234Ala and Leu235Ala) to reduce Fcy receptor and complement binding. Risankizumab was approved by the FDA in April 2019 for the treatment of moderate-to-severe plaque psoriasis in adults.

[0164] Anti-IL-23p19 antibodies

[0165] Anti-IL-23pl9 antibodies of the present disclosure bind to the pl9 subunit of IL-23. Preferably, such antibodies are fully human antibodies and do not bind to the p40 subunit of IL-12.

[0166] In one embodiment, an anti-IL-23pl9 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-23pl9 antibody or antibody fragment thereof can comprise a set of CDRs corresponding to those in one or more of the anti-IL-23pl9 antibodies disclosed in Table 1 (e.g., the CDRs of the Hu-2.18006B antibody).

[0167] In another embodiment, an anti-IL-23pl9 antibody comprises a VL having a set of CDRs (LCDR1, LCDR2, and LCDR3) disclosed in Table 2. For example, an anti-IL-23pl9 antibody or antibody fragment thereof can comprise a set of CDRs corresponding to those in one or more of the anti-IL-23pl9 antibodies disclosed in Table 2 (e.g., the CDRs of the Hu-2.18006B antibody).

[0168] In an alternative embodiment, an anti-IL-23pl9 antibody or antibody fragment thereof comprises a VH having a set of CDRs (HCDR1, HCDR2, and HCDR3) disclosed in Table 1 and a VL having a set of CDRs (LCDR1, LCDR2, and LCDR3) disclosed in Table 2.

[0169] Table 1 ：CDR sequences of human variable heavy chain domains

[0170] Anti-IL-23p19 Ab CDR1 CDR2 CDR3 Hu-2.18006B SEQ ID NO:9 SEQ ID NO:10 SEQ ID NO:11 Hu-4.18006B SEQ ID NO:15 SEQ ID NO:16 SEQ ID NO:17 Hu-5.18006B SEQ ID NO:21 SEQ ID NO:22 SEQ ID NO:23 Hu-6.18006B SEQ ID NO:27 SEQ ID NO:28 SEQ ID NO:29

[0171] Table 2: CDR sequences of human variable light chain domains

[0172] Anti-IL-23p19 Ab CDR1 CDR2 CDR3 Hu-2.18006B SEQ ID NO:12 SEQ ID NO:13 SEQ ID NO:14 Hu-4.18006B SEQ ID NO:18 SEQ ID NO:19 SEQ ID NO:20 Hu-5.18006B SEQ ID NO:24 SEQ ID NO:25 SEQ ID NO:26 Hu-6.18006B SEQ ID NO:30 SEQ ID NO:31 SEQ ID NO:32

[0173] In one embodiment, the antibody can be a monoclonal, chimeric, humanized or human antibody (or an antigen binding portion thereof) that specifically binds human IL-23p19.

[0174] In one embodiment, the anti-IL-23p19 antibody or antibody fragment thereof comprises a VH having a set of complementarity determining regions (CDR1, CDR2, and CDR3) selected from the group consisting of:

[0175] (i) CDR1: SEQ ID NO: 9, CDR2: SEQ ID NO: 10, CDR3: SEQ ID NO: 11;

[0176] (ii) CDR1: SEQ ID NO: 15, CDR2: SEQ ID NO: 16, CDR3: SEQ ID NO: 17;

[0177] (iii) CDR1: SEQ ID NO: 21, CDR2: SEQ ID NO: 22, CDR3: SEQ ID NO: 23; and

[0178] (iv) CDR1: SEQ ID NO: 27, CDR2: SEQ ID NO: 28, CDR3: SEQ ID NO: 29.

[0179] In another embodiment, the anti-IL-23p19 antibody or antibody fragment thereof comprises a VL having a set of complementarity determining regions (CDR1, CDR2, and CDR3) selected from the group consisting of:

[0180] (i) CDR1: SEQ ID NO: 12, CDR2: SEQ ID NO: 13, CDR3: SEQ ID NO: 14;

[0181] (ii) CDR1: SEQ ID NO: 18, CDR2: SEQ ID NO: 19, CDR3: SEQ ID NO: 20;

[0182] (iii) CDR1: SEQ ID NO: 24, CDR2: SEQ ID NO: 25, CDR3: SEQ ID NO: 26; and

[0183] (iv) CDR1: SEQ ID NO: 30, CDR2: SEQ ID NO: 31, CDR3: SEQ ID NO: 32.

[0184] In another embodiment, the anti-IL-23p19 antibody or antibody fragment thereof comprises:

[0185] (a) a VH having a set of complementarity determining regions (CDR1, CDR2, and CDR3) selected from:

[0186] (i) CDR1: SEQ ID NO: 9, CDR2: SEQ ID NO: 10, CDR3: SEQ ID NO: 11;

[0187] (ii) CDR1: SEQ ID NO: 15, CDR2: SEQ ID NO: 16, CDR3: SEQ ID NO: 17;

[0188] (iii) CDR1: SEQ ID NO: 21, CDR2: SEQ ID NO: 22, CDR3: SEQ ID NO: 23; and

[0189] (iv) CDR1: SEQ ID NO: 27, CDR2: SEQ ID NO: 28, CDR3: SEQ ID NO: 29; and

[0190] (b) a VL having a set of complementarity determining regions (CDR1, CDR2, and CDR3) selected from:

[0191] (i) CDR1: SEQ ID NO: 12, CDR2: SEQ ID NO: 13, CDR3: SEQ ID NO: 14;

[0192] (ii) CDR1: SEQ ID NO: 18, CDR2: SEQ ID NO: 19, CDR3: SEQ ID NO: 20;

[0193] (iii) CDR1: SEQ ID NO: 24, CDR2: SEQ ID NO: 25, CDR3: SEQ ID NO: 26; and

[0194] (iv) CDR1: SEQ ID NO: 30, CDR2: SEQ ID NO: 31, CDR3: SEQ ID NO: 32.

[0195] In one embodiment, the antibody comprises a combination of a VH and a VL having a set of complementarity determining regions (CDR1, CDR2, and CDR3) selected from the group consisting of:

[0196] (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;

[0197] (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;

[0198] (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

[0199] (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.

[0200] 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.

[0201] 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 make anti-IL-23p19 antibodies comprising combinations of variable heavy and variable light chains different from the pairs identified above.

[0202] 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 make anti-IL-23p19 antibodies comprising combinations of variable heavy and variable light chains different from the pairs identified above.

[0203] In some embodiments, the anti-IL-23p19 antibody (e.g., antagonist antibody) binds with high affinity to the pl9 subunit of IL-23, but not to the p40 subunit of the related cytokine family member IL-12.

[0204] 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 a Fab, Fab', F(ab)2, Fv, domain antibody (dAbs) 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 specific binding of IL-23 to the polypeptide.

[0205] In some embodiments, the variable region domains of the anti-IL-23p19 antibodies disclosed herein can be covalently linked at the C-terminal amino acid to at least one other antibody domain or fragment thereof. Thus, for example, a VH domain present in a 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. In this manner, for example, the antibody can be a Fab fragment, wherein the antigen binding domain comprises the relevant VH and VL domains, which are covalently linked at their C-termini to CH1 and CK domains, respectively. The CH1 domain can be extended with additional amino acids, for example, to provide a hinge region or a portion of a hinge region domain as present in a Fab' fragment, or to provide other domains, such as antibody CH2 and CH3 domains.

[0206] In some embodiments, the variable region domains of the anti-IL-23p19 antibodies disclosed herein can be covalently linked at the C-terminal amino acid to at least one other antibody domain or fragment thereof. Thus, for example, a VH domain present in a 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. In this manner, for example, the antibody can be a Fab fragment, wherein the antigen binding domain comprises the relevant VH and VL domains, which are covalently linked at their C-termini to CH1 and CK domains, respectively. The CH1 domain can be extended with additional amino acids, for example, to provide a hinge region or a portion of a hinge region domain as present in a Fab' fragment, or to provide other domains, such as antibody CH2 and CH3 domains.

[0207] Thus, in one embodiment, the antibody fragment comprises at least one CDR as described herein. The antibody fragment can comprise at least two, three, four, five, or six CDRs, as described herein. The antibody fragment can further comprise at least one variable region domain of an antibody described herein. The variable region domain can be of any size or amino acid composition and will generally comprise at least one CDR sequence responsible for binding human IL-23p19, such as CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3, as described herein, and which is adjacent to or within a framework sequence(s).

[0208] 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.

[0209] In another aspect, the anti-IL-23p19 antibody or antibody fragment thereof exhibits one or more of the following properties:

[0210] (a) is specific for human IL-23p19 and has the ability to block the binding of IL-23 to its receptor (IL-23R);

[0211] (b) inhibits, interferes with, or modulates the interaction of IL-23p19 with IL-23 receptor signaling;

[0212] (c) inhibits STAT3 activation induced by IL-23;

[0213] (d) inhibits IL-17 production induced by human IL-23 in mouse splenocytes;

[0214] (e) inhibits IL-17 production induced by human IL-23 in activated human T cells in PBMC;

[0215] (f) does not inhibit the interaction of IL-23 with IL-12Rβ1 signaling;

[0216] (g) does not inhibit the production of interferon gamma induced by human IL-12 in human activated T cells (PBMC);

[0217] (h) does not inhibit the production of interferon gamma induced by cynomolgus IL-12 in human activated T cells (PBMC); and

[0218] (i) inhibits skin inflammation induced by human IL-23 in a murine psoriasis model.

[0219] In one embodiment, the anti-IL-23p19 antibody or antibody fragment thereof can reduce, inhibit, interfere with, and / or modulate at least one biological response associated with IL-23, and thus can be used to ameliorate the effects of an IL-23-associated disease or disorder. Such antibodies and antibody fragments thereof can be used, for example, to reduce, inhibit, interfere with, and / or modulate IL-23 signaling, IL-23 activation of Thl7 cells, IL-23 activation of NK cells, or induction of proinflammatory cytokine production.

[0220] In some embodiments, the anti-IL-23p19 antibody or antibody fragment thereof comprises one or more conservative amino acid substitutions. Those of skill in the art will recognize that a conservative amino acid substitution is one in which an amino acid is replaced with another amino acid having similar structural or chemical properties, such as, for example, similar side chains. Exemplary conservative substitutions are described in, e.g., Watson et al., Molecular Biology of the Gene, The Benjamin / Cummings Publication Company, 4th ed. (1987).

[0221] "Conservative modifications" refer to amino acid modifications that do not significantly affect or alter the binding properties of an antibody comprising the amino acid sequence. Conservative modifications include amino acid substitutions, additions, and deletions. Conservative substitutions refer to the replacement of an amino acid by an amino acid residue having similar side chains. Families of amino acid residues having similar side chains are well-defined and include amino acids having 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), amide (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine), and sulfur-containing side chains (cysteine, methionine). In addition, any of the natural residues in a polypeptide can also be substituted with alanine, as in the alanine scanning mutagenesis described previously (MacLennan et al., (1998) Acta Physiol Scand Suppl 643:55-67; Sasaki et al. (1998) Adv Biophys 35:1-24). Amino acid substitutions of the antibodies of the application can be made by known methods, such as by PCR mutagenesis (U.S. Patent No. 4,683,195).

[0222] In one embodiment, the anti-IL-23p19 antibody or antibody fragment thereof comprises all six CDR regions of the Hu-2.18006B, Hu-4.18006B, Hu-5.18006B, or Hu-6.18006B antibody forming a chimeric antibody or a humanized antibody. In other embodiments, the anti-IL-23p19 antibody or antibody fragment thereof comprises all six CDR regions of one of the fully human antibodies disclosed.

[0223] Methods of producing antibodies

[0224] 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 a soluble recombinant human IL-23 protein, or fragments or peptides coupled to carrier proteins thereof. Any suitable immunization method can be used. Such methods can include adjuvants, other immunostimulatory agents, repeated booster immunizations, and use of one or more immunization routes.

[0225] Any suitable source of human IL-23 can be used as an immunogen for the production of non-human or human anti-IL-23p19 antibodies for the compositions and methods disclosed herein.

[0226] Different forms of IL-23 antigens can be used to generate antibodies sufficient to produce biological activity. Thus, the eliciting IL-23 antigen can be a single epitope, multiple epitopes, or the entire protein alone or in combination with one or more immunogenicity enhancers. In some aspects, the eliciting antigen is an isolated soluble full-length protein, or a soluble protein comprising less than the full-length sequence (e.g., immunization with a peptide comprising a particular portion or epitope of IL-23). As used herein, the term "portion" refers to the minimum number of amino acids or nucleic acids suitable to constitute an immunogenic epitope of the antigen of interest. Any genetic vector suitable for transforming the cell of interest can be used, including but not limited to adenoviral vectors, plasmids, and non-viral vectors, such as cationic lipids.

[0227] Monoclonal antibodies (mabs) are desired to be prepared from a variety of mammalian hosts, such as mice, rodents, primates, humans, and the like. Techniques for preparing such monoclonal antibodies are described in, for example, 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, splenocytes from an animal immunized with the desired antigen are immortalized, usually by fusion with myeloma cells. See Kohler and Milstein (196) Eur. J. Immunol. 6:511-519. Alternative methods of 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. Clones produced by individual immortalized cells are screened to produce antibodies having the desired specificity and affinity for the antigen, and the production 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 DNA libraries with the techniques outlined in, for example, Huse et al., Science 246:1275-1281, according to general protocols outlined therein 1275-1281. Thus, monoclonal antibodies can be obtained by a variety of techniques familiar to those skilled in the art.

[0228] Other suitable techniques include selecting antibody libraries in phage, yeast, virus, 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 can be used with or without modification, including chimeric or humanized antibodies. Typically, the polypeptides and antibodies will be labeled by covalent or noncovalent attachment to a substance that provides a detectable signal. A wide variety of labels and conjugation techniques are known by those skilled in the art and have been described in numerous publications. Suitable labels include radionuclides, enzymes, substrates, co-factors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents teaching use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. In addition, recombinant immunoglobulins can 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 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, Alexis, OminiAb, Harbor and other technologies.

[0229] In some embodiments, the ability of the produced antibodies to bind 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 ability of the produced antibodies to inhibit IL-23 blockade of IL-23 receptor beta 1 signaling and inhibition of IL-23p19 and / or IL-23p19-mediated inflammatory microenvironment sequelae, including inhibition of IL-23-induced Stat3 phosphorylation, IL-17 production, and / or IFN-g production, can also be assessed.

[0230] Antibody compositions prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography typically being the purification technique of choice. The suitability of a 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 typically agarose, but other matrices can also be used. A mechanically stable matrix such as controlled pore glass or poly(styrenedivinyl)benzene allows for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, Bakerbond ABX TM resin (J.T. Baker, Phillipsburg, N.J.) is used for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica or on SEPHADEX TM chromatography, anion or cation exchange chromatography (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0231] Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an eluant at a pH between about 2.5 and about 4.5, typically at low salt concentrations (e.g., 0-0.25 M salt).

[0232] Also included are nucleic acids that hybridize under low, medium, and high stringency conditions, as defined herein, to all or a portion of a nucleotide sequence represented by an isolated polynucleotide sequence encoding an antibody or antibody fragment of the present disclosure (e.g., a portion encoding a variable region). The hybridizing portion of a hybridizing nucleic acid is typically at least 15 (e.g., 20, 25, 30, or 50) nucleotides in length. The hybridizing portion of a hybridizing nucleic acid has at least 80%, e.g., at least 90%, at least 95%, or at least 98% identity to a portion or the entire sequence of a nucleic acid encoding an anti-IL-23p19 polypeptide (e.g., a heavy or light chain variable region) or the complement thereof. Hybridizing nucleic acids of the type described herein can be used, e.g., as cloning probes, primers (e.g., PCR primers), or diagnostic probes.

[0233] Polynucleotides, vectors, and cells

[0234] Other embodiments include isolated polynucleotides comprising a sequence encoding an anti-IL-23p19 antibody or antibody fragment thereof, vectors and cells comprising the polynucleotides, and recombinant techniques for producing the antibodies. The isolated polynucleotides can encode any desired form of an 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, minibodies, and multi-specific antibodies formed from antibody fragments.

[0235] 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 one 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.

[0236] In one embodiment, the isolated polynucleotide sequence encodes an antibody or antibody fragment having a light chain and heavy chain variable region comprising the amino acid sequence:

[0237] (a) a variable heavy chain sequence comprising SEQ ID NO: 1 and a variable light chain sequence comprising SEQ ID NO: 2;

[0238] (b) a variable heavy chain sequence comprising SEQ ID NO: 3 and a variable light chain sequence comprising SEQ ID NO: 4;

[0239] (c) a variable heavy chain sequence comprising SEQ ID NO: 5 and a variable light chain sequence comprising SEQ ID NO: 6; or

[0240] (d) a variable heavy chain sequence comprising SEQ ID NO: 7 and a variable light chain sequence comprising SEQ ID NO: 8.

[0241] In one embodiment, the isolated polynucleotide sequence encodes an antibody or antibody fragment having a light chain and heavy chain variable region comprising the amino acid sequence:

[0242] (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;

[0243] (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;

[0244] (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

[0245] (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.

[0246] As known in the art, a polynucleotide comprising 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 can be contained in a suitable expression vector or cell. Each polynucleotide molecule encoding a heavy or light chain variable domain can be independently fused to a polynucleotide sequence encoding a constant domain, such as a human constant domain, enabling production of a complete antibody. Alternatively, the polynucleotides or portions thereof can be fused together to provide a template for production of a single chain antibody.

[0247] For recombinant production, the polynucleotide encoding the antibody is inserted into a replicable vector for cloning (amplification of the DNA) or for expression. Numerous suitable vectors for the expression of 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.

[0248] The anti-IL-23p19 antibody or antibody fragment thereof can also be produced as a fusion polypeptide, wherein 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 heterologous signal sequence selected is typically one that is recognized and processed by the cell into which the fusion polypeptide is introduced. For prokaryotic cells, the signal sequence can be a prokaryotic signal sequence. For eukaryotic cells, the signal sequence can be a eukaryotic signal sequence. The signal sequence can be, for example, that of alkaline phosphatase, penicillinase, lipoprotein, heat-stable enterotoxin II, or the like. For yeast secretion the native signal sequence can be substituted with the lead sequences obtained from yeast invertase alpha-factor (including Saccharomyces and Kluyveromyces alpha factor leader sequences), acid phosphatase, C. albicans glucoamylase, or the signal 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. The DNA for such a precursor region is ligated in reading frame with DNA encoding an anti-IL-23p19 antibody.

[0249] Expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected cells. Generally, in cloning vectors this sequence is the origin of replication, or autonomously replicating sequence, enabling the vector to replicate independent of the host chromosome. Numerous bacterial, yeast, and viral sequences capable of implementing this function are known. The origin of replication of the plasmids pBR322 is suitable for most Gram-negative bacteria, the 2-μ plasmid origin is suitable for yeast, and various viral origins (SV40, papilloma virus, adenovirus, VSV and BPV) are useful for cloning in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may be used, however, solely for the ease of detection, as it confers resistance to the drug mycophenolic acid).

[0250] Expression and cloning vectors can contain a gene encoding a selectable marker to facilitate recognition of expression. Typical selection marker genes encode proteins that confer resistance to antibiotics or other toxins (e.g., ampicillin, neomycin, methotrexate or tetracycline), or alternatively, genes that complement auxotrophic deficiencies, or yet further, genes that provide a specific nutrient that is not present in complex media, such as the gene encoding D-alanine racemase for Bacilli.

[0251] Non-therapeutic uses

[0252] The anti-IL-23p19 antibodies or antibody fragments described herein can be used as affinity purification agents. In this process, the antibody is immobilized on a solid phase using methods well known in the art, such as protein A resin. The immobilized antibody is contacted with a sample containing the IL-23p19 protein (or fragment thereof) to be purified, and then the support is washed with a suitable solvent that will remove essentially all of the material in the sample except for the IL-23p19 protein bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the IL-23p19 protein from the antibody.

[0253] The anti-IL-23p19 antibodies or antibody fragments can also be used in diagnostic assays to detect and / or quantify IL-23p19 protein, e.g., to detect expression of IL-23p19 in a particular cell, tissue, or serum. Anti-IL-23p19 antibodies can be used diagnostically, e.g., to monitor the development or progression of a disease as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment and / or prophylaxis regimen. The anti-IL-23p19 antibodies can be conjugated to a detectable substance to facilitate detection. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See, e.g., U.S. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostic agents according to the present disclosure.

[0254] The anti-IL-23p19 antibodies or antibody fragments can be used in methods of diagnosing an IL-23p19-associated disease (e.g., a disease characterized by abnormal expression of IL-23p19), or for determining whether a subject has an increased risk of developing an IL-23p19-associated disease. 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. A "biological sample" refers to any biological sample obtained from an individual, cell line, tissue culture, or other source of cells that can express IL-23p19. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.

[0255] In some embodiments, the method can further comprise comparing the level of IL-23p19 in the patient sample to a control sample (e.g., a subject without an IL-23p19-associated condition) to determine whether the patient has, or is at risk of developing, an IL-23p19-associated condition.

[0256] In some embodiments, it can be advantageous to label the antibodies with a detectable moiety for diagnostic purposes, for example. A number of detectable labels are available, including radioisotopes, fluorescent labels, enzyme substrate labels, and the like. The labels can be indirectly coupled to the antibodies using a variety of known techniques. For example, the antibodies can be coupled to biotin, and any of the three general classes of labels described above can be coupled to avidin, or vice versa. Biotin selectively binds to avidin, and thus the labels can be bound to the antibodies in this indirect manner. Alternatively, to achieve indirect coupling of labels to antibodies, the antibodies can be coupled to a small hapten (such as digoxin), and one of the different types of labels described above can be coupled to an anti-hapten antibody (e.g., an anti-digoxin antibody). Thus, indirect coupling of labels to antibodies can be achieved.

[0257] Exemplary radioisotopes include 35 S, 14 C, 125 I, 3 H and 131 I. Antibodies can be labeled using radioisotopes, techniques for which are described in, for example, Current Protocols in Immunology, Vols. 1 and 2, 1991, Coligen et al. eds., Wiley-Interscience, New York, N.Y. Publication. For example, radioactivity can be measured by scintillation counting.

[0258] Exemplary fluorescent labels that can be used include labels derived from rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lissamine, phycoerythrin, and Texas Red. Fluorescent labels can be coupled to antibodies by known techniques, for example, those disclosed in Current Protocols in Immunology. Fluorescence can be quantitated using a fluorometer.

[0259] There are a variety of well-characterized enzyme-substrate labels known in the art (see, e.g., U.S. Patent No. 4,275,149). The enzyme typically catalyzes a chemical alteration of the substrate that can be measured either by spectrophotometric monitoring of the alteration, by a color change, or by the use of an enzyme substrate that is linked to a signal generator. For example, one enzyme / substrate combination used is malate dehydrogenase / oxaloacetate, which can be measured by monitoring the oxidation of NADPH at 340 nm. Other examples of spectrophotometrically measured enzyme / substrate combinations include glucose oxidase with glucose as the substrate and horseradish peroxidase with benzoine or ABTS as the substrate. Alterations of the substrate by the enzyme can also be monitored by a color change of the medium, using dye-linked enzymes such as lactate dehydrogenase with lactate as the substrate, or alcohol dehydrogenase with alcohol as the substrate. Yet other techniques for monitoring the presence of enzyme substrates include radioactivity measurements and fluorescence measurements.

[0260] Examples of enzyme labels include luciferases such as Gaussia luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidases (such as horseradish peroxidase (HRPO)), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, saccharide oxidizing enzymes (such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidizing enzymes such as uricase and xanthine oxidase, lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are described in, for example, 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, N.Y., 73: 147-166.

[0261] Examples of enzyme substrate combinations include, for example: horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor such as o-phenylenediamine (OPD) or 3,3,5,5-tetramethylbenzidine hydrochloride (TMB); alkaline phosphatase (AP) with p-nitrophenyl phosphate as a chromogenic substrate; and beta-D-galactosidase (beta-D-Gal) with a chromogenic substrate such as p-nitrophenyl-beta-D-galactoside or the fluorescent substrate 4-methylumbelliferyl-beta-D-galactoside.

[0262] In another embodiment, the anti-IL-23p19 antibody or antibody fragment thereof used is unlabeled and is detected with a labeled antibody that binds to the anti-IL-23p19 antibody or antibody fragment thereof.

[0263] The antibodies and antibody fragments thereof described herein can be used in any known assay method, such as competitive-binding, 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).

[0264] Anti-IL-23p19 antibodies or antibody fragments thereof can be used to inhibit the binding of a ligand to an IL-23 receptor. Such methods include administering an anti-IL-23p19 antibody to a cell (e.g., a mammalian cell) or a cellular environment, thereby inhibiting signaling mediated by the IL-23 receptor. These methods can be performed in vitro or in vivo. A "cellular environment" refers to the tissue, medium, or extracellular matrix surrounding a cell.

[0265] Compositions and methods of treatment

[0266] The present disclosure also provides compositions, including, for example, pharmaceutical compositions comprising an anti-IL-23p19 antibody or antibody fragment thereof. Such compositions have a variety of therapeutic uses, for treating, preventing, or ameliorating a disease or disorder (e.g., a disease or disorder involving biological activities mediated by the IL-23 / IL-23 receptor signaling axis), such as an immune-mediated inflammatory disorder or an autoimmune disease.

[0267] The anti-IL-23p19 antibodies or antibody fragments thereof disclosed herein can be used to treat a variety of diseases or disorders, such as an immune-mediated inflammatory disorder (IMID) or an autoimmune disease. Methods of treating IL-23-related disorders include administering to a subject in need thereof a therapeutically effective amount of an anti-IL-23p19 antibody or antibody fragment thereof. The IMID can be selected from psoriasis, psoriatic arthritis, inflammatory bowel disease (i.e., ulcerative colitis or Crohn's disease), ankylosing spondylitis, systemic lupus erythematosus, hidradenitis suppurativa, atopic dermatitis, and asthma.

[0268] The present disclosure also provides methods for treating or preventing an 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.

[0269] The disclosed antibodies can also be used in methods of treating cancer, either alone (e.g., as a monotherapy) or in combination with other immunotherapies and / or chemotherapies.

[0270] The antibodies can be administered alone, or in combination with other compositions that can be used to treat immune-mediated inflammatory disorders or autoimmune diseases. In some embodiments, a composition comprising an anti-IL-23p19 antibody (including, for example, a pharmaceutical composition) can further comprise a therapeutic agent coupled or uncoupled to a binding agent.

[0271] In some aspects, a composition (e.g., a pharmaceutical composition) is provided comprising one or more antibodies disclosed herein. The pharmaceutical composition can be formulated in accordance with conventional techniques, e.g., those techniques disclosed in Remington: The Science and Practice of Pharmacy, 19th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1995, with a pharmaceutically acceptable carrier or diluent and any other known adjuvants and excipients.

[0272] Generally, the composition for injection is a solution in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical also can include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or aqueous solution in a single vial. Where the pharmaceutical is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0273] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or topical (e.g., by injection or infusion) administration. Depending on the route of administration, the active compound (i.e., the antibody, bispecific and multispecific molecules) can be coated in a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound.

[0274] The composition can be administered by a variety of methods known in the art. As will be appreciated by those of ordinary skill in the art, the route and / or mode of administration will vary depending upon the desired results. The active compound can be prepared with a carrier that protects the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for

[0275] Dosage levels of the active ingredients in the pharmaceutical compositions can be varied over a wide range to obtain the desired therapeutic response for a particular subject, composition, and mode of administration. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular compositions employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds, and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0276] The pharmaceutical compositions described herein can be administered in an effective amount. An "effective amount" refers to the amount that alone, or together with other doses, achieves the desired response or desired effect. In the context of treating a particular disease or particular condition, the desired response preferably involves inhibition of the disease course. This includes slowing the progression of the disease, in particular interrupting or reversing the progression of the disease.

[0277] In some aspects, the compositions described herein are administered to a patient (e.g., in vivo) to treat or prevent a variety of disorders, such as those described herein. Preferred patients include human patients having a disorder that can be corrected or ameliorated by administration of an agent that modulates the biological activity of the IL-23 / IL-23 receptor signaling axis.

[0278] In some aspects, conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding the antibodies or derivatives thereof described herein in mammalian cells or target tissues. Such methods can be used to administer the nucleic acids encoding the antibodies to cells in vitro. In some embodiments, the nucleic acids encoding the antibodies or derivatives thereof are administered for in vivo or ex vivo gene therapy uses. In other embodiments, the gene delivery techniques are used to study the activity of the antibodies 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, which have episomal or integrated genomes upon delivery to cells. Such methods are well known in the art.

[0279] Non-viral delivery methods of nucleic acids encoding the engineered polypeptides of the present disclosure include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycations or lipid:nucleic acid conjugates, naked DNA, artificial virions, and reagent-enhanced DNA uptake. Lipofection methods and lipofection reagents are well known in the art (e.g., Transfectam TM and Lipofectin TM). Cationic and neutral lipids suitable for efficient receptor-mediated lipofection of polynucleotides include those of Feigner, WO 91 / 17424, WO 91 / 16024. Delivery can be to cells (ex vivo administration) or to a target tissue (in vivo administration). Preparation of lipid: nucleic acid complexes comprising targeted liposomes (e.g., immunolipoplexes) is well known to those skilled in the art.

[0280] Delivery of nucleic acids encoding the antibodies described herein using RNA or DNA virus-based systems takes advantage of a highly evolved process for targeting a virus to a specific cell in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to a patient (in vivo), or they can be used to treat cells in vitro and the modified cells administered to a patient (ex vivo). Conventional virus-based systems for delivering the polypeptides of the present disclosure can include retroviral, lentiviral, adenoviral, adeno-associated viral, and herpes simplex viral 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 with retroviral, lentiviral, and adeno-associated viral 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, by way of example only, methodologies which can 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 a

[0281] To the extent not already indicated, those of ordinary skill in the art will appreciate that any of the various embodiments described and exemplified herein can be further modified to incorporate features shown in any of the other embodiments disclosed herein.

[0282] The broad scope of the present disclosure can be best understood with reference to the following examples, which are not intended to limit the present disclosure to particular examples. The particular embodiments described herein are provided by way of example only, and the present disclosure is to be limited only in keeping with the terms of the appended claims and equivalents thereof.

[0283] Examples

[0284] General Methods

[0285] Methods for protein purification including immunoprecipitation, chromatography, and electrophoresis are described. Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York. Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and glycosylation of proteins are described. See, e.g., 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, N.Y., 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, N.J., 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, N.Y.; Harlow and Lane, supra.

[0286] Hybridoma supernatants were purified by HiTrap Protein G columns (GE, Cat# 17040401) according to the manufacturer’s method. Briefly, Protein G columns were equilibrated with 5 CV of DPBS (Gibco, Cat# 14190-136) and hybridoma supernatants were loaded by syringe / infusion pump (Legato 200, KDS) at ambient temperature with a residence time of 3 minutes. Columns were washed with 5 CV of DPBS and eluted using 4 CV of pH 2.8 elution buffer (Fisher Scientific, Cat# PI21004). Elutions were fractionated and fractions were neutralized with 1 M Tris-HCL, pH 8.5 (Fisher Scientific, Cat# 50-843-270) and measured by A280 (DropSense96, Trinean). Peak fractions were pooled and buffer exchanged into DPBS. Centrifugal filters (EMD Millipore, Cat# UFC803024) were equilibrated in DPBS at 4,000 x g for 2 minutes. Purified samples were loaded, DPBS was added and samples were spun at 4,000 x g for 5 to 10 minutes until the total DPBS volume reached > 6 DV. Final pools were analyzed by A280.

[0287] Standard methods of molecular biology are described. Maniatis et al. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Sambrook and Russell (2001) Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif. Standard methods are also found in Ausbel et al. (2001) Current Protocols in Molecular Biology, Vols. 1 to 4, John Wiley and Sons, Inc. New York, N.Y., which describes cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), carbohydrate conjugates and protein expression (third volume), and bioinformatics (Vol. 4).

[0288] The sequences of the heavy and light chain variable regions of hybridoma clones were determined as follows. The sequences were obtained from 1–2 × 10⁻⁶ units using the RNeasy Plus mini kit from Qiagen (Germantown, MD, USA). 6 Total RNA was extracted from hybridoma cells. 5' RACE reactions were performed to generate cDNA using the SMARTer RACE 5' / 3' kit from Takara (Mountainview, CA, USA). PCR was performed using gene-specific primers targeting the 3' mouse constant region of suitable immunoglobulins with Takara Universal Primer Mix, and high-fidelity DNA polymerase Q5 from NEB (Ipswitch, MA, USA) was used to amplify the variable regions of the heavy and light chains. The amplified heavy and light chain variable regions were run on 2% agarose gels, appropriate bands were excised, and the gels were 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) and transformed into Stellar competent E. coli cells from Takara, plated on LB agar plates with 50 μg / ml kanamycin. Direct colony Sanger sequencing was performed using GeneWiz (South Plainfield, NJ, USA). Nucleotide sequences obtained were analyzed using IMGT V-QUEST to identify productive rearrangements and analyze translated protein sequences. CDR determination was based on IMGT numbering.

[0289] Flow cytometry methods, including fluorescence activated cell sorting and detection systems, can be used. See, e.g., Owens et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, N.J.; Givan (2001) Flow Cytometry, 2ndEd.; Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, N.J. Fluorescent reagents suitable for modifying nucleic acids (including nucleic acid primers and probes), polypeptides, and antibodies are available for use as, e.g., diagnostic reagents. Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.

[0290] Positive controls (PC1 and PC2), IL-23p19 and IL-12 / IL-23p40 specific antibodies were prepared by CRO (Biointron). Control antibodies can be prepared by any suitable expression method. For example, by cloning the antibody heavy and light chain variable regions into 293F or ExpiCHO TM Expression System (ThermoFisher Scientific, Waltham, MA). These antibodies were used as controls to establish the binding and functional assays described in the examples and were tested alongside the newly generated specific anti-IL-23p19 antibodies disclosed. The term “PC1” refers to a reference antibody that was synthesized based on the VH and VL sequences reported in US 7,935,344 (VH SEQ ID NO: 106 and VL SEQ ID NO: 116 in the ‘344 patent) and is known to be specific for the human IL-23p19 subunit (Biointron, Lot: 20180926A04). The term “PC2” refers to a reference antibody that was synthesized based on the VH and VL sequences reported in US 6,902,734 (VH SEQ ID NO: 7 and VL SEQ ID NO: 8 in the ‘734 patent) and is known to be specific for the human IL-12 / IL-23p40 subunit (BIOINTRON Lot: 20180925A07).

[0291] Control antibodies can be prepared by standard methods. For example, mammalian systems (293F or ExpiCHO TM) according to the manufacturer's protocol. On day 1, cells were incubated at 37°C and 8% C02, then after transfection, cells were incubated at 32°C and 5% C02 in the media provided with the kit. ExpiCHO TM The culture media was clarified, thereby purifying the antibody. Then, the supernatant was filtered using a 0.45 pm filter, followed by a 0.22 pm filter. Subsequently, the supernatant was affinity purified using a Protein A / G resin (Life Technologies, Carlsbad, CA; Cat. No. 20424) according to the manufacturer's protocol. Prior to ELISA purification, the antibody titer in the culture media was roughly determined to ensure that the amount of media loaded was less than 80% of the resin binding capacity. After incubation, the resin was washed with PBS and eluted with elution buffer (Life Technologies, Cat. No. 21004). Immediately, the elution fractions were adjusted to physiological pH by the addition of Tris buffer (pH 8.0). Subsequently, the purified antibody was buffer exchanged and protein concentrated in PBS buffer using Amicon Ultra-15 centrifugal filter units (Life Technologies, Cat. No. UFC900324). Antibody concentration was determined using the BCA protein assay. SDS-PAGE and Coomassie staining were used to test antibody purity. Purified protein was aliquoted and stored at -80°C for long-term storage or at 4°C for immediate use.

[0292] The integrity of the antibody can be verified by SDS-PAGE followed by Coomassie staining under non-reducing and reducing conditions; under non-reducing conditions, one major band is observed around 150 kDa, while under reducing conditions, two bands are observed, 50 kDa and 25 kDa. Standard techniques for characterizing ligand / receptor interactions can be used. See, e.g., Coligan et al. (2001) Current Protocols in Immunology, Volume 4, John Wiley, Inc., New York. Standard methods for antibody functional characterization suitable for characterizing antibodies with a particular mechanism of action are also well known to those skilled in the art.

[0293] Software packages and databases are available for determining, e.g., antigenic fragments, leader sequences, protein folding, functional domains, CDR annotations, glycosylation sites, and sequence alignments.

[0294] Example 1: Generation of Anti-IL-23p19 Antibodies

[0295] By immunizing human Ig transgenic mice (see, e.g., WO 2013 / 063391, mice) with human IL-23p19.

[0296] Immunization: By immunizing human Ig transgenic mice (see, e.g., WO 2013 / 063391, mice) with human IL-23p19.

[0297] Selection of mice producing anti-IL-23p19 antibodies: To select Trianni mice that produce p19-specific antibodies, immune mouse sera are screened for binding to recombinant human IL-23 by ELISA. Briefly, ELISA plates coated with recombinant human IL-23 are incubated with dilutions of sera from immune mice for 1 hour at room temperature, the plates are washed, and then HRP-labeled anti-mouse IgG antibodies are used to detect specific antibody binding. Plates are read using an ELISA plate reader (Biotek).

[0298] Generation of hybridomas: To generate hybridomas that produce human antibodies of the present disclosure, spleen cells and draining lymph node cells harvested from immunized mice are fused with a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas are screened for production of p19-specific antibodies. For example, single cell suspensions of spleen and lymph node cells from immunized mice are fused by electrofusion with an equal number of Sp2 / 0 non-mouse IgG-secreting myeloma cells (ATCC, CRL 1581). Cells are plated in flat-bottom 96-well tissue culture plates and then incubated in selective media (HAT media) for about 2 weeks, then switched to hybridoma culture media. About 10-14 days after plating, supernatants from individual wells are screened by ELISA as described above. Antibody-secreting hybridomas are transferred to 24-well plates, screened again, and if they remain positive for anti-P19 activity, positive hybridomas are subcloned by limiting dilution or sorting using a single cell sorter. Stable subclones are then cultured in vitro to produce small amounts of antibody for purification and characterization.

[0299] Screening of hybridomasThe hybridoma supernatants were tested for specific binding of IL-23 by ELISA using the same assay as described above for monitoring immune response in immunized mice, using human IL-23, human IL-12 and human p19 / mouse p40.

[0300] Example 2: Binding of anti-IL-23p19 specific antibodies

[0301] 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 on anti-mouse or human Fc chips, which were immobilized on a CM5 chip using an amine coupling kit (GE Healthcare, Cat. No: BR-1000-50, Lot No: 2087295).

[0302] Control antibodies used in the BIAcore binding assay included: PC1 (known as p19 specific antibody, Biointron, Lot No: 20180926A04); PC2 (reference antibody known to be specific for the p40 subunit of human IL-12 and IL-23, Biointron, Lot No: 20180925A07); human IgG isotype control (Invitrogen, Cat. No: 02-7102, Lot No: TJ276309); mouse IgG2a isotype control (manufactured by Novarock Biotherapeutics) and human IgG4 isotype control (Dendritics, Cat. No: DDXCH04P-100; Lot: DDXCH04-028) as negative controls.

[0303] Next, serial dilutions of IL-23 recombinant protein, human p19 / mouse p40 heterodimer protein, human IL-12 protein and human p40 subunit protein in running buffer containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween20, pH 7.4 were injected over the immobilized antibodies at 50 μl / min for 1 min, followed by a 2 min dissociation phase. Each injection was followed by a regeneration step using a 60 sec pulse of 10 mM Glycine-HCl, pH 1.7 buffer. Experimental data were fitted using BIAevaluation software (GE Healthcare) to a Langmuir 1 :1 model to determine apparent binding.

[0304] The binding profile of purified antibodies is shown in Figure 2. Figure 2A The binding profile of Hu-2 18006B (purified from hybridoma) is shown. Figure 2BBinding profiles of Hu-5 18006B (purified from hybridoma) are shown. Figure 2C Binding profiles of Hu-6 18006B* (recombinant mlgG2a) are shown. Figure 2D Binding profiles of Hu-4 18006B* (recombinant, mlgG2a) are shown. Figure 2E Binding profiles of Hu-4 18006B** (recombinant, hlgG4) are shown. Table 3 summarizes anti-IL-23p19 antibodies and their binding specificity to human IL-23 and human p19 / mouse p40 heterodimeric protein as determined by BIAcore. Recombinant antibodies are indicated by asterisks in the table.

[0305] Table 3: Binding of IL-23p19 specific antibodies as determined by BIAcore

[0306]

[0307]

[0308] * = IL-23p19 recombinant antibodies; PC1 and PC2 are recombinant antibodies; the rest of the IL-23p19 Abs were purified from hybridomas

[0309] The results show that anti-IL-23p19 antibodies bind to human IL-23 and human p19 / mouse p40 heterodimer, but not to human IL-12 or human p40 subunit (Figure 2 and Table 3).

[0310] PC1 was positive for human IL-23, human p19 / mouse p40 and negative for human IL-12 and human p40 subunit (data not shown). PC2 was positive for human IL-23, human IL-12, human p40 subunit and negative for human p19 / mouse p40 heterodimer. Isotype control mlgG2a and hlgG did not bind hIL-23, hp19 / mp40, hIL-12 and hp40 subunit.

[0311] Results: Anti-IL-23p19 specific antibodies are characterized by their binding to human IL-23 and recombinant protein comprising a heterodimer consisting of human p19 and mouse p40 subunit. These antibodies do not bind to human IL-12 and human p40 subunit as determined by BIAcore.

[0312] The binding specificity of the disclosed anti-IL-23p19 antibodies was also assessed by ELISA. Briefly, biotinylated IL-23 was captured by streptavidin-coated ELISA plates. Human p19 / mouse p40, human IL-12 and human p40 subunit were coated directly onto the ELISA plates. Purified antibodies were then added to the plates, followed by detection by goat anti-mouse IgG-HRP (Jackson ImmunoResearch, Cat. No: 115-036-071, Lot No: 147271). After the addition of ABTS substrate (Moss Inc., Cat. No: ABTS-1000, Lot No: 03086202), the ELISA plates were read using an ELISA plate reader (Biotek). Controls are depicted in Figure 3: PC1 refers to a reference antibody (known to be a p19 specific antibody, Biointron, Lot No: 20180926A04); PC2 refers to a reference antibody (known to be a p40 specific antibody, Biointron, Lot No: 20180925A07); PC3 refers to a reference antibody (MT155, known as a p19 specific antibody from Mabtech, Cat. No: 3457-6-100, Lot No: 3457-6-1000), negative controls are human IgG4 (Dendritics, Cat. No: DDXCHO4P-100, Lot No: DDXCH04-028) and mouse IgG2a (produced by Novarock Biotherapeutics).

[0313] Figure 3 shows the binding activity of the disclosed p19 specific antibodies. Figure 3A and Figure 3B Hu-418006B** (hlgG4) and Hu-518006B (mIgG2b), Hu-618006B* (mIgG2a), Hu-418006B* (mIgG2a) and Hu-218006B (mIgGl) bind human IL-23 in a dose dependent manner; positive control PC1 binds IL-23 in a dose dependent manner Figure 3A ). Figure 3C These selected representative anti-IL-23p19 antibodies, H-2 1800B (mIgGl), Hu-418006B (mIgG2c), Hu-418006B* (mIgG2a) and H-6 18006B* (mIgG2a) bind human p19 / mouse p40 heterodimer in a dose dependent manner. Figure 3D These anti-IL-23p19 antibodies do not bind hIL-12, whereas positive control antibody PC2 binds hIL-12 in a dose dependent manner. Figure 3EThe results indicate that the anti-IL-23p19 specific antibodies do not bind to the human p40 subunit, while the positive control PC2 binds to the human p40 subunit in a dose responsive manner.

[0314] Figures 3A-3E The results indicate that the anti-IL-23p19 specific antibodies are characterized by binding to human IL-23 and a recombinant protein comprising a heterodimer consisting of a human pl9 combined with a mouse p40 subunit by ELISA. These antibodies do not bind to human IL-12 and human p40 subunit.

[0315] 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 antibodies to recombinant human IL-23 were determined by surface plasmon resonance (SPR) using a BIAcore 3000 system interfaced to a CM5 chip previously immobilized with anti-mouse IgG antibody (GE Cat. No. BR-1008-38) by amine coupling chemistry. Flow cell 1 was left unmodified and used as a reference cell to subtract system instrument noise and drift. The Fc2-1 assay used a double blank (Fc1 and blank analyte buffer) run. Antibody samples were diluted to 50 μg / mL in HBS-EP (GE, Cat. No: BR1001-88) and injected at a flow rate of 10 uL / min for 1 min. This was followed by hIL-23 (R&D Systems, Cat. No: 1290-IL / CF) diluted to 0.156-40 nM injected at 50 uL / min for 2 min, followed by a 10 min dissociation period. Data were analyzed in BIAEvalution software (GE Healthcare) by a 1 : 1 binding model with global fitting to determine apparent binding kinetics.

[0316] Binding kinetics data for the disclosed anti-IL-23p19 antibodies are provided in Table 4. The results indicate that the anti-IL-23p19 specific antibodies bind to human recombinant IL-23 with a KD ranging from 3.84E-11 to 6.62E-11 M. PC1 (known to be a specific antibody to pl9, Biointron, Lot: 20180926A04) KD values ranged from 3.77E-10 to 1.10E-11 in multiple runs.

[0317] Table 4: SPR binding kinetics

[0318] Anti-IL-23p19 mAb ka(1 / Ms) kd(1 / s) KD(M) Hu-2 18006B 1.57E+06 6.54E-05 4.16E-11 Hu-5 18006B 1.49E+06 9.89E-05 6.62E-11 Hu-6 18006B* 4.44E+06 1.71E-04 3.84E-11 Hu-4 18006B** 2.50E+06 1.43E-04 5.72E-11

[0319] Example 3: Blocking the interaction of IL-23 with IL-23 receptor

[0320] The ability of the disclosed p19-specific antibody to block the binding of IL-23 to its homologous 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 then a serially diluted buffer of purified anti-IL-23p19 antibody was premixed with recombinant human IL-23 (50 ng / mL) and added to the plate. After incubation for 30 minutes, the plate was washed. Then, biotinylated anti-p40 antibody (Invitrogen reference number: 13-7129-85, batch number: 2028761, 1 / 3000 dilution) was added to the plate. After incubation for 30 minutes, the plate was washed and then detected with streptavidin HRP. After adding ABTS substrate, the plate was read using a microplate reader (OD 405 nM). The positive control antibody PC1 used in this blocking assay was a reference antibody (known as a p19-specific antibody, Biointron, batch number: 20180926A04). Negative controls were mouse IgG1 (Novus, catalog number: NBP1-97005, batch number: 35613), mIgG2a (manufactured by NovaRock Biotherapeutics), and hIgG4 (Dendritics, catalog number: DDXCHO4P-100, batch number: DDXCH04-028).

[0321] Results : Figure 4 Data showed that the disclosed anti-p19 antibodies (Hu-2 18006B, Hu-6 18006B*, Hu-5 18006B, and Hu-4 18006B**) blocked the interaction between human IL-23 and its 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.

[0322] To demonstrate the specificity of the disclosed p19-specific antibody, a blocking assay was designed to assess the antibody's ability to block the binding of IL-23 to IL-12 receptor β1.

[0323] In summary, human IL-12 receptor β1 was coated onto 96-well plates (2 μg / ml), and then serially diluted buffer of purified anti-IL-23p19 antibody premixed with recombinant human IL-23 (50 ng / ml) was added to the plates. The positive control antibody used in the blocking assay was PC2 (a reference antibody known to be specific to the p40 subunit of IL-12 / IL-23p40, Biointron, batch number: 20180925A07). The negative control was mouse IgG1 (Novus, catalog number: NBP1-97005, batch number 35613).

[0324] After incubation for 30 minutes, wash the plate. Then, add biotinylated anti-p40 antibody (Invitrogen reference number: 13-7129-85, batch number: 2028761, 1 / 3000 dilution) to the wells containing anti-IL-23p19 antibody; add biotinylated anti-p19 antibody (Mabtech, catalog number: MT155, serial number: 3457-6-1000) to the wells containing PC2 antibody. After incubation for 30 minutes, wash the plate and then detect with streptavidin HRP. Read the plate using a microplate reader (OD 405 nM) after adding ABTS substrate.

[0325] Results : Figure 5 Data showed that none of the three selected p19-specific antibodies (Hu-6 18006B*, Hu-4 18006B*, and Hu-4 18006B**) blocked the IL-23 / IL-12 receptor β1 binding interaction. Similarly, the PC1 antibody also failed to block the IL-23 / IL-12 receptor β1 interaction (data not shown). However, consistent with expectations, the p40 control antibody (PC2) blocked the IL-23 / IL-12β1 interaction.

[0326] Example 4: Inhibition of IL-17 production in mouse spleen cell assays

[0327] It is well known that human IL-23 binds to mouse IL-23R and induces the production of mouse IL-17 in mouse spleen cells. In the presence of IL-2, human IL-23 stimulates the production of IL-17 in mouse spleen cells at very low (picomolar) concentrations, which can be inhibited by co-incubation with inhibitors targeting p40 or p19 (Aggarwal, S. et al., 2003, J Biol Chem; 278: 1910-4; Singh et al., 2015, MAbs, July-August; 7(4): 778-791).

[0328] The ability of the published anti-IL-23p19 specific antibody to inhibit human IL-23-induced IL-17 production was evaluated in a mouse spleen cell assay (MSA). The efficacy of inhibiting human IL-23-induced IL-17 production was determined.

[0329] In summary, mouse spleen cells were isolated from C57 / BL-6 mice using a glass homogenizer and the Ficoll Pague Cell Isolation Kit (Ge Healthcare, catalog number: 17-5442-02) according to the manufacturer's instructions. The spleen cells were treated with IL-2 (20 ng / ml, 5 × 10⁻⁶). 6Splenocytes (5 x 106cells / ml) were activated for 5 minutes, then human IL-23 (1.5 ng / ml) was added to the splenocytes. Activated splenocytes were placed into 96 well plates, 100 ul / well. 100 ul / well of 4 purified pl9 antibodies: hu-6 18006B* (mIgG2a), Hu-4 18006B* (mIgG2a), Hu-4 18006** (hIgG4), and Hu-2 18006B (mIgG1) were added to the plates.

[0330] After 72 hours of incubation, supernatants were transferred from the plates and IL-17 quantification was performed using quantikine ELISA kits (R&D, M1700 or SM1700). Controls were included in the IL-17 MSA: PC1 (known as reference antibody for pl9 specific antibodies, Biointron lot: 20180926A04) as positive control; mouse IgGl (Novus, Cat# NBP1-97005) and human IgG4 (Dendritics, Cat# DDXCHO4P-100, lot: DDXCH04-028) as negative controls.

[0331] Results Figure 6A and Figure 6B The data presented in Example 4 support the conclusion that the disclosed antibodies selectively neutralize the binding of IL-23 to IL-23R, and therefore inhibit the production of IL-17 in a dose-dependent manner.

[0332] Example 5: Inhibition of STAT3 activation by reporter cell assay

[0333] The receptor for IL-23 is known to comprise the IL-12Rpl subunit shared with the IL-12 receptor, and the cognate IL-23R. IL-23pl9 selectively binds to IL-23R, signals through IL-23R, induces Janus kinase 2 (JAK2), which activates STAT3, leading to upregulation of RORyt and subsequent increased production of the inflammatory cytokine IL-17 (Parham et al., J. Immunol. 168: 5699-5708, 2002). To determine whether the disclosed anti-pl9 antibodies can inhibit STAT3 activation, antibodies were evaluated in IL-23-induced STAT3 activation by a reporter cell assay. Human IL-23 induces STAT3 phosphorylation by binding to IL-23R on the surface of human lymphoma DB cells (US 2013 / 0172272 Example 13, and Desmet, J. et al., Nat. Commun. 5:5237 (2014)).

[0334] ​DB cells are derived from a human B-cell lymphoma cell line, DB cells express endogenous IL-23 receptor and STAT3 providing a fully functional IL-23 signaling pathway. DB assay cells are generated by stable transfection of DB cells with pGL4.47[luc2p / SIE / Hybro] which allows quantitative detection of biologically active human IL23 using a luciferase reporter system.

[0335] The DB assay is used to measure the inhibitory activity of the disclosed anti-IL-23 antibodies on human IL-23 induced STAT3 activation. Briefly, DB cells (ATCC, CRL-2289) are cultured for 2 days in growth media (RPMI + 10% FBS). On the day of the experiment, cells are harvested and resuspended in growth media. Serial dilutions of test antibodies are prepared in growth media in low-binding 384-well plates (Thermo Scientific 264574), then human IL-23 is added and incubated for 30 minutes at room temperature. Then, the mixture of test antibodies and human IL-23 is added to the plates. The signaling assay plates are incubated for 16 hours in a humidified 37°C / 5% CO2 incubator. OneGlo reagent is added and the mixture is incubated for 2 minutes at room temperature. Luminescence is read on a BioTek Neo2 (BioTek. Winooski. VT) and IC50 values are determined using GraphPad Prism® software (GraphPad Software Inc., San Diego, California, USA) where the ratio is plotted against the log-transformed antibody concentration, IC50 values are determined using a non-linear regression (curve fit) S-shaped dose response.

[0336] 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 maximum inhibition of 99-100%. The positive control (PC1) had IC50 values ranging from 24.30 pM to 117.20 pM with inhibition of 98-99% across multiple experiments.

[0337] Table 5: IL-23 / IL-23 receptor-mediated STAT3 activation Inhibition

[0338]

[0339] ​

[0340] Figure 7 of Results The study indicated that the four selected anti-IL-23p19 antibodies (hu-4 18006B, hu-4-18006B*, hu-6 18006B, and hu-6 18006B*) inhibited STAT3 activation in a dose-dependent manner. As expected, the positive control (PC1) also showed dose-responsive inhibition of STAT3 activation.

[0341] Example 6: Inhibition of IFN-γ production in IL-12-dependent human PBMCs

[0342] It is known that IL-12 stimulation of PBMCs stimulates NK cells and T cells to produce IFN-γ. To determine whether publicly available anti-p19 antibodies can inhibit IFN-γ production, representative publicly available human anti-p19 antibodies were analyzed in a PMBC IL-12 stimulation assay.

[0343] In summary, human PBMCs were thawed from their cryogenic stock solution and resuspended in RPMI + 10% FBS containing 50 ng / ml IL-18 (R&D, 9124-IL / CF) and placed in 384-well plates. Serial dilutions of the test antibody were prepared in growth medium in the low-binding 384-well plates. Human IL-12 (25 ng / ml) or cynomolgus monkey IL-12 (25 ng / ml) was transferred to each well and incubated at room temperature for 30 minutes. The mixture (test antibody + IL-12) was then placed into the PBMC cell plate. Cells were cultured in a humidified 37°C / 5% CO2 incubator for 48 hours. IFN-γ production was measured using an AlphaLISA (PerkinElmer, AL217C) according to the manufacturer's operating protocol.

[0344] The control antibodies used in the human PBMC assay were: PC1 (a reference antibody known to be specific to p19, Biointron, batch number: 20180926A04), PC2 (a reference antibody known to be specific to the p40 subunit of IL-12 and IL-23, Biointron, batch number: 20180925A07), anti-IL-23 p40 subunit Mab (Hu-1918006*, internally generated); mIgG2a (manufactured by NovaRock Biotherapeutics) and human IgG4 (Dendritics, catalog number: DDXCHO4P-100, batch number: DDXCH04-028) as negative control antibodies.

[0345] Results As expected, such asFigure 8 and Figure 9 As shown in Figure 6, anti-IL-23p19 antibodies (Hu-6 18006B*and Hu-418006B*and PC1) did not inhibit human IL-12( Figure 8 ) and cynomolgus IL-12( Figure 9 ) mediated IFN-g production by human PBMC, while the positive controls PC2 and Hu-19 18006*inhibited human( Figure 8 ) and cynomolgus( Figure 9 ) PBMC in a dose dependent manner. This data supports the conclusion that the disclosed p19 antibodies are specific for p19.

[0346] Example 7: In vivo efficacy of specific antibodies against p19 in IL-23 induced murine skin inflammation model

[0347] The role of the IL-23 / IL-17 pathway as a key driver of human psoriasis (PsO) is 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 assess the pharmacology of novel small molecules / biologies in the treatment of PsO (Stephen B. Gauld et al., J. Dermatological Science, 92 (2018) 45-53).

[0348] Human IL-23 is known to bind to the murine IL-23 receptor and induce mIL-17 production and inflammation in mice. Intradermal injection of human IL-23 into mouse ears to induce mouse ear inflammation has been used in psoriasis models for the characterization of biopharmaceuticals for human psoriasis (PsO) (Aggarwal et al., J Biol Chem 2003; 278: 1910-4; Singh et al., MAbs 2015 July-August; 7(4): 77-791).

[0349] To assess the ability of Hu-4 18006B (mIgG2c), Hu-4 18006B** (hIgG4), hu-5 18006B (mIgG2b) and Hu-6 18006B* (mIgG2a) p19-specific antibodies to block IL-23 function in vivo, the antibodies were tested in a human IL-23 induced murine skin inflammation model. The ability of these representative antibodies to reduce the inflammatory response was evaluated.

[0350] In this model, recombinant human IL-23 (3 μg / 10 μl / mouse / day) was injected into the skin of the right ear of mice for 8 consecutive days (D0-D7) to induce a psoriasis-like inflammatory skin response, characterized by erythema and induration with histological evidence of epidermal hyperplasia, parakeratosis and local inflammatory infiltration.

[0351] According to two protocols, mice were treated with two intraperitoneal (ip) injections of IL-23 p19 antibody (hu-418006B, hu-4 18006B**, hu-5 18006B, or hu-6 18006B*) or PC1 (a known reference antibody for p19 specificity) (Biointron, batch number: 20180926A04). In the first protocol, mice received either a PBS control (loador) or the antibody. The first injection was given one day before the IL-23 injection, and the second injection was given three days after the IL-23 injection. In the second protocol, mice received either a PBS control or the antibody. The first injection was given one hour before the IL-23 injection, and the second injection was given three days after the IL-23 injection. Figure 10 ).

[0352] Mice were measured daily for body weight, ear thickness, and ear inflammation scores. Ear inflammation scores were calculated on days 0, 2, 4, 6, and 8, based on the following criteria: auricle shape (relatively normal -0; minimal change -1; moderate to significant change -2; severe swelling and deformity -3); skin color (relatively normal -0; minimal hyperplasia -1; slight hyperplasia -2; severe hyperplasia -3); and white scaling (relatively normal -0; minimal -1; slight -2; significant -3). Right ear thickness was measured and photographed for each mouse on days 0, 2, 4, 6, and 8.

[0353] On the last day of the experiment (day 8), the animals were treated with carbon dioxide, blood samples were collected, and serum was separated (stored in a -80°C freezer). Modeled ears were collected 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.

[0354] Data are presented as mean ± SEM. A p-value less than 0.05 is considered statistically significant.

[0355] The data provided in Tables 6 and 7 summarize the total scores of the injected ears (with scores added for auricle shape, skin color, microvascular changes, and white scaling). The results indicate that mice treated with a representative p19-specific antibody experienced a reduced inflammatory response compared to the IL-23 injection model group. The effect began on day 4 and persisted until day 8. The effect was statistically significant. This conclusion is evident from both the total score and ear thickness values.

[0356] Table 6: Total score of injected ears (x ± s, n = 10)

[0357]

[0358]

[0359] *p < 0.05, **p < 0.01, ***p < 0.001 compared to model.

[0360] Table 7: Total score of IL-23 injected ears (x ± s, n = 8)

[0361]

[0362] **p < 0.01, ***p < 0.001 compared to model

[0363] As shown in Tables 8 and 9, treatment with selected anti-IL-23p19 antibodies reduced the thickness of mouse ears compared to model (IL-23 treated). The effect of treatment (inhibition of cutaneous inflammatory immune response in vivo) started at day 4 and lasted until day 8, the effect being statistically significant (p < 0.001 compared to model (IL-23 treated)).

[0364] Table 8: Ear thickness of injected ears (x ± s, n = 10) from day 0 to day 8

[0365]

[0366] *p < 0.05, **p < 0.01, ***p < 0.001 compared to model.

[0367] Table 9: Ear thickness of IL-23 injected ears (x ± s, n = 8)

[0368]

[0369] **p < 0.01, ***p < 0.001 compared to model

[0370] Figure 10 The data provided in Table 10 show that the disclosed anti-p19 specific antibodies hu-4 18006B** and hu-6 18006B* caused a statistically significant reduction of ear thickness compared to untreated control (model receiving human IL-23 treatment only).

[0371] Figure 11A , Figure 11B , Figure 11C and Figure 11DThe data provided determined the effect of anti-p19 specific antibodies on inflammatory skin reactions, as determined by H&E histopathology scoring of the epidermis thickness ( Figure 11A ), dermis thickness ( Figure 11B ), inflammatory cell infiltration ( Figure 11C ) and hyper- or hypo-keratinization ( Figure 11D ), as obtained during the course of the experiment, and represented by the scoring system described below.

[0372] Briefly, at day 8, mouse ears were collected and observed under a microscope. Tissues were fixed in 10% neutral buffered formalin. After fixation, tissues were trimmed, dehydrated, embedded, sectioned and stained with hematoxylin / eosin (H&E) according to the relevant SOP. Histopathological evaluation was performed by a study pathologist by light microscopy. Microscopic findings were classified using a five-step grading system (relatively normal, minimal, mild, moderate to marked, severe).

[0373] Results : Treatment with anti-p19 antibody hu4 18006B resulted in a significant reduction of the swelling reaction and inflammation score induced by IL-23 injection compared to the model. Hu-4 18006B showed a superior inhibitory effect than PC1 on 3 scores (epidermis thickness ( Figure 11A ), dermis thickness ( Figure 11B ) and inflammatory cell infiltration ( Figure 11C )). Hu-4 also showed an inhibition of hyperkeratinization ( Figure 11D ).

[0374] Figure 12 provides representative pictures of H&E stained ear sections obtained at day 8 post-treatment. H&E staining method is described above.

[0375] Results : Selected published anti-IL-23p19 antibody treatment (Hu-4 18006B) significantly inhibited mouse skin inflammation compared to the model.

[0376] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that can 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.

[0377] Notwithstanding that the numerical ranges and parameters setting forth the broadest scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0378] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the disclosure (especially in the context of following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The description of a range of values herein is merely a shorthand method of referring individually to each numerical value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited 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 and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0379] Groupings of alternative elements or embodiments of the disclosure disclosed herein are not to be construed as limitations. Each group member can be used independently and the grouping can be used as a whole to provide a variety of alternative embodiments. One or more members of a group can be utilized in a combination of groups to provide variations of that combination. When combinations are utilized, the various members of the combinations can be modified and / or combined in a variety of ways to provide a variety of alternative embodiments. When any such modifications or combinations are made to a group, each member of the group can be modified even if only a subset of members of the group are so modified. Each member of a group can be used in a combination of groups even if the combination is not specifically described herein. To the extent that any combinations are recited herein, they are intended to be included in the description of each embodiment in which they are used. The description of groups herein is intended to be a mere description of some embodiments of the disclosure and is not intended to be limiting of the disclosure.

[0380] Certain embodiments of the disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art once the nature of the disclosure has been disclosed herein. Alterations and further modifications of the described embodiments are also included as further embodiments of the disclosure, and each alteration and further modification shall fall within the scope of the disclosure. Accordingly, though specifically described herein, the disclosure of the disclosure is understood and intended to include all possible alterations and further modifications to the embodiments as described herein. Moreover, although the disclosure is described in the context of certain embodiments, it is not intended to be limited to the embodiments set forth herein, but is to be accorded the full scope consistent with the claims, and equivalents thereof.

[0381] The specific embodiments disclosed herein can be further limited by the following claims using the term "comprising". When using the term "comprising" in the claims, the transition term "consisting of" is not intended to be used in a way that would limit the scope of the claims to the specified materials or steps only. The use of the term "consisting of" in the claims will be used in a way that would limit the scope of the claims to the specified materials or steps and those that are implicit to these materials or steps. Such embodiments of the disclosure as set forth in this specification are inherently or explicitly described and enabled herein.

[0382] It is to be understood that the embodiments of the present disclosure disclosed herein are illustrative of the principles of the present disclosure. Other modifications that can be employed are within the scope of the present disclosure. As such, the present disclosure is not to be limited to the precise examples and conditions described herein but rather can utilize alternative configurations in accordance with the teachings herein. Accordingly, the present disclosure is not limited to that precisely as shown and described.

[0383] While the present disclosure has been described and illustrated herein by reference to various specific materials, methods and exemplary embodiments, it will be appreciated that the present disclosure is not limited to the particular combinations of materials and methods set forth herein but is intended to cover any and all equivalents within the scope of the present disclosure. Numerous variations and modifications can be made to the embodiments described herein without departing from the true spirit and scope of the present disclosure. The specification and examples given herein are to be considered exemplary only, with the true scope and spirit of the disclosure indicated by the appended claims. SEQUENCE LISTING <110> Newsham Biopharma LLC <120> Antibodies against interleukin 23 pl9 and methods of use thereof <130> 122863-5002 <160> 32 <170> PatentIn version 3.5 <210> 1 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Hu-2_VH <400> 1 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Lys Tyr Ala Gin Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Trp Arg Ala Phe Tyr Tyr Tyr Gly Leu Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 2 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Hu-2_VL <400> 2 Asp Ile Val Met Thr Gin Thr Pro Leu Ser Ser Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gin Ser Leu Glu His Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Ser Trp Leu Gln Gln Arg Pro Asn Gln Pro 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Thr Gln Ala 85 90 95 Thr Gln Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 3 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Hu-4_VH <400> 3 Gln Val His Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr 20 25 30 Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Ser Gly Asn Thr Asp Tyr Ser Gln His Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ser Ala Asn Trp Tyr Asp Tyr Phe Asp Pro Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 4 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Hu- -4_VL <400> 4 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Gly Gly Ser 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Thr Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile His Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys His Gin Ser Ser lie Leu Pro Tyr 85 90 95 Thr Phe Gly Gin Gly Thr Lys Leu Glu lie Lys 100 105 <210> 5 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Hu-5_VH <400> 5 Gln Leu Gin Leu Gin Gin Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser lie Ser Ser Ser 20 25 30 Ser Phe Tyr Trp Gly Trp lie Arg Gin Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp lie Gly Ser lie Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr lie Ser Val Asp Thr Ser Lys Asn Gin Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe 85 90 95 Cys Ala Arg His Gly Val Arg Gly Val lie Pro His Phe Asp Tyr Trp 100 105 110 Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 6 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Hu-5_VL <400> 6 Gln Thr Val Leu Thr Gin Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Leu Asn Ser Gly Ser Val Ser Thr lie 20 25 30 Tyr Tyr Pro Ser Trp Tyr Gin Gin Thr Pro Gly Gin Ala Pro Arg Ala 35 40 45 Leu lie Tyr Ser Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser lie Leu Gly Asn Lys Ala Ala Leu Thr lie Thr Gly Ala 65 70 75 80 Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Val Leu Phe Leu Gly Ser 85 90 95 Gly Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 7 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Hu-6_VH <400> 7 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Tyr Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Leu Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Gly Gln Lys Val 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Thr Glu Asn Ile Asn Trp Arg Asp Ala Phe Asp Ile Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 8 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Hu-6_VL <400> 8 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Gly Ser Ser 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Ala Tyr Tyr Cys His Gln Ser Ser Ser Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 9 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Hu-2_VH-CDR1 <400> 9 Gly Tyr Thr Phe Thr Thr Tyr Gly 1 5 <210> 10 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Hu-2_VH-CDR2 <400> 10 Ile Ser Ala Tyr Asn Gly Asn Thr 1 5 <210> 11 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Hu-2_VH-CDR3 <400> 11 Ala Arg Glu Trp Arg Ala Phe Tyr Tyr Tyr Gly Leu Asp Val 1 5 10 <210> 12 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Hu-2_VL-CDR1 <400> 12 Gln Ser Leu Glu His Ser Asp Gly Asn Thr Tyr 1 5 10 <210> 13 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Hu-2_VL-CDR2 <400> 13 Lys Val Ser 1 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Hu-2_VL-CDR3 <400> 14 Thr Gin Ala Thr Gin Phe Pro Leu Thr 1 5 <210> 15 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Hu-4_VH-CDR1 <400> 15 Gly Tyr Thr Phe Ser Ser Tyr Gly 1 5 <210> 16 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Hu-4_VH-CDR2 <400> 16 Ile Ser Ala Tyr Ser Gly Asn Thr 1 5 <210> 17 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Hu-4_VH-CDR3 <400> 17 Ala Arg Ala Ser Ala Asn Trp Tyr Asp Tyr Phe Asp Pro 1 5 10 <210> 18 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Hu- -4_VL-CDR1 <400> 18 Gln Thr Ile Gly Gly Ser 1 5 <210> 19 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Hu-4_VH-CDR1 <400> 19 Tyr Ala Ser 1 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Hu-4_VL-CDR1 <400> 20 His Gin Ser Ser lie Leu Pro Tyr Thr 1 5 <210> 21 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Hu-5_VH-CDR1 <400> 21 Gly Gly Ser lie Ser Ser Ser Ser Phe Tyr 1 5 10 <210> 22 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Hu-5_VH-CDR2 <400> 22 Ile Tyr Tyr Ser Gly Ser Thr 1 5 <210> 23 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Hu-5_VH-CDR3 <400> 23 Ala Arg His Gly Val Arg Gly Val Ile Pro His Phe Asp Tyr 1 5 10 <210> 24 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Hu-5_VL-CDR1 <400> 24 Ser Gly Ser Val Ser Thr Ile Tyr Tyr 1 5 <210> 25 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Hu-5_VL-CDR2 <400> 25 Ser Thr Asn 1 <210> 26 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Hu-5_VL-CDR3 <400> 26 Val Leu Phe Leu Gly Ser Gly Leu Val 1 5 <210> 27 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Hu-6_VH-CDR1 <400> 27 Gly Tyr Thr Phe Thr Tyr Tyr Gly 1 5 <210> 28 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Hu-6_VH-CDR2 <400> 28 Ile Ser Ala Tyr Asn Gly Asn Thr 1 5 <210> 29 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Hu-6_VH-CDR3 <400> 29 Val Thr Glu Asn Ile Asn Trp Arg Asp Ala Phe Asp Ile 1 5 10 <210> 30 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Hu-6_VL-CDR1 <400> 30 Gln Thr Ile Gly Ser Ser 1 5 <210> 31 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Hu-6_VL-CDR2 <400> 31 Tyr Ala Ser 1 <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Hu-6_VL-CDR3 <400> 32 His Gln Ser Ser Ser Leu Pro Tyr Thr 1 5

Claims

1. An anti-IL-23p19 antibody, comprising: The heavy chain variable region containing CDR1: SEQ ID NO: 15, CDR2: SEQ ID NO: 16 and CDR3: SEQ ID NO: 17; and the light chain variable region containing CDR1: SEQ ID NO: 18, CDR2: SEQ ID NO: 19 and CDR3: SEQ ID NO:

20.

2. The anti-IL-23p19 antibody according to claim 1, wherein the antibody comprises: The heavy chain variable region sequence containing SEQ ID NO: 3 and the light chain variable region sequence containing SEQ ID NO:

4.

3. The anti-IL-23p19 antibody according to claim 1, wherein the antibody is a full-length antibody.

4. The anti-IL-23p19 antibody according to claim 1, wherein the antibody is an antibody fragment.

5. The anti-IL-23p19 antibody according to claim 4, wherein the antibody fragment is selected from: Fab, Fab', F(ab)2, Fv, scFv-Fc fragment, single-chain antibody, microantibody, and biantibody.

6. The anti-IL-23p19 antibody according to claim 1, wherein the antibody is a monoclonal antibody.

7. The anti-IL-23p19 antibody according to claim 1, wherein the antibody is a human antibody.

8. The anti-IL-23p19 antibody according to claim 1, wherein the antibody is a chimeric antibody.

9. The anti-IL-23p19 antibody according to claim 1, wherein the antibody is an anti-human IL-23p19 antibody.

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

11. A bispecific antibody comprising the anti-IL-23p19 antibody of claim 1.

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

13. Use of the anti-IL-23p19 antibody according to any one of claims 1-10, the bispecific antibody according to claim 11, or the pharmaceutical composition according to claim 12 in the preparation of a medicament for treating psoriasis.

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