Anti-interleukin-23 P19 antibody and method of use thereof

Abstract

The present disclosure provides an antibody that binds to IL-23p19 and an antibody fragment thereof. The disclosed antibody and its antibody fragment can modulate the biological activity of the IL-23 receptor signaling axis and are therefore useful for the treatment of immune-mediated inflammatory disorders.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This international patent application claims priority to U.S. provisional application No. 62 / 951,231 filed on December 20, 2019, the entire text of which is incorporated herein by reference.

[0003] Sequence list

[0004] The present application includes a list of sequences submitted electronically in ASCII format, the full text of which is incorporated herein by reference. The name of the ASCII copy created on December 16, 2019 is "122863-5002-WO_NVRB-004-001_ST25.TXT" and its size is 13 kilobytes.

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

[0006] The interleukin-12 (IL-12) family of regulatory cytokines includes a distinct group of cytokines (IL-12, IL-23, IL-27, IL-35, and IL-39) containing covalently bonded heteromeric subunits. The heteromeric IL-12 family cytokine members consist of an α-chain (p19, p28, or p35) and a β-chain (p40 or Ebi3).

[0007] IL-23 is a heterodimeric cytokine comprising a unique p19 subunit linked to a p40 subunit shared with IL-12. The primary sources of IL-23 are tissue-resident or recruited dendritic cells and macrophages. The biological action of IL-23 is hypothesized to occur via a receptor complex composed of the following two parts: i.) IL-12Rβ-1, a part common to 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 inducing innate and adaptive immune responses. These regulatory cytokines function by altering the function and fate of many immune cell populations, inducing the development of T-cell subpopulations and triggering adaptive immune responses toward infections, inflammation, and the outcomes of autoimmune diseases. IL-12 and IL-23 are primarily pro-inflammatory / anti-stimulating cytokines involved in the development of Th1 and Th17 cells, respectively.

[0009] The functional IL-23 receptor is a heterodimer of the IL-12Rβ1 subunit that is shared with the IL-12 receptor and partners with the signaling chain IL-23R (p19 subunit binding). The receptor for IL-23 is constantially associated with Janus kinase 2 (Jak2) and primarily activates STAT3. Expression of the IL-23 receptor is detected mainly in memory T-cells and NK cells. Monocytes, macrophages, and dendritic cells also express the IL-23 receptor at low levels.

[0010] There is substantial evidence that IL-23-responsive cells are associated with autoimmune inflammatory diseases and cancer, and that the regulation of IL-23 activity may provide promising therapeutic approaches. In particular, abnormal regulation of IL-23 is associated with immune-mediated inflammatory diseases (IMIDs) such as psoriasis, psoriatic arthritis, Crohn's disease, and ulcerative colitis. Furthermore, the balance of pro-inflammatory cytokines, including IL-23 and IL-12, plays a crucial role in shaping the development of anti-tumor or tumor immunity.

[0011] The IL-23 / IL-12 pathway is involved in cellular mechanisms related to the pathophysiology of multisystem inflammatory diseases. Numerous therapeutic strategies have been designed to inhibit IL-23 activity, and there is a continued need for therapies targeting the pro-inflammatory IL-23 / IL-23 receptor signaling axis to treat immune-mediated inflammatory disorders. More specifically, there remains a need for selective IL-23p19 antagonist antibodies that bind with high affinity to the p19 subunit of IL-23, particularly human IL-23, but do not bind to the p40 subunit of IL-12, a member of the related cytokine family.

[0012] The present disclosure addresses the above need by providing an antibody and an antibody fragment that bind to the cytokine p19 subunit of IL-23. The antibody and the antibody fragment are useful for the treatment of immune-mediated inflammatory diseases (IMIDs) (e.g., autoimmune diseases and inflammatory disorders) alone (e.g., monotherapy) or in combination with other immunotherapies.

[0013] In some embodiments, the anti-IL-23p19 antibody or a fragment of the antibody binds to the cytokine p19 subunit of human IL-23. In further embodiments, the antibody is entirely human.

[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 its antibody fragment 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 another embodiment, the anti-IL-23p19 antibody or its antibody fragment comprises a variable light chain sequence selected from the group consisting of SEQ ID NOs 2, 4, 6, and 8.

[0020] In another embodiment, the anti-IL-23p19 antibody or the 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 including SEQ ID NO. 3 and a variable light chain sequence including 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 including SEQ ID NO. 7 and a variable light chain sequence including SEQ ID NO. 8.

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

[0027] In some embodiments, the anti-IL-23p19 antibody or antibody fragment thereof exhibits one or more of the following features: (a) specific to human IL-23p19 and capable of blocking IL-23 binding to its receptor (IL-23R); (b) inhibiting, interfering with, or modulating IL-23p19 interactions with IL-23 receptor signaling; (c) inhibiting IL-23-induced STAT3 activation in DB cells; (d) inhibiting human IL-23-induced IL-17 production in mouse splenocytes; (e) inhibiting human IL-23-induced IL-17 production in activated human PBMCs; (f) not inhibiting IL-23 interactions with IL-12Rβ1 signaling; and (g) not inhibiting human IL-12-induced interferon gamma production in human activated T-cells (PBMCs). (h) without inhibiting cynomolgus monkey IL-12-induced interferon gamma production in human activated T-cells (PBMCs); (i) inhibiting skin inflammation induced by human IL-23 in a murine psoriasis-like model.

[0028] In one aspect, the initiated antibody and the isolated antigen-binding agent may be used to inhibit the IL-23p19-induced IL-23 receptor signaling network (e.g., an inflammatory microenvironment that promotes autoimmune diseases).

[0029] The anti-IL-23p19 antibody or its antibody fragment may exhibit one or more of the following characteristics.

[0030] (a) specific to human IL-23p19 and capable of blocking IL-23 binding to the IL-23 receptor (e.g., blockers);

[0031] (b) inhibiting, interfering with, or modulating IL-23 / IL-23 receptor-mediated signaling;

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

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

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

[0035] (f) without inhibiting IL-12Rβ1 signaling and IL-23 interaction;

[0036] (g) without blocking human IL-12-induced interferon-γ production in human PBMCs;

[0037] (h) Cynomolgus monkey IL-12-induced interferon gamma production in human PBMCs without inhibiting;

[0038] (i) Inhibits IL-23-induced skin inflammation in a murine psoriasis-like model.

[0039] In some embodiments, the anti-IL-23p19 antibody or antibody fragment thereof comprises a combination of a variable heavy chain sequence selected from the group consisting of SEQ ID NOs 1, 3, 5, and 7, and a CDR sequence from 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 its antibody fragments 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 derived from the VH or VL domain of a single anti-IL-23p19 antibody disclosed herein. For example, the binder may comprise all six CDR regions of the anti-IL-23p19 antibody named Hu-2.18006B (human antibody). In a representative example, the antibody or antibody fragment thereof may comprise the amino acid sequences of SEQ ID NOs 9 to 11 and SEQ ID NOs 12 to 14 representing CDR1, CDR2, and CDR3 of the variable light chain region and CDR1, CDR2, and CDR3 of the variable heavy 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 Fab, Fab', F(ab')2, Fd, Fv, scFv and scFv-Fc fragments, single-chain antibodies, minibodies, and diabodies.

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

[0048] The foregoing summary, as well as the following detailed description of the disclosure, will be better understood when read together with the accompanying drawings. For the purpose of illustrating the disclosure, the drawings are currently preferred embodiments. However, it should be understood that the disclosure is not limited to the exact arrangement, examples, and means illustrated. FIGS. 1a through 1d provide the amino acid sequences of the VH and VL domains of the anti-IL-23p19 antibody and their respective CDR sequences. Sequence identifiers are provided, and the CDRs are underlined in relation to the variable domain sequences. Figures 2a, 2b, 2c, 2d, and 2e show the binding characteristics of the anti-IL-23p19 antibody against human IL-23, a recombinant cytokine containing human p19 and murine p40 subunits, human IL-12, and human p40 subunits, as determined by BIAcore. Figures 3a, 3b, and 3c show the dose-dependent binding of selected representative IL-23p19 antibodies to recombinant cytokines containing human IL-23, human p19, and murine p40 subunits as determined by ELISA. Figures 3d and 3e do not show the binding of selected representative anti-IL-23p19 antibodies to human IL-12 and human p40 subunits determined by ELISA. Figure 4 shows the blockade of IL-23 / IL-23 receptor interaction by four IL-23p19 antibodies determined by ELISA. Figure 5 shows two representative IL-23p19 antibodies that do not block IL-23 / IL-12 receptor β1 interaction. Figures 6a and 6b show the inhibition of IL-23-induced IL-17 production by three representative anti-IL-23p19 antibodies in mouse splenocyte analysis (MSA). Figure 7 shows the inhibition of IL-23-induced STAT3 activation in reporter cell analysis by two representative anti-IL-23p19 antibodies. Figure 8 shows the non-inhibition of human IL-12-induced IFN-γ production in human PBMCs by two representative anti-IL-23p19 specific antibodies. Figure 9 shows the non-inhibition of IL-12-induced IFN-γ production in cynomolgus monkeys in human PBMCs by two representative anti-IL-23p19 antibodies. Figure 10 shows the in vivo inhibition of an IL-23-mediated inflammatory response (ear thickness) by two representative anti-IL-23p19 antibodies in the murine skin inflammation model described in Example 7. Figures 11a, 11b, 11c, and 11d provide graphs of the expression of the effect of two anti-IL-23p19 antibodies on the pathological score (H&E staining of frozen ear tissue) on day 8 after treatment in mice treated in the murine skin inflammation model presented in Example 7. Figures 12a, 12b, 12c, and 12d show representative photographs of hematoxylin and eosin (H&E) staining of frozen ear tissue collected on the last day (day 8) of the in vivo study from mice treated in the murine skin inflammation model presented in Example 7. As presented in Example 7. Specific details for implementing the invention

[0049] IL-23 is a pro-inflammatory heterodimeric cytokine containing a p19 subunit that binds to the IL-23 receptor. Targeting the pro-inflammatory IL-23 / IL-23 receptor signaling axis is an area of ​​intensive therapeutic research. The present disclosure provides antibodies and antibody fragments thereof that inhibit the human IL-23 / IL-23 receptor signaling axis and can be used for the treatment or prevention of IMID. Advantageously, the anti-IL-23p19 antibodies disclosed herein enable complete inhibition of IL-23p19, produce lower-dose formulations, result in less frequent and / or more effective administration, and lead to reduced cost and increased efficiency.

[0050] The anti-IL-23p19 antibody and its antibody fragment disclosed herein specifically bind to human IL-23p19 and antagonize the IL-23 / IL-23 receptor signaling axis. In one aspect, the disclosed antibody and its antibody fragment bind to human IL-23 with high affinity and block downstream signaling pathways by preventing its interaction with IL-23R. In a specific aspect, the antibody or its antibody fragment inhibits IL-23-stimulated production of IL-17 from mouse splenocytes and human PBMCs. In another aspect, the antibody or its antibody fragment does not bind to or antagonize IL-12.

[0051] To facilitate a better understanding of the present disclosure, specific technical and scientific terms are defined below. Unless otherwise specifically defined elsewhere in this document, all other technical and scientific terms used in this specification have the meaning generally understood by a person skilled in the art to which the present disclosure pertains.

[0052] Throughout this disclosure, the following abbreviations are used:

[0053] mAb or Mab or MAb - monoclonal antibody.

[0054] CDR - Complementarity-Determining Region of the Immunoglobulin Variable Region.

[0055] VH or VH - Immunoglobulin heavy chain variable region.

[0056] VL or VL - variable region of immunoglobulin light chain.

[0057] FR - Immunoglobulin variable region excluding antibody framework region and CDR region

[0058] As used herein, the term “interleukin-23 (used interchangeably with IL-23)” refers to a human IL-23 heteromer comprising, for example, a protein subunit having the amino acid sequence provided in UniProt List UniProtKB-P29460, identified as an amino IL-23 subunit (p40), which is disulfide-linked to a protein subunit having the amino acid sequence provided in UniProt List UniProtKB-Q9NPF7, identified as interleukin-23 subunit alpha (p19).

[0059] As used herein, the terms “IL-12R complex” and “IL-12R” refer to a high-affinity IL-12 cytokine receptor complex comprising IL-12Rβ1 and IL-12Rβ2 subunits.

[0060] As used herein, the terms “IL-23R complex” and “IL-23R” refer to a high-affinity IL-23 cytokine receptor comprising IL-12Rβ1 (common to the IL-12R complex) and an IL-23R subunit.

[0061] As used herein, the term "interleukin-12" (used interchangeably with "IL-12" throughout this disclosure) refers to a human IL-12 heterodimer comprising or composed of a protein subunit having the amino acid sequence provided in UniProt Listing UniProtKB-P29459 (identified as interleukin-12 subunit alpha) that is disulfide-linked to a protein subunit having the amino acid sequence provided in UniProt Listing UniProtKB-P29460, for example. This term includes a heterodimer protein comprising a 35 kD subunit (p35) and a 40 kD subunit (p40) linked together by disulfide crosslinking. The heterodimer protein is referred to as the "p70 subunit." The structure of human IL-12 is, for example, [Kobayashi, et al. It is further described in

[1989] J. Exp Med. 170:827-845 and Ling, et al. (1995) J. Exp Med. 154:116-127). The term human IL-12 is intended to include recombinant human IL-12 (rh IL-12) that can be produced by standard recombinant expression methods.

[0062] As used herein, the term “Interleukin 17” is also referred to as “IL-17” or “IL-17A” and is a glycosylated homodimeric protein of 20 to 30 kD comprising, for example, a protein subunit having the amino acid sequence provided in UniProt list UniProtKB-Q16552 or a homodimeric protein composed thereof. The human IL-17 gene codes for a 155-amino acid protein having a 19-amino acid signal sequence and a 136-amino acid maturation fragment. IL-17 is secreted by activated T-cells at the site of inflammation but is generally not present in the systemic circulation. IL-17 binds to a type I transmembrane receptor called IL-17R, which is a large, widely expressed protein that does not exhibit significant sequence similarity to other well-known cytokine receptors. Human IL-17 exhibits 62.5% and 58% amino acid sequence identity with respect to mouse and rat IL-17 sequences, respectively. Human IL-17 exhibits 97.4% amino acid sequence identity with cyanobacteria IL-17.

[0063] In this specification, the term "antibody" is used in the broadest sense and includes various antibody structures, including monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), but is not limited to the following.

[0064] Exemplary antibodies, such as IgG, comprise two heavy chains and two light chains. Each heavy chain consists of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated as VL herein) and a light chain constant region. The VH and VL regions may be further subdivided into a supervariable region called a complementarity-determining region (CDR), which is interspersed with a more conserved region called a framework region (FR). Each VH and VL consists of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus.

[0065] The hypervariable region generally consists of about 24 to 34 amino acid residues (LCDR1; "L" indicates light chain), 50 to 56 (LCDR2), and 89 to 97 (LCDR3) in the light chain variable region, and about 31 to 35B (HCDR1; "H" indicates heavy chain), 50 to 65 (HCDR2), and 95 to 102 (HCDR3) in the heavy chain variable region; literature [Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)] and / or includes amino acid residues from the literature [Chothia and Lesk (1987) J. Mol. Biol. 196:901-917] that form a supervariable loop in the light chain variable region (e.g., residues 26 to 32 (LCDR1), 50 to 52 (LCDR2) and 91 to 96 (LCDR3) and in the heavy chain variable region 26 to 32 (HCDR1), 53 to 55 (HCDR2) and 96 to 101 (HCDR3).

[0066] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, and, for example, the individual antibodies comprising the population bind to the same and / or the same epitope, except for naturally occurring mutations or possible variant antibodies that arise during the production of the monoclonal antibody preparation, and such variants are generally present in small amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies derived for different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is derived for a single determinant on the antigen. Accordingly, the modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be interpreted as implying that the antibody must be produced by any means. For example, the monoclonal antibody used according to the present invention may be produced by various techniques including, but not limited to, a hybridoma method, a recombinant DNA method, a phage display method, and a method using a transgenic animal comprising all or part of a human immunoglobulin locus, such methods for producing the monoclonal antibody described herein, and other exemplary methods.

[0067] The term "chimeric" antibody refers to a recombinant antibody in which part of the heavy chain and / or light chain is derived from a specific source or species, and the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0068] "Human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and / or produced using any technique for producing a human antibody known to those skilled in the art. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies are [Cole et al, Monoclonal Antibodies and Cancer Therapy , Alan R. Liss, p. 77 (1985); Boerner et al, J. Immunol It can be produced using various techniques known in the art, including the method described in [van Dijk and van de Winkel, 147(I):86-95 (1991)]. Also, in the literature [van Dijk and van de Winkel, Curr. Opin. Pharmacol See , 5: 368-74 (2001)]. Human antibodies were applied to transgenic animals modified to produce these antibodies in response to antigen challenge experiments but with endogenous loci inactivated, e.g., immunized HuMab mice (e.g., see literature on HuMab mice [Nils Lonberg et al., 1994, Nature 368:856-859], WO 98 / 24884, WO 94 / 25585, WO 93 / 1227, WO 92 / 22645, WO 92 / 03918, and WO 01 / 09187), xenomice (e.g., see U.S. Patents 6,075,181 and 6,150,584 for XENOMOUSE™ technology), or Trianni mice (e.g., see WO 2013 / 063391, WO 2017 / 035252, and WO 2017 / 136734). It can be manufactured by administering an antigen.

[0069] The term "humanized antibody" refers to an antibody engineered to include one or more human framework regions in a variable region, along with the complementarity-determining regions (CDRs) of the heavy and / or light chains of a non-human (e.g., mouse, rat, or hamster) animal. In certain embodiments, the humanized antibody comprises a sequence that is entirely human except for the CDR regions. Humanized antibodies generally have lower immunogenicity to humans compared to non-humanized antibodies, and thus offer therapeutic benefits in certain situations. Those skilled in the art will recognize humanized antibodies and the techniques suitable for their generation. For example, the literature [Hwang, WYK, et al., Methods 36:35, 2005; Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033, 1989; Jones et al., Nature , 321:522-25, 1986; Riechmann et al., Nature , 332:323-27, 1988; Verhoeyen et al., Science , 239:1534-36, 1988; Orlandi et al., Proc. Natl. Acad. Sci. USA [Refer to , 86:3833-37, 1989; US Pat. 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], the full text of each of the above documents is incorporated herein by reference.

[0070] 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, many of which can be subdivided into subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The heavy chain constant domains corresponding to different classes of immunoglobulins are denoted as α, δ, ε, γ, and μ, respectively.

[0071] The term "antigen-binding domain" (or simply "binding domain") or a similar term refers to one or more fragments of an antibody that possess the ability to specifically bind to an antigen complex. Examples of binding fragments included in the term "antigen-binding portion" of an antibody are: (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH domains; (ii) an F(ab')2 fragment, a divalent fragment comprising two Fab fragments connected by disulfide crosslinking at a hinge region; (iii) an Fd fragment consisting of VH and CH domains; (iv) an Fv fragment consisting of VL and VH domains of a single arm of the antibody; and (v) a dAb fragment consisting of a VH domain (reference [Ward et al., (1989) Nature 341: 544-546]). (vi) isolated complementarity-determining regions (CDRs), and (vii) combinations of two or more isolated CDRs that can be optionally linked by a synthetic linker.

[0072] As used herein, the term “complementary-determining region” or “CDR” refers to a short polypeptide sequence within a variable region of both heavy and light chain polypeptides that serves to mediate specific antigen recognition. Each V L and each V H There are 3 CDRs (named CDR1, CDR2, and CDR3) within.

[0073] As understood by those skilled in the art, the exact numbering and arrangement of CDRs may differ between different numbering systems. However, it should be understood that the initiation of a variable heavy chain and / or variable light chain sequence includes the initiation of the associated CDR. Thus, the initiation of each heavy chain variable region is the initiation of vhCDRs (e.g., vhCDR1, vhCDR2, and vhCDR3), and the initiation of each light chain variable region is the initiation of vlCDRs (e.g., vlCDR1, vlCDR2, and vlCDR3).

[0074] In certain embodiments, the CDR of the antibody is [Lefranc MP, (1999) The Immunologist 7: 132-136 and Lefranc MP et al, (1999) Nucleic Acids Res It may be determined according to the IMGT numbering system as described in [27: 209-212], the full text of each of the said documents is incorporated herein by reference. Unless otherwise stated in this specification, references to residue numbers in the variable domain of an antibody refer to residue numbers according to the IMGT numbering system.

[0075] In another embodiment, the CDR of the antibody is [MacCallum RM et al, (1996) J Mol Biol It may be determined according to [262: 732-745], the full text of which is incorporated herein by reference. Reference [Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001)], the full text of which is incorporated herein by reference. In another embodiment, the CDR of the antibody may be determined according to an AbM numbering scheme designating the AbM hypervariable domain, which represents a compromise between the Kavat CDR and the Chotia structural loop and is used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.), the full text of which is incorporated herein by reference.

[0076] "Framework," "Framework Region," or "FR" refers to variable domain residues other than the hypervariable region (HVR) residues. The FRs of the variable domain generally consist of four FR domains (FR1, FR2, FR3, and FR4).

[0077] The "Human Common Framework" is a framework representing the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is made from subgroups of variable domain sequences. Generally, the subgroups of sequences are the subgroups in the literature [Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), Vols. 1-3]. In one embodiment, for VL, the subgroups are [Kabat et al., supra As in ], it is subgroup Kappa I. In one embodiment, for VH, the subgroup is Kabat et al., supra It is subgroup III as in ].

[0078] The "hinge region" is generally defined as strands 216 to 238 (EU number) or 226 to 251 (Kavat number) of human IgG1. The hinge can be subdivided into three distinct regions: upper, middle (e.g., core), and lower hinge.

[0079] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. The term includes the natural sequence Fc region and the variant Fc region. In one embodiment, the human IgG heavy chain Fc region extends from Cys226, or Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified in this specification, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index, as described in the literature [Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)].

[0080] "Blocking" antibodies or "antagonist" antibodies inhibit or reduce the biological activity of the antigen to which they bind. Certain blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0081] The term "effector function" refers to biological activity attributed to the Fc region of an antibody that varies depending on the antibody isoform. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent T-cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B-cell receptors); and B-cell activation.

[0082] An "antibody binding to the same epitope as the reference antibody" refers to an antibody that comes into contact with an overlapping set of amino acid residues of the antigen compared to the reference antibody, or blocks the binding of the reference antibody to the antigen by 50% or more in a competitive analysis. The amino acid residues of the antibody in contact with the antigen can be determined, for example, by determining the crystal structure of the antibody complexing with the antigen, or by performing hydrogen / deuterium exchange. In some embodiments, antibody residues within 5 Å of the antigen are considered to come into contact with the antigen. In some embodiments, the antibody binding to the same epitope as the reference antibody blocks the binding of its antigen to the reference antibody by 50% or more in a competitive analysis, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competitive analysis.

[0083] The term "antibody fragment" refers to a molecule other than a complete antibody that contains a portion of a complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab)2; diabodies; linear antibodies; and single-chain antibody molecules (e.g., scFv). Papain digestion of an antibody produces two identical antigen-binding fragments called the "Fab" fragment and a residue "Fc" fragment that serves as an indicator of the ability to crystallize easily. The Fab fragment consists of the entire light chain (L) along with the variable region domain (VH) of the heavy chain (H) and the first constant region (CH1) of one heavy chain. Pepsin treatment of the antibody produces a single large F(ab)2 fragment that roughly corresponds to two disulfide-linked Fab fragments that possess divalent antigen-binding activity and are still capable of cross-linking antigens. The Fab fragment differs from the Fab' fragment in that it has a few additional residues at the carboxy terminus of the CH1 domain containing one or more cysteines in the antibody hinge region. Fab'-SH is the designation in this specification for Fab' in which the cysteine ​​residue(s) of the constant domain possess free thiol groups. The F(ab')2 antibody fragment was originally generated from pairs of Fab' fragments with a hinge cysteine ​​between them. Other chemical linkages of antibody fragments are also known.

[0084] "Fv" consists of a dimer of one heavy chain and one light chain variable region domain in strong non-covalent bonds. From the folding of these two domains, six supervariable loops (three loops in the H and L chains, respectively) are generated that are involved in amino acid residues for antigen binding and confer antigen-binding specificity to the antibody.

[0085] "Single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising VH and VL antibody domains linked to a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form a structure suitable for antigen-binding. For a review of sFv, refer to the literature [Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)].

[0086] The term “antigen-binding domain” (or simply “binding domain”) or a similar term refers to one or more fragments of an antibody that possess the ability to specifically bind to an antigen complex. Examples of binding fragments included in the term “antigen-binding portion” of an antibody include (i) a monovalent fragment consisting of a Fab fragment, VL, VH, CL, and CH domains; (ii) a divalent fragment consisting of an F(ab’)2 fragment, two Fab fragments connected by disulfide crosslinking at a hinge region; (iii) a Fd fragment consisting of VH and CH domains; (iv) a Fv fragment consisting of VL and VH domains of a single arm of the antibody; (v) a dAb fragment consisting 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 that can be selectively linked by a synthetic linker.

[0087] The term “multispecific antibody” is used in its broadest sense and specifically includes antibodies comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH-VL unit has multiple epitope specificity (e.g., can bind to two different epitopes on one biological molecule or each epitope on different biological molecules). Such multiple specific antibodies include, but are not limited to, full-length antibodies, antibodies having two or more VL and VH domains, bispecific diabodies, and triabodidies. “Multispecific epitope specificity” refers to the ability to specifically bind to two or more different epitopes on the same or different target(s).

[0088] “Bispecificity” or “dual-specificity” refers to the ability to specifically bind to two different epitopes on the same or different target(s). However, unlike bispecific antibodies, dual-specific antibodies have two antigen-binding arms with identical amino acid sequences, and each Fab arm can recognize 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, a multispecific antibody in the form of IgG1 binds to each epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0.1 μM to 0.001 pM. "Single-specificity" refers to the ability to bind to only one epitope. Multi-specific antibodies may have a structure similar to the entire immunoglobulin molecule and may include an Fc region, e.g., an IgG Fc region. Such structures may include, but are not limited to, IgG-Fv, IgG-(scFv)2, DVD-Ig, (scFv)2-(scFv)2-Fc, and (scFv)2-Fc-(scFv)2. In the case of IgG-(scFv)2, the scFv may be attached to the N-terminus or C-terminus of the heavy or light chain.

[0089] As used herein, the term “bispecific antibody” refers to a monoclonal, often human or humanized antibody having binding specificity to at least two different antigens. In the present invention, one of the binding specificities may be induced to IL-12 or IL-23, and the other may be any other antigen, e.g., a cell surface protein, a receptor, a receptor subunit, a tissue-specific antigen, a virus-derived protein, a virus-encoded envelope protein, a bacterial-derived protein, or a bacterial surface protein.

[0090] As used herein, the term “diabody” refers to a divalent antibody comprising two polypeptide chains, each polypeptide chain comprising VH and VL domains connected by very short linkers (e.g., a linker consisting of 5 amino acids), so that intramolecular binding of VH and VL domains is possible within the same peptide chain. In this stereochemical configuration, each domain forms a pair with a complementary domain of the other polypeptide chain to form a homodimeric structure. Thus, the term “triabody” refers to a trivalent antibody comprising three peptide chains, each comprising one VH domain and one VL domain connected by very short linkers (e.g., a linker consisting of 1 to 2 amino acids), so that intramolecular binding of VH and VL domains is possible within the same peptide chain.

[0091] When used to describe the various antibodies disclosed herein, the term “isolated antibody” means an antibody identified, isolated, and / or recovered from the cell or cell culture in which it is expressed. Contamination components of the natural environment are generally substances that interfere with the diagnostic or therapeutic use of polypeptides and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, antibodies are purified to a purity greater than 95% or 99%, as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric point electrophoresis (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) approaches. For a review of methods for evaluating antibody purity, refer, for example, to the literature [Flatman et al., J. Chromatogr. B 848:79-87 (2007)]. In a preferred embodiment, the antibody will be purified homogeneously by SDS-PAGE under non-reducing or reducing conditions using (1) a rotary cup sequencer to a sufficient degree to obtain at least 15 residues of the N-terminal or internal amino acid sequence, or (2) Coomassie blue or preferably a silver dye.

[0092] With respect to the binding of antibodies to target molecules, the terms "specific binding," "to bind specifically to," or "specific" for a specific polypeptide or an epitope on a specific polypeptide target refer to a binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule in comparison to the binding of a control molecule. For example, specific binding can be determined by competition between a target, for example, an excess of an unlabeled target, and a similar control molecule. In this case, if the binding of the labeled target and the probe is competitively inhibited by an excess of the unlabeled target, it is designated as specific binding. As used herein, the terms “specific binding,” “to bind specifically to,” or “specific” with respect to an epitope on a specific polypeptide or a specific polypeptide target may be represented, for example, by a Kd of 10⁻⁴ M or less, alternatively 10⁻⁵ M or less, alternatively 10⁻⁶ M or less, alternatively 10⁻⁷ M or less, alternatively 10⁻⁸ M or alternatively 10⁻⁹ M or alternatively 10⁻¹⁰ M or alternatively 10⁻¹¹ M or alternatively 10⁻¹² M for a target, or by a molecule having a Kd in the range of 10⁻⁴ M to 10⁻⁶ M or 10⁻⁶ M to 10⁻¹⁰ M or 10⁻⁷ M to 10⁻⁹ M. As understood by those skilled in the art, affinity and KD values ​​are inversely proportional. High affinity for an antigen is measured by a low KD value. In one embodiment, the term “specific binding” refers to a binding in which a molecule binds to a specific polypeptide or an epitope on a specific polypeptide without substantially binding to any other polypeptide or polypeptide epitope. As used herein, the terms “specific binding,” “specifically bind,” and “selectively bind” refer to an antibody that binds to an epitope of human interleukin-23 p19.

[0093] As used herein, the term "affinity" refers to the binding strength of an antibody to an epitope. The affinity of an antibody is given by the dissociation constant Kd and is defined as [Ab]X[Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody, and [Ag] is the molar concentration of the unbound antigen. The affinity constant Ka is defined as 1 / Kd. Methods for determining the affinity of an mAb are described in the literature [Harlow, et al., Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., eds., Current Protocols in Immunology , Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol It can be found in . 92:589-601 (1983), the full text of which is incorporated herein by reference. One standard method well known in the art for determining the affinity of mAbs is to use surface plasmon resonance (SPR) screening (e.g., analysis using a BIAcore™ SPR analyzer).

[0094] "Epitope" is a technical term referring to the site or regions of interaction between an antibody and its antigen(s). [Janeway, C, Jr., P. Travers, et al. (2001). Immunobiology: the immune system in health and disease. As stated in [Part II, Section 3-8. New York, Garland Publishing, Inc.]: "Antibodies generally recognize only small regions on the surface of large molecules, such as proteins. [A specific epitope] is likely composed of amino acids from different parts of an [antigen] polypeptide chain that are joined together by protein folding. This type of epitope is known as stereotypic or discontinuous epitope because it consists of protein fragments that are discontinuous in the amino acid sequence of the antigen but are joined together in a three-dimensional structure. In contrast, epitopes composed of a single fragment of a polypeptide chain are referred to as continuous or linear epitopes (Janeway, C. Jr., P. Travers, et al. (2001). Immunobiology: the immune system in health and disease. Part II, Section 3-8. New York, Garland Publishing, Inc.])

[0095] As used herein, the term "KD" is intended to refer to the dissociation constant of a specific antibody-antigen interaction. This is calculated by the following formula: Koff / Kon = KD

[0096] As used herein, the term “IC50” is intended to refer to the effective concentration of the antibody of the present invention required to neutralize 50% of the biological activity of IL-23 against human lymphoma DB cells in the bioassay described in Example 5: Inhibition of STAT3 activation in human DB cell analysis.

[0097] "EC50" regarding a formulation and a specific activity (e.g., binding to cells, inhibition of enzyme activity, activation or inhibition of immune cells) refers to the effective concentration of the formulation that produces 50% of the maximum response or effect for such activity. "EC100" regarding a formulation and a specific activity refers to the effective concentration of the formulation that produces a substantially maximum response for such activity.

[0098] As used herein, the terms “antibody-based immunotherapy” and “immunotherapy” are used to broadly refer to any form of therapy that relies on the targeting specificity of fusion proteins, including anti-IL-23p19 antibodies, bispecific molecules, multispecific molecules, binders, or IL-23p19 specific binders, to mediate direct or indirect effects on cells characterized by abnormal expression of IL-23p19. The term includes naked antibodies, bispecific antibodies (including T-cell binding, NK cell binding, and other immune cell / effector cell binding forms), antibody-drug conjugates, cell therapies including T-cells (CAR-T) or NK cells (CAR-NK) engineered to contain IL-23p19-specific chimeric antigen receptors, oncolytic viruses including IL-23p19-specific binders, and therapeutic methods using gene therapy by antigen-binding sequence delivery of anti-IL-23p19 antibodies and in vivo expression of corresponding antibody fragments.

[0099] 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 result of 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. All organ systems may be affected by IMID, and individuals may face a significant reduction in quality of life, significant morbidity, and reduced life expectancy (see [Bunte, K and Beikler, T, Int. J. Mol. Sci., 20: 3394(2019)]). Note that as used herein and in the appended claims, the singular forms (“a,” “an,” and “the”) include plural subjects unless the context clearly indicates otherwise.

[0100] IL-12 / IL-23 receptor signaling axis

[0101] The p19 subunit of IL-23 (also referred to herein as “IL-23p19” and “p19 subunit”) is an 189-amino acid polypeptide containing a 21-amino acid leader sequence (Reference [(Oppmann et al. Immunity 13:715) 2000)]). The biological activity of the p19 subunit is detected only when it forms IL-23 by pairing with the IL-12 p40 subunit. Both IL-12 and IL-23 exist only as secreted heteromeric cytokines, and the IL-12 p35 and IL-23p19 subunits are not secreted without intracellular covalent bonding with p40. The p40 subunit shared by IL-12 and IL-23 cytokines binds to the common IL-12Rβ1 component of the receptor, while signal specificity is determined by the unique p35 (IL-12) and p19 (IL-23) subunits that bind to the IL-12Rβ2 and IL-23R components of the respective high-affinity receptors. The interactions between IL-12 and IL-23 and their cognate receptors form part of a complex regulatory network that coordinates innate and adaptive immune responses.

[0102] IL-12 is hypothesized to play an important role in the development of protective immune responses against many intracellular pathogens and viruses and in tumor immune surveillance. Literature [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., NatureRefer to [2006 442: 461-5]. Therefore, IL-23 specific inhibition (saving IL-12 or the shared p40 subunit) may have potentially superior safety characteristics compared to dual inhibition of IL-12 and IL-23.

[0103] The receptor for IL-23 contains an IL-12Rβ1 subunit, which is commonly shared with the IL-12 receptor and forms a pair with a unique subunit called IL-23R (Reference [Parham et al. J. Immunol. 168:5699 (2002)]). IL-23R has been reported to bind to IL-23 with high affinity (KD=44 ±3 nM). In contrast, IL-23 binds to the IL-12Rβ1 subunit with lower affinity (KD=2 ± 1 uM). The binding of IL-23R to IL-23 promotes the binding of IL-12Rβ1 to IL-23 with very high affinity (KD=25 ± 5 nM) (Reference [Bloch et al, Immunity, 48, 45-58 (2018)]). IL-23R is expressed by various cells (natural killer cells, macrophages, dendritic cells, memory T-cells, and keratinocytes). IL-23 production induces the expression of IL-23R, creating a positive feedback loop that enhances IL-23 expression.

[0104] IL-23 binds to the IL-23 receptor complex, which is produced by activated antigen-presenting cells and expressed on NK cells and T-cells. IL-23 has been shown to promote the production of IL-17A, IL-17F, IL-6, and tumor necrosis factor α (TNFα), which are pro-inflammatory cytokines known to be involved in the inflammatory response in IMID disorders, either alone or in combination with other cytokines (e.g., IL-1β).

[0105] The binding of IL-23p19 to IL-23R triggers a restructuring process of the IL-23p19 helical domain, which enables the binding of IL-12 p40 and IL-12Rβ1 (Bloch, Y et al. Immunity. 2018; 48(1):45-58). This process activates JAK2 and TYK2, ultimately inducing the formation of STAT3 and STAT4, which function as transcription factors (Parham, C. et al., Immunol. 168(11):5699-5708 (2002)). IL-23 plays a key role in the late stages of differentiation of naive CD4+ T-cells into Th17 cells (Gaffen, SL et al., Nat Rev Immunol. 14(9):585-600 (2014)). Naive T-cells in the absence of IL-23R require other cytokines, such as transforming growth factor (TGF)-β and IL-6, to regulate the early stages of differentiation. These cytokines promote the expression of IL-23R by inducing the expression of retinoic acid receptor-associated orphan receptor-γt as a transcription factor. Immature Th17 cells induced by TGF-β and IL-6 must be exposed to IL-23 to acquire pathogenicity. Upon maturation, Th17 cells can produce IL-17 and TNF-α (Reference [Kashani, A et al., Gastroenterology & Hepatology 15(5):255-265 (2019)]).

[0106] Despite the structural similarities between the two cytokines, the biological activity / function of IL-23 is distinct from that of IL-12. IL-23 supports the differentiation and maintenance of naive CD4+ T-cells into a novel cell subset called Th17 cells, which is distinct from conventional Th1 and Th2 cells. Th17 cells produce interleukin-17A (IL-17A) and interleukin-17F (IL-17F). Th17 cells produce various other factors known to induce inflammatory responses, including tumor necrosis factors known to induce inflammatory responses, such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), CXCL1, and CCL20. Innate lymphocytes such as lymphoid tissue-induced (LTi)-like cells and NK cells respond to IL-23 by expressing IL-23 receptors and retinoic acid-associated orphan receptor (ROR) gamma and producing IL-17. IL-1β and IL-23 also co-stimulate gamma-delta T-cells to induce IL-17 production without T-cell receptor binding.

[0107] Importantly, IL-23 maintains the differentiation and expansion of naive T-cells into distinct Th17 cell lineages. In the absence of IL-23, the Th17 phenotype is lost. Because IL-23 plays a crucial role in maintaining cytotoxic Th17 cells that generate pro-inflammatory cytokine characteristics, it has been described as the "master regulator" of the immune inflammatory response in IMID. The pathogenicity of IL-23 depends in part on the uncontrolled production of IL-17A, IL-17F, and IL-22, which provides the basis for targeting the IL-23 / IL-23R axis for immunotherapy.

[0108] Targeting of the pro-inflammatory IL-23 / IL-23 receptor signaling axis

[0109] Anti-IL-12 / IL-23 antibodies reported to confer therapeutic benefits in vivo include the antibodies ustekinumab (CNTO1275) and briakinumab (ABT-874). Both antibodies target a common IL-12 p40 subunit in the region of the p40 subunit critical for IL-12Rβ1 binding (Clarke, A. et al. mAbs 2(5):539-549 (2010)]).

[0110] Anti-IL-23 selective antibodies reported to confer therapeutic benefits in vivo include guselkumab (TREMFYA®), tildrakizumab (ILUMYA®), risankizumab (SKYRIZI®), brazikumab (MEDI2070), and mirakizumab (Ly3074828); all of these are specific to the p19 subunit of IL-23. Data from randomized, placebo- and active-controlled Phase 3 clinical trials indicate that tildrakizumab, guselkumab, and risankizumab exhibit favorable risk-benefit characteristics in patients with moderate to severe psoriasis. No significant safety issues were observed with any of these IL-23 p19 inhibitors.

[0111] Although IL-12-induced Th1 cells were previously thought to be a pathogenic T-cell subset in many autoimmune diseases, more recent animal studies in models of inflammatory bowel disease, psoriasis, inflammatory arthritis, and multiple sclerosis have evaluated the individual involvement of IL-12 versus IL-23, demonstrating that IL-23, rather than IL-12, is the key inducer of autoimmune / inflammatory diseases (Literature [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)]).

[0112] The role of IL-23 in immune-mediated inflammatory responses is also supported by genetic studies. Genome-wide related studies (GWAS) have linked IL-23R polymorphisms to a predisposition for autoimmune pathologies such as psoriasis and psoriatic arthritis (Literature [Liu et al., PLoS Genet. 4(3)e1000041 (2008), Reveille, et al., Nat. Genet . 42(2): 123-127 (2010), and Duerr et al., Science 314(5804):1461-1463 (2006)]). The binding between CD and rs11209026, a single-nucleotide polymorphism (SNP) of the IL-23R gene, has been established (Reveille, JD et al.). This variant was shown to be protective against CD and UC. The protective properties of rs11209026 were confirmed in a meta-analysis showing that possession of this SNP variant reduced disease risk in a control cohort of over 75,000 cases (Jostins, L. Nature 491(7422):119-124 (2012)]). This SNP variant, along with a few other coding variants of IL-23R, causes a decrease in IL-23R expression and thus reduces immune responses mediated through the IL-23 axis (Reference [ J Biol Chem. 291(16):8673-8685 (2016)]).

[0113] Cytokines such as IL-6 and TGF-β1 can promote the differentiation of RORγt+ Th17 cells from naive CD4+ T-cells, but IL-23 is required for the full inflammatory function of these cells. Additionally, the binding of IL-23 to receptors on activated RORγt+ Th17 cells induces the additional expression of the 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).

[0114] There is strong evidence that the IL-23 / IL-17 axis plays a critical role in the development of chronic inflammation, and genetic studies have revealed possible links between the IL-23 receptor (IL-23R) or its ligands and numerous 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 research in IMIDs, including psoriasis, psoriatic arthritis, inflammatory bowel diseases (ulcerative colitis and Crohn's disease), ankylosing spondylitis, and systemic lupus erythematosus (SLE).

[0115] Generally speaking, IL-23 specific antibodies such as guselkumab, tildrakizumab, risankizumab, brazikumab, or miracizumab selectively bind to IL-23p19 and inhibit IL-23 from binding to its receptor; thus, by antagonizing the action of IL-23, they induce and maintain T helper (Th) 17 cells, innate lymphocytes, γδT-cells, and natural killer (NK) cells involved in tissue inflammation, destruction, and / or abnormal tissue repair associated with IMID.

[0116] Plaque psoriasis, or psoriasis (PsO), is a chronic inflammatory T-cell-mediated skin disorder characterized by complex pathophysiology. Its incidence in developed countries is 1 to 4 percent. Psoriasis is the most widespread 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 percent of patients. Although the pathogenesis of psoriasis is not fully understood, numerous environmental factors, T-cells, dendritic cells, numerous cytokines, and 45 identified loci all interact to produce the systemic psoriatic disease state and ultimately plaque psoriasis (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 effect of genetic and environmental factors, along with the interaction of innate and adaptive immunity, ultimately leads to abnormal keratinocyte proliferation and the formation of psoriatic lesions (Literature [Chan, J. R, et al. ., J. Exp. Med , 203(12)2577-2587 (2006)]).

[0117] PsO plaques are typically well-defined erythematous scaly skin lesions characterized by epidermal thickening. The causative keratinocytes activate dendritic cells, migrate to local lymph nodes, and release multiple cytokines, including interleukins IL-12 and IL-23, which activate type 1 T helper (Th1) and type 17 T helper (Th17) cells, respectively. T lymphocytes and other cell types release additional cytokines, including tumor necrosis factor (TNF)-α, IL-22, and IL-17, which increase keratinocyte activation and initiate a self-driving inflammatory cycle (Lowes MA et al., Trends Immunol.34(4):174-81 (2013)). Histologically, there is significant epidermal proliferation accompanied by parakeratosis and mixed dermal infiltration, including CD4+ T-cells, dendritic cells, macrophages, and mast cells.

[0118] Early publications reported the presence of elevated levels of tumor necrosis factor-α and the IL-12 p40 subunit, accompanied by the overexpression of IL-12 p40 and IL-23 p40 messenger RNAs in psoriatic skin lesions. These findings suggested that inhibiting IL-12 and IL-23 with neutralizing antibodies against IL-12 / 23 p40 subunit proteins could provide an effective therapeutic approach for the treatment of psoriasis (Reference [Piskin G, et al., J Immunol 2006, 176: 1908-15]). Psoriasis was initially considered a Th-1 mediated disease (T helper type 1 cells: based on the characteristic cytokine secretion properties of interleukin-2, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ).

[0119] The fundamental role of IL-23 in the pathogenesis of psoriasis has been clarified and is related to the biology of the Th17 lineage. The initial differentiation of naive T lymphocytes into Th17s requires the presence of TGF-β1, IL-6, and IL-1, and IL-23 is necessary for the activation and maintenance of Th17s to secrete the pro-inflammatory cytokines IL-17, IL-22, IL-21, and tumor necrosis factor-α, which consequently contributes to the formation of psoriatic skin lesions (Reference [Fotaidou, C. et al., Psoriasis: Targets and Therapy 8: 1-5(2018)]).

[0120] Therefore, although both IL-12 and IL-23 are known to be involved in the development of the Th1 immune response in psoriasis, IL-23 is currently recognized as a key inducer of Th17 cell differentiation and survival. The primary cytokines produced by Th17 cells belong to the pro-inflammatory IL-17 family, including IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. IL-17A and IL-17F are similar and bind to the same IL-17 receptor, which is a heterodimer composed of IL-17RA and IL-17RC subunits.

[0121] While early treatment strategies targeted Th1 cells as the central cell type in the pathogenesis of psoriasis, a novel model focused on the IL-23 / Th17 axis (Reference [Lowes MA, et al. Trends Immunol. 34(4):174-81(2013)]). The rationale for this novel focus is based on the conviction that IL-17 plays a key role in the pathogenesis of psoriasis and the information that IL-23 induces Th17 cell activation. Furthermore, IL-23 stimulates the production of other Th17 cytokines (e.g., IL-22) by other cell types, including innate lymphocyte type 3 cells and γδ T-cells (Reference [Ward, NL, J Investig Dermatol. 134: 2305-2307(2014)]). It has been suggested that the inhibition of IL-23 blocks the downstream production of IL-17A and IL-22 by Th17 cells, and that this effect is interpreted as an antagonistic action in the development of psoriasis immunopathology.

[0122] The IL-23 / IL-17 axis is currently considered important in the pathogenesis of psoriasis, and selective IL-23p19 inhibition may yield multiple benefits in relation to IL-12 / 23p40 inhibition or terminal blockade of IL-17A or its receptor (Reference [Torres, T Drugs 77:1493-1503 (2017)]). To date, three IL-23p19-subunit specific monoclonal antibodies (i.e., guselkumab, tildrakizumab, and risankizumab) have received approval from the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of moderate to severe plaque psoriasis in adults who are candidates for systemic therapy or phototherapy. In July 2020, guselkumab was approved by the FDA as a treatment for active psoriatic arthritis in adults.

[0123] The high efficacy of IL-23 blockade in psoriasis has been demonstrated in early proof-of-concept tests and Phase I clinical trials. According to Phase I studies, a single administration of guselkumab produced a significant clinical response in patients with moderate to severe plaque psoriasis (Literature [Sofen H, et al., J Allergy Clin Immunol; 133:1032-1040 (2014)]). Phase I studies also reported that the selective antagonism of interleukin-23 and guselkumab resulted in clinical improvement of psoriasis characterized by a reduction in epidermal thickness, T-cell and dendritic cell infiltration, psoriasis-related gene expression, and serum IL-17A levels. The reported results of measurable clinical responses in patients with moderate to severe psoriasis following a single administration of guselkumab support a novel theory that the selective neutralization of IL-23 is a promising treatment option.

[0124] A rapid onset of guselkumab activity was also observed in a Phase II dosing study (NCT01483599) evaluating the use of guselkumab over a wide range of administrations and two different dosing intervals for up to 40 weeks of continuous treatment. Efficacy was evident at the initial assessment (4 weeks). Many guselkumab regimens were associated with significantly superior response rates compared to regimens associated with adalimumab, a biological agent commonly used for the treatment of psoriasis (reference [Gordon, KB et al., N Engl J Med 373:136-144 (2015)]). The efficacy of guselkumab continued to increase after week 16 (primary endpoint assessment) and was maintained until week 40. Furthermore, the majority of patients in the 100 mg guselkumab group achieved complete clearance of psoriasis after 40 weeks of continuous treatment, as indicated by a 100% improvement in PGA scores from baseline (54% of patients) and a PGA score of 0 (62% of patients). Regulatory approvals by the FDA and EMA were partly due to three important Phase 3 clinical trials, VOYAGE 1, (Literature [Blauvelt, A et al. J. Am. Acad. Dermatol. 76: 405-417 (2017)]) VOYAGE (Literature[Reich, K et al. J Am. Acad. Dermatol. ,76: 418-431 (2017)]) and NAVIGATE (Literature[Langley, RG et al., Brit. J. Dermatol. It relied on the results of 178:114-123 (2017)]).

[0125] VOYAGE 1 (NCT02207231) was a Phase III, randomized, double-blind, placebo- and active comparator-controlled trial conducted at 101 total sites (December 2014 to April 2016). The study consisted of an active-comparator period (0 to 48 weeks) and a placebo-controlled period (0 to 16 weeks) in which guselkumab was compared to adalimumab, after which patients receiving placebo were crossed over and provided with guselkumab until 48 weeks. Guselkumab was superior to placebo and / or adalimumab for the co-primary endpoint and all key secondary endpoints (all P < .001). Compared to placebo, a significantly higher proportion of patients treated with guselkumab achieved IGA 0 / 1 (6.9% vs. 85.1%) and PASI 90 (2.9% vs. 73.3%) at week 16. Similarly, PASI 100 responses in the guselkumab group were significantly higher than in those in the adalimumab group at weeks 24 and 48 (P < .001). After initiating guselkumab at week 16, patients in the placebo crossover group achieved responses similar to those observed in the guselkumab group. VOYAGE 1 confirms the role of IL-23 in the pathogenesis of psoriasis. Compared to TNF-α blockade, selective targeting of the IL-23 pathway provides greater inhibition of psoriasis-specific cytokines with a higher degree of efficacy while maintaining favorable safety characteristics (Literature [Blauvelt, A, et al ., J Investig Dermatol. , 135: 1946-1953 (2015)]).

[0126] VOYAGE 1 was an extended-label trial that followed patients for four years after the initial trial. Patients were initially randomized to receive either Tremfya or a placebo, but at week 16, everyone received Tremfya. According to the VOYAGE 1 study, in the combined group of subjects who were initially given either Tremfya or a placebo and then switched to Tremfya at week 16, 82% of the patients who received Tremfya showed at least 90% improvement in the Psoriasis Area Severity Index (PASI 90) at week 204 (four years), and the investigator's comprehensive assessment (IGA) score was eliminated (0) or minimal disease (1).

[0127] Psoriatic arthritis (PsA) is a chronic inflammatory musculoskeletal disease occurring in up to 40% of patients with psoriasis. Consequently, PsA can be considered a disease within a disease that shares many common pathogenic pathways with psoriasis. Psoriasis generally develops before PsA in 70% of patients, inflammatory skin and joint diseases occur simultaneously in 15% of patients, and inflammatory arthritis develops before skin disease in the remaining patients. Consequently, almost all patients with PsA will develop psoriasis; however, the clinical outcomes and course of PsA are highly heterogeneous, and five distinct patterns of PsA based on the distribution of affected joints have been described (Dobbin-Sears, I et al. Ther Adv Chronic Dis ,. 9(10) 191 -198 (2018)]).

[0128] PsA is a heterogeneous pathological condition with joint and extra-articular manifestations, including a combination of peripheral arthritis, axial disease, enthesitis, onychomycosis, and cutaneous and nail diseases (Quireo, R and Coto-Sequra, P, Expert Opinion On Biological Therapy, 18:9, 931-935 (2018)). Genetic, immunological, and environmental factors that activate both innate and adaptive immune responses appear to play a significant role in the pathogenesis of PsA. As the disease progresses, patients may exhibit multiple patterns and are not limited to a single subset of arthritis. Approximately two-thirds of PsA patients experience progressive joint damage often associated with loss of function and disability.

[0129] The pro-inflammatory 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 considered to play a crucial role in the immunopathogenesis of both psoriasis and PsA. IL-23 / IL-23-R interactions induce IL-23-dependent differentiation and activation of Th-17 cells, while the production and secretion of IL-17 and IL-22 induce synovial and cutaneous inflammation and bone reconstruction. IL-17, which is particularly associated with PsA pathology, promotes bone erosion through the upregulation of RANKL. Analysis of mixed data showed that ustekinumab-treated patients (regardless of dosage) significantly inhibited the progression of radiographic joint damage in patients with active PsA (Kavanaugh, A et al. Ann. Rheum. Dis. 73(6):1000-1006 (2014)). This supports the role of IL-23 and downstream Th17 pathways in radiological damage occurring in most PsA patients.

[0130] Crohn's disease (CD) and ulcerative colitis (UC), the major human inflammatory bowel diseases (IBD), are both chronic, relapsing diseases characterized by chronic tissue inflammation that alters the integrity and function of the intestine. Increased levels of interleukin (IL)-23 and T helper (Th) 17 cell cytokines were observed in the intestinal mucosa, plasma, and serum of patients with inflammatory bowel diseases (IBD) (e.g., Crohn's disease (CD) and ulcerative colitis (UC)).

[0131] Variants in multiple genes encoding elements of the IL-23 and IL-17 cellular 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 IBD risk due to decreased STAT3 signaling and reduced Th17 cellular response upon IL-23 exposure (Literature [Barrett, JC et al. Nat. Genet . 40:955-962 (2008), Duerr, R.H. et al. Science 314:1461-1463 (2006), Allocca, M et al. Best Practice & Res. Clin . Gastro. 32-33:95-102 (2018)]).

[0132] Crohn's disease (CD) is a chronic immune-mediated condition characterized by recurrent and involuntarily affecting the gastrointestinal system. CD is characterized by dysregulation of innate and adaptive immune responses. Although the pathophysiological mechanisms are not fully understood, the disease is likely the result of interactions between the gut commensal flora and host microbial defenses in genetically predisposed individuals, which can consequently lead to intestinal inflammatory responses in Crohn's disease (Deepak, P and Loftus, E, Drug Design, Development and Therapy (10) 3685-3698) (2016)). Persistent intestinal inflammatory responses over a long period often lead to the development of strictures and / or fistulas requiring hospitalization and / or surgery. Since the discovery of the IL-23 / IL-17 pathway, the treatment paradigm for CD has shifted from non-specific immunosuppressive therapy (i.e., methotrexate) to immunotherapy targeting the IL-2 and / or IL-17 pathways.

[0133] Ulcerative colitis (UC) is a chronic, relapsing-to-remission inflammatory bowel disease that causes persistent mucosal inflammation of the large intestine, resulting in small, open ulcers or ulcers that produce pus and mucus. In the United States, there are approximately 1 million people with ulcerative colitis, and it is estimated that UC affects 2.6 million people in Europe. The disease can occur in people of any race or ethnic group; although men are more likely to be diagnosed than women, it is more common among Caucasians. The etiology of UC is not well understood, but it is considered to be partly due to an abnormal immune response to the microbiome (microbial flora and pathogens) in subjects with a genetic predisposition to chronic inflammation in the colon. Ulcerative colitis is known to exhibit Th2-type cytokine characteristics.

[0134] IL-23-specific p19 antagonists currently under clinical investigation for IBD include brazicumab (MEDI2070), risankizumab (BI 655066), mirikizumab (LY3074828), and guselkumab (Tremfya, Janssen). To date, the anti-p19 (anti-IL-23) antibodies brazicumab and risankizumab have been reported to be effective in moderate to severe CD in Phase II trials.

[0135] In Phase II trials, mirikizumab was recently shown to be effective for moderate to severe UC. Across all studied doses, 11.5 to 22.6 percent of patients treated with mirikizumab achieved clinical remission compared to 4.8 percent of patients treated with placebo. Additionally, a proportion of patients treated with mirikizumab achieved endoscopic and symptomatic remission at week 12 compared to placebo. Currently, there are no p-19 selective antibodies approved for the treatment of IBD. Phase 2 and 3 clinical trials of anti-p19 agents (risankizumab, brazicumab, and guselkumab) are underway and are expected to provide additional information regarding efficacy and safety itself, as well as the evolving therapeutic concept of combination therapy with multiple biological agents and head-to-head efficacy compared to existing biological agents.

[0136] Ankylosing spondylitis (AS), similar to psoriatic arthritis, is another spondyloarthropathy genetically linked to the IL-23 pathway; it is a painful condition associated with spinal inflammation that can lead to irreversible vertebral fusion. AS generally does not respond to conventional disease-modifying antirheumatic drugs (DMARDs), and systemic therapy for AS consists of non-steroidal anti-inflammatory drugs (NSAIDs) and tumor necrosis factor inhibitors.

[0137] IL-23 has been identified as a promising therapeutic target for AS in several lines of evidence (Reference [Paine A, et al. Curr. Opin. Rheumatol. 28:359-67 (2016)]). At the genetic level, four-line-control genome-wide studies have demonstrated that IL-23 receptor (IL-23R) polymorphisms are associated with an increased risk of developing AS (Reference [Reveille JD, et al Genet 42:123-7 (2010)]). Furthermore, a protective effect of the IL-23R R381Q polymorphism is observed in AS (Reference [Sarin R, et al. Proc Natl Acad Sci USA; 108:9560-58 (2011)]). An increased number of IL-23-producing cells was found in the facet joints of AS patients (Reference [Appel H, et al. Arthritis Rheum; 65:1522-9 (2013)]), and the number of IL-23-responsive T helper (Th) 22 (Th22), Th17, and gamma / delta T-cells is increased in the blood from AS patients (Reference [Zhang L, et al. PLoS One (7):e31000 (2012)]).

[0138] The recent approval of secukinumab, an IL-17A inhibitor for the treatment of AS (Baeten D. et al., N Engl. J. Med. 373:2534-48 (2015)), supported the clinical hypothesis that direct, specific inhibition of IL-23 would provide therapeutic benefits to AS patients. However, a recent publication reporting the results of a randomized, double-blind, placebo-controlled, proof-of-concept, dose-finding Phase 2 study evaluating the efficacy of risankizumab in active AS patients (NCT02047110) concluded that risankizumab treatment failed to meet the study's primary endpoint and did not demonstrate evidence of clinically significant improvement compared to placebo in active AS patients (Baeten D, et al., Annals of the Rheumatic Diseases 77: 1295-1302 (2018)]).

[0139] IL-23p19 antagonist

[0140] The IL-23 receptor complex consists of IL-12Rβ1, which forms a pair with the signaling chain IL-23R (p19 subunit binding). IL-23 mediates cell activity by sequentially binding to two receptor chains expressed as the IL-12Rβ1 / IL-23R receptor complex on the surface of T-cells and natural killer (NK) cells.

[0141] Murine, humanized, and phage-displaying antibodies selected for the inhibition of recombinant IL-23 are described; see, for example, 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.

[0142] Monoclonal antibodies or their antigen-binding domains that bind with high affinity to the p19 subunit of the IL-23 cytokine can neutralize its activity and consequently block its downstream effects. To date, three (3) anti-p19 antibodies—guselkumab (TREMFYA®), tildrakizumab (ILUMYA®), and risankizumab (SKYRIZI®)—have been FDA approved for the treatment of IMID. Two other IL-23 p19 subunit-specific antibodies are MEDI2070 (brazikumab, AstraZeneca / Medimmue) and Ly3074828 (mirikizumab, Eli Lilly), which are currently in late clinical development. Mirikizumab is a humanized IgG4 monoclonal antibody. By blocking IL-23, anti-p19-specific antibodies attenuate the inflammatory response by suppressing the release of pro-inflammatory cytokines and chemokines.

[0143] IL-23 antagonists in this group do not affect IL-12 activity because they target the p19 subunit of IL-23 rather than the p40 subunit. This function distinguishes ustekinumab (STELARA) from IL-23 receptor antagonists, which target the common p40 subunit shared by IL-12 and IL-23. Despite the efficacy and favorable safety characteristics of ustekinumab, drug development for IMIDs has shifted its focus to the development of agents that selectively antagonize the IL-23 / IL-17 pathway.

[0144] Guselkumab (CNTO1959) is a fully human monoclonal IgG1, λ antibody that binds to the p19 subunit of human IL-23 with high affinity. Guselkumab is the first FDA-approved anti-p19-specific antibody / IL-23 antagonist. It was approved on July 13, 2017, following an expedited regulatory review, as a treatment for moderate to severe plaque psoriasis in adults. It has also been approved in Canada, the European Union, Japan, and numerous other countries worldwide. Guselkumab is marketed by Janssen as TREMFYA (U.S. Patents No. 7,935,344 and 7,993,645).

[0145] Guselkumab inhibits the physiological activity of human IL-23 by preventing IL-23 from binding to IL-23 receptor proteins expressed on the surface of immune cells. More specifically, guselkumab binds to the human IL-23 cytokine via the p19 subunit and prevents the formation of the IL-23-IL-23R complex and subsequent intracellular signaling of the partner receptor chain.

[0146] The TREMFYA® development program currently includes a Phase III trial evaluating the efficacy of TREMFYA® for treating active psoriatic arthritis, a Phase IIb / III study for Crohn's disease, a Phase IIb / III study for ulcerative colitis, and another clinical trial evaluating guselkumab for hidradenitis suppurativa.

[0147] Janssen recently announced plans to further expand the clinical development of guselkumab to include familial adenomatous polyposis (FAP), a gastrointestinal disease. Janssen has initiated a Phase 1b proof-of-concept clinical trial (NCT03649971) to evaluate the efficacy and safety of guselkumab versus placebo in approximately 72 patients. FAP syndrome is the most common adenomatous polyposis syndrome. It is an autosomal dominant genetic disorder characterized by the early development of hundreds to thousands of adenomatous polyps throughout the colon. FAP affects approximately 1 in 8,300 people worldwide and occurs equally in men and women; if left untreated, patients with this syndrome have the highest risk of developing colorectal cancer. Additionally, there is an increased risk of developing other malignancies. Removal of the colon is currently the only way to prevent colorectal cancer in these patients.

[0148] Tildrakizumab (MK322) is a humanized monoclonal IgG1,κ antibody marketed by Merck & Co. / Sun Pharmaceutical as ILUMYA (U.S. Patent No. 8,404,813). Tildrakizumab selectively binds to the p19 subunit to inhibit the interaction between IL-23 and its receptor, thereby suppressing the release of IL-23-mediated pro-inflammatory cytokines. In March 2018, it received its first worldwide approval from the FDA for use in adult patients with moderate to severe plaque psoriasis.

[0149] Risankizumab (BI 655066) is a humanized monoclonal IgG1, κ marketed as SKYRIZI by AbbVie / Boehringer Ingelheim (U.S. Patent No. 8,778,346). Risankizumab was developed as a high-affinity antibody antagonist of IL-23.

[0150] Risankizumab selectively binds to the p19 subunit of interleukin-23 (IL-23p19) with high affinity (dissociation constant less than 10 pmol / L) (Reference [Singh, S, et al., MAbs 7(4)77-791 (2015)]). It selectively targets the p19 subunit of IL-23 and potently inhibits IL-23-induced IL-17 production (human IL-23 produced by THP-1 cells) in mouse splenocyte analysis, with an IC50 value of approximately 2 pM (Reference [Singh, S, et al., MAbs 7(4)77-791 (2015)]). The framework region of risankizumab was engineered with two mutations in the Fc region to reduce FcγR receptor and complement binding. More specifically, some of the Fc variants of risankizumab have two alternative mutations (Leu234Ala and Leu235Ala) that reduce Fcγ receptor and complement binding. Risankizumab was approved by the FDA in April 2019 as a treatment for moderate to severe plaque psoriasis in adults.

[0151] Anti-IL-23p19 antibody

[0152] The anti-IL-23p19 antibody of the present disclosure binds to the p19 subunit of IL-23. Preferably, such antibody is entirely human and does not bind to the p40 subunit of IL-12.

[0153] In one embodiment, the anti-IL-23p19 antibody or its antibody fragment comprises a VH having a set of CDRs (HCDR1, HCDR2, and HCDR3) disclosed in Table 1. For example, the anti-IL-23p19 antibody or its antibody fragment may comprise a set of CDRs corresponding to the CDRs in one or more of the anti-IL-23p19 antibodies disclosed in Table 1 (e.g., the CDRs of the Hu-2.18006B antibody).

[0154] In another embodiment, the anti-IL-23p19 antibody comprises a VL having a set of CDRs (LCDR1, LCDR2, and LCDR3) as disclosed in Table 2. For example, the anti-IL-23p19 antibody or its antibody fragment may comprise a set of CDRs corresponding to the CDRs in one or more of the anti-IL-23p19 antibodies disclosed in Table 2 (e.g., the CDR of the Hu-2.18006B antibody).

[0155] In an alternative embodiment, the anti-IL-23p19 antibody or antibody fragment thereof comprises VH having a set of CDRs (HCDR1, HCDR2, and HCDR3) as disclosed in Table 1, and VL having a set of CDRs (LCDR1, LCDR2, and LCDR3) as disclosed in Table 2.

[0156] CDR sequence of the human variable heavy chain domain Anti-IL-23p19 Ab CDR1 CDR2 CDR3 Hu-2. 18006B Sequence number 9 Sequence number 10 Sequence number 11 Hu-4. 18006B Sequence number 15 Sequence number 16 Sequence number 17 Hu-5. 18006B Sequence number 21 Sequence number 22 Sequence number 23 Hu-6. 18006B Sequence number 27 Sequence number 28 Sequence number 29

[0157] CDR sequence of the human variable light chain domain Anti-IL-23p19 Ab CDR1 CDR2 CDR3 Hu-2. 18006B Sequence No. 12 Sequence No. 13 Sequence No. 14 Hu-4. 18006B Sequence No. 18 Sequence No. 19 Sequence number 20 Hu-5. 18006B Sequence No. 24 Sequence number 25 Sequence number 26 Hu-6. 18006B Sequence number 30 Sequence No. 31 Sequence No. 32

[0158] In an embodiment, the antibody may be a monoclonal, chimeric, humanized, or human antibody (or its antigen-binding portion) that specifically binds to human IL-23p19.

[0159] In an embodiment, the anti-IL-23p19 antibody or its antibody fragment is

[0160] (i) CDR1: Sequence No. 9, CDR2: Sequence No. 10, CDR3: Sequence No. 11;

[0161] (ii) CDR1: Sequence No. 15, CDR2: Sequence No. 16, CDR3: Sequence No. 17;

[0162] (iii) CDR1: Sequence No. 21, CDR2: Sequence No. 22, CDR3: Sequence No. 23; and

[0163] (iv) CDR1: Sequence No. 27, CDR2: Sequence No. 28, CDR3: Sequence No. 29

[0164] It includes a VH having a set of complementarity-determining regions (CDR1, CDR2, and CDR3) selected from a group consisting of

[0165] In another embodiment, the anti-IL-23p19 antibody or its antibody fragment is

[0166] (i) CDR1: Sequence No. 12, CDR2: Sequence No. 13, CDR3: Sequence No. 14;

[0167] (ii) CDR1: Sequence No. 18, CDR2: Sequence No. 19, CDR3: Sequence No. 20;

[0168] (iii) CDR1: sequence number 24, CDR2: sequence number 25, CDR3: sequence number 26; and

[0169] (iv) CDR1: Sequence No. 30, CDR2: Sequence No. 31, CDR3: Sequence No. 32

[0170] It includes a VL having a set of complementarity-determining regions (CDR1, CDR2, and CDR3) selected from a group consisting of

[0171] In another embodiment, the anti-IL-23p19 antibody or its antibody fragment is

[0172] (a)

[0173] (i) CDR1: Sequence No. 9, CDR2: Sequence No. 10, CDR3: Sequence No. 11;

[0174] (ii) CDR1: Sequence No. 15, CDR2: Sequence No. 16, CDR3: Sequence No. 17;

[0175] (iii) CDR1: Sequence No. 21, CDR2: Sequence No. 22, CDR3: Sequence No. 23; and

[0176] (iv) CDR1: Sequence No. 27, CDR2: Sequence No. 28, CDR3: Sequence No. 29

[0177] VH having a set of complementarity-determining regions (CDR1, CDR2, and CDR3) selected from a group consisting of; and

[0178] (b)

[0179] (i) CDR1: Sequence No. 12, CDR2: Sequence No. 13, CDR3: Sequence No. 14;

[0180] (ii) CDR1: Sequence No. 18, CDR2: Sequence No. 19, CDR3: Sequence No. 20;

[0181] (iii) CDR1: sequence number 24, CDR2: sequence number 25, CDR3: sequence number 26; and

[0182] (iv) CDR1: Sequence No. 30, CDR2: Sequence No. 31, CDR3: Sequence No. 32

[0183] VL having a set of complementarity-determining regions (CDR1, CDR2, and CDR3) selected from the group consisting of

[0184] Includes

[0185] In an embodiment, the antibody is

[0186] (i) VH: CDR1: Sequence No. 9, CDR2: Sequence No. 10, CDR3: Sequence No. 11, VL: CDR1: Sequence No. 12, CDR2: Sequence No. 13, CDR3: Sequence No. 14;

[0187] (ii) VH: CDR1: Sequence No. 15, CDR2: Sequence No. 16, CDR3: Sequence No. 17, VL: CDR1: Sequence No. 18, CDR2: Sequence No. 19, CDR3: Sequence No. 20;

[0188] (iii) VH: CDR1: sequence number 21, CDR2: sequence number 22, CDR3: sequence number 23, VL: CDR1: sequence number 24, CDR2: sequence number 25, CDR3: sequence number 26; and

[0189] (iv) VH: CDR1: Sequence No. 27, CDR2: Sequence No. 28, CDR3: Sequence No. 29, VL: CDR1: Sequence No. 30, CDR2: Sequence No. 31, CDR3: Sequence No. 32

[0190] It includes a combination of VH and VL having a set of complementarity-determining regions (CDR1, CDR2 and CDR3) selected from a group consisting of

[0191] In an embodiment, the anti-IL-23p19 antibody or its antibody fragment 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.

[0192] In an embodiment, the anti-IL-23p19 antibody or the antibody fragment thereof comprises a pair of variable heavy chain 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 skilled in the art will understand that the variable light chain and variable heavy chain may be selected independently or mixed and matched to produce an anti-IL-23p19 antibody comprising a combination of variable heavy chain and variable light chain that is distinct from the pairs identified above.

[0193] In an embodiment, the anti-IL-23p19 antibody or the antibody fragment thereof comprises a pair of variable heavy chain and variable light chain sequences selected from the following combinations: a variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO. 1 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO. 2; a variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO. 3 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO. 4; a variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO. 5 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO. 6; and a variable heavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO. 7 and a variable light chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO. 8. Those skilled in the art will understand that the variable light chain and variable heavy chain may be selected independently or mixed and matched to produce an anti-IL-23p19 antibody comprising a combination of variable heavy chain and variable light chain that is distinct from the pairs identified above.

[0194] In some embodiments, the anti-IL-23p19 antibody (e.g., antagonist antibody) binds with high affinity to the p19 subunit of IL-23 and does not bind to the p40 subunit of IL-12, a member of the related cytokine family.

[0195] In some embodiments, the antibody is a full-length antibody. In other embodiments, the antibody is an antibody fragment selected from the group consisting of, for example, Fab, Fab', F(ab)2, Fv, domain antibodies (dAbs), and complementarity-determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies, triabdies, tetrabodies, miniantibodies, and polypeptides comprising at least a portion of immunoglobulin sufficient to confer IL-23 specific binding to the polypeptide.

[0196] In some embodiments, the variable region domain of the anti-IL-23p19 antibody disclosed herein may be attached by covalent bonding to at least one other antibody domain or fragment thereof at the C-terminal amino acid. Thus, for example, the VH domain present in the variable region domain may be linked to the immunoglobulin CH1 domain or fragment thereof. Similarly, the VL domain may be linked to the CK domain or fragment thereof. In this way, for example, the antibody may be a Fab fragment comprising the associated VH and VL domains in which the antigen-binding domain is covalently linked to the CH1 and CK domains at their respective C-terminuses. The CH1 domain may be extended by additional amino acids to provide, for example, a hinge region or part of the hinge region domain present in the Fab fragment, or to provide additional domains such as the antibody CH2 and CH3 domains.

[0197] In some embodiments, the variable region domain of the anti-IL-23p19 antibody may be attached by covalent bonding to at least one other antibody domain or fragment thereof at the C-terminal amino acid. Thus, for example, the VH domain present in the variable region domain may be linked to the immunoglobulin CH1 domain or fragment thereof. Similarly, the VL domain may be linked to the CK domain or fragment thereof. In this way, for example, the antibody may be a Fab fragment comprising the associated VH and VL domains in which the antigen-binding domain is covalently linked to the CH1 and CK domains at their respective C-terminuses. The CH1 domain may be extended by additional amino acids to provide, for example, a hinge region or part of the hinge region domain present in the Fab' fragment, or to provide additional domains such as the antibody CH2 and CH3 domains.

[0198] Accordingly, in one embodiment, the antibody fragment comprises at least one CDR as described herein. The antibody fragment may comprise at least two, three, four, five, or six CDRs as described herein. The antibody fragment may further comprise at least one variable region domain of the antibody described herein. The variable region domain may be of any size or amino acid composition and generally comprises at least one CDR sequence involved in binding to human IL-23p19, e.g., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3 described herein, which is adjacent to or within one or more framework sequences.

[0199] 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 an alternative embodiment, 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.

[0200] In additional aspects, the anti-IL-23p19 antibody or its antibody fragment exhibits one or more of the following characteristics:

[0201] (a) specific to human IL-23p19 and capable of blocking IL-23 from binding to its receptor (IL-23R);

[0202] (b) inhibiting, interfering with, or modulating IL-23p19 interactions with IL-23 receptor signaling;

[0203] (c) inhibiting STAT3 activation induced by IL-23;

[0204] (d) inhibiting human IL-23-induced IL-17 production in mouse splenocytes;

[0205] (e) inhibiting human IL-17 production induced by human IL-23 in human T-cells activated in PBMCs;

[0206] (f) without inhibiting IL-12Rβ1 signaling and IL-23 interaction;

[0207] (g) without inhibiting human IL-12-induced interferon gamma production in human activated T-cells (PBMCs);

[0208] (h) Cynomolgus monkey IL-12-induced interferon gamma production in human activated T-cells (PBMCs) without inhibiting;

[0209] (i) Inhibits skin inflammation induced by human IL-23 in a murine psoriasis-like model.

[0210] In one embodiment, an anti-IL-23p19 antibody or a fragment of the antibody thereof may reduce, inhibit, interfere with, and / or modulate at least one of the biological responses associated with IL-23, and thus be useful for improving the effects of IL-23-related diseases or disorders. Such antibodies and fragments of the antibody thereof may be used, for example, to reduce, inhibit, interfere with, and / or modulate IL-23 signaling, IL-23 activation in Th17 cells, IL-23 activation in NK cells, or to induce the production of pro-inflammatory cytokines.

[0211] In some embodiments, the anti-IL-23p19 antibody or antibody fragments thereof comprise one or more conservative amino acid substitutions. Those skilled in the art will recognize that a conservative amino acid substitution is a substitution of one amino acid to another amino acid having similar structural or chemical properties, such as a similar side chain. Exemplary conservative substitutions are described in the art, for example, in the literature [Watson et al., Molecular Biology of the Gene, The Benjamin / Cummings Publication Company, 4th Ed. (1987)].

[0212] "Conservative modifications" refer to amino acid modifications that do not significantly affect or alter the binding properties of antibodies containing amino acid sequences. Conservative modifications include amino acid substitutions, additions, and deletions. A conservative substitution is the replacement of an amino acid with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are well defined and include 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), non-charged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., tryptophenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine), and sulfur-containing side chains (cysteine, It includes amino acids having methionine. Furthermore, as previously described for alanine scanning mutagenesis, any natural residue of the polypeptide may also be substituted with alanine (References [MacLennan et al. (1998) Acta Physiol Scand Suppl 643: 55-67; ​​Sasaki et al. (1998) Adv Biophys 35: 1-24]). Amino acid substitution for the antibody of the present invention may be achieved by known methods, for example, by PCR mutagenesis (U.S. Patent No. 4,683,195).

[0213] 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 formed as a chimeric or humanized antibody. In another embodiment, the anti-IL-23p19 antibody or antibody fragment thereof comprises all six CDR regions of one of the disclosed complete human antibodies.

[0214] Antibody generation method

[0215] Anti-IL-23p19 antibodies or antibody fragments thereof may be prepared by any method known in the art. For example, the receptor may be immunized with a soluble recombinant human IL-23 protein, or a fragment or peptide conjugated to a carrier protein thereof. Suitable immunization methods may be used. Such methods may include the use of an adjuvant, other immunostimulators, repetitive booster immunization, and one or more immunization pathways.

[0216] Any suitable source of human IL-23 may be used as an immunogen for the production of non-human or human anti-IL-23p19 antibodies of the compositions and methods disclosed herein.

[0217] Different forms of IL-23 antigens can be used to generate antibodies sufficient to produce biological activity. Thus, the inducing IL-23 antigen may be a single epitope, multiple epitopes, or the whole protein alone, or it may be in combination with one or more immunogenicity enhancers. In some aspects, the inducing antigen is an isolated soluble full-length protein, or a soluble protein containing less than the full-length sequence (e.g., immunized with a peptide containing a specific portion or epitope of IL-23). ​​As used herein, the term “some” refers to, where appropriate, a minimum number of amino acids or nucleic acids to constitute the immunogenic epitope of the target antigen. Any genetic vector suitable for the transformation of target cells may be used, including, but not limited to, adenovirus vectors, plasmids, and non-viral vectors such as cationic lipids.

[0218] It is desirable to produce monoclonal antibodies (mAbs) from various mammalian hosts such as mice, rodents, primates, and humans. Descriptions of techniques for producing such monoclonal antibodies are provided in, for example, the literature [Sties et al. (eds.) BASIC AND CLINICAL IMMUNOLOGY (4th ed.) Lance Medical Publication, Los Altos, CA] and the references cited herein; literature [Harlow and Lane (1988) ANTIBODIES: A LABORATORY MANUAL CSH Press; Goding (1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2 nd It can be found in [ed.] Academic Press, New York, NY. Generally, splenocytes of animals immunized with appropriate antigens are typically immortalized by fusion with myeloma cells. Literature [Kohler and Milstein (196) Eur. J. Immunol.Refer to [6:511-519]. Alternative methods of immortalization include transformation using Epstein-Barr virus, oncogenes, or retroviruses, or other methods known in the art. For example, refer to the literature [Doyle et al. (eds. 1994 and periodic supplements) CELL AND TISSUE CULTURE: LABORATORY PROCEDURES, John Wiley and Sons, New York, NY]. Colonies arising from a single immortalized cell are screened for the production of antibodies with appropriate specificity and affinity for the antigen, and the yield of monoclonal antibodies produced by these cells can be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, for example, refer to the literature [Huse et al., (1989) Science DNA sequences encoding monoclonal antibodies or their antigen-binding fragments can be isolated by screening a DNA library from human B cells according to the general protocol summarized in [246: 1275-1281]. Thus, antibodies can be obtained by various techniques familiar to those skilled in the art.

[0219] Other suitable techniques include the selection of antibody libraries from phages, yeasts, viruses, or similar vectors. For example, the literature [Huse et al., supra; and Ward et al., (1989) NatureRefer to [341:544-546]. The polypeptides and antibodies disclosed herein may be used in the presence or absence of modifications, including chimeric or humanized antibodies. Often, polypeptides and antibodies will be labeled by linking substances that provide a detectable signal by covalent or non-covalent bonds. Various labeling and conjugation techniques are known and have been extensively reported in both scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moiety, chemiluminescent moiety, magnetic particles, etc. Patents teaching the use of such labels include U.S. Patents No. 3,817,837; No. 3,850,752; No. 3,996,345; No. 4,277,437; No. 4,275,149; and No. 4,366,241. In addition, recombinant immunoglobulins may be produced (see literature [Cabilly U.S. Patent No. 4,816,567; and Queen et al. (1989) Proc. Nat'l Acad. Sci. USA [See 86: 10029-10023]); produced in transgenic mice (literature [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)], see Medarex, Trianni, Abgenix, Ablexis, OminiAb, Harbour and other technical references).

[0220] In some embodiments, the ability of the generated antibody to bind to IL-23p19 may be evaluated using standard binding assays such as surface plasmon resonance (SPR), octet (BLI), ELISA, Western blot, immunofluorescence, flow cytometry, chemotaxis assay, and cell migration assay. In some aspects, the generated antibody may also be evaluated for the ability to inhibit the blockade of IL-23 receptor β1 signaling by IL-23, including the inhibition of IL-23-induced Stat3 phosphorylation, IL-17 production, and / or IFN-γ production, and for the inhibition of the continuity effects of IL-23p19 and / or IL-23p19-mediated inflammatory microenvironment.

[0221] Antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a representative purification technique. The suitability of protein A as an affinity ligand depends on the species and isoform 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 (e.g., see Lindmark et al., 1983 J. Immunol. Meth. 62:1-13). Protein G is recommended for all mouse isoforms and human γ3 (e.g., see Guss et al., 1986 EMBO J. 5:1567-1575). The matrix to which the affinity ligand is attached is mostly agarose, but other matrices may also be used. Mechanically stable matrices, such as controlled porous glass or poly(styrene-divinyl)benzene, enable faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reverse-phase HPLC, silica chromatography, heparin chromatography, SEPHAROSE™ chromatography on anion or cation exchange resins (e.g., polyaspartate columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the antibody to be recovered.

[0222] After any preliminary purification step(s), a mixture containing the target antibody and contaminants can be applied to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5 to 4.5, typically performed at a low salt concentration (e.g., about 0 to 0.25 M salt).

[0223] Additionally, the invention comprises a nucleic acid that hybridizes to all or part of a nucleotide sequence (e.g., a portion encoding a variable region) represented by isolated polynucleotide sequence(s) encoding the antibody or antibody fragment of the invention, under low, medium, and high severity conditions as defined herein. The hybridized portion of the hybridized nucleic acid is typically at least 15 nucleotides in length (e.g., 20, 25, 30, or 50). The hybridized portion of the hybridized nucleic acid is at least 80%, e.g., at least 90%, at least 95%, or at least 98% identical to the sequence of all or part of the nucleic acid encoding the anti-IL-23p19 polypeptide (e.g., heavy or light chain variable region) or its complement. Hybridized nucleic acids of the type described herein may be used, for example, as cloning probes, primers, e.g., PCR primers, or diagnostic probes.

[0224] Polynucleotides, vectors, and cells

[0225] Other embodiments include an isolated polynucleotide comprising a sequence encoding an anti-IL-23p19 antibody or an antibody fragment thereof, a vector and a cell comprising the polynucleotide, and a recombinant technology for generating the antibody. The isolated polynucleotide may encode any suitable form of an anti-IL-23p19 antibody, for example, a full-length monoclonal antibody, Fab, Fab', F(ab')2, and Fv fragments, a diabadi, a linear antibody, a single-chain antibody molecule, a miniantibody, and a multispecific antibody formed from an antibody fragment.

[0226] Some embodiments include an isolated polynucleotide comprising a sequence encoding a light chain variable region of an antibody or antibody fragment having any one of the amino acid sequences 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 sequences of SEQ ID NOs. 1, 3, 5, and 7.

[0227] In an embodiment, the isolated polynucleotide sequence(s) are

[0228] (a) a variable heavy chain sequence comprising SEQ ID NO. 1 and a variable light chain sequence comprising SEQ ID NO. 2;

[0229] (b) a variable heavy chain sequence including SEQ ID NO. 3 and a variable light chain sequence including SEQ ID NO. 4;

[0230] (c) a variable heavy chain sequence comprising SEQ ID NO. 5 and a variable light chain sequence comprising SEQ ID NO. 6; or

[0231] (d) a variable heavy chain sequence including SEQ ID NO. 7 and a variable light chain sequence including SEQ ID NO. 8

[0232] Encodes an antibody or antibody fragment having a light chain and a heavy chain variable region containing the amino acid sequence of

[0233] In another embodiment, the isolated polynucleotide sequence(s) are

[0234] (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;

[0235] (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;

[0236] (c) a variable heavy chain sequence 90%, 95%, or 99% identical to SEQ ID NO. 5 and a variable light chain sequence 90%, 95%, or 99% identical to SEQ ID NO. 6; or

[0237] (d) 90%, 95%, or 99% identical variable heavy chain sequences in SEQ ID NO. 7 and 90%, 95%, or 99% identical variable light chain sequences in SEQ ID NO. 8

[0238] Encodes an antibody or antibody fragment having a light chain and a heavy chain variable region containing the amino acid sequence of

[0239] Polynucleotide(s) comprising a sequence encoding an anti-IL-23p19 antibody or a fragment thereof may be fused to one or more regulatory or control sequences as known in the art and may be incorporated into a suitable expression vector or cell as known in the art. Each polynucleotide molecule encoding a heavy or light chain variable domain may be independently fused to a polynucleotide sequence encoding a constant domain, such as a human constant domain, to enable the production of an intact antibody. Alternatively, polynucleotides or parts thereof may be fused together to provide a template for the production of a single-chain antibody.

[0240] For recombinant generation, a polynucleotide encoding an antibody is inserted into a replicatable vector for cloning (DNA amplification) or expression. Many suitable vectors are available for expressing recombinant antibodies. Vector components generally include, but are not limited to, one or more of a signal sequence, a replication origin, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0241] The anti-IL-23p19 antibody or its antibody fragment can also be produced as a fusion polypeptide, in which the antibody or fragment is fused with a heterogeneous polypeptide, such as another polypeptide or signal sequence having a specific cleavage site at the amino terminus of the mature protein or polypeptide. The selected heterogeneous signal sequence is typically one that is recognized and processed by the cell (i.e., cleaved by a signal peptidase). For prokaryotic cells that do not recognize and process the anti-IL-23p19 antibody signal sequence, the signal sequence may be substituted with a prokaryotic signal sequence. The signal sequence may be, for example, alkaline phosphatase, penicillinase, lipoprotein, heat-stable enterotoxin II leader, etc. In the case of yeast secretion, the natural signal sequence may be, for example, yeast invertase alpha-factor (Saccharomyces ( Saccharomyces ) and Cluiveromyces( Kluyveromyces ) including α-factor leader), acid phosphatase, C. albicans( C. albicans ) It can be substituted with a glucoamylase, or a leader sequence obtained from the signal described in WO 90 / 13646. In mammalian cells, as well as mammalian signal sequences, a viral secretion leader, e.g., the herpes simplex gD signal, can be used. The DNA of these precursor regions is linked to the DNA encoding the anti-IL-23p19 antibody in the reading frame.

[0242] Expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected cells. Generally, in cloning vectors, this sequence allows the vector to replicate independently of host chromosomal DNA and includes a replication origin or self-replication sequence. These sequences are well known for various bacteria, yeasts, and viruses. The replication origin of the plasmid pBR322 is suitable for 2-υ, which is a majority of Gram-negative bacteria. Plasmid-derived origins are suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV, and BPV) are useful for cloning vectors in mammalian cells. Generally, the replication origin component is not required for mammalian expression vectors (although the SV40 origin can be used because it typically contains an early promoter).

[0243] Expression and cloning vectors may include genes encoding selectable markers to facilitate the verification of expression. Typical selectable marker genes encode proteins that confer resistance to antibiotics or other toxins (e.g., ampicillin, neomycin, methotrexate, or tetracycline), or alternatively, confer complement nutrient deficiency, or alternatively, supply specific nutrients not present in the complex medium; for example, the gene is Bacillus ( Bacilli Encrypts D-alanine racemase for ).

[0244] Non-therapeutic use

[0245] The anti-IL-23p19 antibody or antibody fragment described herein is useful as an affinity purification agent. In this process, the antibody is immobilized on a solid phase, such as a protein A resin, using a method well known in the art. After contacting the immobilized antibody with a sample containing the IL-23p19 protein (or a fragment thereof) to be purified, the support is washed with a suitable solvent to remove substantially all material from the sample except for the IL-23p19 protein that binds to the immobilized antibody. Finally, the support is washed with another suitable solvent that releases the IL-23p19 protein from the antibody.

[0246] Anti-IL-23p19 antibodies or antibody fragments are also useful for diagnostic assays to detect and / or quantify IL-23p19 proteins, e.g., to detect IL-23p19 expression in specific cells, tissues, or serum. Anti-IL-23p19 antibodies may be used diagnostically to monitor the onset or progression of a disease, for example, as part of a clinical trial procedure to determine the efficacy of a specific treatment and / or prophylactic regimen. Detection can be facilitated by conjugating anti-IL-23p19 antibodies to detectable substances. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, radioactive substances, positron-emitting metals used in various positron emission tomography, and non-radioactive paramagnetic metal ions. For example, refer to U.S. Patent No. 4,741,900 for metal ions that can be conjugated to antibodies for use as a diagnosis according to the present disclosure.

[0247] Anti-IL-23p19 antibodies or antibody fragments may be used in a method to diagnose IL-23p19-related disorders (e.g., disorders characterized by abnormal expression of IL-23p19) or to determine that a subject has an increased risk of developing an IL-23p19-related disorder. Such a method comprises the step of contacting a biological sample of a subject with an anti-IL-23p19 antibody or a fragment thereof and detecting the binding of the antibody to IL-23p19. "Biological sample" means any biological sample obtained from an individual, cell line, tissue culture, or other cell source potentially expressing IL-23p19. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.

[0248] In some embodiments, the method further includes the step of determining whether a patient has an IL-23p19-related disorder or is at risk of developing an IL-23p19-related disorder by comparing the level of IL-23p19 in a patient sample with that of a control sample (e.g., a subject without an IL-23p19-related disorder).

[0249] In some embodiments, for example, for diagnostic purposes, it may be advantageous to label the antibody with a detectable moiety. Numerous detectable labels may be used, including radioisotopes, fluorescent labels, enzyme substrate labels, etc. The label may be indirectly conjugated to the antibody using various known techniques. For example, the antibody may be conjugated to biotin, and any of the three broad categories of labels mentioned above may be conjugated to avidin, or vice versa. Since biotin selectively binds to avidin, the label can be conjugated to the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label and the antibody, the antibody may be conjugated to a small hapten (e.g., digoxin), and one of the other types of labels mentioned above may be conjugated to an anti-hapten antibody (e.g., anti-dioxin antibody). Thus, indirect conjugation of the antibody and the label can be achieved.

[0250] An exemplary radioactive isotope label is 35 S, 14 C, 125 I, 3 H, and 131 Includes I. Antibodies may be labeled with radioisotopes using techniques described, for example, in the literature [Current Protocols in Immunology, Volumes 1 and 2, 1991, Coligen et al., Ed. Wiley-Interscience, New York, NY, Pubs]. Radioactivity may be measured, for example, by scintillation counting.

[0251] Exemplary fluorescent labels include labels derived from rare earth chelates (europium chelate) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lissamine, phycoerythrin, and Texas Red. Fluorescent labels may be conjugated to antibodies via known techniques, such as those disclosed in the literature [Current Protocols in Immunology], for example. Fluorescence may be quantified using a fluorescence analyzer.

[0252] There are various enzyme-substrate labels well known in the art (see, for example, U.S. Patent No. 4,275,149). Enzymes generally catalyze chemical changes in chromogenic substrates that can be measured using various techniques. For example, the change may be a change in the color of the substrate that can be measured by a spectrophotometer. Alternatively, enzymes may change the fluorescence or chemiluminescence of the substrate. Techniques for quantifying changes in fluorescence are described above. Chemiluminescent substrates may be electronically excited by a chemical reaction and emit light that can be measured, for example, using a chemiluminometer, or provide energy to a fluorescence receptor.

[0253] Examples of enzyme labels include luciferases such as firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazindione, maleate dehydrogenase, urease, peroxidase, e.g. horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocycle oxidases (e.g., ureicase and xanthine oxidase), lactoperoxidase, and microperoxidase. The technique of conjugating enzymes to antibodies is, for example, described in the literature [O'Sullivan et al., 1981, Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. It is reported in [J. Langone & H. Van Vunakis, eds.], Academic press, NY, 73: 147-166.

[0254] Examples of enzyme-substrate combinations include, for example, horseradish peroxidase (HRPO) with hydrogen peroxide as a substrate, where hydrogen peroxide is orthophenylenediamine (OPD) or 3,3,5,5-tetramethylbenzidine hydrochloride (TMB); alkaline phosphatase (AP) having para-nitrophenyl phosphate as a chromogenic substrate; and β-D-galactosidase (β-D-Gal) having a chromogenic substrate such as p-nitrophenyl-β-D-galactosidase or fluorescent substrate 4-methylumbelliferyl-β-D-galactosidase.

[0255] In another embodiment, the anti-IL-23p19 antibody or its antibody fragment is used in an unlabeled state and is detected as a labeled antibody that binds to the anti-IL-23p19 antibody or its antibody fragment.

[0256] The antibodies and antibody fragments described herein may be used in any known analytical method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. For example, refer to the literature [Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987)].

[0257] Anti-IL-23p19 antibodies or antibody fragments thereof may be used to inhibit the binding of a ligand to the IL-23 receptor. Such methods include the step of administering an anti-IL-23p19 antibody to a cell (e.g., mammalian cell) or the cellular environment to inhibit signal transduction mediated by the IL-23 receptor. These methods may be performed in vitro or in vivo. "Cellular environment" refers to the tissue, medium, or extracellular matrix surrounding the cell.

[0258] Therapeutic composition and method

[0259] The present disclosure also provides compositions comprising, for example, pharmaceutical compositions comprising an anti-IL-23p19 antibody or a fragment thereof. Such compositions have numerous therapeutic uses for the treatment, prevention, or improvement of diseases or disorders such as immune-mediated inflammatory disorders or autoimmune diseases (e.g., diseases or disorders involving biological activity mediated by the IL-23 / IL-23 receptor signaling axis).

[0260] The anti-IL-23p19 antibody or antibody fragment thereof disclosed herein is useful for treating various diseases or disorders such as immune-mediated inflammatory disorders (IMID) or autoimmune diseases. A method for treating IL-23-related disorders comprises the step of administering a therapeutically effective amount of the anti-IL-23p19 antibody or antibody fragment thereof to a subject in need. IMIDs may 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, and asthma.

[0261] The present disclosure also provides a method for treating or preventing IMID, comprising the step of administering to a subject in need a composition or formulation comprising an anti-IL-23p19 antibody or a fragment of the antibody thereof, and optionally another immune-based therapy.

[0262] The disclosed antibody is also useful for methods of treating cancer alone (e.g., monotherapy) or in combination with other immunotherapies and / or chemotherapy.

[0263] The antibody may be administered alone or in combination with other compositions useful for treating immune-mediated inflammatory disorders or autoimmune diseases. In some embodiments, for example, a composition comprising a pharmaceutical composition containing an anti-IL-23p19 antibody may further comprise a therapeutic agent that is conjugated to or unconjugated to a binder.

[0264] In some aspects, a composition comprising one or more antibodies disclosed herein, e.g., a pharmaceutical composition, is provided. The pharmaceutical composition may be formulated with any other known adjuvant and excipient, as well as a pharmaceutically acceptable carrier or diluent, according to conventional techniques such as those disclosed in the literature [Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995].

[0265] Typically, the composition for administration by injection is a solution in a sterile isotonic aqueous buffer. If necessary, the agent may include a solubilizer and a local anesthetic, such as lignocaine, to relieve pain at the injection site. Generally, the components are mixed together, either individually or in unit doses, and are provided as a dry lyophilized powder or an anhydrous concentrate in a completely sealed container, such as an ampoule or sachet, that indicates the amount of active agent. When the agent is administered by injection, it may be dispensed with an infusion bottle containing sterile pharmaceutical-grade water or saline. When the agent is administered by injection, an ampoule of sterile water or saline for injection may be provided to allow the components to be mixed prior to administration.

[0266] As used herein, "pharmaceutically acceptable carriers" include any physiologically mutually compatible solvents, dispersion media, coating agents, antimicrobial and antifungal agents, isotonic agents, absorption retardants, etc. Preferably, the carriers are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., injection or infusion). Depending on the route of administration, the active compound, i.e., antibodies, bispecific and multispecific molecules, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0267] The composition may be administered by various methods known in the art. As understood by those skilled in the art, the route and / or mode of administration will vary depending on the appropriate result. The active compound may be prepared with a carrier that protects the compound from rapid release, such as in controlled-release formulations including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Methods for preparing such formulations are generally known to those skilled in the art. For example, refer to the literature [Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978].

[0268] The dosage level of the active ingredient in a pharmaceutical composition may vary to obtain an amount of the active ingredient effective in achieving an appropriate therapeutic response for a specific subject, composition, and mode of administration without toxicity to the subject. The selected dosage level depends on various pharmacokinetic factors, including the activity of the specific composition used, the route of administration, the time of administration, the elimination rate of the specific compound used, the duration of treatment, other drugs, compounds, and / or substances used in combination with the specific composition used, the age, sex, weight, condition, general health, and medical history of the patient being treated, and similar factors widely known in the medical field.

[0269] The pharmaceutical compositions described herein may be administered in an effective amount. "Effective amount" refers to an amount that achieves an appropriate response or appropriate effect, either alone or in combination with additional doses. In the case of the treatment of a specific disease or specific pathological condition, an appropriate response preferably relates to the inhibition of the disease course. This includes slowing the progression of the disease, particularly hindering or reversing the progression of the disease.

[0270] In some aspects, the compositions described herein are administered to a patient, for example in vivo, to treat or prevent various disorders such as those described herein. Preferred patients include human patients having disorders that can be corrected or improved by administering agents that modulate the biological activity of the IL-23 / IL-23 receptor signaling axis.

[0271] In some aspects, nucleic acids encoding antibodies or derivatives thereof as described herein may be introduced into mammalian cells or target tissues using conventional viral and non-viral-based gene delivery methods. These methods may be used to administer nucleic acids encoding antibodies into cells in vitro. In some embodiments, nucleic acids encoding antibodies or derivatives thereof are administered for in vivo or in vitro gene therapy applications. In other embodiments, gene delivery technology is used to study antibody activity in cell-based or animal models. Non-viral vector delivery systems include nucleic acids conjugated with delivery vehicles such as DNA plasmids, naked nucleic acids, and liposomes. Viral vector delivery systems include DNA and RNA viruses containing episomes or incorporated genomes after delivery to cells. These methods are well known in the art.

[0272] Non-viral delivery methods of nucleic acids encoding engineered polypeptides of the present disclosure include lipofection, microinjection, biolistic, virosome, liposome, immunoliposome, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and formulation-enhanced absorption of DNA. Lipofection methods and lipofection reagents are well known in the art (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO 91 / 17424 and WO 91 / 16024. Delivery may be made to cells (in vitro administration) or target tissues (in vivo administration). The preparation of lipid:nucleic acid conjugates comprising targeted liposomes, such as immunolipid conjugates, is well known to those skilled in the art.

[0273] The use of RNA or DNA virus-based systems for the delivery of nucleic acids encoding antibodies described herein takes advantage of highly evolved processes that target viruses to specific cells in the body and transport viral contents to the nucleus. Viral vectors can be used for direct administration to a patient (in vivo) or to treat cells in vitro, or for administering modified cells to a patient (in vitro). Conventional virus-based systems for the delivery of polypeptides of this disclosure may include retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors for gene delivery. Viral vectors are currently the most efficient and versatile gene delivery methods for target cells and tissues. Incorporation of the host genome is possible with retroviral, lentiviral, and adeno-associated virus gene delivery methods and often results in long-term expression of the inserted transgenic gene. Furthermore, high transduction efficiencies have been observed in various cell types and target tissues. All identified patents and publications are expressly incorporated herein by reference for the purpose of describing and disclosing methodologies described in such publications that may be used, for example, in connection with the present disclosure. Such publications are provided only for disclosures prior to the filing date of this application. In this regard, it should not be construed in any case that the inventors do not prior art or for any other reason to such disclosures. Any statements regarding dates or expressions in the contents of this document are based on information available to the applicant and do not constitute an acknowledgment that the dates or contents of such document are accurate.

[0274] It will be understood by those skilled in the art that, to the extent not yet presented, any of the various embodiments described and illustrated in this specification may be further modified by incorporating features provided in any of the other embodiments disclosed in this specification.

[0275] The broad scope of the present disclosure is best understood by referring to the following examples, which are not intended to limit the present disclosure to specific embodiments. The specific embodiments described herein are provided merely as examples, and the present disclosure is limited to the full scope of equivalents accompanying the appended claims.

[0276] Examples

[0277] General method

[0278] Protein purification methods including immunoprecipitation, chromatography, and electrophoresis are described. Reference [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, generation of fusion proteins, and glycosylation of proteins are described. For example, the literature [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 BiologyRefer to [ , Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, NJ, pp. 384-391]. The generation, purification, and fragmentation of polyclonal and monoclonal antibodies are described. See the literature [Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra].

[0279] The hybridoma supernatant was purified using a HiTrap Protein G column (GE, catalog number 17040401) according to the manufacturer's procedure. Briefly, the Protein G column was equilibrated with 5 CV DPBS (Gibco, catalog number 14190-136), and the hybridoma supernatant was loaded via a syringe / infusion pump (Legato 200, KDS) at ambient temperature with a retention time of 3 minutes. The column was washed with 5 CV DPBS, and elution was performed using 4 CV pH 2.8 elution buffer (Fisher Scientific, catalog number PI21004). The eluent was fractionated, the fractions were neutralized with 1 M Tris-HCl, pH 8.5 (Fisher Scientific, catalog number 50-843-270), and analyzed using an A280 (DropSense96, Trinean). Peak fractions were pooled, and the buffer was replaced with DPBS. A centrifuge filter (EMD Millipore, catalog number UFC803024) was equilibrated in DPBS at 4,000 xg for 2 minutes. The purified sample was loaded, DPBS was added, and the sample was spun at 4,000 xg for 5 to 10 minutes until the total DPBS volume reached 6 DV or more. The final pool was analyzed with A280.

[0280] Standard methods in molecular biology are described. Maniatis et al., (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif. Standard methods also appear in Ausbel et al., (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4)].

[0281] The sequences of the heavy and light chain variable regions for the hybridoma clones were determined as described below. 1 to 2 x 10⁻⁶ were used with the Qiagen (Germantown, MD, USA) RNeasy Plus Mini kit. 6Total RNA was extracted from hybridoma cells. cDNA was generated by performing a 5' race reaction using the SMARTer RACE 5' / 3' kit from Takara (Mountain View, CA, USA). PCR was performed using Q5 High-Fidelity DNA Polymerase from NEB (Ipswich, MA, USA) to amplify the variable regions of the heavy and light chains using the Takara Universal Primer Mix, combined with gene-specific primers for the 3' mouse constant regions of the appropriate immunoglobulins. The amplified variable regions for the heavy and light chains were run on a 2% agarose gel, appropriate bands were cleaved, and the gel was purified using the Qiagen Mini Elute Gel Extraction kit. The purified PCR products were cloned using the Zero Blunt PCR Cloning kit from Invitrogen (Carlsbad, CA, USA), transformed into Takara's Stellar Competent E. coli cells, and plated on LB agar + 50 μg / ml kanamycin plates. Direct colony Sanger sequencing was performed using GeneWiz (South Plainfield, NJ, USA). The generated nucleotide sequences were analyzed using IMGT V-QUEST to confirm productive rearrangements and to analyze the translated protein sequences. CDR determination was based on the IMGT number.

[0282] Flow cytometry methods including a Fluorescence-Activated Cell Classification Detection System (FACS®) may be used. For example, see the literature [Owens et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2nd ed.; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ]. Fluorescent reagents suitable for nucleic acid modifications, for example, for use as diagnostic reagents, may be used, including nucleic acid primers and probes, polypeptides, and antibodies. See the literature [Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo].

[0283] Positive controls (PC1 and PC2), IL-23p19 and IL-12 / IL-23 p40 specific antibodies were prepared by a CRO (Biointron). For example, control antibodies can be prepared by any suitable expression method by cloning antibody heavy and light chain variable regions into the 293F or ExpiCHO™ expression system (ThermoFisher Scientific, Waltham, MA). These antibodies were used as controls to establish the binding and functional analyses described in the Examples and were tested in conjunction with the disclosed newly generated anti-IL-23p19-specific antibody. "PC1" refers to a reference antibody synthesized based on the VH and VL sequences reported in US 7,935,344 (VH sequence number 106 and VL sequence number 116 of the '344 patent) and known to be specific to the human IL-123p19 subunit (Biointron, entry number 20180926A04). The term "PC2" refers to a reference known to be specific to the human IL-12 / IL-23 p40 subunit (BIOINTRON item number 20180925A07) synthesized based on the VH and VL sequences reported in US 6,902,734 (VH sequence number 7 and VL sequence number 8 of the '734 patent).

[0284] Control antibodies can be prepared by standard methods. For example, a plasmid containing the sequence of the control antibody can be transfected using the mammalian system (293F or ExpiCHO™) (Catalog No. A29133, ThermoFisher Scientific, USA) according to the manufacturer's protocol. Cells are cultured at 37°C and 8% CO2 on Day 1, and then cultured at 32°C and 5% CO2 after transfection in the medium provided in the kit. The antibody is purified by centrifuging the ExpiCHO™ culture medium at 1,000 g for 10 minutes, followed by centrifugation at 5,000 g for 30 minutes. The supernatant is then filtered using a 0.45 μm filter followed by a 0.22 μm filter. Subsequently, the supernatant is applied to affinity purification using protein A / G resin (Life Technologies, Carlsbad, CA; Catalog# 20424) according to the manufacturer's protocol. Before ELISA purification, the antibody titer of the culture medium is roughly determined to ensure that the loaded medium accounts for less than 80% of the resin binding capacity. After incubation, the resin is washed with PBS and eluted with elution buffer (Life Technologies, Catalog # 21004). The elution fraction is immediately adjusted to physiological pH by adding Tris buffer at pH 8.0. The purified antibody is subsequently subjected to buffer replacement and protein concentration in PBS buffer using an Amicon Ultra-15 centrifuge filter unit (Life Technologies, Catalog # UFC900324). Antibody concentration is determined by BCA protein analysis. Antibody purity is tested by performing SDS-PAGE and Coomassie staining. The purified protein is aliquoted and stored at -80°C for long-term storage or at 4°C for immediate use.

[0285] The integrity of the antibody can be verified by Coomassie staining under non-reducing versus reducing conditions following SDS-PAGE; under non-reducing conditions, a single distinct band is observed at approximately 150 kDa, while under reducing conditions, two bands are observed at 50 kDa and 25 kDa. Standard techniques can be used to characterize ligand / receptor interactions. For example, refer to the literature [Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York]. Standard methods for antibody functional characterization suitable for characterizing antibodies with specific mechanisms of action are also well known to those skilled in the art.

[0286] For example, software packages and databases are available to determine antigen fragments, leader sequences, protein folding, functional domains, CDR annotations, glycosylation sites, and sequence alignment.

[0287] Example 1: Production of anti-IL-23p19 antibody

[0288] Human anti-IL-23p19 specific antibodies were generated by immunizing human Ig transgenic mice (see, e.g., WO 2013 / 063391, TRIANNI® mouse).

[0289] Immunization: Trianni® mice were immunized with human IL-23 recombinant protein or a combination of human IL-23 protein and a heterodimer of human p19 and mouse p40 proteins by intraperitoneal (IP), subcutaneous (SC), or paw pad or tail base injection. The immune response was monitored by retroorbital hemorrhage. Plasma was screened for binding activity to the human IL-23 heterodimer by ELISA (as described below). Mice with sufficient titers were used for fusion. Mice were boosted with immunogens before sacrifice and removal of the spleen and drainage lymph nodes.

[0290] Selection of mice that produce anti-IL-23p19 antibodiesTo select Trianni mice that produce p19-specific antibodies, serum from immunized mice was screened by ELISA for binding to recombinant human IL-23. In summary, ELISA plates coated with recombinant human IL-23 were incubated with serum dilutions from immunized mice at room temperature for 1 hour, the assay plates were washed, and specific antibody binding was detected using HRP-labeled anti-mouse IgG antibodies. The plates were read using an ELISA reader (Biotek).

[0291] Generation of hybridomas To generate a hybridoma that produces the human antibody of the present disclosure, splenocytes and drained lymph node cells harvested from immunized mice were fused to a suitable immortalized cell line, such as a mouse myeloma cell line. The generated hybridoma was screened for the production of p19-specific antibodies. For example, a single-cell suspension of splenocytes and lymph node cells from immunized mice was fused by electrofusion to an equal number of Sp2 / 0 non-mouse IgG-secreting myeloma cells (ATCC, CRL 1581). The cells were plated on flat-bottom 96-well tissue culture plates and incubated in select medium (HAT medium) for about 2 weeks, after which the medium was replaced with hybridoma culture medium. Approximately 10 to 14 days after cell plating, the supernatant from individual wells was screened by ELISA as described above. Antibody-secreting hybridomas were transferred to 24-well plates, screened again, and if still positive for anti-p19 activity, subcloned by sorting the hybridomas using restriction dilution or a single-cell sorter. Stable subclones were then cultured in vitro to generate small amounts of antibodies for use in purification and characterization.

[0292] Hybridoma screeningAs described above, hybridoma supernatants were tested for IL-23 specific binding using ELISA with human IL-23, human IL-12, and human p19 / mouse p40.

[0293] Example 2: Binding of anti-IL-23p19 specific antibody

[0294] The binding of anti-IL-23p19-specific antibodies to IL-23 proteins was analyzed by surface plasmon resonance (SPR) measured by BIAcore. Briefly, serial dilutions of anti-IL-23p19 antibodies or control antibodies were captured on anti-mouse or human Fc chip(s) immobilized on a CM5 chip using an amine linkage kit (GE Healthcare, Catalog NO: BR-1000-50, Item No. 2087295).

[0295] The control antibodies used in the BIAcore binding assay were PC1 (known as a p19-specific antibody, Biointron, entry number 20180926A04); PC2 (reference antibody known to be specific to the p40 subunit of human IL-12 and IL-23, Biointron, entry number 20180925A07); human IgG isotype control (Invitrogen, catalog number 02-7102, entry number TJ276309); mouse IgG2a isotype control (manufactured by Novarok Biotherapeutics); and human IgG4 isotype control (Dendritics, catalog number DDXCH04P-100; batch: DDXCH04-028) were used as negative controls.

[0296] Next, serial dilutions of IL-23 recombinant protein, human p19 / mouse p40 heteromer protein, human IL-12 protein, and human p40 subunit protein were injected into the immobilized antibody at a rate of 50 μl / min for 1 minute in a drive buffer containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20, and pH 7.4, followed by dissociation for 2 minutes. After each injection, a regeneration step was performed using a 60-second pulse in 10 mM glycine-HCl, pH 1.7 buffer. Experimental data were analyzed using BIAevaluation software (GE Healthcare) and applied to a Langmuir 1:1 model to determine apparent binding.

[0297] The binding characteristics of the purified antibodies are illustrated in Fig. 2. Fig. 2a shows the binding characteristics of Hu-2 18006B (purified from the hybridoma). Fig. 2b shows the binding characteristics of Hu-5 18006B (purified from the hybridoma). Fig. 2c shows the binding characteristics of Hu-6 18006B* (recombinant mIgG2a). Fig. 2d shows the binding characteristics of Hu-4 18006B* (*recombinant, mIgG2a). Fig. 2e shows the binding characteristics of Hu-4 18006B** (**recombinant, hIgG4). Table 3 summarizes the binding specificity of the anti-IL-23p19 antibody and human IL-23 and human p19 / mouse p40 heterodimer proteins by BIAcore. Recombinant antibodies are designated with an asterisk in the table.

[0298] Binding of IL-23p19 specific antibodies by BIAcore antibodies isomorphic hIL23 hp19 / mp40 hIL12 HP40 Hu-2 18006B mIgG1, kappa + + - - Hu-5 18006B mIgG2b, lambda + + - - Hu-6 18006B* mIgG2a, kappa + + - - Hu-4 18006B* mIgG2a, kappa + + - - Hu-4 18006B** hIgG4, kappa + + - - PC1 (p19 specific) hIgG1, lambda + + - - PC2 (p40 specific) hIgG1, kappa + - + + hIgG hIgG - - - - mIgG2a mIgG2a - - - -

[0299] *=IL-23p19 recombinant antibody; PC1 and PC2 are recombinant antibodies; the remaining IL-23p19 Ab is purified from hybridoma.

[0300] The results indicate that the anti-IL-23p19 antibody binds to human IL-23 and the human p19 / mouse p40 heterodimer but does not bind to human IL-12 or the human p40 subunit (Fig. 2 and Table 3).

[0301] PC1 was positive for human IL-23 and human p19 / mouse p40, and negative for human IL-12 and human p40 subunits (data not shown). PC2 was positive for human IL-23, human IL-12, and human p40 subunits, and negative for human p19 / mouse p40 heterodimers. The isomorphic controls mIgG2a and hIgG did not bind to hIL-23, hp19 / mp40, hIL-12, and hp40 subunits.

[0302] result: Anti-IL-23p19-specific antibodies were characterized by binding to human IL-23 and recombinant proteins containing a heterodimer composed of human p19 and mouse p40 subunits. These antibodies did not bind to human IL-12 and human p40 subunits by BIAcore.

[0303] The binding specificity of the initiated anti-IL-23p19 antibody was also evaluated by ELISA. Briefly, biotinylated IL-23 was captured via streptavidin-coated ELISA plates. Human p19 / mouse p40, human IL-12, and human p40 subunits were directly coated onto the ELISA plates. The purified antibodies were then added to the plates and detected with goat-anti-mouse IgG-HRP (Jackson ImmunoResearch, catalog number 115-036-071, entry number 147271). After adding the ABTS substrate (Moss Inc., catalog number ABTS-1000, entry number 03086202), the ELISA plates were read using an ELISA plate reader (Biotek). Controls shown in Fig. 3: PC1 refers to a reference antibody (known as a p19-specific antibody, Biointron, entry number 20180926A04); PC2 refers to a reference antibody (known as a p40-specific antibody, Biointron, entry number 20180925A07); PC3 refers to a reference antibody (MT155, known as a p19-specific antibody of Mabtech, catalog number 3457-6-100, code: 3457-6-1000); and negative controls are human IgG4 (Dendritics, catalog number DDXCHO4P-100, entry number DDXCH04-028) and mouse IgG2a (produced by Novarock Biotherapeutics).

[0304] Figure 3 shows the binding activity of the disclosed p19-specific antibodies. Figures 3a and 3b show that Hu-4 18006B** (hIgG4) and Hu-5 18006B (mIgG2b), Hu-6 18006B* (mIgG2a), Hu-4 18006B* (mIgG2a) and Hu-2 18006B (mIgG1) bind to human IL-23 in a dose-dependent manner; and the positive control PC1 binds to IL-23 in a dose-dependent manner (Figure 3a). Figure 3c shows that these selected representative anti-IL-23p19 antibodies, H-2 1800B (mIgG1), Hu-4 18006B (mIgG2c), Hu-4 18006B* (mIgG2a), and H-6 18006B* (mIgG2a), bind to the human p19 / mouse p40 heterodimer in a dose-dependent manner. Figure 3d shows that these anti-IL-23p19 antibodies do not bind to hIL-12, while the positive control antibody PC2 binds to hIL-12 in a dose-dependent manner. Figure 3e shows that these anti-IL-23p19 antibodies do not bind to the human p40 subunit, while the positive control PC2 binds to the human p40 subunit in a dose-responsive manner.

[0305] of FIGS. 3a to 3e result It was found that anti-IL-23p19-specific antibodies are characterized by binding to human IL-23 and a recombinant protein containing a heterodimer composed of human p19 bound to a mouse p40 subunit by ELISA. These antibodies do not bind to human IL-12 and human p40 subunits.

[0306] To ensure accurate measurements of KD and IC50 endpoints in binding and functional analyses, the antibodies were purified from the hybridoma culture supernatant prior to the test. The binding kinetics of the anti-IL-23p19-specific antibody initiated against recombinant human IL-23 were determined by surface plasmon resonance (SPR) using a BIAcore 3000 system in contact with a previously immobilized CM5 chip via amine linkage chemistry with an anti-mouse IgG antibody (GE catalog number BR-1008-38). Flow cell 1 was maintained in an unmodified state to serve as a reference cell to exclude phylogenetic instrument noise and migration. Fc2-1 detection was driven by double blanking (Fc1 and blank analyte buffer). Antibody samples were diluted to 50 μg / mL in HBS-EP (GE, catalog number BR1001-88) and injected at a flow rate of 10 μL / min for 1 minute. Next, hIL-23 (R&D Systems, catalog number 1290-IL / CF) diluted to 0.156 to 40 nM was injected at a rate of 50 μL / min for 2 minutes and then dissociated for 10 minutes. The data were analyzed using a 1:1 binding model with full fitness in BIAEevaltion software (GE Healthcare) to determine the apparent binding kinetics.

[0307] Binding kinetics data for the disclosed anti-IL-23p19 antibodies are provided in Table 4. The results indicate that the anti-IL-23p19-specific antibody binds to human recombinant IL-23 with a KD in the range of 3.84E-11 to 6.62E-11 M. PC1 (known as a p19-specific antibody, Biointron, entry number 20180926A04) showed KD values ​​in the range of 3.77E-10 to 1.10E-11 over multiple runs.

[0308] SPR coupled dynamics 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

[0309] Example 3: Blocking the interaction between the IL-23 receptor and IL-23

[0310] The ability of the initiated p19-specific antibody to block IL-23 binding 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 serial dilutions of a pre-mixed purified anti-IL-23p19 antibody with recombinant human IL-23 (50 ng / mL) were added to the plate. After 30 minutes of incubation, the plate was washed. Next, a biotinylated anti-p40 antibody (Invitrogen ref: 13-7129-85, entry 2028761, 1 / 3000 dilution) was added to the plate. After 30 minutes of incubation, the plate was washed and detected with streptavidin HRP. After adding the ABTS substrate, the plate was read using a plate reader (OD 405 nM). The positive control antibody PC1 used in this blocking assay was a reference antibody (known as a p19-specific antibody, Biointron, entry number 20180926A04). The negative controls were mouse IgG1 (Novus, catalog number NBP1-97005, entry number 35613), mIgG2a (NovaRock Biotherapeutics product), and hIgG4 (Dendritics, catalog number DDXCHO4P-100, entry number DDXCH04-028).

[0311] result: The data in Figure 4 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 the human IL-23 receptor in a dose-dependent manner. The positive control PC1 also blocked the IL-23 / IL-23 receptor interaction in a dose-response manner. The negative controls hIgG4 and mIgG2a were negative in the analysis.

[0312] To demonstrate the specificity of the initiated p19-specific antibody, a blocking assay was designed to evaluate the antibody's ability to block IL-23 binding to IL-12 receptor β1.

[0313] Briefly, human IL-12 receptor β1 was coated onto 96-well plates (2 μg / ml), and then serial dilutions of purified anti-IL-23p19 antibody, pre-mixed with recombinant human IL-23 (50 ng / ml), were added to the plates. The positive control antibody used for the blocking assay was PC2 (a reference antibody known for specificity to the p40 subunit of IL-12 / IL-23 p40, Biointron entry number 20180925A07). The negative control was mouse IgG1 (Novus, catalog number NBP1-97005, entry 35613).

[0314] After 30 minutes of incubation, the plate was washed. Then, biotinylated anti-p40 antibody (Invitrogen ref: 13-7129-85, entry 2028761, 1 / 3000 dilution) was added to wells containing anti-IL-23p19 antibody; and biotinylated anti-p19 antibody (Mabtech, catalog number MT155, code: 3457-6-1000) was added to wells containing PC2 antibody. After 30 minutes of incubation, the plate was washed and then detected with streptavidin HRP. After adding the ABTS substrate, the plate was read using a plate reader (OD 405 nM).

[0315] result:The data in Figure 5 showed that none of the three selected p19-specific antibodies (Hu-6 18006 B*, Hu-4 18006 B*, and Hu-4 18006 B**) blocked the IL-23 / IL-12 receptor β1 binding interaction. Similarly, the PC1 antibody also did not block the IL-23 / IL-12 receptor β1 interaction (data not shown). However, the p40 control antibody (PC2) blocked the IL-23 / IL-12 β1 interaction as expected.

[0316] Example 4: Inhibition of IL-17 production in mouse splenocyte analysis

[0317] It is widely known that human IL-23 binds to murine IL-23R and induces the production of murine IL-17 in mouse splenocytes. In the presence of IL-2, human IL-23 stimulates the production of IL-17 in murine splenocytes at very low (picomolar) concentrations, which can be inhibited by co-incubation with inhibitors of p40 or p19 (Reference [Aggarwal, S., et al., 2003, J Biol Chem ; 278: 1910-4; Singh et al., 2015; MAbs , July-Aug; 7(4): 778-791]).

[0318] The ability of the initiated anti-IL-23p19-specific antibody to inhibit human IL-23-induced IL-17 production was evaluated in a murine splenocyte assay (MSA). The efficacy for inhibiting human IL-23-induced IL-17 production was determined.

[0319] Briefly, mouse splenocytes 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 procedure. Splenocytes were heated for 5 minutes with IL-2 (5 x 10⁶ 6After activating the cells (20 ng / ml for cells / ml), human IL-23 (1.5 ng / ml) was added to the splenocytes. The activated splenocytes were plated in a 96-well plate at a density of 100 µl / well. 100 µl / well of four purified p19 antibodies, hu-6 18006B* (mIgG2a), hu-4 18006B* (mIgG2a), hu-4 18006** (hIgG4), and hu-2 18006B (mIgG1), were added to the plate.

[0320] After 72 hours of incubation, the supernatant was transferred out of the plate for IL-17 quantitative analysis using a quantikine ELISA kit (R&D, M1700 or SM1700). Controls included in the IL-17 MSA: PC1 (reference antibody known as the p19-specific antibody, Biointron entry number 20180926A04) was used as a positive control; mouse IgG1 (Novus, catalog number NBP 1-97005) and human IgG4 (Dendritics, Cat: DDXCHO4P-100, entry DDXCH04-028) were used as negative controls.

[0321] result: The data presented in Figures 6a and 6b support the conclusion that the disclosed antibody selectively neutralizes the binding of IL-23 and IL-23R and thus inhibits IL-17 production in a dose-dependent manner.

[0322] Example 5: Inhibition of STAT3 activation by reporter cell analysis

[0323] The receptor for IL-23 is known to contain the IL-12Rβ1 subunit, which is commonly shared with the IL-12 receptor that pairs with IL-23R. IL-23p19 selectively binds to IL-23R, and signaling via IL-23R induces Janus kinase 2 (JAK2), which activates STAT3, thereby inducing the upregulation of RORγgt and increasing the production of the inflammatory cytokine IL-17 (Parham et al., J. Immunol.168:5699-5708, 2002). To determine whether the initiated anti-p19 antibody could inhibit STAT3 activation, the antibody was evaluated against IL-23-induced STAT3 activation by reporter cell analysis. Human IL-23 induces STAT3 phosphorylation upon IL-23R binding on the surface of human lymphoma DB cells (US2013 / 0172272 Example 13, and literature [Desmet, J. et al., Nat. Commun. 5:5237 (2014)]).

[0324] DB cells were derived from a human B-cell lymphoma cell line that expresses endogenous IL-23 receptors and STAT3, providing a fully functional IL-23 signaling pathway. DB analysis cells were generated by stably transfecting DB cells with pGL4.47 [luc2p / SIE / Hybro], which enabled the quantitative detection of bioactive human IL23 using a luciferase reporter system.

[0325] This DB analysis is intended to measure the inhibitory activity of an initiated anti-IL-23 antibody against STAT3 activation induced by human IL-23. In summary, DB cells (ATCC, CRL-2289) were cultured in growth medium (RPMI + 10% FBS) for 2 days. On the day of the experiment, cells were harvested and resuspended in the growth medium. Serial dilutions of the antibody to be tested were prepared in the growth medium of a low-conjugation 384-well plate (ThermoScientific 264574), human IL-23 was added, and the mixture was incubated at room temperature for 30 minutes. Subsequently, a mixture of the antibody to be tested and human IL-23 was added to the plate. The signal transduction analysis plates were incubated in a humidified 37°C / 5% CO2 incubator for 16 hours. OneGlo reagent was added, and the mixture was incubated at room temperature for 2 minutes. Luminescence was read on a BioTek Neo2 (BioTek. Winooski.VT) and IC50 values ​​were determined using GraphPad® software (GraphPad Software Inc., San Diego, California, USA), ratios were graphed against log-transformed antibody concentrations, and IC50 values ​​were determined using nonlinear regression (curve fitting) of the sigmoid dose-response. The control antibodies used in the STAT3 activation assay included PC1 as a positive control (PC1 is a reference antibody known to be specific to p19, Biointron, entry number 20180926A04) and mIgG2a (self-generated) as a negative control.

[0326] result: As shown in Table 5, the anti-IL-23p19-specific antibodies evaluated in the analysis inhibited STAT3 activation with IC50 values ​​ranging from 35.2 pM to 264.6 pM, with up to 99 to 100% inhibition. The positive control (PC1) had IC50 values ​​ranging from 24.30 pM to 117.20 pM and showed 98 to 99% inhibition in multiple experiments.

[0327] Inhibition of IL-23 / IL-23 receptor-mediated STAT3 activation Anti-IL-23p19 mAb IC50 pM Top inhibition % Hu-2 18006B 157.7 97% Hu-4 18006B 168.5 99% Hu-5 18006B 226.4 99% Hu-6 18006B 60.5 99% Hu-6 18006B* 35.2 99% Hu-4 18006B* 264.6 100% Hu-4 18006B** 157.4 100%

[0328] The results of Figure 7 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. The positive control (PC1) also showed inhibition of STAT3 activation in a dose-responsive manner as expected.

[0329] Example 6: Inhibition of IL-12-dependent IFN-γ production by human PBMCs

[0330] IL-12 stimulation of PBMCs is known to stimulate the production of IFN-γ by NK cells and T cells. To determine whether the initiated anti-p19 antibody can inhibit IFN-γ production, representative initiated human anti-p19 antibodies were analyzed in the PMBC IL-12 stimulation assay.

[0331] Briefly, human PBMCs were thawed from the frozen stock and resuspended in RPMI + 10% FBS containing 50 ng / ml IL-18 (R&D, 9124-IL / CF) in 384-well plates. Serial dilutions of the test antibody were prepared in the growth medium of a low-binding 384-well plate. Human IL-12 (25 ng / ml) or cyanobacteria IL-12 (25 ng / ml) were transferred to each well and incubated at room temperature for 30 minutes. The mixture (test antibody + IL-12) was plated onto PBMC cell plates. Cells were incubated for 48 hours in a humidified 37°C / 5% CO2 incubator. IFN-γ production was measured by AlphaLISA (PerkinElmer, AL217C) according to the manufacturer's protocol.

[0332] Control antibodies used in the human PBMC analysis: PC1 (reference antibody known as a p19-specific antibody, Biointron, entry number 20180926A04), PC2 (reference antibody known to be specific for the p40 subunit of IL-12 and IL-23, Biointron, entry number 20180925A07), anti-IL-23 p40 subunit Mab (Hu-19 18006*, self-generated); mIgG2a (generated by NovaRock Biotherapeutics) and human IgG4 (Dendritics, catalog DDXCHO4P-100, entry DDXCH04-028) were used as negative control antibodies.

[0333] result: As shown in Figures 8 and 9, the anti-IL-23p19 antibodies (Hu-6 18006B* and Hu-4 18006B* and PC1) did not inhibit human IL-12 (Figure 8) and cynomolgus monkey IL-12 (Figure 9)-mediated IFN-γ production by human PBMCs, whereas the positive controls PC2 and Hu-19 18006* inhibited human (Figure 8) and cynomolgus monkey (Figure 9) PBMCs in a dose-dependent manner as expected. These data support the conclusion that the disclosed p19 antibodies are specific to p19.

[0334] Example 7: In vivo efficacy of anti-p19-specific antibody in IL-23-induced murine skin inflammation model

[0335] The role of the IL-23 / IL-17 pathway as a major inducer of human psoriasis (PsO) has been well characterized and clinically validated. Animal models of psoriasis (PsO) are important for understanding the pathophysiology of the human disease. Intradermal injection of IL-23 has been used to study the IL-23 pathway in rodents and can be used to evaluate the pharmacology of novel small molecule / biological agents in the treatment of PsO (Reference [Stephen B. Gauld et al., J. Dermatological Science, 92(2018) 45-53]).

[0336] Human IL-23 is known to bind to murine IL-23 receptors, inducing mIL-17 production and inflammation in mice. Intradermal injection of human IL-23 into the mouse ear to induce ear inflammation was used in a psoriasis model to characterize biological drugs for human psoriasis (PsO) (Reference [Aggarwal et al., J Biol Chem 2003; 278: 1910-4; Singh et al., MAbs 2015 July-Aug; 7(4): 77-791]).

[0337] To evaluate the ability of Hu-4 18006 B (mIgG2c), Hu-4 18006 B** (hIgG4), hu-5 18006 B (mIgG2b), and Hu-6 18006 B* (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. These representative antibodies were evaluated for their ability to reduce the inflammatory response.

[0338] In this model, recombinant human IL-23 (3 μg / 10 μl / mouse / day) was injected into the skin of the right ear of a mouse (i.e., intradermally) for 8 consecutive days (D0 - D7) to induce a psoriasis-like inflammatory skin response characterized by erythema and induration, along with histological evidence of epidermal proliferation, parakeratosis, and local inflammatory infiltration.

[0339] Mice were treated twice by intraperitoneal (IP) injection according to two protocols with either the reference antibody PC1, known to be a p19-specific antibody (Biointron, item number 20180926A04), or the IL-23p19 antibody (hu-4 18006B, hu-4 18006B**, hu-5 18006B, or hu-6 18006B*). In Protocol 1, mice were administered a PBS control (vehicle) or the antibody. The first injection was given the day before the IL-23 injection, and the second injection was given on day 3 after the IL-23 injection. In Protocol 2, mice were administered a PBS control or the antibody. The first injection was given 1 hour before the IL-23 injection, and the second injection was given on day 3 after the IL-23 injection (Fig. 10).

[0340] Body weight, ear thickness, and ear inflammation score of the mice were measured daily. The ear inflammation score was calculated on days 0, 2, 4, 6, and 8 according to the following criteria: auricular shape (relatively normal-0, minimal change-1, moderate to significant change-2, edema and severe malformation-3). Skin color (relatively normal-0; minimal hypertrophy-1; mild hypertrophy-2; severe hypertrophy-3) and white scales (relatively normal-0, minimal-1, mild-2, obvious-3). The thickness of the right ear of each mouse was measured and photographed on days 0, 2, 4, 6, and 8.

[0341] On the last day of the experiment (Day 8), the animals were treated with carbon dioxide, blood samples were collected, and serum was isolated (stored in a -80°C freezer). Modeling 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.

[0342] Data were provided as mean ± SEM. Statistical significance was considered when the P value was less than 0.05.

[0343] The data provided in Tables 6 and 7 summarize the overall scores for the injected ears (adding scores for auricular shape, skin color, microvascular changes, and white scales). The results indicated 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 continued until day 8. The effect was statistically significant. This conclusion is evident in both the summary score values ​​and the ear thickness values.

[0344] Total score of the injected ear (x±s, n=10) group Total Score## D0 D2 D4 D6 D8 PBS 0.0±0.00 0.0±0.00 0.0±0.00 0.3±0.95 0.2±0.42 IL-23 0.0±0.00 0.0±0.00 1.9±1.37 9.6±2.37 10.8±1.93 PC1 (10 mg / kg) 0.0±0.00 0.0±0.00 0.6±0.97* 1.3±1.34*** 2.0±2.05*** Hu-4 18006B (10 mg / kg) 0.0±0.00 0.0±0.00 0.6±1.26* 1.7±1.83*** 3.2±3.05*** Hu-5 18006B (10 mg / kg) 0.0±0.00 0.0±0.00 0.8±1.23 2.1±1.60*** 4.1±2.33***

[0345] * p <0.05, ** p <0.01, *** p <0.001 vs. model.

[0346] Total score of the ear injected with IL-23 ( x ± s , n=8) group Total Score## D0 D2 D4 D6 D8 PBS 0±0 0±0 0±0*** 0±0*** 0±0*** IL-23 0±0 0±0 1.88±0.3 5.63±0.6 7.75±0.53 PC1 3 mg / kg 0±0 0±0 0.5±0.19** 1.38±0.26*** 2.25±0.25*** PC-1 6 mg / kg 0±0 0±0 0.38±0.18** 1±0.33*** 1.75±0.25*** Hu-4 18006B** 3 mg / kg 0±0 0±0 0.5±0.27** 1.25±0.37*** 2.13±0.3*** Hu-4 18006B** 6 mg / kg 0±0 0±0 0.25±0.16** 1.13±0.3*** 1.88±0.23*** Hu-6 18006B* 3 mg / kg 0±0 0±0 0.38±0.18** 1.13±0.35*** 2.13±0.23*** Hu-6 18006B* 6 mg / kg 0±0 0±0 0.25±0.16** 1±0.27*** 1.75±0.31***

[0347] ** p <0.01, *** p <0.001 vs. model

[0348] As shown in Tables 8 and 9, mouse ear thickness was reduced by treatment with selected anti-IL-23p19 antibodies compared to the model (IL-23 treatment). The therapeutic effect (in vivo suppression of the inflammatory immune response in the skin) began on day 4 and continued until day 8, and the effect was statistically significant (p<0.001 vs. model, IL-23 treatment).

[0349] Ear thickness of the injected ear from day 0 to day 8 (x±s, n=10) group Ear thickness (mm) D0 D2 D4 D6 D8 PBS 0.22±0.02 0.19±0.01 0.22±0.01 0.23±0.02 0.26±0.02 IL-23 0.23±0.02 0.25±0.01 0.37±0.06 0.56±0.09 0.65±0.12 PC1 (10 mg / kg) 0.23±0.02 0.23±0.01 0.28±0.08** 0.35±0.04*** 0.33±0.03*** hu-4 18006B (10 mg / kg) 0.23±0.01 0.22±0.02 0.31±0.04* 0.35±0.03*** 0.43±0.07*** hu-5 18006B (10 mg / kg) 0.23±0.01 0.23±0.02 0.35±0.02 0.42±0.11** 0.50±0.15*

[0350] * p <0.05, ** p <0.01, *** p <0.001 vs. model.

[0351] Ear thickness of the ear injected with IL-23 ( x ± s , n=8) group Ear thickness (mm) D0 D2 D4 D6 D8 PBS 0.19±0 0.2±0*** 0.24±0*** 0.23±0*** 0.23±0.01*** IL-23 0.19±0 0.24±0.01 0.39±0.01 0.48±0.02 0.51±0.02 PC1 3 mg / kg 0.19±0 0.23±0 0.35±0** 0.33±0.01*** 0.34±0.01*** PC1 6 mg / kg 0.19±0 0.23±0 0.34±0*** 0.33±0.01*** 0.33±0.01*** Hu-4 18006B** 3 mg / kg 0.18±0 0.23±0 0.36±0.01** 0.35±0.01*** 0.36±0.01*** Hu-4 18006B** 6 mg / kg 0.18±0 0.23±0 0.35±0.01** 0.34±0.01*** 0.35±0.01*** Hu-6 18006B* 3 mg / kg 0.19±0 0.23±0 0.36±0** 0.33±0.01*** 0.35±0*** Hu-6 18006B* 6 mg / kg 0.19±0 0.23±0 0.34±0*** 0.33±0.01*** 0.34±0.01***

[0352] ** p <0.01, *** p <0.001 vs. model

[0353] The data provided in Figure 10 showed that the disclosed anti-p19-specific antibodies hu-4 18006B** and hu-6 18006B* induced a statistically significant reduction in ear thickness compared to the untreated control group (a model treated only with human IL-23).

[0354] Figures 11a, 11b, 11c, and 11d provide data establishing the effect of an anti-p19-specific antibody on inflammatory skin reactions determined by H&E pathology staining scores, such as epidermal thickness (Fig. 11a), dermal thickness (Fig. 11b), infiltration of inflammatory cells (Fig. 11c), and hyperkeratosis or insufficiency (Fig. 11d), obtained throughout the experimental process and displayed with a scoring system as described below.

[0355] In short, mouse ears were collected on day 8 and examined under a microscope. Tissues were fixed in 10% neutral buffered formalin. After fixation, the tissues were trimmed, dehydrated, embedded, sectioned onto slides, and stained with hematoxylin / eosin (H&E) according to relevant SOPs. The research pathologist performed histopathological evaluation using a light microscope. Microscopic findings were classified using a 5-level grading system (relatively normal, minimal, mild, moderate to significant, severe).

[0356] result:Treatment with the anti-p19 antibody hu4 18006B resulted in a significant reduction in both the IL-23 injection-induced edema response and inflammation scores compared to the model. Hu-4 18006B demonstrated superior inhibitory effects compared to PC1 in three scores: epidermal thickness (Fig. 11a), dermal thickness (Fig. 11b), and inflammatory cell infiltration (Fig. 11c). Hu-4 also showed inhibition of hyperkeratosis (Fig. 11d).

[0357] Figure 12 provides a representative photograph of an H&E-stained ear section obtained on the 8th day after treatment. The H&E staining procedure is as described above.

[0358] result: The selected initiated anti-IL-23p19 antibody treatment (Hu-4 18006B) significantly inhibited mouse skin inflammation compared to the model.

[0359] Unless otherwise specified, all numerical values ​​representing the amounts of ingredients, characteristics such as molecular weights, reaction conditions, etc., used in the specification and claims should be understood as being modified by the term “about” in all cases. Accordingly, unless otherwise indicated, numerical parameters described in the specification and appended claims are approximations that may vary depending on the appropriate characteristics to be obtained by the present disclosure. At the very least, without attempting to limit the application of the equivalents principle to the claims, each numerical parameter should be interpreted by applying general rounding techniques in terms of at least the reported significant digits.

[0360] Although the numerical ranges and parameters describing the broad scope of this disclosure are approximations, the numerical values ​​described in specific embodiments are reported as accurately as possible. However, any numerical values ​​contain certain errors that inevitably occur due to the standard deviations observed in each test measurement.

[0361] In connection with the description of this disclosure (particularly with respect to the following claims), the singular forms (“a,” “an,” “the”) and similar terms used shall be interpreted to include both singular and plural forms unless otherwise specified in this specification or otherwise evident from the context. References to ranges of values ​​in this specification serve only as a shortened method of individually referring to each individual value within the range. Unless otherwise indicated in this specification, each individual value is included in this specification as if individually enumerated in this specification. All methods described in this specification may be performed in any appropriate order unless otherwise indicated in this specification or otherwise evident from the context. Any use of any example or exemplary language provided in this specification (e.g., “e.g., for example”) is merely for the purpose of better describing this disclosure and does not limit the scope of the disclosure as otherwise claimed. No language in this specification shall be interpreted as representing any unclaimed element essential to the execution of this disclosure.

[0362] The grouping of alternative elements or embodiments of the disclosures disclosed herein shall not be construed as a limitation. Each member of a group may be referred to and claimed individually or in any combination with other members of the group or other elements present in this specification. One or more members of a group may be included in or removed from a group for reasons of convenience and / or patentability. Where such inclusion or removal occurs, this specification shall be considered to include the modified group and satisfy all written descriptions of the Markush group used in the appended claims.

[0363] Specific embodiments of the present disclosure are described herein, including optimal modes known to the inventors for carrying out the present disclosure. Of course, variations of these described embodiments will be apparent to those skilled in the art when reading the foregoing description. The inventors expect that a person skilled in the art will make appropriate use of such variations, and the inventors intend that the present disclosure be practiced differently from what is specifically described herein. Accordingly, the present disclosure includes all variations and equivalents of the subject matter cited in the claims appended to this specification, as permitted by applicable law. Furthermore, unless otherwise indicated in this specification or otherwise clearly contradictory in the context, any combination of the foregoing elements in all possible variations is included in the present disclosure.

[0364] Certain embodiments disclosed herein may be further limited in claims using the language “consisting of” or “essentially consisting of”. When used in claims, the transitional term “consisting of” excludes elements, steps, or components not specified in the claims, regardless of whether they were added by application or modification. The transitional term “essentially consisting of” limits the claims to those that do not substantially affect the specified materials or steps and the basic and novel properties. Embodiments of the disclosures so claimed are described and made possible in this specification, either essentially or explicitly.

[0365] It should be understood that the embodiments of the disclosures set forth in this specification are illustrative of the principles of the disclosures. Other variations that may be adopted are within the scope of the disclosures. Accordingly, alternative configurations of the disclosures may be used in accordance with the teachings of this specification as examples rather than limitations. Accordingly, the disclosures are not precisely limited as illustrated and described.

[0366] Although the present disclosure is described and illustrated herein with reference to various specific materials, procedures, and embodiments, it is understood that the present disclosure is not limited to any specific combination of materials and procedures selected for the purposes thereof. Numerous variations of these details may be implied as understood by those skilled in the art. The present specification and embodiments are to be regarded merely as illustrative, and the true scope and spirit of the present disclosure are derived by the following claims. All references, patents, and patent applications mentioned in this application are incorporated herein by reference in their entirety.

Claims

Claim 1 (a) a heavy chain variable region comprising CDR1: SEQ ID NO. 9, CDR2: SEQ ID NO. 10, and CDR3: SEQ ID NO. 11; and a light chain variable region comprising CDR1: SEQ ID NO. 12, CDR2: SEQ ID NO. 13, and CDR3: SEQ ID NO. 14; (b) a heavy chain variable region comprising CDR1: SEQ ID NO. 15, CDR2: SEQ ID NO. 16, and CDR3: SEQ ID NO. 17; and a light chain variable region comprising CDR1: SEQ ID NO. 18, CDR2: SEQ ID NO. 19, and CDR3: SEQ ID NO. 20; (c) a heavy chain variable region comprising CDR1: SEQ ID NO. 21, CDR2: SEQ ID NO. 22, and CDR3: SEQ ID NO. 23; and a light chain variable region comprising CDR1: SEQ ID NO. 24, CDR2: SEQ ID NO. 25, and CDR3: SEQ ID NO. 26; or (d) an anti-IL-23p19 antibody comprising a heavy chain variable region including CDR1: SEQ ID NO 27, CDR2: SEQ ID NO 28, and CDR3: SEQ ID NO 29; and a light chain variable region including CDR1: SEQ ID NO 30, CDR2: SEQ ID NO 31, and CDR3: SEQ ID NO 32. Claim 2 The anti-IL-23p19 antibody according to claim 1, wherein the antibody comprises (a) the heavy chain variable region sequence of SEQ ID NO. 1 and the light chain variable region sequence of SEQ ID NO. 2; (b) the heavy chain variable region sequence of SEQ ID NO. 3 and the light chain variable region sequence of SEQ ID NO. 4; (c) the heavy chain variable region sequence of SEQ ID NO. 5 and the light chain variable region sequence of SEQ ID NO. 6; or (d) the heavy chain variable region sequence of SEQ ID NO. 7 and the light chain variable region sequence of SEQ ID NO.

8. Claim 3 In claim 1, the antibody is an anti-IL-23p19 antibody, which is an anti-human IL-23p19 antibody. Claim 4 In claim 1, the antibody is an anti-IL-23p19 antibody that is a full-length antibody. Claim 5 In claim 1, the antibody is an antigen-binding fragment, and the antigen-binding fragment comprises: (a) a heavy chain variable region comprising CDR1: SEQ ID NO. 9, CDR2: SEQ ID NO. 10, and CDR3: SEQ ID NO. 11; and a light chain variable region comprising CDR1: SEQ ID NO. 12, CDR2: SEQ ID NO. 13, and CDR3: SEQ ID NO. 14; (b) a heavy chain variable region comprising CDR1: SEQ ID NO. 15, CDR2: SEQ ID NO. 16, and CDR3: SEQ ID NO. 17; and a light chain variable region comprising CDR1: SEQ ID NO. 18, CDR2: SEQ ID NO. 19, and CDR3: SEQ ID NO. 20; and (c) a heavy chain variable region comprising CDR1: SEQ ID NO. 21, CDR2: SEQ ID NO. 22, and CDR3: SEQ ID NO. 23; and a light chain variable region comprising CDR1: SEQ ID NO 24, CDR2: SEQ ID NO 25, and CDR3: SEQ ID NO 26; or (d) a heavy chain variable region comprising CDR1: SEQ ID NO 27, CDR2: SEQ ID NO 28, and CDR3: SEQ ID NO 29; and a light chain variable region comprising CDR1: SEQ ID NO 30, CDR2: SEQ ID NO 31, and CDR3: SEQ ID NO 32, an anti-IL-23p19 antibody. Claim 6 In paragraph 5, the antigen-binding fragment is an anti-IL-23p19 antibody selected from the group consisting of Fab, Fab', F(ab)2, Fv, scFv, single-chain antibodies, minibodies, and diabodies. Claim 7 In claim 1, the antibody is an anti-IL-23p19 antibody, which is a monoclonal antibody. Claim 8 In paragraph 1, the antibody is an anti-IL-23p19 antibody, which is a human antibody. Claim 9 In claim 1, the antibody is an anti-IL-23p19 antibody, which is a murine antibody. Claim 10 In claim 1, the antibody is an anti-IL-23p19 antibody, which is a chimeric antibody. Claim 11 In claim 1, the antibody is an anti-IL-23p19 antibody, which is a bispecific antibody. Claim 12 In paragraph 1, the antibody is an anti-IL-23p19 antibody, which is a humanized antibody. Claim 13 In claim 1, the antibody is an anti-IL-23p19 antibody that does not bind to the p40 subunit of IL-12. Claim 14 A pharmaceutical composition for use in the treatment of IL-23-mediated inflammatory diseases, comprising the anti-IL-23p19 antibody of claim 1 and a pharmaceutically acceptable carrier. Claim 15 An isolated polynucleotide comprising a sequence encoding an anti-IL-23p19 antibody according to claim 1. Claim 16 An isolated polynucleotide encoding an anti-IL-23p19 antibody, wherein the isolated polynucleotide comprises: (a) a heavy chain variable region comprising CDR1: SEQ ID NO. 9, CDR2: SEQ ID NO. 10, and CDR3: SEQ ID NO. 11; and a light chain variable region comprising CDR1: SEQ ID NO. 12, CDR2: SEQ ID NO. 13, and CDR3: SEQ ID NO. 14; (b) a heavy chain variable region comprising CDR1: SEQ ID NO. 15, CDR2: SEQ ID NO. 16, and CDR3: SEQ ID NO. 17; and a light chain variable region comprising CDR1: SEQ ID NO. 18, CDR2: SEQ ID NO. 19, and CDR3: SEQ ID NO. 20; (c) a heavy chain variable region comprising CDR1: SEQ ID NO. 21, CDR2: SEQ ID NO. 22, and CDR3: SEQ ID NO. 23; and a light chain variable region comprising CDR1: SEQ ID NO 24, CDR2: SEQ ID NO 25, and CDR3: SEQ ID NO 26; or (d) a heavy chain variable region comprising CDR1: SEQ ID NO 27, CDR2: SEQ ID NO 28, and CDR3: SEQ ID NO 29; and a light chain variable region comprising CDR1: SEQ ID NO 30, CDR2: SEQ ID NO 31, and CDR3: SEQ ID NO 32. Claim 17 A vector containing a polynucleotide according to paragraph 15. Claim 18 Isolated host cell comprising a polynucleotide according to paragraph 15. Claim 19 A method for producing an anti-IL-23p19 antibody according to claim 1, wherein the method comprises the step of culturing isolated host cells comprising an isolated polynucleotide encoding the anti-IL-23p19 antibody, and wherein the anti-IL-23p19 antibody comprises: (a) a heavy chain variable region comprising CDR1: SEQ ID NO. 9, CDR2: SEQ ID NO. 10, and CDR3: SEQ ID NO. 11; and a light chain variable region comprising CDR1: SEQ ID NO. 12, CDR2: SEQ ID NO. 13, and CDR3: SEQ ID NO. 14; (b) a heavy chain variable region comprising CDR1: SEQ ID NO. 15, CDR2: SEQ ID NO. 16, and CDR3: SEQ ID NO. 17; and a light chain variable region comprising CDR1: SEQ ID NO. 18, CDR2: SEQ ID NO. 19, and CDR3: SEQ ID NO. 20; (c) a heavy chain variable region comprising CDR1: SEQ ID NO. 21, CDR2: SEQ ID NO. 22, and CDR3: SEQ ID NO. 23; and a light chain variable region comprising CDR1: SEQ ID NO. 24, CDR2: SEQ ID NO. 25, and CDR3: SEQ ID NO. 26; or (d) a heavy chain variable region comprising CDR1: SEQ ID NO. 27, CDR2: SEQ ID NO. 28, and CDR3: SEQ ID NO. 29; and a light chain variable region comprising CDR1: SEQ ID NO. 30, CDR2: SEQ ID NO. 31, and CDR3: SEQ ID NO.

32. Claim 20 A pharmaceutical composition according to claim 14, wherein the disease comprises one or more of psoriasis, psoriatic arthritis, inflammatory bowel disease, ankylosing spondylitis, systemic lupus erythematosus, hidradenitis suppurativa, atopic dermatitis, and asthma.

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