WO2013009535A1 - Il-23 antagonists for treatment or prevention of skin rash associated with treatment with p13k/akt pathway inhibitors - Google Patents

Il-23 antagonists for treatment or prevention of skin rash associated with treatment with p13k/akt pathway inhibitors Download PDF

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WO2013009535A1
WO2013009535A1 PCT/US2012/045363 US2012045363W WO2013009535A1 WO 2013009535 A1 WO2013009535 A1 WO 2013009535A1 US 2012045363 W US2012045363 W US 2012045363W WO 2013009535 A1 WO2013009535 A1 WO 2013009535A1
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antibody
antagonist
seq
phenyl
sequence
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PCT/US2012/045363
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French (fr)
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Svetlana Sadekova
Robert H PIERCE
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Merck Sharp & Dohme Corp.
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Priority to EP12811568.0A priority Critical patent/EP2729174A1/en
Publication of WO2013009535A1 publication Critical patent/WO2013009535A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule

Definitions

  • the present invention relates generally to methods of treating and/or preventing side effects of treatment with PI3K/AKT pathway inhibitors, such as skin rash resulting from cancer therapy.
  • Cancer is often treated with agents that selectively disrupt the proliferation of malignant cells. Such agents, however, can cause unwanted side effects. Such side effects can be dose-limiting if the agent must be dosed at sub-optimal levels to avoid unacceptable side effects. Such dose-limiting toxicities (“DLT”) may prevent treatment of cancer patients at high enough doses to be efficacious, reducing the usefulness of an otherwise effective drug.
  • DLT dose-limiting toxicities
  • PI3K/AKT pathway provide one example.
  • the PI3K/AKT pathway appears to be important for regulating cell survival/cell death (Kulik et al. (1997) Mol. Cell. Biol. 17:1595- 1606; Franke et al. (1997) Cell 88:435-437; Kauffmann Zeh et al. (1997) Nature 385:544- 548; Hemmings (1997) Science 275:628-630; Dudek et al. (1997) Science, 275:661-665).
  • PI3K platelet derived growth factor
  • NGF nerve growth factor
  • IGF-1 insulin like growth factor 1
  • PI3K or dominant negative Akt mutants abolish survival promoting activities of these growth factors or cytokines. It has been previously disclosed that inhibitors of PI3K (LY294002 or wortmannin) blocked the activation of Akt by upstream kinases. In addition, introduction of constitutively active PI3K or Akt mutants promotes cell survival under conditions in which cells normally undergo apoptotic cell death (Kulik et al. (1997); Dudek et al. (1997)). These and other observations suggest that the PI3K/Akt pathway plays an important role in regulating cell survival or apoptosis in tumorigenesis. See WO 2008/070016.
  • PI3K/AKT pathway inhibitors have been proposed as therapeutic agents for the treatment of cancer, but their use has been hampered by dose-limiting skin rash. See, e.g., Tolcher et al. (2009) J. Clin. Oncol. 27: 15s (abstract 3503) (MK-2206, Merck); Edelman et al. (2010) J. Clin. Oncol. 28: 15s (abstract 3004) (XL147/SAR245408, Exilixis, sanofi- aventis, Merck KGaA) and Baselga et al. (2010) J. Clin. Oncol. 28:15s (abstract 3003) (BKM120, Novartis).
  • the invention provides methods of treatment comprising determining whether a subject is suffering from a skin rash that may be due to administration of a PI3K/AKT pathway inhibitor, and administering an effective amount of an IL-23 antagonist to treat the rash only if such rash is present.
  • the invention provides methods of treatment comprising administering an IL-23 antagonist to a subject to be treated with a PI3K/AKT pathway inhibitor specifically for the purpose of preventing or treating a skin rash, and thereafter optionally monitoring the subject's skin for signs and symptoms of a skin rash.
  • the PI3K/AKT pathway inhibitor is an AKT inhibitor, such as a substituted naphthyridine compound, including MK-2206 and AKTi-X.
  • the AKT inhibitor is perifosine (KRX-0401), VQD-002 (API-2, TCN), SR13668, GSK690693, GSK2110183, GSK2141795, AZD5363 or XL418.
  • the AKT inhibitor is dosed orally.
  • the AKT inhibitor is dosed in combination with one or more other cancer drugs, e.g. a MEK inhibitor such as selumetinib or GSKl 120212.
  • treatment with an IL-23 antagonist permits dosing with AKT inhibitors at levels that might otherwise be difficult or impossible to achieve due to dose limiting skin rash, such as weekly dosing of 250, 300, 350, 400, 450, 500, 600, 700, 750, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg or more, either as a single agent or in combination with another cancer drug or drugs.
  • the dosing interval differs from once every week, such as TID, BID, daily, Q2D, Q3D, Q4D, Q5D, Q6D, Q2W, Q3W, Q4W, but nonetheless results in a total weekly dose of 250, 300, 350, 400, 450, 500, 600, 700, 750, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg or more.
  • the AKT inhibitor is dosed to a human subject at 150 - 500 mg once every week, such as 300 mg once every week, or at 45 - 150 mg every other day.
  • the AKT inhibitor is dosed to a human subject as part of combination therapy at 150 - 500 mg once every week, such as 300 mg once every week, or at 150 - 500 mg once every three weeks.
  • the PI3K/AKT pathway inhibitor is a PI3K inhibitor, such as wortmannin, LY294002, BEZ235, BGT226, BKM120, XL765, XL147, GDC0941, SF1126, GSK1059615, PX-866 or CAL-101.
  • the PI3K/AKT pathway inhibitor is an EGF/EGFR inhibitor, such as cetuximab, gefitnib, erlotinib or panitumumab.
  • the anti-IL-23 antibody, or antigen binding fragment thereof comprises an antibody light chain variable domain, or antigen binding fragment thereof, having at least one, two or three CDRs selected from the group consisting of SEQ ID NOs: 32-46.
  • antibody or fragment comprises a light chain variable domain comprising at least one CDRLl selected from the group consisting of SEQ ID NOs: 32-36; at least one CDRL2 selected from the group consisting of SEQ ID NOs: 37-41; and at least one CDRL3 selected from the group consisting of SEQ ID NOs: 42-46.
  • the anti-IL-23 antibody, or antigen binding fragment thereof comprises an antibody heavy chain variable domain, or antigen binding fragment thereof, having at least one, two or three CDRs selected from the group consisting of SEQ ID NOs: 15-31.
  • the antibody or fragment comprises a heavy chain variable domain comprising at least one CDRH1 selected from the group consisting of SEQ ID NOs: 15-19; at least one CDRH2 selected from the group consisting of SEQ ID NOs: 20-26; and at least one CDRH3 selected from the group consisting of SEQ ID NOs: 27-31.
  • the antibody or fragment comprises a light chain variable domain and a heavy chain variable domain, or the antigen binding fragments thereof, described in the preceding two paragraphs.
  • the antibody or fragment comprises a framework region, wherein the amino acid sequence of the framework region is all or substantially all of the corresponding region of a human immunoglobulin amino acid sequence.
  • the light chain and/or heavy chain variable domains comprise a variant of one or more of the CDRs.
  • the variant domain comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservatively modified amino acid residues relative to the sequence of the respective SEQ ID NOs. Conservative amino acid substitutions are provided at Table 1.
  • the light chain variable domain comprises residues 1-
  • the heavy chain variable domain comprises a sequence selected from the group consisting of residues 1-116 of SEQ ID NOs: 6-8, such as SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.
  • the variant variable domain comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or 50 or more conservatively modified amino acid residues relative to the sequence of the respective SEQ ID NOs.
  • the antibody or fragment comprises a light chain variable domain and a heavy chain variable domain, or the antigen binding fragments thereof, described in this paragraph.
  • the antibody or fragment comprises a light chain sequence of SEQ ID NO: 14 and/or a heavy chain sequence selected from the group consisting of SEQ ID NOs: 6-8. In another embodiment the antibody or fragment comprises two light chain sequences and two heavy chain sequences.
  • the antibody or fragment comprises a light chain variable domain, or an antigen binding fragment thereof, consisting essentially of residues 1- 108 of SEQ ID NO: 14, and/or a heavy chain variable domain, or an antigen binding fragment thereof, consisting essentially of a sequence selected from the group consisting of residues 1- 116 of SEQ ID NOs: 6-8, such as SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.
  • the antibody or fragment comprises a light chain variable domain, or an antigen binding fragment thereof, having at least 50%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence homology with residues 1-108 of SEQ ID NO: 14, and/or a heavy chain variable domain, or an antigen binding fragment thereof, having at least 50%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence homology with a sequence selected from the group consisting of residues 1-116 of SEQ ID NOs: 6-8, such as SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.
  • the antibody or fragment binds to human IL-23pl9 (SEQ ID NO: 1]
  • the IL-23pl9 binding compound binds to an epitope comprising some or all of residues K20, T23, W26, S27, P30, E82, S95, L96, L97, P98, D99, P101, G103, Q104, H106, A107 and LI 10, and optionally residues L24, L85, T91, S100 and V102.
  • the epitope for an antibody of interest is determined by obtaining an X-ray crystal structure of an antibody: antigen complex and determining which residues on IL-23pl9 are within a specified distance of residues on the antibody of interest, wherein the specified distance is, e.g., 4A or 5A.
  • the epitope is defined as a stretch of 11 or more contiguous amino acid residues along the IL-23 l9 sequence in which at least 30%, 40%, 45%), 50%o or 54% of the residues are within the specified distance of the antibody.
  • the antibody or fragment further comprises a heavy chain constant region, wherein the heavy chain constant region comprises a ⁇ , ⁇ 2, ⁇ 3, or ⁇ 4 human heavy chain constant region, or a variant thereof.
  • the light chain constant region comprises a lambda or a kappa human light chain constant region, or a variant thereof.
  • the anti-IL-23 antibodies or fragments thereof are polyclonal, monoclonal, chimeric, humanized or fully human antibodies or fragments thereof.
  • the present invention also contemplates that the antigen binding fragment is an antibody fragment selected from the group consisting of, e.g., Fab, Fab', Fab'-SH, Fv, scFv, F(ab') 2 , and a diabody.
  • FIG. 1 shows comparisons of mouse anti-human IL-23pl9 antibody clone heavy chain variable domain sequences. Sequences are provided for clones mlAl 1, ml 1C1, m5F5, m21Dl, ml3B8, hl3B8a, hl3B8b and hl3B8c. CDRs are indicated. In both figures, an "m” prefix connotes a murine antibody and an "h” connotes a humanized antibody. The suffixes "a”, “b” and “c” refer to sequence variants of the humanized 13B8 heavy chain variable domain, as discussed in greater detail below.
  • FIG. 2 shows comparisons of mouse anti-human IL-23pl9 antibody clone light chain variable domain sequences. Sequence are provided for clones mlAl 1, ml 1C1, m5F5, m21Dl, ml3B8, hl3B8. CDRs are indicated.
  • FIG. 3 shows the levels of expression of the genes encoding the subunits of IL-
  • the leftmost (white) bar in each graph which is essentially zero for the IL-23 l9 and IL-12/23p40 plots, represents control cells treated with vehicle.
  • the next (black) bar represents cells treated with lipopolysaccharide (LPS).
  • the next two bars represent cells treated with LPS and MK-2206 (0.1 ⁇ and 0.5 ⁇ , respectively), as indicated.
  • the right most two bars represent cells treated with LPS and AKTi-X (0.1 ⁇ and 0.5 ⁇ , respectively, as indicated).
  • LPS enhances the expression of IL-23pl9 and IL-12/23p40, but reduces the expression of IL-12p35.
  • AKT inhibitors MK- 2206 and AKTi-X both further enhance expression of IL-23pl9 and IL-12/23p40, but enhance the expression of IL-12p35 less so, if at all.
  • the results demonstrate that in the presence of LPS, AKT inhibitors preferentially enhance the expression of the genes encoding the subunits of IL-23 rather than IL-12. See Example 3.
  • FIG. 4 shows the levels of expression of the genes encoding the pl9 and p40 subunits of IL-23 in primary CD 14+ monocytes isolated from PBMC from two different human donors as determined by TAQMAN ® real time quantitative polymerase chain reaction analysis. Data are presented for three time points (3, 6 and 24 hours) after addition of LPS, or LPS and AKT inhibitor AKTi-X, as indicated. Bars for untreated cells (indicated by arrows) are provided with the three hour time points, and are essentially zero. For each time point, the left (black) bars are data for treatment with LPS alone, and the right (white) bars are data for treatment with LPS and AKT inhibitor AKTi-X.
  • AKTi-X an AKT inhibitor specifically upregulates expression of genes encoding IL-23 subunits in primary CD 14+ monocytes isolated from PBMC.
  • IL- 12p35 is not expressed at detectable levels at three and six hours, and is expressed at only low levels at 24 hours and does not change with addition of AKT inhibitor AKTi-X (data not shown). See Example 4.
  • FIG. 5 shows IL-23 protein levels in primary CD 14+ monocytes isolated from
  • TLR toll-like receptor
  • LTA lipoteichoic acid
  • AKT inhibitors MK-2206 or AKTi-X at 0.1 ⁇ or 0.5 ⁇ .
  • the leftmost bars for vehicle controls are not visible. Black bars show results for treatment with TLR agonist alone; white bars for treatment with TLR agonist and MK-2206 (at 0.1 ⁇ or 0.5 ⁇ ); and gray bars for treatment with TLR agonist and AKTi-X (at 0.1 ⁇ or 0.5 ⁇ ), as indicated.
  • FIGS. 6A and 6B compare expression of several genes in IL-23-treated and
  • MK-2206-treated monkeys Specifically, gene expression levels are compared between IL- 23-treated cynomolgus monkey skin (black symbols, top panels) and MK-2206-treated rhesus monkey skin (black symbols, bottom panels), as well an (untreated) controls (gray symbols).
  • Each of the genes for which data are presented (BD2, S100A8-calgranulin, CCL3-MIPl , Ebi3, IL- ⁇ , IL-6, IL-23pl9, IL-22) was selected based on its prior known association with IL-23 -induced skin inflammation. Both IL-23 and AKT inhibitor increase the expression of each gene shown. See Example 6.
  • PI3K refers to PI3 kinase.
  • PI3 kinase/AKT pathway inhibitor or " PI3K/AKT pathway inhibitor” refers to any agent that inhibits signaling via the PI3 kinase/AKT pathway, including but not limited to inhibitors of PI3 kinase and inhibitors of AKT. It also includes compounds that act "upstream" of PI3K in the PI3 kinase/AKT pathway, such as epidermal growth factor or its receptor (EGF/EGFR), since such compounds can inhibit signaling via the PI3 kinase/AKT pathway by removing an upstream triggering signal.
  • EGF/EGFR epidermal growth factor or its receptor
  • PI3 kinase/AKT pathway inhibitors encompass inhibitors of any activity that signals through or within the PI3 kinase/AKT pathway since any of these inhibitors may give rise to a rash that can benefit from the methods of the present invention.
  • AKTi refers to an AKT inhibitor generally.
  • AKTi-X refers to a specific PI3 kinase/AKT pathway inhibitor.
  • AKT inhibitor tra/75-3-amino-3- ⁇ 4-[l-(difluoromethyl)-8-phenyl[ 1,2,4] triazolo[4,3-a]-l,5- naphthyridin-7-yl]lphenyl ⁇ -l-methylcyclobutanol), as described herein.
  • MK-2206 refers to a specific AKT inhibitor, 8-[4-(l-aminocyclobutyl)phenyl]-9-phenyl[l,2,4]triazolo[3,4-f]-l,6- naphthyridin-3(2H)-one, as described herein.
  • AKT is also known as protein kinase B (PKB).
  • IL-23 antagonist refers to any agent that inhibits signaling by IL-23.
  • antagonists of the IL-23 receptor complex are also IL-23 antagonists.
  • a "non-specific IL-23 antagonist” is an agent that blocks the activity of both IL-23 and IL-12.
  • An "IL-23 -specific antagonist” is an agent that selectively blocks the activity of IL-23 but does not block the activity of IL-12.
  • IL-23 and “IL-12” as used herein refer to human IL-23 and human IL-12. The antibodies disclosed herein bind to human IL-23.
  • Proliferative activity encompasses an activity that promotes, that is necessary for, or that is specifically associated with, e.g., normal cell division, as well as cancer, tumors, dysplasia, cell transformation, metastasis, and angiogenesis.
  • administering refers to contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid.
  • administering can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell.
  • administering also means in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding composition, or by another cell.
  • Treatment refers to therapeutic treatment, prophylactic or preventative measures, to research and diagnostic applications.
  • Treatment as it applies to a human, veterinary, or research subject, or cell, tissue, or organ, encompasses contact of an agent with animal subject, a cell, tissue, physiological compartment, or physiological fluid.
  • “Treatment of a cell” also encompasses situations where the agent contacts IL-23 receptor (IL-23R/IL-12Rpi heterodimer), e.g., in the fluid phase or colloidal phase, but also situations where the agonist or antagonist does not contact the cell or the receptor.
  • the term "antibody” refers to any form of antibody that exhibits the desired biological activity. Thus, it is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, fully human antibodies, etc. so long as they exhibit the desired biological activity.
  • an antibody or fragment is an antibody or fragment that specifically binds to IL-23.
  • IL-23 binding fragment As used herein, the terms “IL-23 binding fragment,” “binding fragment thereof,” “antigen binding fragment thereof or “fragment” (when used with reference to an antibody) encompass a fragment or a derivative of an antibody that still substantially retains its biological activity of inhibiting IL-23pl9 activity. Therefore, the term “antibody fragment” or “IL-23 binding fragment” refers to a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., sc-Fv; and multispecific antibodies formed from antibody fragments.
  • a binding fragment or derivative retains at least 10% of its IL-23 inhibitory activity.
  • a binding fragment or derivative retains at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% (or more) of its IL-23 inhibitory activity, although any binding fragment with sufficient affinity to exert the desired biological effect will be useful.
  • an IL-23 binding fragment can include variants having conservative amino acid substitutions that do not substantially alter its biologic activity.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).
  • the "monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581- 597, for example.
  • Antibodies, and antigen binding fragments thereof may be described herein as comprising a light and/or heavy chain variable domain, or as comprising a light and/or heavy chain. As used herein, and consistent with their plain meanings, such descriptions encompass embodiments in which the antibody, or antigen binding fragment thereof, includes more than one light and/or heavy chain variable domain and/or chain, such as two light and/or heavy chain variable domains and/or chains.
  • the monoclonal antibodies herein specifically include “chimeric” antibodies
  • immunoglobulins in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
  • a “domain antibody” is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain.
  • two or more V H regions are covalently joined with a peptide linker to create a bivalent domain antibody.
  • the two V H regions of a bivalent domain antibody may target the same or different antigens.
  • a “bivalent antibody” comprises two antigen binding sites. In some instances, the two binding sites have the same antigen specificities. However, bivalent antibodies may be bispecific (see below).
  • single-chain Fv or "scFv” antibody refers to antibody fragments comprising the V H and V L domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the sFv to form the desired structure for antigen binding.
  • the monoclonal antibodies herein also include camelized single domain antibodies. See, e.g., Muyldermans et al. (2001) Trends Biochem. Sci. 26:230; Reichmann et al. (1999) J. Immunol. Methods 231 :25; WO 94/04678; WO 94/25591; U.S. Pat. No.
  • the present invention provides single domain antibodies comprising two V H domains with modifications such that single domain antibodies are formed.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (V H ) connected to a light chain variable domain (V L ) in the same polypeptide chain (V H -V L or V L - V H ).
  • V H heavy chain variable domain
  • V L light chain variable domain
  • the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are described more fully in, e.g., EP 404,097; WO 93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448. For a review of engineered antibody variants generally see Holliger and Hudson (2005) Nat.
  • humanized antibody refers to forms of antibodies that contain sequences from non-human ⁇ e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the humanized forms of rodent antibodies will generally comprise the same CDR sequences of the parental rodent antibodies, although certain amino acid substitutions may be included to increase affinity, increase stability of the humanized antibody, or for other reasons. [0049]
  • the antibodies of the present invention also include antibodies with modified
  • Alterations of the Fc region include amino acid changes (substitutions, deletions and insertions), glycosylation or deglycosylation, and adding multiple Fc. Changes to the Fc can also alter the half-life of antibodies in therapeutic antibodies, and a longer half-life would result in less frequent dosing, with the concomitant increased convenience and decreased use of material. See Presta (2005) J. Allergy Clin. Immunol.116:731 at 734-35.
  • Fully human antibody refers to an antibody that comprises human immunoglobulin protein sequences only.
  • a fully human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell.
  • mouse antibody refers to an antibody which comprises mouse immunoglobulin sequences only.
  • a fully human antibody may be generated in a human being, in a transgenic animal having human immunoglobulin germline sequences, by phage display or other molecular biological methods.
  • hypervariable region refers to the amino acid residues of an antibody that are responsible for antigen-binding.
  • the hypervariable region comprises amino acid residues from a "complementarity determining region” or "CDR" ⁇ e.g. residues 24-34 (CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3) in the light chain variable domain and residues 31-35 (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) in the heavy chain variable domain (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed.
  • CDR complementarity determining region
  • Binding compound refers to a molecule, small molecule, macromolecule, polypeptide, antibody or fragment or analogue thereof, or soluble receptor, capable of binding to a target.
  • Binding compound also may refer to a complex of molecules, e.g., a non- covalent complex, to an ionized molecule, and to a covalently or non-covalently modified molecule, e.g., modified by phosphorylation, acylation, cross-linking, cyclization, or limited cleavage, which is capable of binding to a target.
  • binding compound refers to both antibodies and antigen binding fragments thereof.
  • Binding refers to an association of the binding composition with a target where the association results in reduction in the normal Brownian motion of the binding composition, in cases where the binding composition can be dissolved or suspended in solution.
  • Binding composition refers to a molecule, e.g. a binding compound, in combination with a stabilizer, excipient, salt, buffer, solvent, or additive, capable of binding to a target.
  • Constantly modified variants or “conservative substitution” refers to substitutions of amino acids that may be made, as known by those of skill in the art, with little or no impact on the biological activity of the resulting molecule, even in essential regions of the polypeptide. Such exemplary substitutions are preferably made in accordance with those set forth in Table 1 as follows:
  • a binding compound that consists essentially of a recited amino acid sequence may also include one or more amino acids, including substitutions of one or more amino acid residues, that do not materially affect the properties of the binding compound.
  • Effective amount encompasses an amount sufficient to ameliorate or prevent a symptom or sign of the medical condition. Effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • An effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration and the severity of side affects. See, e.g., U.S. Pat. No. 5,888,530.
  • An effective amount can be the maximal dose or dosing protocol that avoids significant side effects or toxic effects.
  • the effect will result in an improvement of a diagnostic measure or parameter by at least 5%, usually by at least 10%, more usually at least 20%, most usually at least 30%>, preferably at least 40%>, more preferably at least 50%, most preferably at least 60%, ideally at least 70%, more ideally at least 80%>, and most ideally at least 90%>, where 100%) is defined as the diagnostic parameter shown by a normal subject. See, e.g., Maynard et al. (1996) A Handbook of SOPs or Good Clinical Practice, Interpharm Press, Boca Raton, FL; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK.
  • the expressions "cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny.
  • the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.
  • samples or assays comprising a given, e.g., protein, gene, cell, or organism, are treated with a potential activating or inhibiting agent and are compared to control samples without the agent.
  • Control samples i.e., not treated with agent, are assigned a relative activity value of 100%.
  • Inhibition is achieved when the activity value relative to the control is about 90%> or less, typically 85% or less, more typically 80%> or less, most typically 75% or less, generally 70%> or less, more generally 65%> or less, most generally 60%> or less, typically 55% or less, usually 50% or less, more usually 45% or less, most usually 40% or less, preferably 35% or less, more preferably 30%) or less, still more preferably 25% or less, and most preferably less than 25%.
  • Activation is achieved when the activity value relative to the control is about 110%, generally at least 120%, more generally at least 140%, more generally at least 160%, often at least 180%, more often at least 2-fold, most often at least 2.5-fold, usually at least 5-fold, more usually at least 10-fold, preferably at least 20-fold, more preferably at least 40-fold, and most preferably over 40-fold higher.
  • Endpoints in activation or inhibition can be monitored as follows. Activation, inhibition, and response to treatment, e.g., of a cell, physiological fluid, tissue, organ, and animal or human subject, can be monitored by an endpoint.
  • the endpoint may comprise a predetermined quantity or percentage of, e.g., an indicia of inflammation, oncogenicity, or cell degranulation or secretion, such as the release of a cytokine, toxic oxygen, or a protease.
  • the endpoint may comprise, e.g., a predetermined quantity of ion flux or transport; cell migration; cell adhesion; cell proliferation; potential for metastasis; cell differentiation; and change in phenotype, e.g., change in expression of gene relating to inflammation, apoptosis, transformation, cell cycle, or metastasis (see, e.g., Knight (2000) Ann. Clin. Lab. Sci. 30: 145- 158; Hood and Cheresh (2002) Nature Rev. Cancer 2:91-100; Timme et al. (2003) Curr. Drug Targets 4:251-261; Robbins and ltzkowitz (2002) Med. Clin. North Am. 86: 1467-1495; Grady and Markowitz (2002) Annu. Rev. Genomics Hum. Genet. 3:101-128; Bauer, et al. (2001) Glia 36:235-243; Stanimirovic and Satoh (2000) Brain Pathol. 10:113-126).
  • An endpoint of inhibition is generally 75% of the control or less, preferably
  • an endpoint of activation is at least 150% the control, preferably at least two times the control, more preferably at least four times the control, and most preferably at least 10 times the control.
  • a specified ligand binds to a particular receptor and does not bind in a significant amount to other proteins present in the sample.
  • an antibody is said to bind specifically to a polypeptide comprising a given sequence (in this case IL-23pl9) if it binds to polypeptides comprising the sequence of IL-23pl9 but does not bind to proteins lacking the sequence of IL-23pl9.
  • an antibody that specifically binds to a polypeptide comprising IL-23pl9 may bind to a FLAG ® -tagged form of IL-23pl9 but will not bind to other FLAG ® -tagged proteins.
  • the antibody, or binding composition derived from the antigen-binding site of an antibody, of the contemplated method binds to its antigen with an affinity that is at least two fold greater, preferably at least ten times greater, more preferably at least 20-times greater, and most preferably at least 100-times greater than the affinity with unrelated antigens.
  • the antibody will have an affinity that is greater than about 10 9 liters/mol, as determined, e.g., by Scatchard analysis. Munsen et al. (1980) Analyt. Biochem. 107:220-239.
  • the PI3K/AKT signaling pathway is the most frequently hyperactivated pathway in cancer. Inhibition of PI3K/AKT pathway is expected to have a significant impact on cancer cell survival and proliferation.
  • drugs targeting this pathway are currently in clinical trials (Liu et a/.(2009) Nat. Rev. Drug Disc. 8:627) including MK-2206, an allosteric inhibitor of AKT. Maculopapular skin rash is the dose limiting toxicity for MK-2206 in the clinic. Tolcher et a/. (2009) J. Clin. Oncol. 27:15s (abstract 3503).
  • the rash appears 7-10 days after the first dose and resolves completely after discontinuation of treatment with the drug. Skin rash is most prevalent at doses above 60 mg QOD, such as 75 mg and 90 mg (and presumably higher doses as well).
  • the present invention relates to the discovery that IL-23 is involved in skin rash that results from treatment with PI3K/AKT pathway inhibitors, e.g. for treatment of cancer.
  • This skin rash has been a dose-limiting toxicity for the AKT inhibitor MK-2206 in the clinic.
  • the invention involves antagonism of IL-23, such as treatment with an IL-23 antagonist, to ameliorate this rash. Elimination or reduction of the rash will have value as a matter of comfort to the patient, and may improve compliance. In addition, elimination or reduction of the rash may allow for elevated dosing and concomitant increased anti-tumor efficacy.
  • the present invention is based in part of studies of this MK-2206-induced skin rash.
  • Pathological examination of skin rash biopsies reveals marked perivascular infiltrates with T cells and abundant macrophages.
  • psoriasiform dermatitis is observed with epidermal hyperplasia and increased keratinocyte proliferation.
  • Other investigational drugs targeting the PI3K/AKT pathway have similar side effects ⁇ e.g., Edelman et al. (2010) J. Clin. Oncol. 28:15s (abstract 3004) and Baselga et al. (2010) J. Clin. Oncol. 28: 15s (abstract 3003)), suggesting that skin rashes are mechanism-based.
  • TLR agonists were included in these experiments based on the hypothesis that AKT inhibitor-induced skin rash is TLR dependent, and thus that TLR agonists should be present in culture to mimic the presence of microbial TLR agonists on natural skin. As illustrated in FIGS. 3 and 4, inhibition of AKT results in significant increase in IL-23 subunit mRNA.
  • FIG. 5 shows AKT inhibitor-induced elevation of IL-23 at the protein level as well.
  • Similarities include similar morphology (perivascular dermal infiltrate with T cell and macrophage components); abnormal stratum corneum, including occasional neutrophilic abscesses, loss of "basket weave” appearance, and abnormal filaggrin staining; epidermal hyperplasia; and increased basal keratinocyte proliferation. Both human and rhesus monkey rashes involve T cell and macrophage infiltrates, and both involve
  • TAQMAN ® real time quantitative polymerase chain reaction analysis was also performed on skin derived from MK-2206- and IL-23-treated monkeys.
  • 32 genes were selected for measurement based on their previous association with IL-23 -induced skin inflammation, as well as eight other genes of interest (VEGFA, CD4, CD8a, CD68, IL-10, TLR2, TLR4, VEGFR1).
  • Exemplary results are provided in Table 2 for two control rhesus monkeys and one MK-2206- treated rhesus monkey. Data are presented for two or three distinct skin samples obtained from each monkey. Values in bold- face are elevated at least two-fold higher than control levels,. As used herein, these two-fold changes constitute upregulation of gene expression. Of the 32 genes previously associated with IL-23-induced skin inflammation, seven were not detected in the monkey samples (IL-24, IL-17A, GM-CSF, IL-12p40, IL-23R, RANKL, IL- 26).
  • Control Rhesus 1 Control Rhesus 2 MK-2206-treated Rhesus
  • CD4 14.0 18.4 16.0 9.8 12.5 12.5 8.9
  • VEGFR1 13.9 11.3 11.6 8.7 8.9 7.0 8.3
  • FIGS. 6 A and 6B also provide exemplary gene expression data for
  • AKT inhibitor-induced rash AKT inhibitors induce IL-23 subunit gene expression and increase IL-23 levels in cells in vitro.
  • MK-2206 also induces a rash in monkeys that is histologically very similar to the clinically observed MK-2206-induced rash in human subjects, and is also very similar to skin inflammation induced by injection of IL-23 into monkey skin.
  • Many of the same genes induced by injection of IL-23 into primate skin are also induced in the primate model of MK-2206-induced rash, effectively mimicking the "gene expression profile" of IL-23 skin inflammation.
  • IL-23 appears to be involved in mediating AKT inhibitor-induced skin rash
  • inhibition of IL-23 -induced skin inflammation is promising approach to prevent and/or treat this rash.
  • Such treatment may eliminate the dose limiting toxicity of PI3K/AKT pathway inhibitors, such as MK-2206, and permit higher and potentially more efficacious dosing, e.g. for the treatment of cancer.
  • This potential increase in the therapeutic window for PI3K/AKT pathway inhibitors may allow for effective treatment with compounds that would otherwise face dose-limiting toxicity (rash) that limited their effectiveness.
  • Antagonism of IL-23 may also find use in treatment of rashes caused by other drugs that disrupt signaling via the PI3K/AKT pathway.
  • Epidermal grown factor (EGF) signals via the epidermal grown factor receptor (EGFR), which is overexpressed in many tumors. EGRF then acts, at least in part, via the PI3K/AKT pathway.
  • EGF/EGFR signaling have been proposed for the treatment of cancer, e.g. cetuximab
  • PI3K/AKT pathway Other receptors that signal via the PI3K/AKT pathway include human epidermal growth factor receptor 2 (HER2/neu, ErbB-2), hepatocyte growth factor receptor (HGFR, MET) and other receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). Inhibitors of signaling through these receptors may also induce the same rash that is observed with PI3K and AKT inhibitors, and thus may similarly benefit from the methods of the present invention.
  • HER2/neu, ErbB-2 human epidermal growth factor receptor 2
  • HGFR hepatocyte growth factor receptor
  • RTKs receptor tyrosine kinases
  • GPCRs G protein-coupled receptors
  • IL-23 has been show to promote tumor incidence and growth an animal models (Langowski et al. (2006) Nature 442:461) and antagonism of IL-23 has been proposed as a method to inhibit tumor growth (US 7,282,204). This advantage is not shared by antagonists of both IL-23 and IL-12, such as anti-p40 antibodies like ustekinumab and briakinumab. See Langowski et al. (2006) Nature 442:461 at Figs. 4(a), (f) and (g).
  • IL-23 Specific inhibition of IL-23 (but not IL-12) should also reduce the risk of various infections that would otherwise be elevated by an IL- 12/IL-23 antagonist, such as an anti-p40 antibody. Bowman et al. (2006) Curr. Op. Infect. Dis. 19:245. The reduced tumor and infection risks will be of great benefit to cancer patients being treated for PI3K/AKT pathway inhibitor-induced skin rash by the methods of the present invention involving IL-23 -specific antagonists.
  • the present invention involves antagonism of IL-23 activity as a means of ameliorating skin rash associated with cancer therapy using PI3K/AKT pathway inhibitors.
  • the method may be of use in treating a skin rash resulting from treatment with any
  • the AKT inhibitor of the present invention is a substituted naphthyridine compound, for example as disclosed in WO 2008/070016 (to Merck & Co., Inc. and Banyu Pharmaceutical Co., Ltd.).
  • the AKT inhibitor is 8- [4-( 1 -aminocyclobutyl)phenyl] -9-phenyl[ 1 ,2,4]triazolo [3 ,4-f] - 1 ,6-naphthyridin-3 (2H)-one (MK-2206) or a pharmaceutically acceptable salt thereof, as disclosed and claimed at US 7,576,209. See also WO 2008/070041 (to Merck & Co., Inc.), which is hereby incorporated by reference in its entirety.
  • the AKT inhibitor is a substituted [l,2,4]triazolo[4,3-a]-
  • 1,5 -naphthyridine compound e.g. tra/?5-3-amino-3- ⁇ 4-[l-(difluoromethyl)-8-phenyl[ 1,2,4] triazolo[4,3-a]-l,5-naphthyridin-7-yl]lphenyl ⁇ -l-methylcyclobutanol
  • AKTi-X alpha-1-naphthyridine compound, e.g. tra/?5-3-amino-3- ⁇ 4-[l-(difluoromethyl)-8-phenyl[ 1,2,4] triazolo[4,3-a]-l,5-naphthyridin-7-yl]lphenyl ⁇ -l-methylcyclobutanol
  • Additional exemplary AKT inhibitors include, but are not limited to: 1 - ⁇ 4-[3-(l -methyl-lH-imidazol-4-yl)-9-phenyl[ 1 ,2,4]triazolo[3,4- J-l ,6-naphthyridin-8- yl]phenyl ⁇ cyclobutanamine;
  • a reaction scheme for the production of exemplary AKT inhibitors of the present invention is provided at Example 2.
  • the AKT inhibitor of the present invention is an AKT allosteric inhibitor selected from among the compounds disclosed in WO 2005/100344;
  • the AKT inhibitor of the present invention is selected from among the compounds disclosed in US 2008/076763 (to Glaxo SmithKline LLC);
  • the AKT inhibitor is perifosine (KRX-0401) (Keryx); VQD-002 (API-2, TCN) (VioQuest); SR13668 (SRI
  • the AKT inhibitor is dosed in combination with one or more other cancer drugs.
  • the other cancer drug is a MEK inhibitor such as AZD6244 (selumetinib/ARRY-886)(AstraZeneca) or GSK1120212
  • the PI3K/AKT pathway inhibitors of the present invention may be administered to mammals, including humans, either alone or, in combination with pharmaceutically acceptable carriers, excipients or diluents, in a pharmaceutical composition, according to standard pharmaceutical practice.
  • the inhibitors (compounds) can be administered orally or parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration.
  • compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.
  • Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, microcrystalline cellulose, sodium crosscarmellose, corn starch, or alginic acid; binding agents, for example starch, gelatin, polyvinyl-pyrrolidone or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc.
  • the tablets may be uncoated or they may be coated by known techniques to mask the unpleasant taste of the drug or delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water soluble carrier such as polyethyleneglycol or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
  • water soluble carrier such as polyethyleneglycol or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
  • Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl- cellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbito
  • the aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p- hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame.
  • preservatives for example ethyl, or n-propyl p- hydroxybenzoate
  • coloring agents for example ethyl, or n-propyl p- hydroxybenzoate
  • flavoring agents for example ethyl, or n-propyl p- hydroxybenzoate
  • sweetening agents such as sucrose, saccharin or aspartame.
  • Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation.
  • These compositions may be preserved by the addition of an anti-oxidant such as butylated hydroxyanisol or alpha- tocopherol.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.
  • the pharmaceutical compositions of the invention may also be in the form of an oil-in-water emulsion.
  • the oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these.
  • Suitable emulsifying agents may be naturally-occurring phosphatides, for example soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions may also contain sweetening, flavouring agents, preservatives and antioxidants.
  • Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, flavoring and coloring agents and antioxidant.
  • sweetening agents for example glycerol, propylene glycol, sorbitol or sucrose.
  • Such formulations may also contain a demulcent, a preservative, flavoring and coloring agents and antioxidant.
  • compositions may be in the form of sterile injectable aqueous solutions.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • the sterile injectable preparation may also be a sterile injectable oil-in-water microemulsion where the active ingredient is dissolved in the oily phase.
  • the active ingredient may be first dissolved in a mixture of soybean oil and lecithin. The oil solution then introduced into a water and glycerol mixture and processed to form a microemulation.
  • the injectable solutions or microemulsions may be introduced into a patient's blood-stream by local bolus injection. Alternatively, it may be advantageous to administer the solution or microemulsion in such a way as to maintain a constant circulating
  • a continuous intravenous delivery device may be utilized.
  • An example of such a device is the Deltec CADD-PLUSTM model 5400 intravenous pump.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension for intramuscular and subcutaneous administration.
  • This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • Compounds of Formula A may also be administered in the form of suppositories for rectal administration of the drug.
  • These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary
  • Such materials include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol.
  • creams, ointments, jellies, solutions or suspensions, etc., containing the compound of Formula A are employed.
  • topical application shall include mouth washes and gargles.
  • the compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles and delivery devices, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in the art.
  • the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
  • Compounds of the present invention may also be delivered as a suppository employing bases such as cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol.
  • composition according to this invention When a composition according to this invention is administered into a human subject, the daily dosage will normally be determined by the prescribing physician with the dosage generally varying according to the age, weight, and response of the individual patient, as well as the severity of the patient's symptoms.
  • the dosage regimen utilizing the compounds of the instant invention can be selected in accordance with a variety of factors including type, species, age, weight, sex and the type of cancer being treated; the severity (i.e., stage) of the cancer to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed.
  • An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to treat, for example, to prevent, inhibit (fully or partially) or arrest the progress of the disease.
  • compounds of the instant invention can be administered in a total daily dose of up to 10,000 mg.
  • Compounds of the instant invention can be administered once daily (QD), or divided into multiple daily doses such as twice daily (BID), and three times daily (TID).
  • Compounds of the instant invention can be administered at a total daily or weekly dosage of up to 10,000 mg, e.g., 2,000 mg, 3,000 mg, 4,000 mg, 6,000 mg, 8,000 mg or 10,000 mg, which can be administered in one daily dose or can be divided into multiple daily doses as described above.
  • compounds of the instant invention can be administered in a total daily dose of up to 1 ,000 mg.
  • Compounds of the instant invention can be administered once daily (QD), or divided into multiple daily doses such as twice daily (BID), and three times daily (TID).
  • compounds of the instant invention can be administered less frequently, such as once a day (QD), once every other day (QOD), once every week (QW), or once every other week (QOW).
  • Compounds of the instant invention can be administered at a total daily or weekly dosage of up to 1,000 mg, e.g., 200 mg, 300 mg, 400 mg, 600 mg, 800 mg or 1 ,000 mg, which can be administered in one daily dose or can be divided into multiple daily doses as described above.
  • the administration can be continuous, i.e., every day, or intermittently.
  • intermittent administration of a compound of the instant invention may be administration one to six days per week or it may mean administration in cycles (e.g. daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week) or it may mean administration on alternate days.
  • the compounds of the instant invention may be administered according to any of the schedules described above, consecutively for a few weeks, followed by a rest period.
  • the compounds of the instant invention may be administered according to any one of the schedules described above from two to eight weeks, followed by a rest period of one week, or twice daily at a dose of 100 - 500 mg for three to five days a week.
  • the compounds of the instant invention may be
  • Preparations of the monohydrochloride salt of MK-2206 may be prepared as a tablet involving roller compression granulation followed by milling, mixing with the other inactive ingredients, compression, and film coating.
  • Some of the diluents or fillers for use in this formulation are preferably swellable agents, and may include, but are not limited to, various grades of microcrystallme cellulose, such as Avicel PH101, Avicel PH102, & Avicel PH200. If microcrystallme cellulose is added, it is preferably from about 50 to 180 microns in size, more preferably about 100.
  • Avicel PH 101 has a mean particle size of about 50
  • Avicel PH 102 has a mean particle size of about 100
  • Avicel PH 200 has a mean particle size of about 190 microns.
  • the preferred microcrystallme cellulose is Avicel PH 102.
  • the edible calcium salts suitable for use herein include but are not limited to, dibasic calcium phosphate dihydrate, calcium phosphate anhydrous, and tribasic calcium phosphate; or mixtures thereof.
  • a preferred edible calcium salt is the dibasic calcium phosphate anhydrous, which also provides good compressibility.
  • microcrystallme cellulose Dibasic calcium phosphate, dihydrate, from about 2 to about 4: 1, preferably from about 2.6-3.1 : 1;
  • microcrystallme cellulose Calcium phosphate, anhydrous from about 1 to about 3: 1, preferably from about 1.6:
  • microcrystallme cellulose Tribasic calcium phosphate, from about 2 to about 4: 1, preferably from about 3.1 : 1.
  • a preferred disintegrating agent is sodium croscarmellose.
  • the sodium croscarmellose is present in an amount of about 2 to about 5% w/w.
  • a preferred lubricant is magnesium stearate.
  • An aspect of the present invention is a process for preparing a tablet formulation which comprises:
  • step (b) roller compression granulation of the mixture of step (a) for the purpose of preparing granules
  • step (d) lubricating the granulation from steb (b); d) compacting the lubricated granulates of step (c) into concave tablet; and e) film coating tablets from step (d).
  • Interleukin-23 is a heterodimeric cytokine comprised of two subunits, pl9 which is unique to IL-23, and p40, which is shared with IL-12.
  • the pl9 subunit is structurally related to IL-6, granulocyte-colony stimulating factor (G-CSF), and the p35 subunit of IL-12.
  • IL-23 mediates signaling by binding to a heterodimeric receptor, comprised of IL-23R and IL-12 i, which is shared by the IL-12 receptor.
  • Therapeutic antibodies may be used to block cytokine activity.
  • the most significant limitation in using antibodies as a therapeutic agent in vivo is the immunogenicity of the antibodies.
  • Most monoclonal antibodies are derived from rodents, repeated use in humans results in the generation of an immune response against the therapeutic antibody.
  • Such an immune response results in a loss of therapeutic efficacy at a minimum and a potential fatal anaphylactic response at a maximum.
  • mice injected with hybrids of human variable regions and mouse constant regions develop a strong anti-antibody response directed against the human variable region, suggesting that the retention of the entire rodent Fv region in such chimeric antibodies may still result in unwanted immunogenicity in patients.
  • CDR loops of variable domains comprise the binding site of antibody molecules. Therefore, the grafting of rodent CDR loops onto human frameworks (i.e., humanization) was attempted to further minimize rodent sequences. Jones et al. (1986) Nature 321 :522; Verhoeyen et al. (1988) Science 239:1534. However, CDR loop exchanges still do not uniformly result in an antibody with the same binding properties as the antibody of origin. Changes in framework residues (FR), residues involved in CDR loop support, in humanized antibodies also are required to preserve antigen binding affinity. Kabat et al. (1991) J. Immunol. 147: 1709.
  • the present invention involves antagonism of IL-23 activity as a means of ameliorating skin rash associated with treatment with PI3K/AKT pathway inhibitors, such as cancer therapy.
  • Any suitable method of antagonizing IL-23 activity can be used in the methods of the present invention. Such methods include blocking the expression or activity of IL-23 or its receptor.
  • the method of antagonizing IL-23 activity is a method that does not antagonize the activity of IL-12, e.g. by use of an IL-23 -specific antagonist.
  • Such methods of antagonizing IL-23 may involve blocking of the activity of the pl9 subunit of IL-23, rather than the p40 subunit, since the pl9 subunit is specific to IL-23 (pi 9 + p40) whereas the p40 subunit is shared with IL-12 (p35 + p40).
  • Such methods of antagonizing IL- 23 may also involve blocking of the activity of the IL-23R subunit of the IL-23 receptor complex (IL-23R + IL-12Rpi), rather than the IL-12Rpi subunit that is shared with the IL-12 receptor (IL-12Rpi + IL-12RP2).
  • IL-23 antagonists include, but are not limited to, small molecule compounds, antisense nucleic acids, small interfering nucleic acids, aptamers, antibodies or antigen binding fragments thereof, and soluble forms of IL-23 receptor.
  • the IL-23 antagonist is an IL-23 -specific antagonist.
  • Exemplary IL-23 -specific antagonists include an antibody that binds specifically to IL-23p40 but not IL-12p40 (US 7,247,711 to Centocor) or an antibody that makes contacts with both the pl9 and p40 subunits of IL-23 (WO 2011/056600 to Amgen, Inc.). Fibronectin-derived IL-23 antagonists are disclosed at WO 2011/103105 (developed at Adnexus Therapeutics Inc., now part of Bristol-Myers Squibb Co.).
  • the IL-23 -specific antagonist binds to pi 9.
  • IL-23 -specific antagonists that bind to pl9 include multimerized IL-23 receptors
  • IL-23 -specific antagonists that bind to pl9 include antibodies or antigen-binding fragments thereof that specifically bind to the pl9 subunit of IL-23, as disclosed at
  • Anti-IL-23pl9 antibodies that may be suitable for use in the methods of the present invention also include, but are not limited to, Merck's SCH 900222/MK-3222; Eli Lilly's LY2525623, and Centocor's CNTO 1959, all of which have entered human clinical trials. Specifically, the sequences of SEQ ID NOs: 48 and 52 (heavy chain variable domains), 57 (light chain variable domain), 28-37-40 (light chain CDRs 1-2-3, respectively) and 3-8-19 (light chain CDRs 1-2-3, respectively) of EP 1937721 Bl (to Eli Lilly and Company) are hereby incorporated by reference.
  • Centocor Centocor
  • the anti-IL-23pl9 antibodies, or antigen binding fragments thereof are based on antibody 13B8 of commonly assigned WO 2008/103432, the disclosure of which is hereby incorporated by reference in its entirety.
  • the anti-human IL- 23pl9 antibody may comprise one, two, three, four, five or six of the CDR sequences, or the heavy and light chain variable domains, of the humanized antibodies disclosed in commonly assigned WO 2008/103432, for example antibodies hul3B8a, b or c.
  • the anti-human IL-23pl9 antibody competes with antibody hul3B8a, b or c for binding to human IL-23.
  • the anti-human IL-23pl9 antibody binds to the same epitope on human IL-23 as hul3B8a, b or c.
  • a hybridoma expressing antibody 13B8 was deposited pursuant to the
  • the anti-human IL-23pl9 antibody is able to block binding of human IL-23pl9 to the antibody produced by the hybridoma deposited with accession number PTA-7803 in a cross-blocking assay.
  • the anti- human IL-23pl9 antibody binds to the same epitope as the antibody produced by the hybridoma deposited with ATCC under accession number PTA-7803.
  • the anti-human IL-23pl9 antibody comprises the same CDR sequences as the antibody produced by the hybridoma deposited with ATCC with accession number PTA- 7803.
  • the IL-23 -specific antagonist binds to IL-23R.
  • Exemplary IL-23 -specific antagonists that bind to IL-23R include anti-IL-23R antibodies (WO 2008/106134 and WO 2010/027767 to Schering Corp.); multimerized and multimerized polypeptides that binds to IL-23R (U.S. Pat. App. Pub. No. 2011/0086806 to Anaphore, Inc.); and IL-23 receptor antagonist peptides (WO 2009/007849 to Valorisation HSJ and Societe en Commandite), such as APG2305 (Allostera Pharma, Inc.).
  • the IL-23 antagonist is a non-specific IL-23 antagonist
  • Exemplary non-specific IL-23 antagonists include antibodies that bind to the p40 subunit of IL-23 and IL-12, such as ustekinumab (CNTO 1275) and briakinumab (ABT-874, J-695).
  • Ustekinumab is marketed by Centocor for the treatment of psoriasis, and is described at US 6,902,734 and US 7,166,285 (to Centocor, Inc.), the disclosures of which are hereby incorporated by reference in their entireties.
  • sequences of SEQ ID NOs: 7 (heavy chain variable domain) and 8 (light chain variable domain), of US 6,902,734 are hereby incorporated by reference.
  • SEQ ID NOs: 4-5-6 and 1-2-3 of US 6,902,734 are also incorporated by reference.
  • Sequences for ustekinumab are also provided at SEQ ID NOs: 51 - 60 of the sequence listing of the present application.
  • Briakinumab was developed by Abbott, and is described at US 6,914,128 and US 7,504,485, the disclosures of which are hereby incorporated by reference in their entireties. Specifically, the sequences of SEQ ID NOs: 31 (heavy chain variable domain), 32 (light chain variable domain) SEQ ID NOs; 30- 28-26 (light chain CDRs 1-2-3, respectively) and 29-27-25 (heavy chain CDRs 1-2-3, respectively) of US 6,914,128 are hereby incorporated by reference. Sequences for briakinumab are also provided at SEQ ID NOs: 61 - 70 of the sequence listing of the present application.
  • exemplary non-specific IL-23 antagonist antibodies that bind to the p40 subunit of IL-23 and IL-12 are disclosed at Clarke et al. (2010) mAbs 2: 1-11 (Cephalon Australia, Pty., Ltd.).
  • FM202 Femta Pharmaceuticals
  • FM202 is also a monoclonal antibody that binds to the p40 subunit of both IL-12 and IL-23, as are the antibodies disclosed at
  • WO 2010/017598 (Arana Therapeutics, Ltd.).
  • Apilimod mesylate (STA-5326, Synta Pharmaceuticals Corp.), an oral non-specific IL-23 antagonist, may also be used in some embodiments of the present invention.
  • Still further exemplary non-specific IL-23 antagonists include antibodies that bind to the IL-12Rpi subunit of both the IL-12 and IL-23 receptor complexes (WO 2010/112458 to Novartis AG).
  • amino acid sequence variants of the human or humanized anti-IL-23 antibody will have an amino acid sequence having at least 75% amino acid sequence identity with the original human or humanized antibody amino acid sequences of either the heavy or the light chain more preferably at least 80%, more preferably at least 85%, more preferably at least 90%), and most preferably at least 95, 98, or 99%.
  • Identity or homology with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the human or humanized anti-IL-23 residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. None of N-terminal, C-terminal, or internal extensions, deletions, or insertions into the antibody sequence shall be construed as affecting sequence identity or homology.
  • the human or humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE.
  • the antibody is an IgG antibody.
  • Any isotype of IgG can be used, including IgGi, IgG 2 , IgG 3 , and IgG 4 .
  • Variants of the IgG isotypes are also contemplated.
  • the human or humanized antibody may comprise sequences from more than one class or isotype. Optimization of the necessary constant domain sequences to generate the desired biologic activity is readily achieved by screening the antibodies in the biological assays described below.
  • either class of light chain can be used in the compositions and methods herein.
  • kappa, lambda, or variants thereof are useful in the present compositions and methods.
  • any suitable portion of the CDR sequences from the non-human antibody can be used.
  • the CDR sequences can be mutagenized by substitution, insertion or deletion of at least one residue such that the CDR sequence is distinct from the human and non-human antibody sequence employed. It is contemplated that such mutations would be minimal.
  • at least 75% of the humanized antibody residues will correspond to those of the non-human CDR residues, more often 90%, and most preferably greater than 95%.
  • any suitable portion of the FR sequences from the human antibody can be used.
  • the FR sequences can be mutagenized by substitution, insertion or deletion of at least one residue such that the FR sequence is distinct from the human and non-human antibody sequence employed. It is contemplated that such mutations would be minimal.
  • at least 75% of the humanized antibody residues will correspond to those of the human FR residues, more often 90%, and most preferably greater than 95, 98, or 99%.
  • CDR and FR residues are determined according to the standard sequence definition of Kabat. Kabat et al. (1987) Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda Md.
  • SEQ ID NOs: 1-5 show the heavy chain variable domain sequences of various mouse anti-human IL-23pl9 antibodies, and SEQ ID NOs: 9-13 depict the light chain variable domain sequences.
  • FIGS. 1 and 2 provide sequence lineups of heavy and light chain variable domains of the various antibodies of the present invention.
  • CDRs are indicated in the figures, and the individual CDR sequences are each presented with unique Sequence Identifiers as indicated in Table 4.
  • 13B8 sequence (with kappa constant region) is provided at SEQ ID NO: 14, and the light chain variable domain comprises residues 1-108 of that sequence.
  • Three versions of the humanized heavy chain 13B8 sequence (with ⁇ constant regions) are provided at SEQ ID NOs: 6-8, and the heavy chain variable domain comprises residues 1-116 of those sequences.
  • the 13B8 heavy chains variants are illustrated at Table 3, with differences from the parental sequence noted in bold.
  • the Met (M) was modified to Lys (K) to avoid the potential for oxidation of the residue and inactivation of the antibody.
  • the substitution of AQKLQ for NEMFE is a replacement of the murine CDR sequence with the human germline sequence from the human framework selected to humanize the antibody.
  • Antibody CDRH2 Sequence SEQ ID NO: ml3B8, hl3B8-a QIFPASGSADYNEMFEG 24
  • Humanized forms of the other antibodies disclosed herein may be created by simply substituting the parental rodent antibody CDRs into the light and heavy chain sequences for humanized 13B8 provided at SEQ ID NOs: 14 and 6. This approach is most likely to be successful for antibody chains with CDRs having high homology with the CDRs of antibody 13B8, e.g. clone 1 ICl on the heavy chain and clones 1 ICl and 21D1 on the light chain.
  • the murine antibodies may be independently humanized using the approaches outlines herein, e.g. at Example 2.
  • CDRs include variants of any single sequence CDR disclosed herein (SEQ ID NOs: 15-46), in which the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions relative to the disclosed sequence, as determined using the data of Table 1.
  • chimeric antibodies comprise a portion of the heavy and/or light chain identical with, or homologous to, corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. See U.S. Pat. No. 4,816,567; and
  • Bispecific antibodies are also useful in the present methods and compositions.
  • bispecific antibody refers to an antibody, typically a monoclonal antibody, having binding specificities for at least two different antigenic epitopes, e.g., IL- 23pl9 and IL-17.
  • the epitopes are from the same antigen.
  • the epitopes are from two different antigens.
  • Methods for making bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly using the co-expression of two immunoglobulin heavy chain/light chain pairs. See, e.g., Milstein et al. (1983) Nature 305: 537-39. Alternatively, bispecific antibodies can be prepared using chemical linkage. See, e.g., Brennan et al. (1985) Science 229:81.
  • Bispecific antibodies include bispecific antibody fragments. See, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6444-48, Gruber et al. (1994) J. Immunol. 152:5368.
  • different constant domains may be appended to the humanized V L and V R regions provided herein. For example, if a particular intended use of an antibody (or fragment) of the present invention were to call for altered effector functions, a heavy chain constant domain other than IgGl may be used.
  • IgGl antibodies provide for long half-life and for effector functions, such as complement activation and antibody-dependent cellular cytotoxicity, such activities may not be desirable for all uses of the antibody.
  • an IgG4 constant domain for example, may be used.
  • Antibodies having the characteristics identified herein as being desirable in a humanized anti-IL-23 antibody can be screened for inhibitory biologic activity in vitro or suitable binding affinity.
  • ANTIBODIES A LABORATORY MANUAL, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988) can be performed.
  • Antibodies that bind to the same epitope are likely to cross-block in such assays, but not all cross-blocking antibodies will necessarily bind at precisely the same epitope since cross-blocking may result from steric hindrance of antibody binding by antibodies bind at overlapping epitopes, or even nearby non-overlapping epitopes.
  • Biol. Chem. 270: 1388-1394 can be performed to determine whether the antibody binds an epitope of interest.
  • "Alanine scanning mutagenesis,” as described by Cunningham and Wells (1989) Science 244: 1081-1085, or some other form of point mutagenesis of amino acid residues in human IL-23 may also be used to determine the functional epitope for an anti-IL- 23 antibody of the present invention. Mutagenesis studies, however, may also reveal amino acid residues that are crucial to the overall three-dimensional structure of IL-23 but that are not directly involved in antibody-antigen contacts, and thus other methods may be necessary to confirm a functional epitope determined using this method.
  • the epitope bound by a specific antibody may also be determined by assessing binding of the antibody to peptides comprising fragments of human IL-23pl9 (SEQ ID NO: 47).
  • the sequence of the p40 subunit of IL-12 and IL-23 is found at GenBank Accession No. P29460.
  • a series of overlapping peptides encompassing the sequence of IL-23pl9 may be synthesized and screened for binding, e.g. in a direct ELISA, a competitive ELISA (where the peptide is assessed for its ability to prevent binding of an antibody to IL-23pl9 bound to a well of a microtiter plate), or on a chip.
  • Such peptide screening methods may not be capable of detecting some discontinuous functional epitopes, i.e. functional epitopes that involve amino acid residues that are not contiguous along the primary sequence of the IL-23pl9 polypeptide chain.
  • the epitope bound by antibodies of the present invention may also be determined by structural methods, such as X-ray crystal structure determination (e.g., WO2005/044853), molecular modeling and nuclear magnetic resonance (NMR)
  • crystallization may be accomplished using any of the known methods in the art (e.g. Giege et al. (1994) Acta Crystallogr.
  • a protein preparation having a concentration of at least about 1 mg/mL and preferably about 10 mg/mL to about 20 mg/mL.
  • Crystallization may be best achieved in a precipitant solution containing polyethylene glycol 1000-20,000 (PEG; average molecular weight ranging from about 1000 to about 20,000 Da), preferably about 5000 to about 7000 Da, more preferably about 6000 Da, with concentrations ranging from about 10% to about 30% (w/v). It may also be desirable to include a protein stabilizing agent, e.g. glycerol at a concentration ranging from about 0.5%) to about 20%>.
  • a suitable salt such as sodium chloride, lithium chloride or sodium citrate may also be desirable in the precipitant solution, preferably in a concentration ranging from about 1 mM to about 1000 mM.
  • the precipitant is preferably buffered to a pH of from about 4.0 to about 10.0, often from about 7.0 to 8.5, e.g. pH 8.0.
  • Specific buffers useful in the precipitant solution may vary and are well-known in the art. Scopes, Protein Purification: Principles and Practice, Third ed., (1994) Springer-Verlag, New York. Examples of useful buffers include, but are not limited to, HEPES, Tris, MES and acetate. Crystals may be grow at a wide range of temperatures, including 2°C, 4°C, 8°C and 26°C.
  • Antibody antigen crystals may be studied using well-known X-ray diffraction techniques and may be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see e.g. Blundell & Johnson (1985) Meth. Enzymol. 114 & 115, H. W. Wyckoff et al. eds., Academic Press; U.S. Patent Application Publication No. 2004/0014194), and BUSTER (Bricogne (1993) Acta Cryst. D49:37-60; Bricogne (1997) Meth. Enzymol. 276A:361-423, Carter & Sweet, eds.; Roversi et al. (2000) Acta Cryst. D56: 1313-1323).
  • X-PLOR Yale University, 1992, distributed by Molecular Simulations, Inc.; see e.g. Blundell & Johnson (1985) Meth. Enzymol. 114 & 115
  • Additional antibodies binding to the same epitope as an antibody of the present invention may be obtained, for example, by screening of antibodies raised against IL-23 for binding to the epitope, or by immunization of an animal with a peptide comprising a fragment of human IL-23 comprising the epitope sequence.
  • Antibodies that bind to the same functional epitope might be expected to exhibit similar biological activities, such as blocking receptor binding, and such activities can be confirmed by functional assays of the antibodies.
  • Antibody affinities ⁇ e.g. for human IL-23) may be determined using standard analysis.
  • Preferred humanized antibodies are those which bind human IL-23pl9 with a Ka
  • the antibodies and fragments thereof useful in the present compositions and methods are biologically active antibodies and fragments.
  • biologically active refers to an antibody or antibody fragment that is capable of binding the desired the antigenic epitope and directly or indirectly exerting a biologic effect. Typically, these effects result from the failure of IL-23 to bind its receptor.
  • specific refers to the selective binding of the antibody to the target antigen epitope.
  • Antibodies can be tested for specificity of binding by comparing binding to IL-23 to binding to irrelevant antigen or antigen mixture under a given set of conditions. If the antibody binds to IL-23 at least 10, and preferably 50 times more than to irrelevant antigen or antigen mixture then it is considered to be specific.
  • An antibody that binds to IL-12 is not an IL-23- specific antibody.
  • An antibody that "specifically binds" to IL-23pl9 does not bind to proteins that do not comprise the IL-23pl9-derived sequences, i.e. "specificity" as used herein relates to IL-23pl9 specificity, and not any other sequences that may be present in the protein in question.
  • an antibody that "specifically binds" to IL-23pl9 will typically bind to FLAG ® -hIL-23pl9, which is a fusion protein comprising IL-23pl9 and a FLAG peptide tag, but it does not bind to the FLAG peptide tag alone or when it is fused to a protein other than IL-23pl9.
  • IL-23 -specific binding compounds of the present invention such as inhibitory
  • IL-23pl9 specific antibodies can inhibit its biological activity in any manner, including but not limited to production of IL- ⁇ and TNF by peritoneal macrophages and IL-17 by T R 17 T cells. See Langrish et al. (2004) Immunol. Rev. 202:96-105. Anti-IL-23pl9 antibodies will also be able to inhibit the gene expression of IL-17A, IL-17F, CCL7, CCL17, CCL20, CCL22, CCR1, and GM-CSF. See Langrish et al. (2005) J. Exp. Med. 201 :233-240.
  • IL-23-specific binding compounds of the present invention such as anti IL-23pl9 antibodies, will also block the ability of IL-23 to enhance proliferation or survival of T H 17 cells.
  • compositions including IL-23pl9 antibody the antibody (or antigen binding fragment thereof) is admixed with a
  • Formulations of therapeutic and diagnostic agents may be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions.
  • physiologically acceptable carriers e.g., lyophilized powders, slurries, aqueous solutions or suspensions.
  • Toxicity and therapeutic efficacy of the antibody compositions, administered alone or in combination with an immunosuppressive agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 5 o (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio of LD 50 to ED 50 .
  • Antibodies exhibiting high therapeutic indices are preferred.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the mode of administration is not particularly important. Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal
  • parenteral delivery including intramuscular, intradermal, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • Administration of antibody used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral, intraarterial or intravenous injection.
  • the liposomes will be targeted to and taken up selectively by the afflicted tissue.
  • an administration regimen for a therapeutic depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells in the biological matrix.
  • an administration regimen maximizes the amount of therapeutic delivered to the patient consistent with an acceptable level of side effects.
  • the amount of biologic delivered depends in part on the particular entity and the severity of the condition being treated. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available. See, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub.
  • Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects.
  • Important diagnostic measures include those of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced.
  • a biologic that will be used is substantially derived from the same species as the animal targeted for treatment (e.g. a humanized antibody for treatment of human subjects), thereby minimizing any immune response to the reagent.
  • Antibodies, antibody fragments, and cytokines can be provided by continuous infusion, or by doses at intervals of, e.g., one day, 1-7 times per week, one week, two weeks, monthly, bimonthly, quarterly, every 4 or 6 months, annually, etc.
  • Doses may be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular,
  • a preferred dose protocol is one involving the maximal dose or dose frequency that avoids significant undesirable side effects.
  • a total weekly dose is generally at least 0.05 ⁇ g/kg, 0.2 ⁇ g/kg, 0.5 ⁇ g/kg, 1 ⁇ g/kg, 10 ⁇ g/kg, 100 ⁇ g/kg, 0.2 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 10 mg/kg, 25 mg/kg, 50 mg/kg body weight or more. See, e.g., Yang et al. (2003) New Engl. J. Med. 349:427-434; Herald et al. (2002) New Engl. J. Med. 346:1692-1698; Liu et al.
  • a small molecule therapeutic e.g., a peptide mimetic, natural product, or organic chemical
  • a small molecule therapeutic e.g., a peptide mimetic, natural product, or organic chemical, is about the same as for an antibody or polypeptide, on a moles/kg basis.
  • inhibit or “treat” or “treatment” includes a postponement of development of the symptoms associated with skin rash induced by PI3K/AKT pathway inhibitors and/or a reduction in the severity of such symptoms that will or are expected to develop.
  • the terms further include ameliorating existing uncontrolled or unwanted side effects, such as skin rash, and preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms.
  • the terms denote that a beneficial result has been conferred on a subject with a sign or symptom, or with the potential to develop such a sign or symptom.
  • Prevent refers to a reduction or complete elimination of a side effect, such as a skin rash, prior to it manifesting at all. Neither treatment nor prevention, as used herein, requires complete elimination of signs and symptoms. Prevention and treatment refer to any clinically meaningful improvement of the relevant condition, such as reduction in the signs or symptoms of skin rash associated with treatment with PI3K/AKT inhibitors.
  • the term "therapeutically effective amount” or “effective amount” refers to an amount of an IL-23 antagonist, e.g. an IL-23pl9-specific antibody, or antigen binding fragment thereof, that when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject is effective to prevent or ameliorate the drug-induced side effect, such as skin rash.
  • a therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • An effective amount of therapeutic will decrease the symptoms typically by at least 10%; usually by at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably by at least 50%.
  • Typical veterinary, experimental, or research subjects include monkeys, dogs, cats, rats, mice, rabbits, guinea pigs, horses, and humans.
  • the nucleic acids encoding the two chains are isolated and inserted into one or more replicable vectors for further cloning (amplification of the DNA) or for expression.
  • DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
  • Many vectors are available.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
  • both the light and heavy chains of the humanized anti-IL-23pl9 antibody of the present invention are expressed from the same vector, e.g. a plasmid or an adenoviral vector.
  • Antibodies of the present invention may be produced by any method known in the art.
  • antibodies are expressed in mammalian or insect cells in culture, such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) 293 cells, mouse myeloma NSO cells, baby hamster kidney (BHK) cells, Spodoptera frugiperda ovarian (Sf9) cells.
  • CHO Chinese hamster ovary
  • HEK human embryonic kidney
  • BHK baby hamster kidney
  • Spodoptera frugiperda ovarian (Sf9) cells In one embodiment, antibodies secreted from CHO cells are recovered and purified by standard chromatographic methods, such as protein A, cation exchange, anion exchange, hydrophobic interaction, and hydroxyapatite chromatography. Resulting antibodies are concentrated and stored in 20 mM sodium acetate, pH 5.5.
  • the antibodies of the present invention are produced in yeast, e.g. according to the methods described in WO 2005/040395. Briefly, vectors encoding the individual light or heavy chains of an antibody of interest are introduced into different yeast haploid cells, e.g. different mating types of the yeast Pichia pastoris, which yeast haploid cells are optionally complementary auxotrophs. The transformed haploid yeast cells can then be mated or fused to give a diploid yeast cell capable of producing both the heavy and the light chains. The diploid strain is then able to secret the fully assembled and biologically active antibody.
  • the relative expression levels of the two chains can be optimized, for example, by using vectors with different copy number, using transcriptional promoters of different strengths, or inducing expression from inducible promoters driving transcription of the genes encoding one or both chains.
  • the respective heavy and light chains of a plurality of different anti-IL-23pl9 antibodies are introduced into yeast haploid cells to create a library of haploid yeast strains of one mating type expressing a plurality of light chains, and a library of haploid yeast strains of a different mating type expressing a plurality of heavy chains.
  • These libraries of haploid strains can be mated (or fused as spheroplasts) to produce a series of diploid yeast cells expressing a combinatorial library of antibodies comprised of the various possible permutations of light and heavy chains.
  • the combinatorial library of antibodies can then be screened to determine whether any of the antibodies has properties that are superior (e.g. higher affinity for IL-23) to those of the original antibodies. See. e.g., WO 2005/040395.
  • antibodies of the present invention are human domain antibodies in which portions of an antibody variable domain are linked in a polypeptide of molecular weight approximately 13 kDa. See, e.g., U.S. Pat. Publication No. 2004/0110941.
  • Such single domain, low molecular weight agents provide numerous advantages in terms of ease of synthesis, stability, and route of administration.
  • the present invention provides methods for preventing and/or treating skin rash that may occur as a side effect of treatment with PI3K/AKT pathway inhibitors.
  • the methods of the present invention involve antagonism of IL-23, e.g. by treatment with an IL- 23 antagonist, in any subject being treated with a PI3K/AKT pathway inhibitor for any reason.
  • PI3K/AKT pathway inhibitors may find use not only in the treatment of cancer or tumors, but also in the treatment of diseases in which angiogenesis is implicated, such as ocular neovascular diseases like diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, age-related macular degeneration (WO 00/30651); atherosclerosis, osteoarthritis (WO 03/035048), psoriasis, obesity, and Alzheimer's disease.
  • PI3K/AKT pathway inhibitors may find use in treating hyperproliferative disorders like restenosis (WO 03/032809), inflammation, autoimmune diseases and allergy/asthma.
  • IL-23 antagonists may find use in the manufacture of medicaments for preventing and/or treating skin rash that may occur as a side effect of treatment with a PI3K/AKT pathway inhibitor.
  • the present invention further relates to an IL-23 antagonist, e.g. an anti-IL-23 antibody, for use in preventing and/or treating skin rash that may occur as a side effect of treatment with a PI3K/AKT pathway inhibitor.
  • the invention further relates to prevention and/or treatment of a disorder, such as cancer or a tumor, by administering to a subject an IL-23 antagonist and a PI3K/AKT inhibitor.
  • the IL-23 antagonist is administered prior to, concurrently with, or after administration of the PI3K/AKT inhibitor.
  • Administration of the IL-23 antagonist prior to or concurrently with the PI3K/AKT inhibitor may be helpful in preventing a PI3K/AKT inhibitor-induced rash, whereas administration of the IL-23 antagonist after the onset of a PI3K/AKT inhibitor-induced rash may be helpful in treatment of the rash or prevention of a rash before is has occurred.
  • the IL-23 antagonist is administered, e.g., 1 hour, 12 hours, 1, 2, 4, 7, 10, 14, 21, 30 or more days prior to administration of the PI3K/AKT inhibitor.
  • the IL-23 antagonist is an anti-IL-23pl9 antibody or antigen binding fragment thereof.
  • the PI3K/AKT inhibitor is naphthyridine compound, e.g., 8-[4-(l-aminocyclobutyl)phenyl]-9- phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-3(2H)-one (MK-2206).
  • PI3K/AKT inhibitors of the present invention may be any PI3K/AKT inhibitors.
  • the methods of the present invention find particular value at doses that are high enough to otherwise induce a rash in subjects.
  • 8-[4-(l- aminocyclobutyl)phenyl]-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-3(2H)-one may be administered at 150 - 200 mg every week, such as 200 mg every week, or at doses that increase the risk of skin rash, such as 250, 300, 350, 400, 450, 500, 600, 700, 750, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg or more.
  • IL-23 antagonist of the present invention may be administered at any dose effective to prevent or treat a rash induced by a PI3K/AKT pathway inhibitor.
  • Anti-IL-23 antibodies such as anti-IL-23pl9 and anti-IL-23p40 antibodies, made be administered, e.g., at 0.01, 0.03, 0.1, 0.3, 0.5, 1, 3, 5, 10, 30, 50 or 100 mg/kg or more.
  • the anti-IL-23 antibody (or fragment) is administered at 0.1, 0.3, 1 or 3 mg/kg.
  • the anti-IL-23 antibody (or fragment) is administered weekly, biweekly, monthly, bimonthly, every three months or at other intervals.
  • the IL-23 antibody (or fragment) is administered only once during a given course of treatment with a PI3K AKT pathway inhibitor, or is administered as needed based on an evaluation of the condition of a specific subject, e.g. based on the presence and severity of a skin rash.
  • Anti- IL-23 antibodies (or fragments thereof) may be administered by any suitable means, including but not limited to subcutaneous injection and intravenous infusion.
  • Exemplary substituted naphthyridine compounds for use as AKT inhibitors in the methods of the present invention may be synthesized as follow. See, e.g., WO 2008/070016 (Schemes 1C and IE).
  • Trifluoroacetic acid (17.4 mL, 234 mmol) was added carefully to a stirred mixture of Boc aldehyde 1-7 (20 g, 78.1 mmol) and dichloromethane (60 mL) keeping the temperature below 25 °C. The solution was warmed to 35 °C, aged overnight (vigorous off- gassing) and then cooled to room temperature. 25 mL of MTBE was added and the resulting white slurry was aged for one hour, filtered, and the filter cake rinsed with MTBE (10 mL x 2). Solid 1-8 TFA salt was dried under vacuum. tert-butyl ⁇ l-[4-(5-chloro-3-phenyl-l,6-naphthyridin-2-yl)phenyl]cyclobutyl ⁇ carbamate (1-9)
  • a stirred slurry of chloronapthyridine 1-9 (1.8 g), methyl hydrazine carboxylate (0.318 g) and isopropanol (20 L) is warmed to 66 °C before becoming homogeneous.
  • 5-6 N HC1 in IPA (0.05 ml) is added and the temperature is increased to 70 °C for 16 hours and then is cooled to RT.
  • 45wt% potassium hydroxide solution (0.52 mL) is mixed with water (5.5 mL) and added over 15 minutes. After 30 minutes, aqueous acetic acid (0.7 mL in 6 mL water) is added followed by water (2 mL).
  • a mono-HCl version of MK-2206 was also produced via dissolution in water.
  • THP-1 levels were determined as follows. THP-1 monocytic cells were cultured at 1 x 10 6 cells/ml in 24-well plates in RPMI 1640 complete medium. Cells were pre-treated with either of two AKT inhibitors (MK-2206 and AKTi-X) at 0.1 ⁇ or 0.5 ⁇ for 1 hour.
  • MK-2206 and AKTi-X AKT inhibitors
  • IL-23 is a heterodimeric complex of p40 and pl9
  • IL-12 is a heterodimeric complex of p40 and p35 subunits. Results are shown in FIG. 3.
  • AKT inhibitor specifically enhances IL-23 subunit gene expression above the level induced by LPS alone. This result holds true for both AKT inhibitors (MK-2206 and AKTi-X). These results are consistent with a downstream role for IL-23 in AKT inhibitor-induced skin rash.
  • CD 14+ cells were isolated from healthy human donors (4 donors total).
  • PBMC were isolated from buffy coats using Ficoll gradient procedure followed by negative selection of CD 14+ cells using
  • Monocyte Isolation Kit II (Miltenyi Biotec Cat # 130-091-153). CD14+ cells were grown in complete DMEM medium supplemented with antibiotics. Cells were pre-treated with either of two AKT inhibitors (MK-2206 and AKTi-X) at 0.1 ⁇ or 0.5 ⁇ for lh. Ten (10) ⁇ lipopolysaccharide (LPS) was then added for 3h, 6h or 24h, at which time cells were harvested for R A extraction and TAQMAN ® real-time quantitative polymerase chain reaction gene expression analysis. Results are shown in FIG. 4. Supernatants were also collected for IL-23 protein determination. See Example 5.
  • AKT inhibitors MK-2206 and AKTi-X
  • LPS lipopolysaccharide
  • AKT inhibitor specifically enhances IL-23 subunit gene expression above the level induced by LPS alone. This result holds true for both donors, and at time points ranging from 3 to 6 to 24 hours post-treatment (other than the single datapoint for Donor 2 at 24 hours). These results are consistent with a downstream role for IL-23 in AKT inhibitor-induced skin rash.
  • PBMCs as a function of treatment with TLR agonists, with or without AKT inhibitor was determined using culture supernatants prepared as described in Example 4.
  • IL-23 was detected in an electrochemilummescence assay (ECL) using an anti-human IL-23pl9 capture antibody and a ruthenium labeled anti-human IL-12/23p40 detection antibody. Results are shown in FIG. 5.
  • the levels of expression of 24 genes known to be modulated by IL-23 were determined in IL-23 -treated cynomolgus monkeys and also in MK-2206-treated rhesus monkeys as follows.
  • IL-23 -induced skin inflammation model 10 or 100 ⁇ g of human IL-23 was intradermally injected to the skin of cynomolgus monkeys each day for 14 days to induce skin inflammation.
  • AKTi skin rash model 54 or 108 mg of MK-2206 was delivered orally to rhesus monkeys daily for 14 - 28 days to induce skin rash. Skin samples were then collected from monkeys for histological and gene expression analyses.
  • Gene expression results for the AKTi skin rash model are provided at Table 2, and representative gene expression data for selected genes in both the IL-23 and AKTi models are provided in FIGS. 6A and 6B.
  • Anti-IL-23pl9 treatment is repeated at approximately 28-day intervals over the course of MK-2206 treatment. Treatment with MK-2206 is continued until the desired therapeutic endpoint is reached, or dose limiting toxicity arises.
  • Grade 2 skin rash involves macular or papular eruption or erythema with pruritis or other associated symptoms, and localized desquamation or other lesions covering less than half of the body surface area.
  • Grade 3 skin rash involves severe, generalized erythroderma or macular, popular or vesicular eruption, and desquamation covering half of the body surface area or more.
  • Grade 4 rash involves generalized exfoliative, ulcerative, of bulluous dermatitis.
  • Anti-IL-23pl9 treatment if necessary, is repeated at approximately 28-day intervals over the course of MK-2206 treatment. Treatment with MK-2206 is continued until the desired therapeutic endpoint is reached, or dose limiting toxicity arises.
  • Anti-IL-23pl9 treatment is repeated at approximately 28-day intervals over the course of MK-2206 treatment. Treatment with MK-2206 is continued until the desired therapeutic endpoint is reached, or dose limiting toxicity arises.
  • Grade 2 skin rash involves macular or papular eruption or erythema with pruritis or other associated symptoms, and localized desquamation or other lesions covering less than half of the body surface area.
  • Grade 3 skin rash involves severe, generalized erythroderma or macular, popular or vesicular eruption, and desquamation covering half of the body surface area or more.
  • Grade 4 rash involves generalized exfoliative, ulcerative, of bulluous dermatitis.
  • Anti-IL-23pl9 treatment if necessary, is repeated at approximately 28-day intervals over the course of MK-2206 treatment. Treatment with MK-2206 is continued until the desired therapeutic endpoint is reached, or dose limiting toxicity arises.
  • Table 4 provides a brief description of the sequences in the sequence listing.

Abstract

The present invention provides methods for use of interleukin-23 (IL-23) antagonists to treat or prevent skin rash associated with treatment with PI3K/AKT pathway inhibitors, such as treatment of cancer.

Description

IL-23 ANTAGONISTS FOR TREATMENT OR PREVENTION OF SKIN RASH ASSOCIATED WITH TREATMENT WITH PI3K/AKT PATHWAY INHIBITORS
FIELD OF THE INVENTION
[0001] The present invention relates generally to methods of treating and/or preventing side effects of treatment with PI3K/AKT pathway inhibitors, such as skin rash resulting from cancer therapy.
BACKGROUND OF THE FNVENTION
[0002] Cancer is often treated with agents that selectively disrupt the proliferation of malignant cells. Such agents, however, can cause unwanted side effects. Such side effects can be dose-limiting if the agent must be dosed at sub-optimal levels to avoid unacceptable side effects. Such dose-limiting toxicities ("DLT") may prevent treatment of cancer patients at high enough doses to be efficacious, reducing the usefulness of an otherwise effective drug.
[0003] Inhibitors of the phosphatidylinositol 3' -OH kinase (PI3K)/Akt pathway
("PI3K/AKT pathway") provide one example. The PI3K/AKT pathway appears to be important for regulating cell survival/cell death (Kulik et al. (1997) Mol. Cell. Biol. 17:1595- 1606; Franke et al. (1997) Cell 88:435-437; Kauffmann Zeh et al. (1997) Nature 385:544- 548; Hemmings (1997) Science 275:628-630; Dudek et al. (1997) Science, 275:661-665). Survival factors, such as platelet derived growth factor (PDGF), nerve growth factor (NGF) and insulin like growth factor 1 (IGF-1), promote cell survival under various conditions by inducing the activity of PI3K (Kulik et al. (1997); Hemmings (1997)). Activated PI3K leads to the production of phosphatidylinositol (3,4,5) triphosphate (Ptdlns(3,4,5) P3), which in turn binds to, and promotes the activation of, the serine/threonine kinase Akt, which contains a pleckstrin homology (PH) domain (Franke et al. (1995) Cell 81 :727-736; Hemmings (1997) Science 277:534; Downward (1998) Curr. Opin. Cell Biol. 10:262-267; Alessi et al. (1996) EMBO J. 15: 6541-6551). Specific inhibitors of PI3K or dominant negative Akt mutants abolish survival promoting activities of these growth factors or cytokines. It has been previously disclosed that inhibitors of PI3K (LY294002 or wortmannin) blocked the activation of Akt by upstream kinases. In addition, introduction of constitutively active PI3K or Akt mutants promotes cell survival under conditions in which cells normally undergo apoptotic cell death (Kulik et al. (1997); Dudek et al. (1997)). These and other observations suggest that the PI3K/Akt pathway plays an important role in regulating cell survival or apoptosis in tumorigenesis. See WO 2008/070016.
[0004] PI3K/AKT pathway inhibitors have been proposed as therapeutic agents for the treatment of cancer, but their use has been hampered by dose-limiting skin rash. See, e.g., Tolcher et al. (2009) J. Clin. Oncol. 27: 15s (abstract 3503) (MK-2206, Merck); Edelman et al. (2010) J. Clin. Oncol. 28: 15s (abstract 3004) (XL147/SAR245408, Exilixis, sanofi- aventis, Merck KGaA) and Baselga et al. (2010) J. Clin. Oncol. 28:15s (abstract 3003) (BKM120, Novartis). The need exists for methods of treating and/or preventing dose- limiting side effects of treatment with PI3K/AKT pathway inhibitors, such as prevention of skin rash resulting from cancer treatment with AKT inhibitors.
SUMMARY OF THE INVENTION
[0005] The present invention provides methods and agents for treating and/or preventing side effects of treatment with PI3K/AKT pathway inhibitors, such as cancer treatment. In one embodiment, cancer treatment is directed to blocking signaling via the PI3K/AKT pathway. In some embodiments the cancer treatment blocks signaling via the PI3K/AKT pathway by blocking the activity of epidermal growth factor receptor (EGFR), PI3K, AKT and/or mTor. In select embodiments the cancer therapy involves blocking the activity of AKT. In further embodiments the cancer treatment involves blocking signaling via the PI3K/AKT pathway using a chemical compound, such as a chemical compound that blocks the activity of EGF/EGFR, PI3K, AKT and/or mTor. In yet a further embodiment, the chemical compound blocks the activity of AKT.
[0006] In some embodiments the side effect is skin rash.
[0007] In some embodiments the method or agent for treating and/or preventing side effects of treatment with PI3K/AKT pathway inhibitors, such as cancer treatment, is a method or agent for antagonizing the activity of interleukin-23 (IL-23). In one embodiment, the method or agent for treating and/or preventing side effects of treatment with PI3K/AKT pathway inhibitors, such as cancer treatment, is a chemical compound that antagonizes the activity of IL-23, referred to herein as an IL-23 antagonist. In various embodiments the IL-23 antagonist is an agent that specifically binds to the pl9 subunit of IL-23, or that specifically binds to the IL-23R subunit of the IL-23 receptor complex. In some embodiments the IL-23 antagonist is an antibody, or antigen binding fragment thereof, that binds to IL-23, including but not limited to an antibody, or antigen binding fragment thereof, that specifically binds to the pl9 subunit of IL-23.
[0008] In various embodiments the invention provides methods of treatment comprising determining whether a subject is suffering from a skin rash that may be due to administration of a PI3K/AKT pathway inhibitor, and administering an effective amount of an IL-23 antagonist to treat the rash only if such rash is present. In various other
embodiments the invention provides methods of treatment comprising administering an IL-23 antagonist to a subject to be treated with a PI3K/AKT pathway inhibitor specifically for the purpose of preventing or treating a skin rash, and thereafter optionally monitoring the subject's skin for signs and symptoms of a skin rash.
[0009] In some embodiments the PI3K/AKT pathway inhibitor is an AKT inhibitor, such as a substituted naphthyridine compound, including MK-2206 and AKTi-X. In other embodiments the AKT inhibitor is perifosine (KRX-0401), VQD-002 (API-2, TCN), SR13668, GSK690693, GSK2110183, GSK2141795, AZD5363 or XL418. In some embodiments the AKT inhibitor is dosed orally. In some embodiments the AKT inhibitor is dosed in combination with one or more other cancer drugs, e.g. a MEK inhibitor such as selumetinib or GSKl 120212. In some embodiments of the present invention, treatment with an IL-23 antagonist permits dosing with AKT inhibitors at levels that might otherwise be difficult or impossible to achieve due to dose limiting skin rash, such as weekly dosing of 250, 300, 350, 400, 450, 500, 600, 700, 750, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg or more, either as a single agent or in combination with another cancer drug or drugs. In some embodiments the dosing interval differs from once every week, such as TID, BID, daily, Q2D, Q3D, Q4D, Q5D, Q6D, Q2W, Q3W, Q4W, but nonetheless results in a total weekly dose of 250, 300, 350, 400, 450, 500, 600, 700, 750, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg or more. In some embodiments the AKT inhibitor is dosed to a human subject at 150 - 500 mg once every week, such as 300 mg once every week, or at 45 - 150 mg every other day. In other embodiments the AKT inhibitor is dosed to a human subject as part of combination therapy at 150 - 500 mg once every week, such as 300 mg once every week, or at 150 - 500 mg once every three weeks. [0010] In other embodiments the PI3K/AKT pathway inhibitor is a PI3K inhibitor, such as wortmannin, LY294002, BEZ235, BGT226, BKM120, XL765, XL147, GDC0941, SF1126, GSK1059615, PX-866 or CAL-101.
[0011] In other embodiments the PI3K/AKT pathway inhibitor is an EGF/EGFR inhibitor, such as cetuximab, gefitnib, erlotinib or panitumumab.
[0012] In some embodiments the anti-IL-23 antibody, or antigen binding fragment thereof, comprises an antibody light chain variable domain, or antigen binding fragment thereof, having at least one, two or three CDRs selected from the group consisting of SEQ ID NOs: 32-46. In one embodiment, antibody or fragment comprises a light chain variable domain comprising at least one CDRLl selected from the group consisting of SEQ ID NOs: 32-36; at least one CDRL2 selected from the group consisting of SEQ ID NOs: 37-41; and at least one CDRL3 selected from the group consisting of SEQ ID NOs: 42-46.
[0013] In other embodiments, the anti-IL-23 antibody, or antigen binding fragment thereof, comprises an antibody heavy chain variable domain, or antigen binding fragment thereof, having at least one, two or three CDRs selected from the group consisting of SEQ ID NOs: 15-31. In one embodiment, the antibody or fragment comprises a heavy chain variable domain comprising at least one CDRH1 selected from the group consisting of SEQ ID NOs: 15-19; at least one CDRH2 selected from the group consisting of SEQ ID NOs: 20-26; and at least one CDRH3 selected from the group consisting of SEQ ID NOs: 27-31.
[0014] In other embodiments the antibody or fragment comprises a light chain variable domain and a heavy chain variable domain, or the antigen binding fragments thereof, described in the preceding two paragraphs.
[0015] In some embodiments, the antibody or fragment comprises a framework region, wherein the amino acid sequence of the framework region is all or substantially all of the corresponding region of a human immunoglobulin amino acid sequence.
[0016] In some embodiments the light chain and/or heavy chain variable domains comprise a variant of one or more of the CDRs. In various embodiments the variant domain comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservatively modified amino acid residues relative to the sequence of the respective SEQ ID NOs. Conservative amino acid substitutions are provided at Table 1.
[0017] In some embodiments the light chain variable domain comprises residues 1-
108 of SEQ ID NO: 14 or a variant thereof. In some embodiments the heavy chain variable domain comprises a sequence selected from the group consisting of residues 1-116 of SEQ ID NOs: 6-8, such as SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. In various embodiments the variant variable domain comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 or 50 or more conservatively modified amino acid residues relative to the sequence of the respective SEQ ID NOs. In yet a further embodiment, the antibody or fragment comprises a light chain variable domain and a heavy chain variable domain, or the antigen binding fragments thereof, described in this paragraph.
[0018] In one embodiment the antibody or fragment comprises a light chain sequence of SEQ ID NO: 14 and/or a heavy chain sequence selected from the group consisting of SEQ ID NOs: 6-8. In another embodiment the antibody or fragment comprises two light chain sequences and two heavy chain sequences.
[0019] In other embodiments the antibody or fragment comprises a light chain variable domain, or an antigen binding fragment thereof, consisting essentially of residues 1- 108 of SEQ ID NO: 14, and/or a heavy chain variable domain, or an antigen binding fragment thereof, consisting essentially of a sequence selected from the group consisting of residues 1- 116 of SEQ ID NOs: 6-8, such as SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.
[0020] In other embodiments the antibody or fragment comprises a light chain variable domain, or an antigen binding fragment thereof, having at least 50%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence homology with residues 1-108 of SEQ ID NO: 14, and/or a heavy chain variable domain, or an antigen binding fragment thereof, having at least 50%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence homology with a sequence selected from the group consisting of residues 1-116 of SEQ ID NOs: 6-8, such as SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.
[0021] In one embodiment, the antibody or fragment binds to human IL-23pl9 (SEQ
ID NO: 47) at an epitope comprising residues 20-30, or residues 82-110, or both. In another embodiment the IL-23pl9 binding compound binds to an epitope comprising some or all of residues K20, T23, W26, S27, P30, E82, S95, L96, L97, P98, D99, P101, G103, Q104, H106, A107 and LI 10, and optionally residues L24, L85, T91, S100 and V102. In various embodiments the epitope for an antibody of interest is determined by obtaining an X-ray crystal structure of an antibody: antigen complex and determining which residues on IL-23pl9 are within a specified distance of residues on the antibody of interest, wherein the specified distance is, e.g., 4A or 5A. In some embodiments, the epitope is defined as a stretch of 11 or more contiguous amino acid residues along the IL-23 l9 sequence in which at least 30%, 40%, 45%), 50%o or 54% of the residues are within the specified distance of the antibody.
[0022] In one embodiment, the anti-IL-23 antibodies or fragments are able to block the binding of a binding compound of the present invention to human IL-23 in a cross- blocking assay. In another embodiment, the anti-IL-23 antibodies or fragments are able to block IL-23 -mediated activity, such activities including but not limited to, binding to its receptor and promoting the proliferation or survival of TR17 cells.
[0023] In some embodiments, the antibody or fragment further comprises a heavy chain constant region, wherein the heavy chain constant region comprises a γΐ, γ2, γ3, or γ4 human heavy chain constant region, or a variant thereof. In various embodiments the light chain constant region comprises a lambda or a kappa human light chain constant region, or a variant thereof.
[0024] In various embodiments the anti-IL-23 antibodies or fragments thereof are polyclonal, monoclonal, chimeric, humanized or fully human antibodies or fragments thereof. The present invention also contemplates that the antigen binding fragment is an antibody fragment selected from the group consisting of, e.g., Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, and a diabody.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 shows comparisons of mouse anti-human IL-23pl9 antibody clone heavy chain variable domain sequences. Sequences are provided for clones mlAl 1, ml 1C1, m5F5, m21Dl, ml3B8, hl3B8a, hl3B8b and hl3B8c. CDRs are indicated. In both figures, an "m" prefix connotes a murine antibody and an "h" connotes a humanized antibody. The suffixes "a", "b" and "c" refer to sequence variants of the humanized 13B8 heavy chain variable domain, as discussed in greater detail below.
[0026] FIG. 2 shows comparisons of mouse anti-human IL-23pl9 antibody clone light chain variable domain sequences. Sequence are provided for clones mlAl 1, ml 1C1, m5F5, m21Dl, ml3B8, hl3B8. CDRs are indicated.
[0027] FIG. 3 shows the levels of expression of the genes encoding the subunits of IL-
23 and IL-12 in THP-1 cells in culture as determined by TAQMAN® real time quantitative polymerase chain reaction analysis. The leftmost (white) bar in each graph, which is essentially zero for the IL-23 l9 and IL-12/23p40 plots, represents control cells treated with vehicle. The next (black) bar represents cells treated with lipopolysaccharide (LPS). The next two bars (white) represent cells treated with LPS and MK-2206 (0.1 μΜ and 0.5 μΜ, respectively), as indicated. The right most two bars (gray) represent cells treated with LPS and AKTi-X (0.1 μΜ and 0.5 μΜ, respectively, as indicated). LPS enhances the expression of IL-23pl9 and IL-12/23p40, but reduces the expression of IL-12p35. AKT inhibitors MK- 2206 and AKTi-X both further enhance expression of IL-23pl9 and IL-12/23p40, but enhance the expression of IL-12p35 less so, if at all. The results demonstrate that in the presence of LPS, AKT inhibitors preferentially enhance the expression of the genes encoding the subunits of IL-23 rather than IL-12. See Example 3.
[0028] FIG. 4 shows the levels of expression of the genes encoding the pl9 and p40 subunits of IL-23 in primary CD 14+ monocytes isolated from PBMC from two different human donors as determined by TAQMAN® real time quantitative polymerase chain reaction analysis. Data are presented for three time points (3, 6 and 24 hours) after addition of LPS, or LPS and AKT inhibitor AKTi-X, as indicated. Bars for untreated cells (indicated by arrows) are provided with the three hour time points, and are essentially zero. For each time point, the left (black) bars are data for treatment with LPS alone, and the right (white) bars are data for treatment with LPS and AKT inhibitor AKTi-X. These data show that in the presence of LPS, addition of an AKT inhibitor (in this case AKTi-X) specifically upregulates expression of genes encoding IL-23 subunits in primary CD 14+ monocytes isolated from PBMC. IL- 12p35 is not expressed at detectable levels at three and six hours, and is expressed at only low levels at 24 hours and does not change with addition of AKT inhibitor AKTi-X (data not shown). See Example 4.
[0029] FIG. 5 shows IL-23 protein levels in primary CD 14+ monocytes isolated from
PBMCs from various donors as a function of treatment with toll-like receptor (TLR) agonists LPS, lipoteichoic acid (LTA), or combinations of LPS or LTA with AKT inhibitors MK-2206 or AKTi-X (at 0.1 μΜ or 0.5 μΜ). The leftmost bars for vehicle controls are not visible. Black bars show results for treatment with TLR agonist alone; white bars for treatment with TLR agonist and MK-2206 (at 0.1 μΜ or 0.5 μΜ); and gray bars for treatment with TLR agonist and AKTi-X (at 0.1 μΜ or 0.5 μΜ), as indicated. These data show that addition of an AKT inhibitor (MK-2206 or AKTi-X, at 0.1 μΜ or 0.5 μΜ) specifically upregulates expression of IL-23 at the protein level in primary CD 14+ monocytes isolated from PBMC. See Example 5.
[0030] FIGS. 6A and 6B compare expression of several genes in IL-23-treated and
MK-2206-treated monkeys. Specifically, gene expression levels are compared between IL- 23-treated cynomolgus monkey skin (black symbols, top panels) and MK-2206-treated rhesus monkey skin (black symbols, bottom panels), as well an (untreated) controls (gray symbols). Each of the genes for which data are presented (BD2, S100A8-calgranulin, CCL3-MIPl , Ebi3, IL-Ιβ, IL-6, IL-23pl9, IL-22) was selected based on its prior known association with IL-23 -induced skin inflammation. Both IL-23 and AKT inhibitor increase the expression of each gene shown. See Example 6.
DETAILED DESCRIPTION
[0031] As used herein, including the appended claims, the singular forms of words such as "a," "an," and "the," include their corresponding plural references unless the context clearly dictates otherwise. Table 4 below provides a listing of sequence identifiers used in this application. All references cited herein are incorporated by reference to the same extent as if each individual publication, patent application, or patent, was specifically and individually indicated to be incorporated by reference. Citation of the references herein is not intended as an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.
I. Definitions
[0032] "PI3K" refers to PI3 kinase.
[0033] "PI3 kinase/AKT pathway inhibitor" or " PI3K/AKT pathway inhibitor " refers to any agent that inhibits signaling via the PI3 kinase/AKT pathway, including but not limited to inhibitors of PI3 kinase and inhibitors of AKT. It also includes compounds that act "upstream" of PI3K in the PI3 kinase/AKT pathway, such as epidermal growth factor or its receptor (EGF/EGFR), since such compounds can inhibit signaling via the PI3 kinase/AKT pathway by removing an upstream triggering signal. It is intended that the "PI3 kinase/AKT pathway inhibitors" of the present invention encompass inhibitors of any activity that signals through or within the PI3 kinase/AKT pathway since any of these inhibitors may give rise to a rash that can benefit from the methods of the present invention. [0034] "AKTi" refers to an AKT inhibitor generally. "AKTi-X" refers to a specific
AKT inhibitor, tra/75-3-amino-3-{4-[l-(difluoromethyl)-8-phenyl[ 1,2,4] triazolo[4,3-a]-l,5- naphthyridin-7-yl]lphenyl}-l-methylcyclobutanol), as described herein. MK-2206 refers to a specific AKT inhibitor, 8-[4-(l-aminocyclobutyl)phenyl]-9-phenyl[l,2,4]triazolo[3,4-f]-l,6- naphthyridin-3(2H)-one, as described herein. AKT is also known as protein kinase B (PKB).
[0035] "IL-23 antagonist" refers to any agent that inhibits signaling by IL-23.
Because IL-23 signals via the IL-23 receptor complex, antagonists of the IL-23 receptor complex are also IL-23 antagonists. A "non-specific IL-23 antagonist" is an agent that blocks the activity of both IL-23 and IL-12. An "IL-23 -specific antagonist" is an agent that selectively blocks the activity of IL-23 but does not block the activity of IL-12. Unless otherwise indicated, or clear from the context, "IL-23" and "IL-12" as used herein refer to human IL-23 and human IL-12. The antibodies disclosed herein bind to human IL-23.
[0036] "Proliferative activity" encompasses an activity that promotes, that is necessary for, or that is specifically associated with, e.g., normal cell division, as well as cancer, tumors, dysplasia, cell transformation, metastasis, and angiogenesis.
[0037] "Administration" and "treatment," as it applies to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration" and "treatment" can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell. "Administration" and "treatment" also means in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding composition, or by another cell. "Treatment," as it applies to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, to research and diagnostic applications. "Treatment" as it applies to a human, veterinary, or research subject, or cell, tissue, or organ, encompasses contact of an agent with animal subject, a cell, tissue, physiological compartment, or physiological fluid. "Treatment of a cell" also encompasses situations where the agent contacts IL-23 receptor (IL-23R/IL-12Rpi heterodimer), e.g., in the fluid phase or colloidal phase, but also situations where the agonist or antagonist does not contact the cell or the receptor. [0038] As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity. Thus, it is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, fully human antibodies, etc. so long as they exhibit the desired biological activity. As used herein, unless otherwise indicated an antibody or fragment is an antibody or fragment that specifically binds to IL-23.
[0039] As used herein, the terms "IL-23 binding fragment," "binding fragment thereof," "antigen binding fragment thereof or "fragment" (when used with reference to an antibody) encompass a fragment or a derivative of an antibody that still substantially retains its biological activity of inhibiting IL-23pl9 activity. Therefore, the term "antibody fragment" or "IL-23 binding fragment" refers to a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., sc-Fv; and multispecific antibodies formed from antibody fragments. Typically, a binding fragment or derivative retains at least 10% of its IL-23 inhibitory activity. Preferably, a binding fragment or derivative retains at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% (or more) of its IL-23 inhibitory activity, although any binding fragment with sufficient affinity to exert the desired biological effect will be useful. It is also intended that an IL-23 binding fragment can include variants having conservative amino acid substitutions that do not substantially alter its biologic activity.
[0040] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581- 597, for example.
[0041] Antibodies, and antigen binding fragments thereof, may be described herein as comprising a light and/or heavy chain variable domain, or as comprising a light and/or heavy chain. As used herein, and consistent with their plain meanings, such descriptions encompass embodiments in which the antibody, or antigen binding fragment thereof, includes more than one light and/or heavy chain variable domain and/or chain, such as two light and/or heavy chain variable domains and/or chains.
[0042] The monoclonal antibodies herein specifically include "chimeric" antibodies
(immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. U.S. Pat. No. 4,816,567; Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81 : 6851-6855.
[0043] A "domain antibody" is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more VH regions are covalently joined with a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody may target the same or different antigens.
[0044] A "bivalent antibody" comprises two antigen binding sites. In some instances, the two binding sites have the same antigen specificities. However, bivalent antibodies may be bispecific (see below).
[0045] As used herein, the term "single-chain Fv" or "scFv" antibody refers to antibody fragments comprising the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer- Verlag, New York, pp. 269-315.
[0046] The monoclonal antibodies herein also include camelized single domain antibodies. See, e.g., Muyldermans et al. (2001) Trends Biochem. Sci. 26:230; Reichmann et al. (1999) J. Immunol. Methods 231 :25; WO 94/04678; WO 94/25591; U.S. Pat. No.
6,005,079). In one embodiment, the present invention provides single domain antibodies comprising two VH domains with modifications such that single domain antibodies are formed.
[0047] As used herein, the term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL- VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, e.g., EP 404,097; WO 93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448. For a review of engineered antibody variants generally see Holliger and Hudson (2005) Nat.
Biotechnol. 23:1126-1136.
[0048] As used herein, the term "humanized antibody" refers to forms of antibodies that contain sequences from non-human {e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human
immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The prefix "hum", "hu" or "h" is added to antibody clone designations when necessary to distinguish humanized antibodies (e.g. huml3B8) from parental rodent antibodies (e.g. mouse 13B8, or ml3B8). The humanized forms of rodent antibodies will generally comprise the same CDR sequences of the parental rodent antibodies, although certain amino acid substitutions may be included to increase affinity, increase stability of the humanized antibody, or for other reasons. [0049] The antibodies of the present invention also include antibodies with modified
(or blocked) Fc regions to provide altered effector functions. See, e.g., U.S. Pat. No.
5,624,821; WO 2003/086310; WO 2005/120571; WO 2006/0057702; Presta (2006) Adv. Drug Delivery Rev. 58:640-656. Such modification can be used to enhance or suppress various reactions of the immune system, with possible beneficial effects in diagnosis and therapy. Alterations of the Fc region include amino acid changes (substitutions, deletions and insertions), glycosylation or deglycosylation, and adding multiple Fc. Changes to the Fc can also alter the half-life of antibodies in therapeutic antibodies, and a longer half-life would result in less frequent dosing, with the concomitant increased convenience and decreased use of material. See Presta (2005) J. Allergy Clin. Immunol.116:731 at 734-35.
[0050] The term "fully human antibody" refers to an antibody that comprises human immunoglobulin protein sequences only. A fully human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, "mouse antibody" refers to an antibody which comprises mouse immunoglobulin sequences only. A fully human antibody may be generated in a human being, in a transgenic animal having human immunoglobulin germline sequences, by phage display or other molecular biological methods.
[0051] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" {e.g. residues 24-34 (CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3) in the light chain variable domain and residues 31-35 (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) in the heavy chain variable domain (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.) and/or those residues from a "hypervariable loop" {i.e. residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (HI), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain (Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917). As used herein, the term "framework" or "FR" residues refers to those variable domain residues other than the hypervariable region residues defined herein as CDR residues. The residue numbering above relates to the Kabat numbering system and does not necessarily correspond in detail to the sequence numbering in the accompanying Sequence Listing. [0052] "Binding compound" refers to a molecule, small molecule, macromolecule, polypeptide, antibody or fragment or analogue thereof, or soluble receptor, capable of binding to a target. "Binding compound" also may refer to a complex of molecules, e.g., a non- covalent complex, to an ionized molecule, and to a covalently or non-covalently modified molecule, e.g., modified by phosphorylation, acylation, cross-linking, cyclization, or limited cleavage, which is capable of binding to a target. When used with reference to antibodies, the term "binding compound" refers to both antibodies and antigen binding fragments thereof. "Binding" refers to an association of the binding composition with a target where the association results in reduction in the normal Brownian motion of the binding composition, in cases where the binding composition can be dissolved or suspended in solution. "Binding composition" refers to a molecule, e.g. a binding compound, in combination with a stabilizer, excipient, salt, buffer, solvent, or additive, capable of binding to a target.
[0053] "Conservatively modified variants" or "conservative substitution" refers to substitutions of amino acids that may be made, as known by those of skill in the art, with little or no impact on the biological activity of the resulting molecule, even in essential regions of the polypeptide. Such exemplary substitutions are preferably made in accordance with those set forth in Table 1 as follows:
Table 1
Exemplary Conservative Amino Acid Substitutions
Figure imgf000016_0001
Figure imgf000017_0001
[0054] In addition, those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity. See, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Edition).
[0055] The phrase "consists essentially of," or variations such as "consist essentially of or "consisting essentially of," as used throughout the specification and claims, indicate the inclusion of any recited elements or group of elements, and the optional inclusion of other elements, of similar or different nature than the recited elements, that do not materially change the basic or novel properties of the specified dosage regimen, method, or composition. As a non-limiting example, a binding compound that consists essentially of a recited amino acid sequence may also include one or more amino acids, including substitutions of one or more amino acid residues, that do not materially affect the properties of the binding compound.
[0056] "Effective amount" encompasses an amount sufficient to ameliorate or prevent a symptom or sign of the medical condition. Effective amount also means an amount sufficient to allow or facilitate diagnosis. An effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration and the severity of side affects. See, e.g., U.S. Pat. No. 5,888,530. An effective amount can be the maximal dose or dosing protocol that avoids significant side effects or toxic effects. The effect will result in an improvement of a diagnostic measure or parameter by at least 5%, usually by at least 10%, more usually at least 20%, most usually at least 30%>, preferably at least 40%>, more preferably at least 50%, most preferably at least 60%, ideally at least 70%, more ideally at least 80%>, and most ideally at least 90%>, where 100%) is defined as the diagnostic parameter shown by a normal subject. See, e.g., Maynard et al. (1996) A Handbook of SOPs or Good Clinical Practice, Interpharm Press, Boca Raton, FL; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK.
[0057] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.
[0058] To examine the extent of inhibition of IL-23 activity, for example, samples or assays comprising a given, e.g., protein, gene, cell, or organism, are treated with a potential activating or inhibiting agent and are compared to control samples without the agent. Control samples, i.e., not treated with agent, are assigned a relative activity value of 100%. Inhibition is achieved when the activity value relative to the control is about 90%> or less, typically 85% or less, more typically 80%> or less, most typically 75% or less, generally 70%> or less, more generally 65%> or less, most generally 60%> or less, typically 55% or less, usually 50% or less, more usually 45% or less, most usually 40% or less, preferably 35% or less, more preferably 30%) or less, still more preferably 25% or less, and most preferably less than 25%. Activation is achieved when the activity value relative to the control is about 110%, generally at least 120%, more generally at least 140%, more generally at least 160%, often at least 180%, more often at least 2-fold, most often at least 2.5-fold, usually at least 5-fold, more usually at least 10-fold, preferably at least 20-fold, more preferably at least 40-fold, and most preferably over 40-fold higher.
[0059] Endpoints in activation or inhibition can be monitored as follows. Activation, inhibition, and response to treatment, e.g., of a cell, physiological fluid, tissue, organ, and animal or human subject, can be monitored by an endpoint. The endpoint may comprise a predetermined quantity or percentage of, e.g., an indicia of inflammation, oncogenicity, or cell degranulation or secretion, such as the release of a cytokine, toxic oxygen, or a protease. The endpoint may comprise, e.g., a predetermined quantity of ion flux or transport; cell migration; cell adhesion; cell proliferation; potential for metastasis; cell differentiation; and change in phenotype, e.g., change in expression of gene relating to inflammation, apoptosis, transformation, cell cycle, or metastasis (see, e.g., Knight (2000) Ann. Clin. Lab. Sci. 30: 145- 158; Hood and Cheresh (2002) Nature Rev. Cancer 2:91-100; Timme et al. (2003) Curr. Drug Targets 4:251-261; Robbins and ltzkowitz (2002) Med. Clin. North Am. 86: 1467-1495; Grady and Markowitz (2002) Annu. Rev. Genomics Hum. Genet. 3:101-128; Bauer, et al. (2001) Glia 36:235-243; Stanimirovic and Satoh (2000) Brain Pathol. 10:113-126).
[0060] An endpoint of inhibition is generally 75% of the control or less, preferably
50% of the control or less, more preferably 25% of the control or less, and most preferably 10% of the control or less. Generally, an endpoint of activation is at least 150% the control, preferably at least two times the control, more preferably at least four times the control, and most preferably at least 10 times the control.
[0061] "Specifically" or "selectively" binds, when referring to a ligand/receptor, antibody/antigen, or other binding pair, indicates a binding reaction which is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies. Thus, under designated conditions, a specified ligand binds to a particular receptor and does not bind in a significant amount to other proteins present in the sample. As used herein, an antibody is said to bind specifically to a polypeptide comprising a given sequence (in this case IL-23pl9) if it binds to polypeptides comprising the sequence of IL-23pl9 but does not bind to proteins lacking the sequence of IL-23pl9. For example, an antibody that specifically binds to a polypeptide comprising IL-23pl9 may bind to a FLAG®-tagged form of IL-23pl9 but will not bind to other FLAG®-tagged proteins.
[0062] The antibody, or binding composition derived from the antigen-binding site of an antibody, of the contemplated method binds to its antigen with an affinity that is at least two fold greater, preferably at least ten times greater, more preferably at least 20-times greater, and most preferably at least 100-times greater than the affinity with unrelated antigens. In a preferred embodiment the antibody will have an affinity that is greater than about 109 liters/mol, as determined, e.g., by Scatchard analysis. Munsen et al. (1980) Analyt. Biochem. 107:220-239.
II. General
[0063] The PI3K/AKT signaling pathway is the most frequently hyperactivated pathway in cancer. Inhibition of PI3K/AKT pathway is expected to have a significant impact on cancer cell survival and proliferation. Several drugs targeting this pathway are currently in clinical trials (Liu et a/.(2009) Nat. Rev. Drug Disc. 8:627) including MK-2206, an allosteric inhibitor of AKT. Maculopapular skin rash is the dose limiting toxicity for MK-2206 in the clinic. Tolcher et a/. (2009) J. Clin. Oncol. 27:15s (abstract 3503). The rash appears 7-10 days after the first dose and resolves completely after discontinuation of treatment with the drug. Skin rash is most prevalent at doses above 60 mg QOD, such as 75 mg and 90 mg (and presumably higher doses as well).
[0064] The present invention relates to the discovery that IL-23 is involved in skin rash that results from treatment with PI3K/AKT pathway inhibitors, e.g. for treatment of cancer. This skin rash has been a dose-limiting toxicity for the AKT inhibitor MK-2206 in the clinic. The invention involves antagonism of IL-23, such as treatment with an IL-23 antagonist, to ameliorate this rash. Elimination or reduction of the rash will have value as a matter of comfort to the patient, and may improve compliance. In addition, elimination or reduction of the rash may allow for elevated dosing and concomitant increased anti-tumor efficacy.
[0065] The present invention is based in part of studies of this MK-2206-induced skin rash. Pathological examination of skin rash biopsies reveals marked perivascular infiltrates with T cells and abundant macrophages. In more severe cases psoriasiform dermatitis is observed with epidermal hyperplasia and increased keratinocyte proliferation. Other investigational drugs targeting the PI3K/AKT pathway have similar side effects {e.g., Edelman et al. (2010) J. Clin. Oncol. 28:15s (abstract 3004) and Baselga et al. (2010) J. Clin. Oncol. 28: 15s (abstract 3003)), suggesting that skin rashes are mechanism-based.
[0066] Studies of cells in culture were used to further study the effects of MK-2206 treatment. One of the proteins that is up-regulated upon MK-2206 treatment of THP-1 monocytic cells, as well as primary monocytes isolated from PBMCs, is IL-23. TLR agonists were included in these experiments based on the hypothesis that AKT inhibitor-induced skin rash is TLR dependent, and thus that TLR agonists should be present in culture to mimic the presence of microbial TLR agonists on natural skin. As illustrated in FIGS. 3 and 4, inhibition of AKT results in significant increase in IL-23 subunit mRNA. FIG. 5 shows AKT inhibitor-induced elevation of IL-23 at the protein level as well. These in vitro results thus are consistent with a role for IL-23 in MK-2206-induced skin rash. [0067] A rhesus monkey model was then used to determine whether IL-23 is involved in MK-2206-induced skin rash in vivo. It was first necessary to perform a comparative study to confirm that the rhesus model recapitulates the human rash that is observed in the clinic. Histopatho logical examination of clinical biopsy material from human subjects experiencing MK-2206-induced rash and biopsies taken from rhesus monkeys (dosed orally at 54 and 108 mg/kg, daily) revealed significant similarities between MK-2206 induced rashes in humans and monkeys. Similarities include similar morphology (perivascular dermal infiltrate with T cell and macrophage components); abnormal stratum corneum, including occasional neutrophilic abscesses, loss of "basket weave" appearance, and abnormal filaggrin staining; epidermal hyperplasia; and increased basal keratinocyte proliferation. Both human and rhesus monkey rashes involve T cell and macrophage infiltrates, and both involve
compromised skin barrier function as evidenced by abnormal stratum corneum.
[0068] This validated rhesus monkey model was then used in experiments designed to look for similarities between IL-23 -induced and MK-2206-induced rashes. See Example 6. Skin samples from IL-23 treated cynomolgus monkeys and MK-2206 treated rhesus monkeys were compared. Histopathological evaluation revealed significant similarities between the two groups including morphology of the rash (dermal perivascular infiltrates with T cells and abundant macrophages), stratum corneum abnormalities, increased angiogenesis and epidermal hyperplasia.
[0069] TAQMAN® real time quantitative polymerase chain reaction analysis was also performed on skin derived from MK-2206- and IL-23-treated monkeys. For the rhesus monkey model of MK-2206-induced skin rash, 32 genes were selected for measurement based on their previous association with IL-23 -induced skin inflammation, as well as eight other genes of interest (VEGFA, CD4, CD8a, CD68, IL-10, TLR2, TLR4, VEGFR1).
Exemplary results are provided in Table 2 for two control rhesus monkeys and one MK-2206- treated rhesus monkey. Data are presented for two or three distinct skin samples obtained from each monkey. Values in bold- face are elevated at least two-fold higher than control levels,. As used herein, these two-fold changes constitute upregulation of gene expression. Of the 32 genes previously associated with IL-23-induced skin inflammation, seven were not detected in the monkey samples (IL-24, IL-17A, GM-CSF, IL-12p40, IL-23R, RANKL, IL- 26). As is evident from Table 2, 22 of the 25 detected genes were upregulated in MK-2206- treated rhesus monkeys compared with untreated control animals (all detected genes except CXCR4, IL-22R and TNFRSFl la). The upregulation of many of the same genes upregulated in IL-23-induced rash (the IL-23 gene expression "signature") suggests that MK-2206 induces IL-23 -mediated skin inflammation.
Table 2
Gene Expression in MK-2206-Treated R lesus Monkeys
Control Rhesus 1 Control Rhesus 2 MK-2206-treated Rhesus
Gene Sample 1 Sample 2 Sample 1 Sample 2 Sample 1 Sample 2 Sample 3
IL-19 0.0 0.0 0.0 0.8 121 81.1 102
IL-24 0.0 0.0 0.0 0.0 0.1 0.4 0.0
IL-22 138 128 109 91.3 45.6 39.9 36.6
TNFRSFl la 2.8 2.1 14.2 3.7 1.6 0.8 0.7
Ebi3 1.7 1.2 0.6 0.8 3.7 2.3 3.4
IL-17A 0.0 0.0 0.0 0.0 0.1 0.0 0.0
IL-17F 0.6 0.5 0.9 1.4 1.8 1.8 1.5
TNFa 0.5 0.6 0.4 0.5 1.3 0.8 1.3
IL-6 1.3 0.8 2.2 1.8 17.7 12.7 17.3
CXCL1 10.8 11.4 8.1 7.1 116.2 89.1 114
CXCL3 0.1 0.2 0.1 0.1 3.3 2.1 2.6
G-CSF 0.1 0.1 0.0 0.0 1.9 1.2 1.8
GM-CSF 0.3 0.1 0.3 0.2 0.8 0.2 0.7
BD2(Defb2) 2.6 4.0 8.5 40.3 1390 2360 1830
S100A8 62.5 84.1 130.4 312 14570 14400 17900
IL-22 0.0 0.0 0.0 0.0 4.4 3.8 4.5
IL-lb 0.4 0.1 0.4 0.2 26.4 18.7 22.3
IL-12p35 0.1 0.3 0.8 0.7 0.9 2.2 1.3
IL-12p40 0.1 0.0 0.1 0.2 0.1 0.1 0.2
IL-23 l9 0.7 1.8 1.2 1.1 8.2 10.3 9.1
IL-23R 0.3 0.2 0.5 0.1 0.3 0.3 0.2
CCL2 5.4 7.0 26.1 4.8 116 113 86.7
CCL3 0.1 0.0 0.7 0.0 8.5 4.8 6.3
VEGFA 45.9 35.5 35.1 23.6 44.7 40.8 39.9
CD4 14.0 18.4 16.0 9.8 12.5 12.5 8.9
CD8a 3.2 1.5 3.5 2.1 7.5 8.3 6.1
CD68 36.4 55.1 57.7 32.6 33.0 32.1 25.0 Control Rhesus 1 Control Rhesus 2 MK-2206-treated Rhesus
Gene Sample 1 Sample 2 Sample 1 Sample 2 Sample 1 Sample 2 Sample 3
IL-10 1.1 1.3 2.0 0.8 1.5 1.1 0.7
CXC 4 6.0 10.1 10.2 6.0 8.4 7.3 6.1
TLR2 3.9 6.7 5.5 4.0 33.6 25.3 24.7
TLR4 3.0 2.4 3.8 2.3 6.6 7.0 5.9
IL-21R 0.1 0.3 0.2 0.4 1.6 1.7 1.2
RANKL 0.1 0.1 0.2 0.0 0.3 0.3 0.4
S100A9 5.1 14.2 28.6 61.0 3080 3100 3250
IL-20 0.1 0.0 0.2 0.0 4.3 3.2 4.9
IL-26 0.0 0.0 0.1 0.0 0.6 0.9 1.1
IL-la 7.7 6.8 8.9 6.1 23.1 24.4 36.4
MMP3 0.7 1.0 2.5 6.6 108.9 108 119
VEGFR1 13.9 11.3 11.6 8.7 8.9 7.0 8.3
IL-IRN 9.8 8.0 10.4 10.7 22.5 20.2 21.2
[0070] FIGS. 6 A and 6B also provide exemplary gene expression data for
(cynomolgus) monkeys treated with IL-23 and (rhesus) monkeys treated with MK-2206. The similarly elevated expression of these genes in IL-23- and MK-2206-induced skin
inflammation is consistent with a model in which MK-2206-induced skin rash in mediated by IL-23.
[0071] Taken together, the results presented herein suggest a pivotal role for IL-23 in
AKT inhibitor-induced rash. AKT inhibitors induce IL-23 subunit gene expression and increase IL-23 levels in cells in vitro. MK-2206 also induces a rash in monkeys that is histologically very similar to the clinically observed MK-2206-induced rash in human subjects, and is also very similar to skin inflammation induced by injection of IL-23 into monkey skin. Many of the same genes induced by injection of IL-23 into primate skin are also induced in the primate model of MK-2206-induced rash, effectively mimicking the "gene expression profile" of IL-23 skin inflammation. Because IL-23 appears to be involved in mediating AKT inhibitor-induced skin rash, inhibition of IL-23 -induced skin inflammation is promising approach to prevent and/or treat this rash. Such treatment may eliminate the dose limiting toxicity of PI3K/AKT pathway inhibitors, such as MK-2206, and permit higher and potentially more efficacious dosing, e.g. for the treatment of cancer. This potential increase in the therapeutic window for PI3K/AKT pathway inhibitors may allow for effective treatment with compounds that would otherwise face dose-limiting toxicity (rash) that limited their effectiveness.
[0072] Antagonism of IL-23 may also find use in treatment of rashes caused by other drugs that disrupt signaling via the PI3K/AKT pathway. Epidermal grown factor (EGF) signals via the epidermal grown factor receptor (EGFR), which is overexpressed in many tumors. EGRF then acts, at least in part, via the PI3K/AKT pathway. Inhibitors of
EGF/EGFR signaling have been proposed for the treatment of cancer, e.g. cetuximab
(ERBITUX®), panitumumab (VECTIBLX®), gefitnib (IRESSA®) and erlotinib
(TARCEVA®). Skin rash has also been observed as a side effect of treatment with EGFR inhibitors (see, e.g., US 7,645,494 and Rothenberg et al. (2005) J. Clin. Oncol. 23:9265 at Table 4). This rash, which has been consistently observed in all trials of EGFR inhibitors, has even been suggested as a surrogate indicator of therapeutic efficacy (Cohen et al. (2003) J Clin Oncol. 21 : 1980-7). Vitamin K has been proposed as a treatment for EGFR antagonist- induced skin rash (WO 2006/113479 to Albert Einstein College of Medicine of Yeshiva University). The facts that the EGFR-inhibitor-induced rash is thought to be mechanism- based, and that EGFR signals via the PI3K/AKT pathway suggests that the methods of the present invention relating to inhibition of skin rash using IL-23 antagonists will find use in treatment of EGFR-inhibitor-induced rash as well.
[0073] Other receptors that signal via the PI3K/AKT pathway include human epidermal growth factor receptor 2 (HER2/neu, ErbB-2), hepatocyte growth factor receptor (HGFR, MET) and other receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). Inhibitors of signaling through these receptors may also induce the same rash that is observed with PI3K and AKT inhibitors, and thus may similarly benefit from the methods of the present invention.
[0074] Aside from the benefit afforded by reduction or elimination of AKTi-induced skin rash, treatment with an IL-23 antagonist will also have inherent anti-tumor benefits. IL- 23 has been show to promote tumor incidence and growth an animal models (Langowski et al. (2006) Nature 442:461) and antagonism of IL-23 has been proposed as a method to inhibit tumor growth (US 7,282,204). This advantage is not shared by antagonists of both IL-23 and IL-12, such as anti-p40 antibodies like ustekinumab and briakinumab. See Langowski et al. (2006) Nature 442:461 at Figs. 4(a), (f) and (g). Specific inhibition of IL-23 (but not IL-12) should also reduce the risk of various infections that would otherwise be elevated by an IL- 12/IL-23 antagonist, such as an anti-p40 antibody. Bowman et al. (2006) Curr. Op. Infect. Dis. 19:245. The reduced tumor and infection risks will be of great benefit to cancer patients being treated for PI3K/AKT pathway inhibitor-induced skin rash by the methods of the present invention involving IL-23 -specific antagonists.
III. PI3K/AKT Pathway Inhibitors
[0075] The present invention involves antagonism of IL-23 activity as a means of ameliorating skin rash associated with cancer therapy using PI3K/AKT pathway inhibitors. The method may be of use in treating a skin rash resulting from treatment with any
PI3K/AKT pathway inhibitor.
[0076] PI3K inhibitors that may induce rashes preventable or treatable with the methods of the present invention include wortmannin and LY294002, as well as; BEZ235, BGT226 and BKM120 (Novartis); XL765 and XL147 (Exelixis); GDC0941 (Genentech); SF1126 (Semafore); GSK1059615 (GlaxoSmithKlme); PX-866 (Oncothyreon); and CAL-101 (Calistoga). See Liu et al. (2009) Nat. Rev. Drug Discovery 8:627 (disclosing the structures of many of these PI3K inhibitors), which is incorporated herein by reference in its entirety.
[0077] In some embodiments the AKT inhibitor of the present invention is a substituted naphthyridine compound, for example as disclosed in WO 2008/070016 (to Merck & Co., Inc. and Banyu Pharmaceutical Co., Ltd.). In one embodiment, the AKT inhibitor is 8- [4-( 1 -aminocyclobutyl)phenyl] -9-phenyl[ 1 ,2,4]triazolo [3 ,4-f] - 1 ,6-naphthyridin-3 (2H)-one (MK-2206) or a pharmaceutically acceptable salt thereof, as disclosed and claimed at US 7,576,209. See also WO 2008/070041 (to Merck & Co., Inc.), which is hereby incorporated by reference in its entirety.
[0078] In other embodiments the AKT inhibitor is a substituted [l,2,4]triazolo[4,3-a]-
1,5 -naphthyridine compound, e.g. tra/?5-3-amino-3-{4-[l-(difluoromethyl)-8-phenyl[ 1,2,4] triazolo[4,3-a]-l,5-naphthyridin-7-yl]lphenyl}-l-methylcyclobutanol) (AKTi-X). See WO 2009/148887 (to Merck & Co., Inc.), the disclosure of which is hereby incorporated by reference in its entirety, compound 10-4.
[0079] Additional exemplary AKT inhibitors include, but are not limited to: 1 - {4-[3-(l -methyl-lH-imidazol-4-yl)-9-phenyl[ 1 ,2,4]triazolo[3,4- J-l ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1 -[4-(9-phenyl-3-pyrimidin-2-yl-[ 1 ,2,4]triazolo[3,4- J- 1 ,6-naphthyridin-8- yl)phenyl] cyclobutanamine;
1 -[4-(9-phenyl-3-[ 1 ,2,4]triazolo[ 1 ,5-a]pyrimidin-2-yl[ 1 ,2,4]triazolo[3,4-/]-l ,6-naphthyridin- 8-yl)phenyl]cyclobutanamine;
1 -[4-(9-phenyl-3-pyrazolo[ 1 ,5-a]pyrimidin-3-yl[ 1 ,2,4]triazolo[3,4- J- 1 ,6-naphthyridin-8- yl)phenyl] cyclobutanamine;
1 -[4-(3-imidazo[2, 1 -b] [ 1 ,3]thiazol-6-yl-9-phenyl[ 1 ,2,4]triazolo[3,4- J- 1 ,6-naphthyridin-8- yl)phenyl] cyclobutanamine;
1 - {4-[9-phenyl-3-(lH-l ,2,3-triazol-4-yl)[ 1 ,2,4]triazolo[3,4- J-l ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1 -[4-(9-phenyl[ 1 ,2,4]triazolo[3,4- J- 1 ,6-naphthyridin-8-yl)phenyl] cyclobutanamine;
8-[4-(l -aminocyclobutyl)phenyl]-9-phenyl[ 1 ,2,4]triazolo[3,4- J- 1 ,6-naphthyridin-3-ol; l-[4-(3-methyl-9-phenyl[l,2,4]triazolo[3,4- J-l,6-naphthyridin-8-yl)phenyl]cyclobutanamm^ 1 - {4-[3-(difluoromethyl)-9-phenyl[ 1 ,2,4]triazolo[3,4- J- 1 ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
{8-[4-(l -aminocyclobutyl)phenyl]-9-phenyl[ 1 ,2,4]triazolo[3,4- J- 1 ,6-naphthyridin-3- yl} methanol;
1 - {4-[3-(methoxymethyl)-9-phenyl[ 1 ,2,4]triazolo[3,4- J- 1 ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1- {8-[4-(l-aminocyclobutyl)phenyl]-9-phenyl[l,2,4]triazolo[3,4- J-l,6-naphthyridin-3- yl}ethanol;
2- {8-[4-(l-aminocyclobutyl)phenyl]-9-phenyl[l,2,4]triazolo[3,4- J-l,6-naphthyridin-3- yl}propan-2-ol;
l-[4-(3-cyclopropyl-9-phenyl[l,2,4]triazolo[3,4: ]-l,6-naphthyridin-8- yl)phenyl] cyclobutanamine;
l-[4-(3-cyclohexyl-9-phenyl[l,2,4]triazolo[3,4: ]-l,6-naphthyridin-8- yl)phenyl] cyclobutanamine;
l-{4-[9-phenyl-3-(tetrahydro-2H-pyran-2-yl)[l,2,4]triazolo[3,4- J-l,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1 - (4-[3-(4-methylmorpholin-2-yl)- 9-phenyl- [ 1 ,2,4]triazolo[3,4- J- 1 ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
l-{4-[3-(2-methyltetrahydro-2H-pyran-2-yl)- 9-phenyl-[l,2,4]triazolo[3,4: ]-l,6- naphthyridin-8-yl]phenyl} cyclobutanamine; l-{4-[9-phenyl-3-(tetrahydro-2H-pyran-3-yl)[l,2,4]triazolo[3,4- J-l,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
l-{4-[9-phenyl-3-(tetrahydro-2H-pyran-4-yl)[l,2,4]triazolo[3,4- J-l,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1 -[4-(3-imidazo[ 1 ,2-a]pyrazin-2-yl-9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl)phenyl] cyclobutanamine;
l-{4 3-(l-methyl H ,2,3-triazol-4-yl)-9-phenyl[l,2,4]triazolo[3,4-f] ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1 -[4-(3-imidazo[ 1 ,2-a]pyridin-2-yl-9-phenyl[ 1 ,2,4]triazolo[3,4-f]-l ,6-naphthyridin-8- yl)phenyl] cyclobutanamine;
l-{4-[9-phenyl-3-(lH-pyrazol-4-yl)[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
l-[4-(9-phenyl-3-pyrazolo[l,5-a]pyrimidin-2-yl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8^ yl)phenyl] cyclobutanamine;
l-(4-{3-[(2-hydroxyethyl)carbamoyl]-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8- yl} phenyl)cyclobutanamine;
1 - {4-[3-(4-hydroxyphenyl)-9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1 -(4- {3-[4-(hydroxymethyl)phenyl]-9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl} phenyl)cyclobutanamine;
1 -[4-(9-phenyl-3-pyridin-3-yl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl)phenyl] cyclobutanamine;
1 -[4-(9-phenyl-3-pyridin-4-yl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl)phenyl] cyclobutanamine;
l-[4-(9-phenyl-3-pyridazin-3-yl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8- yl)phenyl] cyclobutanamine;
1 -[4-(9-phenyl-3-pyrimidin-5-yl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl)phenyl] cyclobutanamine;
l-[4-(9-phenyl-3-pyrazin-2-yl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8- yl)phenyl] cyclobutanamine;
3- {8-[4-(l -aminocyclobutyl)phenyl]-9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-3-yl} -5- oxo-4,5-dihydro-lH-l,2,4-triazole;
1 - {4-[3-(6-hydroxypyridin-2-yl)-9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1 - {4-[3-(2-hydroxyphenyl)-9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl]phenyl} cyclobutanamine; 1 - {4-[3-(6-morpholin-4-ylpyridin-3-yl)-9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1 - {4-[3-(3-hydroxypyridin-2-yl)-9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1 - {4-[3-(6-hydroxypyridin-3-yl)-9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
l-(4-{3-[5-(methoxycarbonyl)pyridin-2-yl]-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8 yl} phenyl)cyclobutanamine;
l-{4-[3-(5-hydroxypyrazin-2-yl)-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1 - {4-[3-(4-hydroxypyrimidin-5-yl)-9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
l-[4-(3-carbamoyl-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8- yl)phenyl] cyclobutanamine;
4-({8-[4-(l-ammoniocyclobutyl)phenyl]-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-3- yl} methyl)morpholine;
8- [4-( 1 -ammoniocyclobutyl)phenyl] -9-phenyl-3 -( 1 H-tetrazol- 1 -ylmethyl) [ 1 ,2,4]triazolo [3 ,4- f]- 1 ,6-naphthyridine;
8-[4-(l-ammoniocyclobutyl)phenyl]-9-phenyl-3-(lH-l,2,4-triazol-l- ylmethyl)[l,2,4]triazolo[3,4-f]-l,6-naphthyridine;
8- [4-( 1 -ammoniocyclobutyl)phenyl] -9-phenyl-3 -( 1 H-pyrazol- 1 -ylmethyl)[ 1 ,2,4]triazolo [3 ,4- f]- 1 ,6-naphthyridine;
1 - {4-[3-(azetidin- 1 -ylmethyl)-9-phenyl[ 1 ,2,4]triazolo[3,4-f]-l ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1 -[4-(3- {[ethyl(methyl)amino]methyl} -9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl)phenyl] cyclobutanamine;
l-{4-[9-phenyl-3-(pyrrolidin-l-ylmethyl)[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1- (4-{3-[(diethylamino)methyl]-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8- yl} phenyl)cyclobutanamine;
2- [({8-[4-(l-aminocyclobutyl)phenyl]-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-3- yl} methyl)(methyl)amino] ethanol;
l-{4-[9-phenyl-3-(piperidin-l-ylmethyl)[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1 - {4-[9-phenyl-3-(piperazin- 1 -ylmethyl)[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl]phenyl} cyclobutanamine; (3R)- 1 -( {8-[4-(l -aminocyclobutyl)phenyl]-9-phenyl[ 1 ,2,4]triazolo[3,4-f]-l ,6-naphthyridin-3- yl} methyl)pyrrolidin-3 -ol;
(3 S)- 1 -( { 8- [4-( 1 -aminocyclobutyl)pheny
yl} methyl)pyrrolidin-3 -ol;
l-[4-(3-{[(2-methoxyethyl)(methyl)amino]methyl}-9-phenyl[l,2,4]triazolo[3,4-i^
naphthyridin-8-yl)phenyl]cyclobutanamine;
2,2'-[({8-[4-(l-aminocyclobutyl)phenyl]-9-phenyl[l,2,4]triazolo[3,4-i^-l,6-naphthyri yl} methyl)imino] diacetonitrile;
4-({8-[4-(l-aminocyclobutyl)phenyl]-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-3- yl} methyl)piperazin-2-one;
1- ({8-[4-(l-aminocyclobutyl)phenyl]-9-phenyl[l,2,4]triazolo[3,4-i^-l,6-naphthyridin-3- yl} methyl)piperidin-3 -ol;
1 -(4- {3-[(4-fluoropiperidin- 1 -yl)methyl]-9-phenyl[ 1 ,2,4]triazolo[3,4-f]-l ,6-naphthyridin-8- yl} phenyl)cyclobutanamine;
2- [({8-[4-(l-aminocyclobutyl)phenyl]-9-phenyl[l,2,4]triazolo[3,4-i^-l,6-naphthyridin-3- yl} methyl)(propyl)amino] ethanol;
(3R)- 1 -( {8-[4-(l -aminocyclobutyl)phenyl]-9-phenyl[ 1 ,2,4]triazolo[3,4-f]-l ,6-naphthyridin-3- yl} methyl)-N,N-dimethylpyrrolidin-3 -amine;
[ 1 -( {8-[4-(l -aminocyclobutyl)phenyl]-9-phenyl[ 1 ,2,4]triazolo[3,4-f]-l ,6-naphthyridin-3- yl}methyl)piperidin-2-yl]methanol;
[ 1 -( {8-[4-(l -aminocyclobutyl)phenyl]-9-phenyl[ 1 ,2,4]triazolo[3,4-f]-l ,6-naphthyridin-3- yl} methyl)piperidin-3 -yljmethanol;
1 -[4-(3- {[(2R)-2-(methoxymethyl)pyrrolidin- 1 -yl]methyl} -9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6- naphthyridin-8-yl)phenyl]cyclobutanamine;
[ 1 -( {8-[4-(l -aminocyclobutyl)phenyl]-9-phenyl[ 1 ,2,4]triazolo[3,4-f]-l ,6-naphthyridin-3- yl}methyl)piperidin-4-yl]methanol;
2-[({8-[4-(l-aminocyclobutyl)phenyl]-9-phenyl[l,2,4]triazolo[3,4-i^-l,6-naphthyridin-3- yl} methyl)(butyl)amino] ethanol;
1- ({8-[4-(l-aminocyclobutyl)phenyl]-9-phenyl[l,2,4]triazolo[3,4-i^-l,6-naphthyridin-3- yl}methyl)piperidine-4-carboxamide;
1 -(4- {3-[(4-acetylpiperazin- 1 -yl)methyl]-9-phenyl[ 1 ,2,4]triazolo[3,4-f]-l ,6-naphthyridin-8- yl} phenyl)cyclobutanamine;
2- [l-({8-[4-(l-aminocyclobutyl)phenyl]-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-3- yl} methyl)piperidin-4-yl] ethanol;
l-{4 3-(3,4-dihydroisoquinolin-2(lH)-ylmethyl)-9-phenyl[l,2,4]triazolo[3,4-f]-l,6- naphthyridin-8-yl]phenyl}cyclobutanamine; 1 -[4-(3- {[methyl(2-phenylethyl)amino]methyl} -9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6- naphthyridin-8-yl)phenyl]cyclobutanamine;
1 -(4- {9-phenyl-3-[(propylamino)methyl] [ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl} phenyl)cyclobutanamine;
1 -(4- {3-[(benzylamino)methyl]-9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl} phenyl)cyclobutanamine;
1 -(4- {3-[(methylamino)methyl^
yl} phenyl)cyclobutanamine;
l-{4-[9-phenyl-3-(lH-l,2,4-triazol-3-yl)[l,2^
yl]phenyl} cyclobutanamine;
1 - {4-[3-(5-hydroxypyridin-2-yl)-9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
l-{4-[3-(ammoniomethyl)-9-phenyl[l,2,4]triazolo[3,4-i^-l,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
8- [4-( 1 -ammoniocyclobutyl)phenyl] -3 - [( 1 -methyl- 1 H-imidazol-4-yl)methyl] -9- phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridine;
1 -[4-(9-phenyl-3-pyridin-2-yl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl)phenyl] cyclobutanamine;
1 -[4-(9-phenyl-3-pyrimidin-4-yl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl)phenyl] cyclobutanamine;
1 - {4-[3-(3-hydroxyphenyl)-9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1 -(4- {3-[hydroxy(methoxy)methyl]-9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl} phenyl)cyclobutanamine;
l-[4-(3,9-diphenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8-yl)phenyl]cyclobutanamine;
1- [4-(3-cyclobutyl-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8- yl)phenyl] cyclobutanamine;
1 - {4-[3-(cyclopropylmethyl)-9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
2- {8-[4-(l-ammoniocyclobutyl)phenyl]-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-3- yl}piperidine;
l-[4-(9-phenyl-3-propyl[l,2,4]triazolo[3,4-i^-l,6-naphthyridin-8-yl)phenyl]cyclobutanamine; 1 -[4-(3-ethyl-9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8-yl)phenyl]cyclobutanamine; l-[4-(3-ethoxy-9-phenyl[l,2,4]triazolo[3,4-i^-l,6-naphthyridin-8-yl)phenyl]cyclobutanamine; l-({8-[4-(l-ammoniocyclobutyl)phenyl]-9-phenyl[l,2,4]triazolo[3,4-i^-l,6-naphthyridin-3- yl} methyl)-4-hydroxypiperidine; l-[4-(3-isobutyl-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8- yl)phenyl] cyclobutanamine;
1 - {4-[3-(2-hydroxy-2-methylpropyl)-9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
l-{4-[3-(l-hydroxypropyl)-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
l-{4 9-phenyl-3-(lH-pyrazol-5-yl)[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
l-{4 3-(l-methyl-lH-pyrazol-3-yl)-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
l-{4 3-(5-methyl-lH-pyrazol-3-yl)-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
l-{4-[9-phenyl-3-(4-pyridazinyl)[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1 - {4-[3-(3-methyl-lH- 1 ,2,4-triazol-5-yl)-9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
l-{4 3-(3-methyl-lH-pyrazol-4-yl)-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1 - {4-[3 -( 1 -methyl- 1 H-pyrazol-4-yl)-9-phenyl[ 1 ,2,4]triazolo [3 ,4-f] - 1 ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1 - {4-[9-phenyl-3-(l ,4,5,6-tetrahydrocyclopenta[c]pyrazol-4-yl)[ 1 ,2,4]triazolo[3,4-f]- 1 ,6- naphthyridin-8-yl]phenyl} cyclobutanamine;
l-{4-[3-(2-methyl-l,3-thiazol-4-yl)-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
l-{4-[3-(l-benzothien-2-yl)-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
l-{4-[3-(lH-indol-2-yl)-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
l-{4-[3-(lH-indol-3-yl)-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1 - {4-[3-(5-methyl-4-isoxazolyl)-9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
l-{4-[3-(2-methyl-3-furyl)-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1- {4-[3-(l ,3-oxazol-4-yl)-9-phenyl[l ,2,4]triazolo[3,4-f]-l ,6-naphthyridin-8- yl]phenyl} cyclobutanamine; 1 - {4-[9-phenyl-3-(3-thienyl)[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
4- {8-[4-(l -aminocyclobutyl)phenyl]-9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-3-yl} - 1 H-pyrazol-3 -amine;
l-{4 3-(lH-indazol-3-yl)-9-phenyl[l,2,4]triazolo[3,4-il-l,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1 - {4-[3-(5-methyl-3-isoxazolyl)-9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1 - {4-[3-(5-methyl-l ,2,3-thiadiazol-4-yl)-9-phenyl[ 1 ,2,4]triazolo[3,4-f]- 1 ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1 - {4-[9-phenyl-3-(l ,2,5-thiadiazol-3-yl)[ 1 ,2,4]triazolo[3,4-f]-l ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
l-{4 3-(l,2,5-oxadiazol-3-yl)-9-phenyl[l,2,4]triazolo[3,4-q-l,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
l-{4-[3-(l-methyl-lH-imidazol-4-yl)-9-(4-fluorophenyl)[l,2,4]triazolo[3,4- J-l,6- naphthyridin-8-yl]phenyl} cyclobutanamine;
l-{4 3-methyl-9-(4-fluorophenyl)[l,2,4]triazolo[3,4- J-l,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1 - {4-[3-(ethylamino)-9-phenyl[ 1 ,2,4]triazolo[3,4- J- 1 ,6-naphthyridin-8- yl]phenyl} cyclobutanamine;
1 - [2-( { 8- [4-( 1 -aminocyclobutyl)phenyl] -9-phenyl[ 1 ,2,4]triazolo [3 ,4-f - 1 ,6-naphthyridin-3 - yl} amino)ethyl] - 1 -methylpiperidine;
8-[4-(l -aminocyclobutyl)phenyl]-N-cyclohexyl-9-phenyl[ 1 ,2,4]triazolo[3,4- J- 1 ,6- naphthyridin-3 -amine;
8-[4-(l -aminocyclobutyl)phenyl]-N-(tert-butyl)-9-phenyl[ 1 ,2,4]triazolo[3,4- J- 1 ,6- naphthyridin-3 -amine;
N'- {8-[4-(l -aminocyclobutyl)phenyl]-9-phenyl[ 1 ,2,4]triazolo[3,4- J- 1 ,6-naphthyridin-3-yl} - N,N-dimethylpropane-l,3-diamine;
l-{4 3-(l-methyl-lH-imidazol-4-yl)-9-phenyl[l,2,4]triazolo[3,4- J-l,6-naphthyridin-8- yl]phenyl} cyclopentanamine;
l-{4 3-methyl-9-(4-fluorophenyl)[l,2,4]triazolo[3,4- J-l,6-naphthyridin-8- yl]phenyl} cyclopentanamine;
1 - {4-[3-( 1 -Methyl- lH-imidazol-4-yl)-9-phenyl[ 1 ,2,4]triazolo[3 ,4- J- 1 ,6-naphthyridin-8- yl]phenyl} cyclohexanamine;
l-{4-[3-Methyl-9-phenyl[l,2,4]triazolo[3,4: ]-l,6-naphthyridin-8- yl]phenyl} cyclohexanamine; 8- [4-( 1 -aminocyclohexyl)phenyl] -N-ethyl-9-phenyl[ 1 ,2,4]triazolo [3 ,4-f - 1 ,6-naphthyridin-3 - amine;
1 -[4-(2-phenyl-9-pyridin-4-yl[ 1 ,2,4]triazolo[4',3': 1 ,6]pyrido[2,3-£]pyrazin-3-yl)phenyl] cyclobutanamine;
l-[4-(2-phenyl-9-pyridin-3-yl[l,2,4]triazolo[4 3^ 1,6]pyrido[2,3-¾]pyrazin-3-yl)phenyl] cyclobutanamine;
1 -[4-(2-Phenyl-9-pyrazin-2-yl[ 1 ,2,4]triazolo[4',3': 1 ,6]pyrido[2,3-¾]pyrazin-3-yl)phenyl] cyclobutanamine; and
l-[4-(2-phenyl-9-propyl[l,2,4]triazolo[4',3^ 1,6]pyrido[2,3-¾]pyrazin-3-yl)phenyl] cyclobutanamine;
or a pharmaceutically acceptable salt or stereoisomer thereof.
[0080] A reaction scheme for the production of exemplary AKT inhibitors of the present invention is provided at Example 2.
[0081] In other embodiments the AKT inhibitor of the present invention is an AKT allosteric inhibitor selected from among the compounds disclosed in WO 2005/100344;
WO 2005/100356; WO 2004/096135; WO 2004/096129; WO 2004/096130;
WO 2004/096131; WO 2006/091395; WO 2008/070134; WO 2009/148916;
WO 2004/041162; WO 2006/068796; WO 2006/065601; WO 2006/110638;
WO 2003/086394; WO 2003/086403; WO 2003/086404; WO 2003/086279;
WO 2002/083139; WO 2002/083675; WO 2006/036395; WO 2002/083138;
WO 2006/135627; WO 2002/083140; WO 2010/088177; WO 2011/075383;
WO 2010/104705; WO 2010/104933; and WO 2010/114780 (all to Merck & Co., Inc.), the disclosures of which are hereby incorporated by reference in their entireties.
[0082] In further embodiments the AKT inhibitor of the present invention is selected from among the compounds disclosed in US 2008/076763 (to Glaxo SmithKline LLC);
WO 2011/050016 and US 2009/221633 (to Eli Lilly & Company); US 2005/272708 (to
Georgetown University); WO 2007/076704 (to Central South University - China); and
WO 2010/091824 (to Bayer Schering Pharma AG.). In other embodiments the AKT inhibitor is perifosine (KRX-0401) (Keryx); VQD-002 (API-2, TCN) (VioQuest); SR13668 (SRI
International); GSK690693, GSK2110183 or GSK2141795 (GlaxoSmithKlme); AZD5363
(AstraZeneca); or XL418 (Exelixis).
[0083] In yet further embodiments the AKT inhibitor is dosed in combination with one or more other cancer drugs. In some embodiments the other cancer drug is a MEK inhibitor such as AZD6244 (selumetinib/ARRY-886)(AstraZeneca) or GSK1120212
(GlaxoSmithKline), or another cancer drug such as lapatinib; bicalutamide; paclitaxel;
goserelin, anastrozole, letrozole, or exemestane; bendamustine hydrochloride; trastuzumab; rituximab; and nelfmavir.
IV. Pharmaceutical Compositions of PI3K/AKT Pathway Inhibitors
[0084] The PI3K/AKT pathway inhibitors of the present invention, such as the AKT inhibitors, may be administered to mammals, including humans, either alone or, in combination with pharmaceutically acceptable carriers, excipients or diluents, in a pharmaceutical composition, according to standard pharmaceutical practice. The inhibitors (compounds) can be administered orally or parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration.
[0085] The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, microcrystalline cellulose, sodium crosscarmellose, corn starch, or alginic acid; binding agents, for example starch, gelatin, polyvinyl-pyrrolidone or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to mask the unpleasant taste of the drug or delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a water soluble taste masking material such as hydroxypropylmethyl-cellulose or hydroxypropylcellulose, or a time delay material such as ethyl cellulose, cellulose acetate buryrate may be employed. [0086] Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water soluble carrier such as polyethyleneglycol or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
[0087] Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl- cellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p- hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame.
[0088] Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as butylated hydroxyanisol or alpha- tocopherol.
[0089] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.
[0090] The pharmaceutical compositions of the invention may also be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring phosphatides, for example soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavouring agents, preservatives and antioxidants.
[0091] Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, flavoring and coloring agents and antioxidant.
[0092] The pharmaceutical compositions may be in the form of sterile injectable aqueous solutions. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
[0093] The sterile injectable preparation may also be a sterile injectable oil-in-water microemulsion where the active ingredient is dissolved in the oily phase. For example, the active ingredient may be first dissolved in a mixture of soybean oil and lecithin. The oil solution then introduced into a water and glycerol mixture and processed to form a microemulation.
[0094] The injectable solutions or microemulsions may be introduced into a patient's blood-stream by local bolus injection. Alternatively, it may be advantageous to administer the solution or microemulsion in such a way as to maintain a constant circulating
concentration of the instant compound. In order to maintain such a constant concentration, a continuous intravenous delivery device may be utilized. An example of such a device is the Deltec CADD-PLUS™ model 5400 intravenous pump.
[0095] The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension for intramuscular and subcutaneous administration. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
[0096] Compounds of Formula A may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary
temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol.
[0097] For topical use, creams, ointments, jellies, solutions or suspensions, etc., containing the compound of Formula A are employed. (For purposes of this application, topical application shall include mouth washes and gargles.)
[0098] The compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles and delivery devices, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in the art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen. Compounds of the present invention may also be delivered as a suppository employing bases such as cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol.
[0099] When a composition according to this invention is administered into a human subject, the daily dosage will normally be determined by the prescribing physician with the dosage generally varying according to the age, weight, and response of the individual patient, as well as the severity of the patient's symptoms.
[00100] The dosage regimen utilizing the compounds of the instant invention can be selected in accordance with a variety of factors including type, species, age, weight, sex and the type of cancer being treated; the severity (i.e., stage) of the cancer to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to treat, for example, to prevent, inhibit (fully or partially) or arrest the progress of the disease. For example, compounds of the instant invention can be administered in a total daily dose of up to 10,000 mg.
Compounds of the instant invention can be administered once daily (QD), or divided into multiple daily doses such as twice daily (BID), and three times daily (TID). Compounds of the instant invention can be administered at a total daily or weekly dosage of up to 10,000 mg, e.g., 2,000 mg, 3,000 mg, 4,000 mg, 6,000 mg, 8,000 mg or 10,000 mg, which can be administered in one daily dose or can be divided into multiple daily doses as described above.
[00101] For example, compounds of the instant invention can be administered in a total daily dose of up to 1 ,000 mg. Compounds of the instant invention can be administered once daily (QD), or divided into multiple daily doses such as twice daily (BID), and three times daily (TID). Alternatively, compounds of the instant invention can be administered less frequently, such as once a day (QD), once every other day (QOD), once every week (QW), or once every other week (QOW). Compounds of the instant invention can be administered at a total daily or weekly dosage of up to 1,000 mg, e.g., 200 mg, 300 mg, 400 mg, 600 mg, 800 mg or 1 ,000 mg, which can be administered in one daily dose or can be divided into multiple daily doses as described above.
[00102] In addition, the administration can be continuous, i.e., every day, or intermittently. The terms "intermittent" or "intermittently" as used herein means stopping and starting at either regular or irregular intervals. For example, intermittent administration of a compound of the instant invention may be administration one to six days per week or it may mean administration in cycles (e.g. daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week) or it may mean administration on alternate days.
[00103] In addition, the compounds of the instant invention may be administered according to any of the schedules described above, consecutively for a few weeks, followed by a rest period. For example, the compounds of the instant invention may be administered according to any one of the schedules described above from two to eight weeks, followed by a rest period of one week, or twice daily at a dose of 100 - 500 mg for three to five days a week. In another particular embodiment, the compounds of the instant invention may be
administered three times daily for two consecutive weeks, followed by one week of rest. Pharmaceutical preparations comprising MK-2206
[00104] Preparations of the monohydrochloride salt of MK-2206 may be prepared as a tablet involving roller compression granulation followed by milling, mixing with the other inactive ingredients, compression, and film coating.
[00105] Some of the diluents or fillers for use in this formulation are preferably swellable agents, and may include, but are not limited to, various grades of microcrystallme cellulose, such as Avicel PH101, Avicel PH102, & Avicel PH200. If microcrystallme cellulose is added, it is preferably from about 50 to 180 microns in size, more preferably about 100. Avicel PH 101 has a mean particle size of about 50; Avicel PH 102 has a mean particle size of about 100; and Avicel PH 200 has a mean particle size of about 190 microns. Preferably, the preferred microcrystallme cellulose is Avicel PH 102.
[00106] The edible calcium salts suitable for use herein include but are not limited to, dibasic calcium phosphate dihydrate, calcium phosphate anhydrous, and tribasic calcium phosphate; or mixtures thereof. A preferred edible calcium salt is the dibasic calcium phosphate anhydrous, which also provides good compressibility.
[00107] Suitable ratios for particular diluents however, are described below: For microcrystallme cellulose: Dibasic calcium phosphate, dihydrate, from about 2 to about 4: 1, preferably from about 2.6-3.1 : 1; For microcrystallme cellulose: Calcium phosphate, anhydrous from about 1 to about 3: 1, preferably from about 1.6: 1, microcrystallme cellulose: Tribasic calcium phosphate, from about 2 to about 4: 1, preferably from about 3.1 : 1.
[00108] A preferred disintegrating agent is sodium croscarmellose. Preferably, the sodium croscarmellose is present in an amount of about 2 to about 5% w/w.
[00109] A preferred lubricant is magnesium stearate.
[00110] An aspect of the present invention is a process for preparing a tablet formulation which comprises:
a) blending together to form an intragranular mixture of the active
monohydrochloride salt of MK-2206, microcrystallme cellulose, an edible calcium salt, disintegrant, and lubricant;
b) roller compression granulation of the mixture of step (a) for the purpose of preparing granules;
c) lubricating the granulation from steb (b); d) compacting the lubricated granulates of step (c) into concave tablet; and e) film coating tablets from step (d).
V. IL-23 Antagonists
[00111] Interleukin-23 (IL-23) is a heterodimeric cytokine comprised of two subunits, pl9 which is unique to IL-23, and p40, which is shared with IL-12. The pl9 subunit is structurally related to IL-6, granulocyte-colony stimulating factor (G-CSF), and the p35 subunit of IL-12. IL-23 mediates signaling by binding to a heterodimeric receptor, comprised of IL-23R and IL-12 i, which is shared by the IL-12 receptor. A number of early studies demonstrated that the consequences of a genetic deficiency in p40 (p40 knockout mouse; p40KO mouse) were more severe than those found in a p35KO mouse. Some of these results were eventually explained by the discovery of IL-23, and the finding that the p40KO prevents expression of not only IL-12, but also of IL-23 (see, e.g., Oppmann et al. (2000) Immunity 13:715-725; Wiekowski et al. (2001) J. Immunol. 166:7563-7570; Parham et al. (2002) J. Immunol. 168:5699-708; Frucht (2002) Sci STKE 2002, E1-E3; Elkins et al. (2002) Infection Immunity 70: 1936-1948).
[00112] Recent studies, through the use of p40 KO mice, have shown that blockade of both IL-23 and IL-12 is an effective treatment for various inflammatory and autoimmune disorders. IL-23 is known to play a central role in psoriasis, and the IL-23/IL-12 antagonist antibody ustekinumab (anti-IL-12/23p40 mAb) has been approved in the U.S. and Europe for the treatment of psoriasis. However, the blockade of IL-12 through p40 appears to have various systemic consequences such as increased susceptibility to opportunistic microbial infections. Bowman et al. (2006) Curr. Opin. Infect. Dis. 19:245.
[00113] Therapeutic antibodies may be used to block cytokine activity. The most significant limitation in using antibodies as a therapeutic agent in vivo is the immunogenicity of the antibodies. As most monoclonal antibodies are derived from rodents, repeated use in humans results in the generation of an immune response against the therapeutic antibody. Such an immune response results in a loss of therapeutic efficacy at a minimum and a potential fatal anaphylactic response at a maximum. Initial efforts to reduce the
immunogenicity of rodent antibodies involved the production of chimeric antibodies, in which mouse variable regions were fused with human constant regions. Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-43. However, mice injected with hybrids of human variable regions and mouse constant regions develop a strong anti-antibody response directed against the human variable region, suggesting that the retention of the entire rodent Fv region in such chimeric antibodies may still result in unwanted immunogenicity in patients.
[00114] It is generally believed that complementarity determining region (CDR) loops of variable domains comprise the binding site of antibody molecules. Therefore, the grafting of rodent CDR loops onto human frameworks (i.e., humanization) was attempted to further minimize rodent sequences. Jones et al. (1986) Nature 321 :522; Verhoeyen et al. (1988) Science 239:1534. However, CDR loop exchanges still do not uniformly result in an antibody with the same binding properties as the antibody of origin. Changes in framework residues (FR), residues involved in CDR loop support, in humanized antibodies also are required to preserve antigen binding affinity. Kabat et al. (1991) J. Immunol. 147: 1709. While the use of CDR grafting and framework residue preservation in a number of humanized antibody constructs has been reported, it is difficult to predict if a particular sequence will result in the antibody with the desired binding, and sometimes biological, properties. See, e.g., Queen et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029, Gorman et al. (1991) Proc. Natl. Acad. Sci. USA 88:4181, and Hodgson (1991) Biotechnology (NY) 9:421- 5. Moreover, most prior studies used different human sequences for animal light and heavy variable sequences, rendering the predictive nature of such studies questionable. Sequences of known antibodies have been used or, more typically, those of antibodies having known X- ray structures, antibodies NEW and KOL. See, e.g., Jones et al, supra; Verhoeyen et al, supra; and Gorman et al, supra. Exact sequence information has been reported for a few humanized constructs.
[00115] The present invention involves antagonism of IL-23 activity as a means of ameliorating skin rash associated with treatment with PI3K/AKT pathway inhibitors, such as cancer therapy. Any suitable method of antagonizing IL-23 activity can be used in the methods of the present invention. Such methods include blocking the expression or activity of IL-23 or its receptor.
[00116] In some embodiments the method of antagonizing IL-23 activity is a method that does not antagonize the activity of IL-12, e.g. by use of an IL-23 -specific antagonist. Such methods of antagonizing IL-23 may involve blocking of the activity of the pl9 subunit of IL-23, rather than the p40 subunit, since the pl9 subunit is specific to IL-23 (pi 9 + p40) whereas the p40 subunit is shared with IL-12 (p35 + p40). Such methods of antagonizing IL- 23 may also involve blocking of the activity of the IL-23R subunit of the IL-23 receptor complex (IL-23R + IL-12Rpi), rather than the IL-12Rpi subunit that is shared with the IL-12 receptor (IL-12Rpi + IL-12RP2).
[00117] In various embodiments the method of antagonism of IL-23 involves administration of an IL-23 antagonist. IL-23 antagonists include, but are not limited to, small molecule compounds, antisense nucleic acids, small interfering nucleic acids, aptamers, antibodies or antigen binding fragments thereof, and soluble forms of IL-23 receptor.
[00118] In some embodiments the IL-23 antagonist is an IL-23 -specific antagonist.
Exemplary IL-23 -specific antagonists include an antibody that binds specifically to IL-23p40 but not IL-12p40 (US 7,247,711 to Centocor) or an antibody that makes contacts with both the pl9 and p40 subunits of IL-23 (WO 2011/056600 to Amgen, Inc.). Fibronectin-derived IL-23 antagonists are disclosed at WO 2011/103105 (developed at Adnexus Therapeutics Inc., now part of Bristol-Myers Squibb Co.).
[00119] In other embodiments the IL-23 -specific antagonist binds to pi 9. Exemplary
IL-23 -specific antagonists that bind to pl9 include multimerized IL-23 receptors
(US 2011/0052585 to Genzyme Corp.); protein constructs against IL-23pl9
(WO 2010/142534 and WO 2009/068627 to Ablynx NV); an IL-23 aptamer (US
2006/0193821 to Archemix); and monoclonal antibody FM303 (Femta Pharmaceuticals). Further IL-23 -specific antagonists that bind to pl9 include antibodies or antigen-binding fragments thereof that specifically bind to the pl9 subunit of IL-23, as disclosed at
WO 2008/103432, US 2007/0048315 and WO 2008/103473 (to Schering Corp.); US
7,491,391, US 7,935,344 and EP 1971366 A2 (to Centocor Ortho Biotech, Inc.); US
7,872,102 (to Eli Lilly and Co.); WO 2007/147019, WO 2008/134659 and WO 2009/082624 (to Zymogenetics); US 2009/0311253 (to Abbott Bioresearch); and US 2009/0123479 and WO 2010/115786 (to Glaxo SmithKline), the disclosures of which are hereby incorporated by reference in their entireties.
[00120] Anti-IL-23pl9 antibodies that may be suitable for use in the methods of the present invention also include, but are not limited to, Merck's SCH 900222/MK-3222; Eli Lilly's LY2525623, and Centocor's CNTO 1959, all of which have entered human clinical trials. Specifically, the sequences of SEQ ID NOs: 48 and 52 (heavy chain variable domains), 57 (light chain variable domain), 28-37-40 (light chain CDRs 1-2-3, respectively) and 3-8-19 (light chain CDRs 1-2-3, respectively) of EP 1937721 Bl (to Eli Lilly and Company) are hereby incorporated by reference. In addition, the sequences of SEQ ID NOs: 106 (heavy chain variable domain), 116 (light chain variable domain), 50-56-73 (light chain CDRs 1-2-3, respectively) and 5-20-44 (light chain CDRs 1-2-3, respectively) of US 7,935,344 (to
Centocor) are also hereby incorporated by reference.
[00121] In some embodiments, the anti-IL-23pl9 antibodies, or antigen binding fragments thereof, are based on antibody 13B8 of commonly assigned WO 2008/103432, the disclosure of which is hereby incorporated by reference in its entirety. The anti-human IL- 23pl9 antibody may comprise one, two, three, four, five or six of the CDR sequences, or the heavy and light chain variable domains, of the humanized antibodies disclosed in commonly assigned WO 2008/103432, for example antibodies hul3B8a, b or c. In another embodiment the anti-human IL-23pl9 antibody competes with antibody hul3B8a, b or c for binding to human IL-23. In another embodiment the anti-human IL-23pl9 antibody binds to the same epitope on human IL-23 as hul3B8a, b or c.
[00122] A hybridoma expressing antibody 13B8 was deposited pursuant to the
Budapest Treaty with American Type Culture Collection (ATCC - Manassas, Virginia, USA) on August 17, 2006 under Accession Number PTA-7803. All restrictions on the accessibility of this deposit will be irrevocably removed upon the granting of a U.S. patent based on the present application. In other embodiments, the anti-human IL-23pl9 antibody is able to block binding of human IL-23pl9 to the antibody produced by the hybridoma deposited with accession number PTA-7803 in a cross-blocking assay. In yet further embodiments, the anti- human IL-23pl9 antibody binds to the same epitope as the antibody produced by the hybridoma deposited with ATCC under accession number PTA-7803. In still further embodiments, the anti-human IL-23pl9 antibody comprises the same CDR sequences as the antibody produced by the hybridoma deposited with ATCC with accession number PTA- 7803.
[00123] In other embodiments the IL-23 -specific antagonist binds to IL-23R.
Exemplary IL-23 -specific antagonists that bind to IL-23R include anti-IL-23R antibodies (WO 2008/106134 and WO 2010/027767 to Schering Corp.); multimerized and multimerized polypeptides that binds to IL-23R (U.S. Pat. App. Pub. No. 2011/0086806 to Anaphore, Inc.); and IL-23 receptor antagonist peptides (WO 2009/007849 to Valorisation HSJ and Societe en Commandite), such as APG2305 (Allostera Pharma, Inc.). [00124] In some embodiments the IL-23 antagonist is a non-specific IL-23 antagonist
Exemplary non-specific IL-23 antagonists include antibodies that bind to the p40 subunit of IL-23 and IL-12, such as ustekinumab (CNTO 1275) and briakinumab (ABT-874, J-695). Ustekinumab is marketed by Centocor for the treatment of psoriasis, and is described at US 6,902,734 and US 7,166,285 (to Centocor, Inc.), the disclosures of which are hereby incorporated by reference in their entireties. Specifically, the sequences of SEQ ID NOs: 7 (heavy chain variable domain) and 8 (light chain variable domain), of US 6,902,734 are hereby incorporated by reference. SEQ ID NOs: 4-5-6 and 1-2-3 of US 6,902,734 are also incorporated by reference. Sequences for ustekinumab are also provided at SEQ ID NOs: 51 - 60 of the sequence listing of the present application. Briakinumab was developed by Abbott, and is described at US 6,914,128 and US 7,504,485, the disclosures of which are hereby incorporated by reference in their entireties. Specifically, the sequences of SEQ ID NOs: 31 (heavy chain variable domain), 32 (light chain variable domain) SEQ ID NOs; 30- 28-26 (light chain CDRs 1-2-3, respectively) and 29-27-25 (heavy chain CDRs 1-2-3, respectively) of US 6,914,128 are hereby incorporated by reference. Sequences for briakinumab are also provided at SEQ ID NOs: 61 - 70 of the sequence listing of the present application.
[00125] Further exemplary non-specific IL-23 antagonist antibodies that bind to the p40 subunit of IL-23 and IL-12 are disclosed at Clarke et al. (2010) mAbs 2: 1-11 (Cephalon Australia, Pty., Ltd.). FM202 (Femta Pharmaceuticals) is also a monoclonal antibody that binds to the p40 subunit of both IL-12 and IL-23, as are the antibodies disclosed at
WO 2010/017598 (Arana Therapeutics, Ltd.). Apilimod mesylate (STA-5326, Synta Pharmaceuticals Corp.), an oral non-specific IL-23 antagonist, may also be used in some embodiments of the present invention. Still further exemplary non-specific IL-23 antagonists include antibodies that bind to the IL-12Rpi subunit of both the IL-12 and IL-23 receptor complexes (WO 2010/112458 to Novartis AG).
[00126] Other potential IL-23 antagonists for use in the methods of the present invention include the peptides disclosed in WO 2011/033493 (Peptinov SAS), the variant pl9 polypeptides disclosed at WO 2011/011797 (Eleven Biotherapeutics, Inc. and Stanford University), and the oxidized lipid compounds disclosed at WO 2004/106486 and
US 7,625,882 (Vascular Biogenics, Ltd.), such as VBL-201 (VBL Therapeutics). VI. Anti-IL-23p 19 Antibodies
[00127] In some embodiments involving anti-IL-23 l9 antibodies, of antigen binding fragments thereof, amino acid sequence variants of the human or humanized anti-IL-23 antibody will have an amino acid sequence having at least 75% amino acid sequence identity with the original human or humanized antibody amino acid sequences of either the heavy or the light chain more preferably at least 80%, more preferably at least 85%, more preferably at least 90%), and most preferably at least 95, 98, or 99%. Identity or homology with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the human or humanized anti-IL-23 residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. None of N-terminal, C-terminal, or internal extensions, deletions, or insertions into the antibody sequence shall be construed as affecting sequence identity or homology.
[00128] The human or humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE. Preferably, the antibody is an IgG antibody. Any isotype of IgG can be used, including IgGi, IgG2, IgG3, and IgG4. Variants of the IgG isotypes are also contemplated. The human or humanized antibody may comprise sequences from more than one class or isotype. Optimization of the necessary constant domain sequences to generate the desired biologic activity is readily achieved by screening the antibodies in the biological assays described below.
[00129] Likewise, either class of light chain can be used in the compositions and methods herein. Specifically, kappa, lambda, or variants thereof are useful in the present compositions and methods.
[00130] For some embodiments involving humanized anti-IL-23pl9 antibodies, any suitable portion of the CDR sequences from the non-human antibody can be used. The CDR sequences can be mutagenized by substitution, insertion or deletion of at least one residue such that the CDR sequence is distinct from the human and non-human antibody sequence employed. It is contemplated that such mutations would be minimal. Typically, at least 75% of the humanized antibody residues will correspond to those of the non-human CDR residues, more often 90%, and most preferably greater than 95%.
[00131] For some embodiments involving humanized anti-IL-23pl9 antibodies, any suitable portion of the FR sequences from the human antibody can be used. The FR sequences can be mutagenized by substitution, insertion or deletion of at least one residue such that the FR sequence is distinct from the human and non-human antibody sequence employed. It is contemplated that such mutations would be minimal. Typically, at least 75% of the humanized antibody residues will correspond to those of the human FR residues, more often 90%, and most preferably greater than 95, 98, or 99%.
[00132] CDR and FR residues are determined according to the standard sequence definition of Kabat. Kabat et al. (1987) Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda Md. SEQ ID NOs: 1-5 show the heavy chain variable domain sequences of various mouse anti-human IL-23pl9 antibodies, and SEQ ID NOs: 9-13 depict the light chain variable domain sequences. FIGS. 1 and 2 provide sequence lineups of heavy and light chain variable domains of the various antibodies of the present invention. CDRs are indicated in the figures, and the individual CDR sequences are each presented with unique Sequence Identifiers as indicated in Table 4.
[00133] Humanized forms of antibody 13B8 are provided. The humanized light chain
13B8 sequence (with kappa constant region) is provided at SEQ ID NO: 14, and the light chain variable domain comprises residues 1-108 of that sequence. Three versions of the humanized heavy chain 13B8 sequence (with γΐ constant regions) are provided at SEQ ID NOs: 6-8, and the heavy chain variable domain comprises residues 1-116 of those sequences. The 13B8 heavy chains variants are illustrated at Table 3, with differences from the parental sequence noted in bold. The Met (M) was modified to Lys (K) to avoid the potential for oxidation of the residue and inactivation of the antibody. The substitution of AQKLQ for NEMFE is a replacement of the murine CDR sequence with the human germline sequence from the human framework selected to humanize the antibody.
Table 3
Antibody 13B8 CDRH2 Variants
Antibody CDRH2 Sequence SEQ ID NO: ml3B8, hl3B8-a QIFPASGSADYNEMFEG 24
hl3B8-b QIFPASGSADYNEKFEG 25
hl3B8-c QIFPASGSADYAQKLQG 26 [00134] Humanized forms of the other antibodies disclosed herein may be created by simply substituting the parental rodent antibody CDRs into the light and heavy chain sequences for humanized 13B8 provided at SEQ ID NOs: 14 and 6. This approach is most likely to be successful for antibody chains with CDRs having high homology with the CDRs of antibody 13B8, e.g. clone 1 ICl on the heavy chain and clones 1 ICl and 21D1 on the light chain. Alternatively, the murine antibodies may be independently humanized using the approaches outlines herein, e.g. at Example 2.
[00135] In one embodiment, CDRs include variants of any single sequence CDR disclosed herein (SEQ ID NOs: 15-46), in which the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions relative to the disclosed sequence, as determined using the data of Table 1.
[00136] Also contemplated are chimeric antibodies. As noted above, typical chimeric antibodies comprise a portion of the heavy and/or light chain identical with, or homologous to, corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. See U.S. Pat. No. 4,816,567; and
Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81 : 6851-6855.
[00137] Bispecific antibodies are also useful in the present methods and compositions.
As used herein, the term "bispecific antibody" refers to an antibody, typically a monoclonal antibody, having binding specificities for at least two different antigenic epitopes, e.g., IL- 23pl9 and IL-17. In one embodiment, the epitopes are from the same antigen. In another embodiment, the epitopes are from two different antigens. Methods for making bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly using the co-expression of two immunoglobulin heavy chain/light chain pairs. See, e.g., Milstein et al. (1983) Nature 305: 537-39. Alternatively, bispecific antibodies can be prepared using chemical linkage. See, e.g., Brennan et al. (1985) Science 229:81.
Bispecific antibodies include bispecific antibody fragments. See, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6444-48, Gruber et al. (1994) J. Immunol. 152:5368. [00138] In yet other embodiments, different constant domains may be appended to the humanized VL and VR regions provided herein. For example, if a particular intended use of an antibody (or fragment) of the present invention were to call for altered effector functions, a heavy chain constant domain other than IgGl may be used. Although IgGl antibodies provide for long half-life and for effector functions, such as complement activation and antibody-dependent cellular cytotoxicity, such activities may not be desirable for all uses of the antibody. In such instances an IgG4 constant domain, for example, may be used.
VII. Biological Activity of Humanized Anti-IL-23p 19 Antibodies
[00139] Antibodies having the characteristics identified herein as being desirable in a humanized anti-IL-23 antibody can be screened for inhibitory biologic activity in vitro or suitable binding affinity. To screen for antibodies that bind to the epitope on human IL-23 (i.e. the pl9 subunit) bound by an antibody of interest {e.g., those that block binding of the cytokine to its receptor), a routine cross-blocking assay such as that described in
ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. Antibodies that bind to the same epitope are likely to cross-block in such assays, but not all cross-blocking antibodies will necessarily bind at precisely the same epitope since cross-blocking may result from steric hindrance of antibody binding by antibodies bind at overlapping epitopes, or even nearby non-overlapping epitopes.
[00140] Alternatively, epitope mapping, e.g., as described in Champe et al. (1995) J.
Biol. Chem. 270: 1388-1394, can be performed to determine whether the antibody binds an epitope of interest. "Alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science 244: 1081-1085, or some other form of point mutagenesis of amino acid residues in human IL-23 may also be used to determine the functional epitope for an anti-IL- 23 antibody of the present invention. Mutagenesis studies, however, may also reveal amino acid residues that are crucial to the overall three-dimensional structure of IL-23 but that are not directly involved in antibody-antigen contacts, and thus other methods may be necessary to confirm a functional epitope determined using this method.
[00141] The epitope bound by a specific antibody may also be determined by assessing binding of the antibody to peptides comprising fragments of human IL-23pl9 (SEQ ID NO: 47). The sequence of the p40 subunit of IL-12 and IL-23 is found at GenBank Accession No. P29460. A series of overlapping peptides encompassing the sequence of IL-23pl9 may be synthesized and screened for binding, e.g. in a direct ELISA, a competitive ELISA (where the peptide is assessed for its ability to prevent binding of an antibody to IL-23pl9 bound to a well of a microtiter plate), or on a chip. Such peptide screening methods may not be capable of detecting some discontinuous functional epitopes, i.e. functional epitopes that involve amino acid residues that are not contiguous along the primary sequence of the IL-23pl9 polypeptide chain.
[00142] The epitope bound by antibodies of the present invention may also be determined by structural methods, such as X-ray crystal structure determination (e.g., WO2005/044853), molecular modeling and nuclear magnetic resonance (NMR)
spectroscopy, including NMR determination of the H-D exchange rates of labile amide hydrogens in IL-23 when free and when bound in a complex with an antibody of interest (Zinn- Justin et al. (1992) Biochemistry 31 :11335-11347; Zinn-Justin et al. (1993)
Biochemistry 32:6884-6891).
[00143] With regard to X-ray crystallography, crystallization may be accomplished using any of the known methods in the art (e.g. Giege et al. (1994) Acta Crystallogr.
D50:339-350; McPherson (1990) Eur. J. Biochem. 189: 1-23), including microbatch (e.g. Chayen (1997) Structure 5:1269-1274), hanging-drop vapor diffusion (e.g. McPherson (1976) J. Biol. Chem. 251 :6300-6303), seeding and dialysis. It is desirable to use a protein preparation having a concentration of at least about 1 mg/mL and preferably about 10 mg/mL to about 20 mg/mL. Crystallization may be best achieved in a precipitant solution containing polyethylene glycol 1000-20,000 (PEG; average molecular weight ranging from about 1000 to about 20,000 Da), preferably about 5000 to about 7000 Da, more preferably about 6000 Da, with concentrations ranging from about 10% to about 30% (w/v). It may also be desirable to include a protein stabilizing agent, e.g. glycerol at a concentration ranging from about 0.5%) to about 20%>. A suitable salt, such as sodium chloride, lithium chloride or sodium citrate may also be desirable in the precipitant solution, preferably in a concentration ranging from about 1 mM to about 1000 mM. The precipitant is preferably buffered to a pH of from about 4.0 to about 10.0, often from about 7.0 to 8.5, e.g. pH 8.0. Specific buffers useful in the precipitant solution may vary and are well-known in the art. Scopes, Protein Purification: Principles and Practice, Third ed., (1994) Springer-Verlag, New York. Examples of useful buffers include, but are not limited to, HEPES, Tris, MES and acetate. Crystals may be grow at a wide range of temperatures, including 2°C, 4°C, 8°C and 26°C. [00144] Antibody: antigen crystals may be studied using well-known X-ray diffraction techniques and may be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see e.g. Blundell & Johnson (1985) Meth. Enzymol. 114 & 115, H. W. Wyckoff et al. eds., Academic Press; U.S. Patent Application Publication No. 2004/0014194), and BUSTER (Bricogne (1993) Acta Cryst. D49:37-60; Bricogne (1997) Meth. Enzymol. 276A:361-423, Carter & Sweet, eds.; Roversi et al. (2000) Acta Cryst. D56: 1313-1323).
[00145] Additional antibodies binding to the same epitope as an antibody of the present invention may be obtained, for example, by screening of antibodies raised against IL-23 for binding to the epitope, or by immunization of an animal with a peptide comprising a fragment of human IL-23 comprising the epitope sequence. Antibodies that bind to the same functional epitope might be expected to exhibit similar biological activities, such as blocking receptor binding, and such activities can be confirmed by functional assays of the antibodies.
[00146] Antibody affinities {e.g. for human IL-23) may be determined using standard analysis. Preferred humanized antibodies are those which bind human IL-23pl9 with a Ka
-7 -8
value of no more than about 1x10" ; preferably no more than about 1x10" ; more preferably no more than about lxlO"9; and most preferably no more than about lxlO"10 or even lxlO"11 M.
[00147] The antibodies and fragments thereof useful in the present compositions and methods are biologically active antibodies and fragments. As used herein, the term
"biologically active" refers to an antibody or antibody fragment that is capable of binding the desired the antigenic epitope and directly or indirectly exerting a biologic effect. Typically, these effects result from the failure of IL-23 to bind its receptor. As used herein, the term "specific" refers to the selective binding of the antibody to the target antigen epitope.
Antibodies can be tested for specificity of binding by comparing binding to IL-23 to binding to irrelevant antigen or antigen mixture under a given set of conditions. If the antibody binds to IL-23 at least 10, and preferably 50 times more than to irrelevant antigen or antigen mixture then it is considered to be specific. An antibody that binds to IL-12 is not an IL-23- specific antibody. An antibody that "specifically binds" to IL-23pl9 does not bind to proteins that do not comprise the IL-23pl9-derived sequences, i.e. "specificity" as used herein relates to IL-23pl9 specificity, and not any other sequences that may be present in the protein in question. For example, as used herein, an antibody that "specifically binds" to IL-23pl9 will typically bind to FLAG®-hIL-23pl9, which is a fusion protein comprising IL-23pl9 and a FLAG peptide tag, but it does not bind to the FLAG peptide tag alone or when it is fused to a protein other than IL-23pl9.
[00148] IL-23 -specific binding compounds of the present invention, such as inhibitory
IL-23pl9 specific antibodies, can inhibit its biological activity in any manner, including but not limited to production of IL-Ιβ and TNF by peritoneal macrophages and IL-17 by TR17 T cells. See Langrish et al. (2004) Immunol. Rev. 202:96-105. Anti-IL-23pl9 antibodies will also be able to inhibit the gene expression of IL-17A, IL-17F, CCL7, CCL17, CCL20, CCL22, CCR1, and GM-CSF. See Langrish et al. (2005) J. Exp. Med. 201 :233-240. IL-23- specific binding compounds of the present invention, such as anti IL-23pl9 antibodies, will also block the ability of IL-23 to enhance proliferation or survival of TH17 cells. Cua and Kastelein (2006) Nat. Immunol. 7:557-559..
VIII. Pharmaceutical Compositions of anti-IL-23pl9 Antibodies
[00149] To prepare pharmaceutical or sterile compositions including IL-23pl9 antibody, the antibody (or antigen binding fragment thereof) is admixed with a
pharmaceutically acceptable carrier or excipient. See, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984).
[00150] Formulations of therapeutic and diagnostic agents may be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions. See, e.g., Hardman et al. (2001) Goodman and Gilman 's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and
Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY.
[00151] Toxicity and therapeutic efficacy of the antibody compositions, administered alone or in combination with an immunosuppressive agent, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD5o (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio of LD50 to ED50. Antibodies exhibiting high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
[00152] The mode of administration is not particularly important. Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal
administration; parenteral delivery, including intramuscular, intradermal, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Administration of antibody used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral, intraarterial or intravenous injection.
[00153] Alternately, one may administer the antibody in a local rather than systemic manner, for example, via injection of the antibody directly into a tumor or cancerous organ, often in a depot or sustained release formulation. Furthermore, one may administer the antibody in a targeted drug delivery system, for example, in a liposome coated with a tissue- specific antibody, targeting, for example, a tumor cell. The liposomes will be targeted to and taken up selectively by the afflicted tissue.
[00154] Selecting an administration regimen for a therapeutic depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells in the biological matrix. Preferably, an administration regimen maximizes the amount of therapeutic delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of biologic delivered depends in part on the particular entity and the severity of the condition being treated. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available. See, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al.
(2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341 : 1966- 1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343: 1594-1602.
[00155] Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced. Preferably, a biologic that will be used is substantially derived from the same species as the animal targeted for treatment (e.g. a humanized antibody for treatment of human subjects), thereby minimizing any immune response to the reagent.
[00156] Antibodies, antibody fragments, and cytokines can be provided by continuous infusion, or by doses at intervals of, e.g., one day, 1-7 times per week, one week, two weeks, monthly, bimonthly, quarterly, every 4 or 6 months, annually, etc. Doses may be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular,
intracerebrally, intraspinally, or by inhalation. A preferred dose protocol is one involving the maximal dose or dose frequency that avoids significant undesirable side effects. A total weekly dose is generally at least 0.05 μg/kg, 0.2 μg/kg, 0.5 μg/kg, 1 μg/kg, 10 μg/kg, 100 μg/kg, 0.2 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 10 mg/kg, 25 mg/kg, 50 mg/kg body weight or more. See, e.g., Yang et al. (2003) New Engl. J. Med. 349:427-434; Herald et al. (2002) New Engl. J. Med. 346:1692-1698; Liu et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456; Portielji et al. (20003) Cancer Immunol. Immunother. 52: 133-144. The desired dose of a small molecule therapeutic, e.g., a peptide mimetic, natural product, or organic chemical, is about the same as for an antibody or polypeptide, on a moles/kg basis.
[00157] As used herein, "inhibit" or "treat" or "treatment" includes a postponement of development of the symptoms associated with skin rash induced by PI3K/AKT pathway inhibitors and/or a reduction in the severity of such symptoms that will or are expected to develop. The terms further include ameliorating existing uncontrolled or unwanted side effects, such as skin rash, and preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result has been conferred on a subject with a sign or symptom, or with the potential to develop such a sign or symptom. "Prevent" or "prevention" refers to a reduction or complete elimination of a side effect, such as a skin rash, prior to it manifesting at all. Neither treatment nor prevention, as used herein, requires complete elimination of signs and symptoms. Prevention and treatment refer to any clinically meaningful improvement of the relevant condition, such as reduction in the signs or symptoms of skin rash associated with treatment with PI3K/AKT inhibitors.
[00158] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of an IL-23 antagonist, e.g. an IL-23pl9-specific antibody, or antigen binding fragment thereof, that when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject is effective to prevent or ameliorate the drug-induced side effect, such as skin rash. A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. An effective amount of therapeutic will decrease the symptoms typically by at least 10%; usually by at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably by at least 50%.
[00159] Typical veterinary, experimental, or research subjects include monkeys, dogs, cats, rats, mice, rabbits, guinea pigs, horses, and humans.
IX. Antibody Production
[00160] In one embodiment, for recombinant production of the antibodies of the present invention, the nucleic acids encoding the two chains are isolated and inserted into one or more replicable vectors for further cloning (amplification of the DNA) or for expression. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. In one embodiment, both the light and heavy chains of the humanized anti-IL-23pl9 antibody of the present invention are expressed from the same vector, e.g. a plasmid or an adenoviral vector.
[00161] Antibodies of the present invention may be produced by any method known in the art. In one embodiment, antibodies are expressed in mammalian or insect cells in culture, such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) 293 cells, mouse myeloma NSO cells, baby hamster kidney (BHK) cells, Spodoptera frugiperda ovarian (Sf9) cells. In one embodiment, antibodies secreted from CHO cells are recovered and purified by standard chromatographic methods, such as protein A, cation exchange, anion exchange, hydrophobic interaction, and hydroxyapatite chromatography. Resulting antibodies are concentrated and stored in 20 mM sodium acetate, pH 5.5.
[00162] In another embodiment, the antibodies of the present invention are produced in yeast, e.g. according to the methods described in WO 2005/040395. Briefly, vectors encoding the individual light or heavy chains of an antibody of interest are introduced into different yeast haploid cells, e.g. different mating types of the yeast Pichia pastoris, which yeast haploid cells are optionally complementary auxotrophs. The transformed haploid yeast cells can then be mated or fused to give a diploid yeast cell capable of producing both the heavy and the light chains. The diploid strain is then able to secret the fully assembled and biologically active antibody. The relative expression levels of the two chains can be optimized, for example, by using vectors with different copy number, using transcriptional promoters of different strengths, or inducing expression from inducible promoters driving transcription of the genes encoding one or both chains.
[00163] In one embodiment, the respective heavy and light chains of a plurality of different anti-IL-23pl9 antibodies (the "original" antibodies) are introduced into yeast haploid cells to create a library of haploid yeast strains of one mating type expressing a plurality of light chains, and a library of haploid yeast strains of a different mating type expressing a plurality of heavy chains. These libraries of haploid strains can be mated (or fused as spheroplasts) to produce a series of diploid yeast cells expressing a combinatorial library of antibodies comprised of the various possible permutations of light and heavy chains. The combinatorial library of antibodies can then be screened to determine whether any of the antibodies has properties that are superior (e.g. higher affinity for IL-23) to those of the original antibodies. See. e.g., WO 2005/040395.
[00164] In another embodiment, antibodies of the present invention are human domain antibodies in which portions of an antibody variable domain are linked in a polypeptide of molecular weight approximately 13 kDa. See, e.g., U.S. Pat. Publication No. 2004/0110941. Such single domain, low molecular weight agents provide numerous advantages in terms of ease of synthesis, stability, and route of administration.
X. Uses
[00165] The present invention provides methods for preventing and/or treating skin rash that may occur as a side effect of treatment with PI3K/AKT pathway inhibitors. The methods of the present invention involve antagonism of IL-23, e.g. by treatment with an IL- 23 antagonist, in any subject being treated with a PI3K/AKT pathway inhibitor for any reason. PI3K/AKT pathway inhibitors may find use not only in the treatment of cancer or tumors, but also in the treatment of diseases in which angiogenesis is implicated, such as ocular neovascular diseases like diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, age-related macular degeneration (WO 00/30651); atherosclerosis, osteoarthritis (WO 03/035048), psoriasis, obesity, and Alzheimer's disease. In addition, PI3K/AKT pathway inhibitors may find use in treating hyperproliferative disorders like restenosis (WO 03/032809), inflammation, autoimmune diseases and allergy/asthma.
[00166] IL-23 antagonists may find use in the manufacture of medicaments for preventing and/or treating skin rash that may occur as a side effect of treatment with a PI3K/AKT pathway inhibitor. The present invention further relates to an IL-23 antagonist, e.g. an anti-IL-23 antibody, for use in preventing and/or treating skin rash that may occur as a side effect of treatment with a PI3K/AKT pathway inhibitor.
[00167] The invention further relates to prevention and/or treatment of a disorder, such as cancer or a tumor, by administering to a subject an IL-23 antagonist and a PI3K/AKT inhibitor. In various embodiments, the IL-23 antagonist is administered prior to, concurrently with, or after administration of the PI3K/AKT inhibitor. Administration of the IL-23 antagonist prior to or concurrently with the PI3K/AKT inhibitor may be helpful in preventing a PI3K/AKT inhibitor-induced rash, whereas administration of the IL-23 antagonist after the onset of a PI3K/AKT inhibitor-induced rash may be helpful in treatment of the rash or prevention of a rash before is has occurred. In various embodiments, the IL-23 antagonist is administered, e.g., 1 hour, 12 hours, 1, 2, 4, 7, 10, 14, 21, 30 or more days prior to administration of the PI3K/AKT inhibitor. In various embodiments the IL-23 antagonist is an anti-IL-23pl9 antibody or antigen binding fragment thereof. In various embodiments, the PI3K/AKT inhibitor is naphthyridine compound, e.g., 8-[4-(l-aminocyclobutyl)phenyl]-9- phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-3(2H)-one (MK-2206).
[00168] Although the PI3K/AKT inhibitors of the present invention may be
administered at any effective dose, the methods of the present invention find particular value at doses that are high enough to otherwise induce a rash in subjects. For example, 8-[4-(l- aminocyclobutyl)phenyl]-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-3(2H)-one, may be administered at 150 - 200 mg every week, such as 200 mg every week, or at doses that increase the risk of skin rash, such as 250, 300, 350, 400, 450, 500, 600, 700, 750, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg or more.
[00169] IL-23 antagonist of the present invention may be administered at any dose effective to prevent or treat a rash induced by a PI3K/AKT pathway inhibitor. Anti-IL-23 antibodies, such as anti-IL-23pl9 and anti-IL-23p40 antibodies, made be administered, e.g., at 0.01, 0.03, 0.1, 0.3, 0.5, 1, 3, 5, 10, 30, 50 or 100 mg/kg or more. In some embodiments the anti-IL-23 antibody (or fragment) is administered at 0.1, 0.3, 1 or 3 mg/kg. In some embodiments the anti-IL-23 antibody (or fragment) is administered weekly, biweekly, monthly, bimonthly, every three months or at other intervals. In other embodiments the IL-23 antibody (or fragment) is administered only once during a given course of treatment with a PI3K AKT pathway inhibitor, or is administered as needed based on an evaluation of the condition of a specific subject, e.g. based on the presence and severity of a skin rash. Anti- IL-23 antibodies (or fragments thereof) may be administered by any suitable means, including but not limited to subcutaneous injection and intravenous infusion.
[00170] The broad scope of this invention is best understood with reference to the following examples, which are not intended to limit the inventions to the specific
embodiments. The specific embodiments described herein are offered by way of example only, and the invention is to be limited by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. EXAMPLES
Example 1
General Methods
[00171] 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, CA. 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).
[00172] Methods for protein purification including immunoprecipitation,
chromatography, electrophoresis, centrifugation, and crystallization are described. Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York. Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, glycosylation of proteins are described. See, e.g., Coligan et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, 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, N.J., pp. 384-391. Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described. Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra. Standard techniques for characterizing ligand/receptor interactions are available. See, e.g., Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York.
[00173] Methods for flow cytometry, including fluorescence activated cell sorting detection systems (FACS®), are available. See, e.g., 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 modifying nucleic acids, including nucleic acid primers and probes, polypeptides, and antibodies, for use, e.g., as diagnostic reagents, are available. Molecular Probes (2003) Catalogue,
Molecular Probes, Inc., Eugene, OR; Sigma-Aldrich (2003) Catalogue, St. Louis, MO.
[00174] Standard methods of histology of the immune system are described. See, e.g.,
Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt, et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins, Phila, PA; Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY.
[00175] Software packages and databases for determining, e.g., antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments, are available. See, e.g., GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, MD); GCG Wisconsin Package (Accelrys, Inc., San Diego, CA); DeCypher® (TimeLogic Corp., Crystal Bay, Nevada); Menne et al. (2000) Bioinformatics 16: 741-742; Menne et al. (2000) Bioinformatics Applications Note 16:741-742; Wren et al. (2002) Comput. Methods Programs Biomed. 68: 177-181; von Heijne (1983) Eur. J. Biochem. 133: 17-21; von Heijne (1986) Nucleic Acids Res. 14:4683-4690.
Example 2
Reaction Scheme for Substituted Naphthyridine AKT Inhibitors
[00176] Exemplary substituted naphthyridine compounds for use as AKT inhibitors in the methods of the present invention, such as (MK-2206), may be synthesized as follow. See, e.g., WO 2008/070016 (Schemes 1C and IE).
Reaction Scheme I
Figure imgf000060_0001
1 -10 MK-2206
1 -(4-bromophenyl)cyclobutanecarbonitrile (1-2)
[00177] TBAB (1.61 g, 0.5 mmol), dibromopropane (22.2 g, 110 mmol), and nitrile 1-
1 (19.6 g, 100 mmol) were added to a stirred solution of KOH (31.17 g, 500 mmol) in a mixture of 15 mL of water and 200 mL of toluene (temperature maintained between 72 and 79 °C). The mixture was heated by steam and was stirred at 99-108 °C for 2.5 h. The mixture was cooled to 80 °C and 200 mL of heptane was added. After the resulting mixture was cooled to RT with stirring, the top clear solution was filtered, washed with water (3X30 mL) and concentrated in vacuo to give oily product 1-2.
1 -(4-bromophenyl)cyclobutanecarboxamide (1-3)
[00178] H2O2 (30% 11.3 mL, 118 mmol) was added over 3 h to a stirred mixture of nitrile 1-2 (13.88 g, -58.9 mmol) and K2C03 (1.62 g, 11.8 mol) in 59 mL of DMSO at 40-87 °C, cooling with a water bath. The resulting mixture was cooled to 27 °C and water (100 mL) was added over 30 min. Crystalline product 1-3 formed. More water (100 mL) was added over 1 h. The resulting slurry was aged at RT for 16 h before filtration. The cake was rinsed with 100 mL of water and then with 100 mL of heptane. After drying in a vacuum oven at 50 °C, product 1-3 was obtained as a white solid. tert-butyl [l-(4-bromophenyl)cyclobutyl] carbamate (1-4)
[00179] Pb(OAc)4 (25.7 g, 25.7 mmol) was added to a stirred solution of amide 1-3
(12.7 g, 50 mmol) in 64 mL of t-BuOH at 57 °C to 86 °C cooling with a water bath. The resulting mixture was stirred at 65-86 °C for 0.5 h. The mixture was cooled to 26 °C and 12.7 g of Na2CC"3 were added followed by 65 mL MTBE. After 10 min, the mixture was filtered.
The cake was rinsed with 10 L of MTBE and the combined filtrate was washed with 20 mL of water and the organic layer was then washed with 3 X 10 mL of 10% KHCO3 (caution:
bubbling) dried over Na2S04 and concentrated in vacuo. The resulting solid was rinsed with 8 mL of IP Ac and 8 mL of heptane and dried in a vacuum oven at 40 °C to give product 1-4 as a grey solid. tert-butyl [l-(4-cyanophenyl)cyclobutyl] carbamate (1-5)
[00180] A stirred slurry of Pd2dba3 (101 mg; 1 mol%) and dppf (122 mg; 2 mole%) in
DMF (25 mL) was sparged with nitrogen for 5 min and then warmed to 65 °C and aged for 30 min. At this temp was added the aryl bromide 1-4 (3.6 g, 11 mmol), zinc powder (51 mg; 6 mol%) and the zinc cyanide (777 mg; 0.60 equiv) rinsing with DMF (5 mL). The solution was heated to 92-95 °C and aged for 4 h. The solution was cooled to RT overnight and filtered through a pad of Solka Floe, rinsing the cake with DMF (5 mL). Water (30 mL) was added over 3.5 h at 25-33 °C, along with seed. After aging overnight at RT, the resulting crystalline solution was filtered and washed with aqueous methanol and dried overnight to yield 1-5 as a yellow solid. tert-butyl {l-[4-phenylacetyl)phenyl]cyclobutyl} carbamate (1-6)
[00181] Benzyl Grignard (19 mL, 38.5 mmol) was added to a stirred, slightly cloudy solution of nitrile 1-5 (3 g, 11 mmol) in THF (25 mL) cooled to ca. -20 °C at a rate such that the reaction temperature did not warm above -10 °C. The solution was aged for 3-4 hours keeping the reaction temperature between -10 °C and -20 °C. The stirred solution was cooled to -30 °C and added to a 15 wt% aqueous citric acid solution (60 mL) which was previously cooled to 5-10 °C, maintaining the temperature below 15 °C. The layers were separated and the aqueous layer was washed with MTBE. [00182] The organic layers were combined, washed with half saturated brine (60 mL), and concentrated under reduced pressure. Heptane was added and the mixture was concentrated to a slurry which was filtered, washed with heptane (15 mL) and dried under nitrogen to give 1-6.
4-Amino-2-chloronicotinaldehyde (1-8)
[00183] Trifluoroacetic acid (17.4 mL, 234 mmol) was added carefully to a stirred mixture of Boc aldehyde 1-7 (20 g, 78.1 mmol) and dichloromethane (60 mL) keeping the temperature below 25 °C. The solution was warmed to 35 °C, aged overnight (vigorous off- gassing) and then cooled to room temperature. 25 mL of MTBE was added and the resulting white slurry was aged for one hour, filtered, and the filter cake rinsed with MTBE (10 mL x 2). Solid 1-8 TFA salt was dried under vacuum. tert-butyl {l-[4-(5-chloro-3-phenyl-l,6-naphthyridin-2-yl)phenyl]cyclobutyl} carbamate (1-9)
[00184] 45wt% Potassium hydroxide solution (18 mL; 5 equiv) was added dropwise over 20 minutes to a stirred mixture of chloropyridine TFA salt 1-8 (19.5 g), cyclobutylamino ketone 1-6 (26 g) and isopropanol (200 mL) keeping the temperature below 24 °C. After 1 h, water (100 mL) was added and after a further 1 h the resulting slurry was filtered, washing with 2: 1 IP A/water (30 mL, then 24 mL) then with water (80 mL then 2 x 60 mL). The solid was dried under nitrogen flow to afford 1-9 as an off-white solid. tert-butyl { 1 -[4-(3-oxo-9-phenyl-2,3-dihydro[ 1 ,2,4]triazolo[3,4- ][l,6]naphthyridin-8-yl)phenyl]cyclobutyl} carbamate (1-10)
[00185] A stirred slurry of chloronapthyridine 1-9 (1.8 g), methyl hydrazine carboxylate (0.318 g) and isopropanol (20 L) is warmed to 66 °C before becoming homogeneous. 5-6 N HC1 in IPA (0.05 ml) is added and the temperature is increased to 70 °C for 16 hours and then is cooled to RT. After cooling to RT, 45wt% potassium hydroxide solution (0.52 mL) is mixed with water (5.5 mL) and added over 15 minutes. After 30 minutes, aqueous acetic acid (0.7 mL in 6 mL water) is added followed by water (2 mL). The resulting slurry is aged at RT for three hours, filtered and washed with 1 : 1 IP A/water (2 x 2.4 mL). The product is dried under nitrogen flow then slurried in methylene chloride at 20 °C for 4 hours, filtered and dried under nitrogen flow to afford 1-10 as an off-white solid. 8-[4-(l -Aminocyclobutyl)phenyl]-9-phenyl[ 1 ,2,4]triazolo[3,4- J- 1 ,6- naphthyridin-3(2H)-one (MK-2206)
[00186] A solution of aqueous concentrated HC1 (12.1 M, 1.64 mL) in ethanol (2.0 mL) was added dropwise over 30 min to a stirred slurry of 1-10 (500 mg, 0.985 mol) in ethanol (1.7 mL) and water (0.2 mL) at 50°C. After 3 hours following acid addition, the mixture was seeded and aged overnight at 50°C, cooled to room temperature and filtered. Acetyl chloride (0.5 g, 7 mmol) was added over 1 h to ethanol (2 mL) at 0°C. The solution was then cooled to room temperature and aged for 30 minutes. The filter cake was washed with this solution (1 mL x 2), then with ethyl acetate (4 mL x 2) and dried, finally in a vacuum oven at 75.0°C with nitrogen sweep (50 torr) to afford MK-2206 as the bis-HCl salt.
[00187] A mono-HCl version of MK-2206 was also produced via dissolution in water.
After 6 hours, the aqueous slurry turns light yellow and is filtered. Silver chloride titration of this solid reveals the presence of one equivalent of chloride.
Example 3
IL-12 and IL-23 Gene Expression in THP-1 Cells
[00188] The levels of IL-12 and IL-23 subunit gene expression in THP-1 cells as a function of treatment with TLR agonist (LPS), with or without AKT inhibitor, were determined as follows. THP-1 monocytic cells were cultured at 1 x 10 6 cells/ml in 24-well plates in RPMI 1640 complete medium. Cells were pre-treated with either of two AKT inhibitors (MK-2206 and AKTi-X) at 0.1 μΜ or 0.5 μΜ for 1 hour. Ten (10) μ^πιΐ lipopolysaccharide (LPS) was then added for 3h or 6h, at which time cells were harvested for RNA extraction, and TAQMAN® real-time quantitative polymerase chain reaction gene expression analysis was performed to assess the levels of expression of pi 9, p40 and p35 subunits. IL-23 is a heterodimeric complex of p40 and pl9, whereas IL-12 is a heterodimeric complex of p40 and p35 subunits. Results are shown in FIG. 3.
[00189] The data demonstrate that AKT inhibitor specifically enhances IL-23 subunit gene expression above the level induced by LPS alone. This result holds true for both AKT inhibitors (MK-2206 and AKTi-X). These results are consistent with a downstream role for IL-23 in AKT inhibitor-induced skin rash. Example 4
IL-12 and IL-23 Gene Expression in Primary CD 14+ Monocytes Isolated from PBMCs
[00190] The level of IL-12 and IL-23 subunit gene expression in primary CD 14+ monocytes isolated from PBMCs as a function of treatment with TLR agonist (LPS), with or without AKT inhibitor (AKTi-X), was determined as follows. CD 14+ cells were isolated from healthy human donors (4 donors total). First, PBMC were isolated from buffy coats using Ficoll gradient procedure followed by negative selection of CD 14+ cells using
Monocyte Isolation Kit II (Miltenyi Biotec Cat # 130-091-153). CD14+ cells were grown in complete DMEM medium supplemented with antibiotics. Cells were pre-treated with either of two AKT inhibitors (MK-2206 and AKTi-X) at 0.1 μΜ or 0.5 μΜ for lh. Ten (10) μ^πιΐ lipopolysaccharide (LPS) was then added for 3h, 6h or 24h, at which time cells were harvested for R A extraction and TAQMAN® real-time quantitative polymerase chain reaction gene expression analysis. Results are shown in FIG. 4. Supernatants were also collected for IL-23 protein determination. See Example 5.
[00191] The data demonstrate that AKT inhibitor specifically enhances IL-23 subunit gene expression above the level induced by LPS alone. This result holds true for both donors, and at time points ranging from 3 to 6 to 24 hours post-treatment (other than the single datapoint for Donor 2 at 24 hours). These results are consistent with a downstream role for IL-23 in AKT inhibitor-induced skin rash.
Example 5
IL-23 Protein Levels in Primary CD 14+ Monocytes Isolated from PBMCs
[00192] The levels of IL-23 protein in primary CD 14+ monocytes isolated from
PBMCs as a function of treatment with TLR agonists, with or without AKT inhibitor, was determined using culture supernatants prepared as described in Example 4. IL-23 was detected in an electrochemilummescence assay (ECL) using an anti-human IL-23pl9 capture antibody and a ruthenium labeled anti-human IL-12/23p40 detection antibody. Results are shown in FIG. 5.
[00193] The data demonstrate that AKT inhibitor enhances IL-23 expression above the level induced by LPS alone. This result holds true for all donors, both TLR agonists, and both AKT inhibitors. These results are consistent with a downstream role for IL-23 in AKT inhibitor-induced skin rash. Example 6
Gene Expression in IL-23 and MK-2206 Treated Monkey Skin
[00194] The levels of expression of 24 genes known to be modulated by IL-23 were determined in IL-23 -treated cynomolgus monkeys and also in MK-2206-treated rhesus monkeys as follows. For the IL-23 -induced skin inflammation model, 10 or 100 μg of human IL-23 was intradermally injected to the skin of cynomolgus monkeys each day for 14 days to induce skin inflammation. For the AKTi skin rash model, 54 or 108 mg of MK-2206 was delivered orally to rhesus monkeys daily for 14 - 28 days to induce skin rash. Skin samples were then collected from monkeys for histological and gene expression analyses. Gene expression results for the AKTi skin rash model are provided at Table 2, and representative gene expression data for selected genes in both the IL-23 and AKTi models are provided in FIGS. 6A and 6B.
[00195] Comparison of the gene expression changes in IL-23 -treated and MK-2206- treated monkeys shows that MK-2206 induces most of the same genes induced by IL-23, which is consistent with the hypothesis that IL-23 is involved in AKT inhibitor-induced skin rash.
Example 7
Prevention of Skin Rash in MK-2206-Treated Human Subjects with Anti-IL-23pl9 Antibody SCH 900222/MK-3222
[00196] Human patients with advanced, metastatic, or recurrent breast cancer are selected for treatment with the AKT inhibitor MK-2206. Patients must have histologically or cytologically confirmed breast cancer, with diagnoses or suspected metastatic, inoperable locally advanced breast cancer, or inoperable locally recurrent breast cancer. Patients are treated by subcutaneous injection of 200 mg anti-IL-23pl9 antibody SCH 900222/MK-3222 on day 0. On day 7, patients are started on MK-2206 at 300 mg orally once a week.
[00197] Anti-IL-23pl9 treatment is repeated at approximately 28-day intervals over the course of MK-2206 treatment. Treatment with MK-2206 is continued until the desired therapeutic endpoint is reached, or dose limiting toxicity arises. Example 8
Treatment of Skin Rash in MK-2206-Treated Human Subjects with Anti-IL-23pl9 Antibody SCH 900222/MK-3222
[00198] Human patients with advanced, metastatic, or recurrent breast cancer are selected for treatment with the AKT inhibitor MK-2206. Patients must have histologically or cytologically confirmed breast cancer, with diagnoses or suspected metastatic, inoperable locally advanced breast cancer, or inoperable locally recurrent breast cancer.
[00199] On day 0, patients are started on MK-2206 at 300 mg orally once a week.
Patients are monitored for occurrence of skin rash during the MK-2206 treatment regimen. If a Grade 2, 3 or 4 skin rash occurs, the patient is treated by subcutaneous injection of 200 mg anti-IL-23pl9 antibody SCH 900222/MK-3222. Grade 2 skin rash involves macular or papular eruption or erythema with pruritis or other associated symptoms, and localized desquamation or other lesions covering less than half of the body surface area. Grade 3 skin rash involves severe, generalized erythroderma or macular, popular or vesicular eruption, and desquamation covering half of the body surface area or more. Grade 4 rash involves generalized exfoliative, ulcerative, of bulluous dermatitis.
[00200] Anti-IL-23pl9 treatment, if necessary, is repeated at approximately 28-day intervals over the course of MK-2206 treatment. Treatment with MK-2206 is continued until the desired therapeutic endpoint is reached, or dose limiting toxicity arises.
Example 9
Treatment or Prevention of Skin Rash in MK-2206-Treated Human Subject
with Ustekinumab
[00201] Skin rash is prevented or treated as in Examples 7 and 8, respectively, except that ustekinumab is used in place of SCH 900222/MK-3222, and it is dosed at 45 mg every 28 days by subcutaneous injection for patients weighing 100 kg or less, and 90 mg for patients weighing more than 100 kg. See, e.g., Prescribing Information (STELARA®), U.S. license no. 1864, revised August 2011. Example 10
Treatment or Prevention of Skin Rash in MK-2206-Treated Human Subject
with Briakinumab
[00202] Skin rash is prevented or treated as in Examples 7 and 8, respectively, except that briakinumab is used in place of SCH 900222/MK-3222. See, e.g., Gottleib et al. (2011) British J. Dermatol. 165:652 and Strober et al. (2011) British J. Dermatol. 165:661.
Example 11
Prevention of Skin Rash in MK-2206-Treated Human Subjects with High Dose Anti-IL-23pl9 Antibody SCH 900222/MK-3222
[00203] Human patients with advanced, metastatic, or recurrent breast cancer are selected for treatment with the AKT inhibitor MK-2206. Patients must have histologically or cytologically confirmed breast cancer, with diagnoses or suspected metastatic, inoperable locally advanced breast cancer, or inoperable locally recurrent breast cancer. Patients are treated by intravenous infusion of anti-IL-23pl9 antibody SCH 900222/MK-3222 at
10 mg/kg on day 0. On day 7, patients are started on MK-2206 at 300 mg orally once a week.
[00204] Anti-IL-23pl9 treatment is repeated at approximately 28-day intervals over the course of MK-2206 treatment. Treatment with MK-2206 is continued until the desired therapeutic endpoint is reached, or dose limiting toxicity arises.
Example 12
Treatment of Skin Rash in MK-2206-Treated Human Subjects with High Dose Anti-IL-23pl9 Antibody SCH 900222/MK-3222
[00205] Human patients with advanced, metastatic, or recurrent breast cancer are selected for treatment with the AKT inhibitor MK-2206. Patients must have histologically or cytologically confirmed breast cancer, with diagnoses or suspected metastatic, inoperable locally advanced breast cancer, or inoperable locally recurrent breast cancer.
[00206] On day 0, patients are started on MK-2206 at 300 mg orally once a week.
Patients are monitored for occurrence of skin rash during the MK-2206 treatment regimen. If a Grade 2, 3 or 4 skin rash occurs, the patient is treated by intravenous infusion of anti-IL- 23pl9 antibody SCH 900222/MK-3222 at 10 mg/kg. Grade 2 skin rash involves macular or papular eruption or erythema with pruritis or other associated symptoms, and localized desquamation or other lesions covering less than half of the body surface area. Grade 3 skin rash involves severe, generalized erythroderma or macular, popular or vesicular eruption, and desquamation covering half of the body surface area or more. Grade 4 rash involves generalized exfoliative, ulcerative, of bulluous dermatitis.
[00207] Anti-IL-23pl9 treatment, if necessary, is repeated at approximately 28-day intervals over the course of MK-2206 treatment. Treatment with MK-2206 is continued until the desired therapeutic endpoint is reached, or dose limiting toxicity arises.
[00208] Table 4 provides a brief description of the sequences in the sequence listing.
Table 4
Sequence Identifiers
SEQ ID NO: Description
1 ml Al l VH
2 ml lCl VH
3 m5F5 VH
4 m21Dl VH
5 ml3B8 VH
6 huml3B8 HC-a
7 huml3B8 HC-b
8 huml3B8 HC-c
9 ml Al l VL
10 ml lCl VL
11 m5F5 VL
12 m21Dl VL
13 ml3B8 VL
14 huml3B8 LC
15 ml Al l CDRH1
16 ml lCl CDRH1
17 m5F5 CDRH1
18 m21Dl CDRH1
19 ml3B8 CDRH1 SEQ ID NO: Description
20 ml All CDRH2
21 mllCl CDRH2
22 m5F5 CDRH2
23 m21Dl CDRH2
24 ml3B8 CDRH2-a
25 hl3B8 CDRH2-b
26 hl3B8 CDRH2-C
27 ml All CDRH3
28 mllCl CDRH3
29 m5F5 CDRH3
30 m21Dl CDRH3
31 ml3B8 CDRH3
32 ml All CDRL1
33 mllCl CDRLl
34 m5F5 CDRLl
35 m21Dl CDRLl
36 ml3B8 CDRLl
37 ml Al 1 CDRL2
38 mllCl CDRL2
39 m5F5 CDRL2
40 m21Dl CDRL2
41 ml3B8 CDRL2
42 ml Al 1 CDRL3
43 mllCl CDRL3
44 m5F5 CDRL3
45 m21Dl CDRL3
46 ml3B8 CDRL3
47 human IL-23 l9
48 mouse IL-23 l9
49 huml3B8-b HC DNA SEQ ID NO: Description
50 huml3B8 LC DNA
51 ustekinumab CDRH1
52 ustekinumab CDRH2
53 ustekinumab CDRH3
54 ustekinumab CDRL1
55 ustekinumab CDRL2
56 ustekinumab CDRL3
57 ustekinumab VH
58 ustekinumab VL
59 ustekinumab HC
60 ustekinumab LC
61 briakinumab CDRH1
62 briakinumab CDRH2
63 briakinumab CDRH3
64 briakinumab CDRL1
65 briakinumab CDRL2
66 briakinumab CDRL3
67 briakinumab VH
68 briakinumab VL
69 briakinumab HC
70 briakinumab LC

Claims

CLAIMS WHAT IS CLAIMED IS:
1. A method for preventing skin rash in a subject being treated with a PI3K/AKT pathway inhibitor comprising administering an antagonist of IL-23.
2. A method for treating skin rash in a subject being treated with a PI3K/AKT pathway inhibitor comprising administering an antagonist of IL-23.
3. A method of treating a subject having cancer comprising administering to the subject a PI3K/AKT pathway inhibitor and an IL-23 antagonist.
4. The method of any of Claims 1 - 3 further comprising:
a) determining whether the subject has a skin rash prior to administering the IL-23 antagonist; and
b) administering the IL-23 antagonist only if the subject has the skin rash.
5. The method of any of Claim 4 further comprising:
a) determining whether the subject has a skin rash after administration of the PI3K/AKT inhibitor and the IL-23 antagonist; and
b) administering one or more additional doses of the IL-23 antagonist only if the subject has the skin rash.
6. Use of an antagonist of IL-23 in the manufacture of a medicament for treating or preventing a skin rash in a subject being treated with a PI3K/AKT pathway inhibitor.
7. An antagonist of IL-23 for use in for treating or preventing a skin rash in a subject being treated with a PI3K/AKT pathway inhibitor.
8. The method, use or antagonist of any of the preceding claims wherein the PI3K/AKT pathway inhibitor is an AKT inhibitor.
9. The method, use or antagonist of Claim 8 wherein the AKT inhibitor is 8-[4-(l- aminocyclobutyl)phenyl]-9-phenyl[l,2,4]triazolo[3,4-f]-l,6-naphthyridin-3(2H)-one (MK- 2206).
10. The method, use or antagonist of Claim 9 wherein the MK-2206 is administered to a human subject at a dose of 60 mg or higher.
11. The method, use or antagonist of any of the preceding claims wherein the IL-23 antagonist is an antibody, or antigen binding fragment thereof, that specifically binds to either the pl9 subunit of IL-23 or the IL-23R subunit of the IL-23 receptor complex.
12. The method, use or antagonist of Claim 11 wherein the IL-23 antagonist is an antibody, or antigen binding fragment thereof, that specifically binds to the pl9 subunit of IL- 23.
13. The method, use or antagonist of Claim 12 wherein the antibody, or antigen binding fragment thereof, that specifically binds to the pl9 subunit of IL-23 binds to human IL-23 at an epitope comprising residues 20-30 or residues 82-110 of SEQ ID NO: 47.
14. The method, use or antagonist of Claim 12 wherein the antibody, or antigen binding fragment thereof, that specifically binds to the pl9 subunit of IL-23 binds to an epitope comprising residues 20-30 and residues 82-110 of SEQ ID NO: 47.
15. The method, use or antagonist of Claim 12 wherein the antibody, or antigen binding fragment thereof, that specifically binds to the pl9 subunit of IL-23 binds to an epitope comprising residues K20, T23, W26, S27, P30, E82, S95, L96, L97, P98, D99, P101, G103, Q104, H106, A107 and LI 10 of SEQ ID NO: 47.
16. The method, use or antagonist of any of the preceding claims wherein the IL-23 antagonist is an antibody, or antigen binding fragment thereof, comprising: a) an antibody light chain variable domain, or antigen binding fragment thereof, comprising CDRL1, CDRL2 and CDRL3, wherein:
i) CDRL1 comprises the sequence of SEQ ID NO: 36;
ii) CDRL2 comprises the sequence of SEQ ID NO: 41; and
iii) CDRL3 comprises the sequence of SEQ ID NO: 46,
and
b) an antibody heavy chain variable domain, or antigen binding fragment thereof, comprising CDRH1, CDRH2 and CDRH3, wherein:
i) CDRH1 comprises the sequence of SEQ ID NO: 19;
ii) CDRH2 comprises a sequence selected from the group consisting of SEQ ID NOs: 24-26; and
iii) CDRH3 comprises the sequence of SEQ ID NO: 31.
17. The method, use or antagonist of Claim 16 wherein:
a) the antibody light chain variable domain, or antigen binding fragment thereof, comprises residues 1 - 108 of SEQ ID NO: 14; and
b) the antibody heavy chain variable domain, or antigen binding fragment thereof, comprises a sequence selected from the group consisting of residues 1 - 116 of SEQ ID NOs: 6-8.
18. The method, use or antagonist of Claim 17 wherein the antibody, or antigen binding fragment thereof, comprises:
a) an antibody light chain comprising the sequence of SEQ ID NO: 14; and b) an antibody heavy chain comprises a sequence selected from the group consisting of SEQ ID NOs: 6-8.
19. The method, use or antagonist of any of the preceding claims wherein the IL-23 antagonist is an antibody, or antigen binding fragment thereof, that is able to block binding of the antibody produced by the hybridoma Accession No. PTA-7803 to human IL-23 in a cross- blocking assay.
20. The method, use or antagonist of any of Claims 1 - 10 wherein the IL-23 antagonist is an antibody, or antigen binding fragment thereof, that specifically binds to the p40 subunit of IL-23.
21. The method, use or antagonist of Claim 20 wherein the IL-23 antagonist is an antibody, or antigen binding fragment thereof, comprising:
a) an antibody light chain variable domain, or antigen binding fragment thereof, comprising CDRL1, CDRL2 and CDRL3, wherein:
i) CDRL1 comprises the sequence of SEQ ID NO: 54;
ii) CDRL2 comprises the sequence of SEQ ID NO: 55; and
iii) CDRL3 comprises the sequence of SEQ ID NO: 56,
and
b) an antibody heavy chain variable domain, or antigen binding fragment thereof, comprising CDRH1, CDRH2 and CDRH3, wherein:
i) CDRH1 comprises the sequence of SEQ ID NO: 51;
ii) CDRH2 comprises the sequence of SEQ ID NO: 52; and
iii) CDRH3 comprises the sequence of SEQ ID NO: 53.
22. The method, use or antagonist of Claim 21 wherein:
a) the antibody light chain variable domain, or antigen binding fragment thereof, comprises the sequence of SEQ ID NO: 58; and
b) the antibody heavy chain variable domain, or antigen binding fragment thereof, comprises the sequence of SEQ ID NO: 57.
23. The method, use or antagonist of Claim 22 wherein the antibody, or antigen binding fragment thereof, comprises:
a) an antibody light chain comprising the sequence of SEQ ID NO: 60; and b) an antibody heavy chain comprising the sequence of SEQ ID NO: 59.
24. The method, use or antagonist of Claim 20 wherein the IL-23 antagonist is an antibody, or antigen binding fragment thereof, comprising: a) an antibody light chain variable domain, or antigen binding fragment thereof, comprising CDRL1, CDRL2 and CDRL3, wherein:
i) CDRL1 comprises the sequence of SEQ ID NO: 64;
ii) CDRL2 comprises the sequence of SEQ ID NO: 65; and iii) CDRL3 comprises the sequence of SEQ ID NO: 66,
and
b) an antibody heavy chain variable domain, or antigen binding fragment thereof, comprising CDRH1, CDRH2 and CDRH3, wherein:
i) CDRH1 comprises the sequence of SEQ ID NO: 61;
ii) CDRH2 comprises the sequence of SEQ ID NO: 62; and iii) CDRH3 comprises the sequence of SEQ ID NO: 63.
25. The method, use or antagonist of Claim 24 wherein:
a) the antibody light chain variable domain, or antigen binding fragment thereof, comprises the sequence of SEQ ID NO: 68; and
b) the antibody heavy chain variable domain, or antigen binding fragment thereof, comprises the sequence of SEQ ID NO: 67.
26. The method, use or antagonist of Claim 25 wherein the antibody, or antigen binding fragment thereof, comprises:
a) an antibody light chain comprising the sequence of SEQ ID NO: 70; and b) an antibody heavy chain comprising the sequence of SEQ ID NO: 69.
27. The method, use or antagonist of any of the preceding claims wherein the IL-23 antagonist is administered prior to administration of the PI3K/AKT pathway inhibitor.
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