(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property
`Organization
`International Bureau
`
`(43) International Publication Date
`18 September 2014 (18.09.2014) WI PC I P CT
`
`\9
`
`(10) International Publication Number
`
`WO 2014/144903 A1
`
`(51) International Patent Classification:
`A61K 38/18 (2006.01)
`C07K 14/00 (2006.01)
`A61K 38/00 (2006.01)
`_
`_
`_
`(21) International Appllcatlon Number:
`
`PCT/US201 4/029502
`
`14 March 2014 (14.03.2014)
`
`(72)
`
`Inventors: PADHI, Desmond; C/o Amgen Inc., One Am-
`gen Center Drive, Thousand Oaks, CA 91320-1799 (US).
`HAN, Huiquan; C/o Amgen 1nc., One Amgen Center
`Drive, Thousand Oaks, CA 91320-1799 (US). HAQQ,
`.
`.
`,
`.
`.
`.
`Christopher, Mlchael, C/o Pmta Brotherapeutics,
`Inc.,
`3260 Bayshore Blvd., Brisbane, CA 94005
`(US).
`CIECHANOVER, Isaac; C/o Pinta Biotherapeutics, Inc.,
`3260 Bayshore Blvd., Brisbane, CA 94005 (US).
`
`(22) International Filing Date:
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English (74) Agents: HUBL, Susan T. et al., FenWick & West LLP,
`,
`801 California Street, Mountain View, California 94041
`English
`(US).
`
`(30) Priority Data:
`US
`15 March 2013 (15.03.2013)
`61/799,928
`(71) Applicants: AMGEN INC. [US/US]; One Amgen Center
`Drive, Thousand Oaks, CA 91320-1799 (US). PINTA
`BIOTHERAPEUTICS, INC.
`[US/US]; 3260 Bayshore
`Blvd., Brisbane, CA 94005 (US).
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`A0» AT: AU» AZ» BA» BB» BG» BH» BN9 BR» BW» BY»
`32, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR,
`KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME,
`MG, MK, MN, MW, Mx, MY, Mz, NA, NG, N1, NO, NZ,
`OM, PA, PE, PG, PH, PL, PT, QA, Ro, RS, RU, RW, SA,
`SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM,
`TN, TR, TT, TZ, UA, UG, US, UZ, vc, VN, ZA, ZM,
`ZW.
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`
`[Continued on nextpage]
`
`(54) Title: MYOSTATIN ANTAGONISM IN HUMAN SUBJECTS
`
`Percent Change from Baeeline Total Lean Body Mase
`
`on‘3
`
`e a:3
`
`10-h-oaeafi%pfBaselin Nas
`
`l
`“‘1
`
`lll1xlla
`
`
`
`(57) Abstract: Disclosed are methods of treating or modu-
`lating cachexia and/or increasing lean body mass and/or in-
`creasing lower extremity muscle size in a prostate cancer pa-
`tient comprising administering a therapeutically effective
`amount of a myostatin antagonist. Further disclosed is the
`peptibody sequence of the myostatin antagonist, and the for-
`mulation of the peptibody.
`
`503
`
`FUP
`
`VlSlt
`3.0 m Ikg so AMG 745
`m Place
`
`Fig.1?»
`
`Apotex Inc. et al. v. Amgen Inc. et al., IPR2016-01542
`
`Amgen Exhibit 2027
`
`Page 1
`
`
`
`W02014/144903A1|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
`
`Amgen Exhibit 2027
`Apotex Inc. et al. v. Amgen Inc. et al., IPR2016-01542
`Page 1
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`

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`WO 2014/144903 A1 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
`
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ,
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU,
`LV, MC, MK, MT, NL, NO, PL, PT, R0, RS, SE, SI, SK,
`SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, KM, ML, MR, NE, SN, TD, TG).
`
`Published:
`
`with international search report (Art. 21(3))
`
`before the expiration of the time limit for amending the
`claims and to be republished in the event of receipt of
`amendments (Rule 48.2(h))
`
`with sequence listing part of description (Rule 5.2(a))
`
`Page 2
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`WO 2014/144903
`
`PCT/US2014/029502
`
`MYOSTATIN ANTAGONISM IN HUMAN SUBJECTS
`
`CROSS REFERENCE TO RELATED APPLICATIONS
`
`[0001]
`
`This application claims the benefit of US. Provisional Application No.
`
`61/799,928, filed MARCH 15, 2013, which is hereby incorporated in its entirety by
`
`reference.
`
`STATEMENT REGARDING FEDERALLY SPONSORED RESARCH OR
`DEVELOPMENT
`
`[0002]
`
`Not applicable.
`
`SEQUENCE LISTING
`
`[0003]
`
`The instant application contains a Sequence Listing which has been submitted via
`
`EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on
`
`March 13, 2014, is named 26324PCT_sequencelisting.txt, and is 200,000 bytes in size.
`
`FIELD OF THE INVENTION
`
`[0004]
`
`The invention relates to methods of using myostatin antagonists, e.g., myostatin
`
`binding peptibodies, for treatment of cachexia in prostate cancer patients.
`
`BACKGROUND
`
`[0005]
`
`The transforming growth factor (TGF) [3 superfamily of growth factors consists of
`
`a large number of growth and differentiation factors that regulate muscle tissue development
`
`and homeostasis. Myostatin, a member of the TGF-B superfamily, is expressed almost
`
`exclusively in skeletal muscle, and acts as a negative regulator of muscle growth (Roth and
`
`Walsh, 2004; Thomas et al, 2000). Myostatin inhibits myoblast proliferation by causing up-
`
`regulation of cyclin—dependent kinase (CDK) inhibitors (e.g., p21), which in turn results in
`
`down-regulation of CDK2 and in Go/G1 cell cycle arrest.
`
`In addition, myostatin negatively
`
`regulates myoblast differentiation through decreased expression of MyoD (Langley et al,
`
`2002).
`
`[0006]
`
`Observations from mice and cattle with loss—of-function mutations in the
`
`myostatin gene (Roth and Walsh, 2004; Grobet et al, 1998; Szabo et al, 1998; Grobet et al,
`
`1997; Kambadur et a1, 1997; McPherron and Lee, 1997; McPherron et al, 1997), as well as a
`
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`WO 2014/144903
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`PCT/US2014/029502
`
`recent case report describing a human child with loss—of-function mutations affecting both
`
`myostatin alleles (Schuelke et al, 2004), provide strong evidence that myostatin plays an
`
`important role in regulating perinatal skeletal muscle development. In adult mouse muscle,
`
`myostatin appears to inhibit the activation of regenerative satellite cells (McCroskery et al,
`
`2003). Of particular interest, by a muscle—specific conditional myostatin gene inactivation
`
`approach, general muscle hypertrophy can be induced post-natally in mice, to an extent
`
`similar to that in constitutively myostatin-deficient knockout mice (Grobet et al, 2003).
`
`[0007]
`
`Skeletal muscle wasting is prevalent and clinically impactful in a variety of
`
`conditions and disease states, such as cancer cachexia, androgen deprivation, renal cachexia
`
`due to end stage renal disease, chronic obstructive pulmonary disease, cardiac cachexia,
`
`HIV/AID S, steroid induced myopathy, disuse atrophy, sarcopenia of the elderly and
`
`postoperative immobilization (Muscaritoli et al, 2006; Alibhai et al, 2006; Morley et al, 2006;
`
`MacDonald et al, 2003; Roubenoff et al, 1997). Skeletal muscle wasting results in reduced
`
`muscle strength, physical and psychological disability, and impaired quality of life
`
`(Muscaritoli et al, 2006; Roubenoff et al, 1997). Current treatment options used for muscle
`
`wasting in settings of illness or immobility, including appetite stimulants, nutritional support,
`
`corticosteroids, anabolic steroids, and growth hormone, are limited in their utility and can be
`
`associated with significant systemic side effects (Muscaritoli et al, 2006; MacDonald et al,
`
`2003)
`
`[0008]
`
`Prostate cancer is the most common malignancy in men and the second most
`
`common cause of cancer-related death in men in the US (American Cancer Society, 2005).
`
`Androgen deprivation therapy (ADT) by administration of gonadotropin—releasing hormone
`
`(GnRH) agonists is the mainstay of treatment for metastatic prostate cancer. (Sharafi et al
`
`JAMA 2005) Neoadjuvant/adjuvant ADT improves survival for men receiving radiation
`
`therapy for intermediate—risk and high—risk early stage prostate cancer. Adjuvant ADT is
`
`also associated with improved survival after prostatectomy for men with node-positive
`
`disease
`
`In contemporary clinical practice, chronic treatment with a GnRH agonist,
`
`commonly for biochemical relapse, is the most common form of androgen deprivation
`
`therapy. (Sharafi et al JAMA 2005
`
`[0009]
`
`ADT has a variety of adverse effects including weight gain, increased fat mass,
`
`decreased lean body mass, and fatigue. (Hematol Oncol Clin North Am, 2006
`
`Aug;20(4):909—23. In prospective clinical studies, ADT is associated with decreased lean
`
`body mass and muscle size and increased fat mass. (Smith et al, 2002; Smith et al, 2001).
`
`2
`
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`Changes in body composition are apparent within the first six months of treatment and appear
`
`to continue during long term therapy. (Smith et al JCO 2012). Decreased muscle mass and
`
`strength may contribute to the overall fatigue and to decreased quality of life in men with
`
`prostate cancer. Treatment-related changes in body composition may also contribute to ADT
`
`decreased insulin sensitivity and greater risk for diabetes associated with ADT. (Smith et al
`
`2006 JCEM; Keating et al 2006 JCO; Braga-Basaria et al 2006).
`
`[0010]
`
`AMG 745 is a novel anti-myostatin peptibody. Structurally, it is a fusion protein
`
`with a human PC at the N—terminus and a myostatin-neutralizing bioactive peptide at the C-
`
`terminus. AMG 745 and/or AMG 745/Mu-S, a murine surrogate of AMG 745, have been
`
`tested in a variety of mouse models, including normal mice, immune—deficient mice, MDX
`
`mice (Duchenne muscular dystrophy model), Colon—26 tumor—bearing mice (cancer cachexia
`
`model), hind limb suspended mice (disuse atrophy model), and orchiectomized mice
`
`(androgen—deficiency model). Effects of AMG 745 and/or AMG 745/Mu—S in these models
`
`have included increased body weight gain, increased or improved maintenance of, skeletal
`
`muscle mass, and increased strength compared to control mice. A preclinical study in
`
`orchiectomized mice, a disease model of hypogonadism that features muscle loss and fat
`
`accumulation related to androgen deficiency, demonstrated that administration of AMG
`
`745/Mu—S markedly attenuated loss of lean body mass and accumulation of fat, as assessed
`
`by nuclear magnetic resonance (NMR) imaging, and furthermore, demonstrated that in vivo
`
`myostatin inhibition may enhance skeletal muscle growth via an androgen-independent
`
`mechanism.
`
`[0011]
`
`Myostatin antagonists and their uses are described in International patent
`
`application no. PCT/US2003/040781, published as WO/2004/058988 and filed on December
`
`19, 2003 and PCT/US2006/046546, published as WO2007/067616 and filed on December 6,
`
`2006 and the related national phase patent applications.
`
`SUMMARY
`
`[0012]
`
`Described herein are methods of treating or modulating cachexia and/or increasing
`
`lean body mass and/or decreasing fat mass and/or increasing lower extremity muscle size in a
`
`human subject in need thereof comprising administering a therapeutically effective amount of
`
`a myostatin antagonist in admixture with a pharmaceutically acceptable carrier to the subject,
`
`wherein the human subject has prostate cancer and is receiving androgen deprivation therapy;
`
`the myostatin antagonist consists of a peptibody comprising a polypeptide consisting of the
`
`3
`
`Page 5
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`WO 2014/144903
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`PCT/US2014/029502
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`amino acid sequence of SEQ ID NO:635 (MDKTHTCPPC PAPELLGGPS VFLFPPKPKD
`
`TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST
`
`YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY
`
`TLPPSRDELT KNQVSLTCLV KGFYP SDIAV EWESNGQPEN NYKTTPPVLD
`
`SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGKGG
`
`GGGAQLADHG QCIRWPWMCP PEGWE);
`
`the myostatin antagonist is formulated in 10
`
`mM sodium acetate, 9% (w/v) sucrose, 0.004% (w/v) polysorbate 20, pH 4.75; and the
`
`myostatin antagonist is administered subcutaneously at doses of 0.3 mg/kg, 1.0 mg/kg, or 3.0
`
`mg/kg once weekly for 4 weeks.
`
`[0013]
`
`Also described are methods of treating or modulating cachexia and/or increasing
`
`lean body mass and/or decreasing fat mass and/or increasing lower extremity muscle size in a
`
`human subject in need thereof comprising administering a therapeutically effective amount of
`
`a myostatin antagonist in admixture with a pharmaceutically acceptable carrier to the subject,
`
`wherein the human subject has prostate cancer and is receiving androgen deprivation therapy
`
`and the myostatin antagonist comprises a polypeptide consisting of the amino acid sequence
`
`set forth in SEQ ID N023] l (LADHGQCIRWPWMCPPEGWE). In some embodiments,
`
`the
`
`myostatin antagonist consists of a peptibody comprising a polypeptide consisting of the
`
`amino acid sequence set forth in SEQ ID NO:635 (MDKTHTCPPC PAPELLGGPS
`
`VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT
`
`KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA
`
`KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN
`
`NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK
`
`SLSLSPGKGG GGGAQLADHG QCIRWPWMCP PEGWE). In other embodiments, the
`
`myostatin antagonist consisting of a peptibody consisting of an amino acid sequence that is at
`
`least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the
`
`amino acid sequence set forth in SEQ ID NO:635.
`
`[0014]
`
`The myostatin antagonist used in the method can be a peptibody expressed in
`
`insoluble inclusion bodies in E coli and isolated via cell harvesting, cell lysing, solubilizing
`
`of inclusion bodies, refolding, concentrating, and chromatographic purifying.
`
`[0015]
`
`In some embodiments, the myostatin antagonist is conjugated to an additional
`
`compound.
`
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`[0016]
`
`In some embodiments, the myostatin antagonist is formulated in a pharmaceutical
`
`composition. Examples include but are not limited to a pharmaceutical composition
`
`comprising a buffer, an antioxidant, a low molecular weight molecule, a drug, a protein, an
`
`amino acid, a carbohydrate, a lipid, a chelating agent, a stabilizer, or an excipient. For
`
`example, the formulation can be 10 mM sodium acetate, 9% (w/v) sucrose, 0.004% (w/v)
`
`polysorbate 20, pH 4.75.
`
`[0017]
`
`The method can use administration that is, e.g., parenteral or oral or subcutaneous.
`
`[0018]
`
`In some embodiments, the myostatin antagonist is administered at a dose between
`
`0.01 to 10.0 mg/kg, inclusive or at a dose of0.3 to 3.0 mg/kg, inclusive or at a dose of 0.3,
`
`1.0, or 3.0 mg/kg. The myostatin antagonist can be administered, e.g., twice daily, once
`
`daily, twice weekly, once weekly, twice monthly, or once monthly. In some embodiment the
`
`myostatin antagonist is administered once weekly for 4 weeks.
`
`[0019]
`
`In some embodiments, the myostatin antagonist is co-administered with an
`
`additional agent, e.g., an anti-prostate cancer agent.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`[0020]
`
`Figurc 1 shows myostatin activity as mcasurcd by cxprcsscd lucifcrasc activity (y-
`
`axis) vs. concentration (x-axis) for the TN8-l9 peptide QGHCTRWPWMCPPY (SEQ ID
`
`NO: 32) and the TN8-l9 peptibody (pb) to determine the IC50 for each using the C2C12
`
`prLARE luciferase assay described in the Examples below. The peptibody has a lower IC50
`
`value compared with the peptide.
`
`[0021]
`
`Figure 2 is a graph showing the increase in total body weight for CD1 nu/nu mice
`
`treated with increasing dosages of the 1x mTN8-l9-2l peptibody over a fourteen day period
`
`compared with mice treated with a hch control, as described in Example 8.
`
`[0022]
`
`Figure 3A shows the increase in the mass of the gastrocnemius muscle mass at
`
`necropsy of the mice treated in Figure 2 (Example 8). Figure 3B shows the increase in lean
`
`mass as determined by NMR on day 0 compared with day 13 of the experiment described in
`
`Example 8.
`
`[0023]
`
`Figure 4 shows the increase in lean body mass as for CD1 nu/nu mice treated with
`
`biweekly injections of increasing dosages of 1x mTN8-l9—32 peptibody as determined by
`
`NMR on day 0 and day 13 of the experiment described in Example 8.
`
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`PCT/US2014/029502
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`[0024]
`
`Figure 5A shows the increase in body weight for CD1 nu/nu mice treated with
`
`biweekly injections of lx mTN8—l9—7 compared with 2x mTN8—l9—7 and the control animal
`
`for 35 days as described in Example 8. Figure 5B shows the increase in lean carcass weight
`
`at necropsy for the lx and 2x versions at 1 mg/kg and 3 mg/kg compared with the animals
`
`receiving the vehicle (hch) (controls).
`
`[0025]
`
`Figure 6A shows the increase in lean muscle mass vs. body weight for aged mdx
`
`mice treated with either affinity matured 1x mTN8-l9-33 peptibody or hch vehicle at 10
`
`mg/kg subcutaneously every other day for three months. Figure 6B shows the change in fat
`
`mass compared to body weight as determined by NMR for the same mice after 3 months of
`
`treatment.
`
`[0026]
`
`Figure 7 shows the change in body mass over time in grams for collagen—induced
`
`arthritis (CIA) animals treated with the peptibody 2x mTN8—l9-2 l/mch or mch vehicle, as
`
`well as normal non—CLA animals.
`
`[0027]
`
`Figure 8 shows the relative body weight change over time in streptozotocin
`
`(STZ)—induced diabetic mice treated with the peptibody 2x mTN8-l9-21/mch or the mch
`
`vehicle control.
`
`[0028]
`
`Figure 9 shows creatine clearance rate in streptozotocin (STZ)—induced diabetic
`
`mice and age-matched normal mice after treatment with peptibody 2x mTN8-l9-2 l/mch or
`
`the mch vehicle.
`
`[0029]
`
`Figure 10A shows urine albumin excretion in streptozotocin (STZ)—induced
`
`diabetic mice and age-matched normal mice after treatment with peptibody 2x mTN8-l9-
`
`2l/mch or the mch vehicle. Figure lOB shows the 24 hour urine volume in streptozotocin
`
`(STZ)—induced diabetic mice and age—matched normal mice after treatment with peptibody 2x
`
`mTN8—l9-2l/mch or the mch vehicle.
`
`[0030]
`
`Figure 11 shows body weight change over time for 4 groups of C57Bl/6 mice; 2
`
`groups pretreated for 1 week with peptibody 2x mTN8- 1 9-2 l/mch, then treated with 5—
`
`fluoruracil (5—Fu) or vehicle (PBS); and 2 groups pretreated for 2 weeks with 2x mTN8—l9—
`
`2l/mch, and then treated with 5-fluorouracil or vehicle (PBS). The triangles along the
`
`bottom of the Figure show times of administration of 2 week pretreatment with 2x mTN8—l9—
`
`2l/mch, times of administration of 1 week pretreatment with 2x mTN 8-19-2 l/muFc, and
`
`times of administration of 5-Fu.
`
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`[0031]
`
`Figure 12 shows the survival rate percentages the animals described in Figure 11
`
`above, showing normal mice not treated, animals treated with 5-Fu only, animals pretreated
`
`with 2x mTN8-l9-2l/mch for 1 week and then treated with S-Fu, and animals pretreated
`
`with 2x mTN8-19-2l/mch for 2 weeks and then treated with 5-Fu.
`
`[0032]
`
`Figure 13 shows the percent change from baseline of total lean body mass in
`
`human subjects treated with AMG 745 or placebo. The placebo groups are on the left in each
`
`of EOS and FUP; the AMG 745 groups are on the right in each of EOS and FUP.
`
`DETAILED DESCRIPTION
`
`[0033]
`
`The present invention provides methods of treating cachexia in prostate cancer
`
`patients receiving androgen therapy by administration of a myostatin antagonist comprising
`
`the myostatin binding peptide SEQ ID NO:311, e. g., a peptibody consisting of SEQ ID
`
`NO:635.
`
`Myostatin
`
`[0034]
`
`Myostatin, a growth factor also known as GDF—8, is a member of the TGF—B
`
`family. Myostatin known to be a negative regulator of skeletal muscle tissue. Myostatin is
`
`synthesized as an inactive preproprotein which is activated by proteolyic cleavage (Zimmers
`
`ct al., supra (2002)). The precursor protein is cleaved to produce an NHz—tcrminal inactivc
`
`prodomain and an approximately 109 amino acid COOH-terminal protein in the form of a
`
`homodimer of about 25 kDa, which is the mature, active form (Zimmers et al, supra (2002)).
`
`It is now believed that the mature dimer circulates in the blood as an inactive latent complex
`
`bound to the propeptide (Zimmers et al, supra (2002)).
`
`[0035]
`
`As used herein the term “full-length myostatin” refers to the full-length human
`
`preproprotein sequence described in McPherron et al. PNAS USA 94, 12457 (1997), as well
`
`as related full—length polypeptides including allelic variants and interspecies homologs
`
`(McPherron et al. supra ( 1997)). As used herein, the term “prodomain” or “propeptide”
`
`refers to the inactive NHz—terminal protein which is cleaved off to release the active COOH—
`
`terminal protein. As used herein the term “myostatin” or “mature myostatin” refers to the
`
`mature, biologically active COOH-terminal polypeptide, in monomer, dimer, multimeric
`
`form or other form. “Myostatin” or “mature myostatin” also refers to fragments of the
`
`biologically active mature myostatin, as well as related polypeptides including allelic
`
`variants, splice variants, and fusion peptides and polypeptides. The mature myostatin
`
`7
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`COOH-terminal protein has been reported to have 100% sequence identity among many
`
`species including human, mouse, chicken, porcine, turkey, and rat (Lee et al., PNAS 98, 9306
`
`(2001)). Myostatin may or may not include additional terminal residues such as targeting
`
`sequences, or methionine and lysine residues and /or tag or fusion protein sequences,
`
`depending on how it is prepared.
`
`Myostatin Antagonists
`
`[0036]
`
`The methods of treatment described herein use myostatin antagonists comprising
`
`the myostatin binding peptide SEQ ID NO:311, e. g., a peptibody comprising at least one
`
`polypeptide consisting of SEQ ID NO:635, e. g., the peptibody AMG—745.
`
`[0037]
`
`As used herein the term “myostatin antagonist” is used interchangeably with
`
`“myostatin inhibitor”. A myostatin antagonist according to the present invention inhibits or
`
`blocks at least one activity of myostatin, or alternatively, blocks expression of myostatin or
`
`its receptor. Inhibiting or blocking myostatin activity can be achieved, for example, by
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`employing one or more inhibitory agents which interfere with the binding of myostatin to its
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`receptor, and/or blocks signal transduction resulting from the binding of myostatin to its
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`receptor. Antagonists include agents which bind to myostatin itself, or agents which bind to a
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`myostatin receptor.
`
`[0038]
`
`Other examples of myostatin antagonists include but are not limited to follistatin,
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`the myostatin prodomain, growth and differentiation factor 1 l (GDF—l l) prodomain,
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`prodomain fusion proteins, antagonistic antibodies that bind to myostatin, antagonistic
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`antibodies or antibody fragments that bind to the activin type IIB receptor, soluble activin
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`type IIB receptor, soluble activin type IIB receptor fusion proteins, soluble myostatin analogs
`
`(soluble ligands), oligonucleotides, small molecules, peptidomimetics, and myostatin binding
`
`agents. These are described in more detail below.
`
`[0039]
`
`Follistastin inhibits myostatin, as described, for example, in Amthor et al., Dev
`
`Biol 270, 19-30 (2004), and US patent 6,004,937, which is herein incorporated by reference.
`
`Other inhibitors include, for example, TGF-B binding proteins including growth and
`
`differentiation factor-associated serum protein-1 (GASP) as described in Hill et al., MOI.
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`Endo. 17 (6): 1144—1154 (2003). Myostatin antagonists include the propeptide region of
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`myostatin and related GDF proteins including GDF-l 1, as described in PCT publication WO
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`02/09641, which is herein incorporated by reference. Myostatin antagonists further include
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`modified and stabilized propeptides including Fc fusions of the prodomain as described, for
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`example, in Bogdanovisch et al, FASEB J 19, 543-549 (2005). Additional myostatin
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`antagonists include antibodies or antibody fragments which bind to and inhibit or neutralize
`
`myostatin, including the myostatin proprotein and/or mature protein, which in monomeric or
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`dimeric form. Such antibodies are described, for example, in US patent application US
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`2004/01423 83, and US patent application 2003/ 103 8422, and PCT publication WO
`
`2005/094446, PCT publication WO 2006/116269, all of which are incorporated by reference
`
`herein. Antagonistic myostatin antibodies further include antibodies which bind to the
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`myostatin proprotein and prevent cleavage into the mature active form.
`
`[0040]
`
`As used herein, the term “antibody” refers to refers to intact antibodies including
`
`polyclonal antibodies (see, for example Antibodies: A Laboratory Manual, Harlow and Lane
`
`(eds), Cold Spring Harbor Press, (1988)), and monoclonal antibodies (see, for example, US.
`
`Patent Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993, and Monoclonal Antibodies: A
`
`New Dimension in Biological Analysis, Plenum Press, Kennett, McKearn and Bechtol (eds.)
`
`(1980)). As used herein, the term “antibody” also refers to a fragment of an antibody such as
`
`F(ab), F(ab’), F(ab’)2, Fv, Fc, and single chain antibodies, or combinations of these, which are
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`produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact
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`antibodies. The term “antibody” also refers to bispecific or bifunctional antibodies which are
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`an artificial hybrid antibody having two different heavy/light chain pairs and two different
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`binding sites. Bispeciflc antibodies can be produced by a variety of methods including fusion
`
`of hybridomas or linking of Fab’ fragments. (See Songsivilai et al, Clin. Exp. Immunol.
`
`79:315—321 (1990), Kostelny et al., J. Immunol.148:1547-1553 (1992)). As used herein the
`
`term “antibody” also refers to chimeric antibodies, that is, antibodies having a human
`
`constant antibody immunoglobulin domain is coupled to one or more non-human variable
`
`antibody immunoglobulin domain, or fragments thereof (see, for example, US. Patent No.
`
`5,595,898 and US. Patent N 0. 5,693,493). The term “antibodies” also refers to “humanized”
`
`antibodies (see, for example, US. Pat. No. 4,816,567 and WO 94/103 32), minibodies (WO
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`94/09817), single chain Fv-Fc fusions (Powers et al., JImmunol. Methods 251: 123-135
`
`(2001)), and antibodies produced by transgenic animals, in which a transgenic animal
`
`containing a proportion of the human antibody producing genes but deficient in the
`
`production of endogenous antibodies are capable of producing human antibodies (see, for
`
`example, Mendez et al., Nature Genetics 15:146-156 (1997), and US. Patent No. 6,300,129).
`
`The term “antibodies” also includes multimeric antibodies, or a higher order complex of
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`proteins such as heterodimeric antibodies. “Antibodies” also includes anti—idiotypic
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`antibodies.
`
`[0041]
`
`Myostatin antagonists further include soluble receptors which bind to myostatin
`
`and inhibit at least one activity. As used herein the term “soluble receptor” includes
`
`truncated versions or fragments of the myostatin receptor, modified or otherwise, capable of
`
`specifically binding to myostatin, and blocking or inhibiting myostatin signal transduction.
`
`These truncated versions of the myostatin receptor, for example, includes naturally occurring
`
`soluble domains, as well as variations due to proteolysis of the N— or C-termini. The soluble
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`domain includes all or part of the extracellular domain of the receptor, alone or attached to
`
`additional peptides or modifications. Myostatin binds activin receptors including activin type
`
`HB receptor (ActRIIB) and activin type TIA receptor (ActRIIA), as described in Lee et al,
`
`PNAS 98 (16), 9306-9311 (2001). Soluble receptor fusion proteins can also act as
`
`antagonists, for example soluble receptor PC as described in US patent application publication
`
`2004/0223966, and PCT publication WO 2006/012627, both of which are herein incorporated
`
`by reference.
`
`[0042]
`
`Myostatin antagonists further include soluble ligands which compete with
`
`myostatin for binding to myostatin receptors. As used herein the term “soluble ligand
`
`antagonist” refers to soluble peptides, polypeptides or peptidomimetics capable of binding the
`
`myostatin activin type IIB receptor (or ActRlIA) and blocking myostatin-receptor signal
`
`transduction by competing with myostatin. Soluble ligand antagonists include variants of
`
`myostatin, also referred to as “myostatin analogs” that maintain substantial homology to, but
`
`not the activity of the ligand, including truncations such an N- or C-terminal truncations,
`
`substitutions, deletions, and other alterations in the amino acid sequence, such as substituting
`
`a non-amino acid peptidomimetic for an amino acid residue. Soluble ligand antagonists, for
`
`example, may be capable of binding the receptor, but not allowing signal transduction. For
`
`the pu1poses of the present invention a protein is “substantially similar” to another protein if
`
`they are at least 80%, preferably at least about 90%, more preferably at least about 95%
`
`identical to each other in amino acid sequence.
`
`[0043]
`
`Myostatin antagonists further includes polynucleotide antagonists. These
`
`antagonists include antisense or sense oligonucleotides comprising a single—stranded
`
`polynucleotide sequence (either RNA or DNA) capable of binding to target mRNA (sense) or
`
`DNA (antisense) sequences. Antisense or sense oligonucleotides, according to the invention,
`
`comprise fragments of the targeted polynucleotide sequence encoding myostatin or its
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`receptor, transcription factors, or other polynucleotides involved in the expression of
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`myostatin or its receptor. Such a fragment generally comprises at least about 14 nucleotides,
`
`typically from about 14 to about 30 nucleotides. The ability to derive an antisense or a sense
`
`oligonucleotide, based upon a nucleic acid sequence encoding a given protein is described in,
`
`for example, Stein and Cohen, Cancer Res. 48:2659, 1988, and van der Krol et al.
`
`BioTechnz'ques 6:958, 1988. Binding of antisense or sense oligonucleotides to target nucleic
`
`acid sequences results in the formation of duplexes that block or inhibit protein expression by
`
`one of several means, including enhanced degradation of the mRNA by RNAse H, inhibition
`
`of splicing, premature termination of transcription or translation, or by other means. The
`
`antisense oligonucleotides thus may be used to block expression of proteins. Antisense or
`
`sense oligonucleotides further comprise oligonucleotides having modified sugar—
`
`phosphodiester backbones (or other sugar linkages, such as those described in WO 91/06629)
`
`and wherein such sugar linkages are resistant to endogenous nucleases. Such
`
`oligonucleotides with resistant sugar linkages are stable in vivo (i.e., capable of resisting
`
`enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide
`
`sequences. Other examples of sense or antisense oligonucleotides include those
`
`oligonucleotides which are covalently linked to organic moieties, such as those described in
`
`W0 90/ 10448, and other moieties that increases affinity of the oligonucleotide for a target
`
`nucleic acid sequence, such as poly- (L)—lysine. Further still, intercalating agents, such as
`
`ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense
`
`oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for
`
`the target nucleotide sequence.
`
`[0044]
`
`Antisense or sense oligonucleotides may be introduced into a cell containing the
`
`target nucleic acid by any gene transfer method, including, for example, lipofection, CaPO4-
`
`mediated DNA transfection, electroporation, or by using gene transfer vectors such as
`
`Epstein-Barr virus or adenovirus. Sense or antisense oligonucleotides also may be
`
`introduced into a cell containing the target nucleic acid by formation of a conjugate with a
`
`ligand—binding molecule, as described in WO 91/04753. Suitable ligand binding molecules
`
`include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other
`
`ligands that bind to cell surface receptors. Preferably, conjugation of the ligand-binding
`
`molecule does not substantially interfere with the ability of the ligand-binding molecule to
`
`bind to its corresponding molecule or receptor, or block entry of the sense or antisense
`
`oligonucleotide or its conjugated version into the cell. Alternatively, a sense or an antisense
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`oligonucleotide may be introduced into a cell containing the target nucleic acid by formation
`
`of an oligonucleotide-lipid complex, as described in W0 90/ 10448. The sense or antisense
`
`oligonucleotide—lipid complex is preferably dissociated within the cell by an endogenous
`
`lipase.
`
`[0045]
`
`Additional methods for preventing expression of myostatin or myostatin receptors
`
`is RNA interference (RNAi) produced by the introduction of specific small interfering RNA
`
`(s