(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) WIPO| PCT
`
`\a
`
`(10) International Publication Number
`WO 2014/144903 Al
`
`(51) International Patent Classification:
`A61K 38/18 (2006.01)
`C0O7K 14/00 (2006.01)
`A6LK 38/00 (2006.01)
`.
`sas
`:
`(21) International Application Number: PCT/US2014/029502
`,
`(22) International Filing Date:
`
`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 Inc., One Amgen Center
`Drive, Thousand Oaks, CA 91320-1799 (US). HAQQ,
`Christopher, Michael; C/o Pinta Biotherapeutics,
`Inc.,
`3260 Bayshore Blvd., Brisbane, CA 94005
`(US).
`CIECHANOVER,Isaac; C/o Pinta Biotherapeutics, Inc.,
`3260 Bayshore Blvd., Brisbane, CA 94005 (US).
`
`(25) Filing Language:
`
`(26) Publication Language:
`(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).
`
`English (74) Agents: HUBL, Susan T. et al.; Fenwick & West LLP,
`.
`801 California Street, Mountain View, California 94041
`English
`(US).
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW,BY,
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EF, 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, NL 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 next page]
`
`(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.
`
`(54) Title: MYOSTATIN ANTAGONISM IN HUMAN SUBJECTS
`
`Percent Change from Baseline Total Lean Body Mass
`
`:
`~
`
`Ecessananaunnsnns;
`
`iij|
`4j
`ri
`
`a
`
`EOS
`
`FUP
`
`Visit
`-+« 3.0 mg/kg SC AMG 745
`-- Place
`
`Fig.13
`
`Amgen Exhibit 2027
`Apotex Inc. et al. v. Amgen Inc. et al., IPR2016-01542
`Page |
`
`
`
`Wo2014/144903A1|IITTIMNTNNIINIMMTUTNNITTIANACMTATAYMATAT
`
`Amgen Exhibit 2027
`Apotex Inc. et al. v. Amgen Inc. et al., IPR2016-01542
`Page 1
`
`

`

`WO 2014/144903 A2 IINMIININIUAMANTTTAATAA
`
`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, RO,RS, SE, SL SK,
`SM, TR), OAPI (BE, 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
`
`Page 2
`
`

`

`WO 2014/144903
`
`PCT/US2014/029502
`
`MYOSTATIN ANTAGONISM IN HUMAN SUBJECTS
`
`CROSS REFERENCE TO RELATED APPLICATIONS
`
`[0001
`
`This application claims the benefit of U.S. Provisional Application No.
`
`61/799,928, filed MARCH 15, 2013, which is hereby incorporatedin its entirety by
`
`reference.
`
`STATEMENT REGARDING FEDERALLY SPONSORED RESARCH OR
`DEVELOPMENT
`
`[0002
`
`Not applicable.
`
`SEQUENCELISTING
`
`[0003]
`
`Theinstant application contains a Sequence Listing which has been submitted via
`
`EFS-Webandis hereby incorporated by referencein its entirety. Said ASCII copy, created on
`
`March 13, 2014, is named 26324PCT_sequencelisting.txt, and is 200,000 bytesin 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 cancerpatients.
`
`BACKGROUND
`
`[0005]
`
`The transforming growth factor (TGF) 8 superfamily of growth factors consists of
`
`a large numberof 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; Thomaset 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/G, cell cycle arrest.
`
`In addition, myostatin negatively
`
`regulates myoblast differentiation through decreased expression of MyoD (Langleyetal,
`
`2002).
`
`[0006]
`
`Observations from mice andcattle with loss-of-function mutations in the
`
`myostatin gene (Roth and Walsh, 2004; Grobetet al, 1998; Szabo et al, 1998; Grobetet al,
`
`1997; Kambaduret al, 1997; McPherron and Lee, 1997; McPherronet al, 1997), as well as a
`
`Page 3
`
`Page 3
`
`

`

`WO 2014/144903
`
`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 (Grobetet 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/AIDS,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; Roubenoffet al, 1997). Skeletal muscle wasting results in reduced
`
`muscle strength, physical and psychological disability, and impaired quality oflife
`
`(Muscaritoli et al, 2006; Roubenoff et al, 1997), Current treatment options used for muscle
`
`wasting in settings ofillness 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 etal,
`
`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 ADTis
`
`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]
`
`ADThasa 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
`
`Page 4
`
`Page 4
`
`

`

`WO 2014/144903
`
`PCT/US2014/029502
`
`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 mayalso contribute to ADT
`
`decreased insulin sensitivity and greater risk for diabetes associated with ADT. (Smith etal
`
`2006 JCEM;Keating et al 2006 JCO; Braga-Basaria et al 2006).
`
`[0010]
`
`AMG745is a novel anti-myostatin peptibody. Structurally, it is a fusion protein
`
`with a humanFc 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-Sin these models
`
`have included increased body weight gain, increased or improved maintenanceof, 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 offat, 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 andfiled on December
`
`19, 2003 and PCT/US2006/046546, published as WO2007/067616 and filed on December6,
`
`2006 and the related national phase patent applications.
`
`SUMMARY
`
`[0012]
`
`Described herein are methodsof 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
`
`Page 5
`
`

`

`WO 2014/144903
`
`PCT/US2014/029502
`
`amino acid sequence of 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);
`
`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 methodsof 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 acceptablecarrierto 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 NO:311 (LADHGQCIRWPWMCPPEGWE). In some embodiments,
`
`the
`
`myostatin antagonist consists of a peptibody comprising a polypeptide consisting of the
`
`amino acid sequenceset 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 sequencethat is at
`
`least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the
`
`amino acid sequenceset 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.
`
`Page 6
`
`Page 6
`
`

`

`WO 2014/144903
`
`PCT/US2014/029502
`
`[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 of 0.3 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]
`
`Figure 1 shows myostatin activity as mcasured by expressed luciferase activity (y-
`
`axis) vs. concentration (x-axis) for the TN8-19 peptide QGHCTRWPWMCPPY(SEQ ID
`
`NO: 32) and the TN8-19 peptibody (pb) to determine the ICso for each using the C2C12
`
`pMARE luciferase assay described in the Examples below. The peptibody has a lower ICs
`
`value compared with the peptide.
`
`[0021]
`
`Figure 2 is a graph showingthe increase in total body weight for CD1 nu/nu mice
`
`treated with increasing dosagesof the 1x mTN8-19-21 peptibody over a fourteen day period
`
`compared with mice treated with a huFc control, as described in Example 8.
`
`[0022]
`
`Figure 3A showsthe increase in the mass of the gastrocnemius muscle mass at
`
`necropsy of the mice treated in Figure 2 (Example 8). Figure 3B showsthe 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 showsthe increase in lean body mass as for CD1 nu/nu micetreated with
`
`biweekly injections of increasing dosages of 1x mTN&-19-32 peptibody as determined by
`
`NMRon day 0 and day 13 of the experiment described in Example 8.
`
`Page 7
`
`Page 7
`
`

`

`WO 2014/144903
`
`PCT/US2014/029502
`
`[0024]
`
`Figure 5A showsthe increase in body weight for CD1 nu/nu mice treated with
`
`biweekly injections of 1x mTN8-19-7 compared with 2x mTN8-19-7 and the control animal
`
`for 35 days as described in Example 8. Figure 5B showsthe increase in lean carcass weight
`
`at necropsy for the 1x and 2x versions at | mg/kg and 3 mg/kg compared with the animals
`
`receiving the vehicle (huFc) (controls).
`
`[0025]
`
`Figure 6A showsthe increase in lean muscle mass vs. body weight for aged mdx
`
`mice treated with either affinity matured 1x mTN8-19-33 peptibody or huFc vehicle at 10
`
`mg/kg subcutaneously every other day for three months. Figure 6B showsthe changein fat
`
`mass compared to body weight as determined by NMRfor the same mice after 3 months of
`
`treatment.
`
`[0026]
`
`Figure 7 showsthe change in body mass overtime in grams for collagen-induced
`
`arthritis (CIA) animals treated with the peptibody 2x mTN&-19-21/muFc or muFc vehicle, as
`
`well as normal non-CIA animals.
`
`[0027]
`
`Figure 8 showsthe relative body weight change over time in streptozotocin
`
`(STZ)-induced diabetic mice treated with the peptibody 2x mTN8-19-21/muFc or the muFe
`
`vehicle control.
`
`[0028]
`
`Figure 9 showscreatine clearance rate in streptozotocin (STZ)-induced diabetic
`
`mice and age-matched normal miceafter treatment with peptibody 2x mTN8-19-21/muFc or
`
`the muFcvehicle.
`
`[0029]
`
`Figure 10A shows urine albumin excretion in streptozotocin (STZ)-induced
`
`diabetic mice and age-matched normal miceafter treatment with peptibody 2x mTN8-19-
`
`21/muFc or the muFc vehicle. Figure 10B shows the 24 hour urine volumein streptozotocin
`
`(STZ)-induced diabetic mice and age-matched normal mice after treatment with peptibody 2x
`
`mTN8-19-21/muFc or the muFc vehicle.
`
`[0030}
`
`Figure 11 shows body weight change over time for 4 groups of C57BI/6 mice; 2
`
`groupspretreated for 1 week with peptibody 2x mTN8-19-21/muFc, then treated with 5-
`
`fluoruracil (5-Fu) or vehicle (PBS); and 2 groups pretreated for 2 weeks with 2x mTN8-19-
`
`21/muFc, 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 mTN8-19-
`
`21/muFc, times of administration of 1 week pretreatment with 2x mTN8-19-21/muFc, and
`
`times of administration of 5-Fu.
`
`Page 8
`
`Page 8
`
`

`

`WO 2014/144903
`
`PCT/US2014/029502
`
`[0031]
`
`Figure 12 showsthe survival rate percentages the animals described in Figure 11
`
`above, showing normal mice nottreated, animals treated with 5-Fu only, animals pretreated
`
`with 2x mTN8-19-21/muFc for 1 week and then treated with 5-Fu, and animals pretreated
`
`with 2x mTN8-19-21/muFc for 2 weeks and then treated with 5-Fu.
`
`[0032]
`
`Figure 13 showsthe percent change from baseline of total lean body mass in
`
`human subjects treated with AMG 745 or placebo. The placebo groupsare on the left in each
`
`of EOS and FUP; the AMG 745 groupsare on the right in each of EOS and FUP.
`
`DETAILED DESCRIPTION
`
`[0033]
`
`The present invention provides methodsof treating cachexia in prostate cancer
`
`patients receiving androgen therapy by administration of a myostatin antagonist comprising
`
`the myostatin binding peptide SEQ ID NO:31], e.g., a peptibody consisting of SEQ ID
`
`NO:635.
`
`Myostatin
`
`[0034]
`
`Myostatin, a growth factor also known as GDF-8, is a memberof the TGF-B8
`
`family. Myostatin knownto be a negative regulator of skeletal muscle tissue. Myostatin is
`
`synthesized as an inactive preproprotein which is activated by proteolyic cleavage (Zimmers
`
`et al., supra (2002)). The precursor protcin is cleaved to produce an NH)-terminal inactive
`
`prodomain and an approximately 109 amino acid COOH-terminalprotein in the form of a
`
`homodimerof about 25 kDa, which is the mature, active form (Zimmersetal, supra (2002)).
`
`It is now believed that the mature dimercirculates in the blood as an inactive latent complex
`
`boundto the propeptide (Zimmerset al, supra (2002)).
`
`[0035]
`
`Asused herein the term “full-length myostatin” refers to the full-length human
`
`preproprotein sequence described in McPherronet al. PNAS USA 94, 12457 (1997), as well
`
`as related full-length polypeptides includingallelic variants and interspecies homologs
`
`(McPherronet al. supra (1997)). As used herein, the term “prodomain”or“propeptide”
`
`refers to the inactive NH>-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
`
`Page 9
`
`Page 9
`
`

`

`WO 2014/144903
`
`PCT/US2014/029502
`
`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 howit is prepared.
`
`Myostatin Antagonists
`
`[0036]
`
`The methodsof 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
`
`employing one or more inhibitory agents which interfere with the binding of myostatintoits
`
`receptor, and/or blocks signal transduction resulting from the binding of myostatinto its
`
`receptor. Antagonists include agents which bind to myostatin itself, or agents which bind to a
`
`myostatin receptor.
`
`[0038]
`
`Other examples of myostatin antagonists include but are not limitedto follistatin,
`
`the myostatin prodomain, growth and differentiation factor 11 (GDF-11) prodomain,
`
`prodomain fusion proteins, antagonistic antibodies that bind to myostatin, antagonistic
`
`antibodies or antibody fragments that bind to the activin type IIB receptor, soluble activin
`
`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 Amthoret 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., Mol.
`
`Endo. 17 (6): 1144-1154 (2003). Myostatin antagonists include the propeptide region of
`
`myostatin and related GDF proteins including GDF-11, as described in PCT publication WO
`
`02/09641, which is herein incorporated by reference. Myostatin antagonists further include
`
`modified and stabilized propeptides including Fc fusions of the prodomain as described, for
`
`8
`
`Page 10
`
`Page 10
`
`

`

`WO 2014/144903
`
`PCT/US2014/029502
`
`example, in Bogdanovischet al, FASEB J 19, 543-549 (2005). Additional myostatin
`
`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
`
`dimeric form. Such antibodies are described, for example, in US patent application US
`
`2004/0142383, and USpatent application 2003/1038422, 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 bindto the
`
`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, Harlowand Lane
`
`(eds), Cold Spring HarborPress, (1988)), and monoclonal antibodies (see, for example, U.S.
`
`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’), Fv, Fc, and single chain antibodies, or combinations of these, which are
`
`produced by recombinant DNAtechniques or by enzymatic or chemical cleavage ofintact
`
`antibodies. The term “antibody”also refers to bispecific or bifunctional antibodies which are
`
`an artificial hybrid antibody having two different heavy/light chain pairs and two different
`
`binding sites. Bispecific antibodies can be produced by a variety of methods including fusion
`
`of hybridomasorlinking of Fab’ fragments. (See Songsivilai et al, Clin. Exp. Immunol.
`
`79:3 15-321 (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, U.S. Patent No.
`
`5,595,898 and U.S. Patent No. 5,693,493). The term “antibodies”also refers to “humanized”
`
`antibodies (see, for example, U.S. Pat. No. 4,816,567 and WO 94/10332), minibodies (WO
`
`94/09817), single chain Fv-Fc fusions (Powerset al., Jdmmunol. Methods 251:123-135
`
`(2001)), and antibodies produced by transgenic animals, in which a transgenic animal
`
`containing a proportion of the humanantibody producing genesbut deficient in the
`
`production of endogenous antibodies are capable of producing humanantibodies(see, for
`
`example, Mendezetal., Nature Genetics 15:146-156 (1997), and U.S. Patent No. 6,300,129).
`
`The term “antibodies” also includes multimeric antibodies, or a higher order complex of
`
`Page 11
`
`Page 11
`
`

`

`WO 2014/144903
`
`PCT/US2014/029502
`
`proteins such as heterodimeric antibodies. “Antibodies”also includes anti-idiotypic
`
`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
`
`domain includesall or part of the extracellular domain of the receptor, alone or attached to
`
`additional peptides or modifications. Myostatin binds activin receptors including activin type
`
`IIB receptor (ActRIUB)and activin type ITA receptor (ActRITA), as described in Leeet al,
`
`PNAS 98 (16), 9306-9311 (2001). Soluble receptor fusion proteins can also act as
`
`antagonists, for example soluble receptor Fc 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 IB receptor (or ActRITA) 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
`
`notthe 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 purposes of the present inventiona proteinis “substantially similar” to anotherproteinif
`
`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
`
`10
`
`Page 12
`
`Page 12
`
`

`

`WO 2014/144903
`
`PCT/US2014/029502
`
`receptor, transcription factors, or other polynucleotides involved in the expression of
`
`myostatin or its receptor. Such a fragment generally comprisesat 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 describedin,
`
`for example, Stein and Cohen, Cancer Res. 48:2659, 1988, and van der Krolet al.
`
`BioTechniques 6:958, 1988. Binding of antisense or sense oligonucleotides to target nucleic
`
`acid sequencesresults 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 sugarlinkages are resistant to endogenous nucleases. Such
`
`oligonucleotides with resistant sugar linkages are stable in vivo(i.e., capable ofresisting
`
`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
`
`WO 90/10448, and other moieties that increases affinity of the oligonucleotide for a target
`
`nucleic acid sequence, such as poly- (L)-lysine. Furtherstill, 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, CaPO,.-
`
`mediated DNAtransfection, 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
`
`1]
`
`Page 13
`
`Page 13
`
`

`

`WO 2014/144903
`
`PCT/US2014/029502
`
`oligonucleotide may be introducedinto a cell containing the target nucleic acid by formation
`
`of an oligonucleotide-lipid complex, as described in WO 90/10448. The senseor 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 RNAinterference (RNAj) producedby the introduction of specific small interfermg RNA
`
`(siRNA), as described, for example in Bosheret al., Nature Cell Biol 2, E31-E36 (2000).
`
`[0046]
`
`Myostatin antagonists further include small molecule antagonists which bind to
`
`either myostatin or its receptor. Small molecules are selected by screening for binding to
`
`myostatin or its receptor followed by specific and non-specific