USOO8716232B2

(12) United States Patent (10) Patent No.: US 8,716,232 B2

Fe hali-Bostwick et al. 45 Date of Patent: Ma 6 a 2014

(54) USE OF ENDOSTATIN PEPTIDES FOR THE FOREIGN PATENT DOCUMENTS TREATMENT OF FIBROSIS / EP 1 985 302 10 2008 (71) Applicants.Carol. _ At Fegliali._ BostWick,. Pittsburgh,- KRJP 10_2000_00061752007084459 A 4/20079/2001 PA (US), Yukie Yamaguchi, Kanagawa W0 W0 02/068457 9 QOOZ (JP) W0 W0 02068457 A2 * 9/2002 W0 WO 2008/014564 2/2008 (72) Inventors: Carol A. Feghali-Bostwick, Pittsburgh, PA (US); Yukie Yamaguchi, Kanagawa OTHER PUBLICATIONS JP ( ) Chufan et al., “Amidation ofBioactive Peptides: The Structure of the (73) Assignee; University of Pittsburgh_ 0f the Lyase Domain 0ftheAmidating Enzyme,” Structure 17:965-973 (Jul. Commonwealth System of Higher 15, 2009) Education, pinsburgh’ PA (Us) Hui et al., “Effect of Recombinant Adenovirus Vector Expressing Human Endostatin 0n Endothelial Cell Proliferation,” Journal of ( * ) Notice: Subject to any disclaimer, the term of this Regagggggive Tissue Engineering Reseamh 12(50): 9986' . . ec. , . gage? 115 Sixgeltdeg g; adJuSted under 35 International Search Report from parent PCT Application No. PCT/ ' ' ' ( ) y W USZOlO/53831, 8 pages, (mailed on Jan. 7, 2011). _ Isobe et al., “Inhibition of Endostatin/ XVIII Deteriorates (21) Appl' NO" 13/939’058 Left Ventricular Remodeling and Heart Failure in Rat Myocardial _ _ Infarction Model,” Circulation Journal 74: 109-119 (Jan. 2010). (22) Flled' JUL 10’ 2013 Kim and Seong, “Peptide Amidation: Production of Peptide Hor _ _ _ mones in vivo and in vitro,” Biotechnol Bioprocess Engineering (65) Prior Publication Data 61244451 (Aug 2001), Us NOV et al., “Endostatin Inhibits Endothelial and Tumor Cellular ' ’ Invasion by Blocking the Activation and Catalytic Activity of Matrix . . 2,” CancerResearch 60:5410-5413 (Oct. 1,2000). Related U's' Apphcatlon Data Morbidelli et al., “Angiosuppressive and Angiostimulatory Effects (62) Division of application No. 13/ 503,339, ?led as Exeited by Synthetic Partial Sequences of Endostatin,” Clinical Can application No. PCT/US2010/053831 on Oct. 22, Gel" Reseamh 91 5358-5369 (NOV 12, 2003) 2010’ HOW pat NO_ 8,507,441_ Richter et al., “Soluble Endostatin is a Novel Inhibitor of Epithelial Repair in Idiopathic Pulmonary Fibrosis,” Thorax 64:156-161 (60) Provisional application No. 61/261,280, ?led on Nov. (2009), 13, 2009, provisional application NO. 61/ 254, 143, Rodriguez et al., “ as a Potential Therapeutic Target,” ?led on Oct. 22, 2009. Drug News Perspect 21(4): 218-224 (2008). Szauter et al, “Lysyl Oxidase in Development, Aging and Pathologies (51) Int, Cl, 0fthe Skin,” Pathologie Biologie 53: 448-456 (2005). A61K 38/00 (200601) Written Opinion from parent PCT Application No. PCT/US2010/ A611) 1 7/00 (200601) 53831, 8 pages, (mailed on Jan. 7, l). I I A 61K 48/00 (200601) Yamaguchi et al., “Endostatin Inhibits VEGF-induced Endothelial C12N 15/00 (2006.01) Cell Mi g ration and Tumor Growth Inde P endentl Y of Zinc Bindin g, ” EMBO 18:4414-4423 (Aug. 16, 1999). C12N 5/00 (2006.01) 555%??? $0338 *Ci‘edbyexaminer C12N 5/10 (2006.01) C07K 5/00 (2006.01) Primary Examiner * Cecilia J Tsang C07K 7/00 (2006-01) Assistant Examiner * Zachary J Miknis C07K 16/00 (200601) (74) Attorney, Agent, or Firm * Klarquist Sparkman, LLP C07K 17/00 (2006.01) C07H 21/02 (2006.01) (52) us. Cl. (57) ABSTRACT

USPC ...... 514/18.6; 514/44 R; 435/3201; . . . . . 435/325; 435/348; 435/375; 530/324; 536/235 C-terminal 'endostatin' polypeptides are disclosed herein. 58 F M f Cl _? _ s h Polynucleotides encoding these polypeptide, host cells trans ( ) Nle 0 as“ canon earc formed With the polynucleotides, and methods of using these S one 1, _ ?l f 1 h h, polypeptides and polynucleotides are disclosed. Uses of these ee app Icanon e or comp ete seam lstory' polypeptide, polynucleotides and expression vectors include (56) References Cited the treatment of ?brosis in a subject. Thus, methods are pro vided for treating ?brosis, including ?brosis of the skin and/ or U.S. PATENT DOCUMENTS the lung.

6,764,995 B2 7/2004 O’Reilly et al. 2006/0193830 A1 8/2006 Hauswiith et al. 23 Claims, 17 Drawing Sheets US. Patent May 6, 2014 Sheet 1 0117 US 8,716,232 B2

FN

CGH oz?

GAPDH FIG. 13 NL 886 IPF m m), m RB TGF-{? V YE 5-3 {5-2 E-S {3-4 V YE 55-1 E-2 E—S E-4 V 1E 54 5-2 E-S E-4

FN US. Patent May 6, 2014 Sheet 2 0f 17 US 8,716,232 B2

FIG. 10 Morphea 880

V E~1 E-2 E-3 54 V rE E4 E-2 E-3 E-4

FN ......

Cono?FN Comm * '

FIG. 1E 5-4 v E E1 E-2 E-3 5-4 s 10 20 v FN COM (:11

GAPDH "

FIG. 1F

TGF-B V V E E—‘l E—2 E3 E4 a—SMA _ ......

GAPDH US. Patent M y 6, 2014 Sheet 3 0f 17 US 8,716,232 B2

FIG TGF

FIG. ZB rE (uq/ml)

FIG. 20 US. Patent May 6, 2014 Sheet 4 0f 17 US 8,716,232 B2

FIG. 3A TGF-B V TGF-{S rE (mg/mi) rE (Eug/mi) rE (mug/ml)

(units)Skinthickness US. Patent May 6, 2014 Sheet 5 0f 17 US 8,716,232 B2

FIG. 4B

Skinthickness(units)

V V {E E-1 E—Z E-S E-4 ma), US. Patent May 6, 2014 Sheet 6 0f 17 US 8,716,232 B2

FIG. 5A

TGF—B + 151 1 Q 10 20

1 5 10720 TGF-? + E-4 FIG. 5B

i k 2

I"? 6 W ‘ 'E 3 5 3 a) 4 C .x .9E a

.E 2 ‘ X m 1 0 VV151020151020 US. Patent May 6, 2014 Sheet 7 0f 17 US 8,716,232 B2

(units)Skinthickness US. Patent May 6, 2014 Sheet 8 0f 17 US 8,716,232 B2

FIG. YB

%controi cordformation 0.4 “ US. Patent May 6, 2014 Sheet 9 0f 17 US 8,716,232 B2

+ E-4 Bleo FIG.8B

V

88%qu ums

BleoE-4+

FIG.8A Bleo U S. Patent May 6, 2014 Sheet 10 0f 17 US 8,716,232 B2

FIG. 9 TG F B V E ('P).

**

E-4L E-1L (IP) (IP) TG F-B US. Patent M y 6, 2014 Sheet 11 0f 17 US 8,716,232 B2

FIG. 10A Bleo Bleo vs,vi 0...... 3..

Bleo + E4 Bleo + E-4L Bleo + E-4 Bleo + E-4L

FIG. 108

A 8 9: 6 A m1 4 V

V Bleo Bleo Bleo U S. Patent May 6, 2014 Sheet 12 0f 17 US 8,716,232 B2

FIG .10C Bleo

Trichrome

Ih

B m O + E 4 Bleo + E-4L

Bleo

H& E

Bleo + E4 Bleo + E-4L U S. Patent May 6, 2014 Sheet 13 0f 17 US 8,716,232 B2

FIG .11

Bleo

Trichrome

20x Blgg; E 41. (IP) Bleo + E 4L (IT) Bleo Trichrome

1. 0 Au X Bleo + E-4L (IP) Bleo + 5-41. (IT) U S. Patent May 6, 2014 Sheet 14 0f 17 US 8,716,232 B2

FIG .12

u...."k U S. Patent May 6, 2014 Sheet 15 0f 17 US 8,716,232 B2

FIG. 13

OX

a Tubulin

VE4V E4

Pro-MMP2 Active-MMP2 U S. Patent May 6, 2014 Sheet 16 0f 17 US 8,716,232 B2

FIG. 15

m 211 HRNALEVEL Q. 1%,

Egr 1 , “ -

Collagen |

a Tubulin - ._ _ US. Patent May 6, 2014 Sheet 17 0117 US 8,716,232 B2

FIG. 17A TGF-B V V E-‘I L E-4L

SkinThickness(units) US 8,716,232 B2 1 2 USE OF ENDOSTATIN PEPTIDES FOR THE 10443-10448) and show a noncovalently held dimer at high TREATMENT OF FIBROSIS concentration required for crystallization (Ding et al. (1998) Proc Natl Acad Sci USA, 95: 10443-10448). The presence of CROSS REFERENCE TO RELATED two disul?de bonds results in a highly folded structure. APPLICATION Endo statin binds one atom of Zinc per monomer via the three histidines in the N-terminus of the molecule (histidines 1, 3, This is a divisional of US. patent application Ser. No. and 11) and asparatic 76. The binding property of 13/503,339, ?led Apr. 20, 2012, which is the US. national endostatin is mediated by noncontiguous arginines clustered stage of PCT Application No. PCT/US2010/053831, ?led over the three dimensional globular surface of the molecule Oct. 22, 2010, which was published in English under PCT (Sasaki et al. (1999) Embo J, 18: 6240-6248). Article 21(2), which claims the bene?t of US. Provisional Excessive deposition of extra cellular matrix (ECM) com Application No. 61/261,280, ?led Nov. 13, 2009 and US. ponents such as ?bronectin (FN) and Provisional Application No. 61/254,143, ?led Oct. 22, 2009. (Collotl) by organ ?broblasts is de?ned as ?brosis. Organ The prior applications are incorporated by reference herein in ?brosis is the ?nal common pathway for many diseases that their entirety. result in end-stage organ failure. However, effective therapy STATEMENT OF GOVERNMENT SUPPORT for organ ?brosis is still unavailable (see, for example, Bjo raker et al., Am. J. Respir. Crit. Care. Med 2000; 157:199 This invention was made with government support under 203). Uncontrollable wound-healing responses, including grant AR050840 awarded by the National Institutes of 20 acute and chronic in?ammation, , activation of Health; the government has certain rights in the invention. resident cells, and ECM remodeling, are thought to be involved in the pathogenesis of ?brosis (Wynn, J Clin Invest FIELD 2007; 117:524-29; Kalluri et al., Curr Opin Nephrol Hyper tens 2000; 9:413-8). TGF-[3 is the prototype ?brotic cytokine This relates to the ?eld of ?brosis, speci?cally to the use of 25 that is increased in ?brotic organs and contributes to the C-terminal polypeptides of endostatin for the treatment of development of ?brosis by stimulating the synthesis of ECM ?brosis. molecules, activating ?broblasts to (x-smooth muscle actin (0t-SMA)-expressing myo?broblasts, and downregulating BACKGROUND matrix (MMPs) (see, for example, Bran 30 ton et al., Microbes Infect 1999; 1:1349-65). Despite high Endostatin, a 183 amino acid proteolytic cleavage frag expectations, a clinical trial of a monoclonal anti-TGF-[3 anti ment corresponding to the C-terminus of collagen 18, has body in patients with early SSc failed to show any ef?cacy anti-tumor activity with no toxic side effects (O’Reilly et al. (Varga et al., Nature Reviews Rheumatology 2009; 5:200-6). (1997) Cell, 88: 277-285.; Kisker et al. (2001) Cancer Res, Thus, a need remains for other treatments of ?brosis. 61:7669-7674; Dhanabal et al. (1999) Cancer Res, 59: 189 35 197; Yoon et al. (1999) Cancer Res, 59: 6251-6256; Folkman SUMMARY and Kalluri, (2003) Cancer Medicine, 6th edition, pp. 161 194. Hamilton: B. C. Decker Inc.). A number of anti-angio C-terminal endostatin polypeptides are disclosed herein genic activities have been reported for this , such as that have anti-?brotic activity. In some embodiments, these inhibition of endothelial cell proliferation, migration, and 40 polypeptides include, consist essentially of or consist of (1) at tube formation. This activity has been localized to the N-ter least 40 consecutive amino acids of the amino acid sequence minal region of endostatin. Endostatin also suppresses vas set forth as amino acids 133-180 of SEQ ID NO: 2, SEQ ID cular endothelial growth factor (VEGF)-induced vascular NO: 13 or SEQ ID NO: 4; (2) at least 40 consecutive amino permeability (Takahashi et al. (2003) Faseb J, 17: 896-898). acids of the amino acid sequence set forth as amino acids Endostatin inhibits endothelial cell migration by inhibiting 45 133-180 ofSEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 4, phosphorylation of focal adhesion kinase via binding to (x5 [31 with at most 5 amino acid substitutions, (3) the amino acid integrin (Wickstrom et al. (2002) Cancer Res, 62: 5580 sequence set forth as amino acids 133-180 of SEQ ID NO: 2, 5589). It also has been shown that cell surface glypicans are SEQ ID NO: 13 or SEQ ID NO: 4; or (4) the amino acid low-af?nity endostatin receptors (Karumanchi et al. (2001) sequence set forth as amino acids 133-180 of SEQ ID NO: 2, Mol Cell, 7: 811-822). Endostatin has been implicated in 50 SEQ ID NO: 13 or SEQ ID NO: 4 with at most 5 amino acid several signaling pathways, such as downregulation of c-myc substitutions. These polypeptides have anti-?brotic activity (Shichiri and Hirata (2001) Faseb J, 15: 1044-1053), cyclin and do not include amino acids 1-92 of SEQ ID NO: 2, SEQ D1 (Hanai et al. (2002) J Biol Chem, 277. 16464-16469) and ID NO: 13, or SEQ ID NO: 4, respectively. Polynucleotides RhoA activity (Wickstrom et al. (2003) J Biol Chem, 278: encoding these polypeptides, host cells transformed with the 37895-37901), blockage of VEGF signaling (Hajitou et al. 55 polynucleotides, and methods of using these polypeptides (2002) Faseb J, 16: 1802-1804; Kim et al. (2002) J Biol and polynucleotides are disclosed. In one example, the Chem, 277: 27872-27879), and inhibition of the wnt-signal polypeptide includes a modi?cation of the carboxy terminal ing pathway (Hanai et al. (2002) J Cell Biol, 158: 529-539). polypeptide to include an amide. Furthermore, endostatin has been shown to bind and inacti In some embodiments, methods are disclosed for inhibit vate metalloproteinases (Kim et al. (2000) Cancer Res, 60: 60 ing ?brosis in vivo or in vitro. In additional embodiments, 5410-5413; Nyberg et al. (2003) J Biol Chem, 278: 22404 methods are disclosed for the treatment of ?brosis in a sub 22411; Lee et al. (2002) FEBS Lett, 519: 147-152) and to ject. In some speci?c non-limiting examples, the subject has regulate a spectrum of genes which suppress angiogenesis scleroderrna or pulmonary ?brosis. (Abdollahi et al. (2004) Mol Cell, 13: 649-663). The foregoing and other features and advantages will The crystal structures of both murine and human endosta 65 become more apparent from the following detailed descrip tin have been resolved (Hohenester et al. (1998) Embo J, 17: tion of several embodiments, which proceeds with reference 1656-1664; Ding et al. (1998) Proc Natl Acad Sci USA, 95: to the accompanying ?gures. US 8,716,232 B2 3 4 BRIEF DESCRIPTION OF THE FIGURES of dermal thickness data shown in A. DMSO was used as a vehicle control. Experiments were conducted in duplicate, FIGS. 1A-1F. ECM production in recombinant endo statin and dermal thickness was measured in 6 ?elds from each and endostatin-derived peptide-treated ?broblasts in combi section. Mann-Whitney U test was used for statistical analy nation with TGF-[3 stimulation. A: FN and Col 1 (x1 expression sis. * P<0.02, ** P<0.01. in human normal lung ?broblasts (NL) treated with vehicle FIGS. 6A-6B. The effect of endostatin polypeptides in the (V), rE alone, or both with prior TGF-[3 stimulation. development of ?brosis in vivo in mouse skin. A: Mice were were detected Western blot. GAPDH was used as a loading injected intradermally with vehicle, 10 ng/ml TGF-[3 alone, or control for lysates. B: FN and Collal expression of endosta E-l, E-2, E-3, and E-4 (10 ug/ml) in combination with TGF-[3 tin polypeptide-treated lung ?broblasts following TGF-[3 (10 ng/ml). Skin was harvested after 1 week post-injection. stimulation in primary pulmonary ?broblasts from a healthy Sections were stained with H&E. Magni?cation, 20x. B: control, a patient with SSc, and a patient with IPF. C: Graphi cal summary of FN and Collal expression in lung ?broblasts Graphical summary of dermal thickness data shown in A. obtained using ?broblasts from 4 healthy controls (NL), 3 Data represent four independent experiments, each done in patients with SSc, and 3 patients with IPF. Intensity of bands duplicate. Mann-Whitney U test was used for statistical was normalized to that of GAPDH and expressed as a ratio to analysis. * P<0.04, ** P<0.01. Vehicle (V). Paired-t test was used for statistical analysis. * FIGS. 7A-7B. Capacity of endostatin polypeptide to P<0.04, ** P<0.01. D: Representative result of FN and inhibit tubular formation in MATRIGEL®. A, Representative Collal levels in human skin ?broblasts obtained from a images of MATRIGEL® cultures of HUVECs treated with patient with morphea and a patient with SSc. E: Representa 20 vehicle, rE (50 nM), or E4 (50 nM). An equivalent amount of tive result of FN and Collal expression in ?brotic ?broblasts DMSO was used as vehicle. Magni?cation 40x. B, Image obtained from a patients with IPF treated with V, 5 t1ng of quanti?cation of the cord formation shown in A. Data shown rE, or endostatin polypeptides alone (left). IPF ?broblasts summarize results of three independent experiments. * were treated with different concentrations (5, 10, and 20 P<0.05, one-way ANOVA followed by Bonferroni’s test. ug/ml) of E4. DMSO (V) was added in a volume equivalent to 25 FIGS. 8A-8B. The effect of endostatin E-4 on bleomycin that in the lane corresponding to 20 ug/ml of E4 (right). F: induced dermal ?brosis in vivo. A: Mice were injected sub (x-SMA levels in normal lung ?broblasts treated with cutaneously with l><) of mouse lung shown in FIG. 9