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`
`US007138370B2
`
`(IO) Patent No.: US 7,138,370 B2
`
`c12) United States Patent
`(45)Date of Patent:
`
`Nov. 21, 2006
`
`
`Oliner et al.
`
`(54)SPECIFIC BINDING AGENTS OF HUMAN
`
`ANGIOPOIETIN-2
`
`
`
`
`
`5,643,755 A 7/1997 Davis et al.
`
`
`
`
`
`5,650,490 A 7/1997 Davis et al.
`
`
`5,814,464 A 9/1998 Davis et al.
`(75)Inventors: Jonathan Daniel Oliner, Newbury
`
`
`
`5,879,672 A 3/1999 Davis et al.
`
`Park, CA (US); Hosung Min, Newbury
`
`
`
`5,955,291 A 9/1999 Alitalo et al.
`Park, CA (US)
`
`
`5,972,338 A 10/1999 Godowski et al.
`
`
`6,030,831 A 2/2000 Godowski et al.
`
`
`6,166,185 A 12/2000 Davis et al.
`
`
`6,291,646 Bl 9/2001 Sledziewski et al.
`
`
`
`6,323,323 Bl 11/2001 Sledziewski et al.
`
`6,455,035 Bl 9/2002 Suri et al.
`
`2003/0236193 Al 12/2003 Oliner et al.
`
`(73)Assignee: Amgen Inc., Thousand Oaks, CA (US)
`
`( *) Notice: Subject to any disclaimer, the term of this
`
`
`
`
`
`patent is extended or adjusted under 35
`
`U.S.C. 154(b) by 619 days.
`
`(21)Appl. No.: 10/269,695
`
`
`
`(22)Filed:Oct. 10, 2002
`
`FOREIGN PATENT DOCUMENTS
`
`(65)
`
`
`
`Prior Publication Data
`
`
`
`US 2003/0229023 Al Dec. 11, 2003
`
`
`
`
`
`Related U.S. Application Data
`
`WO
`WO
`WO
`WO
`
`
`WO 95/13387 5/1995
`
`WO 95/21866 8/1995
`
`WO 98/05779 2/1998
`
`WO 199933865 7 /1999
`
`(60)Provisional application No. 60/414,155, filed on Sep.
`
`
`
`
`
`27, 2002, provisional application No. 60/328,624,
`filed on Oct. 11, 2001.
`
`(Continued)
`
`OTHER PUBLICATIONS
`
`(51)Int. Cl.
`A61K 38/08 (2006.01)
`A61K 38/10(2006.01)
`A61K 38/16 (2006.01)
`
`
`
`
`(52)U.S. Cl. .............................. 514/2; 514/12; 514/13;
`(Continued)
`
`514/15; 514/16; 514/17; 530/324; 530/325;
`
`Primary Examiner-Anish Gupta
`
`
`
`
`
`530/326; 530/327; 530/328; 530/329; 530/330
`
`
`
`
`
`(58)Field of Classification Search ..................... None
`Bernstein
`
`
`
`See application file for complete search history.
`
`Putaporntip et al. Diversity in the Thrombospondin-Related Adhe­
`
`
`
`
`
`sive Protein Gene (Trap_of Plasmodium Vivax, Gene 268, pp.
`97-104 (2001).*
`
`(74)Attorney, Agent, or Firm-Raz E. Fleshner; Scott N.
`
`(56)
`
`
`
`References Cited
`
`(57)
`
`ABSTRACT
`
`U.S. PATENT DOCUMENTS
`
`et al.
`
`5,116,964 A 5/1992 Capon
`
`
`
`et al.
`5,428,130 A 6/1995 Capon
`5,447,860 A 9/1995 Ziegler
`
`
`
`
`5,455,165 A 10/1995 Capon et al.
`5,514,582 A 5/1996 Capon
`
`
`et al.
`
`
`et al.
`
`5,521,073 A 5/1996 Davis
`
`Disclosed are peptides that bind to Ang-2. Also disclosed are
`
`
`
`
`
`
`
`
`peptibodies comprising the peptides, methods of making
`
`
`
`such peptides and peptibodies, and methods of treatment
`
`using such peptides and peptibodies.
`
`
`
`25 Claims, 19 Drawing Sheets
`
`700
`
`600
`
`500
`
`M�
`.§. 400
`
`300
`
`200
`
`100
`
`p=<0.0001
`
`10 12 14 16
`
`DAYS
`
`
`-+- 2: TN8-Con4 (C) (1 mg)
`
`
`
`-B-- 4: TN8·Con4 (C) (40 ug)
`
`
`
`---1: Vehicie Control
`
`
`
`--.- 3: TN8-Con4 (C) (200 ug)
`
`
`
`--B-- 5: TN8·Con4 (C) (8 ug)
`
`KASHIV EXHIBIT 1034
`IPR2019-00791
`
`Page 1
`
`

`

`US 7,138,370 B2
`Page 2
`
`FOREIGN PATENT DOCUMENTS
`
`WO99/43801
`WO
`WOOOO6195
`WO
`WO 200023O82
`WO
`WOOO,57901
`WO
`WOOOf 77037
`WO
`WOOO75323
`WO
`WO O1/62891
`WO
`WOO 1/71042
`WO
`WO WO 2003O3O833
`WO WO 2003057134
`
`9, 1999
`2, 2000
`4/2000
`10, 2000
`12/2000
`12/2000
`8, 2001
`9, 2001
`4/2003
`T 2003
`
`OTHER PUBLICATIONS
`Carmeliet, et al. "Angiogenesis in cancer and other diseases' Nature
`407(6801):249-57 (2000).
`Siemeister, et al. “Two independent mechanisms essential for tumor
`angiogensis: inhibition of human melanoma xenograft growth by
`interfering with either the vascular endothelial growth factor recep
`tor pathway or the tie-2 pathway” Cancer Res. 59(13): 3185-3191
`(Jul. 1, 1999).
`Lin Pengnian et al. "Inhibition of tumor angiogensis using a soluble
`receptor establishes a role for Tie2 in pathologic vascular growth”
`J. Clin. Invest. 100(8): 2072-2078 (1997).
`
`Chao et al. (2000), Genomic Sequence for Arabidopsis thaliana
`BAC F1K23 from Chromosome I. Acc. No. Q9SHQe, EMBL/
`GenBank/DDBJ database, EMBL: AC007508. AAF 24.543,
`Accessed Dec. 10, 2003.
`Syed et al. (2001), “The Effects of Angiopoietin-1 and -2 on Tumor
`Growth and Angiogenesis in Human Colon Cancer, Cancer
`Research 61: 1255-1259.
`Coxon et al. (2000), "Inhibition of interleukin-1 but not tumor
`necrosis factor Suppresses neovascularization in rat models of
`corneal angiogenesis and adjuvant arthritis.” Arthritis Rheum.
`46:2604-2612.
`Feige et al. (2000), “Anti-interleukin-1 and anti-tumor necrosis
`factor-O. Synergistically inhibit adjuvant arthritis in Lewis rats,” Cell
`Mol. Life Sci, 57:1457-1470.
`Peacock et al. (1992), "Angiogenesis inhibition Suppresses collagen
`arthritis,” J. Exp. Med. 175:1135-1138.
`Peacock et al. (1995), “A novel angiogenesis inhibitor suppresses
`rat adjuvant arthritis,” Cell Immunol. 160:178-184.
`Walsh et al. (2001), "Angiogenesis in the pathogenesis of inflam
`matory joint and lung diseases.” Arthritis Res. 3:147-153.
`Connell et al. (2001), Ashley Publications Ltd. ISSN 1354-3776, pp.
`1171-1203.
`* cited by examiner
`
`Page 2
`
`

`

`U.S. Patent
`
`Nov. 21, 2006
`
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`
`US 7,138,370 B2
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`
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`
`

`

`U.S. Patent
`U.S. Patent
`
`Nov. 21, 2006
`
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`US 7,138,370 B2
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`

`US 7,138,370 B2
`
`1.
`SPECIFIC BINDING AGENTS OF HUMAN
`ANGOPOETIN-2
`
`This application claims benefit to U.S. Provisional Appli
`cation Ser. No. 60/414,155, filed Sep. 27, 2002, and U.S.
`Provisional Application Ser. No. 60/328,624 filed Oct. 11,
`2001, which are incorporated herein by reference in their
`entirety.
`
`FIELD OF INVENTION
`
`The present invention relates to specific binding agents
`that recognize and bind to angiopoietin-2 (Ang-2). More
`specifically, the invention relates to the production, diag
`nostic use, and therapeutic use of the specific binding agents
`and fragments thereof, which specifically bind Ang-2.
`
`BACKGROUND OF THE INVENTION
`
`10
`
`15
`
`2
`(Davis, S., et al., 1996, supra; Maisonpierre, P. C., et al.,
`1997, supra; Kim, I., J. H. Kim, et al., Oncogene 19(39):
`4549–4552 (2000); Teichert-Kuliszewska, K., P. C. Maison
`pierre, et al., Cardiovascular Research 49(3): 659–70
`(2001)).
`The phenotypes of mouse Tie-2 and Ang-1 knockouts are
`similar and Suggest that Ang-1-stimulated Tie-2 phosphory
`lation mediates remodeling and Stabilization of developing
`vessels in utero through maintenance of endothelial cell
`Support cell adhesion (Dumont, D. J., et al., Genes &
`Development, 8:1897–1909 1994: Sato, T. N., et al.,
`Nature, 376:70–74 (1995: Suri, C., et al., 1996), supra).
`The role of Ang-1 in vessel stabilization is thought to be
`conserved in the adult, where it is expressed widely and
`constitutively (Hanahan, D., Science, 277:48–50 1997:
`Zagzag, D., et al., Experimental Neurology, 159:391-400
`1999). In contrast, Ang-2 expression is primarily limited to
`sites of vascular remodeling, where it is thought to block
`Ang-1 function, thereby inducing a state of vascular plas
`ticity conducive to angiogenesis (Hanahan, D., 1997.
`supra: Holash, J., et al., Science, 284: 1994–1998 1999:
`Maisonpierre, P. C., et al., 1997, supra).
`Numerous published studies have purportedly demon
`strated vessel-selective Ang-2 expression in disease states
`associated with angiogenesis. These pathological conditions
`include, for example, psoriasis, macular degeneration, and
`cancer (Bunone, G., et al., American Journal of Pathology,
`155:1967–1976 (1999: Etoh, T., et al., Cancer Research,
`61:2145–2153 2001; Hangai, M., et al., Investigative Oph
`thalmology & Visual Science, 42:1617–1625 2001;
`Holash, J., et al., 1999 Supra; Kuroda, K., et al., Journal of
`Investigative Dermatology, 116:713–720 2001: Otani, A.,
`et al., Investigative Ophthalmology & Visual Science,
`40:1912–1920 1999: Stratmann, A., et al., American Jour
`nal of Pathology, 153:1459–1466 1998: Tanaka, S., et al.,
`J Clin Invest, 103:34–345 (1999: Yoshida, Y., et al., Inter
`national Journal of Oncology, 15:1221–1225 1999: Yuan,
`K., et al., Journal of Periodontal Research, 35:165-171
`2000; Zagzag, D., et al., 1999 Supra). Most of these
`studies have focused on cancer, in which many tumor types
`appear to display vascular Ang-2 expression. In contrast
`with its expression in pathological angiogenesis, Ang-2
`expression in normal tissues is extremely limited (Maison
`pierre, P. C., et al., 1997, supra; Mezquita, J., et al.,
`Biochemical and Biophysical Research Communications,
`260:492-498 (1999). In the normal adult, the three main
`sites of angiogenesis are the ovary, placenta, and uterus;
`these are the primary tissues in normal (i.e., non-cancerous)
`tissues in which Ang-2 mRNA has been detected.
`Certain functional studies suggest that Ang-2 may be
`involved in tumor angiogenesis. Ahmad et al. (Cancer Res.,
`61: 1255–1259 (2001) describe Ang-2 over-expression and
`show that it is purportedly associated with an increase in
`tumor growth in a mouse Xenograft model. See also Etoh et
`al., Supra, and Tanaka et al., Supra, wherein data is presented
`purportedly associating Ang-2 over expression with tumor
`hypervascularity. However, in contrast, Yu et al. (Am. J.
`Path., 158:563–570 2001) report data to show that over
`expression of Ang-2 in Lewis lung carcinoma and TA3
`mammary carcinoma cells purportedly prolonged the Sur
`vival of mice injected with the corresponding transfectants.
`In the past few years, various publications have Suggested
`Ang-1. Ang-2 and/or Tie-2 as a possible target for anticancer
`therapy. For example, U.S. Pat. Nos. 6,166,185, 5,650,490,
`and 5,814,464 each disclose the concept of anti-Tie-2 ligand
`antibodies and receptor bodies. Lin et al. (Proc. Natl. Acad.
`Sci USA, 95:8829–8834 (1998) injected an adenovirus
`
`Angiogenesis, the formation of new blood vessels from
`existing ones, is essential to many physiological and patho
`logical processes. Normally, angiogenesis is tightly regu
`lated by pro- and anti-angiogenic factors, but in the case of
`diseases such as cancer, ocular neovascular diseases, arthri
`tis, and psoriasis, the process can go awry. Folkman, J., Nat.
`Med., 1:27–31 (1995).
`There are a number of diseases known to be associated
`with deregulated or undesired angiogenesis. Such diseases
`include, but are not limited to, ocular neovascularisation,
`Such as retinopathies (including diabetic retinopathy), age
`related macular degeneration, psoriasis, hemangioblastoma,
`hemangioma, arteriosclerosis, inflammatory disease. Such as
`a rheumatoid or rheumatic inflammatory disease, especially
`arthritis (including rheumatoid arthritis), or other chronic
`inflammatory disorders, such as chronic asthma, arterial or
`post-transplantational atherosclerosis, endometriosis, and
`neoplastic diseases, for example so-called solid tumors and
`liquid (or hematopoietic) tumors (such as leukemias and
`lymphomas). Other diseases associated with undesired
`angiogenesis will be apparent to those skilled in the art.
`Although many signal transduction systems have been
`implicated in the regulation of angiogenesis, one of the
`best-characterized and most endothelial cell-selective sys
`tems involves the Tie-2 receptor tyrosine kinase (referred to
`as “Tie-2 or “Tie-2R (also referred to as “ORK'); murine
`Tie-2 is also referred to as “tek’) and its ligands, the
`angiopoietins (Gale, N. W. and Yancopoulos, G. D., Genes
`Dev. 13:1055–1066 (1999). There are 4 known angiopoi
`etins; angiopoietin-1 ("Ang-1') through angiopoietin-4
`('Ang-4). These angiopoietins are also referred to as “Tie-2
`ligands'. (Davis, S., et al., Cell, 87:1161–1169 1996:
`Grosios, K., et al., Cytogenet Cell Genet, 84:118-120
`1999; Holash, J., et al., Investigative Ophthalmology &
`Visual Science, 42:1617–1625 1999: Koblizek, T.I., et al.,
`Current Biology, 8:529–532 (1998: Lin, P., et al., Proc Natl
`AcadSci USA,95:8829–8834 (1998: Maisonpierre, P. C., et
`al., Science, 277:55–60 1997; Papapetropoulos, A., et al.,
`Lab Invest, 79:213–223 1999: Sato, T. N., et al., Nature,
`375:70–74 (1998: Shyu, K. G., et al., Circulation,
`98:2081-2087 (1998: Suri, C., et al., Cell, 87:1171–1180
`1996; Suri, C., et al., Science, 282:468–471 (1998: Valen
`Zuela, D. M., et al., Proceedings of the National Academy of
`Sciences of the USA, 96:1904–1909 1999: Witzenbichler,
`B., et al., J Biol Chem, 273:18514–18521 1998). Whereas
`Ang-1 binding to Tie-2 stimulates receptor phosphorylation
`in cultured endothelial cells, Ang-2 has been observed to
`both agonize and antagonize Tie-2 receptor phosphorylation
`
`25
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`3
`expressing soluble Tie-2 into mice; the soluble Tie-2 pur
`portedly decreased the number and size of the tumors
`developed by the mice. In a related study, Lin etal (J. Clin.
`Invest., 100:2072–2078 (1997) injected a soluble form of
`Tie-2 into rats; this compound purportedly reduced tumor
`size in the rats. Siemeister et al. (Cancer Res., 59:3185-3189
`1999) generated human melanoma cell lines expressing the
`extracellular domain of Tie-2, injected these cell lines into
`nude mice, and concluded that soluble Tie-2 purportedly
`resulted in a “significant inhibition of tumor growth and
`tumor angiogenesis. In view of this information, and given
`that both Ang-1 and Ang-2 bind to Tie-2, it is not clear from
`these studies whether Ang-1. Ang-2, or Tie-2 would be an
`attractive target for anti-cancer therapy.
`The fusion of certain peptides to a stable plasma protein
`Such as an Ig constant region to improve the half-life of these
`molecules has been described in, for example, PCT publi
`cation WO 00/24782, published May 4, 2000.
`The fusion of a protein or fragment thereof to a stable
`plasma protein such as an Ig constant region to improve the
`half-life of these molecules has been variously described
`(see, for example, U.S. Pat. No. 5,480,981; Zheng et al., J.
`Immunol. 154:5590–5600, (1995); Fisher et al., N. Engl. J.
`Med., 334:1697–1702, (1996); Van Zee, K. et al., J. Immu
`mol. 156:2221–2230, (1996); U.S. Pat. No. 5,808,029,
`25
`issued Sep. 15, 1998; Capon et al., Nature, 337:525-531,
`(1989); Harvillet al., Immunotech. 1:95-105, (1995); WO
`97/23614, published Jul. 3, 1997; PCT/US 97/23183, filed
`Dec. 11, 1997: Linsley, J. Exp. Med., 174:561-569, (1991);
`WO95/21258, published Aug. 10, 1995).
`An effective anti-Ang-2 therapy might benefit a vast
`population of cancer patients because most solid tumors
`require neovascularization to grow beyond 1–2 millimeters
`in diameter. Such therapy might have wider application in
`other angiogenesis-associated diseases as well. Such as ret
`inopathies, arthritis, and psoriasis.
`There is an undeveloped need to identify new agents that
`specifically recognize and bind Ang-2. Such agents would
`be useful for diagnostic screening and therapeutic interven
`tion in disease states that are associated with Ang-2 activity.
`Accordingly, it is an object of the present invention to
`provide specific binding agents of Ang-2 that modulate
`Ang-2 activity. Such agents of the present invention take the
`form of peptibodies, i.e., peptides fused to other molecules
`Such as an Fc domain of an antibody, where the peptide
`moiety specifically binds to Ang-2.
`
`5
`
`10
`
`15
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`30
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`US 7,138,370 B2
`
`4
`In still another embodiment, the invention provides
`nucleic acid molecules encoding the peptibodies, as well as
`variants and derivatives thereof. Optionally, such nucleic
`acid molecules include SEQ ID NO:33–SEQ ID NO:53.
`In still another embodiment, the invention provides a
`method of decreasing a tumor by administering an effective
`amount of the specific binding agents of the present inven
`tion to a subject in need thereof. The invention also provides
`a method of inhibiting angiogenesis in a Subject, comprising
`administering an effective amount of the specific binding
`agents of the present invention to a subject in need thereof.
`The invention further provides a method of treating cancer
`in a subject, comprising an effective amount of the specific
`binding agents of the present invention to a subject in need
`thereof.
`The invention also relates to a polypeptide capable of
`binding Ang-2 wherein the polypeptide comprises the amino
`acid sequence WDPWT (SEQ ID NO: 65), and wherein the
`polypeptide is from 5 to 50 amino acids in length, as well as
`physiologically acceptable salts thereof. The polypeptide
`can also comprise the amino acid sequence:
`WDPWTC
`(SEQ ID NO: 66)
`
`and physiologically acceptable salts thereof. Additionally,
`the polypeptide can comprise the amino acid sequence:
`CZ2WDPWT
`(SEQ ID NO: 67)
`wherein z is an acidic or neutral polar amino acid residue,
`and physiologically acceptable salts thereof. The polypep
`tide can further comprise the amino acid sequence:
`CZ2WDPWTC
`(SEQ ID NO: 68)
`wherein z is an acidic or neutral polar amino acid residue,
`and physiologically acceptable salts thereof.
`In another embodiment, the invention relates to a
`polypeptide capable of binding Ang-2 comprising an amino
`acid sequence of the formula:
`aa2a3CaWDPWTCa2a1a14
`
`(SEQ ID NO: 69)
`
`wherein:
`a', a. and a are each independently amino acid residues;
`a is an amino acid residue.
`a' is absent or an amino acid residue.
`a' is absent or a neutral hydrophobic, neutral polar, or a
`basic amino acid residue;
`a' is a neutral hydrophobic or neutral polar amino acid
`residue;
`and physiologically acceptable salts thereof. In a pre
`ferred embodiment:
`a' is V. I. P. W. G. S., Q, N, E, K, R, or H:
`a is V. P. M. G. S., Q, D, E, K, R, or H:
`a is A. V. P. M. F. T., G, D, E, K, or H:
`a is A. V. G., Q, N, D, or E:
`a' is S, Q, N, D, E, K, or R:
`a' is L., T, or H; and
`a' is V, L, I, W, or M.
`In a more preferred embodiment, a' is Q, a is E; a is E:
`a is D or E; a' is D or E; a' is H; and a' is M.
`It will be appreciated that the use of lower case letters
`with superscripted numbers herein (such as a' and b) are
`intended to identify amino acid positions, and are not meant
`to indicate the single letter abbreviations for a given amino
`acid. Single letter amino acid abbreviations are given in
`upper case letters herein.
`
`SUMMARY OF THE INVENTION
`
`The present invention is directed in one embodiment to
`peptides (also referred to as polypeptides herein) that bind to
`Ang-2. Also embodied in the present invention are variants
`and derivatives of Such peptides.
`In another embodiment, the peptides and variants and
`derivatives thereof of the present invention are attached to
`vehicles.
`In another embodiment, the peptides may be fused to Fc
`domains, thereby providing peptibodies. Optionally, the
`peptibodies comprise at least one peptide of for example,
`SEQ ID NO:3 SEQ ID NO:6, or SEQ ID NO:76 SEQ ID
`NO:157, as well as variants and derivatives thereof. Further,
`the peptides may comprise at least one peptide according to
`the formulae set forth in SEQ ID NO:65-SEQ ID NO:75,
`and SEQ ID NO:158.
`In yet another embodiment, the invention provides
`nucleic acid molecules encoding the specific binding agents,
`and variants and derivatives thereof.
`
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`

`5
`The invention further relates to a polypeptide capable of
`binding Ang-2 comprising an amino acid sequence of the
`formula:
`blb2bbbbéCb8wDPWTCb1b16b 7b18b 19520
`
`(SEQ ID NO: 70)
`
`wherein:
`b' is absent or an amino acid residue;
`b is absent or a neutral hydrophobic, neutral polar, or a
`basic amino acid residue;
`b,b,b, and b are each independently absent or amino
`acid residues;
`b is an amino acid residue.
`b' is absent or an amino acid residue.
`b' is absent or a neutral hydrophobic, neutral polar, or a
`basic amino acid residue;
`b'' is absent or a neutral hydrophobic or neutral polar
`amino acid residue;
`b', b', and bare each independently absent or amino
`acid residues; and physiologically acceptable salts
`thereof. In a preferred embodiment:
`b' is absent, or A. V. L. P. W. F. T. G. S., Q, N, K, R, or
`H;
`b' is absent, or A. V. L., I, P. W. M. T. G., S.Y. N, K, R,
`or H:
`b is absent, or A. L., I, P. W. M. T. G. S. Q, N, E, R, or
`H;
`b is V, I, P. W. G. S. Q, N, E, K, R, or H;
`b is V. P. M. G. S., Q, D, E, K, R, or H:
`b is A. V. P. M., F, T, G, D, E, K, or H;
`b is A. V. G., Q, N, D, or E:
`b' is S. Q, N, D, E, K, or R:
`b' is L., T, or H:
`b7 is V. L., I, W, or M:
`b' is absent, or A. V. L. P. W. F. T. G. Y. Q, D, E, or
`R;
`b' is absent, or V. L., I, P. T. G. S. Y. Q, N, D, E, or R:
`and
`b' is absent, or V. L. P. W. M. T. G. S. Y, Q, N, D, K,
`or R.
`In a more preferred embodiment, b" is absent, or P or T.
`b is absent, or I, or N; b is absent, or R, or I; b is Q. b
`is E; b is E; b is D or E; b' is D or E; b' is H: b7 is M:
`b' is absent, or W, or P; b is absent, or G, or E; and b
`is absent, or V, or K.
`It will also be appreciated that the invention preferably
`relates to a polypeptide comprising at least one amino acid
`sequence selected from the group consisting of SEQID NO:
`4, and SEQ ID NO: 76 to SEQ ID NO: 118, inclusive,
`wherein the polypeptide is capable of binding to Ang-2, as
`well as physiologically acceptable salts thereof. The peptide
`sequences are set forth below:
`
`TABLE 1.
`
`PEPTIDE
`
`SEQ ID NO. PEPTIDE SEQUENCE
`
`10
`
`15
`
`25
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`30
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`35
`
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`
`US 7,138,370 B2
`
`6
`
`TABLE 1-continued
`
`PEPTIDE
`
`SEQ ID NO. PEPTIDE SEQUENCE
`
`Con4-41
`Con4-36
`Con4-34
`Con4-28
`Com4-39
`Con4-25
`Com4-50
`Con4-38
`Con4-29
`Con4-47
`Con4-20
`Con4-45
`Com4-37
`Con4-33
`AC2-Con4
`Con4-32
`Con4-17
`Con4-8
`AC4-Con4
`Con4-1
`Con4-C1
`Con4-21
`Con4-C2
`Con4-18
`Con4-19
`Con4-16
`Con4-11
`Con4-C4
`Con4-23
`Con4-15
`Con4-9
`TN8-Co4*
`
`88
`89
`90
`91
`92
`93
`94
`95
`96
`97
`98
`99
`OO
`O1
`O2
`O3
`O4
`05
`O6
`O7
`O8
`09
`10
`11
`12
`13
`14
`15
`16
`17
`18
`4
`
`NVRQEKCEWDPWTCEHMPVR
`KSGQVECNWDPWTCEHMPRN
`VKTQEHCDWDPWTCEHMREW
`AWGQEGCDWDPWTCEHMLPM
`PVNQEDCEWDPWTCEHMPPM
`RAPQEDCEWDPWTCAHMDIK
`HGQNMECEWDPWTCEHMFRY
`PRLQEECVWDPWTCEHMPLR
`RTTQEKCEWDPWTCEHMESQ
`QTSQEDCVWDPWTCDHMVSS
`QVIGRPCEWDPWTCEHLEGL
`WAQQEECAWDPWTCDHMVGL
`LPGQEDCEWDPWTCEHMVRS
`PMNQVECDWDPWTCEHMPRS
`FGWSHGCEWDPWTCEHMGST
`KSTQDDCDWDPWTCEHMVGP
`GPRISTCQWDPWTCEHMDQL
`STIGDMCEWDPWTCAHMQVD
`VLGGQGCEWDPWTCRLLQGW
`VLGGQGCQWDPWTCSHLEDG
`TTIGSMCEWDPWTCAHMQGG
`TKGKSVCOWDPWTCSHMQSG
`TTIGSMCQWDPWTCAHMQGG
`WVNEVVCEWDPWTCNHWDTP
`VVQVGMCQWDPWTCKHMRLQ
`AVGSQTCEWDPWTCAHLVEV
`QGMKMFCEWDPWTCAHIVYR
`TTIGSMCQWDPWTCEHMQGG
`TSQRVGCEWDPWTCQHLTYT
`QWSWPPCEWDPWTCQTVWPS
`GTSPSFCQWDPWTCSHMVQG
`QEECEWDPWTCEHM
`
`It will be appreciated that certain peptides and/or pepti
`bodies may contain the prefix “TN”, “TN8’, or “TN12, and
`that this prefix may or may not be present for a given
`peptibody. Thus, for example, the terms “TN8-Con4 and
`“Con4 are used interchangeably herein.
`In another embodiment, the invention relates to a com
`position of matter having the formula:
`(X)-F-CX),
`and multimers thereof, wherein:
`F" is a vehicle:
`X' and X’ are each independently selected from
`-(L)-P';
`-(L)-P'-(L)-P:
`-(L)-P'-(L)-P’-(L)-P; and
`-(L)-P'-(L), P’-(L)-P-(L)-P'.
`wherein one or more of P', P, P, and P each indepen
`dently comprise a polypeptide as described herein. For
`example, in a preferred embodiment, P', P, P, and P can
`each independently comprise a polypeptide of
`SEQID NO:3 to SEQID NO: 6, and/or SEQ ID NO: 76
`to SEQ ID NO: 157.
`In another embodiment, the composition of matter is of
`the formulae:
`
`Con4-44
`Con4-40
`Con4-4
`Con4-31
`Con4-CS
`Con4-42
`Con4-35
`Con4-43
`Con4-49
`Con4-27
`Con4-48
`Con4-46
`
`76
`77
`78
`79
`8O
`81
`82
`83
`84
`85
`86
`87
`
`PIRQEECDWDPWTCEHMWEV
`TNIQEECEWDPWTCDHMPGK
`WYEQDACEWDPWTCEHMAEV
`NRLQEVCEWDPWTCEHMENV
`AATQEECEWDPWTCEHMPRS
`LRHQEGCEWDPWTCEHMFDW
`VPROKDCEWDPWTCEHMYVG
`SISHEECEWDPWTCEHMQVG
`WAAQEECEWDPWTCEHMGRM
`TWPQDKCEWDPWTCEHMGST
`GHSQEECGWDPWTCEHMGTS
`QHWQEECEWDPWTCDHMPSK
`
`XI-Fl
`
`60
`
`O
`
`F-X2
`and physiologically acceptable salts thereof, where X', F,
`and X’ are as defined herein. In another embodiment, the
`composition of matter is of the formula:
`
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`

`US 7,138,370 B2
`
`7
`and physiologically acceptable salts thereof, where L', F",
`and P' are as defined herein. In yet another embodiment, the
`composition of matter is of the formula:
`
`and physiologically acceptable salts thereof, where L', F,
`P', P, and c and d are as defined herein. In still another
`embodiment the composition of matter is of the formula:
`
`and physiologically acceptable salts thereof. In a preferred
`embodiment, F is an Fc domain or fragment thereof.
`The invention further relates to a polypeptide capable of
`binding Ang-2 comprising an amino acid sequence of the
`formula:
`Pc2Dc Licéc7c8LY
`
`(SEQ ID NO: 71)
`
`d' is M, Q, E, or K:
`d is L or M:
`d is D or E:
`d' is E:
`d'' is Q or E:
`d' is T or R:
`d' Y, D, E, or K:
`d' is Q:
`d'7 is W or F:
`d' is L., I, M, or T:
`d' is L., F, or Y:
`d' is Q, D, or E:
`d' is absent, Q, or H:
`d’ is absent, A, L. G., S, or R.
`In a preferred embodiment, the polypeptide comprises at
`least one amino acid sequence selected from the group
`consisting of SEQ ID NO: 6, and SEQ ID NO: 119 to SEQ
`ID NO: 142, inclusive, wherein the polypeptide is capable of
`binding to Ang-2. SEQ ID NO: 6, and SEQ ID NOS:
`119–142 are set forth below:
`
`10
`
`15
`
`wherein
`c is a neutral hydrophobic amino acid residue
`c is a A, D, or E
`c' is an acidic amino acid residue
`c’ is an amino acid residue; and
`c is a neutral hydrophobic, neutral polar, or basic amino
`acid residue; and physiologically acceptable salts
`thereof. In a preferred embodiment, c is L or M. In
`another preferred embodiment, c' is D or E.
`The invention further relates to a polypeptide capable of
`binding Ang-2 comprising an amino acid sequence of the
`formula:
`did2d3d P6DSLdlodild 12LY
`did 1617, 1818, 19202122
`
`25
`
`30
`
`(SEQ ID NO: 72)
`
`wherein,
`d' is absent, or an amino acid residue.
`d is absent, or a neutral polar, acidic, or a basic amino
`acid residue;
`d is absent, or a neutral hydrophobic or neutral polar
`amino acid residue;
`d' is absent, or an amino acid residue.
`d is a neutral hydrophobic amino acid residue;
`d is a A, D, or E:
`d" is an acidic amino acid residue:
`d' is an amino acid residue:
`d' is a neutral hydrophobic, neutral polar, or basic amino
`acid residue;
`d' is absent, or a neutral polar, acidic, or a basic amino
`acid residue;
`d' is absent, or a neutral polar, acidic, or a basic amino
`acid residue;
`d' is absent, or a neutral hydrophobic, or neutral polar
`amino acid residue;
`d' is absent, or a neutral hydrophobic, or neutral polar
`amino acid residue;
`d' is absent, or a neutral hydrophobic, neutral polar, or
`basic amino acid residue;
`d' is absent, or an amino acid residue.
`d' is absent, or a neutral polar, acidic, or a basic amino
`acid residue;
`d’ is absent, or a neutral hydrophobic, neutral polar, or
`basic amino acid residue;
`and physiologically acceptable salts thereof. In a preferred
`embodiment:
`d' is T, S, Q, R, or H:
`d is T, Q, N, or K:
`d is F:
`
`35
`
`40
`
`45
`
`50
`
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`
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`
`65
`
`Peptide
`
`SEQ ID NO. Peptide Sequence
`
`L1-1
`L1-2
`L1-3
`L1-4
`L1-5
`L1-7
`L1-9
`L1-10
`L1-11
`L1-12
`L1-13
`L1-14
`L1-15
`L1-16
`L1-17
`L1-18
`L1-19
`L1-20
`L1-21
`L1-22
`AC6-L1
`L1-C1
`L1-C2
`L1-C3
`
`19
`2O
`21
`22
`23
`24
`25
`26
`27
`28
`29
`30
`31
`32
`33
`34
`35
`36
`37
`38
`39
`40
`41
`42
`6
`
`QNYKPLDELDATLYEHFIFHYT
`LNFTPLDELEQTLYEQWTLQQS
`TKFNPLDELEQTLYEQWTLQHQ
`VKFKPLDALEQTLYEHWMFQQA
`VKYKPLDELDEILYEQQTFQER
`TNFMPMDDLEQRLYEQFILQQG
`SKFKPLDELEQTLYEQWTLQHA
`QKFQPLDELEQTLYEQFMLQQA
`QNFKPMDELEDTLYKQFLFQHS
`YKFTPLDDLEQTLYEQWTLQHV
`QEYEPLDELDETLYNQWMFHQR
`SNFMPLDELEQTLYEQFMLQHQ
`QKYQPLDELDKTLYDQFMLQQG
`QKFQPLDELEETLYKQWTLQQR
`VKYKPLDELDEWLYHQFTLHHQ
`QKFMPLDELDEILYEQFMFQQS
`QTFQPLDDLEEYLYEQWIRRYH
`EDYMPLDALDAQLYEQFILLHG
`HTFQPLDELEETLYYQWLYDQL
`YKFNPMDELEQTLYEEFLFQHA
`TNYKPLDELDATLYEHWILQHS
`QKFKPLDELEQTLYEQWTLQQR
`TKFQPLDELDQTLYEQWTLQQR
`TNFQPLDELDQTLYEQWTLQQR
`KFNPLDELEETLYEQFTFQQ
`
`The invention also relates to a polypeptide capable of
`binding Ang-2 comprising an amino acid sequence of the
`formula:)
`RPeeeee.7G
`
`(SEQ ID NO: 73)
`
`- - -
`
`wherein
`e is a neutral polar amino acid residue,
`3
`e' is an acidic amino acid residue:
`e is a neutral polar or an acidic amino acid residue.
`e' is a neutral hydrophobic amino acid residue.
`7 e' is a neutral hydrophobic amino acid residue,
`and physiologically acceptable salts thereof. In a preferred
`embodiment, e is Y or C. In another preferred embodiment,
`e' is D or E. In still another preferred embodiment, e” is I or
`M.
`The invention further relates to a polypeptide capable of
`binding Ang-2 compri